CONTENTS 1.

HAEMATOLOGY
a. Instruction for Phlebotomist b. Blood Grouping & RH c. DU testing d. ESR Erythrocyte Sedimentation Rate e. CBC Complete Blood Count f. Urine Complete Analysis g. Malarial Parasite

2. BIOCHEMISTRY
a. Glucose b. HBA1c c. Cholesterol d. Triglycerides e. HDL Cholesterol f. LDL Cholesterol g. Urea h. Creatinine i. Albumin j. Total Protein

k. Albumin/Globulin Ratio l. Alkaline Phosphatase m. Aspartate Amintransferase SGOT n. Alanine Aminotransferase SGPT

o. Total Bilirubin p. Direct Bilirubin q. Indirect Bilirubin r. Folic Acid s. Gamma Glutamyl Transferase

3. SEROLOGY
a. Widal Slide b. Tuberculin Diluted Mantoux c. Serum Electrophoresis d. ANA HEp-2 e. Anti HSV Pool ELISA (IgG) f. Dengue IgG Capture ELISA g. Anti HBc IgM h. Syphilis i. Syphilis Rapid Plasma Reagin (RPR) card test j. Principles of Chemiluminescence

4. HISTOPATHOLOGY

1.

HEAMATOLOGY

INSTRUCTION FOR PHLEBOTOMIST

General Instructions Approach the patient pleasantly, confidently and in a friendly manner. Inform the patient that blood is going to be drawn from him/ her and that it will not hurt and get his /her confidence and co-operation. If possible, speak with the patient during the procedure so that the patient is at ease and will be less focused on the procedure. Always thank the patient and excuse yourself courteously when finished. Collect the Test Requisition Form for individual patients from the front office and verify if the TRF contains all the necessary details (Name, age, sex, SID number of the patient, tests requested, name of the referral doctor & clinical diagnosis date and time of registration). Call the patient by name adding Mr / Mrs / Miss and ensure the person answering the call is same as in Test Requisition Form. Blood Collection Ensure all consumables and materials required for venipuncture are available before starting the procedure. Use only 19 22 G needle for venipuncture and 23 G scalp vein set for children and infants. Ensure that syringe size is determined on the basis of the volume of blood to be drawn. Use either 2cc, 5cc or if necessary 10 cc. Use only disposable syringes and needles. Verify, before collecting the sample, if the patient has followed the instructions regarding preparation, if any, required for the tests, if not inform the patient / attendant suitably after verifying with Master Test List and issue a Test Instruction Slip wherever necessary. Write the ID, Name and Test Requested on the tube/ Container in to which sample is to be collected. Wear gloves before proceeding for vein puncture. Selection of Venipuncture Site Select a vein that looks and feels fullest. Use the median cubital vein at the bend of the elbow or the cephalic or basillic vein in the elbow.

Wrist veins are also acceptable for venipuncture. Do not select the following areas for venipuncture. Healed burn areas. Areas with hematoma. Veins over a scar from surgery or burn. The upper extremity on the side of a previous mastectomy - test results may be affected because of lymphedema. Do not perform veni puncture from a vein with Intravenous fluid line (IV) / blood transfusions IV fluid may dilute the specimen, so collect from the opposite arm if possible. Otherwise, follow the procedure given below to draw satisfactory samples below the IV by following these procedures: Turn off the IV for at least 2 minutes before venipuncture. Apply the tourniquet below the IV site. Select a vein other than the one with the IV.

Preparation for Venipuncture Clean selected site of venipuncture with 70% isopropyl alcohol / rectified spirit and allow it to air dry. Cleanse venipuncture site in a circular fashion, beginning at the site and working outward. Position the patient. The patient should either sit in a chair or lie down or sit up in bed. And then hyperextend the patient's arm. Ask the patient to make a fist. Apply the tourniquet. Ensure that the patient is not hurt by this procedure. Use tourniquet of specified width [1 inch / 2.5 cm] and length [18 inches / 38 cm] and use Velcro type (for adults). Do not apply tourniquet within 4 to 6 inches of the venipuncture site. Ensure that the patient does not pump his / her fist or bend the elbows when tourniquet is used. Do not apply tourniquet for more than one minute while drawing blood. As soon as blood enters the syringe release the tourniquet. Palpate and trace the path of veins with the index finger. Select the vein that looks and feels fullest. If superficial veins are not readily apparent, force blood into the

vein by massaging the arm from wrist to elbow, tap the site with index and second finger, apply a warm, damp washcloth to the site for 5 minutes, or lower the extremity over the bedside to allow the veins to fill. Grasp the patient's arm firmly using thumb to draw the skin taut and anchor the vein. The needle should form a 15 to 30 degree angle with the surface of the arm. Swiftly insert the needle through the skin and into the lumen of the vein. Avoid trauma and excessive probing. Do not ask the patient to pump the fist and do not use tourniquet while drawing blood sample for potassium, calcium or magnesium and Lithium estimation.

Order of draw Always draw blood in the following order to avoid cross-contamination of additives between tubes. The recommended order of draw is: First - blood culture tube. Second - Non-additive tube (Plain red stopper). Third - coagulation tube (light blue stopper). Never draw blood first in a a light blue stopper (sodium citrate).If a coagulation assay is the only test ordered, draw a nonadditive tube (red stopper) first, and then draw the light blue stopper tube. Last draw - additive tubes in this order: EDTA (lavender stopper). Oxalate/fluoride (light gray stopper).

Transfer the blood quickly but gently in to collecting tubes Avoid vigorous suction of blood into the syringe and forceful ejection of blood from the syringe into the collecting tube. Gently but thoroughly mix blood with anti coagulant by inverting 5- 8 times or by using a cyclomixer. Remove the needle from the patient's arm using a swift backward motion. Press down on the gauze once the needle is out of the arm, applying adequate pressure to avoid formation of a hematoma. After blood is drawn cut the needle with needle cutter and dispose it in 1 in 50 Sodium hypochlorite solution.

Specific Instructions If fasting is required for any of the tests verify whether the patient is fasting for a period of 12 hours.

Collect fasting blood sample for all lipid Profile tests, all types of GTT, Fasting Plasma Glucose, Serum Insulin, C-Peptide, Intact PTH). For post prandial plasma glucose, collect blood sample 2 hours after a meal unless the doctor requests 1 hour post prandial plasma glucose. Note the timing of collection of the sample in the Sample Collection Register (PB/ SCR). Ensure that only proper vacutainer tubes are used. Plain tube Red topped K2EDTA tube Lavender topped Sodium Citrate Blue topped Sodium Fluoride / Na2 EDTA Grey topped

For the following tests collect 3 ml of venous blood in Lavender color topped vacutainer tubes containing 5.4 mg of K2EDTA. a. Complete blood count, AEC, Absolute Reticulocyte count, peripheral smear, Granulocyte Count, Haemoglobin, Haemocrit b. MP & MF c. Blood grouping & Rh typing d. Glycosylated Haemoglobin (HbA 1 C) e. Direct Coombs test

For prothrombin time, activated partial thromboplastin time draw 2.7 ml of venous blood in light-blue topped vacutainer tubes containing 0.3 ml of 0.105 M (3.2 %) sodium citrate. For all other tests collect 3 ml of venous blood in Red topped plain vacutainer tubes. For the patients coming for free T3, free T4 and TSH tests for the first time, collect venous blood at random and instruct them on fasting for 12 hours overnight and avoid morning dose of thyroid medication while coming for the follow up. Centrifuge and separate the plasma/serum in collection centre itself for the following tests: PT aPTT Label the tubes with ID number of the patient.

..1 ..2

Specimen handling

After collection, the specimen must be processed in a timely manner to ensure

accurate results.

 After labeling, seal the specimen in the zipper pocket and place in a transport bag
incase, if the specimen has to be transported. Place the test requisition form and any other associated paperwork in the outer pocket. Utilize the tube system or hand-deliver the specimen to the laboratory immediately.

BLOOD GROUPING & RH

To determine the blood group and Rh type in human

Principle: This test is based on the principle of direct haemagglutination. The erythrocytes of a
person contain antigens on the surface of the membrane. When these antigens are allowed to react with the corresponding antibodies, antigen antibody reactions are produced. Normal erythrocytes will clump or agglutinate when mixed with Anti A/ Anti B/ Anti A & B, if they possess A / B / AB antigens respectively.

Primary Sample: Use whole blood as specimen Consumables / Reagents:

Anti A, blood grouping reagent (Commercially available). Anti B, blood grouping reagent (Commercially available). Anti AB, blood grouping reagent (Commercially available). Anti D, blood grouping reagent (Commercially available). Anti A1 Lectin (Commercially available). Glass slides. Applicator sticks. 0.9% isotonic saline.

Slide Agglutination Method: Take a clean, grease free microscope slide. Draw a line in the middle of this slide using a wax glass marking pencil and label the left portion Anti-A and the right as Anti-B. Add a drop of Anti-A to the portion marked as Anti-A, and add a drop of Anti-B to the portion marked as Anti-B. Add one drop of well mixed 3 - 5% cell suspension to each side. With a tooth pick, separate for each side, mix the cells and anti-sera well. Gently rotate the slide for mixing. Place the slide against a white background. After 2 minutes, examine both macroscopically and microscopically for agglutination.

Tube Agglutination Method: Switch on the water bath and adjust the temperature to 37C. Take two tubes of size 12 x 100 mm. Stick a label with patients name, MRD No., Lab No. on each tube. Mark one tube as anti A and the other as Anti B. Add two drops of Anti-A to the tube labelled as Anti-A and two drops of Anti-B to the tube labelled Anti-B. Add two drops of 3 - 5% cell suspension to each tube. Mix well and centrifuge both tubes at 1500 rpm for 1 minute. Remove the tubes and inspect the button of the cells in the bottom. With gentle shaking or tapping of the tube, observe the dislocation of the cell button. Report - Positive if agglutination is observed. Place the tube / tubes in which no agglutination was observed in 37C water bath for 30 minutes. Remove the tubes from the water bath and centrifuge once again and observe the cell button by shaking the tube gently. Report as positive if agglutination is observed. Double check the results microscopically by putting one or two drops from each tube on microscopic slides. Prepare a 3-5% suspension of test red cells. All suspensions may be prepared in autologous serum/ plasma /saline. Place one drop of the appropriate A, B, AB blood grouping reagent on a clean, dry glass slide at room temperature. Add one drop of the prepared 35-45% suspension of red cells to each drop of reagent on the glass slide. Mix the cells and reagent thoroughly over an approximate 20 mm circular area, using a separate, clean applicator stick, one for each reagent - red cell mixture. Rock or rotate slides gently and examine for macroscopic haemagglutination. Agglutination may begin within a few seconds. However observation should not continue beyond 2 minutes.

Interpretation of Results: If agglutination is observed with anti A blood group reagent, then the patients blood
group is A. If agglutination is observed with anti A1 Lectin reagent, then the patients blood group is A1. If agglutination is observed with anti B blood group reagent, then the patient's blood group is B. If agglutination is observed with both anti A and Anti B blood group reagent, then the patient's blood group is AB.

 If agglutination is not observed with both anti A and Anti B blood group reagent then the patients blood group is O. If agglutination is observed with anti D blood group reagent, then the patients Rh type is Positive. All Rh negative is reconfirmed with Du testing.

Precautions:
Thoroughly clean and dry the test slides before use. Ensure reagents and specimens are at room temperature before use.

Safety Precautions:
Handle all reagents with care and avoid contact with eye, mouth and skin. Handle all samples as potentially infectious. Discard used reagents and sample as per disposal procedure.

Potential Source of Variability:

False negative or unexpectedly weak reactions may occur with blood samples of weak A or B subgroups or with cord blood red cells from newborn infants. False negative or unexpectedly weak reactions may occur with red cells or anti sera that have been subject to prolonged storage and / or inappropriate storage conditions.

DU TESTING
The Du factor (a variant of D antigen present in the red cells of individuals of Du blood type) reacts with anti D but does not bring about haemagglutination that is not strong enough to be visualized. This test is done to reconfirm all Rh negative blood groups

Principle: Cells with Du antigen are sensitized with anti D by incubating at 37oC for 30
minutes. This results in the adsorption of anti D on the surface of the cell without producing haemagglutination. The presence of reacted antibody on the surface of Du cells is recognized by using antihuman globulin which reacts with coated antibody and brings about haemagglutination.

Primary Sample :
Use whole blood as specimen.

Consumables / Reagents :
Slides. Pipettes & tubes. Anti D (Monoclonal IgM + IgG). Antihuman globulin. Normal saline.

Procedure :
Prepare 5% suspension of washed red cells with normal saline. Take 1 tube and label it as test (T). Place 2 drops of 5% cell suspension in the tube. Add 1 drop of anti D in the tube labeled T. Place the tube in the water bath at 37oC for 30 minutes. Remove the tubes from the water bath and wash the cells in normal saline 2 3 times. Add 2 drops of anti human globulin to both the tubes. Mix gently. Centrifuge the tubes at 1500 rpm for 1 minute and look for agglutination.

Interpretation of results:
Du positive: If agglutination is present in the tube labeled T. Du negative: If no agglutination seen in the tube T.

ERYTHROCYTE SEDIMENTATION RATE-ESR

Quantitative determination of the rate at which the red cells sediment by the Westergrens Method. ESR is a non-specific test and is influenced by levels of plasma proteins especially globulin, fibrinogen and albumin. ESR is increased in all conditions where there is tissue breakdown or when foreign proteins enter the blood. It reflects changes in plasma protein concentrations, which accompany most of the acute and chronic infections. Conversely, normalization of the ESR in indicates possible recovery from the diseased state. Principle: When the anti coagulated blood is taken in a tube and left undisturbed in a vertical position, the erythrocytes tend to settle down at the bottom. The level of the column of red cells is noted in the beginning (0 hr) and after 1 hr. The first is the stage of aggregation where the red cells form rouleaux. This is followed by the stage of sedimentation in which the falling of the red cells takes place. The larger the aggregation in the first stage, the faster the rate of fall. Two layers are formed, the upper plasma layer and the lower one of red cells. The distance (mm) the column has moved is the erythrocyte sedimentation rate. Performance Specification: The Westergren tube must be exactly in a vertical position while taking the reading and during the sedimentation period. Tilting of the tube from the vertical position may increase the ESR ] Test should be performed within 4- 6 hours of collection as delay will result in rapid decrease in sedimentation Ambient temperature affects ESR values. If the ambient temperature is high, falsely elevated ESR values will be obtained and conversely low values at low temperatures. Hence the specimen has to be brought to room temperature before setting up for the test.

Concentration of anticoagulant affects the ESR results. If the concentration of the anticoagulant is high falsely low ESR values will be reported. Primary Sample: Use citrated whole blood as specimen (sodium citrate anti-coagulant) Reagents / Consumables: Westergrens ESR tubes / Glass tubes 100 mm X 10 mm. Westergren tube. Timer / Stop Watch. Rack or Stand for holding the tube. Procedure: Set the Westergren tube rack / stand exactly vertical on a level surface and away from any vibration or air draft source. Fill the Westergren tube with the blood sample blood exactly up to 0 mark by means of a rubber bulb. Place the Westergren tube up right and exactly vertical in the rack / stand so that the tube fits exactly in to the groove of the stand. Note the time. Start the stop watch. Allow to stand exactly for 60 minutes. Note the level to which the red cell has fallen at the end of 30 minutes & 60 minutes. Report the result in mm in 1 hour. Safety Precautions: Do not perform mouth pipetting of the blood sample in to the ESR tube Handle all reagents with care and avoid contact with eye, mouth and skin. Handle all samples as potentially infectious. Discard used reagents and sample as per disposal procedure Potential Sources of Variability: The Westergren tube must be exactly in a vertical position while taking the reading and during the sedimentation period. Tilting of the tube from the vertical position may increase the ESR Ambient temperature affects ESR values. If the ambient temperature is high, falsely elevated ESR values will be obtained and conversely low values at low temperatures. Hence the specimen has to be brought to room temperature before setting up for the test. Concentration of anticoagulant affects the ESR results. If the concentration of the anticoagulant is high falsely low ESR values will be reported.

Presence of bubbles / fibrin clots in the column of blood in the ESR tube leads to erroneous results and as hemolysis. Improperly cleaned tubes affect ESR results. Use only clean dry tubes washed & rinsed in deionised water.

Reference Range: Male : <10mm/1hour. Female : <20mm/1hour. Interpretation : Increased sedimentation rates are found in: All collagen disease (SLE), Infections, pneumonia, syphilis, inflammatorydisease, toxemia, anemia, nephritis, nephrosis. Normal sedimentation rate ( No increase ) : Polycythemia vera, hyperfibrinogenemia, sickle cell anemia, congestive heart failure, hereditary sperocytosis, pyruvate kinase deficiency.

COMPLETE BLOOD COUNT AUTOMATED HEMATOLOGY ANALYSER

Quantitative enumeration of different formed elements (cells) of blood and Hemoglobin in whole blood by ADVIA 60 OT (Open Tube) automated Hematology cell counter by the principle of Impedance Variation. ADVIA -60 OT Automated Hematology System enumerates the following parameters, o Total RBC count. o Total WBC count. o Differential WBC Count both absolute and % (3 part Differentiation i.e. counts SW Lymphocytes, MW Monocytes, LW Granulocytes). o Hemoglobin. o Packed Cell Volume (PCV). o Mean Corpuscular Volume (MCV). o Mean Corpuscular Hemoglobin (MCH). o Mean Corpuscular Hemoglobin Concentration (MCHC). o Platelet count. o RBC Distribution Width (RDW). It is the enumeration of cellular elements of blood, evaluation of red cells indices and determination of cell morphology by means of stained smears. The various parameters generated from the automated hematology analyzer are of clinical importance to differentiate between diseases of hematological abnormalities. Reference values vary across the life cycle and between sexes and differ in different age groups and in various disease conditions.

Principle:
RBC, WBC, Platelets: White blood cells, red blood cells and platelets are counted by the principle of impedance variation this principle takes advantage of the fact that blood cells (RBC, WBC, Platelets) are less conductive to electrical current than the cell diluting fluid. It is based on the principle that blood cells are less conductive to electric current than the cell diluting fluid. The number of cells is quantitatively determined by passing a metered volume of a cell suspension (by diluting the blood sample in electrolytic diluents) between two electrodes placed on either side of the aperture. The current passes between the two electrodes through the diluent. When the cell passes through the aperture, electric resistance (or impedence) between the two electrodes increases proportionately with the cell volume which is registered as a pulse which is amplified and allows the cell to be counted. The magnitude of each pulse is proportional to the size of the particle. The distribution of magnitude of pulses according to particle size of the cell population is printed as a Histogram. Only electronic impulses of a size falling within a threshold limit are registered as cell particles while pulses from particles with a size beyond the fixed limit are not counted. WBCs are counted after lysing the RBCs by a detergent (ionic or non ionic) which does not lyse the WBCs themselves. After dilution (1: 50 for WBC count and Hb 1: 50,000 for RBC count Indices and platelet count) the diluted sample is directed to two sides of the instrument and particles on the red cells side that are larger than 36 fl are counted as RBCs. Cells from 2- 20 fl are counted as platelets. For WBC counting lysing reagent is added to the sample in the system to lyse RBCs and causes differential shrinking of WBCs, thereby allowing them to be separated in to a partial differential (3 part differential) and white cells are shown in 3 groups Lymphocytes (35- 90 fl) mononuclear cells (90 -160 fl ) and granulocytes ( 160 -450 fl). Hct is calculated based on RBC pulse height value it is measured by height of impulse generated by a passage of cells through a micro aperture, which is directly proportional to the volume of analyzed cells. RBCs are lysed and the freed haemoglobin combines with potassium cyanide to form cynmethhaemoglobin, which is measured by Spectrophotometry at 450 nm. MCV, MCH and MCHC are calculated from RBC count Hb and Hct. MCV: calculated from RBC and Hct. MCH: Calculated from Hb and Hct. MCHC: calculated from Hb and Hct. RDW: Calculated from deviation of RBC Distribution Width.

Performance specification:
Linearity WBC Minimum detection range 80 ( 80, 000 Cells / 0.5 80 (500 to 80,000 Cells / 0.5 (500 Cells / cu Measurement Range

( X 103 Cells / L) RBC ( X 106 Cells / L) Platelets ( X 103 Cells / L) Hb (g/dL) Hct (%)

cu mm)

cu mm)

mm)

7.5 (7.5 million cells 0.2 7.5 ( 0.2 - 7.5 million 0.2 ( 0.2 million cells / cumm) cells / cumm) / cumm) 1000 (10,00,000 10 1000 (10, 000 - 10,00,000 10 (10,000 cells / cu cells / cu mm) cells / cu mm) mm) 23 g /dL 55 2.5 23 g /dL 11.6 - 55 2.5 g /dL 11.6

Primary Sample:
Use whole blood as sample collected in K3 EDTA.

Instrument:
Advia 60 OT Automated Hematology Cell Counter. Hemomixer.

Step by Step Procedure:

Switch on the instrument by pressing the ON/OFF Switch Located in the rear panel of the instrument. Wait for Approximately 3 min for the initialisation process to be completed. Change of Front Panel LED from Red To Green indicates that the initialisation process is completed. Press the start up key and the instrument automatically performs a blank cycle for a background count. Process QC / patient samples when the background limits are within the acceptable range. Run any one of the Blood samples to prime the instrument. Mix the sample thoroughly but gently using the hemomixer just prior to processing the sample. Enter the patient ID or run # using the <ID> keys after verifiying the ID on the sample matches with that on the Requisition Form. Present the sample after gentle and through mixing under the sampling probe. Press the < START> key on the front panel or the sampling bar located behind the sampling needle. The Green - Red LED Starts Blinking. Remove the sample tube when the LED Stops blinking. Result will be displayed on the LCD screen.

Enter the results in the Daily Results Register Haematology.

Quality Control Procedure:

Run all three levels of an assayed QC sample (Bio-rad and ABX PNTRA Three Level Quality Control samples - Low, Normal and High) and verify if the values are close to the Mean and within the reference range mentioned in the QC product insert. If the QC is not within the reference range even for any one level of QC, perform another cleaning procedure and rerun that level of QC.

Reference Range :
Parameter WBC count RBC count Reference Range Adults 4000-10,000 cells/cu.mm

Male : 4.5 to 6.0 x 106cells/cu.mm Female: 4.0 to 4.5 x 10 6 cells/cu.m

Haemoglobin Platelet count Haematocrit MCV MCH

Men 14 18g/100ml Women 12-16 g/ 100ml 250,000 500,000/CU.MM

Male: 42 52 Female 36 48%

70-90 microns 14-18 gm/dl

MCHC

45-55%

Potential Sources of variability: The Following factors will affect cell counting in an
automated hematology analyzer. Visible clots Fibrin micro clots or cryoprecipitate in the specimen. Inadequate lysis of RBC. Inadequate dilution of specimen and inadequate calibration of the analyzer. Non Homogenous suspension of blood cells in the diluting fluid. Deterioration of diluting solution. Presence of dust particles in the diluent.-Ensure the diluents and buffer solutions. Used are free of dust particles especially for platelet counting. Partial or total obstruction of the aperture. Wrong threshold setting of the instrument. Carry over from one to next measurement. Fluctuation of electric current.

Safety Precautions:

Handle all reagents with care and avoid contact with eye, mouth and skin. Handle all samples as potentially infectious. Discard used reagents and sample as per disposal procedure.

URINE COMPLETE ANALYSIS

Combur Test provide semi-quantitative determination of Specific Gravity, pH, Leucocytes, Nitrites, Protein, Glucose, Ketone bodies, Urobilinogen, Bilirubin and Bloods in urine by visual comparison to a color chart or by use of the Combur Test. For evaluation by reflectancephotometry with urisys 1100 and for visual reading. The nature and amount of substance present in urine reflect ongoing physiological process in health and disease status

Principle:
Specific Gravity: The test detects the ion concentration of urine. In the presence of two indicators methyl red & bromothymol blue which give colours changes ranging from blue- green to orange with increasing low ionic concentration. pH: This test is based on the double indicator principle that gives a broad range of colors covering the entire urinary pH range. The test strip contains a combination of methyl-red (pH range 5.0-6.0.) and bromothymol- blue (pH range 8.0-9.6) as indicators. The color gradations extend form orange via green to blue. Depending upon the pH of urine the color of the reaction area changes from orange / yellow / green / blue Leukocyte: The test is for granulocytic leucocytes The test area for granulocytic leucocytes contains an indoxylcarbonic ester and a buffer ester, which is hydrolysed by granulocytic esterases. The indoxyl ester thus liberated then reacts with a diazonium salt to produce a bluepurple color. Nitrite: The p- arsanillic acid contained in the test area reacts in the presence of an acidic buffer with nitrite to form a diazonium compound. Together with a coupling component, this diazonium compound produces a pink color. Any degree of pink is considered positive. protein: The test is based on the color change of the indicator, tetrabromo thymol blue,in the presence of protein . A positive reaction is indicated by a color change from yellow which is negative to a green gold for a trace reaction through green & then to greenish-blue for elevated levels Glucose: The test area is impregnated with glucose-oxidase / peroxidase together with potassium iodide and a blue background dye. The oxygen liberated in the final reaction binds with the dye to produce a series of colour changes 30 seconds after wetting the strip with urine. Depending upon the concentration of glucose in urine the color of test area changes from bluegreen through green,brownish green to brown. Ketone bodies: The test is based on the reaction of ketone with nitroprusside. The resulting color ranges from tan when no reaction takes place to buffy pink through pink to purple for a positive reaction Urobilinogen: A stable diazonium salt 4-methoxybenzen diazonium reacts immediately with urobilinogen in the strong acid environment of the test to effect a change from tan to pink Bilirubin: The test for bilirubin is based on the coupling of bilirubin with a stable diazonium salt (2-4 dichloroanilinediazonium)in the acid environment of the test area of the strip.The color

ranges from tan to to orangish when no bilirubin is present through various shades of tan to reddish brown with increasing levels of billirubin. Blood (Erythrocytes, Haemoglobin): The test is based on the fact that peroxidase present in haemoglobin and myoglobin catalyze the oxidation of a colour indicator 3, 5, 35 Tetra methyl Benzenidine by an organic hydroperoxide to a blue-green dye that appears green on the original yellow test area of the strip.

Performance specifications
Specific gravity: o The specific gravity test permits determination of urine specific gravity between 1.000 and 1.030. In general, the specific gravity test correlates within 0.005 with values obtained with the reflective index method. o Highly buffered alkaline urine may cause low readings relative to other methods. o Elevated specific gravity readings may be obtained in the presence of moderate quantities (100-750 mg/dl) of protein. o The chemical nature of the specific gravity test may cause slightly different results from those obtained with the specific gravity methods when elevated amounts of certain urine constituents are present. pH: o The pH test area permits quantitative differentiation of pH values to one unit within the range of 5-9. pH reading is not affected by variation in the urinary buffer concentration o If proper procedure is not followed and excess urine remains on the strip, a phenomenon known as running over may occur, in which the acid buffer from the protein reagent area run onto the pH area, causing a false lowering in the pH result. Leucocytes: o This test can detect as low as 20-25 WBC/mL. and will not react with erythrocytes or bacteria common in urine o Highly colored urine and the presence of the drugs cephalexin and gentamicin interfere with the test results. o High urinary protein of 500 mg/dl or above diminishes the intensity of the reaction color. o Elevated glucose concentration or high specific gravity may cause decreased results Nitrites: o This test is specific for nitrite and will not react with substances normally excreted in the urine o The test is sensitive to 0.05-1.0mg/dl o This test has sensitivity to sodium nitrite of 0.075 mg/dl. Comparison of the reacted reagent area on a white background may aid in the detection of low levels of nitrite ion, which may otherwise be missed. o The pink color is not quantitative in relation to the number of bacteria present. Any degree of pink coloration should be interpreted as a positive nitrite test suggestive of 105 or more organisms/ml. If the infection is caused by bacteria which do not contain reductase (to convert nitrate to nitrite) the test results will be negative although bacteria are present

Morning specimen of urine is preferred for Nitrite test as only an incubated sample of urine will contain high bacterial content Protein: o The test area is sensitive to 5 -10 mg albumin /dl urine o A color matching any color block greater than trace indicates significant proteinuria o The test area is more sensitive to albumin than to globulin, hemoglobin, Bence-Jones proteins, and mucoprotein; therefore a negative result does not rule out the presence of these other proteins. o For urine with high specific gravity, the test area may most closely match the trace color block even though only normal concentrations of protein are present. Glucose : o The measurement range for glucose is 50-150 mg of glucose / dl of urine and is specific for glucose. o Presence of ascorbic acid in urine (most likely to be present in large amounts in the urine of pregnant women and those taking multivitamin medications) will cause a false-positive result o This test does not detect other reducing sugars, fructose, galactose, etc. and other non reducing substances in the urine as it are specific for glucose. Ketones bodies o The minimum detection limit of ketones by this method is 5 mg/dl for Acetoacetic acid o False Positive results occur in patients receiving Levodopa or with urine containing MESNA or large amounts of phenylketones o This test does not detect the ketone body, beta hydroxyl butyric acid Urobilinogen: o This minimum detection limit of urobilinogen is 0.4 mg/dl in urine. o The absence of urobilinogen cannot be determined with this test o The test area will react with interfering substances known to react with Ehrlichs reagent, such as porphobilinogen and p-aminosalicyclic acid. o This test is not a reliable method for the detection of porphobilinogen o Drugs containing azo-dyes (e.g. Azo Gantrisin) may give a masking golden color. Bilirubin o The test has a sensitivity of 0.5 mg /dl of bilirubin in urine and is specific for bilirubin only o False positive results may obtain with the urine of patients receiving large doses of Chlorpromazine. o False negative results will be obtained when urine contains large amount of Ascorbic acid and Nitrite and when bilirubin is oxidised to Biliverdin. Occult blood o The test has a sensitivity to free hemoglobin of 0.02 to 0.06mg/dL or 5- 20 intact red cells / ul urine. The minimum Detection limit for RBC is about 5 RBCs.ul and free Hb equivalent to 10 RBC/ ul urine. o This test is slightly more sensitive to free hemoglobin and myoglobin than to intact erythrocytes o

o False positive results may be obtained in the presence of Hypochlorite or when the urine has high bacterial count (as in urinary tract infections) as bacteria contain Peroxidase o The sensitivity of the blood test is reduced in urine with high specific gravity and/ or high ascorbic acid content

Primary sample:
Instruct patients to collect a minimum 15 ml of a random sample of urine in a clean dry container (preferably collected from the laboratory). Instruct patients to void directly in to the container and while collecting allow the first portion of urine to escape Use fresh well mixed uncentrifuged urine as specimen for the test Process the samples within 1 hour of sample received. If delay is anticipated instruct patients to store the sample at 2- 8 C in the refrigerator for a maximum of upto 2 hour Do not accept if Insufficient sample is given and samples without an ID number Instrument: : Urisys 1100 Consumables: Combur Test UX Switch on the instrument Self-Test appears in the screen Do calibration if required Insert Strip will appears in the screen
Automatically Serial No. appears on the screen

Dip the test strip and remove excess urine Place the strip in the tray pad facing upwards Press OK Instrument transports the strip to the measuring channel Sequence No. Incremented automatically Now place the Next strip Reports are printed one by one automatically by the instrument

Test procedure :
Ensure the urine sample is at room temperature and mix the urine sample thoroughly before testing. Take a test strip from the pack and close it again immediately. Briefly, no longer than 1 sec, dip the test strip into the urine making sure that all the test areas are moistened. If the strip is dipped for a longer time reagents from the test areas of the strip will dissolve in the urine When removing the strip, wipe the edge of the strip against the rim of the specimen container to remove excess urine or dab the side edge of the strip on a clean absorbent surface. If reading visually compares the test results carefully with the color chart on the label after the appropriate time period (60 to 120 seconds) or place the strip with the pads facing upward on the test strip tray and press ok The report will be printed one by one The test strips must be properly stored and handled before and during testing.Reaction. Perform positive & negative controls everyday and record the details in internal quality control register . If there is any deviation perform necessary corrective action.

Quality control:

Precautions & potential sources of variability: Carry out the examination in a fresh sample with in 2 hours of collection, as delay will cause
leucocytes to be destroyed, casts to decompose and urea will be broken-down due to bacterial action and make the urine alkaline affect test results Do not touch the edges of the strip where the reagent patch is present Tap excess urine from the strip by stroking along the rim of the container after dipping the strip into the sample. Do not expose the strip to the atmospheric air when not in use and keep the bottle closed. Protect the strips from moisture and excessive heat but do not refrigerate. Replace the top on the storage container immediately after removing a strip. Darkening of the enzyme-coated area of the strip indicates loss of sensitivity. Hence do not use discolored strips. Contamination of glassware with Sodium Hypochlorite and Bleaching powder and detergents like sodium phosphate will oxidize and change the color of chromogen in Dipstick. Hence ensure the glassware is free of these chemicals

Reference range : pH : 5-9 Specific Gravity: SG of random urine ranges from 1.010- 1.030 .
Nitrite: Normally no detectable amount of nitrite is present in urine.

Protein: Normally no detectable amount of protein is present in urine Ketone : Normally, no ketones are present in urine. Glucose : Normally no detectable amount of glucose present in urine Bilirubin: Normally, no bilirubin is detectable in urine Urobilinogen: Present in normal amounts Leucocytes : Normal urine yields negative results Pathological leucocyte concentration > 20 leucocytes / l Blood Normally no blood is present in urine. Blood may be in the urine of menstruating females

Safety Precautions: Handle all reagents with care and avoid contact with eye, mouth and skin.
Handle all samples as potentially infectious. Discard used reagents and sample as per disposal procedure Avoid contact with skin &mucous membrane

MALARIAL PARASITE
To prepare a blood film to examine the presence or absence of the malarial parasite. This test helps in identification of the stage of the parasite in lifecycle and also the characteristics of certain species of plasmodium in the diagnosis of malaria.

Principle: The thick and thin blood smear is prepared on a microscopic slide which is dried, fixed and
stained. The thin smear is stained with Leishmanns stain which allows studying the morphological abnormalities of red cells. The thick smear is stained with field stain without fixing in methanol, which removes the red cells by hemolysis and leaves behind the imprints of the parasite as pink dots and other structures.

Performance Specifications: The film must be smooth at the end. There should be no lines extending across or down through the film and it should not contain holes. Primary Sample: Use whole blood as specimen Collect 3 ml blood in a tube containing 0.2ml of 4% K3 EDTA. Mix well gently. Do not use clotted sample for testing. Consumables/Reagents: Leishman stain : It is prepared by dissolving 2g of powdered stain in 1000 ml of (acetone free) methyl alcohol Giemsa stain: Preparation of the solution: Giemsa powder certified (Merck or Sigma) 3.8 g Glycerol 250ml

Absolute Methanol (acetone free)

250ml

Weigh 3.8 g of Giemsa powder and transfer to a mortar. Using dry cylinder measure 10 ml glycerol and add this into the mortar and grind well with Giemsa powder and transfer into a dry brown bottle containing glass beads. Add 240 ml of glycerol and keep the bottle in 60C water bath for 2 hours and allowed to cool at room temperature. Then add 250 ml of absolute methanol slowly with stirring. Keep the bottle in 60 C for water bath for 1 hour. Allow to cool and store at room temperature for 1 week. Decant and remove the sediment. Filter before use. Preparation of Stock Phosphate buffer (pH7) Stock A: 0.2 M sodium dihydrogen phosphate 3.12g in 200 ml of distil water Stock B: 0.2 M disodium hydrogen phosphate 2.33 g in 400 ml of distilled water After preparation the solution is stored at room temperature in dark bottles Working solution of buffer: To14 ml of stock A and 36 ml of Stock B is added and made upto 100 ml with distilled water Preparation of Giemsa stain: Ready to use Dilute 5 ml of the Giemsa stain with 37 ml of the working solution of the phosphate buffer and transfer in a Coplin jar in the incubator at 37C. o Buffered water : Distilled water o Immersion oil o Glass Slides o Cotton o Slide rack Instrument: o Light Microscope. Procedure: Preparation of thick Smear: Arrange 2-3 clean grease free - microscope slides on the work table. Before dislodging the needle from the syringe in which the blood is collected from the patient, place one large drop of blood on each slide. Using the corner of another microscope slide carefully spread the drop of blood over an area approximately the size of a 10 paise coin. Allow the smears to air dry for about 30 minutes. After drying place two drops of distilled water, so that the RBCs will be lysed. Fix the smear in methanol for 5 mins, and stain with Giemsa stain for 30 mins at 37C. After 30 mins the slide is washed off with distilled water by the technician. Examine the smears microscopically using the oil immersion objective to identify the species of Malaria. Preparation of thin smear: Take a clean, dry slide.

Transfer a small drop of blood near the edge of the slide. Place a smaller piece of glass with a width slightly less than that of the slide, Called as spreader at an angle of about 45 to the surface of the slide. Pull back the spreader until it touches the drop of blood. Put the spreader forward to the end of the slide with a smooth movement. Dry the blood smear at room temperature. Staining : Staining of thin smear with Leishman stain: o Place the slide with the smear side up on a staining rack horizontally (without sloping). o Write the patients lab reference No. and the date with a pencil and place it on staining rack, making sure that the smeared surface faces upward for staining. o Add Leishman stain to cover the entire slide. Then dilute it with working buffer. o Fifteen minutes later drain off the stain and wash the slide under running tap water. o Place it on a rack to dry. o Examination of stained smears: The air-dried stained smear is viewed 1st under dry 10X and later under 100X oil immersion objective of a light microscope. Interpretation of results: Look for species of the malarial parasite and various stages of the parasite namely mature and immature Trophozoites, Schizonts, and Gametocytes. Immature trophozoites: seen in all infected cells and appear as undivided nucleated cells with blue colored cytoplasm or as a ring of cytoplasm within the red cells. Mature Trophozoites: Trophozoites without ring structure but with compact cytoplasm and amoeboid Shape. Schizonts: Appear as individual nucleated cells arranged in a circle forming a rosette or distributed throughout the cell. Gametocytes: Appear as round or oval or elongated filling the entire cell. Infected red cell: May appear normal, enlarged, oval shaped with jagged edges or with pink dots called Schuffners dots. Pigment: Some parasites have pigment granules yellow/ brown or black in their cytoplasm. Safety Precautions: Handle all reagents with care and avoid contact with eye, mouth and skin. Handle all samples as potentially infectious. Discard used reagents and sample as per disposal procedure Potential Sources of Variability: Dirty slides do not give an even smear. Smears that are too thick or too thin will not stain well. Ensure the stained smears are translucent Expired stains and old stains may not yield good results

An appropriate size of blood drop should be used. Jerky movement of the spreader slide and the loss of contact between the spreader and the smear slide will yield poor smears.

2.

BIOCHEMISTRY
GLUCOSE

Quantitative estimation of Glucose in plasma and urine by Glucose oxidase and Peroxidase method. Measurement of glucose is important in the diagnosis and treatment of carbohydrate metabolism disorder such as diabetes mellitus, neonatal hypoglycemia, and idiopathic hypoglycemia and of pancreatic islet cell carcinoma. Principle: Glucose is determined after enzymatic oxidation in the presence of glucose oxidase. The hydrogen peroxide produced, reacts catalysed by peroxidase, with phenol and 4aminophenazone to form a red-violet quinoneimine dye as indicator. The intensity of the final colour is directly proportional to the glucose concentration and is measured at 505nm. Glucose + 02 + H20 GOD Gluconic acid + H202 POD 2H202 + 4-aminophenazone + phenol Quinoneimine + 4H20

Performance specifications: Linearity: This kit is linear for Glucose concentrations up to 614 mg/dL Measurement range: This method has a measurement range of 6.04- 614 mg/dL Sensitivity: The minimum detection limit by this kit is 6.04mg/dL Primary Sample: Use plasma or urine as specimen. Collect 2 ml of venous blood from a peripheral vein in a sodium fluoride K3 EDTA tube. Separate the plasma within 2 hours of collection. Separate the plasma by centrifugation at 2500- 3000 rpm for 3 5 minutes. Process the sample on the same day. For Urine ,use 24 hour urine as specimen using thymol as preservative Do not use hemolysed, contaminated samples for testing. Required Equipment and Reagents: Equipment: RX Imola and RX Daytona Clinical Chemistry Analyzer.
Reagents:

 Phosphate buffer: 50 mmol/l, pH 7.0 MOPS Buffer: 50 mmol/l, pH 7.06.2.3 Phenol: 11 mmol/l 4-aminophenazone: 0.77 mmol/l Glucose oxidase: 1.5kU/l Peroxidase : 1.5kU/l Procedure: Before Switching on, check the pre-start inspection. Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode. Check water tank, wash solution, cuvette, and waste tank. Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. (BL/CB/L). Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples. Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface.

Inter the results in the Daily Results Register Biochemistry. Assay condition: Assay type: End Point Wavelength: 510 nm Reaction Time:10 min Reaction Slope: Increase Reagent volume: 180 l Sample volume: 2 l Temperature: 370 C

Calibration procedure: Calibrate using a calibrator material (CAL 3) With every new lot of reagent. If the internal QC is repeatedly out of range. After major equipment maintenance or breakdown. Record the details of calibration in the calibration register. Quality Control Procedure: Run two levels (level 1 & level 2) of an assayed QC sample daily before processing the patient samples and ensure the QC values are with in the Reference Range and close to the mean mentioned in the product insert. If both the QC levels (L 1 & L 2) are out of range on the same day in the same run perform calibration for the analyte. External Quality Assessment Scheme /Inter Laboratory Comparison: BIORAD-EQAS-Monthly Clinical Chemistry. Calculation of Results: The system performs all calculations internally to produce the final reported result. The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor. Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. Reference Range: Fasting: Adult: 74-99 Children: 60-100 Random: 80-120 Post prandial: 90-140

Interpretation of Results: Elevated levels are found in pancreatitis, pituitary and thyroid dysfunction, renal failure and liver diseases. Low glucose levels are found in insulinoma, hypopituitarism, neoplasms, insulin induced hypoglycemia. Safety precautions: Handle all samples as potentially infectious.

 Handle all reagents with care and avoid contact with eye mouth and skin. Do not perform mouth pipetteing. Discard used reagents and sample as per disposable procedure. Potential sources of variability: Lysed serum specimens may give falsely elevated values. Do not use if the reagents is turbid as it indicates contamination of the reagent. Bilirubin in the tested level of 60 mg/dL has no significant interference on the assay. Hemoglobin in the tested level of 2000 mg/dL has no significant interference on the assay. Intralipid in the tested level of 2000 mg/dL has no significant interference on the assay.

HBA1c
Hemoglobin A1c is intended for the percent determination of hemoglobin A 1c in human whole blood using ion-exchange high-performance liquid chromatography (HPLC). Principle : The D-10 Hemoglobin A1c Program utilizes the principle of ion-exchange high performance liquid chromatography (HPLC). The samples are automatically diluted on the D10 and injected into the analytical cartridge. The D10 delivers a programmed buffer gradient of increasing ionic strength to the cartridge where the hemoglobins are separated based on their ionic interactions with the cartridge material. The separated hemoglobins then pass through the flow cell of the filter photometer where changes in absorbance at 415 nm are measured. The D10 software performs reduction of raw data collected from each analysis .Two level calibration is used for quantitation of the HbA1c values.The sample report and a choromatogram are generated for each sample. The A1c area is calculated using an exponentially modified Gaussian (EMG) algorithm that excludes the labile A1C and carbamylated peak areas from the A1c peak area. Performance Specification : Linearity: This method is linear for whole blood (normal) : 3.8 %. whole blood (Diabetis): 18.5 %. Primary Sample: Use EDTA whole blood as specimen for the test. Collect 2 ml of venous blood in Lavender top EDTA tube. Do not use lysed / contaminated sample for testing

Samples are stable at room temperature for 3 days. No sample preparation is required If analysis is not done on the same day / with in 3 hrs of collection store the sample at 2 8 C for up to 7 days. Type Of Container: 1.1 Lavender top EDTA tube. Required Equipment and Reagents: Equipment: Fully automated D10 HbA1C Testing Ion-exchange High-performance liquid chromatography System. Pipettes: 5 L, 0.5 mL, 1 mL, 7 mL. Sample Vial Adapter Reagents: Elution buffer 1: Bis tri phosphate buffer pH 6.0, containing < 0.05 % sodium azide as a preservative. Elution buffer 2: Bis tri phosphate buffer pH 6.7, containing < 0.05 % sodium azide as a preservative. Wash / diluent solution: Deionized water, containing < 0.05 % sodium azide as a preservative.

D-10 Thermal Paper roll. Lyphochek Diabetes Bi-level control Lyphocheck Hemoglobin A1c Linearity set Deionized Water Disposable Gloves Procedure Switch on the Ups & instrument .Press startup automatic self check is done. Allow sample tubes to reach room temperature (15-30 C) before performing the assay. Priming Run: Analytical Cartridge Priming Procedure has to be performed prior to the first use of each recorder pack as given below: Perform the Priming Run as a as a separate. Do not combine the priming run with calibrator, QC or any other sample. Pipette 1 mL of reconstituted Whole Blood Primer into a sample vial. Put the sample vial into a sample vial adapter labeled with a PRIME barcode, then place the adapter into sample rack position 1. Make sure there are no other samples in the rack. Start a run. When the priming run is completed remove the sample rack. Once the priming run is completed the cartridge is ready for calibration and proceed with calibration run according to the recorder pack instruction manual.

When the priming run with Rack Loader is followed by a second rack containing calibrators, controls, or patient samples an error message will be displayed as One calibrator for each type required. After completing the priming run, calibrators, QC, and samples can be processed together using multiple racks within the same analytical run. Load the samples in the rack For low sample volume dilute: The sample 5 micro lit +1.5 ml wash/diluent solution) and Place in the rack loader area. Inside then key in the sample ID and press start key then confirm. After the run has complete record the values and eject the rack then press sleep mode and then shutdown the instrument. Switch off the machine & UPS. Enter the results in the Daily Results Register Biochemistry. Calibration procedure: Perform the calibration run according to the recorder pack instruction manual. Perform calibration as per the following Whenever a new lot of new analytical cartridge is installed and primed After major equipment breakdown or maintenance. whenever the internal QC is repeatedly out of range Record the details in the calibration register. Quality Control : Run Two Levels (Level 1 & Level 2) of an assayed QC Sample daily before processing patient Samples. Process patient samples only if the QC values are within the Reference Range and close to the mean mentioned in the Product insert. If both the QC levels (L 1 & L 2) are out of range on the same day in the same run perform recalibration for the analyte. Calculation of Results: The system performs all calculations internally to produce the final reported result. Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. Reference range : 4-6 % Interpretation of Results: < 6 % - Non Diabetic < 7 % - Ideal in Diabetic individual > 8 % - High risk of developing long-term complications such as retinopathy, nephropathy, neuropathy and cardiopathy. Potential Source of Variability: If the sample area is outside of the expected range, manually predilute the sample. In iron deficiency anaemia and transfusion the average age of erythrocytes is altered. Caution should be used when interpreting the result

Carbamylated hemoglobin levels up to 3.5% have no clinically significant effect on A1c determination Labile A1c levels up to 4% have no clinically significant effect on A1c determination. Lipimia, as indicated by triglyceride concentrations upto 5680 mg/dl, does not interfere with the assay. Icterus, as indicated by bilirubin concentrations upto 20 mg/dl, does not interfere with the assay. Heamoglobin F concentrations upto 10% do not interfere with the assay. Safety Precautions: Consider all samples as potentially infectious. Consider any materials of human origin as infectious and handle them using typical biosafety procedures. Use gloves while handling all the reagents. Do not pipette by mouth. Avoid contact with eye, mouth and skin. Discard used reagents and sample as per disposal procedure.

CHOLESTEROL
Quantitative estimation of Total Cholesterol in human serum by enzymatic endpoint method. Measurement of serum cholesterol is useful in the screening of the lipid status of the individual to detect atherosclerotic risks and in monitoring the response to lipid lowering measures and also in the diagnosis and classification of hyperlipidemias. Principle: Cholesterol Esterase hydrolyses cholesterol esters into free cholesterol and fatty acids. In the second reaction cholesterol oxidase converts cholesterol to cholest-4-en-3-one and hydrogen peroxide. In presence of peroxidase, hydrogen peroxide oxidatively couples with 4-aminoantipyrine and phenol to produce red quinoneimine dye which has absorbance maximum at 510 nm (500-530). The intensity of the red colour is proportional to the amount of total cholesterol in the specimen. Cholesterol Esterase Cholesterol Oxidase

Cholesterol esters + H20 Cholesterol + O2

Cholesterol + Fatty Acids H2O2 + Cholestene-3-one

2H202 + phenol + 4-Aminoantipyrine

Peroxidase

quinoneimine + 4H20

Performance specifications: Linearity: This assay is linear for concentration up to 643.0 mg/dL. Measurement Range: This method has a measurement range of 55.0 643.0 mg/dL. Sensitivity: The analytical sensitivity of this assay is 55.0 mg/dL.

Primary Sample: Use only serum / plasma as specimen for the test. Collect 2 ml of venous blood in a plain red topped vacutainer tube or yellow capped gel tube Allow the tube to stand for 30 minutes at room temperature and separate the serum by centrifugation at 2500 - 3000 rpm for 5 - 10 minutes. Ensure complete clot formation has occurred and there are no fibrin threads. Process all samples on the same day within 2 hours of collection. If processing is done after 8 hours of collection, separate the serum and aliquot it storage vials at 2- 80C. If assay is likely to be done after 48 hours of collection, store the serum at - 200C Do not use hemolysed, contaminated samples for testing. Type of container and additive: Use Red topped Plain vaccutainer tubes / green topped heparinised vaccutainer tubes, yellow capped gel tubes for collecting samples. Do not add any additives / preservatives. Required Equipment and Reagents: Equipment: RX Imola and RX Daytona Clinical Chemistry Analyzer.
Reagents:

Pipes buffer: 80 mmol/l, pH 6.8 4-aminoantipyrine: 0.25 mmol/l Phenol: 6 mmol/l Peroxidase: 0.5 U/ml Cholesterol esterase: 0.15 U/ml Cholesterol oxidase : 0.10 U/ml

Procedure: Before Switching on, check the pre-start inspection. Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode.

Check water tank, wash solution, cuvette, and waste tank. Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. (BL/CB/L). Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples. Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface.

Inter the results in the Daily Results Register Biochemistry. Assay Conditions: Assay type: End Point Reagent volume: 180 l Wavelength: 510 nm Reaction Time:10 min Reaction Slope: Increase Calibration Procedure: Calibrate using a calibrator material With every new lot of reagent. If the internal QC is repeatedly out of range. After major equipment maintenance or breakdown. Record the details of calibration in the calibration register. Quality Control Procedure: Run Two Levels (Level 1 and Level 2) of an assayed QC Sample daily before processing patient Samples. Process patient samples only if the QC values are within the Reference Range and close to the mean mentioned in the Product insert. If both the QC levels (L1 & L2) are out of range on the same day in the same run perform calibration for the analyte. External Quality Assessment Scheme /Inter Laboratory Comparison: BIORAD-EQAS-Monthly Clinical Chemistry Sample volume: 2 l Temperature: 370 C

Calculation of Results: The system performs all calculations internally to produce the final reported result. The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor. Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. Reference Range:
< 200 mg/dl 200-239 mg/dl 240 mg/dl -Desirable blood cholesterol. - Borderline-high blood cholesterol. - High blood cholesterol.

Interpretation of results Increased Serum levels are seen in the following conditions: o Familial hypercholesterolemia. o Nephrotic syndrome. o Biliary obstruction. o Hypothyroidism. o Pregnancy. Decreased Serum levels are seen in the following conditions: o Hyperthyroidism. o Malnutrition. o Chronic anemia. o Thyroiditis. o Severe liver insufficiency. Safety precautions: o Handle all samples as potentially infectious. o Handle all reagents with care and avoid contact with eye mouth and skin. o Do not perform mouth pipetteing. o Discard used reagents and sample as per disposal procedure. Potential sources of variability: o Lysed plasma specimens may give falsely elevated values. o The influence of Ascorbic acid, Bilirubin, Haemoglobin, Glucose, Sodium fluoride, Heparin, EDTA, Creatinine and Uric Acid is negligible. o Falsely elevated results are obtained due to lipemic samples

SERUM TRIGLYCERIDES

Quantitative estimation of Triglycerides in human serum by (GPO-PAP) enzymatic method. Triglyceride measurements are used in the diagnosis and treatment of diseases involving lipid metabolism and various endocrine disorder e.g. diabetes mellitus, nephrosis and liver obstruction.

Principle: Glycerol released from hydrolysis of triglycerides by lipoprotein lipase is converted by glycerol kinase into glycerol-3-phosphate which is oxidized by glycerol phosphate oxidase to dihydroxyacetone phosphate and hydrogen peroxide. In presence of peroxidase, hydrogen peroxide oxidizes phenolic chromogen to a red coloured compound.
Lipase

Triglycerides+ H2O

Glycerol + Fatty Acids

Glycerol + ATP

Glycerol Kinase

Glycerol-3-phosphate + ADP

Glycerol-3-phosphate + O2 H2O2

Glycerol Phosphare Oxidase

Dihydroxyacetone phosphate +

2H2O2 +4-aminophenazone+4 chlorophenol Performance Significance: o o

Peroxidase

Quinoneimine+HCl+4H2 0

Linearity: This assay is linear for concentration up to 12.8mmol/l Measurement Range: This method has a measurement range of <0.13- 12.8mmol/l.

o Sensitivity: The analytical sensitivity of this assay is < 0.13mmol/l. Primary sampling: o Use only serum / plasma as specimen for the test. o Collect 2 ml of venous blood from a peripheral vein in a plain red topped vacutainer tube. Separate the serum within 2 hours of collection. o Allow the tube to stand at room temperature till complete clot formation occurs (for 30 minutes) and then separate the serum by centrifugation at 2500- 3000 rpm for 3 5 minutes. Ensure complete clot formation has occurred and there are no fibrin threads. o Process the sample on the same day. If analysis is not done on the same day, separate the serum and store it at 2-8 C. o If assay is likely to be done after 48 hours of collection, store the serum at - 200C.

Do not use hemolysed, contaminated samples for testing.

Required Equipment and Reagents: Equipment: RX Imola and RX Daytona Clinical Chemistry Analyzer.
Reagents:

Buffer o Pipes Buffer o 4-chloro-phenol o Magnesium ions o Enzyme reagent o 4-aminophenazone o ATP o Lipase o Glycerol-Kinase o Glycerol-3-phosphate oxidase o peroxidase Procedure: o Before Switching on, check the pre-start inspection. o o Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode. Check water tank, wash solution, cuvette, and waste tank. Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. (BL/CB/L). Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples.

o o o o

o o

Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface.

o Inter the results in the Daily Results Register Biochemistry. Assay condition: Assay type: End Point Reagent volume: 180 l Wavelength: 510 nm Reaction Time:10 min Reaction Slope: Increase Sample volume:2 l Temperature: 370 C

Calibration Procedure: Calibrate using a calibrator material o With every new lot of reagent. o Every Three months even with the same lot of reagent. o If the internal QC is repeatedly out of range. o After major equipment maintenance or breakdown. o Record the details of calibration in the calibration register. Quality Control Procedure: o Run Two Levels (Level 1 & Level 2) of an assayed QC Sample daily before processing patient Samples. Process patient samples only if the QC values are within the Reference Range and close to the mean mentioned in the Product insert. o If both the QC levels (L 1 & L 2) are out of range on the same day in the same run perform recalibration for the analyte.

External Quality Assessment Scheme /Inter Laboratory Comparison: BIORAD-EQAS-Monthly Clinical Chemistry. Calculation of Results: o The system performs all calculations internally to produce the final reported result. o The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor. Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. Reference Interval / Range :

Normal: <150 High: 150-199 Very High: >499 Interpretation of results:

Blood tests for triglycerides are usually part of a lipid profile used to identify the risk of developing heart disease. As part of a lipid profile, it may be used to monitor those who have risk factors for heart disease, those who have had a heart attack, or those who are being treated for high lipid and/or high triglyceride levels. Safety Precautions: o Handle all samples as potentially infectious. o Handle all reagents with care and avoid contact with eye, mouth and skin. o Do not perform mouth pipetting. o Discard used reagents and samples as per disposal procedure. Potential source of variability : o Presence of fibrin in the sample may lead to erroneous results. o Glycerol contamination in glasswares leads to errorneous results. o Lipemic Samples may leads to falsely elevated results. o Influence of lipids, haemolysis and bilirubin (upto 8 mg%) is negligible.

HDL-CHOLESTEROL
Quantitative estimation of Direct HDL Cholesterol in human serum and plasma by end point assay. Measurement of Direct HDL cholesterol is of a vital importance when accessing patient risk from coronary heart disease (CHD). Principle: The assay has 2 distinct reaction steps as given below: Reaction 1: Elimination of chylomicron, VLDL-Cholesterol and LDL-Cholesterol by cholesterol esterase, cholesterol oxidase and subsequently catalase. Cholesterol ester cholesterol esterase Cholesterol + Fatty acid Cholesterol + O2 2 H2O2 catalase cholesterol oxidase 2H2O + O2 Cholestenone + H2O2

Reaction 2: Specific measurement of HDL-Cholesterol after release of HDL-Cholesterol by detergents in Reagent 2. Cholesterol ester cholesterol esterase Cholesterol + Fatty acid

Cholesterol + O2

cholesterol oxidase

Cholestenone + H2O2 Peroxidase Quinone pigment + 4 H2O

2 H2O2 + 4- Aminoantipyrine + HDAOS

The intensity of the Quinoneimine dye produced is directly proportional to the cholesterol concentration when measured at 600 nm. In the second reaction catalase is inhibited by sodium azide in the Enzyme reagent 2. Performance specifications: o Linearity: This method is linear for HDL concentrations up to 144 mg/dL of serum. o Measurement range: This method has a measurement range of 1.43 144.0 mg/dL. o Sensitivity: The minimum detection limit is 1.43 mg/dL. Primary Sample: o Use only fasting serum as specimen for the test. o Collect 2 ml of venous blood in a plain red topped vacutainer tube / yellow capped gel tube. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500- 3000 rpm for 5 10 minutes. o Do not use lysed serum for testing as it may give very high reslts. o Do not use contaminated / turbid samples for testing. o Process the sample on the same day within 3 hours of collection. o If analysis is not done on the same day /within 3 hours of collection, separate the serum and store it at 2-8 C for up to 7 days. Required Equipment and Reagents: Equipment: RX Imola and RX Daytona Clinical Chemistry Analyzer.
Reagents:

Enzyme Reagent 1: o N,N-Bis(2-Hydroxyethyl)2-aminoethanesulfonic acid: 100 mM, pH 6.6 o N-(2-hydroxy-3-sulfopropyl)3,5-dimethoxy aniline, Sodium salt (HDAOS) - 0.7 mmol/l N o o o Cholesterol Esterase: 800 U/l Cholesterol Oxidase : 500 U/l Catalase: 300KU/l

o Ascorbate oxidase 3000KU/l Enzyme Reagent 2: o N,N-Bis (2-hydroxyethyl) 2-aminoethanesulfonic acid: 100 mM, pH7.0 o 4-aminoantipyrine: 4 mmol/l o Peroxidase: 3500 U/l o Sodium azide: 0.05 % (w/v) o Surfactants 1.4 % W/V %

Procedure: o o Before Switching on, check the pre-start inspection. Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode. Check water tank, wash solution, cuvette, and waste tank. Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. (BL/CB/L). Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples. Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface.

o o o o

o o

o o

o Inter the results in the Daily Results Register Biochemistry. Assay condition: Assay type: End Point Reagent volume: R1-180 R2-60 Wavelength: 600 nm Reaction Time:9 min Reaction Slope: Increase Sample volume: 3 l Temperature: 370 C

Calibration procedure:

Calibrate using Lipid Calibrator Calibrate the instrument for the assay by using the calibrator specified in the kit. Whenever a new lot of reagent is used. After major breakdown of Machine. Whenever the internal QC is repeatedly out of range. Record the details in the calibration register. Quality Control Procedure: o Run Two Levels (Level 1 & Level 2) of an assayed QC Sample daily before processing patient Samples. Process patient samples only if the QC values are within the Reference Range and close to the mean mentioned in the Product insert. o If both the QC levels (L 1 & L 2) are out of range on the same day in the same run perform calibration for the analyte. External Quality Assessment Scheme /Inter Laboratory Comparison: BIORAD-EQAS-Monthly Clinical Chemistry. Calculation of Results: o The system performs all calculations internally to produce the final reported result. o The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor. Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. Reference Interval / Range : Low: < 40.0 mg/dL. High: 60.0 mg/dL. Interpretation of results: o Values >60 mg/dL are considered a negative risk factor for CHD and are considered protective. Values > or = 80-100 mg/dL may indicate metabolic response to certain medications such as hormone replacement therapy, chronic liver disease, or some form of chronic intoxication, such as with alcohol, heavy metals, industrial chemicals including pesticides. o o Values <40 mg/dL correlate with increased risk for CHD. HDL values < or = 5 mg/dL occur in Tangier disease, in association with cholestatic liver disease, and in association with diminished hepatocyte function.

Safety precautions: o Handle all samples as potentially infectious. o Handle all reagents with care and avoid contact with eye, mouth and skin.

o Do not perform mouth pipetting. o Discard used reagents and samples as per disposal procedure. Potential sources of variability o Lysed serum specimens may give falsely elevated values. o Do not use if the reagent is turbid as it indicates contamination of the reagent or if the absorbance of the blank reagent is more than 0.100.

LDL-CHOLESTEROL
Quantitative estimation of direct LDL Cholesterol in human serum and plasma by end point assay. Measurement of serum LDL cholesterol is of a vital important in therapies which focus on lipid reduction to prevent atherosclerosis or reduce its progress and to avoid plaque rupture. Principle: The assay has 2 distinct reaction steps as given below: Reaction 1: Elimination of chylomicron, VLDL-Cholesterol and HDL-Cholesterol by cholesterol esterase, cholesterol oxidase and subsequently catalase. Cholesterol ester Cholesterol + O2 H2O2 catalase cholesterol esterase cholesterol oxidase 2H2O + O2 Cholesterol + Fatty acid Cholestenone + H2O2

Reaction 2: Specific measurement of LDL-Cholesterol after release of LDL-Cholesterol by detergents in Reagent 2. Cholesterol ester Cholesterol + O2 cholesterol esterase cholesterol oxidase Cholesterol + Fatty acid Cholestenone + H2O2 Peroxidase Quinone pigment + 4 H2O

2 H2O2 + 4- Aminoantipyrine + TOOS

The intensity of the Quinoneimine dye produced is directly proportional to the cholesterol concentration when measured at 600 nm. In the second reaction catalase is inhibited by sodium azide in the Enzyme reagent Performance specifications: o Linearity: This method is linear for LDL concentrations up to 860.0 mg/dL of serum. o Measurement range: This method has a measurement range of 7.30 860.0 mg/dL. o Sensitivity: The minimum detection limit is 7.30 mg/dL. Primary Sample: o Use only fasting serum as specimen for the test

Collect 2 ml of venous blood in a plain red topped vacutainer tube / yellow capped gel tube. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500- 3000 rpm for 5 10 minutes. o Do not use lysed serum for testing as it may give very high results. o Do not use contaminated / turbid samples for testing. o Process the sample on the same day within 3 hours of collection. o If analysis is not done on the same day /within 3 hours of collection, separate the serum and store it at 2-8 C for up to 7 days. o If assay is likely to be done after 48 hours of collection, store the serum at - 200C. Required Equipment and Reagents: Equipment: RX Imola and RX Daytona Clinical Chemistry Analyzer. o
Reagents: o Enzyme Reagent 1:

o o o o

PIPES buffer: 50 mmol/l TOOS: 2.0 mmol/l Cholesterol Esterase: 600U/l Cholesterol Oxidase: 500 U/l

o Catalase: 600 KU/l o Enzyme Reagent 2: o PIPES Buffer:50 mmol/l, pH 7.0 o 4-aminoantipyrine: 4mmol/l o Peroxidase: 4 KU/l o Sodium Azide.n: 0.05% (w/v) Procedure: o o Before Switching on, check the pre-start inspection. Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode. Check water tank, wash solution, cuvette, and waste tank. Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. (BL/CB/L).

o o o o

o o

Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples. Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface.

o o

o Inter the results in the Daily Results Register Biochemistry. Assay condition: Assay type: End Point Reagent volume: R1-180 R2-60 Wavelength: 600 nm Reaction Time:10 min Reaction Slope: Increase Calibration procedure: Calibrate using Calibrator (Lipid calibrator) o Calibrate the instrument for the assay by using the calibrator specified in the kit. o Whenever a new lot of reagent is used. o After major breakdown of Machine. o Whenever the internal QC is repeatedly out of range. o Record the details in the calibration register. Quality Control Procedure: o Run Two Levels (Level 1 & Level 2) of an assayed QC Sample daily before processing patient Samples. Process patient samples only if the QC values are within the Reference Range and close to the mean mentioned in the Product insert. o If both the QC levels (L 1 & L 2) are out of range on the same day in the same run perform recalibration for the analyte. Sample volume: 3 l Temperature: 370 C

External Quality Assessment Scheme /Inter Laboratory Comparison: BIORAD-EQAS-Monthly Clinical Chemistry. Calculation of Results: o The system performs all calculations internally to produce the final reported result.

The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor. Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. o Reference Range: Optimal: < 100 mg/dL Near or above optimal: 100 - 129 mg/dL. 12.3. Borderline High: 130-159 mg/dL. High: 160- 189 mg/dL. Very high: >190 mg/dL. Reportable interval of examination results: 7.30 860.0 mg/dL. Interpretation of results: o Evaluation of cardiovascular risk is based on the following range of values established by the National Cholesterol Education Program (NCEP): Desirable: <100 mg/dL:Low risk: 100 mg/dL to 129 mg/dL:Borderline high: 130 mg/dL to 159 mg/dL:High: 160 mg/dL to 189 mg/dL:Very high: > or =190 mg/dL. Decreased values may indicate hypobetalipoproteinemia. Nondetectable low density lipoprotein cholesterol indicates abetalipoproteinemia. Related polyneuropathy may exist in affected individuals.

o o

Safety precautions: o It is for invitro diagnoistic purposes only. o Handle all reagents with care and avoid contact with eye mouth and skin. E2 contains sodium azide, in case of contact with skin flush affected area with copious amount of water and in case of eye contact or ingested seek immediate medical attention. o Do not perform mouth pipetteing. o Discard used reagents and sample as per disposal procedure. Potential sources of variability: o Lysed serum specimens may give falsely elevated values. o Do not use if the reagent is turbid as it indicates contamination of the reagent or if the absorbance of the blank reagent is more than 0.100.

SERUM-UREA
Quantitative estimation of serum/plasma Urea by UV Kinetic method. Impaired renal function or increased tissue protein breakdown are associated with increased Urea Nitrogen levels, whereas liver damage or pregnancy is associated with decreased levels. Principle: Urea is hydrolysed urease to form ammonium and carbon dioxide. In the second reaction, 2-Oxoglutarate reacts with ammonium in the presence of glutamate-dehydrogenase (GLDH) and co-enzyme NADH to produce L-glutamate. In this reaction two moles of NADH are oxidized to NAD+ for each mole of Urea hydrolyzed. The decrease in NADH absorbance per unit time is proportional to the concentration of urea in the sample is measured photometrically. Urea + 2 H2O Urease 2 NH4+ + CO2 GLDH 2L-Glutamate + 2NAD+ + 2H2O

2-Oxoglutarate + 2NH4+ + 2NADH Performance specifications:

Linearity: This method is linear for urea concentration upto 50.5 mmol/l in serum Measurement Range : This method has a measurement range of 1.46 -50.6 mmol/l for Urea in serum Sensitivity : The minimum detection limit by this kit is 1.46 mmol/l for Urea in serum

Primary Sample : Use serum as specimen for the test. Type of container and additive: If Serum is used as specimen - Use plain Red topped vacutainer tubes. Do not add any additive or anticoagulants. No additive/preservative needed to be added Required Equipment and Reagents: Equipment: RX Imola and RX Daytona Clinical Chemistry Analyzer.
Reagents:

Reagent 1: Capso Buffer.

NADH.

Reagent 2: Bicine Buffer. Urease. GLDH. -oxoglutarate.

Procedure: Before Switching on, check the pre-start inspection. Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode. Check water tank, wash solution, cuvette, and waste tank. Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples, load the Internal Quality Control as per Quality Control procedure. (BL/CB/L). Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples. Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface.

Inter the results in the Daily Results Register Biochemistry. Assay condition: Assay type: Kinetic Reagent volume: 180 l Wavelength: 340nm Sample volume: 5 l

Reaction Time:2 min Reaction Slope: Decrease

Temperature: 370 C

Calibration Procedure: Calibrate using a calibrator material With every new lot of reagent. Every Three months even with the same lot of reagent. If the internal QC is repeatedly out of range. After major equipment maintenance or breakdown. Record the details of calibration in the calibration register.

Quality Control Procedure: Run Two Levels (Level 1 & Level 2) of an assayed QC Sample daily before processing patient Samples. Process patient samples only if the QC values are within the Reference Range and close to the mean mentioned in the Product insert.

If both the QC levels (L 1 & L 2) are out of range on the same day in the same run perform recalibration for the analyte. Calculation of Results: The system performs all calculations internally to produce the final reported result. The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor. Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. Reference Range : Serum : 10-50 mg/dl Reportable interval of examination results: 1.46 -50.6 mmol/l for Urea in serum. Interpretation of Results: Impaired renal function or increased tissue protein breakdown are associated with increased Urea Nitrogen levels.

Safety precautions: Handle all samples as potentially infectious Handle all reagents with care and avoid contact with eye mouth and skin Do not perform mouth pipetteing Discard used reagents and sample as per disposable procedure. Potential sources of variability: Lysed serum specimens may give falsely elevated values Do not use if the reagents is turbid as it indicates contamination of the reagent Billirubin in the tested level of 60 mg/dL has no significant interference on the assay. Hemoglobin in the tested level of 1000 mg/dL has no significant interference on the assay. Ammonium ions may cause erroneously elevated results.

CREATININE
Quantitative estimation of Creatinine in human serum, plasma and urine. Creatinine measurements are used in the diagnosis and treatment of kidney disease. Principle: Creatinine in alkaline solution reacts with picrate to form a coloured complex. The rate of formation of the complex is measured. Performance Significance: Linearity: This assay is linear for concentration up to 10.0 mg/dL. Measurement Range: This method has a measurement range of 0.1 10.0 mg/dL. Sensitivity: The analytical sensitivity of this assay is 0.1 mg/dL. Primary sampling: Use serum / plasma or urine as specimen. Collect 2 ml of venous blood from a peripheral vein in a plain red topped vacutainer tube, yellow caped get tube. Separate the serum within 2 hours of collection. Allow the tube to stand at room temperature till complete clot formation occurs (for 30 minutes) and then separate the serum by centrifugation at 2500- 3000 rpm for 3 5 minutes. Ensure complete clot formation has occurred and there are no fibrin threads. Process the sample on the same day. If analysis is not done on the same day, separate the serum and store it at 2-8 C. For Urine use 24 hour urine as specimen, this is to be collected using boric acid as preservative.

 If assay is likely to be done after 48 hours of collection, store the serum at - 200C. Do not use hemolysed, contaminated samples for testing. Required Equipment and Reagents: Equipment: RX Imola and RX Daytona Clinical Chemistry Analyzer.
Reagents:

Sodium Hydroxide-0.32mol/l Picric Acid-35mmol/l Procedure: Before Switching on, check the pre-start inspection. Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode. Check water tank, wash solution, cuvette, and waste tank. Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. (BL/CB/L). Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples. Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface.

Inter the results in the Daily Results Register Biochemistry. Assay condition: Assay type: Kinetic Reagent volume: 200 l Wavelength: 510 nm Sample volume: 20 l

Reaction Time: 2 min Reaction Slope: Increase

Temperature: 370 C

Calibration Procedure: Calibrate using a calibrator material (CAL 3) With every new lot of reagent. If the internal QC is repeatedly out of range. After major equipment maintenance or breakdown. Record the details of calibration in the calibration register. Quality Control Procedure: Run Two Levels (Level 1and Level 2) of an assayed QC Sample daily before processing patient Samples. Process patient samples only if the QC values are within the Reference Range and close to the mean mentioned in the Product insert. If both the QC levels (L2 & L3) are out of range on the same day in the same run perform calibration for the analyte. External Quality Assessment Scheme /Inter Laboratory Comparison: BIORAD-EQAS-Monthly Clinical Chemistry Calculation of Results: The system performs all calculations internally to produce the final reported result. The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor. Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. Reference Range : Cord: 0.6-1.2 1-4 days: 0.3-1.0 Infant: 0.2-0.4 Children: 0.3-0.7 Adolescent: 0.5-1.0 Adult: 18-60y Male: 0.9-1.3

Female: 0.6-1.1

Reportable interval of examination results: 0.1 10.0 mg/dL. Interpretation of results Increased values are observed in impaired renal function. Increase of serum creatinine indicates a definite damage of renal tissue. Safety Precautions: Handle all samples as potentially infectious. Handle all reagents with care and avoid contact with eye, mouth and skin. Do not perform mouth pipetting. Discard used reagents and samples as per disposal procedure. Potential source of variability : Presence of fibrin in the sample may lead to erroneous results. Lysed and contaminated samples can lead to erroneously high values. The following components were found to interfere with the study: Acetoacetate, Pyruvate, Methyl dopa, Gentisic Acid, Cephalothin, Cefotaxime, Cefoxitin, Cephalosporin, Hemoglobin, Bilirubin, and Lipemia.

ALBUMIN
Quantitative estimation of Albumin in human serum by BCG endpoint method. Albumin measurements are used in the diagnosis and treatment of numerous diseases involving primarily the liver or kidneys. Principle: Albumin combines with Bromocresol green (BCG) Reagent to form a coloured product. The Intensity of the colour formed is directly proportional to the albumin present in the specimen and is measured at 578 nm. Albumin + BCG Albumin-BCG complex (Coloured Complex) Performance Significance: o Linearity: This assay is linear for concentration up to 5.0 g/dl. o Measurement Range: This method has a measurement range of 0.5 5.0 g/dl. o Sensitivity: The analytical sensitivity of this assay is 0.5 g/dl. Primary sampling: o Use only serum as specimen for the test. o Collect 2 ml of venous blood from a peripheral vein in a plain red topped vacutainer tube or yellow capped gel tube, separate the serum within 2 hours of collection.

Allow the tube to stand at room temperature till complete clot formation occurs (for 30 minutes) and then separate the serum by centrifugation at 2500- 3000 rpm for 3 5 minutes. Ensure complete clot formation has occurred and there are no fibrin threads. o Process all samples on the same day within 2 hours of collection. If processing is done after 8 hours of collection, separate the serum and aliquot it storage vials at 2- 80C. o If assay is likely to be done after 48 hours of collection, store the serum at - 200C. o Do not use hemolysed, contaminated samples for testing. Required Equipment and Reagents: Equipment: RX Imola and RX Daytona Clinical Chemistry Analyzer. o
Reagents: o Bromocresol Green o Succinate buffer: 75 mmol/l; pH 4.2. o Bromocresol green: 0.15 mmol/l. o Brij 35. o Preservative.

Step by Step Procedure: o Before Switching on, check the pre-start inspection. o Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode. Check water tank, wash solution, cuvette, and waste tank. Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. (BL/CB/L). Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples. Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface.

o o o o

o o

o o

o Inter the results in the Daily Results Register Biochemistry. Assay Conditions: Assay type: End Point Wavelength: 600 nm Reaction Time:2 min Reaction Slope: Increase Calibration Procedure: Calibrate using a calibrator material o With every new lot of reagent. o If the internal QC is repeatedly out of range. o After major equipment maintenance or breakdown. o Record the details of calibration in the calibration register. Quality Control Procedure: o Run Two Levels (Level 1 & Level 2) of an assayed QC Sample daily before processing patient Samples. Process patient samples only if the QC values are within the Reference Range and close to the mean mentioned in the Product insert. o If both the QC levels (L 1 & L 2) are out of range on the same day in the same run perform calibration for the analyte. External Quality Assessment Scheme /Inter Laboratory Comparison: BIORAD-EQAS-Monthly Clinical Chemistry. Calculation of Results: o The system performs all calculations internally to produce the final reported result. o The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor. Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. Reference Interval / Range :
Adults: 3.50 5.20 g/dl Neonates 3.8 - 4.2 g/dl

Reagent volume: 330 l Sample volume:3 l Temperature: 370 C

Reportable interval of examination results: 0.5 5.0 g/dl. Interpretation of results: o Albumin level in blood increases due to dehydration, reduced plasma water content, stasis during vein puncture which can cause fluid to escape into the extra cellular compartment.

Albumin levels in blood is found to be decreased in conditions like excessive protein loss from kidney, skin, or intestine, decreased synthesis due to dietary, hepatic disease or malabsorption, increased catabolism in fever, untreated diabetes mellitus and hypertension. Safety Precautions: o Handle all samples as potentially infectious. o Handle all reagents with care and avoid contact with eye, mouth and skin. o Do not perform mouth pipetting. o Discard used reagents and samples as per disposal procedure. Potential source of variability : o Lysed and contaminated samples can lead to erroneously high values. o The method is based on Albumin - BCG binding. The BCG binding capacity is strictly correlated to the nature of albumin. o

SERUM TOTAL PROTEIN

Quantitative estimation of Total Protein (TP) in human serum by Biuret endpoint method. Total protein measurements are used in the diagnosis and treatment of diseases involving the liver, kidney or bone marrow, as well as other metabolic or nutritional disorders. Principle: Cupric ions, in an alkaline medium, interact with protein peptide bonds, resulting in the formation of a colored peptide/copper complex whose intensity is proportional to the concentration of proteins in the sample. Protein + Cu++ Alkali Protein-Copper Complex

Performance Significance: o Linearity: This assay is linear for concentration up to 124 g/l o Measurement Range: This method has a measurement range of <5.05g/l -124g/l o Sensitivity: The analytical sensitivity of this assay is <5.05g/l Primary sampling: o Use only serum as specimen for the test. o Collect 2 ml of venous blood from a peripheral vein in a plain red topped vacutainer tube. Separate the serum within 2 hours of collection. o Allow the tube to stand at room temperature till complete clot formation occurs (for 30 minutes) and then separate the serum by centrifugation at 2500- 3000 rpm for 3 5 minutes. Ensure complete clot formation has occurred and there are no fibrin threads. o Process all samples on the same day within 2 hours of collection. If processing is done after 8 hours of collection, separate the serum and aliquot it storage vials at 2- 80C.

o If assay is likely to be done after 48 hours of collection, store the serum at - 200C o Do not use hemolysed, contaminated samples for testing. Type of Container and additive: o Use Red topped Plain vaccutainer tubes / green topped heparinised vaccutainer tubes for collecting samples. o Do not add any additives / preservatives. Required Equipment and Reagents: Equipment: RX Imola and RX Daytona Clinical Chemistry Analyzer.
Reagents:

o Sodium Hydroxide o Na-K-tartarate o Potassium iodide o cupric sulfate Procedure: o Before Switching on, check the pre-start inspection. o Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode. Check water tank, wash solution, cuvette, and waste tank. Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. (BL/CB/L). Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples. Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface. Inter the results in the Daily Results Register Biochemistry.

o o o o

o o

o o

Assay condition: Assay type: End Point Wavelength: 546nm Reaction Time:10 min Reaction Slope: Increase

Reagent volume: 180 l Sample volume: 4 l Temperature: 370 C

Calibration Procedure: Calibrate using a calibrator material o With every new lot of reagent. o Every Three months even with the same lot of reagent. o If the internal QC is repeatedly out of range. o After major equipment maintenance or breakdown. o Record the details of calibration in the calibration register. Quality Control Procedure: o Run Two Levels (Level 1 & Level 2) of an assayed QC Sample daily before processing patient Samples. Process patient samples only if the QC values are within the Reference Range and close to the mean mentioned in the Product insert. o If both the QC levels (L 1 & L 2) are out of range on the same day in the same run perform recalibration for the analyte.

External Quality Assessment Scheme /Inter Laboratory Comparison: BIORAD-EQAS-Monthly Clinical Chemistry. Calculation of Results: o The system performs all calculations internally to produce the final reported result. o The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor.

Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. Reference Interval / Range: Adults: 6.4 - 8.3 g/dl. Reportable interval of examination results: 10 U/l to 600 U/l. Interpretation of results: The following analytes were tested up to the following levels and were found not to interfere. Haemoglobin : 10 g/dl

Bilirubin : Triglycerides : Intralipid : Safety Precautions:

29 mg/dl 20 mg/dl 2%

o Handle all samples as potentially infectious. o Handle all reagents with care and avoid contact with eye, mouth and skin. o Do not perform mouth pipetting. o Discard used reagents and samples as per disposal procedure. Potential source of variability : o Presence of fibrin in the sample may lead to erroneous results. o Lysed and contaminated samples can lead to erroneously high values.

SERUM ALBUMIN/GLOBULIN RATIO

The albumin/globulin ratio is the calculation of serum albumin compare to serum globulin level to determine whether there is an overproduction or underproduction of gamma-globulin. A low A/G ratio may be due to overproduction of gamma-globulin monoclonal/polyclonal gammopathy, multiple myeloma or autoimmune diseases etc.) or due to low albumin (low production as in cirrhosis or excessive loss as in nephrotic syndrome or protein losing enteropathy etc.). In contrast, if the A/G ratio is high then one should look for diseases with low gamma-globulin production such as agammaglobuminemia. Often a serum electrophoresis or immunoelectrophoresis can determine a more specific abnormality.

Principle: Calculation. Procedure: Refer Test Procedures of Albumin & Globulin Calculation of Results: Calculation of results: Albumin Globulin Interpretation of Results:

Low A/G Ratio occur in collagen disease, chronic inflammation, liver diseases macroglobunemia, severe infection, burns, ulcerative, colitis,

Potential Source of Variablity : o Lysed serum specimen may give falsely elevated values.

ALKALINE PHOSPHATASE
Quantitative estimation of Alkaline Phosphatase in human serum. Measurement of alkaline phosphatase are of use in the diagnosis, treatment and investigation of hepatobilary disease and in bone disease associated with increase osteoblastic activity and in parathyroid and intestinal disease. Principle: Alkaline Phosphatase hydrolyses p-nitrophenyl phosphate (p-NPP) in the presence of magnesium ions, to form yellow coloured product, p-nitrophenol and phosphate which is read at 405nm. ALP p-Nitrophenyl phosphate + H2O p-Nitrophenol + Phosphate Mg++ Performance Significance: o Linearity: This assay is linear for concentration up to 843 U/l. o Measurement Range: This method has a measurement range of 18.0 to 843 U/l. o Sensitivity: The analytical sensitivity of this assay is 18.0 IU/l. Primary sampling: o Use only serum as specimen for the test. o Collect 2 ml of venous blood from a peripheral vein in a plain red topped vacutainer tube or yellow capped gel tube; separate the serum within 2 hours of collection. o Allow the tube to stand at room temperature till complete clot formation occurs (for 30 minutes) and then separate the serum by centrifugation at 2500- 3000 rpm for 3 5 minutes. Ensure complete clot formation has occurred and there are no fibrin threads. o Process all samples on the same day within 2 hours of collection. If processing is done after 8 hours of collection, separate the serum and aliquot it storage vials at 2- 80C. If assay is likely to be done after 48 hours of collection, store the serum at - 200C. o Do not use hemolysed, contaminated samples for testing Type of Container and additive: o Use plain Red topped vacutainer tube or yellow capped gel tube for collecting samples. o Do not add any additives / preservatives Required Equipment and Reagents: Equipment: RX Imola Clinical Chemistry Analyzer.

Reagents:

Buffer

o 2-amino-2-methyl-1-propanol: 0.35 mol/l,pH 10.4 o Mg2+ : 2.0 mmol/l o Substrate o p-nitrophenylphsphate 10 mmol/l Step by Step Procedure: o Before Switching on, check the pre-start inspection. o Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode. Check water tank, wash solution, cuvette, and waste tank. Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. (BL/CB/L). Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples. Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface.

o o o o

o o

o o

Assay condition: Assay type: Kinetic Reagent volume: R1:180 l R2: 36 l Wavelength: 415 nm Sample volume: 8 l

Reaction Time: 3 min Reaction Slope: Increase

Temperature: 370 C

Calibration Procedure: Calibrate using a calibrator material (CAL 3) With every new lot of reagent. If the internal QC is repeatedly out of range. After major equipment maintenance or breakdown. Record the details of calibration in the calibration register. Quality Control Procedure: Run Two Levels (Level 1 and Level 2 ) of an assayed QC Sample daily before processing patient Samples. Process patient samples only if the QC values are within the Reference Range and close to the mean mentioned in the Product insert. If both the QC levels (L1 & L2) are out of range on the same day in the same run perform calibration for the analyte. External Quality Assessment Scheme /Inter Laboratory Comparison: BIORAD-EQAS-Monthly Clinical Chemistry. Calculation of Results: o The system performs all calculations internally to produce the final reported result. o The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor. Reference Interval / Range: Adult: 42.0-98 U/l. Children: 47 - 406 U/l.

Reportable interval of examination results: 18.0 to 843 U/l. Interpretation of results: o Increased ALP levels are observed in hepatobiliary diseases, diseases of biliary tract, and primary bone diseases such as osteomalacia, osteogenesis imperfectia, Vitamin D intoxication and primary bone tumors. ALP level is also increased in conditions like acromegaly, multiple myeloma, Renal Insufficiency, hyperthyroidism, sarcoidosis, bone tuberculosis and healing fractures. o Reduced levels of ALP are found in familial hypophasphatasia, hypoparathyroidism, achondroplasia, adynamic bone disease in dialysis patients, pituitary dwarfism, chronic radiation sickness and malnutrition. Safety Precautions: o Handle all samples as potentially infectious.

o Handle all reagents with care and avoid contact with eye, mouth and skin. o Do not perform mouth pipetting. o Discard used reagents and samples as per disposal procedure. Potential source of variability: o Presence of fibrin in the sample may lead to erroneous results. o Lysed and contaminated samples can lead to erroneously high values. o Hemoglobin, Bilirubin, and Lipemia in the samples can cause interference.

SGOT ASPARTATE AMINOTRANSFERASE

Quantitative estimation of Aspartate Aminotransferase (AST) or Serum glutamic oxaloacetic transaminase (SGOT) by enzymatic kinetic method. Aspartate aminotransferase measurements are used in the diagnosis and treatment of certain types of liver and heart disease. Principle: -oxoglutarate react with the L-aspartate in the presence of AST to form L-glutamate plus oxaloacetate. The indicator reaction utilizes the oxaloacetate for a kinetic determination of NADH consumption. AST -oxoglutarate +L-aspartate L-glumate +oxaloacetate

Oxaloacetate + NADH + H+

LDH

L-malate + NAD+

Performance Significance: Linearity: This assay is linear for concentration up to 657 U/l and extends up to 2628U/l. Measurement Range: This method has a measurement range of 18.7 to 657 U/l. Sensitivity: The analytical sensitivity of this assay is 18.7 U/l. Primary sample: Use only serum / plasma as specimen for the test. Collect 2 ml of venous blood in a plain red topped vacutainer tube. Allow the tube to stand for 30 minutes at room temperature and separate the serum by centrifugation at 2500 - 3000 rpm for 5 - 10 minutes. Ensure complete clot formation has occurred and there are no fibrin threads. Process all samples on the same day within 2 hours of collection. If processing is done after 8 hours of collection, separate the serum and aliquot it storage vials at 2- 80C. If assay is likely to be done after 48 hours of collection, store the serum at - 200C. Do not use hemolysed, contaminated samples for testing. Type of Container and additive:

Use Red topped Plain vaccutainer tubes / green topped heparinised vaccutainer tubes for collecting samples. Do not add any additives / preservatives. Required Equipment and Reagents: Equipment: RX Imola and RX Daytona Clinical Chemistry Analyzer.
Reagents:

Buffer/Enzyme Tris buffer: 80 mmol/l, pH 7.5 L-aspartate:240 mmol/l MDH: 1.5 U/ml LD: 2 U/ml -oxoglutarate/Coenzyme -oxoglutarate: 12 mmol/l NADH: 0.18 mmol/l Procedure: Before Switching on, check the pre-start inspection. Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode. Check water tank, wash solution, cuvette, and waste tank. Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. (BL/CB/L). Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples.

Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface.

Inter the results in the Daily Results Register Biochemistry. Assay condition: Assay type: Kinetic Reagent volume: R1- 180 l R2 36 l Wavelength: 340nm Reaction Time:5 min Reaction Slope: Decrease Calibration Procedure: Calibrate using a calibrator material With every new lot of reagent. Every Three months even with the same lot of reagent. If the internal QC is repeatedly out of range. After major equipment maintenance or breakdown. Record the details of calibration in the calibration register. Quality Control Procedure: Run Two Levels (Level 1 & Level 2) of an assayed QC Sample daily before processing patient Samples. Process patient samples only if the QC values are within the Reference Range and close to the mean mentioned in the Product insert. If both the QC levels (L 1 & L 2) are out of range on the same day in the same run perform recalibration for the analyte. External Quality Assessment Scheme /Inter Laboratory Comparison: BIORAD-EQAS-Monthly Clinical Chemistry. Calculation of Results: The system performs all calculations internally to produce the final reported result. The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor. Sample volume: 16 l Temperature: 370 C

Interferences:

Interferences vary depending upon icterus, haemolysis and lipemia status. Reference Interval / Range : Serum for Men: 35 U/l. Reportable interval of examination results: 18.7 to 657 U/l. Interpretation of results Increased Serum levels are seen in the following conditions: Hepatocellular destruction, myocardial infraction, pulmonary infraction, post hepatic conditions, and Musculo skeletal disease. Decreased Serum levels are seen in the following conditions: End stage of liver disease, and Pregnancy.

Safety Precautions: Handle all samples as potentially infectious. Handle all reagents with care and avoid contact with eye, mouth and skin. Do not perform mouth pipetting. Discard used reagents and samples as per disposal procedure. Potential source of variability : Presence of fibrin in the sample may lead to erroneous results. Lysed and contaminated samples can lead to erroneously high values. Lipemia, pyruvate and Bilirubin may interfere with the test. Samples showing evidence of hemolysis should not be used. Hemolysis may cause falsely elevated results. Pyruvate may cause elevated results at a level of 4 mg/dL. Lipemic samples >3+ should be ultra-centrifuged and the analysis performed on the infranate. Drugs, disease and preanalytical variables as mentioned in the references may cause interferences.

SGPT ALANINE AMINOTRANSFERASE (ALT)

Quantitative estimation of Alanine Aminotransferase (ALT) or Serum glutamic pyruvic transaminase (SGPT) in human serum by kinetic method. Alanine aminotransferase measurements are used in the diagnosis and treatment of certain liver diseases (e.g., viral hepatitis and cirrhosis) and heart diseases. Principle:

-oxoglutarate reacts with the L-alanine in the presence of ALT to form L-glutamate plus pyruvate. The indicator reaction utilizes the pyruvate for a kinetic determination of NADH consumption. -oxoglutarate +L-alanine ALT LDH L-glumate +Pyruvate

Pyruvate + NADH + H+

L-lactate + NAD+

Performance Significance: Linearity: This assay is linear for concentration up to 600 U/l and the linearity extends up to 2400 U/l. Measurement Range: This method has a measurement range of 10 U/l to 600 U/l. Sensitivity: The analytical sensitivity of this assay is 10 U/l. Primary sampling: Use only serum / plasma as specimen for the test. Collect 2 ml of venous blood in a plain red topped vacutainer tube. Allow the tube to stand for 30 minutes at room temperature and separate the serum by centrifugation at 2500 - 3000 rpm for 5 - 10 minutes. Ensure complete clot formation has occurred and there are no fibrin threads. Process all samples on the same day within 2 hours of collection. If processing is done after 8 hours of collection, separate the serum and aliquot it storage vials at 2- 80C. If assay is likely to be done after 48 hours of collection, store the serum at - 200C. Do not use hemolysed, contaminated samples for testing. Type of Container and additive: Use Red topped Plain vaccutainer tubes / green topped heparinised vaccutainer tubes for collecting samples. Do not add any additives / preservatives. Required Equipment and Reagents: Equipment: RX Imola and RX Daytona Clinical Chemistry Analyzer.
Reagents:

Buffer/Enzyme Tris buffer: 100 mmol/l, pH 7.5 L-aspartate: 0.5 mmol/l LD: 1.2 U/ml -oxoglutarate/Coenzyme -oxoglutarate: 15 mmol/l NADH: 0.18 mmol/l Procedure:

Before Switching on, check the pre-start inspection. Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode. Check water tank, wash solution, cuvette, and waste tank. Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. (BL/CB/L). Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples. Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface.

Inter the results in the Daily Results Register Biochemistry. Assay condition: Assay type: Kinetic Reagent volume: R1- 180 l R2 36 l Wavelength: 340 nm Reaction Time:5 min Reaction Slope: Decrease Sample volume: 4 l Temperature: 370 C

Calibration Procedure: Calibrate using a calibrator material With every new lot of reagent. Every Three months even with the same lot of reagent.

 If the internal QC is repeatedly out of range. After major equipment maintenance or breakdown. Record the details of calibration in the calibration register. Quality Control Procedure: Run Two Levels (Level 1 & Level 2) of an assayed QC Sample daily before processing patient Samples. Process patient samples only if the QC values are within the Reference Range and close to the mean mentioned in the Product insert. If both the QC levels (L 1 & L 2) are out of range on the same day in the same run perform recalibration for the analyte. External Quality Assessment Scheme /Inter Laboratory Comparison: BIORAD-EQAS-Monthly Clinical Chemistry. Calculation of Results: The system performs all calculations internally to produce the final reported result. The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor. Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. Reference Interval / Range Serum Men (37C) : 45 U/l 8.1 Serum Women(37C): 34 U/l Reportable interval of examination results: 10 U/l to 600 U/l. Interpretation of results Increased values are observed in o Hepatitis o Jaundice o Necrosis o Cirrhosis Decreased values are observed in o End stage liver disease o Renal insufficiency Safety Precautions: Handle all samples as potentially infectious. Handle all reagents with care and avoid contact with eye, mouth and skin. Do not perform mouth pipetting. Discard used reagents and samples as per disposal procedure. Potential source of variability :

Presence of fibrin in the sample may lead to erroneous results. Lysed and contaminated samples can lead to erroneously high values. Samples showing evidence of hemolysis should not be used. Hemolysis may cause falsely elevated results.

BILIRUBIN - TOTAL
Quantitative estimation of Total Bilirubin in human serum and plasma by Jendrassik & Grof Method (1938). Bilirubin measurements are used in the diagnosis and treatment of liver, hemolytic, hematological, and metabolic disorders, including hepatitis and gall bladder block. Principle: Bilirubin reacts with diazotized sulphanilic acid to form an azocompound. The colour which is proportional to the concentration of bilirubin. Total Bilirubin is determined in the presence of caffinne, which release albumin bound bilirubin, by the reaction with diazotized acid. Direct Bilirubin + Diazo + H+ Azobilirubin Caffeine, Benzoate, Acetate Performance Significance: Linearity: This assay is linear for concentration up to 25 mg/dL Measurement Range: This method has a measurement range of 0.1 25 mg/dL Sensitivity: The analytical sensitivity of this assay is 0.1 mg/dL Primary sampling: Use only serum / plasma as specimen for the test. Collect 2 ml of venous blood from a peripheral vein in a plain red topped vacutainer tube or yellow capped gel tube. Separate the serum within 2 hours of collection. Allow the tube to stand at room temperature till complete clot formation occurs (for 30 minutes) and then separate the serum by centrifugation at 2500- 3000 rpm for 3 5 minutes. Ensure complete clot formation has occurred and there are no fibrin threads. Process the sample on the same day. If analysis is not done on the same day, separate the serum and store it at 2-8 C. If assay is likely to be done after 48 hours of collection, store the serum at - 200C. Do not use hemolysed, contaminated samples for testing. Type of Container and additive: Use Red topped Plain vaccutainer tubes / green topped heparinised vaccutainer tubes, yellow capped gel tube for collecting samples. Do not add any additives / preservatives.

Required Equipment and Reagents: Equipment:

RX Imola and RX Daytona Clinical Chemistry Analyzer.

Reagents: R1 Caffeine: 0.26 mmol/l Sodium benzoate :0.52 mmol/l Sulphanilic acid: 29 mmol/l Hydrochloric acid: 170 mmol/l

Sodium nitrite: 385mmol/l Procedure: Before Switching on, check the pre-start inspection. Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode. Check water tank, wash solution, cuvette, and waste tank. Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. (BL/CB/L). Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples. Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface. Inter the results in the Daily Results Register Biochemistry. Assay Conditions: Assay type: End Point Reagent volume: R1-180 l R2-22 l Wavelength: 546 Reaction Time: 10 min Reaction Slope: Increase Calibration Procedure: Calibrate using a calibrator material With every new lot of reagent. If the internal QC is repeatedly out of range. Sample volume: 14 l Temperature: 370 C

 After major equipment maintenance or breakdown. Record the details of calibration in the calibration register. Quality Control Procedure: Run Two Levels (Level 1 and Level 2 ) of an assayed QC Sample daily before processing patient Samples. Process patient samples only if the QC values are within the Reference Range and close to the mean mentioned in the Product insert. If both the QC levels (L1 & L2) are out of range on the same day in the same run perform calibration for the analyte. External Quality Assessment Scheme /Inter Laboratory Comparison: BIORAD-EQAS-Monthly Clinical Chemistry. Calculation of Results: The system performs all calculations internally to produce the final reported result. The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor. Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. Reference Interval / Range :
Total bilirubin: Premature: Cord : <2.0 0-1 D: 1.0-8.0 1-2 days: 6.0-12.0 3-5 days: 10.0-14.0 Full term: Cord: <2.0 0-1day: 2.0-6.0 1-2 days: 6.0-10.0 3-5 days: 4.0-8.0 Adult: 0.0-2.0

Reportable interval of examination results: 0.1 117 mg/dL Interpretation of results: Elevation in serum unconjugated bilirubin levels occur in hemolytic jaundice due to excessive hemolysis. Hepatic Jaundice is associated with increase in both conjugated and unconjugated bilirubin in serum. Safety Precautions: Handle all samples as potentially infectious. Handle all reagents with care and avoid contact with eye, mouth and skin.

 Do not perform mouth pipetting. Discard used reagents and samples as per disposal procedure. Potential source of variability : Presence of fibrin in the sample may lead to erroneous results. Lysed and contaminated samples can lead to erroneously high values. The following can cause interferences Hemoglobin, Lipemia, Azide, Citrate, oxalate Gentisic acid. Drugs, Disease, and preanalytical variables can interfere with the test.

BILIRUBIN DIRECT
Quantitative estimation of Direct Bilirubin in human serum and plasma by colorimetric Method. Bilirubin measurements are used in the diagnosis and treatment of liver, hemolytic, hematological, and metabolic disorders, including hepatitis and gall bladder block. Principle: Bilirubin reacts with diazotized sulphanilic acid to form an azodye. The colour which is proportional to the concentration of bilirubin. Direct Bilirubin + Diazo + H+ Performance Significance: Linearity: This assay is linear for concentration up to 8.11 mg/dL Measurement Range: This method has a measurement range of 0.08 8.11 mg/dL Sensitivity: The analytical sensitivity of this assay is 0.08 mg/dL Primary sampling: Use only serum / plasma as specimen for the test. Collect 2 ml of venous blood from a peripheral vein in a plain red topped vacutainer tube or yellow capped gel tube, Separate the serum within 2 hours of collection. Allow the tube to stand at room temperature till complete clot formation occurs (for 30 minutes) and then separate the serum by centrifugation at 2500- 3000 rpm for 3 5 minutes. Ensure complete clot formation has occurred and there are no fibrin threads. Process the sample on the same day. If analysis is not done on the same day, separate the serum and store it at 2-8 C. If assay is likely to be done after 48 hours of collection, store the serum at - 200C. Do not use hemolysed, contaminated samples for testing. Type of Container and additive: Use Red topped Plain vaccutainer tubes / green topped heparinised vaccutainer tubes, yellow capped gel tubes, for collecting samples. Caffeine, Benzoate, Acetate Azobilirubin

 Do not add any additives / preservatives. Required Equipment and Reagents: Equipment: RX Imola and RX Daytona Clinical Chemistry Analyzer.
Regents: Saline: Nacl : 9g/l Sulphanilic acid: 29 mmol/l Hydrochloric acid: 170 mmol/l

Sodium nitrite: 385mmol/l Procedure: Before Switching on, check the pre-start inspection. Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode. Check water tank, wash solution, cuvette, and waste tank. Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. (BL/CB/L). Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples. Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface. Inter the results in the Daily Results Register Biochemistry.

Assay condition: Assay type: End Point Reagent volume:

R1-180 l R2-22 l Wavelength: 546 Reaction Time: 10 min Reaction Slope: Increase Calibration Procedure: Calibrate using a calibrator material With every new lot of reagent. If the internal QC is repeatedly out of range. After major equipment maintenance or breakdown. Record the details of calibration in the calibration register. Sample volume:14 l Temperature: 370 C

Quality Control Procedure: Run Two Levels (Level 1 and Level 2 ) of an assayed QC Sample daily before processing patient Samples. Process patient samples only if the QC values are within the Reference Range and close to the mean mentioned in the Product insert. If both the QC levels (L1 & L2) are out of range on the same day in the same run perform calibration for the analyte. External Quality Assessment Scheme /Inter Laboratory Comparison: BIORAD-EQAS-Monthly Clinical Chemistry. Calculation of Results: The system performs all calculations internally to produce the final reported result. The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor. Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. Reference Interval / Range :
Direct bilirubin: 0.0-0.2 mg/dL

Reportable interval of examination results: 0.08 8.11 mg/dl. Interpretation of results: Elevation in serum unconjugated bilirubin levels occur in hemolytic jaundice due to excessive hemolysis. Hepatic Jaundice is associated with increase in both conjugated and unconjugated bilirubin in serum. Critical/Alert level values: Not applicable

Safety Precautions: Handle all samples as potentially infectious. Handle all reagents with care and avoid contact with eye, mouth and skin. Do not perform mouth pipetting. Discard used reagents and samples as per disposal procedure. Potential source of variability : Presence of fibrin in the sample may lead to erroneous results. Lysed and contaminated samples can lead to erroneously high values. The following can cause interferences Hemoglobin, Lipemia, Azide, Citrate, oxalate Gentisic acid. Drugs, Disease, and preanalytical variables can interfere with the test.

INDIRECT BILIRUBIN
Quantitative estimation of indirect bilirubin in serum derived from total bilirubin (TBIL) and direct bilirubin (DBIL). Indirect bilirubin is used for evaluating liver and hereditary diseases. Principle: Total and Direct Bilirubin values are calculated on Randox Imola and Randox Dytona Systems, using these measured results and the following equation is used to calculate Indirect Bilirubin: Indirect Bilirubin = Total Bilirubin Direct Bilirubin Reference Range: 0.0 0.6 mg/dL Interpretation of Results: An increased indirect bilirubin occurs in both intrahepatic and extrahepatic biliary tract obstructions. Hepatocellular causes of elevation include hepatitis, cirrhosis, and advanced neoplastic states.

FOLIC ACID
ARCHITECT Folate assay is a chemiluminescent Microparticles immunoassay (CMIA) for the quantitative determination of the Folate in human serum and plasma on the ARCHITECT i system. The ARCHITECT Folate assay is to be used as an aid in monitoring megaloblastic (microcytic) anemia.Folate deficiency caused by low dietary intake,malabsorption due to gasterointestinal diseases,inadequate utilization due to enzyme deficiencies or folate antagonist therapy. Principle:

The ARCHITECT Folate assay is a two step immuno assay for the quantitative determination of the Folate in human serum and plasma using CMIA technology with flexible assay protocols, referred to as chemiflux. Two pre-treatment steps mediate the release of folate from endogenous folate binding protein.In the first step, the sample and Pre-Treatment Reagent 2 (Dithiothreiotol or DTT) are aspirated and dispensed into a reaction vessel (RV).In Pre-treatment step 2, an aliquot of sample/Pre- Treatment Reagent 2 mixture is aspirated and dispensed into a second RV.PreTreatment Reagent 1 (potassium hydroxide or KOH) is then added.An aliquot of the pre-treated sample is transferred into a third RV,followed by the addition of Folate Binding Protein(FBP) coated paramagnetic microparticles and assay specific diluent.Folate present in the sample binds to the FBP coated microparticles. After washing, pteroic acid - acridinium labeled conjugate is added and binds to unoccupied sites on the FBP-coated microparticles.Pre-Trigger and Trigger Solutions are then added to the reaction mixture; the resulting chemiluminescent reaction is measured as relative light unit (RLUs). An inverse relationship exists between the amount of folate in the sample and the RLUs detected by the architect i system optics. Performance Specifications: Linearity: up to 20 ng/mL Lower Detection limit: 3.5 ng/mL Measurement Range: 3.5 to 20 ng/mL

Trueness of measurement: Folate assay values are expressed as ng/ml. A unit is a value related to a World Health Organisation (WHO) Standard 03/178. Analytical Sensitivity: The analytical sensitivity of this assay is < 3.5 ng/mL of Folate in serum. Analytical Specificity: Mean assay specificity is < 12%. Bilirubin > 20 mg/dL, Hemoglobin >100mg/dL, Total Protein >12g/dL and Triglycerides > 3 g/dL were found to interfere with the CA 125 assay. Primary Sample system: Use serum/plasma as specimen for the test. If serum is used as specimen, collect 2 3 mL of venous blood from a peripheral vein in a red topped vacutainer tube. Allow the tube to stand at room temperature till complete clot formation occurs (for 30 minutes) and then separate the serum by centrifugation at 2500- 3000 rpm for 5 minutes.

Separate the serum within 2 hours of collection. Ensure complete clot formation has occurred and there are no fibrin threads. Do not use lysed serum for testing as it may give very high results. Do not use contaminated / turbid samples for testing. Process the sample on the same day. If analysis is not done on the same day, separate the serum and store it at 2 to 8 C for up to 7 days. If the sample is been stored more than 7 days, store it at 20 C.

Type of Container and Additive: If Serum is used as specimen - Use plain Red topped vacutainer tubes. Do not add any additive or anticoagulants. If Plasma is used as specimen - Use green topped Li heparin tube or Lavender topped K3 EDTA tube.

Required Equipment and Reagents: Equipment: Abbott Architect - Fully Automated Immunoassay Analyzer. Reagents: Microparticles: 1 or 4 bottle (s) (6.6 mL for the 100 test bottle/27.0 mL for the 500 test bottle) anti-CA 125 (mouse, monoclonal) coated Microparticles in TRIS buffer with protein (bovine) stabilizers. Preservative: antimicrobial agents. Conjugate: 1 or 4 bottles (5.9 mL for the 100 test bottle/26.3 mL for the 500 test bottle) anti-CA 125 (mouse, monoclonal) acridinium-labeled conjugate in phosphate buffer with protein (bovine) stabilizer. Minimum concentration: 0.075 g/ml. Preservative: Antimicrobial agents.

Other Reagents Pre trigger Solution: Pre trigger solution containing 1.32 %( w/v) hydrogen peroxide. Trigger Solution: Trigger Solution containing 0.35N sodium hydroxide. Wash: Wash buffer containing phosphate buffered saline solution. Preservative: antimicrobial agents.

Procedure: Before Switching on, check the pre-start inspection.

Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a snapshot screen. (Perform daily maintenance) Check Pre trigger, trigger, Assay cup, assay tip tray, solid waste tray, and liquid waste container bottles. Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <F8> key on monitor display, and begins to process patient samples. Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface. Enter the results in the Daily Results Register Biochemistry.

Assay Conditions: Assay Temperature Sample Volume Measuring Point End Point 25C 2 75 L Two steps assay

Total Assay Time 28 minutes

Calibration Procedure: Calibrate the instrument using Elecsys CA 125 CalSet. Use fresh reagent while performing calibration (within 24 hours of registering the reagent on to the analyzer

using bar code). The equipment automatically reads the calibrator from the bar code of the calibrator and stores the reading. During calibration process the original readings are rescaled to establish a valid stored value for the system. Validity of the results of calibrator is checked automatically by the machine with each new calibration and draws attention to any deviations. Carry out Calibration whenever the reagent lot is changed. After major equipment breakdown or maintenance / service. Whenever the internal QC is repeatedly out of range. Once every 28 days using the same reagent lot or after 7 days if the reagents are onboard. Record the details in the calibration register.

Quality Control Procedures: Internal Quality Control Program: Run alternate levels of assayed QC samples for every 25 patient samples and ensure the QC values are with in the Reference Range and close to the mean mentioned in the Internal Quality Control product insert. Internal Quality Control is also run after each new calibration and after every major equipment breakdown or maintenance.

External Quality Assessment Scheme /Inter Laboratory Comparison: BIORAD-EQAS-Monthly Immuno Assay. Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. In patients receiving high doses of Biotin therapy ( > 5 mg/day) no samples should be taken for 8 hours after the last dosage administration. Erroneous findings may be obtained from patients receiving treatment for monoclonal antibodies or have received them for diagnostic purposes.

Calculation of Results: The Architect CA-125 Assay utilizes a four parameter logistic curve fit data reduction method (4 PLC, Y weighted) to generate a calibration curve for measuring the value. Reference interval: Females: <35.0 U/mL.

Reportable interval of examination results: 1.0 to 1000 U/mL 15.0 Laboratory Interpretation: High levels of CA 125 present in the serum in case of residual tumor. 15.2 Low levels of CA 125 occur in patients suffering from residual ovarian cancer. 16.0 Safety Precautions: Follow Material Safety Data Sheet provided by the reagent manufacturer. Handle all samples as potentially infectious Handle all reagents with care and avoid contact with eye mouth and skin Do not perform mouth pipetting. Discard used reagents and sample as per disposal procedure. Potential sources of variability: Presence of fibrin in the sample may lead to erroneous results and hence centrifuge the sample only after complete clot formation. Lysed serum specimens may give falsely elevated values. Patients receiving treatment for HAMA for diagnosis may produce anomalous values. The CA 125 assay value should be used in conjunction with information available from clinical evaluation and other diagnostic procedure this assay should not be used as a cancer screening test. Heterophilic antibodies in human serum can react with reagent immunoglobulins, interfering with invitro immunoassay

GAMMA GLUTAMYL TRANSFERASE

Quantitative estimation of GT activity in human serum/plasma samples by in vitro determination using carboxy substrate. Estimation of Gamma- Glutamyl Transferase (GGT) is useful in the diagnosis and treatment of hepatobiliary disorders. Principle: Gamma-glutamyl transferase catalyzes the transfer of the glutamyl group from -L glutamyl3-carboxy-p-nitroanilide to acceptor glycyl-glycine with the formation of L-glutamylglyclyglycine and 5-amino-2-nitrobenzoate. The rate of 5-amino-2-nitrobenzoate formation is measured at 405 nm. -GT 2.2 L--glutamyl-3-carboxy-4-nitroanilide + glycylglycine L--glutamyl-glycylglycine + 5-amino-2-nitrobenzoate. Performance Specifications:

Linearity: This kit is linear for GT activity up to1280 U/l in serum. Measurement Range: This kit has a measurement range of 8-1280 U/l of GT activity in serum. Sensitivity: The minimum detection limit by this kit is 8 U/l in serum. Primary sample: Use only non haemolysed serum. Only use EDTA plasma or Li Heparinised plasma that is free from haemolysis other anticoagulants interfere with this test. If serum is used as specimen, collect 3 ml of venous blood in a plain red topped vaccutainer tube, or yellow capped gel tube. Do not use hemolysed, contaminated or lipemic sera. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500- 3000 rpm for 5 10 minutes. Do not use contaminated / turbid samples for testing. Process the sample on the same day of collection with in 3 hours of collection. If analysis is not done on the same day separate the serum and store it at 2-8 C till analysis for a maximum of up to 7 days. If it is been stored for more than 7 days store it at -20C. Type of Container / Additive: Use Red topped Plain vaccutainer tubes / green topped heparinised vaccutainer tubes, yellow capped gel tube for collecting samples No additives/preservatives needed to be added Required Equipment and Reagents: Equipment: RX Imola and RX Daytona Clinical Chemistry Analyzer. Reagents: R1 Buffer Glycylglycine 150mmol/l, pH7.7 Reagent 2 :Substrate L--Glutamyl-3-carboxy-4-nitroanilide

6.0 mmol/l.

Procedure: Before Switching on, check the pre-start inspection. Once pre-start inspections are done, Switch ON the Computer, Printer, and the analyzer. Wait for initialization process to be completed. After initialization is completed, ensure that the screen displays a standby mode. Check water tank, wash solution, cuvette, and waste tank.

Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray Log on the system, check system overview, and prepare the system before start routine operation. Daily before processing the patient samples load the Internal Quality Control as per Quality Control procedure. (BL/CB/L). Remove controls from the sample disk or rack once QC is validated. Start routine sample for analysis. Place the barcode samples on the sample disk or enter the patient ID and cross verify SID code on the sample matches with that on the requisition form. Press the <START> key on monitor display, and begins to process patient samples. Once the process is completed, result will be displayed on the monitor screen, and automatically will be transferred to the LIS through interface.

Enter the results in the Daily Results Register Biochemistry. Assay condition: Assay type: Kinetic Reagent volume: R1-180 l R2-36 l Wavelength: 340 nm Reaction Time: 2.5 min Reaction Slope: Increase Sample volume:5 l Temperature: 370 C

Calibration procedure: Calibrate using the recommended calibrator every time a new lot of reagent is used or as follows When changing the lot numbers of assay reagents When replacing the system components When quality control results are repeatedly out of range and when a new vial of control does not correct the problem After major equipment breakdown or maintenance or service Record the details in the calibration register.

The calibration report should be used in conjunction with quality control results to determine the validity of a calibration.

Quality Control Procedure: Run two levels (level 1 & level 2) of an assayed QC sample daily before processing the patient samples and ensure the QC values are with in the Reference Range and close to the mean mentioned in the product insert. If both the QC levels (L1 & L2) are out of range on the same day in the same run perform recalibration for the analyte. Calculation of Results: The system performs all calculations internally to produce the final reported result. The system does not calculate the final result for sample dilutions made by the operator. In these cases the result produced by the operator must be multiplied by the operator. If the dilution factors are entered in to the system during the sample programming by the operator the system automatically calculates the final result by multiplying with the dilution factor.

Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. Reference Interval: Male: <55.0 U/l in serum. Female: <38.0 U/l in serum. Reportable interval of examination results: 8-1280 U/l of GT activity in serum. Interpretation of Result: Elevated level of GGT may be associated with acute hepatitis, chronic hepatitis, alcoholic cirrhosis and primary and secondary liver tumors. Safety precautions: Handle all samples as potentially infectious Handle all reagents with care and avoid contact with eye mouth and skin. Do not perform mouth pipetteing.

Discard used reagents and sample as per disposable procedure. The reagent must be used only for the purpose intended by suitably qualified laboratory personnel, under appropriate laboratory condition. Potential sources of variability: Lysed serum specimens may give falsely elevated values. Do not use if the reagents is turbid as it indicates contamination of the reagent. Hemoglobin in the tested level of 4g/L has no significant interference on the assay.

 Bilirubin in the tested level of 3g/L has no significant interference on the assay. Glucose in the tested level of 4.5g/L has no significant interference on the assay. Cholesterol in the tested level of 5g/L has no significant interference on the assay. Ascorbate in the tested level of 0.4g/L has no significant interference on the assay. Dopamethyl in the tested level of 50mg/L has no significant interference on the assay. Triglyceride in the tested level of 10g/L has no significant interference on the assay.

3.

SEROLOGY
WIDAL (SLIDE)

Enteric fever occurs when pathogenic microorganisms like Sslmonella typhi, S. paratyphi A, S. paratyphi B, S. paratyphi C infect the human body. During the course of disease, the body responds to this antigenic stimulus by producing antibiotics whose titre rises slowly falls till it is undetectable. Antibodies to salmonella organisms may be detected in the patient serum from the second week after onset of infection. Information regarding the titres and whether or not they are rising or falling can be obtained by performing serological tests using TYDAL antigen suspensions. Principle: When the colored, smooth, attenuated TYDAL antigen suspensions are mixed / incubated with patient serum, anti-salmonella antibodies present in the patient serum react with the antigen suspensions to give agglutination. Agglutination is a positive test result, indicating presence of anti-salmonella antibodies in the patient serum. No agglutination is a negative test result indicating absence of anti-salmonella antibodies. Sample type: Serum samples are used.

Procedure: Place one drop of positive control onto a reaction circle of the glass slide. Place 50 L of physiological saline onto the next reaction circle of the glass slide. Place one drop of patients serum to be tested onto each of the required number of reaction circles. Add one drop of appropriate TYDAL antigen suspension to the reaction circles containing positive control and physiological saline. Add one drop of appropriate TYDAL antigen suspensions to the reaction circles containing the patients serum. Mix contents of each circle uniformly over the entire circle with separate mixing sticks. Rock the slide gently back and fourth and observe for agglutination macroscopically at one minute. Use of positive control serum and saline control in parallel with unknown test serum is recommended to assure the antigens used in the test are sensitive as well as specific and to show what results are to be expected in positive and negative specimens. Interpretation of results: Positive: The titre of the patients serum corresponds to the visible agglutination in the tests circle with the smallest amount of serum sample. Negative: The negative control should show no agglutination with any of the TYDAL antigen suspensions.

TUBERCULIN DILUTED
Tuberculin P.P.D., IP for Mantoux test only For in vivo detection of sensitization by Mycobacterium tuberculosis Introduction The Tuberculin skin test (TST) is the most commonly used test to detect exposure to Mycobacterium tuberculosis, being used in epidemiological surveys, clinical evaluation of patients with suspected active tuberculosis, and assessment of anti-tuberculous drug therapy. An intradermal Mantoux test is performed with different dilutions of Tuberculin PPD. In developed countries, dose of 5 or 10 Tuberculin units (TU) of PPD is commonly used. Whereas, in developing countries, such as India, with high prevalence of Tuberculosis, TU is the recommended dose as per the WHO guidelines. International Union against Tuberculosis & lung diseases (IUATLD) has also suggested the dose of 2 TU for tuberculin surveys. SPANs

tuberculin PPD (1TU/2TU/5TU/10TU) is a diluted and ready to use solution for performing Mantoux test. Source material is calibrated against batch RT 23 manufactured by Statens Serum Institute, Denmark. It is diluted with a special buffer containing Tween-80 as a stabilizer. Principle Infection with Mycobacterium tuberculosis results in hyhpersensitivity to tuberculoprotein. Intradermally injected Tuberculin PPD produces erythema and induration of the skin around the point of injection. The diameter of induration is directly proportional to the degree of sensitization. Advantages of SPANs tuberculin PPD Accurate interpretations of response, because every batch is carefully standardized by guinea pig assay. No risk of non-specific reactions Supplied in convenient ready to use form No loss of potency due to adsorption on glass wall of container. Highly economical as supplied in multidose vials containing preservative.

Storage & stability of Reagent Tuberculin PPD (diluted) is stable at 2-8C till the expiry date mentioned on the label on the vial (do not freeze) Precautions Tuberculin PPD has affinity for glass and plastic surfaces. Hence do not transfer Tuberculin PPD solution to other containers and do not hold it in syringes for longer time. A separate sterilized syringe and needle should be used for each individual patient to prevent possible transmission of infectious agents from one person to another. Syringes that have previously been used with histoplasmin, blastomycin or other similar antigens should not be used for Tuberculin PPD. As with any biological products, epinephrine should be immediately available to treat anaphylactoid or acute hypersensitivity reaction, although such reactions are extremely rare with Tuberculin PPD.

Avoid subcutaneous injection, after subcutaneous injection local reaction may not develop, but a general febrile reaction and /or exacerbation or inflammation around old tuberculous lesions may occur in highly sensitive individuals. Avoid vigorous shaking of the container.

Test Procedure Standard method of Mantoux test: Mantoux test is performed by intradermally injecting SPANs Tuberculin PPD of desired strength with tuberculin syringe. The preferred site for the test is the flexor or dorsal surface of the forearm about 4 inches below the elbow joint. Clean the skin at the chosen site with 70% alcohol or spirit and allow to dry. Clean the stopper of the PPD vial with 70% alcohol or spirit and draw 0.1 ml of Tuberculin PPD solution into the sterile tuberculin syringe fitted with a short 26 gauge needle. Inject Tuberculin PPD intradermally by inserting the tip of the needle in to the most superficial layers of the skin with needle bevel pointing upwards. As the solution is injected, a pale white bleb, 6 to 10 mm in diameter will rise at the needle point. This will be quickly absorbed and hence no dressing is required. Note: if the tuberculin PPD is inadvertently injected subcutaneously, no bleb will form, or if a significant part of the dose leaks from the injection site, the test should be repeated immediately at another site, 5 cms away from the first site.

Results & Interpretation Results of Mantoux test should be read between 48 to 72 hours after the injection. Induration only should be considered while interpreting the test. The diameter of induration should be measured transversely to the long axis of the forearm and recorded in millimeters. Erythema (without induration) of less than 10 mm should be disregarded. If the diameter of erythema is greater than 10mm and induration is absent the injection may have been made too deeply and retesting is indicated.

Reactions are classified as Follows: Positive : Induration measuring 10mm or more, this indicates hypersensitivity to tuberculoprotein and indicates past or present infection with Mycobacterium tuberculosis.

Doubtful : induration measuring between 5 and 9mm, Retesting is indicated at another site. This retesting is desirable to rule out cross reactions from other mycobacterial infections. Negative : induration of less than 5 mm. this indicates lack of hypersensitivity to tuberculoprotein and tuberculous infection is highly unlikely. It should be noted that reactivity to tuberculin may be depressed or suppressed for as long as four weeks following viral infections, live-virus vaccines (e.g. measles, smallpox, polio, rubella, mumps etc.) or by the administration or corticosteroids. Malnutrition may also have a similar effect. A negative test should be accepted as proof of absence of hypersensitivity only after normal reactivity to non specific irritants has been demonstrated. Intradermal injection of 0.1 ml % solution of codeine is recommended for the demonstration of non specific cutaneous reactivity. A positive tuberculin reaction does not necessarily diagnose the presence of active disease. Further diagnostic procedures should be carried out before diagnosis of tuberculosis is made. It serves only as an aid in the diagnosis of Tuberculous infection.

SERUM ELECTROPHORESIS
Gel electrophoresis is a method for separation and analysis of macromolecules (DNA,RNA and proteins) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge and/or size (IEF agarose, essentially size independent) and in biochemistry to separate to estimate the size of separate proteins by charge Principle: In serum electrophoresis separates protein into 5 main classes (Albumin, Alpha 1Globulin, Alpha 2_Globulin, Beta Globulin and Gamma globulin) according to charge in an agarose gel The proteins are then stained to allow visualization and quantitative determination and determination. Each of the classical elecrophoretic zones with the exception of albumin, normally contains 2 or more components The relative proportions of these fractions have proven to be useful aids in the diagnosis and prognosis of certain disease states

Primary Sample system:

Use serum/plasma as specimen for the test. If serum is used as specimen, collect 2 3 mL of venous blood from a peripheral vein in a red topped vacutainer tube. Allow the tube to stand at room temperature till complete clot formation occurs (for 30 minutes) and then separate the serum by centrifugation at 2500- 3000 rpm for 5 minutes. Separate the serum within 2 hours of collection. Ensure complete clot formation has occurred and there are no fibrin threads. Do not use lysed serum for testing as it may give very high results. Do not use contaminated / turbid samples for testing. Process the sample on the same day. If analysis is not done on the same day, separate the serum and store it at 2 to 8 C for up to 7 days. If the sample is been stored more than 7 days, store it at 20 C.

Type of Container and Additive: If Serum is used as specimen - Use plain Red topped vacutainer tubes. Do not add any additive or anticoagulants. If Plasma is used as specimen - Use green topped Li heparin tube or Lavender topped K3 EDTA tube.

Required Equipment and Reagents: Equipment: Electrophoresis Apparatus-Helena Biosciences SAS MX Serum Protein gel: Contains agarose in a Tris / Barbital buffer with thiomersol and sodium azide as preservative. The gel is ready use as packaged. Tris/ Barbital buffer concentrate: Contains barbital and sodium barbital with sodium azide as preservative. Dilute the contents of the bottle (100ml) with purified water (400ml) and mix well. Buffer salts may crystallize slightly on standing. Wash any crystals from the bottle with diluted buffer to ensure complete dissolution. Acid Blue Stain concentrates: Contains concentrate Acid blue stain. Dilute the contends of bottle to 700ml with purified water .stir overnight and filter before use. Store in a tightly stopper bottle. Destain solution: Contains concentrated destain solution. Dilute the contents of the bottle in 2litres of purified water. Stored in tightly stopper bottle.

Other Kit Components: Templates and blotters Reagents storage and shelf-life: SAS-MX serum protein gel: Gels Should be stored at 15 Degree To 30 Degree C and are stable until the expiry date do not refrigerate or freeze .Deterioration of the gel may be indicated by 1) crystalline appearance indicating the gel has been frozen, 2) cracking and peeling indicating drying of the gel or 3) visible contamination of the agarose form bacterial or fungal sources. Tris Barbital Buffer: The buffer concentrate should be stored at 15 to 30 Degree Celsius and is stable until the expiry date indicated on the label. Diluted buffer is stable for 2 months at 15 to 30 degree Celsius. Cloudiness or poor performance of the diluted buffer may indicate deterioration. Acid Blue Stain: The stain concentrate should be stored at 15 to 30 Degree Celsius and is stable until expired date indicated on the label. Diluted stain solution is stable for 6 months at 15 to 30 degree Celsius. It is recommended to discard used stain immediately to prevent depletion of staining capability. Poor staining performances may indicate deterioration of the stain solution. Destain solution: The destain concentrate should be stored at 15 to 30 Degree Celsius and is stable for until the expiry date indicated on the label. The diluted destain is stable for 6 months at 15 to 30 Degree Celsius. Cloudiness may indicate deterioration of the destain solution.

Procedure: Dilute each patient sample and control 1:4 (1 part sample + 3 parts buffer) with GEL Serum Protein Buffer. Remove the GEL Serum Protein Gel from the protective packaging. One edge of the agarose gel has been numbered for easy sample placement and identification. Using Blotter A, gently blot the application area of the gel using a slight fingertip pressure on the blotter. Carefully place the GEL Template on the gel, aligning the application slits with the zero Signs (0) on the sides of the gel and trying to avoid trapping any air bubbles under the template. Place a Blotter A over the template and remove any bubbles in the slit area with slight fingertip pressure. Retain the blotter for use in Step 7.

7.5. Place 3.0 L of each sample onto the template slits, spreading the sample completely over the entire slit. Apply the samples as quickly as possible. 6. Wait 4 minutes after the last sample has been applied to allow the samples to diffuse into the agarose. Gently blot the template with the Blotter A retained in Step 4 and then carefully remove the blotter. Wait 30 seconds and then carefully remove the template. Quickly place the gel into the inner section of the chamber, agarose side down, by gently squeezing the gel into place. Position the gel so that the edges of the agarose are in the buffer and the application point is on the cathodic (-) side. Two gels may be electrophoresed at one time. Place the cover on the chamber and insure that the cover does not touch the gel. Electrophoresis the gel(s) at 80 volts for 30minutes.

Evaluation At the end of the electrophoresis period, remove the gel from the chamber and place it in methanol for 5 minutes. Remove the gel from the methanol and lay it on a blotter. Then place it into an I.O.D. or other laboratory drying oven with forced air at 60-70 C for 5 minutes or until dry. The gel may be dried at a lower temperature but additional time will be required. The gel will not destain properly if it is not completely dry. Fill one container of the Staining Set with prepared stain. Fill another container with Fixative/Destain Solution. Remove the gel from the oven and place it in the Staining Rack. Immerse the rack into the stain for 10 minutes. Remove the rack from the stain and allow it to drain on a blotter. Destain the gel by rinsing it in two (2) consecutive washes of destain solution. Allow the gel to remain in each wash for 1 minute. The gel background should be completely clear. If the gel background is not completely clear, a final water wash should be used to remove trace amounts of stain. Place the gel in tap water for 1 minute after destaining it. Wipe the back of the gel with laboratory tissue dampened with methanol to remove any remaining stain. Dry the destained gel by placing it on a blotter and into an I.O.D or other drying oven at 60-70C until dry.

Evaluation of the Protein Band Scan the dried GEL Serum Protein Gel at 595 nm. Quality Control Procedures: Internal Quality Control Program: Run alternate levels of assayed QC samples for every 10 patient samples (for each Gel )and ensure the QC values are with in the Reference Range and close to the mean mentioned in the Internal Quality Control product insert. Interferences: Interferences vary depending upon icterus, haemolysis and lipemia status. In patients receiving high doses of Biotin therapy ( > 5 mg/day) no samples should be taken for 8 hours after the last dosage administration. Erroneous findings may be obtained from patients receiving treatment for monoclonal antibodies or have received them for diagnostic purposes. Reference Range and Laboratory Interpretation: Reference range: The reference values for serum protein electrophoresis on the Serum Protein System are presented. These values are presented as a guideline. Each laboratory should establish its own normal range study. Protein Fraction and % of Total Protein Albumin - 50.8 - 62.1 % Alpha1 Alpha2 Beta 2.5 - 5.0 % 8.8 13.6 % 10.0-15.1 %

Gamma - 11.6- 20.4 % Results on normal individuals will cover age and sex related variations and day-to-day biologic variations. Disease states in which abnormal patterns are observed include inflammatory response, rheumatic disease, liver diseases, protein-loss disorders, monoclonal gammopathies, pregnancy and genetic deficiencies. LIMITATIONS Since all electrophoretic procedures are nonlinear, it is critical to use the recommended volume of undiluted serum to obtain optimal resolution and reproducible results. Noncompliance with the recommended procedure may affect the results.

SPECIFIC PERFORMANCE CHARACTERISTICS Precision: Within-Run and Run-to-Run precision studies yielded CVs of less than 10%. Sensitivity: The sensitivity of the system, using the Amido Black Protein Stain, is 10 g/dL. Comparison: A comparison study of this method to the cellulose acetate method, using a range of 4.48 g/dL- 11.85 g/dL, was excellent yielding a linear regression equation of Y = 0.996X + 0.072 (where X is the GEL method and Y is the cellulose acetate method) and a correlation coefficient of 0.998.

Safety Precautions: Follow Material Safety Data Sheet provided by the reagent manufacturer. Handle all samples as potentially infectious Handle all reagents with care and avoid contact with eye mouth and skin Do not perform mouth pipetting. Discard used reagents and sample as per disposal procedure.

Potential sources of variability: Presence of fibrin in the sample may lead to erroneous results and hence centrifuge the sample only after complete clot formation. Lysed serum specimens may give falsely elevated values. Patients receiving treatment for HAMA for diagnosis may produce anomalous values. The CA 125 assay value should be used in conjunction with information available from clinical evaluation and other diagnostic procedure this assay should not be used as a cancer screening test. Heterophilic antibodies in human serum can react with reagent immunoglobulins, interfering with invitro immunoassay

ANA HEp-2
ANA HEp2 is a solid phase enzyme immunoassay for the combined qualitative detection of IgG antibodies against HEp2 cells in human serum. Each well is coated with lysed HEp2 cells. The

test collectively detects, in one well, total ANAs against double stranded DNA (dsDNA), histones, SS-A (Ro), SS-B (La), Sm, snRNP/Sm, Scl-70, PM-Scl, Jo-1 and centromeric antigens along with sera positive for HEp2 immunofluorescence test (IFT). Clinical application and principle of the assay Anti-nuclear antibodies (ANA) directed against a variety of nuclear and cytoplasmic antigens occur in high frequency in systemic rheumatic diseases and thus are an important tool for the differential diagnosis. For instance, SS-A (Ro) and SS-B (La) antibodies are associated with SLE and Sjogrens syndrome (SS), anti-dsDNA and anti-Sm antibodies with SLE, anti-histone antibodies with SLE and drug-induced lupus, anti-RNP antibodies with mixed connective tissue disease (MCTD) and SLE, anti Scl-70 antibodies with scleroderma (progressive systemic sclerosis (PSS), anti Jo-1 antibodies with polymyositis and dermatomyositis and anti-centromere antibodies with CREST syndrome. Indirect immunofluorescence test (IFT) on eukaryotic cells like HeLa and HEp2 has been the established method for the detection of ANAs. Although the IFT is a sensitive test, it is laborious when testing large number of patient samples and is subject to errors from humn interpretation and from variability in fluorescent microscope. The ELISA test system is an excellent alternative to the IFT for screening patients serum for the presence of ANAs of clinical significance. Single antibody specificities have to be determined by more specific testing using ELISAs employing the specific target antigens for a simple and reliable differentiation of ANAs. Principle of the test Serum samples diluted 1:101 are incubated in the microplates coated with the specific antigen. Patients antibodies, if present in the specimen, bind to the antigen. The unbound fraction is washed off in the following step. Afterwards anti-human immunoglobulins conjugated to horseradish peroxidase (conjugate) are incubated and react with the antigen-antibody complex of the samples in the microplates. Unbound conjugate is washed off in the following step. Addition of TMB substrate generates an enzymatic colorimetric (blue) reaction, which is stopped by diluted acid (color changes to yellow). The rate of color formation from the chromogen is a function of the amount of conjugate bound to the antigen antibody complex and this is proportional to the initial concentration of the respective antibodies in the patient sample. Kit contents 5x sample Buffer - 1 vial, 20ml 5x concentrated (capped white:yellow solution). Containing Tris, NaCl, BSA, sodium azide < 0.1% and thimerosal 0.01% (preservatives). 50x wash buffer : 1 vial, 20 ml 50x concentrated (capped white : green solution) containing Tris, NaCL, Twen, sodium azide <0.1% and thimerosal 0.01% (preservative). Ready to use:

Negative control : 1 vial, 1.5ml (capped green : yellow solution) containing human serum (diluted), sodium azide <0.1% (preservative) Positive control : 1 vial, 1.5ml (capped red:yellow solution) containing human serum (diluted) sodium azide <0.1% (preservative) Cut-off control : 1 vial, 1.5 ml (capped blue:yellow solution) containing human serum (diluted), sodium azide < 0.1% (preservative) Conjugate : 1 vial, 15 ml IgG (capped blue:blue solution) containing anti-human immunoglonulins conjugated to horseradish peroxidase and thimerosal 0.01% (preservative). TMB substrate : 1 vial, 15ml (capped black) containing stabilized TMB/H2O2 Stop solution : 1 vial, 15 ml (capped white:colorless solution) containing 1M hydrochloric acid Microtiterplate : 12 X 8 well strips with breakaway microwells.

Sample collection, handling and storage Use preferentially freshly collected serum samples. Blood withdrawal must follow national requirements. Do not use icteric, lipemic, hemolysed or bacterially contaminated samples. Sera with particles should be cleared by low speed centrifugation (<1000x g). blood samples should be collected in clean, dry and empty tubes. After separation, the serum samples should be used immediately, respectively stored tightly closed at 2-8C/35-46F up to threed days, or frozen at -20C/-4F for longer periods.

Preparations prior to pipetting Dilute concentrated reagents Dilute the concentrated sample buffer 1:5 with distilled water (e.g. 20ml plus 80 ml) Dilute the concentrated wash buffer 1:50 with distilled water (e.g. 20 ml plus 980 ml).

Samples Dilute serum samples 1: 101 with sample buffer (1x) e.g., 1000l sample buffer (1x) + 10l serum, mix well.

Washing

Prepare 20 ml of diluted wash buffer (1x) per 8 wells or 200 ml for 96 wells e.g. 4 ml concentrate plus 196 ml distilled water Automated washing Consider excess volumes required for setting up the instrument and dead volume of robot pipette. Manual washing Discard liquid from wells by inventing the plate. Knock the microwell frame with wells downside vigorously on clean adsorbent paper. Pipette 300 l of diluted wash buffer into each well, wait for 20 seconds. Repeat the whole procedure twice again. Microplates Calculate the number of wells required for the test. Remove unused wells from the frame, replace and store in the provided plastic bag, together with desiccant,seal tightly (2-8C/3546F). Work flow Pipette 100l of each patients diluted serum into the designated microwells. Pipette 100l cut off control and negative and positive controls into the designated wells Incubate for 30 minutes at room temperature (20-26C/64-78.8F). Wash 3x with 300l washing buffer (diluted 1:50) Pipette 100 l conjugate into each well Incubate for 15 minutes at room temperature (20-26C/64-78.8F). Wash 3x with 300l washing buffer (diluted 1:50) Pipette 100l TMB substrate into each well. Incubate for 15 minutes at room temperature (20-26C/64-78.8F) in the dark. Pipette 100 l stop solution into each well, using the same order as pipetting the substrate. Incubate 5 minutes minimum Agitate plate carefully for 5 seconds

Read absorbance at 450 nm (optionally 450/620nm) within 30 minutes.

Semiquantitative interpretation Read the optical density of the cut-off control and the patient samples. Compare patient ODs with the OD of the cut-off control. All samples which are higher than cut-off are considered positive. Negative : Positive : Calibrtors OD patient < OD cutoff OD patient > OD cutoff OD 450/620nm CV % (variation) 2.6 1.8 0.7

Negative control 0.081 Cutoff control Positive control 0.350 1.259

Example of interpretation We recommend pipetting cut-off control in parallel for each run, Cutoff control Patient sample Interpretation 0.35 OD 0.35 OD 0.35 OD 0.35 OD 0.25 OD 0.35 OD 0.40 OD 1.75 OD Negative Borderline Positive positive

We recommend retesting samples that are borderline. Medical laboratories might perform an in-house quality control by using own controls and/or internal pooled sera, as foreseen by EU regulations. Do not use this example for interpreting patients results. For semi-quantification of the results, each patient OD value can be expressed by the indexvalue. The index-value is calculated by dividing the patient OD by the cut-off OD: Index value = OD (patient sample) / OD (cut-off control) Negative: index value <1.0

Positive:

index value >1.0

Technical data Sample material: Sample volume: Total incubation time: Storage: serum 10l of sample diluted 1:101 with 1x sample buffer 60 minutes at room temperature (20-26C/64-78.8F) at 2-8C/35-46F use original vials, only

Number of determinations: 96 tests Performance data Specificity and sensitivity The microplate is coated with lysed HEp2 cells. No crossreactivities to other autoantigens have been found. ANA are not specific for SLE but are found in a variety of rheumatic diseases. Detection of ANA is a very sensitive marker for an active SLE and is positive in >99% of all cases. Linearity Chosen sera have been tested with this kit and found to dilute linearly. However, due to the heterogenous nature of human autoantibodies, there might be samples that do not follow this rule. Sample No 1 Dilution factor 1/100 1/200 1/400 1/800 Measured (OD Ratio) 4.10 2.10 1.00 0.55 concentration Expected concentration Recovery (OD Ratio) (%) 4.2 2.1 1.1 0.5 97.6 100.0 95.2 103.8

1/100 1/200 1/400

6.10 3.00 1.59

6.2 3.1 1.6

98.4 96.8 102.6

1/800

0.79

0.8

102.0

Precision To determine the precision of the assay, the variability (intra and inter-assay) was assessed by examining its reproducibility on three serum samples selected to represent a range over the standard curve. Intra-Assay Inter-Assay

Sample No Mean (OD-Ratio) CV (%) Sample No Mean (OD-Ratio) CV (%) 1 2 3 4.6 2.8 1.4 1.5 2.0 1.8 1 2 3 4.7 3.0 1.2 3.1 2.5 2.4

ANTI HSV POOL ELISA (IgG)

Principle of the test: The ELISA test kit provides a semiquantitative or quantitative in vitro assay for human antibodies of the IgG class against HSV-1 and HSV-2 in serum or plasma. The test kit contains microtiter strips each with 8 break-off reagent wells coated with a pool of HSV-1 and HSV-2 antigens. In the first reaction step, diluted patient samples are incubated in the wells. In the case of positive samples, specific IgG antibodies (also IgA and IgM) will bind to the antigens. To detect the bound antibodies, a second incubation is carried out using an enzyme labeled antihuman IgG (enzyme conjugate) catalyzing a color reaction. Component Color Format 12X8 symbol Strips

Microplate wells coated with antigens : 12 microplate strips each containing 8 individual break-off wells in a frame, ready for use Calibrator 1 : 200RU/ml (IgG, human), ready for use Calibrator 2 : 20 RU/ml (IgG human), ready for use Calibrator 3 : 2RU/ml (IgG, human) ready for use Dark red Red Light red

1X2.0 ml 1X2.0ml 1X2.0 ml

CAL 1 CAL 2 CAL 3

Positive control : (IgG, human) ready for use Negative control : (IgG, human) ready for use

Blue Green

1X2.0ml 1X2.0 ml

POS CONTROL NEG CONTROL CONJUGATE

Enzyme conjugate peroxidase-labelled anti-human Green IgG (rabbit), ready for use Sample buffer ready for use Light blue Colorless

1X12ml

1X100ml

SAMPLE BUFFER WASHBUFFER 10x SUBSTRATE

Wash buffer 10x concentrate

1X100ml

Chromogen/substrate solution TMB/H2O2, ready for Colorless use Stop solution 0.5M sulphuric acid, ready for use Colorless

1X100ml

1X12ml

STOP SOLUTION

Test instruction

1 booklet 1 protocol

Protocol with reference values

Storage and stability : the test kit has to be stored at a temperature between +2C to +8C. Do not freeze. Unopened, all test kit components are stable until the indicated expiry date. Waste disposal : patients samples, calibrators, control and incubated microplate strips should be handled as infectious waste. All reagents must be disposed of in accordance with local disposal regulations. Preparation and stability of the reagents: All reagents must be brought to room temperature (+18C to +25C) approx. 30 minutes before use. After first use, the reagents are stable until the indicated expiry date if stored at +2C to +8C and protected from contamination, unless stated otherwise below. Coated wells: ready for use. Tear open the resealable protective wrapping of the microplate at the recesses above the grip seam. Do not open until the microplate has reached room temperature to prevent the individual strips from moistening. Immediately replace the remaining wells of a partly used microplate in the protective wrapping and tightly seal with the integrated grip seam (do not remove the desiccant bag).

Once the protective wrapping has been opened for the first time, the wells coated with antigens can be stored in a dry place and at a temperature between +2C and +8C for 4 months. Calibrators and controls: ready for use. The reagents must be mixed thoroughly before use. Enzyme conjugate : Ready for use. The enzyme conjugate must be mixed thoroughly before use. Sample buffer : Ready for use. Wash buffer : the wash buffer is a 10x concentrate. If crystallization occurs in the concentrated buffer, warm it to 37C and mix well before diluting. The quantity required should be removed from the bottle using a clean pipette and diluted with deionized or distilled water (1 part reagent plus 9 parts distilled water). For example: for 1 microplate strip, 5 ml concentrate plus 45 ml water. The working strength wash buffer is stable for 4 weeks when stored at +2C to +8C and handled properly. Chromogen/substrate solution: ready for use. Close the bottle immediately after use, As the contents are sensitive to light. The chromogen/substrate solution must be clear on use. Do not use the solution if it is blue coloured. Stop solution: ready for use. Warning : the control sera used have been tested negative for HBsAG, antiHCV, anti HIV-1 and anti HIV-2 using enzyme immunoassays and indirect immunofluorescence methods. Nonetheless, all materials should be treated as being a potential infection hazard and should be handled with care. Some of the reagents contain the toxic agent sodium azide. Avoid skin contact. Preparation and stability of the patient samples Sample material : human serum or EDTA, heparin or citrate plasma. Stability : patient samples to be investigated can generally be stored at +2C to +8C for up to 14 days. Diluted samples should be incubated within one working day. Sample dilution: patient samples are diluted 1:101 sample buffer. For example: dilute 10l serum in 1.0ml sample buffer and mix well by vortexing (sample pipettes are not suitable for mixing). Note : calibrators and controls are prediluted and ready for use, do not dilute them. Incubation Manual test performance

For semiquantitative analysis incubate calibrator 2 along with the positive and negative controls and patient samples. For quantitative analysis incubate calibrators 1,2 and 3 along with the positive and negative controls and patient samples. Sample incubation : (1 step) transfer 100 l of the calibrators, positive and negative controls or diluted patient samples into the individual microplate wells according to the pipetting protocol. Incubate for 30 minutes at room temperature (+18C to +25C). Washing : Manual : empty the wells and subsequently wash 3 times using 300l of working strength wash buffer for each wash. Automatic : wash reagent wells 3 times with 450l of working strength wash buffer (program setting:e.g. TECAN Columbus washer overflow modus). Leave the wash buffer in each well for 30 to 60 seconds per washing cycle, then empty the wells. After washing (manual and automated tests), thoroughly dispose of all liquid from the microplate by tapping it on absorbent paper with the openings facing downwards to remove all residual wash buffer. Attention: residual liquid (> 10l) in the reagent wells after washing can interfere with the substrate and lead to false low extinction values. Insufficient washing (e.g. less than 3 wash cycles, too small wash buffer volumes, or too short reaction times) can lead to false high extinction values. Free positions on the microplate strip should be filled with blank wells of the same plate format as that of the parameter to be investigated. Conjugate incubation : (2 step). Pipette 100l of enzyme conjugate (peroxidase labeled anti human IgG) into each of the microplate wells. Incubate for 30 minutes at room temperature (+18C to 25C). Washing : Empty the wells. Wash as described above. Substrate incubation : (3 step). Pipette 100l of chromogen/substrate solution into each of the microplate wells. Incubate for 15 minutes at room temperature (+18C to 25C) (protect from direct sunlight). Stopping the reaction: pipette 100l of stope solution into each of th emicroplate wells in the same order and at the same speed as the chromogen/substrate solution was introduced. Measurement: Photometric measurement of the color intensity should be made at a wavelength of 450nm and a reference wavelength between 620nm and 650nm within 30 minutes of adding

the stop solution. Prior to measuring, slightly shake the micro-plate to ensure a homogenous distribution of the solution. Test performance using fully automated analysis devices: Sample dilution and test performance are carried out fully automatically using the analysis device. The incubation conditions programmed in the respective software authorized by EUROIMMUN may deviate slightly from the specifications given in the ELISA test instruction. However, these conditions were validated in respect of the combination of the EUROIMMUN analyzer 1 or the DSX from DYNEX and this EUROIMMUN ELIISA. Automated test performance using other fully automated, open system analysis devices is possible, however, the combination should be validated by the user. The pipetting protocol for microtiter strips 1-4 is an example for the semiquantitative analysis of 24 patient samples (P1 to P24). The pipetting protocol for microtiter strips 7-10 is an example for the quantitative analysis of 24 patient samples (P1 to P24). The calibrators (C 1 to C3), the positive (pos.) and negative (neg.) controls, and the patient samples have each been incubated in one well. The reliability of the ELISA test can be improved by duplicate determinations for each sample. The wells can be broken off individually from the strips. This makes it possible to adjust the number of test substrates used to the number of samples to be examined and minimizes reagent wastage. Both positive and negative controls serve as internal controls for the reliability of the test procedure. they should be assayed with each test run. Calculation of results Semiquantitative : Results can be evaluated semiquantitatively by calculating a ratio of the extinction value of the control or patient sample over the extinction value of the calibrator 2. Calculate the ratio according to the following formula :

Extinction of the control or patient sample/Extinction of calibrator 2 = Ratio EUROIMMUN recommends interpreting results as follows: Ratio <0.8: Ratio >0.8 to <1.1: Positive borderline

Ratio >1.1:

Positive

In cases of borderline test results, an additional patient sample should be taken, 7 days later and retested in parallel with the first patient sample. The results of both samples allow proper evaluation of titer changes. Quantitative : the standard curve from which the concentration of antibodies in the patient samples can be taken is obtained by point to point plotting of th extinction values measured for the 3 calibration sera against the corresponding units (linear/linear). Use of point to point plotting for calculation of the standard curve by computer. The following plot is an example of a typical calibration curve. Please do not use this curve for the determination of antibody concentration in patient samples. If the extinction of a serum sample lies above the value of calibrator 1(200 RU/ml). the result should be given as >200RU/ml. it is recommended that the sample be retested at a dilution of 1:400. The result in RU/ml read from the calibration curve for this sample must then be multiplied by a factor of 4. The upper limit of the normal range of non-infected persons (cut off value) recommended by EUROIMMUN is 20 relative units (RU/ml. EUROIMMUN recommends interpreting results as follows: <R16 RU/ml : >16 to <22 RU/ml: >22RU/ml : negative borderline positive

For duplicate determinations the mean of the two values should be taken. If the two values deviate substantially from one another the sample should be retested. For diagnosis, the clinical symptoms of the patient should always be taken into account alongside the serological results. Test Characteristics Calibration : as no international reference serum exists for antibodies against HSV 1 and HSV 2, the calibration is performed in relative units (RU.ml). for every group of tests performed, the extinction values of the calibrators and the relative units and /or ratios determined for the positive and negative controls must lie within the limits stated for the relevant test kit lot. A protocol containing these target values is included. If the values specified for the controls are not achieved, the test results may be inaccurate and the test should be repeated.

The activity of the enzyme used is temperature dependent and the extinction values may vary if a thermostat is not used. The higher the room temperature during substrate incubation, the greater will be the extinction values. Corresponding variations apply also to the incubation times. However, the calibration sera are subject to the same influences, with the result that such variations will be largely compensated in the calculation of the result. Antigen : the microplate wells were coated with a pool of HSV 1 and HSV 2 antigens. The antigen source is provided by inactivated cell lysates of BSC 1 cells infected either with the Macintyre strain of HSV 1 or with the MS strain of HSV 2. Linearity: the linearity of the Anti-HSV pool ELISA (IgG) was determined by assaying 4 serial dilutions of 4 serum samples. The linear regression was calculated and R2 amounts to >0.95 in all samples. The anti HSV pool ELISA (IgG) is linear at least in the tested concentration range (7RU/ml to 104RU/ml). Detection limit : the lower detection limit is defined as the value of an analyte free samples plus three times the standard deviation and is the smallest detectable antibody titer. The lower detection limit of the anti HSV pool ELISA (IgG) is 0.8 RU/ml. Cross reactivity : This ELISA showed no serological cross reactivity with sera positive for antibodies against the following : EBV-CA (n=12). CMV (n=4). VZV (n=7), Adenovirus (n=12). RSV (n=12), Parainfluenza virus types 1-4 (n=12). Influenza virus type A (n=7), influenza virus type B (n=12), Mycoplasma pneumonia (n=7), Mumps virus (n=8), Measles virus (n=11), Rubellia (n=12), Toxoplasma gondii (n=3).] Interference : Haemolytic, lipaemic and icteric samples showed no influence on the result up to a concentration of 10mg/ml for hemoglobin, 20 mg/ml for triglycerides and 0.4mg/ml for bilirubin in this ELISA. Reproducibility : the reproducibility of the test was investigated by determining the intra and inter assay coefficients of variation using 3 sera. The intra assay CVs are based on 20 determinations and the inter-assay CVs on 4 determinations performed in 6 different test runs. INTRA ASSAY VARIATION, N=20 Serum Mean Value (RU/ml) CV (%) 1 2 3 49 75 105 12 10.9 3.7

INTER ASSAY VARIATION, N=4X6 Serum Mean Value (RU/ml) CV (%) 1 2 3 47 67 109 12 6.7 4.9

Specificity and sensitivity : 34 clinically characterized patient samples (Interlaboratory test samples from INSTAND, Germany) were examined with the EUROIMMUN Anti HSV pool ELISA (IgG). The test showed a specificity of 100% and a sensitivity of 100%. N=34 INSTAND Positive Negative ELISA Positive 24 0 10

negative 0

Reference range : the levels of anti HSV antibodies (IgG) were analyzed with this EUROIMMUN ELISA in a panel of 300 healthy blood donors. With a cut off of 20 RU/ml. 76% of the blood donors were anti HSVC positive (IgG) which reflects the known percentage of infections in adults. Clinical significance Herpes simplex is a disease which is characterized by the formation of blisters on the skin and mucous membrances as intermittent, lytic replications in epithelia. It is prevalent world wide and normally proceeds asymptomatically. Complications result when internal organs are also afftected and necrotise. Herpes simplex virus type 1 (HSV 1) infection commonly occurs in the area of the mouth and nose, and Herpes simplex virus type 2 (HSV 2) infection in the genital area. People with a large number of sexual partners are at increased risk of acquiring HSV 2 infections. The presence of pre-existing HSV 1 antibodies may reduce the severity of genital HSV 2 infection. The seroprevalence increases up to the age of 50 years. Provoked relapses are typical. In primary HSV 2 infections, cephalalgia and neck stiffness are frequent symptoms, where as meningitis is rarely observed. If newborns are infected with HSV 2 when passing through the

birth canal, blisters on the skin, mouth and eyes are frequently observed, along with hepatomegaly, splenomegaly, renal failure, icterus, neurological damage and encephalitis. Untreated, the disease has a case fatality ration of 60%. Herpes simplex virus infection of the central nervous system is a significant cause of morbidity and often mortality of young people. Changes in central nervous system are results of primary infection or activation of latent HSV 1 or HSV 2. Neurological defects often follow encephalitis herpetica. Antibodies agains HSV can be detected in the serum of nearly all patients after the illness has taken its course. More than 90% of the population are infected with HSV 1. Antibodies against HSV 2 can be found in 7-20% of general population and more than 20% in sexually active adults. The mixed infection of both types is revealed in about 11%. Co-infection of HIV 1 and HSV 2 was detected in 95% in Africa. However, the antibodies are not able to prevent a relapse or re-infection. The HSV 2 meningitis/encephalitis agent specific antibodies of class IgG are produced in CSF. The intrathecal agent specific antibody production is defined by the relative CSF/serum quotient CSQ rel. (Synonym:antibody specificity index). The quotient is calculated from the amount of agent specific antibodies in total CSF IgG in proportion to the amount of agent specific antibodies in total serum IgG. Primary infections with Herpes simplex virus (HSV) and varicella zoster virus (VZV) may lead to severe illness in pregnancy. Both diseases may be associated with transplacental virus transmission and fetal infection. Such infections can lead to intrauterine death, severe malformations and premature birth. Type specific serological diagnosis of Herpes simplex virus (HSV) infections requires assays based on type specific antigens : glycoprotein C1 (gc-1) or glycoprotein G-1 (gG-1) of HSV 1 and glycoprotein G2 (gG-2) of HSV 2.

DENGUE IgG CAPTURE ELISA

For the detection of Secondary Dengue Infection Intended use The Panbio Dengue IgG capture ELISA is for the qualitative presumptive detection of elevated IgG antibodies to dengue virus (Serotype 1-4) in patients with secondary infection. This test is intended as an aid in the clinical laboratoray diagnosis of patients presenting with clinical symptoms consistent with dengue virus infection, and should be used in conjunction with panbio dengue IgM capture ELISA and Dengue early ELISA, which also detects primary dengue virus infection. High IgG levels indicative of secondary dengue are detectable on the panbio dengue IgG capture ELISA as early as 3 days post onset of illness. However, the peak

detection window for accurate secondary diagnosis is 6-15 days following onset of illness. Positive results are presumptive and must be confirmed by virus isolation, paired serum analysis, antigen detection by immune histochemistry or viral nucleic acid detection for confirmation of dengue virus infection. Introduction Dengue virus is a flavivirus found largely in areas of the tropics and sub-tropics. Over half the worlds population lives in regions at risk of potential dengue transmission, making dengue the most important arbovirus disease in humans, in terms of morbidity and mortality. There are four distinct but antigenically related serotypes of dengue viruses, and transmission is via female mosquito, principally Aedes aegypti, Aedes albopictus and Aedes Polynesienses. The clinical manifestations of dengue virus infection are vried, ranging from subclinical through to fatal. The disease is graded according to severity as follows : non-specific febrile illness, classic dengue fever, dengue haemorrhagic fever (DHF) (grades I and II), and dengue shock syndrome (DSS) (grades III and IV). Classic dengue fever is characterized by the sudden onset of fever with two or more of : headache, retro-orbital pain, myalgia. A diphasic febrile course is common as in insomnia and anorexia with loss of taste or bitter taste. DHF and DSS are severe, potentially fatal complications often associated with infection by a second serotype. The majority of adult patients in countries where dengue is endemic will have secondary dengue infection. Detection of specifically elevated IgG antibodies to dengue virus by ELISA is a valuable diagnostic tool, for identification of secondary dengue infections, where the occurrence of complications in patients is higher. Traditionaly, haemagglutination-inhibition (HAI) titres have been used to classify infections as primary or secondary. The current definition depends upon an assay of paired serum specimens separated in time by at least 7 days, with any acute specimen with an HAI titre > 1:1280 defined as coming from a patient with a secondary flavivirus infection. The panbio dengue IgG capture ELISA is an alternative to the HAI assay for the serological diagnosis of secondary dengue infections. As illustrated below, secondary dengue virus infection is characterized by high IgG which may be accompanied by elevated IgM levels. The panbio dengue IgG capture ELISA is set to detect the specific high level of IgG antibodies to dengue infection above this threshold. The assay does not detect low level IgG antibodies from past exposure typically present in many individuals from endemic regions. A high IgG level (>22 panbio units) is therefore indicative of secondary dengue virus infection. Principle Serum antibodies of the IgG class, when present, combine with anti-human IgG antibodies coated on the polystyrene surface of the microwell test strips (assay plate). A concentrated pool

of recombinant dengue 1-4 antigens is diluted to the correct working volume with antigen diluents. The antigens are produced using an insect cell expression system and immunopurified utilizing a specific monoclonal anibody. An equal volume of horseradish peroxidase (HRP)conjugated monoclonal antibody (MAb) is added to the diluted antigen, allowing the formation of anigen MAb complexes. Residual serum is removed from the assay plate by washing, and complexed antigen-MAb is added to the assay plate. These antigen MAb complexes then bind to the serum dengue specific IgG antibodies. After incubation the microwells are washed and a colorless substrate system, tetramethylbenzidine/hydrogen peroxide (TMB chromogen) is added. The substrate is hydrolysed by HRP if present, and the chromogen turns blue. After stopping the reaction with acid, the TMB turns yellow. Color development is indicative of the presence of anti-dengue IgG antibodies in the test sample. Materials provided 1. Anti-human IgG coated Microwells (Assay Plate) : (12X8 wells). Ready for use. Unused microwells should be resealed immediately and stored in the presence of the desiccant. Stable at 2-8C until expiry. 2. Dengue 1-4 Antigens (recombinant) One clear capped vial, 150l (Red) concentrated dengue viral antigens 1,2,3 and 4. Unused diluted antigen must be discarded. Concentrated antigen is stable at 2-8C until expiry. 3. Wash buffer (20x) one bottle, 60ml of 20x concentrate of phosphate buffered saline (pH 7.2-7.6) with Tween 20 and preservative (0.1%) proclin). Crystallisation may occur at low temperatures. To correct, incubate at 37C until clear. Mix well. Dilute one part wash buffer with 19 parts of distilled water. Diluted buffer may be stored for one week at 225C. 4. Sample dilutent Two bottles, 50ml (pink). Ready for use. Tris buffered saline (pH 7.2 7.6) with preservatives (0.1% proclin) and additives. Stable at 2-8C until expiery. 5. Antigen diluent One bottle, 50ml (clear). Ready for use. Phosphate buffer containing preservatives (0.1% proclin and 0.005% gentamycin). Stable at 2-8C until expiry. 6. HRP conjugated Monoclonal antibody tracer one bottle, 7 ml (green). Ready for use. Horseradish peroxidase conjugated monoclonal antibody tracer with preservative (0.1% proclin) and protein stabilizers. Stable at 2-8C until expiry. 7. TMB chromogen (TMB)-one bottle, 15 ml. Ready for use. A mixture of 3,3, 5,5 tetramethylbenzidine and hydrogen peroxide in a citric acid citrate buffer (pH 3.5-3.8). Stable at 2-8C until expiry. 8. Reactive control one Red-capped vial, 200l, human serum (contains 0.1% sodium azide and 0.005% gentamycin sulphate). Stable at 2-8C until expiry.

9. Calibrator one yellow capped vial, 400 l human serum (contains 0.1% sodium azide and 0.005% gentamycin sulphate). Stable at 2-8C until expiry. 10. Negative control one green capped vial 200 l human serum (contains 0.1% sodium azide and 0.005% gentamycin sulphate). Stable at 2-8c until expiry. 11. Stop solution one Red capped bottle, 15ml. Ready to use. 1M phosphoric acid. Stable 225C until expiry. Precautions For in vitro diagnostic use 1. All human source material used in the preparation of controls has been tested for antibody to human immunodeficiency virus 1&2 (HIV 1&2), hepatitis C(HCV) as well as hepatitis B surface antigen and found to be negative. However no test method can offer complete assurance and all human controls and antigen should be handled as potentially infectious material. The centers for disease control and prevention and the national institutes of health (USA) recommend that potentially infectious agents be handled at Biosafety level 2. 2. This test should be performed on serum only. The use of whole blood, plasma or other specimen matrix has not been established. 3. Icteric or lipaemic sera or sera exhibiting haemolysis or microbial growth hsoul d not be used. 4. Do not heat-inactivate sera 5. All reagents must be equilibrated to room temperature (20-25C) before commencing the assay. The assay will be affected by temperature changes. Do not remove imcrowells from closed bag until they have reached room temperature (20-25C) 6. Dispense reagents directly from bottles using clean pipette tips. Transferring reagents may result in contamination. 7. Unused microwells should be resealed immediately and stored in the presence of desiccant. Failure to do this may cause erroneous results. 8. Substrate system: As TMB is susceptible to contamination from metal ions, do not allow the substrate system to come into contact with metal surfaces. Avoid prolonged exposure to direct light

Some detergents may interfere with the performance of the TMB The TMB may have a faint blue color. This will not affect the activity of the substrate or the results of the assay. 9. Some kit components contain sodium azide, which may react with lead or copper plumbing to form highly explosive metal azide compounds. When disposing of these reagents through plumbing fixtures, flush with a large volume of water to prevent azide build up in drains 10. Sodium azide inhibits conjugate activity. Clean pipette tips must be used for the conjugate addition so that sodium azide is not carried over from other reagents. Specimen collection and preparation Blood obtained by venipunture should be left to clot at room temperature (20-25C) and then centrifuged according to the clinical and laboratory standards institute (CLSI) (approved standard Procedures for the collection of Diagnostic Blood specimens by Venipuncture, H3A5, 2003). The serum should be separated as soon as possible and refrigerated (2-8C) or stored frozen (<20C) if not tested within two days. Self defrosting freezers are not recommended for storage. The use of icteric sera or sera exhibiting haemolysis, lipaemia or microbial growth is not recommended. The CLSI provides recommendations for storing blood specimens (Approved Standard-Procedures for the Handling and Processing of Blood specimens, H18-A3, 2004). Test Procedure Note: Ensure all reagents are equilibrated to room temperature (20-25C) before commencing assay. Performing the assay outside the time and temperature ranges provided may produce invalid results. Assays not falling within the established time and temperature ranges must be repeated. Serum Predilution Remove the required number of microwells from the foil sachlet and insert into strip holder. Five microwells are required for Negative Control (N), Reactive Control (R) and Calibrator (CAL) in triplicate. Ensure the remaining unused imcrowells are sealed tightly in the foil sachet. Using suitable test tubes or a microtitre plate, dilute the Negative Control, Reactive Control, Calibrator and patients samples : o o To 10l serum add 1000 ul of sample Diluent. Mix well. Alternatively

To 10l serum add 90l of sample dilutent, take 20l of the diluted serum and add 180 l sample dilutent. Mix well.

ELISA Procedure (a) Antigen a. Determine the required number of wells for your assay. Dilute the antigen 1/250 using the antigen diluents. It is recommended, as a minimum, to dilute 10l of antigen into 2.5ml of antigen diluents. This is sufficient for up to five strips (40 wells). A volume of 0.5ml of diluted antigen is required per strip. Once the antigen is added to the antigen diluent, the solution becomes a pale red color. Ensure the remaining unused concentrated antigen remains at 2-8C. b. Remove the required volume of diluted antigen and mix with an equal volume of MAb tracer in a clean glass or polypropylene vial. Gently mix the antigenMAb tracer solution and leave at room temperature (20-25C) until required (requires 60 minute incubation). Discard the unused diluted antigen. (b) Assay plate a. Within 10 minutes after mixing the MAb tracer and diluted antigen, pipette 100l diluted patient sample and controls into their respective microwells. b. Cover plate and incubate for 60 minutes at 37C 1C. c. Wash six (6) times with diluted wash buffer (refer to washing procedure) d. Mix the antigen-MAb tracer solution before transfer. Pipette 100 l of antigenMAb complexes from the antigen vial to the appropriate wells of the assay plate. e. Cover plate and incubate for 60 minutes at 37C1C. f. Wash six (6) times with diluted wash buffer (refer to washing procedure).

g. Pipette 100 l TMB into each well. h. Incubate for 10 minutes at room temperature (20-25C), timing from the first addition. A blue color will develop. i. Pipette 100 l of stop solution to all wells in the same sequence and timing as the TMB addition. Mix well. The blue color will change to ellow. Within 30 minutes read the absorbance of each well at a wavelength of 450 nm, with a reference filter of 600-650nm.

j.

Note: if a dual wavelength spectrophotometer is available, set the reference filter between 600 650nm. Reading the microwells at 450 nm without a reference filter may result in higher absorbance values due to background. Washing procedure Efficient washing to remove uncomplexed sample or components is a critical requirement of the ELISA procedure. (A) Automated plate Washer a. Completely aspirate all wells b. Fill all wells to rim (350l) during wash cycle c. On completion of six (6) washes, invert plate and tap firmly on absorbent paper towel to ensure all wash buffer is removed. d. Automated plate washers must be well maintained to ensure efficient washing. Manufacturers cleaning instructions should be followed at all time. (B) Manual Washing a. Discard contents of plate in appropriate waste container b. Fill wells with wash buffer using a suitable squeeze bottle. Avoid bubbling of wash buffer as this may reduce wash efficiency. Discard wash buffer from wells immediately. c. Refill wells with wash buffer and discard immediately d. Repeat step (3) another four times. This will make a total of six (6) washes with wash buffer e. After the final wash, discard contents of wells and tap the plate on absorbent paper towel to ensure all wash buffer is removed. Quality Control Each kit contains calibrator, Reactive and Negative controls. Acceptable values for these are found on the accompanying specification sheet. The negative and Reactive controls are intended to monitor for substantial reagent failure. The Reactive control will not ensure precision at the assay cut-off. The test is invalid and must be repeated if the absorbance reading of either the controls or the calibrator do not meet the specifications. If the test is invalid, patient results cannot be reported.

Quality control (QC) requirements must be performed in conformance with local, state and or federal regulations or accreditation requirements and your laboratorys standard QC procedures. It is recommended that the user refer to CLSI C24-A and 42 CFR 493.1256 for guidance on appropriate QC practices. Calculations Note : the calibration factor is batch specific and detailed in the specification sheet. Obtain the calibration factor value before commencing calculations. Calculate the average absorbance of the triplicate of the calibrator and multiply by the calibration factor. This is the cut off value An index value can be calculated by dividing the sample absorbance by the cut off value (calculated in step (1) above).

Alternatively Panbio units can be calculated by multiplying the index value (calculated in step (2) above) by 10.

Index value = sample absorbance/cut off value. Example : Sample A absorbance = 0.949 Sample B absorbance = 0.070

Mean absorbance of calibrator = 0.802 Calibration Factor = 0.62 Cut off value = 0.802X0.62 = 0.497

Sample A (0.949/0.497) = 1.91 index value Sample B (0.070/0.497) = 0.14 index value

Panbio units = index value X 10 Sample A 1.91 X 10 = 19.1 panbio units

Sample B 0.14 X 10 = 1.4 panbio units Interpretation of results The panbio dengue IgG capture ELISA presumptively detects the elevated level of IgG antibodies to dengue virus in a patients serum. A positive result (>22 panbio units) is indicative of active secondary infection. If primary dengue infection is suspected this assay should be used in conjunction with the panbio dengue IgM capture (E-DEN011M) ELISA.

INDEX PANBIO UNITS RESULT <1.8 1.8-2.2 >2.2 <18 18-22 >22 Negative Equivocal Positive

RESULT Negative

INTERPRETATION No detectable elevated IgG antibody levels. The absence of elevated IgG antibodies is presumptive evidence that the patient does not have a secondary dengue infection. Samples with panbio units < 18 should be tested on the panbio dengue IgM capture ELISA (E-DEN01M) to detect primary dengue virus infections. Other dengue assays should be performed to rule out acute infection (eg. Culture, PCR, NS1 antigen detection). Refer to the CDC MMWR Recommendations and Reports 1997: 46 RR-10 P45-46 for confirmatory diagnosis of dengue infections. Equivocal samples should be repeated in duplicate. If both duplicates are above or below the cut off, the specimens may be reported as positive or negative, respectively. If one duplicate is above the cutoff and the other is below the cutoff, or

Equivocal

if either sample remains equivocal after repeat testing it should be reported that elevated dengue virus IgG cannot be determined, and testing should be repeated by an alternative method. Alternatively, a further sample should be collected. Positive Presence of detectable elevated IgG antibodies, presumptive evidence that the patient has been recently exposed to or is currently infected with, dengue virus consistent with secondary infections. The result must be confirmed by current CDC guidelines for diagnosis of this disease. Refer to the CDC MMWR recommendations and reports 1997 : 46 RR-10 p 45-46 for confirmatory diagnosis of dengue infections.

A recommended way of reporting the results obtained. The following results were obtained with the panbio dengue IgG capture ELISA. Values obtained with different methods may not be used interchangeably. The magnitude of the measured result above the cut off value is not indicative of the total amount of antibody present. The result should be reported as positive, negative or equivocal, and not as a numerical value. The reported results should contain an appropriate interpretation. Test Limitations (A) The clinical diagnosis must be interpreted with clinical signs and symptoms of the patient. The results from this kit are not by themselves diagnostic and should be considered in association with other clinical data and patient sysmptoms. (B) Screening of the general population must not be performed. The positive predictive value depends on the likelihood of the virus being present. Testing should only be performed on patients with clinical symptoms or when exposure is suspected. (C) The assay performance characteristics have not been established for visual result determination. (D) Results from immunosuppressed patients must be interpreted with caution.

(E) Serological cross-reactivity across the flavivirus group is common (i.e., between St.Louis encephalitis, Murray Valley encephalitis, Japanese encephalitis, West Nile and ellow fever viruses). These diseases must be excluded before confirmation of diagnosis. (F) Generally primary responders exhibit mainly manotypic antibody responses. However, during successive infections the antibody response broadens to include heterotypic reactivity to other flaviviruses in the same or different antigenic groups. (G) All sera demonstrating a positive result by the panbio dengue IgG capture ELISA test must be referred to a reference laboratory for confirmation and epidemiological recording. (H) This test my be negative with persons that are currently infected with dengue virus. Samples obtained very early in the infection may not have detectable antibodies. Suspected acute infections should be tested with the panbio early ELISA or other assays which detect acute infections. (I) IgG antibody levels in convalescent samples taken from primary dengue infected individuals may rise above the threshold of the assay. It is recommended that samples be tested within 6-15 days for accurate primary and secondary differentiation. Expected Values Secondary dengue virus infection is characteristed by high IgG levels detectable as early as 3 days following the onset of infection, which may be accompanied by elevated IgM levels. The accuracy of the assay is dependent upon the timing following the infection when the sample is collected. Although this assay can detect samples as early as three days following infection, the peak performance of the assay is achieved when samples are taken between 6-15 days following onset of illness. In some secondary infections detectable levels of IgG antibodies may be low. Where symptoms persist, we recommend that patients be re-tested 4-7 days after the first specimen.

ANTI-HBc IgM
Anti HBc IgM assay is a Chemiluminescent Micro particle Immunoassay (CMIA) for the qualitative detection of IgM antibody to hepatitis B core antigen (anti HBc IgM ) in human serum and plasma . Purpose : Anti HBc IgM is a diagnosis of acute or recent hepatitis B viral infection. Anti HBc IgM raise rapidly in patients with acute infection high levels of Anti HBc Ig M have been detected in patients with acute hepatitis B viral infections. Principle: Pre-diluted sample and anti-human IgM (mouse monoclonal) coated paramagnetic micro particles are combined. Human IgM present in the sample binds to the anti-human IgM (mouse monoclonal) coated micro particles. After washing, the anti-HBc specific IgM binds to the acridinium-labeled rHBcAg conjugate that is added in the second step. Following another wash cycle, Pre-Trigger and Trigger Solutions are added to the reaction vessel (RV). The resulting Chemiluminescent reaction is measured as relative light units (RLUs). A direct relationship exists between the amount of anti-HBc IgM in the sample and the RLUs detected by the ARCHITECT I * optical system. The presence or absence of anti-HBc IgM in the specimen is determined by comparing the chemiluminescent signal in the reaction to the cutoff signal determined from a previous ARCHITECT Anti-HBc IgM calibration. If the Chemiluminescent signal of the reaction is greater than or equal to the cutoff signal, the specimen is considered reactive for anti-HBc IgM by the ARCHITECT Anti-HBc IgM assay. Consumables: ARCHITECT I system ARCHITECT Assay CD-Rom Calibrators Controls Pre trigger solution Trigger solution Wash buffer Reaction vessels Sample cups Septum Replacement cap Pipettes Assay Procedure :

Before loading the ARCHITECT Anti-HBc IgM Reagent Kit on the system for the first time, the micro particle bottle requires mixing to re suspend micro particles that may have settled during shipment. Invert the micro particle bottle 30 times. Prepare Calibrators and Controls. Make sure the ARCHITECT Anti-HBc IgM Calibrators and Controls are completely thawed before mixing. Allow sufficient time for thawing. To obtain the recommended volume requirements for the ARCHITECT Anti-HBc IgM Calibrators and Controls, hold the bottles vertically and dispense 5 drops of each calibrator or 5 drops of each control into each respective sample cup. Load samples. Press RUN. The ARCHITECT I System performs the following functions: Moves the sample to the aspiration point. Loads a reaction vessel (RV) into the process path. Aspirates and transfers an aliquot of the sample into the RV. Moves the RV one position and adds ARCHITECT i Wash Buffer to dilute the sample. Aspirates micro particles and an aliquot of the diluted sample and transfers it to a new RV. Mixes, incubates, and washes the reaction mixture. Adds conjugate to the RV. Mixes, incubates, and washes the reaction mixture. Adds Pre-Trigger and Trigger Solutions. Measures Chemiluminescent emission to detect the presence of anti-HBc IgM in the sample. Aspirates contents of RV to liquid waste and unloads RV to solid waste. Calculates the result. Calculation The ARCHITECT i System calculate the cutoff rate (CO) from the mean RLU of three replicates for Calibrator 1 and Calibrator 2 and stores the results. Cutoff RLU = [(Calibrator 2 mean RLU Calibrator 1 mean RLU) x 0.75] +Calibrator 1 mean RLU The cutoff RLU is stored for each reagent lot calibration. Interpretation of Results Specimens with S/CO values < 1.00 are considered nonreactive by the ARCHITECT Anti-HBc IgM assay. Specimens with S/CO values > 1.00 are considered reactive by the ARCHITECT AntiHBc IgM assay. Specificity A total of 1634 random blood donor and hospitalized patient specimens was tested at three clinical sites. None of the 1634 specimens were reactive by ARCHITECT Anti-HBc IgM.

The specificity of ARCHITECT Anti-HBc IgM in this population was 100.00% (1631/1631b) with a 95% confidence interval of 99.77-100.00%. Sensitivity In a total of 212 specimens from patients with acute hepatitis B, all were reactive by ARCHITECT Anti-HBc IgM. The sensitivity was 100.00% (212/212) with a 95% confidence interval of 98.28-100.00%. LIMITATIONS OF THE PROCEDURE If the anti-HBc IgM results are inconsistent with clinical evidence, additional testing is suggested to confirm the result. For diagnostic purposes, results should be used in conjunction with patient history and other hepatitis markers for diagnosis of acute or chronic infection. Specimens that have been frozen and thawed and specimens containing red blood cells, clots, or particulate matter must be centrifuged prior to running the assay. Performance has not been established using cadaver specimens or body fluids other than human serum or plasma. Do not use heat-inactivated specimens. Do not use grossly hemolyzed specimens. Specimens with obvious microbial contamination should not be used. Specimens from heparinized patients may be partially coagulated and erroneous results could occur due to the presence of fibrin. To prevent this phenomenon, draw the specimen prior to heparin therapy. Specimens from patients with high levels of IgM, e.g. specimens from patients with multiple myeloma, may show depressed values when tested with assay kits that use reagents containing anti-human IgM.

SYPHILIS
The syphilis 3.0 test is a solid phase immunochromatographic assay for the qualitative detection of antibodies of all isotypes (IgG, IgM, IgA) against Treponema pallidu7m (TP). This test is intended for professional use as an aid on the diagnosis of syphilis. Treponema pallidum (TP) is the causative agent of the venereal disease syphilis. Syphilis is a disease caused nby the spirochetal bacterium Treponema pallidum (TP). Clinical diagnostic issues related to syphilis are the detection of syphilis antibodies in human blood by immunoassay. Among the existing immunological method, the confirmatory treponemal tests are the agglutination format such as the T.pallidum hemagglutination assay (TPHA) and the immunostaining analysis by fluorescent treponemal antibody adsorption test (FTA-ABS). Recently, the ELISA format and immunochromatography format (rapid) to detect antibody of T.pallidum are available. Since even highly purified antigens from inoculated TP may contain a certain amount of contaminating materials such as flagella of TP, native TP antigen may cause a non-specific reaction in the assay of test serum samples, and this may result in lower sensitivity

and poor reproducibility. To circumvent these potential problems in immunoassays, researchers have constructed TP genes for the expression of recombinant antigens in bacterium systems such as E.coli and focused on TP membrane protein, which are definitely immunogenic. The major immunoreactive antigens of these membrane proteins have been reported to have a MW 47,42,17 and 15KDa based on western blot analysis. The syphilis 3.0 contains a membrane strip, which is pre-coated with recombinant Treponema palladium antigens (17, 15KDa) on test band region. The recombinant Treponema palladium antigens-colloid gold conjugate (17, 1KDa), patient sample and sample diulent moves along the membrane chromatographically to the test region (T) and forms a visible line as the antigenantibody-antigen gold particle complex forms. Therefore, the formation of a visible line in the test region (T) indicates a positive result for the detection of Treponema palladium specific antibodies (igG, IgA, IgM). When the Treponema palladium specific antibodies (IgG, IgM, IgA) are absent in the sample, no visible color band in the test region (T). Materials provided/composition Test strip includes Gold conjugates (as main component): Recombinant Treponema palladium antigens (17, 15KDa) a gold colloid 10.2g Test line (as main component): Recombinant Treponema palladium antigens (17, 15KDa) 0.70.14g control line (as main component) : Goat anti-Treponema palladium serum 0.750.15g. Assay dilutent: 50mMTris-HCL, sodium azide

Precautions/storage and kit stability Store at room temperature The test device is sensitive to humidity and as well as to heat. Perform the test immediately after removing the test device from the foil pouch Do not use it beyond the expiry Do not use the kit if the pouch is damaged or the seal is broken Assay dilutents: it contains sodium azide as a preservative, in case of contact with skin, wash immediately, wear gloves & eye protective.

Specimen collection and storage/precaution whole blood Collect whole blood using the suitable anti-coagulant The whole blood may be used for testing immediately or may be stored at 2-8C up to three days.

Serum or Plasma Centrifuge whole blood to get plasma or serum specimen If specimens are not immediately tested they should be refrigerated at 2-8C. For storage periods greater than three days, freezing is recommended. They should be brought to room temperature prior to use. Specimens containing precipitate may yield inconsistent test results. Such specimens must be clarified prior to assaying.

Precaution Anticoagulants such as heparin, EDTA, and citrate do not affect the test result. Haemolytic samples, rheumatoid factors-contained samples and lipaemic, icteric samples can lead to impair the test results. Use separate disposable pipettes or pipette tips for each sample in order to avoid cross contamination of either samples which could erroneous result.

Warnings For in vitro diagnostic use only. Do not re-use test device. Do not eat or smoke while handling specimens. Wear protective gloves while handling specimens. Avoid splashing or aerosol formation Clean up spills thoroughly using an appropriate disinfectant Decontaminate and dispose of all specimens, reaction kits and potentially contaminated maerials, as if they were infectious waste, in a biohazard container. Do not mix and interchange different specimen Assay diluents contains, sodium azide as a preservative, if these material are to be disposed off through sink or other common plumbing system, flush with generous amount of water to prevent accumulation of potentially explosive compound.

Procedure Remove the test device from the foil pouch, and place it on a flat dry surface Transfer the specimen by a pipette or a dropper

Add 10l of serum or plasma (or 20l of whole blood to the sample well (S) of the test device Add 3-4 drops of assay diluents (approximately 110) and start the timer. As the test begins to work, you will see purple color move across the result window in the center of the test device.\ Interpret test results at 5-20 minutes, a positive result will not change once it has been established at 20 minutes. However, in order to prevent any incorrect results, the result should not be interpreted after 20 minutes. Especially, when you use the whole blood, please interpret the test results within 10 minutes. In this case, do not interpret after 10 minutes. This interpreting time is based on reading the test results at room temperature.

Interpretation of the test A color band will appear in the left section of the result window to show that the test is working properly. This band is the control band. The right section of the result window indicates the test results. If another color band appears in the right section of the result window, this band is the test band.

Negative result : The presence of only one purple color band within the result window indicates a negative result Positive result : the presence of two color bands (T band and C band) within the result window, no matter which band appears first, indicates a positive result for TP antibodies. Invalid result : if the purple color band is not visible within the result window after performing the test, the result is considered invalid. The directions may not have been followed correctly or the test may have deteriorated. It is recommended that the specimen be re-tested. Limitations of the test The syphilis 3.0 test will only indicate the presence of TP antibodies in the specimen and should not be used as the sole criteria for the diagnosis of TP infection. As with all diagnostic tests, all results must be interpreted together with other clinical information available to the physician If the test result is negative and clinical symptom persists, additional testing using other clinical methods is recommended. A negative result does not at any time preclude the possibility of TP infection.

Internal quality control The syphilis test device has a letter of T and C as test Line and Control Line on the surface of the case. Both the test line and control line in result window are not visible before applying any samples. The control line is used for procedural control. Control line should always appear if the test procedure is performed properly and the test reagents of control line are working. Expected Values The syphilis test has been compared with a leading commercial TPHA syphilis test. The overall accuracy is greater or equal to 9.0%. Performance characteristics The syphilis test has tested with positive and negative clinical samples tested by a leading commercial TPHA syphilis test. The result shows that the syphilis test is very accurate to TPHA. Syphilis test Results Positive Negative 152 1 209 210 153 210 363 Total results

Reference Method

TPHA results Positive

Negative 1 Total results 153

Precision : within-run and between run precisions have been determined by the testing 3 replicates of 6 specimens : a negative, a low positive, 2 medium positive and 2 strong positive. All values were correctly identified 110% of the time to evaluate the interference of syphilis test kit with known relevant interfering specimens, the haemolytic samples, rheumatoid factors-contained samples and lipaemic, icteric samples were investigated. In these studies, those specimens did not interfere with the syphilis test. Analytical sensitivity: the limit of detection : the smallest amount of the target maker that can be precisely detected : have been equal or superior to a leading commercial syphilis detection rapid test.

SYPHILIS - RAPID PLASMA REAGIN (RPR) CARD TEST

Rapid Plasma regain (RPR) card test is a qualitative and semi-quantitative screening test for regain antibodies. The test is similar in principle to the classical VDRL test. The reagent used is a modified cardiolipin antigen, coated with microparticulate carbon particles. In addition, it contains a balanced quantity of cholesterol and lecithin. In the presence of regain antibodies, flocculation appears which can be visusalized macroscopically. Contents RPR antigen (A) store at 2-8C Positive control (B) - store at 2-8C Negative control - store at 2-8C Dispensing dropper with measuring needle Disposable 6 well test cards Disposable mixing sticks Insert

Storage and stability Reagents are stable till the expiry date indicated on the labels when stored at store at 2-8C. Do not freeze. Sample collection and Storage Any fresh serum or plasma sample free of contamination and hemolysis may be used. Turbid samples should be clarified by centrifugation before testing. The sample may be stored at store at 2-8C or at -20C if longer storage is required. It is not necessary to heat or inactivate the sample before testing. Precautions Reagents are for in vitro diagnostic use only. Infectious agents may be present in test specimens. All specimens and test materials should be handled and regarded as a potential biohazard. Gently shake the RPR antigen before use to ensure a homogenous suspension. Avoid vigorous shaking. Samples and reagents should be brought to room temperature before use and should be returned to store at 2-8C after use.

To obtain correct results it is necessary to dispense the RPR antigen with the dispensing dropper provided.

Qualitative Procedure Dispense 50l of sample or control on to a circle of the test card using a clean and dry pipette. Using the wooden mixing stick provided, spread the sample over the entire area of the test circle. Gently resuspend the RPR antigen, with the help of the dispensing dropper provided, slowly suck the required quantity of the reagent. Add one drop of the reagent onto each test sample, while holding the dropper in a vertical position. Do not restir the mixture on the test circle Rotate the card for 8 minutes, either manually or on a mechanical rotator at 10 rpm Read the results by visual inspection in good light. No magnification is required.

Interpretation of results A positive result (reactive) is indicated by the development of clearly visible clumps of the carbon particles either in the center or at the edge of the test circle A negative result (non-reactive) is indicated when the carbon particles remain in a homogenous suspension and no aggregates are visible It is recommended to carry out the quantitative estimation to determine the titre of a positive sample

Semi-quantitative estimation Make doubling dilutions of the sample to be tested with 0.9% saline. Test each dilution with RPR antigen till the dilution, where a positive result is obtained. The titre is reported as the reciprocal of the highest dilution which shows a positive result.

Limitations Certain non syphilitic conditions which cause tissue damage can cause result in false positive reactions. Common conditions like malaria, typhoid, leprosy, tuberculosis, certain viral diseases, pregnancy and some autoimmune disorders can lead to low titre positive reactions.

Thus positive samples should be confirmed with specific tests like Treponema palladium Haemagglutination test (TPHA), fluorescent Treponemal antibody test (FTA) etc., False negative reactions can occur in stages of the disease where there is minimal tissue damage, particularly in early infection and in latent stages. Clinical Significance Syphilis is a sexually transmitted disease caused usually by direct but sometimes by indirect contact. Infection is usually rendered evident by the development of a primary lesion or chance. This appears within a month of infection and is accompanied by the enlargement of the local lymph nodes. From 6 to 12 weeks after the appearance of the primary chance, the secondary stage of the disease sets in, during which skin, lymph nodes, bones, joints, eyes and other organs may get affected. Thereafter the disease becomes latent and may only be detected by serological tests. This may persist for several years and may result in lesions of the heart, producing aneurysms or central nervous system producing tabes dorsalis and general paralysis.

HIV BLOT WESTERN BLOT ASSAY

Screening tests are widely available for detecting antibodies to both HIV-1 and HIV-2, the etiologic agents to the acquired immunodeficiency syndrome (AIDS). Such tests can be extremely sensitive but have a potential for being less specific, leading to false positive interpretations. Independent supplemental tests of high specificity are therefore necessary to further confirm the presence of antibodies to HIV-1 and HIV-2. The kit is intended for use as a more specific supplemental test on human serum or plasma specimens found repeatedly reactive using ELISA. The separated specific HIV-1 viral antigens incorporated onto the strips via electrophoretic and electrotransblot procedures, combined with a specific HIV-2 sysnthetic peptide on the same strip allow for further delineation of the antibody responses to specific viral proteins. Each strip also includes an internal sample addition control to minimize the risk of false negatives due to operational errors and to ensure the addition of samples. Principles of the procedure The nitrocellulose strips are incorporated with separated, bound antigenic proteins from partially purified inactivated HIV-1 using electrophoretic blotting, plus a specific HIV-2 synthetic peptide on the same strips. Individual nitrocellulose strips are incubated with diluted serum or plasma and controls. Specific antibodies to HIV-1 and HIV-2 if present in the specimens will bind to the HIV-1 proteins and HIV-2 peptide on the strips. The strips are washed to remove unbound materials. Antibodies that bind specifically to HIV proteins can be visualized using a series of reactions with goat anti-human IgG conjugated with alkaline

phosphatase and the substrate BCIP/NBT. This method has the sensitivity to detect marginal amounts of HIV specific antibodies in serum or plasma.

Components Component Antigen strips Nitrocellulose strips incorporated with HIV-1 viral lysate, a specific HIV-2 envelope peptide and a serum addition control band. Keep dry and away from light Non-rective control inactivated normal human serum nonreactive for Hepatitis B surface antigen (HBsAg), antibodies to HIV1/2, and anti HCV. Contains sodium azide and thimerosal as preservatives Strong Reactive control inactivated human serum with high tittered antibodies to HIV-1 and HIV-2 and nonreactive for HBsAg & anti-HCV. Contains sodium azide and thimerosal as preservatives. Quantity available Available in 18 or 36 strips Quantity provided 108 strips

Control (-)

1 vial (80l)

3 vials (80l)

Control (+)

1 vial (80l)

3 vials (80l)

Control (weak)

Weak reactive control 1 vial (80l) inactivated human serum with low tittered antibodies to HIV-1. Only and nonreactive for HBsAg, anti HIV-2 and anti HCV. Contains sodium azide and thimerosal as preservatives. Stock Buffer concentrate (10x) Tris buffer with heat inactivated normal goat serum. Contains thimerosal as preservative Wash Buffer concentrate (20x) Tris with Tween 20. Contains thimerosal as preservative CONJUGATE Goat anti-human IgG conjugated with alkaline phosphatase. Contains sodium azide as preservative Substrate solution of 5-bromo-4-chloro3-indolyl-phosphate (BCIP) and nitroblue tetrazolium (NBT) Blotting powder Non-fat dry milk 1 bottle (20ml)

3 vials (80l)

BUF STOCK-10x

3 bottles (20ml)

BUF WASH-20x

1 bottle (70ml)

3 bottles (70ml)

CONJUGATE

1 vial (120l)

3 vials (120l)

SUBS BCIP/NBT

1bottle (100ml)

3 bottles (100 ml)

POWDER Blotting

10 packets (1g each)

10x3packets (1g each)

Incubation tray, 9 wells Foreceps

2 or 4 trays

12 trays

1 pair

1 pair

The strong reactive control, weak reactive control and non reactive control contain thimerosal and sodium azide while stock buffer concentrate and wash buffer concentrate contain thimerosal and conjugate contains sodium azide. Sodium azide can react with copper and lead used in some plumbing systems to form explosive salts. The quantities used in this kit are small, nevertheless when disposing of azide containing materials they should be flushed away with relatively large quantities of water to prevent metal azide buildup in plumbing system. The following are the appropriate risk (R) phrases. The substrate contains 5 bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium which is classified per applicable European Economic Community (EEC) Directives as harmful (Xn). Avoid microbial contamination of reagents when opening and removing aliquots from the original vials or bottles. Do not pipette by mouth Handle test specimens, nitrocellulose strips, reactive, weak reactive and non reactive controls as potentially infectious agents. Wear laboratoray coats and disposable gloves while performing the assay. Discard gloves in bio-hazard waste bags. Wash hands thoroughly afterwards. It is highly recommended that this assay be performed in a biohazard cabinet. Keep materials away from food and drink In case of accident or contact with eyes, rinse immediately with plenty of water and seek medical advice. Consult a physician immediately in the event that contaminated materials are ingested or come in contact with open lacerations, or other breaks in the skin. Wipe spills of potentially infectious materials immediately with absorbent paper and swab the contaminated area with 1% sodium hypochlorite solution before work is resumed. Sodium hypochlorite should not be used on acid containing spills unless the area is wiped dry with absorbent paper first. Material used (including disposable gloves) should be disposed off as potentially biohazardous material. Do not autoclave material containing sodium hypochlorite.

Autoclave all used and contaminated materials at 121C at 15 p.s.i for 30 minutes before disposal. Alternatively, decontaminate materials in 5% sodium hypochlorite solution for 30-60 minutes before disposal in biohazard waste-bags. Decontaminate all used chemicals and reagents by adding sufficient volume of sodium hypochlorite to make a final concentration of at least 1%. Leave for 30 minutes to ensure effective decontamination. We do not recommend re-use of incubation trays.

Specimen collection, Transport and Storage Serum or plasma samples collected in EDTA, heparin or sodium citrate may be used. Before storage, ensure that blood clot or blood cells have been separated by centrifugation. Samples should be stored at 2C to 8C if the test is to be run within 7 days of collection or frozen at -20C or colder if the test is to be delayed for more than 7 days. Clear, non-hemolyzed samples are preferred. Lipemic, icteric or contaminated (particulate) samples should be filtered (0.45m) or centrifuged before testing. Patients specimens can be inactivated but this is not a requirement for optimal test performance. Inactivate as follows: Loosen caps of specimen containers Heat specimen to 56C for 30 minutes in a water bath Allow specimen to cool before retightening caps Specimen can be stored frozen until analysis

Preparation of Reagents Diluted wash buffer 1. Diluted wash buffer should be prepared fresh prior to use. 2. Dilute 1 volume of wash buffer concentrate (20x) with 19 volumes of reagent grade water mix wel. Blotting Buffer 1. Blotting buffer should be prepared fresh prior to use

2. Dilute 1 volume of stock buffer concentrate (10x) with 9 volumes of reagent grade water. Mix wel.

PRINCIPLES OF CHEMILUMINESCENCE
Chemiluminescence (sometimes "chemoluminescence") is the emission of light (luminescence), as the result of a chemical reaction. There may also be limited emission of heat. Given reactants A and B, with an excited intermediate, [A] + [B] [] [Products] + light For example, if [A] is luminol and [B] is hydrogen peroxide in the presence of a suitable catalyst we have: luminol + H2O2 3-APA[] 3-APA + light where:

where 3-APA is 3-aminophthalate 3-APA[] is the vibronic excited state fluorescing as it decays to a lower energy level.

The decay of this excited state[] to a lower energy level causes light emission. In theory, one photon of light should be given off for each molecule of reactant. This is equivalent to Avogadro's number of photons per mole of reactant. In actual practice, non-enzymatic reactions seldom exceed 1% QC,quantum efficiency. In a chemical reaction, reactants collide to form a transition state, the enthalpic maximum in a reaction coordinate diagram, which proceeds to the product. Normally, reactants form products of lesser chemical energy. The difference in energy between reactants and products, represented as , is turned into heat, physically realized as excitations in the vibrational state of the normal modes of the product. Since vibrational energy is generally much greater than the thermal agitation, it rapidly disperses in the solvent through molecular rotation. This is how exothermic reactions make their solutions hotter. In a chemiluminescent reaction, the direct product of a reaction is an excited electronic state, which then decays into an electronic ground state through either fluorescence orphosphorescence, depending partly on the spin state of the electronic excited state formed. Chemiluminescence differs from fluorescence in that the electronic excited state is derived from the product of a chemical reaction rather than the more typical way of creating electronic excited states, namely absorption. It is the antithesis of a photochemicalreaction, in which light is used to drive an endothermic chemical reaction. Here, light is generated from a chemically exothermic reaction. A standard example of chemiluminescence in the laboratory setting is the luminol test. Here, blood is indicated by luminescence upon contact with iron in hemoglobin. When

chemiluminescence takes place in living organisms, the phenomenon is called bioluminescence. A light stick emits light by chemiluminescence. Liquid-phase reactions Luminol in an alkaline solution with hydrogen peroxide in the presence of iron or copper,[1] or an auxiliary oxidant,[2] produces chemiluminescence. The luminol reaction is luminol + H2O2 3-APA[] 3-APA + light Gas-phase reactions

One of the oldest known chemoluminescent reactions is that of elemental white phosphorus oxidizing in moist air, producing a green glow. This is a gas-phase reaction of phosphorus vapor, above the solid, with oxygen producing the excited states (PO)2and HPO.[3] Another gas phase reaction is the basis of nitric oxide detection in commercial analytic instruments applied to environmental air-quality testing. Ozone is combined with nitric oxide to form nitrogen dioxide in an activated state. NO+O3 NO2[]+ O2 The activated NO2[] luminesces broadband visible to infrared light as it reverts to a lower energy state. A photomultiplier and associated electronics counts the photons that are proportional to the amount of NO present. To determine the amount of nitrogen dioxide, NO2, in a sample (containing no NO) it must first be converted to nitric oxide, NO, by passing the sample through a converter before the above ozone activation reaction is applied. The ozone reaction produces a photon count proportional to NO that is proportional to NO2 before it was converted to NO. In the case of a mixed sample that contains both NO and NO2, the above reaction yields the amount of NO and NO2 combined in the air sample, assuming that the sample is passed through the converter. If the mixed sample is not passed through the converter, the ozone reaction produces activated NO2[] only in proportion to the NO in the sample. The NO2 in the sample is not activated by the ozone reaction. Though unactivated NO2 is present with the activated NO2[], photons are emitted only by the activated species that is proportional to original NO. Final step: Subtract NO from (NO + NO2) to yield NO2 Enhanced chemiluminescence

Enhanced chemiluminescence is a common technique for a variety of detection assays in biology. A horseradish peroxidaseenzyme (HRP) is tethered to the molecule of interest (usually through labeling an immunoglobulin that specifically recognizes the molecule). This enzyme complex then catalyzes the conversion of the enhanced chemiluminescent substrate into a sensitized reagent in the vicinity of the molecule of interest, which on further oxidation by hydrogen peroxide, produces a triplet (excited) carbonyl, which emits light when it decays to the singlet carbonyl. Enhanced chemiluminescence allows detection of minute quantities of a biomolecule. Proteins can be detected down to femtomole quantities,[5] well below the detection limit for most assay systems. APPLICATIONS Gas analysis: for determining small amounts of impurities or poisons in air. Other compounds can also be determined by this method (ozone, N-oxides, S-compounds). A typical example is NO determination with detection limits down to 1 ppb Analysis of inorganic species in liquid phase Analysis of organic species: useful with enzymes, where the substrate is not directly involved in chemiluminescence reaction, but the product is Detection and assay of biomolecules in systems such as ELISA and Western blots DNA sequencing using pyrosequencing Lighting objects. Chemiluminescence kites,[6] emergency lighting, glow sticks (party decorations). Combustion analysis: Certain radical species (such as CH* and OH*) give off radiation at specific wavelengths. The heat release rate is calculated by measuring the amount of light radiated from a flame at those wavelengths. Children's toys

Electrochemiluminescence Electrochemiluminescence or electrogenerated chemiluminescence (ECL) is a kind of luminescence produced during electrochemical reactions in solutions. In electrogenerated chemiluminescence, electrochemically generated intermediates undergo a highly exergonic reaction to produce an electronically excited state that then emits light upon relaxation to a lower-level state. This wavelength of the emitted photon of light corresponds to the energy gap between these two states.[1] ECL excitation can be caused by energetic electron transfer (redox) reactions of electrogenerated species. Such luminescence excitation is a form of chemiluminescence where one/all reactants are produced electrochemically on the electrodes.[2] ECL is usually observed during application of potential (several volts) to electrodes of electrochemical cell that contains solution of luminescent species (polycyclic aromatic hydrocarbons,[3] metal complexes, Quantum Dots or Nanoparticles[4]) in aprotic organic solvent (ECL composition). In organic solvents both oxidized and reduced forms of luminescent species

can be produced at different electrodes simultaneously or at a single one by sweeping its potential between oxidation and reduction. The excitation energy is obtained from recombination of oxidized and reduced species. In aqueous medium which is mostly used for analytical applications simultaneous oxidation and reduction of luminescent species is difficult to achieve due to electrochemical splitting of water itself so the ECL reaction with the coreactants is used. In the later case luminescent species are oxidized at the electrode together with the coreactant which gives a strong reducing agent after some chemical transformations (the oxidative reduction mechanism). Application ECL proved to be very useful in analytical applications as a highly sensitive and selective method. It combines analytical advantages of chemiluminescent analysis (absence of background optical signal) with ease of reaction control by applying electrode potential. As an analytical technique it presents outstanding advantages over other common analytical methods due to its versatility, simplified optical setup compared with photoluminescence (PL), and good temporal and spatial control compared with chemiluminescence (CL). Enhanced selectivity of ECL analysis is reached by variation of electrode potential thus controlling species that are oxidized/reduced at the electrode and take part in ECL reaction[5] (see electrochemical analysis). It generally uses Ruthenium complexes, especially [Ru (Bpy)3]2+ (which releases a photon at ~620 nm) regenerating with TPA (Tripropylamine) in liquid phase or liquidsolid interface. It can be used as monolayer immobilized on an electrode surface (made e.g. ofnafion, or special thin films made by LangmuirBlogett technique or self-assembly technique) or as a coreactant or more commonly as a tag and used in HPLC, Ru tagged antibody based immunoassays, Ru Tagged DNA probes for PCR etc., NADH or H2O2 generation based biosensors, oxalate and organic amine detection and many other applications and can be detected from picomolar sensitivity to dynamic range of more than six orders of magnitude. Photon detection is done with photomultiplier tubes (PMT) or silicon photodiode or gold coated fiber-optic sensors. ECL is heavily used commercially for many clinical lab applications. Analytical Chemiluminescence Electrochemiluminescence is chemiluminescence arising as a result of electrochemical reactions. It includes electrochemical initiation of ordinary chemiluminescent reactions, electrochemical modification of an analyte enabling it to take part in a chemiluminescent reaction, or electron transfer reactions between radicals or ions generated at electrodes. Prominent in the work done on electrochemiluminescence are reactions involving polyaromatic hydrocarbons or transition metal complexes, especially those of ruthenium, palladium, osmium and platinum. Applications have made use of the sensitivity, selectivity and wide working range of analytical chemiluminescence, but electrochemiluminescence offers additional advantages without adding

much to the inexpensive instrumentation[1]. Electrodes can be designed to achieve maximum detection of the light emitted and electrochemical measurements can be made simultaneously with the light output. Generation of chemiluminescence reagents at electrodes gives control over the course of light producing reactions, which can effectively be switched on and off by alteration of the applied potential; this is particularly useful when using unstable reagents or intermediates. Other possible benefits include generation of reagents from inactive precursors and regeneration of reagents, which permits the use of lower concentrations or immobilization of the reagents on the electrode. Analytes can also be regenerated, so that each analyte molecule can produce many photons, increasing sensitivity, or they can be modified to make them detectable by the chemiluminescence reaction in use. Electrochemiluminescence can be coupled with high performance liquid chromatography or with capillary electrophoresis. The usefulness of tris-(2, 2/-bipyridyl)ruthenium(II)) in electrochemiluminescence rests on its activity with very high efficiency at easily accessible potentials and ambient temperature in aqueous buffer solutions in the presence of dissolved oxygen and other impurities. The reaction sequence that leads to electrochemiluminescence is shown in equations Oxidation: [Ru(bipy)3]2+ e [Ru(bipy)3]3+ Reduction by analyte: [Ru(bipy)3]2+ + e [Ru(bipy)3]+ Electron transfer: [Ru(bipy)3]3+ + [Ru(bipy)3]+ [Ru(bipy)3]2+ + [Ru(bipy)3]2+* Chemiluminescence: [R(bipy)3]2+* [Ru(bipy)3]2+ + light Chemiluminescence: [R(bipy)3]2+* [Ru(bipy)3]2+ + light

Figure A flow injection manifold for measuring electrochemiluminescence. The oxidation occurs electrochemically at the anode, whereas the reduction is brought about chemically by the analyte in the free solution. Electron transfer and subsequent chemiluminescence also occur in the free solution close to the anode, where the [Ru(bipy) 3]3+ is

concentrated. Other analytes, e.g. alkylamines, are oxidized at the anode to form a highly reducing radical intermediate that reacts with [Ru(bipy)3]3+ to form [Ru(bipy)3]2+*, which emits light. Oxalates, on the other hand, are oxidized by [Ru(bipy)3]3+ to radicals that then reduce more [Ru(bipy)3]3+ to give [Ru(bipy)3]2+* and chemiluminescence. Instrumentation for electrochemiluminescence differs from that for other chemiluminescence only in having a flow cell provided with working, counter and reference electrodes, regulated by a potentiostat, which is in turn controlled by the computer that receives input from the photomultiplier or other transducer that receives the light signals. Figure D7.1 shows the usual flow injection manifold used for measuring electrochemiluminescence. The flow cell is in a light-tight box to exclude ambient light. A more portable alternative is a probe containing a set of electrodes and a fibre optic bundle to carry emitted light to a photomultiplier. Ambient light is excluded by means of baffles in the channels that admit the test solution. Because it can be electrochemically regenerated, it is useful to immobilize [Ru(bipy)3]3+ in a cation exchange resin deposited on the electrode to form a sensor that does not need a continual reagent supply.

4.

HISTOPATHOLOGY
Diagnostic Histopathology and Cytopathology

Introduction : The Diagnostic Histopathology and Cytopathology Section, which belongs to the Clinical Laboratory Division, deals with surgical, cytological and autopsy materials, and is responsible for histological and cytological diagnosis. These two sections maintain good communication which allows thorough morphological examination of the materials with a high level of diagnostic accuracy. A newly customized computerassisted pathology system was introduced in May, 1997. The quality, safety and permanence of pathological information has been effectively assured by this system. HISTOLOGY : Introduction : Histology is the study of the structure and function of microscopic anatomy of plants and animals. Microscopic anatomy includes the cells, tissues, organs and organ system of an organism. These studies are performed especially by examining cells and tissues.

First particular part of an organ is selected. Then staining is done followed by an examination under a light or electron microscope. Microscopes are the basic tools used in the histological studies. Histological stains are very important as they enhance the ability of visualizing and differentiating of microscopic structures. Histology is the study of cellular organization of body tissues and organs. The term is derived from the Greek histos meaning web or tissue, and refers to the science of tissues. Reference to Anatomical Structures As Tissues originated with the French surgeon Bichat, who compared the characteristic appearance of different parts of the body to the texture of cloth. The light microscope is the tool used most widely for clinical applications of histology. However, the advent of the electron microscope greatly extended the detail at which sub cellular structure can be studied. Thus, histology now embraces the study of the structures of both tissue and cells, and the relationship between these structures and physiological function. FIXATION: Many techniques have been developed which are designed to preserve the structural integrity of a specimen so that it can be viewed microscopically. The process through which cell structure is preserved is called fixation. since cells rapidly deteriorate after a tissue has been removed from the body; achieving adequate fixation is often the most difficult task confronting a histologist. Artifacts are chan ges to the original structure of cells and tissues that arise from tissue deterioration and from the fixation process itself. Thus, a skilled histologist employs techniques that minimize the formation of artifacts in different types of tissues, and has is the ability to distinguish artifacts from normal cell structures. FIXATION : 1. 2. 3. 4. 10 % Formalin Chloroform Ethanol Methanol

1. 10 % formalin preparation :

40 % Formaldehyde DH2O

: :

10 ml 90 ml

TISSUE GROSSING : Sample Receiving: 1. Receiving sample 2. Check Patient Name , Age/sex , which branch / hospital , bill no, Normal saline/formalin. Grossing: 1. After gross the specimen. 2. The taken bit draw in the diagram note. 3. According to tissue stage if need to be fix, fix the tissues with proper fixative in 10% formalin. 4. Remain the tissues to be process. Decalcification : 1. If bone/nail/any hard tissues are going to be process under the decalcification (5 % Nitric Acid ). 2. According to the stage of biopsy. 3. Calculate the enough time/day for the bone/nail/other tissues.

Decalcification Reagent ( 5 % Nitric Acid) : 1.

2. 3.

10 % formalin DH2O : Con Nitric acid

: 350 ml 150 ml : 5

ml

Grossing with the eye on the embedding mold The grossing sample for embedding should fit the processing cassette, as well as it should not exceed the thickness designated to the processing program. However, the main technical task is that the grossing person should envision the samples embedding to assure that the most informative part appears on the slide. Embedding orientation of biopsies and small specimens has generated a variety of techniques that is evidence that surgical pathology practitioners take orientation seriously ) There is no any universal orientation method. In the end of the day, however, the quality of embedding always depends on the performance at the embedding station. Technical details of manual embedding Lets explore the technical aspects of manual embedding under the angle of automation feasibility in practical surgical pathology. These details are simple but crucial for professional embedding. 1. With some hesitation over triviality of description, it is necessary to define the elementary simple technical embedding procedures: the sample is placed with the surface to be cut facing down at the bottom of the mold with dispensed paraffin according to the instruction, if any. 2. The artisan art includes the race against hardening wax and cooling instruments, although their warming and change is a part of the procedure. Embedding is not a simple task keeping in mind unevenness of the samples surfaces, and, of course, multiple fragments with sometimes different consistency. 3. Sometimes a gentle pressure over the specimen is enough to keep embedding flat, but often it is not enough. There are some gadgets which make the specimen flat. For example, now rarely used, aluminum tissue press, called tamper, can flatten large surfaces. However, in most cases, especially with multiple fragments, this task is carried out by forceps. 4. Anyway, the sample does not fall as a stone at the bottom of the mold, but is managed by the embedding person. Uneven embedding surface inevitably would require trimming and loss of precious diagnostic material or diagnostically informative part of the specimen can be far away from the microtome surface.

MANUAL PROCESSING : Tissue fixation Dehydration I 10 % Formalin 70 % Isopropyl alcohol 80 % Isopropyl alcohol 90 % Isopropyl alcohol 100 % Isopropyl alcohol Dehydration II Xylene Xylene Infiltration I II

Paraffin Wax I Paraffin Wax II Paraffin Wax III

Processing procedure : 1. 10 % formalin 2. 70 % iso prophyl alcohol : 3. 80 % iso prophyl alcohol : 4. 90 % iso prophyl alcohol : 5. 100 % iso prophyl alcohol : 6. Xylene I 7. Xylene II 8. Paraffin wax I 9. Paraffin wax II 10.Paraffin wax III : 1 HOUR

1 HOUR 1 HOUR 1 HOUR 1 HOUR : : : 1 HOUR 1 HOUR 1 HOUR

: 1 HOUR : 1 HOUR

MANUAL AUTOMATIC TISSUE PROCESSOR :

Microprocessor Based Automatic Tissue Processor

Automatic tissue processor is designed to provide a solution to the rapid processing of tissue in histology labs. Atmp17 removes all the water from a tissue sample and replaces it with paraffin wax with optimum speed to curtail damage to the tissue caused by dehydration and shrinkage. Technical aspect: 1. Otis sue processor consists of a highly accurate microprocessor based control. O provision for automatic adjustable delay start. 12 stage timing sequence programmed for duration ranging fro1 min to 9 hrs, 59 min, in steps of 1 min. o precise and accurate Bit controller with pt100 temperature sensor. 2. O the microprocessor based Bit controller maintains temperature between 50c to 65 c. O the main unit proceeds to next stage from 9th onward only after the set temperature is attained, o safety systems functioning at 10th, 11th and 12th stages. If the wax is melted, the tissue processor automatically proceeds from stage 9 to 10 and so on o if the wax is not melted . 3. The tissue processor indicates wax not melted / temperature insufficient. Pecial feature: empower to do most potentially hazardous procedures on just a click. tissues are processed simultaneously with one program. system is prudent and well guarded.

Manual automatic tissue processor : 1 Tissue fixation 2 Dehydration I 10 % Formalin 70 % Isopropyl alcohol 80 % Isopropyl alcohol Isopropyl alcohol I Isopropyl alcohol II Isopropyl alcohol III

3 Dehydration

II

Xylene I Xylene II Xylene III

4 Infiltration Procedure :

Paraffin Wax I Paraffin Wax II

1. 10 % Formalin 2. 10% Formalin 3. 70 % Isopropyl alcohol 4. 80 % Isopropyl alcohol 5. 6. 7. Isopropyl alcohol I Isopropyl alcohol II Isopropyl alcohol III

: : : : : : : : : :

45 mts 45 mts 45 mts 45 mts 45 mts 45 mts 45 mts 45 mts 45 mts 45 mts

8. Xylene I 9. Xylene II 10.Xylene III

11.Paraffin wax I 12.Paraffin wax II

: :

45 mts 45 mts

BLOCK
The paraffin method :

MAKING

All histological procedure can be divided into a similar series of steps . For the paraffin method these steps are as follows . 1. 2. 3. 4. 5. 6. 7. 8. Tissue resection Fixation Dehydration Infiltration and embedding in paraffin Sectioning with a microtome Mounting on microscope slides Clearing and staining Preparation of permanent mounts.

PROCEDURE FOR BLOCK MAKING 1. Open the cassette take the tissue, keep tissue near the L mode. 2. Keep the L mode in square 3. Heat the wax 4. Pour the wax in to the L mode 5. Keep the tissue in to wax 6. Press the tissue with the help of forceps 7. Leave for 8 to 10 minutes for air dry 8. Stick the number on the block 9. Heat the wood 10. Stick the wood 11. Keep the block in ice tray BLOCK STORAGE 1. After diagnose/report the block 2. Store it in number wise in suitable box 3. The blocks are stored for 5 years.

SECTION CUTTING : CUTTING MACHINE : 1. MANUAL ROTORY MICROTOME (WEXWOS) 2. FULLY AUTOMATIC MICROTOME (MEDIMEAS). 1. MANUAL ROTARY MICROTOME (WESWOS) Microtome use steel, glass, or diamond blades depending upon the specimen being sliced and the desired thickness of the sections being cut. Steel blades are used to prepare sections of animal or plant tissues for light microscopy histology. Glass knives are used to slice sections for light microscopy and to slice very thin sections for electron microscopy. Industrial grade diamond knives are used to slice hard materials such as bone, teeth and plant matter for both light microscopy and for electron microscopy. Gem quality diamond knives are used for slicing thin sections for electron microscopy. Microtome is a method for the preparation of thin sections for materials such as bones, minerals and teeth, and an alternative to electro polishing and ion milling. Microtome sections can be made thin enough to section a human hair across its breadth, with section thickness between 50 nm and 100 m. 1. Microtome base plate or stage: A platform which has rails that secure the knife holder base. 2. Knife holder base: A part that anchors the knife holder to the microtome stage. The knife holder base can be moved toward or away from the block, but MUST be stationary and locked during microtome. 3. Knife holder: This part is comprised of several components including the blade clamp that holds the blade, the knife tilt for adjusting the knife angle, and the face

plate that guides that ribbons away from the blade and towards the operator. 4. Cassette clamp or block holder: Holds the paraffin block in place. Typically, the block moves up and down with each revolution while the blade is stationary. The block holder may have knobs that allow the user to manipulate the block face in various directions to bring the tissue in alignment with the blade. 5. Coarse hand wheel: Moves the block holder either toward the knife or away from the knife. 6. Advancement hand wheel: Turns in one direction and advances the block toward the knife at the specified microns. Most hand wheels are equipped with a safety lock to prevent the wheel from releasing and having the block holder come down towards the blade while a block is inserted or removed. The safety lock should be used anytime the microtomist is not actively sectioning paraffin blocks. 7. Micron adjustment: Micron settings for section thickness can range from 1 to 60 microns on most microtome. Procedure to cut sections on a manual rotary Microtome: 1. Move back the coarse feeding mechanism and lock the hand wheel. 2. Clamp cassette in position, orientating the tissue as desired. 3. Ensuring that the knife holder is a safe. Distance from the cassette, carefully place a Microtome : 1. Blade into the holder and tighten securely. Flip the finger guard on top of the blade. Loosen back lever of knife holder . Advance towards the cassette until there is a Microtome:

1. 0.5mm (approx.) interval between Tighten back the lever of knife holder. 2. Flip over the finger guard to expose . Blade :

blade

edge

and

cassette

surface

1. Unlock wheel and trim away surplus wax from tissue surface by advancing the coarse feed mechanism before commencing each turn of the wheel. 2. Trim all tissue carefully until the desired. Surface is exposed : 1. Reduce section thickness Cut tissue sections . MICROTOME KNIFE I recently purchased a rotary microtome on ebay which included a knife but did not include the handle or the bevel/holder normally used to sharpen a microtome knife. This knife was in fair condition so it required honing as well as stropping prior to use. I used decide how to go about sharpening the knife. Since I didn't have the standard holder I constructed my own; the result, which is not at all like the standard holder, is shown below (with strops and hone). The goal of this holder design is to minimize the operator skill needed to sharpen a microtome knife, necessary since I had never seen a microtome knife prior to sharpening this one. Overview The upper block is a guide for the upper end of the hone or strop which fixes the angle between tool and knife. The lower projection (the lowermost layer) is meant to prevent the tool from being rolled over the edge of the knife inadvertently. Note that the edge of the knife is back slightly from the edge of the supporting block this to reduce the danger of contact with the knife edge while it is in the holder. The knife is held in position by a magnet and is prevented from moving sideways by small vertical pins. to 3-5 microns using adjustment knob

In use, the upper end of the hone or strop is kept in contact with the upper block while the abrasive section of the tool is in contact with the knife, thus ensuring that the angle built into the holder is transferred to the knife. CAUTION: Handling or unconsidered movement near any microtome knife is hazardous. The holder described here is NOT SAFE to use and cannot be made safe. Build and use at your own risk. My technique when moving the microtome knife about is to grip it with two hands, one on each end. This keeps both hands occupied and away from the cutting edge while also minimizing the chance of dropping the knife (which could easily sever a toe). I found that I could shave hair from the back of my hand with the knife after honing with 600 grit and the knife got much sharper thereafter! LEATHER STRAP A sharp edge can be restored quickly to a knife with judicious manual stropping on leather straps. Stropping polishes the razor edge and removes fine metal burrs retained along the edge. Made of best quality leather mounted on wooden frame, for final stropping of Microtomes knives. FULLY AUTOMATIC MICROTOME ( MEDIMEAS ) Semi Automatic Microtome Newly Developed Semi Automatic Microtome with motorized feed drive for uniform and flat sections. Fully Automatic Microtome Medimeas Fully Automatic Microtome with motorized feed and sectioning provides

uniform and flat sect ions.

Model

: MRM AT full automatic microtome

Storage location Storage temperature range Humidity Nominal supply voltages : Nominal frequency Power draw Power fuses MICROTOME Section thickness setting : 0 100 m 0 m 0 to 10 m in 1 m increments : : : +5 C to 40 C < 80 %

100/120/230/240 V AC 10 % 50/60 Hz : : < 200 VA 2 x F1A

10 to 20 m in 2 m increments ` Trimming section thickness setting 20 to 100 m in 5 m increments : 0 100 m

0 to 10 m in 1 m increments 10 to 20 m in 2 m increments 20 to 100 m in 5 m increments Object feed Vertical stroke Sectioning modes : 26 mm

: 52 mm / 2.05 inches : 6

Maximum specimen size ( LxW) : Maximum specimen area w/o retraction

50x50 mm

65 mm (with out specimen orientation) 60 mm 6.5 C

Maximum specimen area with retraction : Specimen orientation Horizontal Vertical Repositioning of knife holder base North - south Specimen retraction Electric coarse feed Sectioning speed Return speed Dimensions Basic instrument Width ( with hand wheel ) Width ( without hnad wheel) Depth ( with waste tray) Depth ( with out waste tray) + connecting socket +40 mm : : : 20 m : :

6.5 C

32.5 mm

: 450 m/s 10 % : : 68 150 mm /s 10 % 450 m/s 10 %

470 mm : 35 mm : 610 mm

: 530 mm

Working height ( Knife edge ): 99 mm / 3.9 inches ( measured from the base plate) Working height (Knife edge) : 165 mm/ 6.5 inches ( measured from the table) Weight ( without accessories) : app . 30 kg

FULLY AUTOMATIC MICROTOME BLADE : Low Profile Disposable Microtome Blades by most economical as compared to any other imported blade worldwide.

Excellent reproducible Sections Toughened to cut Hard tissues with ease. 35'angle,80mm length 50 Blades per packet

Range of graduations for cutting of specimen: 1-40um 1-10m in increments of 1m 10-30m in increments of 2m 30-40m in increments of 5m

Distance of vertical movement : 60mm Angle scale for fitting knife older: 0-20' Universal clamp of specimen : 45mm Quick release clamp (Option) : Cassette Type TISSUE FLOATATION BATH : Tissue floatation bath to remove wax

Tissue Floatation Water Bath 1. Our glass Tissue Floatation Water Bath has been developed using microprocessor controls and a modular design that has improved reliability and precision.

2. The unit is low profile with a removable rectangular glass basin for easy cleaning. The glass basin is illuminated by LED light from the side allowing clear observation without glare against the black background.

3. There is an ON/OFF switch on the control panel for the LED light. Temperature settings are controlled by push buttons for accurate and precise control and the easy to read LED displays show both set temperature and actual temperature.

4. The corrosion resistant, stainless steel temperature probe flips down into place. The probe has a thermostat sensor inside that turns off when it is lifted up and re-starts when placed back into the basin. STAINING : Preparation stains : 1. 2. Heamotoxyline 1 % Eosin

3.

1 % A cid Alcohol : Aluminium potassium sulphate Mercuric oxaide Iso prophyl alcohol DH2O Heamotoxyline powder

Heamotoxyline 1. 2. 3. 4. 5. 1 % Eosin :

gms

1. 1 gm 2. 100 ml 1 % Acid Alcohol:

: :

Eosin Powder DH2O

1. Iso prophyl alcohol 2. DH2O 3. Con HCL

: : :

70 ml 29 ml 1 ml

HAEMOTOXYLINE AND EOSIN STAIN : Staining reagents (histology) : 5 gms% Haemotoxyline Powder 1 % Esoin 1 % acid alcohol Xylene Iso prophyl alcohol

H&E Staining Procedure :

1. After Section cutting

2. Incubate the slide 3. Xylene I 4. Xylene II 5. Xylene +Alcohol 6. Alcohol I 7. Alcohol II 8. Wash in tap water 9. Haemotoxyline 10. Wash in tap water 11. 1 % acid alcohol 12. Wash in tap water 13. Bluing in tap water 14. 1 % Eosin 15. Wash in tap water 16. Drying 17. mounting
CYTOLOGY

20 mts 10 dips 10 dips 10 dips

10 dips 10 dips

03 mts

Few seconds

10 mts

- 50 secs

Introduction : Study of structure, function and chemistry of cells is known as cytology. It is a branch of biology which deals directly with the structural and functional

organization of cells and also with the other phenomena such as metabolism, ontogenetic differentiation, heredity, and phyloge Cytology: The medical and scientific study of cells. Cytology refers to a branch of pathology, the medical specialty that deals with making diagnoses of diseases and conditions through the examination of tissue samples from the body. Cytologic examinations may be performed on body fluids (examples are blood, urine, and cerebrospinal fluid) or on material that is aspirated (drawn out via suction into a syringe) from the body. Cytology also can involve examinations of preparations that are scraped or washed (irrigated with a sterile solution) from specific areas of the body. For example, a common example of diagnostic cytology is the evaluation of cervical smears (referred to as the Papanicolaou test or Pap smear). Cytology is that branch of life science, which deals with the study of cells in terms of structure, function and chemistry. Based on usage it can refer to:

Cytopathology - the study of cellular disease and the use of cellular changes for the diagnosis of disease. Cell biology - the study of (normal) cellular anatomy, function and chemistry.

Fine needle aspiration biopsy Fine needle aspiration (FNA) is sometimes considered a cytology test and is sometimes considered a biopsy. Its discussed in the section called Overview of biopsy types. Body fluids Fluids from cavities and spaces in the body can be tested to see if cancer cells are present. Some of the body cavity fluids tested in this way include:

Urine Sputum (phlegm) Spinal fluid, also known as cerebrospinal fluid or CSF (from the space surrounding the brain and spinal cord)

Pleural fluid (from the space around the lungs) Pericardial fluid (from the sac that surrounds the heart) Ascitic fluid.

Vaginal Cytology: Introduction and Index

The vaginal epithelium is responsive to sex steroids, particularly estrogen, and undergoes predictable changes through the cycle in response to changes in blood concentrations of ovarian hormones. Rising levels of estrogen cause the vaginal epithelium to become "cornified" - the surface cells become large and flattened, with small or absent nuclei. In essence, vaginal cytology is a type of endocrine assay. Tracking changes in the morphology of desquamated vaginal epithelial cells provides a convenient means of assaying changes in estrogen levels. The technique is used widely in managing canine breeding programs, which is the focus of this section, but also has utility in evaluating reproductive function in several other species, including cats (if you don't mind losing a little blood) and rats. Core information on canine vaginal cytology is presented in the following topics:

Techniques for preparing a vaginal smear Classification of vaginal epithelial cells Cytologic changes through the canine estrous cycle Practice sets and self evaluations

Pap stain :

1.After fixative slides 2.wash in tap water 3. haemotoxyline - 3 mts 4. wash in tap water 5. OG 6 6. Alcohol I 7. Alcohol II 8. EA 50 9. Alcohol III 10. Alcohol IV - 5 mts - 5 mts - 5 mts - 5 mts - 5 mts - 5 mts

11. Drying / Mounting