TPPA (TREPONEMA PALLIDUM PARTICLE AGGLUTINATION) Principle :

The TPPA test is based on the agglutination of colored gelatin particle carriers sensitized with T.pallidum antigen Sample collection : Human serum is the specimen of choice for this procedure. However EDTA, Sodium Citrate or Heparin plasma may be sued when serum cannot be obtained. Procedure :

1. Place 4 drops (100 l) of sample diluent in well 1, and 1 drop (25 l) in well 2

through 4 using calibrated dropper.

2. Add 25 l of specimen to well 1 using micropipette. 3. Fill the micropipette or a diluter with 25 l of the diluted solution well 1 and mix

well. Transfer 25 l of the mixture of the specimen and sample diluted into well 2. Then mix well and repeat this procedure again with well 2, 3, and 4 to obtain serial doubling dilutions. Discard 25 l of the mixture from the well 4.
4. Place 1 drop (25 l) of unsensitized particles in well 3, 1 drop (25 l) of

sensitized particles in well 4 using droppers supplied in the kit,

5. Mix the contents of wells thoroughly for approximately 30 seconds. Do not use

rotator. Then cover the plate and let it stand at room temperature for example at 15-30oC for 2 hours by reading. The incubation can be extended to overnight without any perceptible difference in patterns.

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Interpretation

: the

Non-reactive - Particles concentrated in the shape of a button in the center of well with a smooth round outer margin (-).

Indetermine - Particles concentrated in the shape of a compact ring with a smooth round outer margin (). Reactive - Definite large ring with a rough multiform outer margin and peripheral

agglutination (+), or, agglutinated particles spread out covering the bottom of the well uniformly (++).

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RPR (Rapid Plasma Reagin) Principles :

RPR Card antigen suspension is a carbon particle cardiolipin antigen which detects reagin, an antibody-like substance present in serum or plasma from syphilitic person and occasionally in serum or plasma of person with other acute or chronic conditions. Procedure :

1. Holding dispenstir in a vertical position directly over the card test area to

which the specimen is to be delivered, squeeze dispenstir device allowing 1 drop of serum to fall onto card. 2. Invert dispenstir device and with stirring end, spread the specimen filling entire surface of circle.
3. Gently shake antigen dispensing bottle before use. Place one free falling

drop onto each test area. Do not re-stir; mixing of antigen and specimen is accomplished during rotation. 4. Rotate for 8 minute, on mechanical rotators at 100 2rpm.
5. Following the rotary and tilting of the card by hand must be made.

Immediately read macroscopically in the wet state under a high intensity incandescent lamp.

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Quantitative card test: 1. For each specimen to be tested, dispense 100L of 0.9% saline into test tubes numbered 2 to 5 using a serological pipette. 2. Using micropipette, place 100L specimen into tube 1. 3. Refill pipette with test specimen and holding in a vertical position, prepare a serial dilutions by drawing mixture up and down pipette 5 to 6 times. Avoid formation of bubbles. Transfer 100 from tube 2, to 3, to 4, to 5. Discard 100L after mixing content in tube 5.
4. Using a new dispenstir for each specimen, start at highest dilution of serum

(tube 5) and draw up specimen and by holding in a vertical position, drop one drop of specimen onto the card test area, invert the dispenstir and spread the specimen filling the entire surface of circle using stirrer. Precede to tubes 4, 3, 2 and 1 and accomplish similar spreading. 5. Gently shake antigen dispensing bottle before use. Place one free falling drops onto each test area. Do not stir. Mixing of antigen and specimen is accomplished during rotation. Rotate for 8 minute (30 second) on mechanical rotator at 1002 rpm. 6. Following the rotation, to help differentiate Non-reactive from minimally reactive results, a brief rotating and tilting of the card by hand (3 to 4 to-andfro motions) must be made. Immediately read macroscopically in the wet state under a high intensity incandescent lamp. Interpretation REACTIVE NON-REACTIVE : - Showing characteristic clumping raging from slight but definite to marked and intense. - Showing no clumping.

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HIV SCREENING Principle :

The ARCHITECT HIV Ag/Ab Combo assay is a two-step immunoassay to determine the presence of HIV p24 antigen and antibodies to HIV-1 and HIV-2 in human serum and plasma using CMIA technology with flexible assay protocols. Sample collection: Human serum(including serum collected in serum separator tubes) Plasma collected in: o Potassium EDTA o Sodium heparin o Lithium heparin o Plasma separator tubes o Sodium citrate o Potassium oxalate Procedure procedures Load samples Run samples o Moves the sample carier to the aspiration point. o Loads a reaction vessel (RV) into the process path. o Aspirates and transfer sample into the RV o Advances the RV one position and transfers assay diluents and microparticles into the RV. o Mixes, incubates, and washes the reaction mixture. o Adds conjugates to the RV
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: Order test

For information on ordering patient specimens, controls and general operating

For information on loading samples

o Mixes, incubates, and washes the reaction mixture. o Adds pre-trigger and trigger solutions. o Measures chemiluminescent emission to detect the presence of HIV p24 antigen and/or HIV-1/HIV-2 antibodies in the sample. o Aspirates contents of RV to liquid waste and unloads RV to solid waste. o Calculates the result. Results :

The ARCHITECT immunoassay System calculates the cutoff (CO) using the mean chemiluminescent signal (RLU) from three replicates of the calibrator 1 and stores the result. Calculation :

The ARCHITECT HIV Ag/Ab Combo Assay calculates results based on a cutoff determined by the following calculation: Cutoff (CO) S/CO = calibrator 1 mean RLU Value x 0.40 = sample RLU / Cutoff RLU

The cutoff RLU is stored for each reagent lot calibration The ARCHITECT immunoassay System calculates a result based on the ratio of the sample RLU(s) to the cutoff RLU for each specimen and control.

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Results

: Specimen with S/CO values < 1.00 are considered nonreactive (NR) Specimen with S/CO values > 1.00 are considered reactive (R)

Note= all specimens that are initially reactive must be centrifuged and retested in duplicate. (Transferred to a sample cup or secondary tube) ARCHITECT HIV Ag/Ab Combo Results Initial results (S/CO) R R Retest result Both test are NR One or both test are NR reactive No retest required NR Final result NR R Interpretation HIV p24 Ag and/or HIV-1/HIV-2 Ab not detected Presumptive evidence of HIV p24 Ag and/or HIV-1/HIV-2 Ab; perform a supplemental assay HIV p24 Ag and / or HIV-1/HIV-2 Ab not detected

MICROTITER PARTICLES AGGLUTINATION TEST KIT

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Principle

The test is based on the use of highly stable coloured polymeric microparticles (3 um in diameter) and recombinant HIV-1 and HIV-2 antigens with high antigenic strength. The particles sensitized with HIV-1 and/or HIV-2 antigens are used in particle agglutination (PA) test. Procedure :

1. Place 3 drops (75 ul) of serum diluent to well 1 and 1 drop (25 ul) to wells 2, 3 and 4 using a micropipette. 2. Add 25 ul of specimen into well 1 using a micropipette, and mix by filling and discharging the micropipette repeatedly for 5 times with fluid in well 1.
3. Then fill the micropipette with 25ul of diluted sample in the well 1 and transfer

it into well 2. Mix as before and transfer 25ul from well 2 in to well 3. Mix again and transfer 25ul from well 3 in to well 4, mix and discard the last 25ul from well 4. Avoid forming bubbles during this dilution process. 4. Add 1 drop (25ul) of Unsensitized Particles to well 2, 1 drop (25 l) of HIV-1 Sensitized Particles in well, 3 and 1 drop (25 l) of HIV-2 Sensitized Particles in well 4 using droppers supplied in the kit (red dropper for USPs, gray droppers for SPs).
5. Mix the content of the wells by rotating the plate with hand on a flat surface or

using a rotary mixer. 6. Cover the plate, place it on a level surface and allow to stand at room temperature for 2 hours. Read and interpret the patterns.

Results

:
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Compare the agglutination patterns with those of the reagent control and interpret according to the criteria shown in the table below: Settling pattern of Particles Particles concentrated in the shape of a button In the center of the well with a smooth round outer margin Particles concentrated in the shape of a compact ring with a smooth round outer margin. Definite larger ring with a rough multiform outer margin and peripheral agglutination Agglutinated particles spread out covering the bottom of the well uniformly + ++ Positive (Reactive) + Inconclusive Reading Interpretation Negative

DETERMINE TM HIV-1/2

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Principle

Determine HIV-1/2 is an immunochromatographic test for the qualitative detection of antibodies to HIV-1 and HIV-2. Materials/ reagent : Determine HIV-1/2 test card precision pipette Lancet Alcohol swab Gauze pad Chase buffer

Specimen collection: Serum/ plasma/ and whole blood collection Method I. II. : Remove the protective foil cover from each test. For serum or plasma samples: a) Apply 50l of sample (precision pipette) to the sample pad (marked by the arrow symbol). b) Wait a minimum of 15 minutes (up to 60 minutes) and read the result III. For whole blood (venipunture) samples: a) Apply 50l of sample (precision pipette) to the sample pad (marked by the arrow symbol). b) Wait one minute, then apply one drop of chase buffer to the sample pad. c) Wait a minimum of 15 minutes (up to 60 minutes) and read the result. IV. For whole blood (fingerstick) samples: a) Apply 50l of sample (by EDTA capillary tube) to the sample pad (marked by the arrow symbol).
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b) Wait until blood is absorbed into the sample pad, then apply one drop of chase buffer to the sample pad. c) Wait a minimum of 15 minutes (up to 60 minutes) and read the result. Interpretation of results Positive (Two Bars) Red bars appear in both the control window (labeled control), and the patient window (labeled patient) of the strip. Any visible red color in the patient window should be interpreted as positive. Negative (One Bars) One red appears in the control window of the strip, (labeled control), and no red bar appears in the patient window of the strip (labeled patient). Invalid (No Bar) If there is no red bar in the control window of the strip, and even if a red bar appears in the patient window of the strip, the result is invalid and should be repeated. NOTES: The test result is positive even if the patient bar appears lighter or darker than the control bar.

ANA ANTI NUCLEIC ANTIBODY

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Principle

This test system is to use as an aid in the detection of antibodies associated with systemic rheumatic disease. Antinuclear antibody (ANA) is a general term used to describe autoantibodies against various cell nuclear proteins. Reagent :

1. Reactive reagents a) Substrate slides b) Homogenous +ve control c) Speckled +ve control d) Nucleolar +ve control e) Centromere +ve control f) SSA (Ro) +ve control g) Titratable control serum h) ve control serum i) fluorescent antibody reagent
2. Non-reactive reagents

a) PBS buffer powder b) Semi-permanent, Mounting medium, Coverslip Procedure :

1. Centrifuge the blood sample received and separate the serum. 2. Transfer the serum into plastic tube and cover the tube with parafilm.
3. Incubate 370C for 30 min to activate the complement in the serum.

4. Dilute the serum with PBS, 1: 40. 5. Add 25l into the well of the substrate slide. 6. Incubate in the moist chamber at room temperature for 30 min. 7. Wash with PBS for 10 min. 8. Add conjugate and incubate at room temperature for 30 min. 9. Wash with PBS for 10 min. 10. Mount and observe under immunofluorescent microscope. Results :
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POSITIVE RESULT = a serum is considered positive if the nucleus shows a clearly discernible pattern of staining in a majority of the interphase cells. 4+ = Brilliant yellow-green (maximal fluorescence), clear-cut cell outline, sharply defined cell center 3+ = Less brilliant yellow-green fluorescence, clear-cut cell outline, sharply defined cell center 2+ = Definite cell pattern but dim fluorescence, cell outline less well defined. 1+ = Very subdued fluorescence, cell outline almost indistinguishable from cell center in most instances.

CHLAMYDIA IF (DIRECT SPECIMEN TEST)

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Principle

The Chlamydia IF Test is a rapid in vitro direct immunofluorescence test for the detection and diagnosis of Chlamydia trachomatis organisms in patient specimens. The fluorescent-labelled mouse monoclonal antibody reagent binds specifically to Chlamydia trachomatis display bright green fluorescence with typical morphology. Specimen Urethral swab Cervical swab Rectal swab Conjunctival swab Nasopharyngeal swab Serum :

Procedure

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1. Apply 25ul of positive control as battles to the appropriate slide well. Do not dilute or pretreat. 2. Apply 25ul of negative control as battles to the appropriate slide well. Do not dilute or pretreat.
3. For each patient sample to be tested, add approximately 25ul of the diluted/

pretreated sample to an appropriate slide well. Make rotations for later identification of each well when reading the results. 4. Incubate the slides in a humid chamber for 90 minutes at 35C to 37C.
5. Remove the slides - gently rinse each slide with a stream of PBS rinse one row at

a time to avoid mixing of specimens. Wash slides by submersing the rinsed slides into a coplin or slide staining jar containing PBS for about 10 minutes. 6. Dip the washed slides briefly in distilled or parified water and allow the slides to air dry. 7. Add approximately 25ul (or one drop from dropper tip) IgM conjugate to each slide well. 8. Incubate slides in a humid chamber for 30 minutes at 35C to 37C 9. Repeat step 6 and 7. 10. Place a few drops of mounting medium on the slide and cover with a 24x50mm coverslip. 11. View well at a final magnification of objective x400 on a properly equipped fluorescent microscope for optimum fluorescent, read slides the same day the assay is performed. If this is not possible store in dark place at 2 to 8C up to 24 hours. Results

: : apple green dots under the IF green dots

Positive

Negative : only the background is visible (orange) with the absence of apple

DENGUE IGM CAPTURE ELISA

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Principle

The principle of this test is is for the qualitative detection of IgM antibodies to dengue antigen in serum, as an aid in the clinical laboratory diagnosis of patients with clinical symptom consistent with dengue fever. Serum IgM antibodies can be detected from dengue patients as early as 3 to 5 days after the onset of fever and generally persist from 30-90 days, although detectable levels may be present 8 months post-infection. Materials :

Anti-human IgM coated microwells (assay plate) Dengue 1-4 antigens Wash buffer concentrate Serum diluent and antigen diluent HRP conjugated monoclonal antibody tracer Tetramethylbenzidine TMB IgM positive control serum IgM calibrator serum IgM negative control Stop solution :

Procedure Serum predilution

1. Remove the required number of microwells from the foil sachet and insert into strip holder. Five microwells are required for negative control (N), positive control (P) and calibrator in triplicate. 2. Using suitable test tube or microtitre plate, dilute the positive control, negative control, calibrator and patients sample: to l serum add 1000 l serum diluent. Mix well.

ELISA 1. Add 10 l antigen to 2.5ml antigen diluent. Mix well.

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2. Remove required volume of diluted antigen. 3. Add equal volume of MAB tracer to diluted antigen. 4. Immediately add 100 l of diluted serum into wells containing anti-human IgM (PLATE) 5. Incubate PLATE for 60 minutes at 37C. 6. Wash PLATE for 6 times. 7. Transfer 100 l of antigen-MAB tracer mixture to PLATE. 8. Incubate PLATE for 60 minutes at 37C. 9. Wash PLATE for 6 times. 10. Add 100 l TMB per well for 10 minutes at 20-25C.
11. Add 100 l per well of stop solution. Read absorbance value at 450nm, reference

630nm Interpretation :

The dengue IgM capture ELISA determines the level of IgM antibodies to dengue in a patients serum. A positive result (>1.1 index) is indicative of either an active primary or secondary dengue infection. Negative (Index value <0.9): No detectable IgM antibody. The result does not rule out dengue infection Equivocal (Index value 0.9-1.1): Equivocal samples should be repeated Positive (Index value > 1.1): Presence of detectable IgM antibody. Other dengue serology assays should be performed to confirm dengue infection

MYCOPLASMA TITRE

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Principle

An invitro diagnostic test for detection antibody to Mycoplasma pneumoniae which is manufactured using artificial gelatin particles sensitized with cell membrane components of M.pneumoniae. Sensitized particles are agglutinated by the presence of antibodies to M.pneumoniae in human serum. Materials Micropipette Microplate Sample diluent Sensitized and unsensitized particles : :

Procedure

1. Add 25 L of sample diluent (4 drops in 1st well and 1 drop in well 2-12) 2. Add 25 L of sample in 1st well and mix well.then, transfer the diluted sample

from 1st well into 2nd well and follow till 12th well. 3. Add control positive while negative (diluent only).
4. Add unsensitized particles into 2nd well and sensitized particles into 3rd till 12th

well. 5. Incubate for 2 hours. Cover microplate and avoid vibration. Results:

Positive Negative

Ring form or carpet form Button form

ASOT Anti Streptolysin O titre

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Principle

To detect Streptolysin O where its used agglutination method Procedure :

1. Add 1 drop of Avitex ASO on a black jubin. 2. Then add 1 drop of sample and mix. 3. Add positive control and negative control. 4. Mix at 75 rpm for 3min. 5. Read the results visually under good light. Results

: Agglutination can be seen.

Positive

Negative No agglutination

Notes: If agglutination seen, we have to do dilution Dilution method: 1. Add 50 L normal saline and 50 L sample and mix well from behind well. 2. Add 1 drop of Avitex ASO and mix at 75 rpm for 3min. 3. Record the results in which ratio, we can see the ring form/carpet form.

Hepatitis B, C and HIV Screening

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Principle

The ARCHITECT machine system uses chemiluminescent microparticle immunoassay principle for the quantitative determination of antibody to hepatitis B, C and HIV Materials :

Paramagnetic micro particles coated with a capture molecules (antigen, antibody or viral particle) specific for the analyte being measured. Acridinium labeled conjugate. Pre-trigger solution and trigger solution :

Procedure

1. Prepare analyzer, reagent and diluent. 2. Perform maintenance daily, weekly and monthly maintenance. 3. Calibrate analyzer and run QC. 4. Check calibration data and QC data in Levey Jenning Chart in analyzer computer and if NOT OK troubleshoot and repeat step 3. 5. Put specimen rack into specimen tray and load to analyzer sample loader (RSD). 6. Highlight 3 module (RSD, 1&2) and touch RUN to run the specimen and release report 7. Specimen rack automatically park at the same place where it was loaded after the analysis. 8. Discard specimen after 2 days the result have been dispatched.

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