UNIT I.

BLOOD COLLECTION
DEFINITION OF TERMS: ANTECUBITAL FOSSA most common site for venous blood collection; located where the elbow ends. ARTERIAL BLOOD GAS (ABG) procedure that involves taking blood from an artery; generally from the radial artery site located near the thumb. ARTERY blood vessel that carries richly oxygenated blood from the heart to the body tissues. CAPILLARIES tiny blood vessels that connect the smallest arteries to the smallest veins. GAUGE measure of the diameter of a needle; larger numbers indicate smaller sizes. HEMATOMA loss of blood from a vessel (capillary, vein, or artery) that results in a lump and discoloration forming near the blood collection site. HEMOCONCENTRATION increase in the concentration of formed elements in the blood caused by lack of fluid in the blood; often caused by a tourniquet too tightly applied to the arm or having the tourniquet left on the arm too long. HEMOLYSIS destruction or lysis of red blood cells; sometimes caused by improper blood drawing technique. LUMEN cavity or channel within a tubular organ, such as blood vessel. PHLEBOTOMY act of entering a vein with a needle for the purpose of obtaining venous blood. THROMBOSIS blood clot in a vessel. TOURNIQUET device often a rubber band at least inch wide, that reduces flow of blood in the veins and allows the veins to become more prominent. VACUUM TUBE SYSTEM system used for blood collection (also called vacutainer system); uses a special disposable needle, a holder, and blood collection tubes that have little or no air. VEIN blood vessel that carries unoxygenated blood from the tissues back to the heart. VENIPUNCTURE procedure where a needle is inserted into a vein to obtain a venous blood sample. ASAP (as soon as possible) physicians order that is request for a clinical specimen to be collected as soon as possible; with not as rapid a turnaround time compared with a STAT order. This tells the staff that the test is not an emergency but the physician is eager to get the results. CENTRIFUGATION high speed separation of a solid material from a liquid using a centrifuge; integral part of specimen preparation for testing. FASTING clinical specimen obtained after a patient has not taken anything by mouth except water for a specified time. POSTPRANDIAL specimen obtained at a specified time after a meal, e.g. one hour postprandial RANDOM SPECIMEN clinical specimen obtained at no specific time. SPECIMEN PROCESSING area in some clinical laboratories where specimens are logged and processed before dispersal to various clinical laboratory departments. STAT order order for a clinical specimen to be collected and a test performed immediately; considerd an emergency. TIMED SPECIMEN specimen obtained at a time specified by the patients physician. The amount of samples will depend on the method to be used. AMOUNT OF SAMPLES NEEDED: 1. Macromethod 1 ml and above 2. Micromethod 0.1 0.9 ml 3. Ultramicromethod 0.01 0.09 ml 4. Nanoliter method 0.001 0.009 ml

METHODS OF BLOOD COLLECTION: The three general procedures/methods for obtaining blood are: 1. SKIN PUNCTURE/PRICK METHOD/CAPILLARY PUNCTURE for micromethod, ultramicromethod, and nanoliter method. Sample collected is a mixture of blood coming from arterioles, venules and capillaries, and correctly called skin punctured blood. It may contain tissue fluids. Site of Collection: a) Infants - plantar heel surface b) Children - plantar surface of the big toe - palmar surface of the last digit of the second, third or fourth finger may be used in older infants - lateral side of the finger adjacent to the nail - earlobe recommended site for arterial blood ADVANTAGES OF USING A CAPILLARY PUNCTURE TO OBTAIN BLOOD FOR TESTING INCLUDE: - Capillary sites are accessible and patients veins are exhausted from repeated punctures. - Some patients prefer the capillary puncture to other options. - Only small amount of blood is needed, and no blood is wasted. DISADVANTAGES OF USING A CAPILLARY PUNCTURE TO OBTAIN BLOOD FOR TESTING INCLUDE: - Tissue fluid, escaping into the sample during squeezing necessary to obtain the specimen, can mix with the blood flow, resulting in a dilution effect. - Squeezing to obtain the capillary specimen can cause hemolysis of the blood (rupture of red blood cells that releases their contents into the liquid portion of the blood than can affect test results). - Capillary punctures can be painful, especially to the fingertips. - Capillary punctures do not provide enough blood when more than a few tests are ordered. Capillary puntures, usually performed on the middle or ring finger of an adult, might be indicated on adults in the following situations: - Absence of palpable veins - Severe burns - Obese patients with thrombotic tendencies - When only microspecimens are needed - Often preferred to geriatric patients due to thinness and less elasticity of the skin. 2. VENIPUNCTURE (Phlebotomy) for macromethod This is considered to be the most commonly used method of blood collection in Clinical Chemistry and immunologic studies. Samples obtained iis called venous blood. Venous blood is deoxygenated blood that Contains substances that come from the metabolic activities of different organs. Sites of Collection: 1. veins in the antecubital fossa (three main veins in this area are the cephalic, median cephalic, and median basilic) cephalic rolls and bruises less easily, but the blood flows more easily, it is also tougher to puncture. basilic this vein tends to roll more easily.

median cephalic used most commonly because it is well anchored, has good blood flow and bruises less easily. 2. femoral vein 3. wrist vein 4. veins on the dorsal portion of the hands GENERAL METHODS OF DOING VENIPUNCTURE 1. Syringe Method used for blood collection only 2. Vacutainer Method used for collection and storage - for single or multiple draws ADVANTAGES OF VACUTAINERS: 1. A prepackaged sterile unit that requires no prior preparation. 2. A wide range of tube size and contained anticoagulant (2, 3, 5, 7, 10 and 15 ml) 3. The avoidance of syringe breakage. 4. The system makes possible direct sampling from a vein, economically and efficiently. 5. Disposable needles eliminate the hazard of serum hepatitis transmission, provided the phlebotomist uses Proper drawing and needle disposal technique. 6. Rubber stoppers are color-coded to distinguish whether the tube contains a specific anticoagulant, is a plain Tube, or is a tube made chemically clean (i.e. no lead, iodine, iron) 7. The self-sealing stopper prevents problems due to evaporation and contamination. The vacutainer blood collection system: 1. A detectable and disposable needle Double pointed needle one end of which is capped and enters the patients vein. The other end punctures the vacuum blood collection tubes. 2. A reusable tube holder. 3. A disposable, color-coded, collecting tube (with or without anticoagulant) COMMONLY USED EVACUATED TUBES Stopper Color Red Additive None Action Allows blood clotting resulting in serum Use Most chemistry, blood bank, and immunology assays Most chemistry tests

Red/Gray Red/Black Lavander

Contains material

separator

Orange

EDTA (versene, sequestrene) (Na2 or K2) Thrombin

Allows blood clotting resulting in serum; material serves as a barrier between serum and cells Anticoagulant; binds Ca++ resulting in whole blood or plasma (chelates calcium) Accelerates clot formation resulting in serum Anticoagulant; binds Ca++ resulting in whole blood plasma Inhibits the glycolytic enzyme enolase and acts as an anticoagulant resulting in whole blood or plasma. Interferes with Na, K, and most BUN (urease) determinations

Blue

Na Citrate

Gray

a) Na fluoride/ K oxalate

CEA, lead, CBC with differential, platelet and reticulocyte counts STAT serum tests (less time needed for clot formation) Coagulation testing; factor assays, fibrinogen, PT, PTT, thrombin time Glucose (OGTT)/Lactate

b) Iodacaetate

Green

Black

Heparin (Na, Li, or NH4); interferes with many ions Buffered sodium citrate

Inhibits the glycolytic enzyme glyceraldehydes-3-phosphate; results in serum; will not interfere with Na, K, or BUN (urease) assays Inhibits thrombin activation resulting in whole blood or plasma Binds calcium

Ammonia, carboxy/methemoglobin, lead Westergren ESR

Integrated serum separator tubes are available for isolating serum from whole blood. During centrifugation, blood is forced into a silicone gel material located at the base of the tube, causing a temporary change in viscosity. The specific gravity of the gel is intermediate to that of the red cells and serum, so that the gel rises and lodges between the packed cells and the top serum layer. The gel hardens and forms an inert barrier. Pediatric-sized tubes are also available with the same concept. ADVANTAGES OF SERUM SEPARATOR TUBES: 1. ease of use 2. shorter processing time through clot activation 3. higher serum yield 4. minimal liberation of potentially hazardous aerosols 5. only one centrifugation step 6. use of the same tube as that into which the patient specimen was drawin 7. use of a single label 8. centrifuged specimens can be transported without disturbing the separation MICROTAINER SYSTEM This system provides a method of collecting, separating, and storing a capillary blood sample all in one unbreakable plastic tube. Microtainer tubes are used to collect and separate capillary blood samples for maximum usable serum yield. The system is composed of a unique capillary collector, a plastic tube with inert silicone material and a red stopper. The collector channels free-flowing capillary blood directly into the unbreakable plastic microtainer tube. Serum can be pipette directly from the collection tubes. Unnecessary transfers and waste of serum sample are eliminated. Sample identification is maintained. GENERAL PRECAUTIONS IN COLLECTING BLOOD SAMPLE: 1. If a fasting specimen is required, confirm that the fasting order has been followed. 2. Always read the request first to see whether the examination is complete or incomplete, to determine the amount of blood needed. 3. Use dry and sterile syringe and needle to avoid hemolysis. Before use, all needles and syringes should be sterilized by one of the following methods: a) autoclaving for 20 min. at 5 pounds pressure b) dry heating in oven at 170C for 2 hours c) boiling in water for 30 min. and drying in an oven 4. Do not extract blood from patients while they are receiving intravenous medications because these solutions may influence the chemical analysis. 5. Never draw out the syringe without removing first the tourniquet to avoid hematoma. 6. Avoid prolonged application of tourniquet to avoid hemoconcentration. 7. Blood specimens obtained must be placed in appropriate containers for each specific test. Ensure prompt and adequate mixture of blood and anticoagulant to prevent formation of unwanted clot.

ADVANTAGES OF VENIPUNCTURE: 1. large amount of blood can be obtained 2. additional and repeated tests can be done 3. blood can be transported and stored for future use after a suitable anticoagulant has been used 4. ideal for blood chemistry determinations 5. it reduces the possibility of error resulting from dilution with tissue juices DISADVANTAGES OF VENIPUNCTURE: 1. requires more time and skill 2. requires more equipment 3. more complications may arise 4. hard to do on infants, children and obese patients COMPLICATIONS OF VENIPUNCTURE I. Immediate Local 1. hemoconcentration 2. failure of blood to enter the syringe 3. hematoma Causes: a) failure to remove the tourniquet before withdrawing the needle b) failure to have the needle completely in the vein c) repeated punctures of the vein d) failure to apply pressure to the wound for a sufficient length of time 4. circulatory failure 5. fainting or syncope II. Local Delayed (Late Local) 1. thrombosis 2. thrombophlebitis III. General Delayed (Late General) 1. serum hepatitis 2. AIDS STEPS IN VENIPUNCTURE: 1. Preparing the materials needed. 2. Applying the tourniquet. 3. Selecting the vein. 4. Applying the antiseptic at the proposed site of injection. Clean the venipuncture site with 70% alcohol or 1% iodine. 5. Inserting the needle. The needle as it enters the skin, is positioned at approximately 15 degree angle to the site with the bevel up. 6. Withdrawing the blood. 7. Releasing the tourniquet. 8. Withdrawing the needle. 9. Prevent further bleeding. RECOMMENDED ORDER OF DRAW Blood culture tubes tubes without anticoagulants or preservatives (red stoppered) containing anticoagulants and preservatives (blue green lavender gray)

tubes

- Blood cultures are always drawn first to reduce bacterial contamination. - Citrate tubes (coagulation tubes) should never be drawn alone or as the first in the sequence of multiple draws. Always draw one other tube before the citrate to reduce contamination by tissue fluids which could initiate clotting mechanism. Citrate tubes must be filled completely. 3. ARTERIAL PUNCTURE (Anaerobic) is used to measure oxygen and carbon dioxide tension, and measure pH. Blood collected is called arterial blood. Arterial blood is oxygenated blood. It is uniform in composition throughout the body. Sites of puncture: a) radial artery preferred sites for b) brachial artery arterial blood collection c) femoral artery d) scalp artery e) umbilical artery best method of arterial blood collection in newborn Materials needed: a) glass syringe b) each site of arterial puncture uses different gauge of needle - brachial artery: 18-20 gauge - radial artery: 23-25 gauge c) cotton (dry and wet) d) alcohol or iodine e) heparin preferred anticoagulant f) warm towel (45C) arterializations Capillary blood may be a suitable substitute for arterial blood determination of pH and pCO 2 provided that the site must be warmed prior to collection. Warming increases blood flow through arterioles and capillaries and result in arterial-riched blood g) iced water bath or other coolant (temperature of 1-5C) preservative for transport to minimize leukocyte consumption of oxygen TYPES OF BLOOD SAMPLE: 1. Whole blood 2. Plasma is the liquid portion of unclotted blood. It is the liquid part of the blood that separates after standing or centrifugation of blood with an anticoagulant. 3. Serum is the liquid portion of clotted blood. It is the liquid portion of the blood that separates after standing or centrifugation of blood without anticoagulant. WHAT ADVANTAGES DOES PLASMA HAVE OVER SERUM? 1. time saving 2. higher yield 3. virtually no interference due to subsequent coagulation 4. results from plasma are more representative of the in-vivo state compared to serum 5. low risk of hemolysis and thrombocytolysis WHAT ARE THE DISADVANTAGES OF PLASMA RELATIVE TO SERUM? 1. Protein electrophoresis is altered 2. Method-dependent interference 3. Cation interference

DIFFERENCE BETWEEN PLASMA AND SERUM: PLASMA: 1. contains fibrinogen 2. presence of lipemia clearing factor 3. optically less clear compared to serum SERUM: 1. no fibrinogen is present 2. lipemia clearing factor is absent, appears to be co-precipitated or destroyed during the clotting process 3. optically clearer compared to plasma Serum is the preferred specimen in Clin. Chem. because: 1) certain substances like the lipemia clearing factor are co-precipitated during clotting 2) clearer than plasma 3) there is potential interference produced by anticoagulants. COMMON DIFFICULTIES ENCOUNTERED DURING COLLECTION AND PROCESSING OF BLOOD: I. Hemolysis destruction of red blood cells This must be avoided because of the following reasons: a. Most constituents, such as SGOT, LDH, acid phosphatase, and potassium are present in large amounts in erythrocytes so that hemolysis will significantly elevate the values of these substances in serum. b. Invalidates determination due to color ranges c. May directly interfere in a chemical determination by inhibiting an enzyme such as lipase d. Hemoglobin may interfere with the diazotization of bilirubin Hemolysis can take place during: A. Collection of blood This can be prevented by: 1. avoid excessive venous stasis through prolonged application of tourniquet 2. avoid moisture or contamination in needles, syringes, or blood containers 3. avoid excessive traction on syringe plunger 4. avoid use of small lumen needles. Use a gauge 20 needle and the blood should be allowed to flow 5. do not allow air to leak into the syringe during blood extraction B. Transfer of blood 1. do not expel blood from syringe through a needle 2. do not allow blood to fall into the container throught the air 3. do not shake blood in container to mix with anticoagulant 4. do not agitate blood that does not contain an anticoagulant 5. clotted blood should not be disturbed for at least 15 to 20 minutes C. Separation of blood 1. do not allow the red blood cells to be in contact with plasma or serum for a long time 2. do not freeze whole blood, because the red blood cells will hemolyze on thawing 3. separation of clot from container should be done by gently rimming or ringing the clot 4. do not overcentrifuge II. Lipemia or Lactescence Milky or lipemic plasma occurs when blood samples are obtained 1 to 2 hours after eating a fatty meal. This is caused by a transient rise in chylomicrons following a meal containing fat. It causes interference with a large number of chemical analyses because of turbidity. Lipemia disturbs the following investigations strongly: amylase, bilirubin, BSP test, protein, GOT and GPT.

III. Concentration changes This may occur through dilution or evaporation. The sources of error are: 1. use of syringe and needles rinsed in saline solution, liquid anticoagulants 2. use of liquid form of anticoagulant 3. allowing the blood to stand in open container 4. centrifugation of blood specimens in open containers IV. Composition changes (Changes that occur in the blood upon long standing). These are brought about by: 1. Bacterial changes such as formation of ammonia from urea This can be prevented by: a. sterile handling of blood b. prompt separation of cells from plasma or serum c. storing the specimen at 4C to 6C or by freezing at -20C 2. Enzymatic changes such as metabolic degradation of glucose and autodegradation (loss of enzyme activity) 3. Blood gas changes such as loss of CO2 4. Extravascular interchange this is due to movement of substance between the cells and plasma or serum 5. Changes in lipid concentration due to lipolysis 6. Changes in phosphates due to hydrolysis of organic phosphate esters SPECIMEN VARIABLES In general, blood for chemical analysis should be drawn while the patient is in the postabsorptive state in order to obtain valid results. An overnight fast (10-14 hours after the last meal) is the usual procedure although a 6hour fast is ample. During this time there is no need to restrict water intake. 1. Postprandial Increased ammonia glucose iron calcium catecholamines 2. Diurnal variation iron acid phosphatase (ACP) catecholamine cortisol 3. Hemolysis Increased ammonia total protein (TP) iron phosphate potassium magnesium alanine aminotransferase (ALT) aspartate aminotransferase (AST) creatine kinase (CK)

Decreased phosphate chloride

growth hormone (GH) parathyroid hormone (PTH) thyroid stimulating hormone (TSH)

Decreased albumin bilirubin sodium

lactate dehydrogenase (LDH) alkaline phosphatase (ALP) acid phosphatase (ACP) cholesterol triglyceride catecholamine 4. Prolonged tourniquet application Increased ammonia total protein albumin iron cholesterol

Decreased potassium

ANTICOAGULANTS These are chemical substances that interfere in the clotting mechanism. These are chemical substances that prevent clotting of blood. The majority of these act by making calcium unavailable to the clotting mechanism, either by precipitating it as a salt (oxalates) or by binding it in a non-ionized form. 1. HEPARIN a natural anticoagulant a. ideal universal anticoagulant b. present in most body tissue (amount too small to interfere with clot formation) * it exists in highest concentration in the liver and lungs c. least interference with analyses d. high cost, temporary action e. inappropriate for hematology (blue background on Wright-stained blood smear) f. acts as an antiprothrombin and antithrombin thus interfering in the conversion of prothrombin to thrombin g. anticoagulant of choice for blood gas analysis Amount needed 0.2 mg/ml of blood 2. OXALATES such as sodium, potassium, ammonium, lithium or dioxalate a. potassium oxalate (1-2 mg/ml of blood) usually employed b. lithium oxalate is the best oxalate to be used for plasma uric acid test c. sodium salt is used routinely in prothrombin time tests d. unsuitable for analyses of sodium, potassium, ammonia, lithium e. acts as inhibitor of LD, acid phosphatase, amylase f. acts by formation of insoluble complexes with calcium (precipitate calcium as a salt) g. potassium oxalate may cause variable dilution of plasma due to water transport from cells to the plasma NOTE: If the oxalate to be used is dried, it should be dried as a thin film on the wall of the vial to minimize hemolysis. The temperature of drying should not exceed 100C because the oxalate might be converted to carbonate, which has no anticoagulant activity. Amount needed 0.01 ml of aq. 20% solution per ml of blood or 1 gt per 5 ml of blood

3. SODIUM FLUORIDE a. effective anticoagulant but its anticoagulant effect does not last very long. It is issued more as a preservative rather than an anticoagulant b. sodium fluoride added to heparin preserves glucose level by preventing glycolysis (otherwise approximately 7% glucose is destroyed/hour) c. in high concentration will inhibit urease enzyme activity d. inhibits glucose oxidase activity in enzymatic glucose reaction measurements e. increases amylase activity f. diminishes acid phosphatase activity Amount needed 10 mg of sodium fluoride per 1 ml of blood is the usual concentration used 4. SALTS OF ETHYLENE DIAMINE TETRAACETIC ACID (EDTA) a. a chelating agent that is useful particularly for hematologic examinations since it preserves the cellular constituents of the blood b. acts by binding (chelation) of calcium c. inappropriate for enzyme analysis because of the binding of metal cofactor necessary for enzyme function Commercially available as: Versene (disodium salt) in crystal form Sequestrene (dipotassium salt) have a higher degree of stability liquid form Amount needed 1 mg per ml of blood 5. CITRATES a. bind with calcium in non-ionized form (double salt) b. buffered citrates stabilize plasma pH c. available as Na citrate which is commonly used in hematologic experiments d. inhibits amylase activity ACD (Acid Citrate Dextrose) a citrate preparation is used for blood transfusion because it is non-toxic, and rapidly utilized by our body and easily excreted by the kidneys. Dextrose is added as source of energy to prolong the life span of the red blood cells during storage. maintains plasma pH of 7.1 Amount needed 5 mg/ml of blood 6. SODIUM POLYANETHOLSULFONATE this is employed in a concentration of 1 to 2.5 mg/ml of blood Mechanical means of preventing coagulation use of glass beads GUIDE TO SUGGESTED SPECIMEN COLLECTION IN CLINICAL CHEMISTRY 7-ml plain tube (red top) albumin alanine aminotransferase (ALT) alkaline phosphatase

protein triglyceride triiodothyronine

amylase aspartate aminotransferase (AST) bilirubin blood urea nitrogen (BUN) calcium calcium, ionized cholesterol creatine kinase (CK) CKMB creatinine electrolytes chloride potassium sodium free thyroxine (free T 4) glucose lactate dehydrogenase lipase magnesium phosphorus

thyroxine (T4) uric acid heparin (5-ml green top tube) ammonia (on ice) carboxyhemoglobin/O2 saturation methemoglobin hemoglobin, plasma NaF Oxalate (5-ml gray top tube) glucose glucose tolerance lactose tolerance EDTA (versene; 7-ml lavender top tube) carcinoembryonic antigen (CEA) lead

PRESERVATION OF SPECIMEN Preservation of a biological specimen is usually not significant problem in the hospital clinical laboratory for the usually requested metabolites. Those analyses, however, that are not performed daily or are sent to a reference laboratory may require the use of preservation technics to maintain a valid biological specimen. There are additional reasons for storing specimens: a) each sample should be retained for a period of time after analysis to permit a repeat analysis if deemed advisable, and b)if they are time sequence specimens in a long-term study, they should be retained to be assayed at the same time. PHYSICAL PRESERVATION using reduced temperature (refrigeration, freezing) Commonly employed reduced temperatures include the following: 4C ------------------------------------------------------------- refrigeration -5C to -20C ------------------------------------------------- freezer -70C ----------------------------------------------------------- dry ice -142C --------------------------------------------------------- liquid nitrogen CHEMICAL PRESERVATIVES Classification according to function: 1. enzyme inhibitors prevent chemical changes such as glycolysis 2. bacteriostatic agents interfere with and prevent microbial growth The problem of microbial growth enters whenever the sample is to be stored for longer than one day either at room or refrigerator temperature. This can be stored by four alternative courses of action: a) collection and storage under sterile conditions (not always possible and certainly impractical as a routine procedure for all determinations) b) freezing of the sample c) extreme alteration of pH

d) addition of an antibacterial agent Common preservatives: 1. Conc. HCl for catecholamines and VMA 2. Thymol, toluene, CHCl3, boric acid for urine 3. Fluoride prevents glycolysis and other enzyme actions; should not be used when such actions are involved 4. Sodium carbonate for porphyrins 5. Petroleum ether to retard oxidation by formation of a protective layer 6. Antibiotics 1 mg streptomycin base per 10 ml blood for urea determination and hemoglobin 7. Boric acid for adrenal corticosteroids PROCESSING OF BLOOD SAMPLES AFTER COLLECTION: - Whole blood should be refrigerated at 4-5C if not done within an hour after collection - PFF should be prepared at once (30 min. after collection) and refrigerated - Separate serum and plasma after standing and refrigerate at 4-6C or freeze at -20C if analysis is delayed for more than four hours - Specimen for blood ammonia, gases or other unstable substances shoud be processed as soon as possible CLINICAL SPECIMEN HANDLING The individual handling of the specimen (serum or plasma) will depend on the analysis that is to be done and the time that will elapse before the analysis is started. Glucose (Blood Sugar) If test cannot be performed within 2 hours, the blood may be refrigerated for a few hours. PFF (drop of toluol added) may be stored overnight in a refrigerator. The use of fluoride can deter glycolysis for a short time. NPN substances (BUN, Creatinine, Uric Acid, etc.) PFF may be stored overnight in a refrigerator. Cholesterol and Cholesterol Esters serum may be stored in a refrigerator for a few hours. HDL Cholesterol store at room temperature. Chromogens (Urobilinogen, Porphobilinogen, etc.) Only fresh or suitably preserved specimens should be used. Bilirubin, BSP, Chloride, Icterus Index, Na or K, Proteins, Thymol Turbidity Serum may be stored overnight in a refrigerator. IMPORTANT: Bilirubin should always be stored in the dark (sensitive to light; protect from fluorescent light; protect from natural light). Calcium Serum must be separated from the clot within 30 min., otherwise calcium diffuses into the cells. Serum can be kept overnight in a refrigerator. Phosphorus This is very unstable. Serum must be separated from the clot within 30 min. and tested immediately. Cephalin Cholesterol Flocculation Serum CANNOT be preserved. Test must be started within 4 hours of specimen collection. Amylase Amylase activity rapidly decreases after blood withdrawal. If the test cannot be performed immediately, the serum or plasma may be refrigerated for up to 3 hours ONLY. Lipase Test must be performed within 2 hours of blood collection. Phosphatase Serum (small drop of toluol added) may be kept for 2 hours in a refrigerator. Serum for alkaline phosphatase will keep for a few days in a freezer. Acid phosphatase, on the other hand, deteriorates rapidly even in the frozen state. Sodium citrate added to serum and frozen can keep acid phosphatase for a short period. Transaminase (GPT & GOT) Serum may be stored overnight in the freezer. T3 Serum should be frozen until ready to test.

PBI Can be kept at room temperature. Carbon Dioxide Combining Power Plasma (under oil) may be stored in a refrigerator overnight. IMPORTANT: When obtaining blood for CO2 determinations, the tourniquet should be released before the blood is aspirated. Ammonia Place sample at -5C; analyze within 30 min.