Practical Manual of Hematology Girish Kamat
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Collection of Blood and Anticoagulants1

 
WAYS OF OBTAINING BLOOD
 
Capillary/Peripheral Blood
  • Blood can be obtained by pricking following sites, after taking all aseptic precautions:
    • Palmar surface of tip of 3rd or 4th finger.
    • Heel in case of infants—Puncture should be restricted to outer medial and lateral portions of plantar surface.
    • Ear lobe—Now not preferred, because reduced blood flow renders it unrepresentative of circulating blood.
  • Puncture should be about 3 mm deep with needle, lancet or Noll-Bard-Parker blades (Round needles do not bleed freely, so lancets with flat body and cutting edge are used).
  • Area to be punctured should be warmed by massaging or bathing in warm water (otherwise Hb, RBC and WBC counts will be high).
  • Site should be cleaned with spirit and allowed to dry before puncture is made.
  • Wipe off first drop of blood, as it is often mixed with tissue fluid.
  • Never press out the blood.
 
Indications
Indications for obtaining blood by this method are as follows:
  • Infants younger than 1 year
  • Gross obesity
  • For point of care blood tests.2
Note:
  • PCV, RBC count and Hb are slightly higher in capillary blood than venous blood
  • Total WBC count is also increased
  • Platelet count is decreased.
 
Venous Blood
Phlebotomy tray should consist of the following:
  • Syringes and needles—19 or 21 G (23 G for children)
  • Tourniquet
  • Specimen containers—Plain and with anticoagulant
  • Request form
  • 70% isopropyl alcohol swabs
  • Sterile gauze swabs
  • Adhesive dressing
  • Self-sealing plastic bags.
 
Procedure
  • Check the identity of patient and request form.
  • Tourniquet is applied if needed just above the venepuncture site and it should be released as soon as blood begins to flow into the syringe.
  • Needles of gauge less than 22 should be used.
  • Usually anterior cubital vein is preferred.
  • After taking all aseptic precautions, skin over the site of vein is tapped and patient is asked to make fist for few times.
  • Once the vein becomes prominent it is punctured and piston of the syringe is withdrawn very slowly.
  • After collection, patient is asked to apply pressure over puncture site using 3 fingers (3 fingers because, site of skin puncture may not be same as site of venepuncture).
  • Once required amount of blood is filled in syringe, needle is removed and blood is transferred from syringe into container gently.
  • Anticoagulated specimens must be mixed by inverting the container several times.3
  • Specimen should be adequately labeled with patient identification or bar coded. (Blood films are prepared immediately after collection, as artifacts may be produced due to delay in transit to laboratory).
Hemolysis can be avoided in the following manner:
  • Using clean and dry apparatus
  • Withdrawing the blood slowly without much suction
  • Not using too fine needle
  • Delivering blood gently into the receiver (Not through the needle)
  • Avoiding frothing during withdrawal and mixing with anticoagulant
  • Avoiding freezing of blood.
 
Complications of Phlebotomy
  • Immediate
    • Hematoma—It can be avoided by applying gentle pressure
    • Syncope.
  • Late
    • Thrombosis of vein—Resolves upon application of thrombo-phob
    • Transmission of HIV and other diseases.
 
Vacutainer System
  • It consists of a needle, a needle holder and a vacuum tube.
  • Requisite quantity of blood flows automatically into the vacutainer which contains adequate anticoagulant (Vacuum controls the amount of blood that enters).
  • From single venepunture blood can be collected in separate vacutainers like EDTA, oxalate, etc.
  • Type of anticoagulant can be identified by color of the cap.
 
Containers for Venous Blood
  1. Routine (Hb, PCV, ESR, etc.)
    • Penicillin vial containing suitable anticoagulant.4
  2. For tests using serum
    • Clean dry sterile test tube/penicillin vial. Blood is allowed to clot.
  3. Bacteriological work
    • Collect in sterile blood culture bottle containing glucose broth.
 
Storage of Blood Before Tests are Performed
Time limit
Test
< 2 hours
Prothrombin time, PL count, Blood smear
Within 3 hours
ESR
Within 24 hours
RBC count, Hb, PCV, WBC count, Retic count
Store the blood in refrigerator at 4°C (Freezing should be avoided).
 
Plasma for Coagulation Studies
Room temp
within 2 hours
4°C
4 hours
–20°C
2 weeks
–70°C
5 months
 
Effects of Storage on Blood Count
(Changes occur rapidly at higher ambient temperature)
RBC, WBC, PL and Indices are stable for 8 hours (If stored at 4°C then up to 24 hours).
Later on:
  • PCV and MCV start increasing as RBCs are swollen
  • Osmotic fragility increases
  • ESR—Decreases
  • Prothrombin time prolonged.
 
Morphological Changes
(start at 3 hours and striking at 12–18 hours)
Neutrophils-Nuclei stain more homogeneously5
- Nuclear lobes become separated
- Ragged cytoplasmic margin
- Small vacuoles appear in cytoplasm
Monocytes
- Vacuoles appear in cytoplasm
- Nucleus undergoes irregular lobulation
Lymphocytes
- Vacuoles in cytoplasm
- Nucleus stains more homogeneously
- Nucleus undergoes budding
RBCs
- Progressive crenation and sphering.
 
Biochemical Changes in Blood upon Storage
  • Loss of CO2—It diffuses from plasma into atmosphere.
  • Conversion of glucose to lactic acid by glycolysis.
  • Increased plasma inorganic phosphates—Formed from ester phosphates present in cells.
  • Increased ammonia—Formed from nitrogenous substances like urea.
  • Passage of intracellular material of RBC into plasma ex-potassium.
  • Conversion of pyruvate to lactate.
Note: To ensure even dispersal of blood cells, it is essential that specimens are mixed effectively immediately prior to taking a sample for testing.
Two ways:
  • Mechanical rotator mixing for 2 min.
  • Invert the tube for 8–10 times by hand.
 
Serum (Plasma minus Fibrinogen)
  • Blood is transferred to plain sterile tubes
  • It is allowed to clot undisturbed for about 1 hour at room temp (18–25°C)
  • Clot is loosened gently from container wall by means of wooden stick
  • Tube is capped (Some clot activators may be added for accelerated separation of serum)
  • Centrifuge the tube for 10 min at 1200 g6
  • Supernatant serum is then pipetted into another tube and centrifuged again for 10 min at 1200 g
  • Supernatant serum is transferred to tubes for tests or for storage at 4°C
    (–20°C—up to 3 months
    (–40°C—long-term).
Note: If cold agglutinins are to be titrated, the blood must be kept at 37°C until the serum has separated.
 
ANTICOAGULANTS
 
Ethylenediamine Tetra-acetic Acid (EDTA, Sequestrene)
  • It is a powerful calcium chelating agent
  • Used in concentration of 1.5 ± 0.25 mg (anhydrous) per ml of blood (i.e. 4 mmol per ml of blood)
  • Dipotassium salt is preferred over disodium salt
  • Blood collected in EDTA can be used for TLC, PS preparation, Hb and DC.
 
Disadvantages
  • Excess of EDTA causes shrinkage of WBCs and RBCs and induces degenerative changes
  • It is unsuitable for coagulation studies
  • EDTA blood fails to demonstrate basophilic stippling of RBCs in lead poisoning
  • Causes leukoagglutination affecting both neutrophils and lymphocytes
  • Activates naturally occurring antiplatelet auto-antibodies which cause platelet adherence to neutrophils.
 
Trisodium Citrate
  • 3.8% solution (i.e. 109 mmol/liter) is used
  • Citrated blood is used for
    • ESR estimation by Westergren method—for 1 volume of citrate, 4 volume of blood is added
    • Coagulation studies—for 1 volume of citrate, 9 volume of blood is added.7
  • Citrate acts by binding to calcium in blood
  • Citrate blood cannot be used for PCV, Hb, TC and DC, because citrate is used as solution and it alters the concentration of blood.
 
Oxalates
  1. Sodium oxalate, potassium oxalate—2 mg/ml
  2. Double oxalate (Wintrobe's mixture)—2 mg/ml
Consists of:
  • Ammonium oxalate—1.2 gm (3 parts)
  • Potassium oxalate—0.8 gm (2 parts)
  • Water—100 ml
    So it contains 20 mg of oxalate per ml.
    So 0.1 ml contains 2 mg of oxalate.
    0.5 ml is placed in a container and dried, which is sufficient for 5 ml of blood.
  • 2 salts are used because ammonium salt causes swelling of RBCs, while potassium salt causes shrinkage.
  • Oxalated blood is useful in estimation of Hb, PCV, TLC, determination of specific gravity and blood chemistry.
 
Sodium Fluoride
  • It complexes with calcium to form calcium fluoride
  • 30 mg is used for 5 ml of blood
  • It is useful in estimation of blood glucose level (Fluoride prevents glycolysis by blocking phosphorylase enzymes in RBCs).
 
Heparin
  • It inhibits thromboplastin formation and has antithrombin activity
  • 10–0 IU or 0.1–0.2 mg is used for 1 ml of blood
  • Heparinized blood is used in assessing osmotic fragility, chemical estimation (ex-plasma iron), gas analysis and immunophenotyping.8
Disadvantages:
  • Expensive
  • Not useful for TC, DC as leukocytes tend to clump
  • Induces platelet clumping as well
  • Bluish discoloration to background of smears.
 
Blood Specimens for Different Analysis
  1. Whole blood—Glucose, urea, NPN, pH.
  2. Serum—Total protein, albumin, globulin, bilirubin, cholesterol, creatinine, phosphates, uric acid, transaminases (SGPT, SGOT).
  3. Plasma—Chlorides, fibrinogen, ascorbic acid, bicarbonates.
 
Causes of Misleading Results from Discrepancies in Specimen Collection
 
Precollection
  1. Toilet within 30 min; food/water intake within 2 hours
  2. Smoking
  3. Physical activity within 20 min
  4. Stress
  5. Drugs within 8 hours.
 
During Collection
  1. Different times (diurnal variance)
  2. Posture; lying, standing or sitting
  3. Hemoconcentration from prolonged tourniquet pressure
  4. Excessive negative pressure when drawing blood into syringe
  5. Incorrect type of tube
  6. Capillary vs Venous blood.
 
Handling of Specimen
  1. Insufficient or excess anticoagulant
  2. Inadequate mixing of blood with anticoagulant
  3. Error in patient and/or specimen identification
  4. Inadequate specimen storage conditions
  5. Delay in transit to laboratory.