|Dipotassium EDTA||20 gm|
|Distilled water||200 ml|
|Ammonium oxalate||1.2 gm|
|Potassium oxalate||0.8 gm|
|Distilled water||upto 100 ml|
|Trisodium citrate||3.2 gm|
|Distilled water||upto 100 ml|
Use 1:9 (anticoagulant: blood) proportion for coagulation studies; for ESR, 1:4 proportion is recommended.
There are two methods for ABO grouping:
- Cell grouping (forward grouping): Red cells are tested for the presence of A and B antigens employing known specific anti-A and anti-B (and sometimes anti-A, B) sera.
- Serum grouping (reverse grouping): Serum is tested for the presence of anti-A and anti-B antibodies by employing known group A and group B reagent red cells.
Both cell and serum grouping should be done since each test acts as a check on the other.
- Autoagglutination: Presence of IgM autoantibodies reactive at room temperature in patient’s serum can lead to autoagglutination. If autocontrol is not used, blood group in such a case will be wrongly typed as AB. Therefore, for correct result, if autocontrol is also showing agglutination, cell grouping should be repeated after washing red cells with warm saline, and serum grouping should be repeated at 37°C.
- Rouleaux formation: Rouleux formation refers to red cells adhering to each other like a stack of coins and can be mistaken for agglutination. Rouleaux formation is caused by high levels of fibrinogen, immunoglobulins, or intravenous administration of a plasma expander such as dextran. Rouleaux formation (but not agglutination) can be dispersed by addition of normal saline during serum grouping.
- False-negative result due to inactivated antisera: For preservation of potency of antisera, they should be kept stored at 4°-6°C. If kept at room temperature for long, antisera are inactivated and will give false-negative result.
- Age: Infants start producing ABO antibodies by 3-6 months of age and serum grouping done before this age will yield false-negative result. Elderly individuals also have low antibody levels.
- A clean and dry glass slide is divided into two sections with a glass marking pencil. The sections are labeled as anti-A and anti-B to identify the antisera (see Figure 786.2).
- Place one drop of anti-A serum and one drop of anti-B serum in the center of the corresponding section of the slide. Antiserum must be taken first to ensure that no reagents are missed.
- Add one drop of blood sample to be tested to each drop of antiserum.
- Mix antiserum and blood by using a separate stick or a separate corner of a slide for each section over an area about 1 inch in diameter.
- By tilting the slide backwards and forwards, examine for agglutination after exactly two minutes.
Positive (+): Little clumps of red cells are seen floating in a clear liquid.
Negative (–): Red cells are floating homogeneously in a uniform suspension.
- Interpretation: Interpret the result as shown in the Table 786.1 and Figure 786.2.
Separate tubes of auto-control, positive control, and negative control should always be setup along with the test sample tube. Auto-control tube consists of mixture of patient’s red cells and patient’s own serum. This is required to rule out false-positive result due to auto antibodies in patient’s serum causing auto agglutination of patient’s own red cells. Auto-control test is particularly essential when ABO grouping is being done only by forward method and blood group is typed as AB. If there are auto antibodies in recipient’s serum, ABO grouping, Rh typing, antibody screening, and cross matching all will show positive result.
If forward grouping, reverse grouping, and autocontrol tests are all positive, then these results are probably indicative of a cold-reactive autoantibody. Before performing forward typing, red cells should be washed with normal saline to elute the antibody. Before performing reverse grouping, autoantibody should be adsorbed by washed cells till autocontrol is negative.
- Scan the slide in a methodical grid pattern, in order not to cover the same area twice. Counts can be completed quickly under 400×magnification, but if you are also evaluating morphology, 1000×magnification should be used.
- Count a minimum of 100 WBCs.
- Manual or microscopic method
- Automated method
Hemocytometer with cover glass, compound microscope.
HgCl2 0.05 gm
NaSO4 2.5 gm
NaCl 0.5 gm
Distilled water 100 ml
- Wipe finger with cotton soaked with alcohol, with a sterile lancet do small prick on the finger tip. Use pipette. Aspirate blood to 0.5.
- Aspirate diluting Hayem’s solution to the 101 mark. It will give 1:200 dilution of the blood.
- Hold the pipette horizontally and role it with both hands between finger and thumb.
- Place the counting chamber, absolutely free from dust and grease, on the table and lay the cover glass in place over the ruled area.
- Discard the first two or three drops from the pipette. Charge the counting chamber by holding the pipette in an inclined position. Allow 3 minutes for the cells to settle.
- Locate the central square, which is divided into 25 medium sized squares. Each of the medium sized squares is further divided into 16 smallest squares.
- Count the erythrocytes in medium sized squares (80 smallest squares) using high power objective.
- In order to avoid confusion in counting, count all cells wihich touch the upper and left outer double line of the group of 16 squares as if they were inside the square. Neglect all those cells, which touch the lower and right inner line.
= 1/5 sq mm
= 1/5 sqmm x 1/10 mm
= 1/50 cu mm
(1) Increased in numbers of RBC called polycythemia it is due to
• Bone marrow failure
• Erythropoietin deficiency (2ndry to kidney disease)
• Hemolysis (RBC destruction) from transfusion reaction
• Multiple myloma
• Nutritional deficiencies of (Iron, Copper, Folate, Vit B12, B6)
• Newborns: 4.8-7.2 millions
• Children: 3.8-5.5 millions
• Adult ( male): 4.6-6.0 millions
• Adult (Females): 4.2-5.0 millions
• Pregnancy: slightly lower than normal
- Brown, B.A., Haemotology, Principles and Procedures, Lea & Febiger, U.S.A., 1976.
- Hoffbrand, A. V. and Pettit, 1. E., Essential Haemotology, Blackwell Scientific Publication, U.S.A., 1980.
- Kassirsky, I. and Alexeev, G., Clinical Haemotology, Mir Publishers, U.S.S.R., 1972.
- Widmann, F.K., Clinical interpretation of Laboratory tests, F.A. Davis Company, U.S.A., 1985.
- Kirk, C.J.C. et al, Basic Medical Laboratory Technology, Pitman Book Ltd., U.K. 1982.
- Green, J.H., An Introduction to human Physiology, Oxford University Press, U.K., 1980.
Anticoagulated whole blood is centrifuged in a capillary tube of uniform bore to pack the red cells. Centrifugation is done in a special microhematocrit centrifuge till packing of red cells is as complete as possible. The reading (length of packed red cells and total length of the column) is taken using a microhematocrit reader, a ruler, or arithmetic graph paper.
- Microhematocrit centrifuge: It should provide relative centrifugal force of 12000 g for 5 minutes.
- Capillary hematocrit tubes: These are disposable glass tubes 75 mm in length and 1 mm in internal diameter. They are of two types: plain (containing no anticoagulant) and heparinised (coated with a dried film of 2 units of heparin). For plain tubes, anticoagulated venous blood is needed. Heparinised tubes are used for blood obtained from skin puncture.
- Tube sealant like plastic sealant or modeling clay; if not available, a spirit lamp for heat sealing.
- Microhematocrit reader; if not available, a ruler or arithmetic graph paper.
Venous blood collected in EDTA (dipotassium salt) for plain tubes or blood from skin puncture collected directly in heparinised tubes. Venous blood should be collected with minimal stasis to avoid hemoconcentration and false rise in PCV.
- Fill the capillary tube by applying its tip to the blood (either from skin puncture or anticoagulated venous blood, depending on the type of tube used). About 2/3rds to 3/4ths length of the capillary tube should be filled with blood.
- Seal the other end of the capillary tube (which was not in contact with blood) with a plastic sealant. If it is not available, heatseal the tube using a spirit lamp.
- The filled tubes are placed in the radial grooves of the centrifuge with the sealed ends toward the outer rim gasket. Counterbalance by placing the tubes in the grooves opposite to each other.
- Centrifuge at relative centrifu-gal force 12000 g for 5 minutes to completely pack the red cells.
- Immediately remove the tubes from the centrifuge and stand them upright. The tube will show three layers from top to bottom: column of plasma, thin layer of buffy coat, and column of red cells.
- With the microhematocrit reader, hematocrit is directly read from the scale. If hematocrit reader is not available, the tube is held against a ruler and the hematocrit is obtained by the following formula:
Length of total column in mm
- Prolonged application of tourniquet during venepuncture causes hemoconcentration and rise in hematocrit.
- Excess squeezing of the finger during skin puncture dilutes the sample with tissue fluid and lowers the hematocrit.
- Correct proportion of blood with anticoagulant should be used. Excess EDTA causes shrinkage of red cells and falsely lowers the hematocrit.
- Inadequate mixing of blood with anticoagulant, and inadequate mixing of blood before testing can cause false results.
- Low hematocrit can result if there are clots in the sample.
- Centrifugation at lower speed and for less time falsely increases PCV.
- A small amount of plasma is trapped in the lower part of the red cell column which is usually insignificant. Increased amount of plasma is trapped in microcytosis, macrocytosis, spherocytosis, and sickle cell anemia, which cause an artifactual rise in hematocrit. Larger volume of plasma is trapped in Wintrobe tube than in capillary tube.
- As PCV requires whole blood sample, it is affected by plasma volume (e.g. PCV is higher in dehydration, and lower in fluid overload).
- Expression of PCV: Occasionally, PCV is expressed as a percentage. In SI units, PCV is expressed as a volume fraction. Conversion factor from conventional to SI units is 0.1 and from SI to conventional units is 100.
- Rules of 3 and 9: These rules of thumb are commonly used to check the accuracy of results and are applicable only if red cells are of normal size and shape.
• Hemoglobin (gm/dl) × 3 = PCV
• Red cell count (million/cmm) × 9 = PCV
- Automated hematocrit: In automated hematology analyzers, hematocrit is obtained by multiplying red cell count (in millions/cmm) by mean cell volume (in femtoliters).
- Adult males: 40-50%
- Adult females (nonpregnant): 38 45%
- Adult females (pregnant): 36-42%
- Children 6 to 12 years: 37-46%
- Children 6 months to 6 years: 36 42%
- Infants 2 to 6 months: 32-42%
- Newborns: 44-60%
- Packed cell volume: < 20% or > 60%