USES OF ANTICOAGULANTS FOR HEMOTOLOGICAL INVESTIGATIONS

Published in Hemotology
Saturday, 22 July 2017 10:04
Anticoagulants used for hematological investigations are ethylene diamine tetra-acetic acid (EDTA), heparin, double oxalate, and trisodium citrate (Table 791.1).
 
Table 791.1 Salient features of three main anticoagulants used in the hematology laboratory
Salient features of three main anticoagulants used in the hematology laboratory
 
Ethylene Diamine Tetra-acetic Acid (EDTA)
 
Changes occurring due to prolonged storage of blood in EDTAThis is also called as Sequestrene or Versene. This is the recommended anticoagulant for routine hematological investigations. However, it cannot be used for coagulation studies. Disodium and dipotassium salts of EDTA are in common use. International Committee for Standardization in Hematology recommends dipotassium EDTA since it is more soluble. It is used in a concentration of 1.5 mg/ml of blood. Dried form of anticoagulant is used as it avoids dilution of sample. Its mechanism of action is chelation of calcium. Proportion of anticoagulant to blood should be maintained. EDTA in excess of 2mg/ml causes shrinkage of and degenerative changes in red and white blood cells, decrease in hematocrit, and increase in mean corpuscular hemoglobin concentration. Excess EDTA also causess welling and fragmentation of platelets, which leads to erroneously high platelet counts. Prolonged storage of blood in EDTA anticoagulant leads to alterations as shown in Figure 791.1 and Box 791.1. EDTA is used for estimation of hemoglobin, hematocrit, cell counts, making blood films, sickling test, reticulocyte count, and hemoglobin electrophoresis.
 
Preparation
 
Dipotassium EDTA 20 gm
Distilled water 200 ml
 
Mix to dissolve. Place 0.04 ml of this solution in a bottle for 2.5 ml of blood. Anticoagulant should be dried on a warm bench or in an incubator at 37°C before use. For routine hematological investigations, 2-3 ml of EDTA blood is required.
 
Changes in blood cell morphology crenation of red cells separation of nuclear lobes of neutrophil vacuoles in cytoplasm and irregular lobulation of monocyte and lymphocyte nuclei due to storage of blood in EDTA anti
Figure 791.1 Changes in blood cell morphology (crenation of red cells, separation of nuclear lobes of neutrophil, vacuoles in cytoplasm, and irregular lobulation of monocyte and lymphocyte nuclei) due to storage of blood in EDTA anticoagulant for prolonged time
 
Heparin
 
Heparin prevents coagulation by enhancing the activity of anti-thrombin III (AT III). AT III inhibits thrombin and some other coagulation factors. It is used in the proportion of 15-20 IU/ ml of blood. Sodium, lithium, or ammonium salt of heparin is used. Heparin should not be used for total leukocyte count (since it causes leukocyte clumping) and for making of blood films (since it imparts a blue background). It is used for osmotic fragility test (since it does not alter the size of cells) and for immunophenotyping.
 
Double Oxalate (Wintrobe Mixture)
 
This consists of ammonium oxalate and potassium oxalate in 3:2 proportion. This combination is used to balance the swelling of red cells caused by ammonium oxalate and shrinkage caused by potassium oxalate. Mechanism of anticoagulant action is removal of calcium. It is used for routine hematological tests and for estimation of erythrocyte sedimentation rate by Wintrobe method. As it causes crenation of red cells and morphologic alteration in white blood cells, it cannot be used for making of blood films.
 
Preparation
 
Ammonium oxalate 1.2 gm
Potassium oxalate 0.8 gm
Distilled water upto 100 ml
 
Place 0.5 ml of this solution in a bottle for 5 ml of blood. Anticoagulant should be dried in an incubator at 37°C or on a warm bench before use.
 
Trisodium Citrate (3.2%)
 
This is the anticoagulant of choice for coagulation studies and for estimation of erythrocyte sedimentation rate by Westergren method.
 
Preparation
 
Trisodium citrate 3.2 gm
Distilled water upto 100 ml
 
Mix well to dissolve. Store in a refrigerator at 2-8°C.
 
Use 1:9 (anticoagulant: blood) proportion for coagulation studies; for ESR, 1:4 proportion is recommended.
 
ESR should be measured within 4 hours of collection of blood, while coagulation studies should be performed within 2 hours.
 
Further Reading:
 

ABO GROUPING AND Rh D GROUPING

Published in Hemotology
Friday, 21 July 2017 11:48
ABO Grouping

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.
 
There are three methods for blood grouping: slide, tube and microplate. Tube and microplate methods are better and are employed in blood banks.
 
Further Reading:
 

FALSE REACTION IN ABO GROUPING

Published in Hemotology
Friday, 21 July 2017 11:19
  1. 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.
  2. 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.
  3. 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.
  4. 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.

Rh D GROUPING METHOD

Published in Hemotology
Friday, 21 July 2017 10:47
D antigen is the most immunogenic after ABO antigens and therefore red cells are routinely tested for D. Individuals are called as Rh-positive or Rh-negative depending on presence or absence of D antigen on their red cells. Following transfusion of Rhpositive blood to Rh-negative persons, 70% of them will develop anti Rh-D antibodies. This is of particular importance in women of childbearing age as anti-D antibodies can crosss the placenta during pregnancy and destroy Dpositive fetal red cells and cause hemolytic disease of newborn. In other sensitized individuals, reexposure to D antigen can cause hemolytic transfusion reaction.
 
In Rh D grouping, patient’s red cells are mixed with anti-D reagent. Serum or reverse grouping is not carried out because most Rhnegative persons do not have anti-D antibodies; anti-D develops in Rh-negative individuals only following exposure to Rh-positive red cells.
 
Rh typing is done at the same time as ABO grouping. Method of Rh D grouping is similar in principle to ABO grouping. Since serum or reverse grouping is not possible, each sample is tested in duplicate. Dosage effect (stronger antigenantibody reaction in homozygous cells i.e. stronger reaction with DD) is observed with antigens of the Rh system. Autocontrol (patient’s red cell + patient’s serum) and positive and negative controls are included in every test run. Monoclonal IgM anti-D antiserum should be used for cell grouping, which allows Rh grouping to be caried out at the same time as ABO grouping at room temperature. With monoclonal antisera, most weak and variant forms of D antigen are detected and further testing for weak forms of D antigen (Du) is not required. Differences between ABO and Rh grouping are shown in Table 788.1.
 
Table 788.1 Comparison of ABO grouping and Rh typing
Comparison of ABO grouping and Rh typing

Microplate Technique for Rh D Grouping

Published in Hemotology
Friday, 21 July 2017 10:28
Microplate is a polystyrene plate consisting of 96 micro wells of either U- or V-shape. Grouping is carried out in micro wells. This method is sensitive and ideal for large number of samples (see Figure 787.1).
 
Further reading: Rh D GROUPING METHOD

DETERMINATION OF BLOOD GROUP BY SLIDE METHOD

Published in Hemotology
Wednesday, 19 July 2017 13:46
Principle
 
Red cells from the specimen are reacted with reagent antisera (anti-A and anti-B). Agglutination of red cells indicates presence of corresponding antigen (agglutinogen) on red cells.
 
Specimen
 
Capillary blood from finger prick, or venous blood collected in EDTA anticoagulant.
 
Reagents
 
ABO antisera: See box 786.1 and Figure 786.1.
 
BOX ABO antisera
Box 786.1: ABO antisera
 
Anti A and anti B sera used for cell grouping
 Figure 786.1 Anti-A and anti-B sera used for cell grouping
 
Method
 
  1. 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).
  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.
  3. Add one drop of blood sample to be tested to each drop of antiserum.
  4. 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.
  5. By tilting the slide backwards and forwards, examine for agglutination after exactly two minutes.
  6. Result:
    Positive (+): Little clumps of red cells are seen floating in a clear liquid.
    Negative (–): Red cells are floating homogeneously in a uniform suspension.
  7. Interpretation: Interpret the result as shown in the Table 786.1 and Figure 786.2.
 
Table 786.1 Interpretation of cell grouping (forward grouping) by slide test
Anti-A Anti-B Blood Group
+ - A
- + B
+ + AB
- - O
 
Cell grouping by slide method
Figure 786.2 Cell grouping by slide method
 
Slide test is quick and needs only simple equipment. It can be used in blood donation camps and in case of an emergency. However, it is not recommended as a routine test in blood banks since weakly reactive antigens on cells on forward grouping and low titer anti-A and anti-B on reverse grouping may be missed. Also, drying of the reaction mixture at the edges causes aggregation that may be mistaken for agglutination. Results of slide test should always be confirmed by cell and serum grouping by tube method.

DETERMINATION OF BLOOD GROUP BY TUBE METHOD

Published in Hemotology
Wednesday, 19 July 2017 12:53
Test tube method is more reliable than slide test, but takes longer time and more equipment. For cell grouping, patient’s saline-washed red cells are mixed with known antiserum in a test tube; the mixture is incubated at room temperature, and centrifuged. For serum grouping, patient’s serum is mixed with reagent red cells of known group (available commercially or prepared in the laboratory), incubated at room temperature, and centrifuged (See Table). Following centrifugation, a red cell button (sediment) will be seen at the bottom of the tube. Cell button is dislodged by gently tapping the base of the tube and examined for agglutination.
 
Positive (+) Test
 
Clumps of red cells suspended in a clear fluid. Agglutination in tube test is graded from 1+ to 4+ and read macroscopically (See Figure). 
 
Grading of ABO tube test
Grading of ABO tube test. Negative: Uniform suspension of red cells; Grade 1 (1+): Many small clumps of red cells (fine granular appearance); Grade 2 (2+): Many large clumps with many free red cells; Grade 3 (3+): Three or four individual clumps with few free red cells; and Grade 4 (4+): One solid clump of red cells with no free red cells
 
Negative (–) Test
 
Uniform suspension of red cells.

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.
 
In two positive control tubes, anti-A serum is mixed with group. A red cells and anti-B is mixed with group B red cells respectively. In two negative control tubes, anti-A serum is mixed with group B red cells and anti-B serum is mixed with group. A red cells respectively. These controls are necessary to confirm that reagents are working properly.
 
Interpretation of forward (cell) and reverse (serum) grouping
Interpretation of forward cell and reverse serum grouping
 
Why test tube method of blood grouping is more reliable than slide method?
 
Test tube method of blood grouping is more reliable than slide method. This is because centrifugation enhances the reaction by bringing antigen and antibodies closer together and allows detection of weaker antigen antibody reactions; in addition drying is avoided and smaller amounts of reagent are required.
 
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.

DIFFERENTIAL LEUKOCYTE COUNT (MANUAL METHOD)

Published in Hemotology
Wednesday, 19 July 2017 11:45
A WBC differential count gives us information regarding the proportion and numbers of individual leukocytes in the patient’s sample, including significant morphological changes. This can provide useful diagnostic information in cases of inflammation, infection, and antigenic responses.

METHOD
 
Equipment
 
Stained PBS, microscope with 100×objective lens and cell counter.
 
Procedure
 
It is important that examinationand counts be performed withinthe monolayer area of your slide
 
  1. 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.
  2. Count a minimum of 100 WBCs.
 
(If the total WBC Count is increased, 200 cells should be counted to maintain accuracy.)
 
Calculations
 
Relative count:
 
No. of Cell Type Seen = ___%
100
 
Absolute count:
 
Relative (%) x WBC Count (10³/ L) = ___ x 10³/μL
100
 
Note: Check your math:
 
• Relative counts of each cell type should add up to equal 100
• Absolute counts of each cell type should add up to equal your WBC count.

TOTAL ERYTHROCYTE COUNT (MANUAL METHOD)

Published in Hemotology
Tuesday, 18 July 2017 13:44
Erythrocyte (Gr. erythros, red; kytos, cell) or red blood corpuscles are circular, anucleated, highly flexible, biconcave disc-shaped cells with high edges. The sixe of each cell averages 7.2 micrometer in diameter and 2.1 micrometer in thickness. It is 1.0 micrometer thick in the center. A complex membrane surrounds it, which is a bimolecular layer of protein. There is an inner most structure, called stroma, which is composed of lipids and proteins in the form of a fibrous protein. The cell contents are 90% hemoglobin. There are two methods for estimation of erythrocyte count:
 
  • Manual or microscopic method
  • Automated method
 
MANUAL METHOD

Equipment

Hemocytometer with cover glass, compound microscope.
 
 
Hayem’s diluting solution is prepared as follows:

HgCl2               0.05 gm
NaSO4               2.5 gm
NaCl                  0.5 gm
Distilled water     100 ml
 
Specimen
 
EDTA anticoagulated venous blood or blood obtained by skin puncture is used.
 
Method
 
  1. 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.
  2. Aspirate diluting Hayem’s solution to the 101 mark. It will give 1:200 dilution of the blood.
  3. Hold the pipette horizontally and role it with both hands between finger and thumb.
  4. Place the counting chamber, absolutely free from dust and grease, on the table and lay the cover glass in place over the ruled area.
  5. 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.
  6. 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.
  7. Count the erythrocytes in medium sized squares (80 smallest squares) using high power objective.
  8. 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.
 
Calculation
 
You may calculate total number of erythrocytes per cu mm of the blood as shown in the following.
 
Supose number of erythrocytes counted in 5 intermediate squares
 
= E
 
Area of each of the five squares in which cells are counted
 
= 1/25 sq mm
 
Therefore, total area counted
 
= 1/25 sq mm x 5
= 1/5 sq mm
 
Depth of chamber = 1/10 mm
 
Therefore, the volume in which cells are counted
 
= Area x Depth
= 1/5 sqmm x 1/10 mm
= 1/50 cu mm
 
Now, in 1/50 cu mm of diluted blood, the number of erythrocyte counted = E
 
Number of erythrocyte in one cu mm in diluted blood = E x 50
 
Since the dilution of the blood is 1 in 200, the number of erythrocytes in one cu mm of undiluted blood
 
= E x 50 x 200
 
GENERAL NOTES

(1) Increased in numbers of RBC called polycythemia it is due to
 
Congenital heart disease
• Cor pulmonale
Dehydration
• Pulmonary fibrosis
• Polycythemia vera
 
(2) Decreased in numbers of RBC is due to
 
• Anemia
Bone marrow failure
• Erythropoietin deficiency (2ndry to kidney disease)
Hemolysis (RBC destruction) from transfusion reaction
Hemorrhage
• Leukemia
• Multiple myloma
• Nutritional deficiencies of (Iron, Copper, Folate, Vit B12, B6)
 
REFERENCE RANGES

• 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
 
REFERENCES
 
  • 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.

MICRO METHOD FOR ESTIMATION OF PACKED CELL VOLUME (PCV) OR HEMATOCRIT

Published in Hemotology
Monday, 17 July 2017 17:57
Principle

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.

Equipment
 
  1. Microhematocrit centrifuge: It should provide relative centrifugal force of 12000 g for 5 minutes.
  2. 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.
  3. Tube sealant like plastic sealant or modeling clay; if not available, a spirit lamp for heat sealing.
  4. Microhematocrit reader; if not available, a ruler or arithmetic graph paper.
 
Specimen

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.

Method
 
  1. 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.
  2. 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.
  3. 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.
  4. Centrifuge at relative centrifu-gal force 12000 g for 5 minutes to completely pack the red cells.
  5. 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.
  6. 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 red cell column in mm
-------------------------------------------------------
Length of total column in mm
 
To obtain PCV, the above result is multiplied by 100.

GENERAL NOTES
 
  1. Prolonged application of tourniquet during venepuncture causes hemoconcentration and rise in hematocrit.
  2. Excess squeezing of the finger during skin puncture dilutes the sample with tissue fluid and lowers the hematocrit.
  3. Correct proportion of blood with anticoagulant should be used. Excess EDTA causes shrinkage of red cells and falsely lowers the hematocrit.
  4. Inadequate mixing of blood with anticoagulant, and inadequate mixing of blood before testing can cause false results.
  5. Low hematocrit can result if there are clots in the sample.
  6. Centrifugation at lower speed and for less time falsely increases PCV.
  7. 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.
  8. As PCV requires whole blood sample, it is affected by plasma volume (e.g. PCV is higher in dehydration, and lower in fluid overload).
  9. 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.
  10. 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
  11. Automated hematocrit: In automated hematology analyzers, hematocrit is obtained by multiplying red cell count (in millions/cmm) by mean cell volume (in femtoliters).
 
REFERENCE RANGES
 
  • 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%
 
CRITICAL VALUES
 
  • Packed cell volume: < 20% or > 60%
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