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DETERMINATION OF BLOOD GROUP BY SLIDE METHOD

Published in Hemotology
Wednesday, 19 July 2017 08:46
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 07:53
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 06:45
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 08:44
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 12:57
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%

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

Published in Hemotology
Monday, 17 July 2017 12:34
Principle
 
Anticoagulated whole blood is centrifuged in a Wintrobe tube to completely pack the red cells. The volume of packed red cells is read directly from the tube. An advantage with this method is that before performing PCV, test for erythrocyte sedimentation rate can be set up.
 
Equipment
 
  1. Wintrobe tube: This tube is about 110 mm in length and has 100 markings, each at the interval of 1 mm. Internal diameter is 3 mm. It can hold about 3 ml of blood.
  2. Pasteur pipette with a rubber bulb and a sufficient length of capillary to reach the bottom of the Wintrobe tube.
  3. Centrifuge with a speed of 2300 g.
 
Specimen
 
Venous blood collected in EDTA (1.5 mg EDTA for 1 ml of blood) or in double oxalate. Test should be performed within 6 hours of collection.

Method
 
  1. Mix the anticoagulated blood sample thoroughly.
  2. Draw the blood sample in a Pasteur pipette and introduce the pipette up to the bottom of the Wintrobe tube. Fill the tube from the bottom exactly up to the 100 mark. During filling, tip of the pipette is raised, but should remain under the rising meniscus to avoid foaming.
  3. Centrifuge the sample at 2300 g for 30 min (To counterbalance a second Wintrobe tube filled with blood from another patient or water should be placed in the centrifuge).
  4. Take the reading of the length of the column of red cells.
 
Hematocrit can be expressed either as a percentage or as a fraction of the total volume of blood sample.
 
Significance
 
In anemia, PCV is below the lower level of normal range. PCV is raised in dehydration, shock, burns, and polycythemia.

After centrifugation of anticoagulated whole blood, three zones can be distinguished in the Wintrobe tube from above downwards-plasma, buffy coat layer (a small greyish layer of white cells and platelets, about 1 mm thick), and packed red cells. Normal plasma is straw-colored. It is colorless in iron deficiency anemia, pink in the presence of hemolysis or hemoglobinemia, and yellow if serum bilirubin is raised (jaundice). In hypertriglyceridemia, plasma appears milky. Increased thickness of buffy coat layer occur if white cells or platelets are increased in number (e.g. in leukocytosis, thrombocytosis, or leukemia). Smears can be made from the buffy coat layer for demonstration of lupus erythematosus (LE) cells, malaria parasites, or immature cells.

WHAT IS PACKED CELL VOLUME (PCV) OR HEMATOCRIT? USES AND METHODS FOR ESTIMATION OF PACKED CELL VOLUME (PCV)

Published in Hemotology
Monday, 17 July 2017 11:02
Packed cell volume (PCV) is the volume occupied by the red cells when a sample of anticoagulated blood is centrifuged. It indicates relative proportion of red cells to plasma. PCV is also called as hematocrit or erythrocyte volume fraction. It is expressed either as a percentage of original volume of blood or as a decimal fraction.
 
USES OF PCV
 
  • Detection of presence or absence of anemia or polycythemia
  • Estimation of red cell indices (mean cell volume and mean corpuscular hemoglobin concentration)
  • Checking accuracy of hemoglobin value (Hemoglobin in grams/dl × 3 = PCV).
 
There are two methods for estimation of PCV: macro method (Wintrobe method) and micro method (microhematocrit method). Micro method is preferred because it is rapid, convenient, requires only a small amount of blood, capillary blood from skin puncture can be used, and a large number of samples can be tested at one time.

This method is also more accurate as plasma trapping in red cell column is less.

OXYHEMOGLOBIN METHOD FOR ESTIMATION OF HEMOGLOBIN

Published in Hemotology
Sunday, 16 July 2017 19:21
In this method, blood sample is mixed with a weak ammonia solution. Absorbance of this solution is measured in a spectrophotometer at 540 nm or in a photometer using a yellow-green filter. Absorbance of the test sample is compared with that of the standard solution.

This method is rapid and simple. However, no stable standard solution is available, color of oxyhemoglobin solution rapidly fades, and hemoglobin derivatives other than oxyhemoglobin are not measured.

SALT TOLERANCE TEST

Published in Microbiology
Wednesday, 05 April 2017 10:10

Objective: To test organism's ability to tolerate various osmotic concentrations.

Test Procedure
1. Use a sterile loop or needle to inoculate broth tubes with different salt concentrations.
2. Incubate at the optimum temperature for 48-96 hours.

• Interpretation
Positive = growth; Negative = no growth

PENICILLIN DISC TEST

Published in Microbiology
Wednesday, 05 April 2017 09:45

Objective: To test the organism's susceptibility to antibiotic penicillin.

Test Procedure and Interpretation: See the Optochin Disc Test.

Discovery of Penicillin

The discovery of penicillin's antibiotic powers is attributed to Alexander Fleming. The story goes that he returned to his laboratory one day in September 1928 to find a Petri dish containing Staphylococcus bacteria with its lid removed.

The dish had become contaminated by blue-green mold. He noted that there was a clear ring surrounding the mold where the bacteria had been inhibited from growing.

This discovery of the mold - Penicillium notatum - and his recognition of its special powers set the wheels in motion to create one of the most used drugs in medical history.

In March 1942, Anne Miller became the first civilian to be treated successfully with penicillin having almost died from a huge infection following a miscarriage.

Although Fleming often gets the accolade for having invented the first antibiotic, there was a lot of work to do before penicillin could become as commonly used and useful as it is today.

The bulk of the work was eventually carried out by scientists who had a much better-stocked laboratory and a deeper understanding of chemistry than Fleming. Dr. Howard Florey, Dr. Norman Heatley, and Dr. Ernst Chain carried out the first in-depth and focused studies.

Interestingly, and with impressive foresight, Fleming's Nobel Prize acceptance speech warned that the overuse of penicillin might, one day, lead to bacterial resistance.

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