Dayyal Dg.

Dayyal Dg.

Clinical laboratory professional specialized to external quality assessment (proficiency testing) schemes for Laboratory medicine and clinical pathology. Author/Writer/Blogger

Friday, 21 July 2017 05:47

Rh D GROUPING METHOD

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
Friday, 21 July 2017 05:28

Microplate Technique for Rh D Grouping

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

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.

Wednesday, 19 July 2017 07:53

DETERMINATION OF BLOOD GROUP BY TUBE METHOD

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.

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.
Tuesday, 18 July 2017 08:44

TOTAL ERYTHROCYTE COUNT (MANUAL METHOD)

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.

Reagent

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 (Female) 4.2-5.0 millions
  • Pregnancy slightly lower than normal

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%

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.

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.

By this method, the approximate value of hemoglobin is estimated. This method is simple and rapid. This method is most common in the blood bank for the selection of blood donors.

In this method, a drop of the blood sample is allowed to fall in the solution of copper sulfate having specific gravity 1.053 from the altitude of 1 cm. The hemoglobin concentration of 12.5 g/dl is equivalent to the specific gravity of 1.053. The drop of blood gets covered with copper proteinate and remains separate and distinct for 15-20 seconds. If the drop of blood sample sinks within 15-20 seconds, the specific gravity of copper sulfate solution is lower than the specific gravity of blood sample and the approximate value of hemoglobin is more than 12.5 grams/dl and hemoglobin level is acceptable for the donation of blood. If the drop of blood sample floats, hemoglobin value is less than 12.5 grams/dl and unacceptable for blood donation. However, the concentration of plasma proteins and total leukocyte count also influence the specific gravity of whole blood which may lead false-positive result. In the existence of hypergammaglobulinemia (e.g. multiple myeloma) or leukocytosis (e.g. myeloid or lymphoid reaction, chronic myeloid or lymphocytic leukemia), hemoglobin level will be misleadingly high.

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