Clinical laboratory professional specialized to external quality assessment (proficiency testing) schemes for Laboratory medicine and clinical pathology. Author/Writer/Blogger
ABO antisera: See box 786.1 and Figure 786.1.
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.
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.
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:
Hemocytometer with cover glass, compound microscope.
Hayem’s diluting solution is prepared as follows:
EDTA anticoagulated venous blood or blood obtained by skin puncture is used.
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
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.
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.
To obtain PCV, the above result is multiplied by 100.
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.
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.
Hematocrit can be expressed either as a percentage or as a fraction of the total volume of blood sample.
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.
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.