Erythrocyte Indices and Fragility Tests
By using accurately determined red blood cell counts, hematocrits, and hemoglobin values, the size and hemoglobin content of the average red cell in a given blood sample is calculated. The values obtained are the erythrocyte indices which aid in the classification and study of anemias. They consist of the mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).
Calculation of Erythrocyte Indices.
- Mean corpuscular volume (MCV)--The average volume of the individual red blood cell. Femtoliter (fl) or 10-15 liter = 1 fl.
- Mean corpuscular hemoglobin (MCH)--The average weight of hemoglobin of the individual red cell.
- Mean corpuscular hemoglobin concentration (MCHC)--The percent of hemoglobin in the average red cell.
Accurate individual determinations of hemoglobin, hematocrit, and erythrocyte count ensure reliable indices. The following procedures are recommended:
- Erythrocyte count--two separate pipets and two to four counting chambers or electronic cell counting.
- Hemoglobin--precise reagent standards and accurate instrument calibration.
It is useful to compare the calculated indices with a stained peripheral blood smear.
Wintrobe classified anemias into the following groups on the basis of the indices (Figure 7-2).
Figure 7-2. MCV, MCH, and MCHC indices.
The MCHC cannot exceed the normal value since the erythrocyte cannot be supersaturated with hemoglobin. The MCHC is the most valid of the indices since it does not require the erythrocyte count in its deviation. It is a good index of iron deficiency.
The MCV and MCH are increased at birth and fall to low values during childhood. The MCHC is fairly constant for all ages.
- Mean corpuscular volume: 80 to 96 fl.
- Mean corpuscular hemoglobin: 27 to 32 micromicrograms.
- Mean corpuscular hemoglobin concentration: 32 to 36 percent.
ERYTHROCYTE OSMOTIC FRAGILITY TEST (DACIE)
A specific amount of blood is introduced into a series of tubes containing different concentrations of buffered salt solutions. The amount of hemolysis is then determined by examining the supernatant fluid either visually or with a spectrophotometer.
Sources of Error.
- The concentration of the NaCl in the solutions is critical. The salt must be chemically pure and dried before weighing. It is advisable to dry the salt in a 100șC oven and store it in a desiccator. Store the NaCl solutions in a glass-stoppered, tightly sealed bottle.
- Inaccurate preparation of the dilutions causes inaccurate results.
- Maintain the pH of the solution at an interval of 7.35-7.50. A different pH range causes invalid results.
- Rough handling of the blood specimen causes hemolys is which leads to invalid results.
In hypotonic salt solutions, erythrocytes take up water, swell to a spheroid shape and burst. In congenital spherocytic anemia the red cells with defective structure more rapidly rupture at salt concentrations closer to isotonicity (0.85 percent). These cells thus show an increased osmotic fragility. In contrast, the flat or thin but otherwise normal red cells of hypochromic anemia show a decreased osmotic fragility and do not hemolyze until lower salt concentrations are reached.
When hemolysis begins beyond the range of the prepared solutions or when intermediate dilutions are desired, the additional dilutions are readily prepared using the 1 percent sodium chloride stock solution.
In cases where the results of the fragility test are borderline, the following procedure is recommended to enhance any latent abnormality in fragility. Incubate samples of defibrinated blood (control and patient's) at 37șC for 24 hours under sterile conditions and controlled pH (7.35 to 7.50). The test is then performed as described above.
Decreases in pH increase osmotic fragility. The reagents are buffered to maintain a constant pH of 7.35 to 7.50.
This test may also be run visually, with some sacrifice of accuracy, by allowing the blood-saline dilutions to stand at 20șC for 45 minutes. The tubes are then lightly centrifuged (1,000 rpm for 3 minutes) and observed for signs of initial and complete hemolysis. A slight pink coloration of the supernatant fluid indicates initial hemolysis and a clear red solution, free of sediment, indicates complete hemolysis. The salt concentrations in these two tubes are noted and recorded. The control should always be reported along with results of patient's tubes.
- 0.30% saline: 97 to 100 percent hemolysis.
- 0.35% saline: 90 to 99 percent hemolysis.
- 0.40% saline: 50 to 90 percent hemolysis.
- 0.45% saline: 0 to 45 percent hemolysis.
- 0.50% saline: 0 to 5 percent hemolysis.
- 0.55% saline: 0 percent hemolysis.
HAM TEST FOR ERYTHOCYTE FRAGILITY
This test is positive in paroxysmal nocturnal hemoglobinuria (PNH). Erythrocytes in this form of anemia lyse easily in slight variations in the pH (acid). In this test, the erythrocytes are subjected to pH values ranging from 6.5 to 7.0 at 37șC.
- With a positive test the tubes containing acidified sera and patient's cells should show considerable hemolysis.
- Normally no tubes should show hemolysis.
- Occasionally, tubes with unacidified sera and patient's cells may show moderate hemolysis.
- A false positive test is sometimes seen in congenital spherocytic anemia.
- If congenital spherocytic anemia is suspected, the test should be repeated, using acidified serum previously inactivated at 56șC for 30 minutes.
- Since erythrocytes of PNH require complement for hemolysis, the modified test (item 5 above) will be negative in PNH and will remain positive in spherocytosis.
Hemolysis in the acidified tube is indicative of paroxysmal nocturnal hemoglobinuria.