The Kleihauer-Betke test is used to determine the degree of fetal-maternal hemorrhage (FMH) and to help calculate the dosage of Rh immune globulin (RhIG)—Rho(D) RhoGAM IM or Rhophylac IM or IV—to be given in some cases of Rh-negative mothers. Administration of RhIG inhibits formation of Rh antibodies in the mother to prevent Rh disease in future pregnancies with Rh-positive children. The test is also used to resolve the question of whether FMH was the cause of fetal death in the case of stillbirth. A sample of maternal blood should be collected within 1 hr of delivery. A blood film of maternal red blood cells (RBCs) is prepared, treated with an acid buffer, and stained. The acid solution causes hemoglobin to be leached from the maternal cells, giving them a ghostlike appearance. Fetal cells containing hemoglobin F retain their hemoglobin and are stained bright red. Approximately 2,000 cells are examined microscopically and counted. The ratio between maternal cells and fetal cells is determined by dividing the number of fetal cells present by the total number of cells counted. The percentage of fetal cells is determined by multiplying the ratio by 100. For example if 12 fetal cells were identified in a total cell count of 2000, then 12/2000 = 0.006 × 100 = 0.6% fetal cells. The estimated number of fetal cells present in maternal circulation is used to calculate the quantity of fetal bleed in milliliters of fetal whole blood. FMH is calculated by dividing the percentage of fetal cells by 100 (to convert the percentage back to an absolute number) and then multiplying by 5000 (based on the assumption that maternal blood volume is 5 liters or 5000 mL). For example, if the percentage of fetal cells counted is 0.6%, then FMH = (0.6/100) × 5000 = 30 mL. The fetal blood cell volume can also be used to calculate the quantity of fetal bleed in milliliters of fetal packed cells. For example, if the fetal blood cell volume is 30 mL the packed cell volume would be 15 mL, based on the assumption that the hematocrit of fetal whole blood is 50%. The FMH is used to estimate the dosage of RhIG needed. A commonly accepted recommendation is that one 300 mcg dose of RhIG will cover 30 mL of fetal whole blood or 15 mL fetal cells. In the example the FMH was determined to be 30 mL of whole blood. Therefore, 30 mL of fetal blood divided by 30 mL covered per dose indicates that a single dose of RhIG should be administered. In another example if the FMH was determined to be 55 mL then 55/30 = 1.8. When the number after the decimal is 5 or greater the dose would be rounded up to the next whole number plus one, meaning 3 doses would be required. In a third example if the FMH was determined to be 40 mL then 40/30 = 1.3. When the number after the decimal is less than 5 the dose would be rounded down to the next whole number plus one, meaning 2 doses would be required. Calculation of RhIG dosage is based on the calculated size of FMH and should only be done after reviewing the information in the manufacturer’s package insert. Postpartum RhIG should be given within 72 hr of delivery. The test can also be used to distinguish some forms of thalassemia from the hereditary persistence of fetal hemoglobin, but hemoglobin electrophoresis and flow cytometry methods are more commonly used for this purpose.
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