The Kleihauer–Betke ("KB") test, Kleihauer–Betke ("KB") stain, Kleihauer test or acid elution test is a blood test used to measure the amount of fetal hemoglobin transferred from a fetus to a mother's bloodstream. It is usually performed on Rh-negative mothers to determine the required dose of Rho(D) immune globulin (RhIg) to inhibit formation of Rh antibodies in the mother and prevent Rh disease in future Rh-positive children. It is named after Enno Kleihauer and Klaus Betke who described it in 1957.
Test details The KB test is the standard method of quantitating fetal–maternal hemorrhage (FMH). It takes advantage of the differential resistance of fetal hemoglobin to acid. A standard blood smear is prepared from the mother's blood and exposed to an acid bath. This removes adult hemoglobin, but not fetal hemoglobin, from the red blood cells. Subsequent staining, using Shepard's method, makes fetal cells (containing fetal hemoglobin) appear rose-pink in color, while adult red blood cells are only seen as "ghosts". 2,000 cells are counted under the microscope and a percentage of fetal to maternal cells is calculated. In those with positive tests, follow up testing at a postpartum check should be done to rule out the possibility of a false positive. This could be caused by a hemoglobinopathy in the mother which causes persistent elevation of fetal hemoglobin, e.g. sickle cell trait. Comparison with other more expensive or technologically advanced methods such as flow cytometry has shown that the KB stain, like the more advanced methods, is sensitive in its detection of FMH. However, publications have shown that in comparison with flow cytometry methods, the KB Test overestimates FMH due to false positive results when F-cells are present in the maternal blood sample. Background counting errors can result in estimates of as much as 5 mL fetal blood loss when there actually is no such blood loss, but standard methods available in most laboratories admit an extremely low probability of the return of a false positive when more severe FMH has taken place.
Original technique
Method Thin smears are prepared from capillary blood or venous blood collected into anticoagulants such as heparin, oxalate, citrate, or EDTA. Smears are air dried between 10 and 60 minutes, fixed in 80 vol% ethanol for 5 min at 20-22 °C, rinsed with tap water, and air dried. Films are then immersed in the citrate-phosphate buffer for 5 minutes at 37 °C and gently agitated for about 3 minutes. Slides are rinsed with tap water, dried, and stained with Ehrlich's acid hematoxylin for 3 min, rinsed with water, and dried again. They are counterstained with erythrosine for 3 min. After a final rinse, films are dried and examined under light microscopy.
Results of the original method Hemoglobin F cells are densely stained with erythrosine, Hemoglobin A cells appear as ghost cells, while intermediate cells are stained more or less pink. Reticulocytes containing Hemoglobin A may appear as intermediate cells and/or may show intracellular granulation. Inclusion bodies (Heinz bodies, precipitated α-chains or β-chains) are visible in eluted cells as compact inclusions of different size. Hemoglobin A is eluted regardless of whether it is oxyhemoglobin, methemoglobin, cyanmethemoglobin, reduced hemoglobin, or carboxyhemoglobin.
Quantitation of hemoglobin F cells
Methods developed by Schneider and Ludwig and Bartsch' are recommended. For determination of the intracellular distribution of Hemoglobin F, the semi-quantitative method of Shepard, Weatherall, and Conley' may be employed.
Normal values Normal values for hemoglobin F cells in adults as published originally by Kleihauer were below 0.01%; in full-term newborns they are above 90%.
Uses
Fetal–maternal hemorrhage severity estimation To determine if a positive test for FMH indicates the likely cause of fetal death, the percent of total fetal blood volume lost should be calculated, making appropriate adjustments based on the following known relationships:
the size of a fetal red blood cell is 1.22 times that of an adult red blood cell; the KB stain is known to have a mean success rate of 92% in detecting fetal red blood cells; in a woman at or near term in her pregnancy, the mean volume of maternal red blood cells is approximately 1800 ml; the mean fetal hematocrit is 50%; and at stillbirth, the mean fetal blood volume is 150 m l k g {\displaystyle 150{\frac {ml}{kg}}}
These constraints can then be applied to yield the formula
where
P F B {\displaystyle PFB} is the percentage of fetal blood lost;
F C {\displaystyle FC} is the observed number of fetal red blood cells;
M C {\displaystyle MC} is the observed number of maternal red blood cells (N.B. we have that M C = T C − F C {\displaystyle MC=TC-FC} , where T C {\displaystyle TC} is the total observed number of red blood cells, both maternal and fetal);
F W {\displaystyle FW} is the stillbirth weight of the fetus in kilograms.
Number of RhD vials An estimate of the required number of Rho(D) immune globulin vials may assume the following equations:
Volume (mL) of Fetal Blood = % Fetal Cells x 50 Number of Vials of 300 mcg RhIG Required = Volume of Fetal Blood/30mL Combining those two equations results in:
Number of vials = % Fetal Cells x 50 / 30 This is approximately equal to:
Number of vials = % Fetal Cells x 1.7 Practically, if the number to the right of the decimal point is ≥5, it is rounded up to add one vial.
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