Kleihauer–Betke test
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Kleihauer–Betke test | |
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Purpose | measures fetal hemoglobin transfer |
The Kleihauer–Betke ("KB") test, Kleihauer–Betke ("KB") stain, Kleihauer test or acid elution test is a
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,[3] 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.[2]
In those with positive tests, follow up testing at a
Comparison with other more expensive or technologically advanced methods such as
Original Technique
Method
Thin smears are prepared from capillary blood or venous blood collected into anticoagulants such as
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[5] 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
These constraints can then be applied to yield the formula
where
- is the percentage of fetal blood lost;
- is the observed number of fetal red blood cells;
- is the observed number of maternal red blood cells (N.B. we have that , where is the total observed number of red blood cells, both maternal and fetal);
- 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:[6]
- 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:[6]
- 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.[6]
Stillbirth resolution
Suppose that a KB stain is performed and total red blood cells are observed, of which are found to be fetal red blood cells. Suppose further that the stillbirth weight of the fetus under consideration is . Then we would conclude that the total percentage of fetal blood lost is approximately:
to five
Fetal red-blood-cell detection problems
Since fetal and maternal blood cells have the same life expectancy in the maternal bloodstream, it is possible to obtain informative results from a KB stain for a fair period of time after a stillbirth. However, if the mother and fetus are ABO incompatible, it is more crucial to quickly perform the KB stain following a stillbirth, as the fetal red blood cells will be eliminated from the maternal bloodstream quickly, causing the KB stain to underestimate the degree of FMH, if any. Much concern has been raised in the literature concerning false positives when sampling is done after delivery. In general this is not a problem. Delivery does result in higher frequency of detection of micro-hemorrhages but this should not confound interpretation of FMH as a possible cause of stillbirth. It is not necessary to draw the sample before induction, onset of labor, delivery, placental delivery etc. despite what some published literature purports. However, if Caesarean section is to be used, failure to draw the sample prior to that will result in a 2% false positive rate.[citation needed]
Finally, anything which causes persistence of fetal hemoglobin in maternal blood cells will make interpretation much trickier. Certain hemoglobinopathies, the most common of which is sickle cell trait, do this. Overall, somewhere around 1–3% of the time this could result in false interpretation.
All cases of maternal trauma
An article published in 2004 concluded that a Kleihauer-Betke (KB) test is necessary in all cases of maternal trauma, as clinical evaluation is not sensitive enough for determination of risk of pre-term labour. It accurately predicts the risk of preterm labor after maternal trauma whereas the article concluded that clinical assessment does not. With a negative KB test, posttrauma electronic fetal monitoring duration may be limited safely. With a positive KB test, the significant risk of pre-term labour mandates detailed monitoring. KB testing has important advantages to all maternal trauma victims, regardless of Rh status.[7]
See also
- Apt test
References
External links
- [1] The Kleihauer Test