Kleihauer–Betke test

Thin smears are prepared from capillary blood or venous blood collected into anticoagulants such as heparine, oxalate, citrate, or EDTA. Smears are air dried between 10–60 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.[4]

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.

KB stain with green marks at cells counted as fetal (HbF) cells, and red marks at incompletely colored cells at top and a too small cell at right

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 for Hemoglobin F cells in adults as published originally by Kleihauer were below 0.01%; in full-term newborns they are above 90%.

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

${\displaystyle PFB={\frac {(3200)(FC)}{(FW)(MC)}}}$

where

• • ${\displaystyle PFB}$ is the percentage of fetal blood lost;
  • ${\displaystyle FC}$ is the observed number of fetal red blood cells;
  • ${\displaystyle MC}$ is the observed number of maternal red blood cells (N.B. we have that ${\displaystyle MC=TC-FC}$, where ${\displaystyle TC}$ is the total observed number of red blood cells, both maternal and fetal);
  • ${\displaystyle FW}$ is the stillbirth weight of the fetus in kilograms.

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]

Suppose that a KB stain is performed and ${\displaystyle TC=5000}$ total red blood cells are observed, ${\displaystyle FC=200}$ of which are found to be fetal red blood cells. Suppose further that the stillbirth weight of the fetus under consideration is ${\displaystyle FW=2.0kg}$. Then we would conclude that the total percentage of fetal blood lost is approximately:

${\displaystyle PFB={\frac {(3200)(FC)}{(FW)(MC)}}={\frac {(3200)(200)}{(2.0)(4800)}}={\frac {200}{3}}=66.667}$

to five significant digits. We would hence conclude that the fetus under consideration lost 66.667% (two-thirds) of its blood via FMH. Generally, stillbirth is highly probable for any value of ${\displaystyle PFB\geq 20}$, particularly if the fetus abruptly loses this much blood; in this example, we would hence be likely to suspect FMH as the cause of the stillbirth. It is important to note, however, that such a diagnosis is still not completely conclusive; fetuses losing large quantities of blood over long periods of time are able to compensate for this slower blood loss; since the KB stain tells us nothing with regard to the level of acuity of FMH. This means that it is not possible to entirely correlate a positive KB stain and high ${\displaystyle PFB}$ with a stillbirth, though in many cases, given other information, such as known hereditary complications of pregnancy, extremely high positive correlation coefficients ${\displaystyle r\approx +1.000}$ between FMH and stillbirth have been observed.