Kleihauer–Betke test

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Author: Mikael Häggström [notes 1]
The Kleihauer–Betke test stains fetal red blood cells (cells containing HbF) dark reddish-pink, while adult red blood cells will be white to light pink.



Criteria for fetal cells

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.

A fetal blood cell should be:

  • Stained more than approximately half of what is seen in control.
  • Not be nucleated or too big (white blood cell are generally also stained).
  • Not be too small.


This is done for Rh-negative mothers, with the following main indications:

  • Fetal demise
  • Bleeding due to placenta previa
  • Placental abruption

10HPFs (in 40x) are scanned, and fetal RBCs are counted (cells per 10 HPFs, not average per HPF), and classified as:

  • 0 - Negative
  • 1 - Rare
  • 2-5 - Few
  • 6-10 - Moderate
  • >10 - Abundant


This is done for Rh-negative mothers to estimate the number of Rho(D) immune globulin vials to administer.

  • Approximately 2000 cells are counted. It can be done by using a microscopy grid (or micrograph) as follows:
  • Count cells (both adult and fetal) in square by square until reaching 100 cells. Take note of how many squares (or how large micrograph area) were counted (x amount), and how many fetal RBCs were counted.
  • Switch to another area of seemingly similar cell density, and count the cells in the same (x) number of squares (or micrograph area), and how many of them are fetal RBCs.
  • If the count deviates no more than 6-7 from the first count, repeat in another 1-2 areas. If it is more inconsistent, repeat in more areas.
  • If a count of 200-300 cells only shows 0 or 1 stained cell, there only needs to be 1 vial of 300 micrograms Rho(D) immune globulin, and the rest of the steps in this section can be skipped.
  • Calculate the average of the number of cells counted in x number of squares, and assume that number for the rest of the counting.
  • In additional areas, only count the number of fetal cells in x number of squares, until presumably exceeding a total of 2000 cells counted.

Fetal RBCs (given in%) = (Fetal RBC count) / (Presumable total cell count) *100

Assuming that a vial of 300 micrograms of Rho(D) immune globulin will protect against 30 mL of fetal blood, the number of vials needed to compensate for the fetal-maternal transfusion is calculated as following, rounded up,[1] or rounded to the closest full number and then adding 1.[2]

Number of vials = Fetal RBCs in% * 1.7


  1. For a full list of contributors, see article history. Creators of images are attributed at the image description pages, seen by clicking on the images. See Patholines:Authorship for details.

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  1. Diann M. Krywko. Kleihauer Betke Test. StatPearls, National Center for Biotechnology Information. Last update: Last Update: January 20, 2020.
  2. Practice at Danbury Hospital, Danbury, Connecticut, New England.