Kleihauer–Betke test

From patholines.org
Jump to navigation Jump to search

Author: Mikael Häggström [note 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.

Collection

EDTA-containing-tube.

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.

To count, 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.

Semi-quantification

This is done for Rh-positive mothers to estimate the severity of a suspected feto-maternal hemorrhage, which can be suspected by various clinical findings (including neonatal anemia, stillbirth, intrauterine growth restriction, hydrops fetalis, decreased or absent fetal movements, non-reassuring fetal heart rate tracing, sinusoidal fetal tracing, and fetal tachyarrythmias, placenta previa with bleeding, and placental abruption)[1].

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

Quantification

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

2000 cells are counted, in order to give a percentage calculated as:

  • Fetal RBCs (given in%) = (Fetal RBC count) / (Total cell count) *100

Alternatively, an acceptable estimation of at least 2000 cells can be done by using a micrograph (or a microscopy grid) to estimate the mean number of cells in a certain area, and using the same mean to estimate the number of cells in equally sized areas:
1. Count cells (both adult and fetal) until reaching 100 cells (including each cell in square by square if using a microscopy grid). Take note of how large micrograph area (or how many grid squares) were counted (here designated as x amount), and how many fetal RBCs were counted.
2. Pick another random location (you may randomize again if it is of a significantly different cell density, but do not let your decision be influenced by the number of fetal RBCs in the area or near its edge). Count the total number of cells in the same area size (or same x number of squares), and how many of them are fetal RBCs.
3. Pick another location again, and count the number of cells in the same area size, and how many of them are fetal RBCs.

  • If a count of 200-300 cells only shows 0 or 1 fetal RBC, 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.
Standard
deviation
Count cells
in following
number of areas
Up to 6 3
7 4
8 5
9 6-7
10 8
11 10
12 12
  • Calculate how much the count for the second and third areas deviated from 100, and take the average thereof, which will be used as standard deviation.[note 2] If the standard deviation is higher than 12, count a total of 2000 cells regardless of areas and calculate as per formula above, and the rest of the steps can be skipped.
  • Use the table at right to estimate how many areas in total you need to count in order to have a mean number of cells per area with an acceptable confidence interval.

4. After having counted the needed number of areas, calculate the average of the number of cells per area (or per x number of squares), and assume that number for the rest of the counting.
5. Divide 2000 by the number of cells per area, and round that up to know the number of areas you need to perform the next step on in order to presumably have a total of 2000 cells (including previously counted areas).
6. In those additional areas, only count the number of fetal cells per area (or x number of squares), and add that to the fetal cells from previous areas.

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

Example:

The sum of all fetal RBCs in all areas in this example is 45. The presumable total cell count is 103 * 20 = 2060. Thus:

  • Fetal RBCs (given in%) = 45 / 2060 * 100 = 2.2%

Calculation of number of vials

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,[2] or rounded to the closest full number and then adding 1.[3]

Number of vials = Fetal RBCs in% * 1.7

For example, with 2.2% fetal RBCs, the number of vials would be 4[2] or 5[3].

Notes

  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.
  2. 2.0 2.1 Technically, the standard deviation would be calculated as the average deviation from the mean of all areas, but the simplified calculation used in this resource can be regarded to be close enough for practical purposes.

Main page

References

  1. Solomonia N, Playforth K, Reynolds EW (2012). "Fetal-maternal hemorrhage: a case and literature review. ". AJP Rep 2 (1): 7-14. doi:10.1055/s-0031-1296028. PMID 23946896. PMC: 3653511. Archived from the original. . 
  2. 2.0 2.1 Diann M. Krywko. Kleihauer Betke Test. StatPearls, National Center for Biotechnology Information. Last update: Last Update: January 20, 2020.
  3. 3.0 3.1 Practice at Danbury Hospital, Danbury, Connecticut, New England.

Image sources