Kleihauer Betke Test

Article Author:
Diann Krywko
Article Author:
Siva Naga Yarrarapu
Article Editor:
Sara Shunkwiler
9/11/2020 2:34:54 PM
PubMed Link:
Kleihauer Betke Test


Fetomaternal hemorrhage (FMH) occurs when there is a break in the placental barrier, allowing blood from the fetal circulation to enter the maternal circulation.  This disruption in the placental barrier may occur for many reasons, including intra-uterine fetal demise and trauma.  Trauma is the number one cause of pregnancy-associated maternal deaths in the United States.[1]. It occurs in as many as 40% of traumas, increasing in frequency and amount with high-force trauma, blunt force trauma, abdominal trauma, and with anterior placental placement in the uterus.

When FMH occurs, fetal hemoglobin (HgF) is mixed with maternal blood.  In response to this exposure, the maternal immune system is activated, and isoimmunization (formation of Anti-RhD antibodies) may occur if the mother is Rhesus-D protein (RhD) negative and the blood type of the fetus is RhD positive. It takes only 0.01 ml to 0.03 ml of FMH to isoimmunize the mother.   Future pregnancies may be at risk for RhD disease if the fetus is RhD positive. The maternal antibodies bind to fetal RhD +ve erythrocytes, leading to hemolysis, anemia, hydrops fetalis and possible fetal death.  

To prevent the formation of Anti-RhD antibodies, RhoGAM (IgG Anti-D (Anti-Rh)) is indicated.  Before 12-weeks gestational age, in the setting of a RhD negative mother and FMH, a mini-dose of 150 mcg (MICRhoGAM) is given.  This dose will suppress the immune response to 2.5 mL of Rh-positive red blood cells.  After 12-weeks gestational age, a dose of 300 mcg is recommended. This standard dose of RhoGAM (300 mcg) covers FMH up to 15 mL of fetal red cells (30 mL of whole fetal blood).  However, there are times when the additional dose is necessary due to massive red blood cell FMH and subsequent maternal immune response.  This is when the Kleihauer-Betke (KB) test is essential. (See Potential Diagnosis section for preliminary Rosette testing).

Specimen Collection

The specimen is collected from the maternal patient through peripheral venous phlebotomy.


Korber noted in 1864 that HgF was resistant to alkali denaturation with NaOH much more so than adult hemoglobin (HgA). It was further determined that, when immersed in a citrate buffer pH of 3.3, the HgF remained intact and the HgA leaked out.  Utilizing this property, the KB test was first described in 1957 by Enno Kleihauer and Klaus Betker [2]. The KB test is an acid-elution assay performed on maternal blood to determine the amount of HgF that has passed into maternal circulation.  The process exposes maternal blood smear to an acid solution.  HgF, being resistant to the acid, removes intact, whereas HgA is removed. Following this, the smear is stained via Shepard’s method*.  The fetal red blood cells are left rose-pink in color, and the maternal cells appear “ghost-like” due to the absence of staining.  Though manual detection and quantification had been widely used, flow cytometry was shown to be more precise* and now may be utilized.  A total of 2000 cells is counted.  Calculation of the percentage of fetal vs. maternal cells is used to estimate the total amount of FMH.  [3]

Fetal Cells % = Number of Fetal Cells X 100 / Total Number RBCs


KB testing has obstetrical implications in diagnosis and prognosis of preterm labor, fetal demise, and other conditions. 

There is some controversy on KB testing in the setting of trauma in pregnancy. This test has been historically only recommended for the Rh-negative pregnant patient with major trauma. However, the term ‘major’ is not specifically defined by most authors.  Intuitively, however, the risk of FMH would increase with higher magnitude blunt force, anterior placental location, and coagulopathies among other factors. Some advocate its use in all pregnant trauma patients, including those who are RhD negative.  It has been shown that a positive KB test accurately predicts the risk of preterm labor following trauma, whereas clinical assessment does not [4].  Because this is an independent risk factor, many authors recommended routine use in the setting of trauma, regardless of Rh status and regardless of the mechanism or force of the trauma.  The result then is used to guide management and education on prognosis.[5]

Potential Diagnosis

The Rosette test is a qualitative screening test performed on a maternal blood sample to determine if FMH has occurred between a Rh-positive fetus and a Rh-negative mother and serves as a useful screening test.   Qualitative testing may be utilized prior to quantitative testing, i.e., if the Rosette test is positive, a KB test should be performed to confirm and then subsequently quantify the amount of FMH.  In the case of massive trauma, the KB test may be utilized primarily, without the preliminary use of the screening Rosette test.  The Kleihauer Betke test is utilized to determine if there is fetal blood in maternal circulation, with a threshold of 5 mL.

The Rosette test is performed by incubating Rh negative maternal venous whole blood sample with anti-Rho(D) immune globulin.  The maternal cells are left unbound to the Anti-Rho(D) as they are Rho(D) negative.  During this incubation period, any Rh-positive fetal cells in the maternal sample are sensitized to the anti-Rho(D) immune globulin and bound.  Enzyme treated indicator cells are added, only binding to the fetal cells that were present and sensitized, resulting in a process called erythrocyte rosetting, or E-rosetting.  The indicator cells will be at the center of the rosette, while the fetal RBCs will be clustered around the edges, like petals on a flower.  This rosetting pattern may then be viewed under microscopy.

Kleihauer Betke test is utilized to determine if there is fetal blood in maternal circulation, with a threshold of 5 mL.

Normal and Critical Findings

Any value not reported as zero (0) is abnormal and indicated FMH.

Interfering Factors

In the case of maternal persistence of fetal hemoglobin or other maternal hemoglobinopathies that result in elevated HbF, the KB test will be falsely positive and flow cytometry must be used to quantitate the amount of fetal hemorrhage in maternal circulation.


The KB test is highly specific, with low sensitivity, having a threshold of 5 mL of FMH to be positive.  Keep in mind that the amount of FMH to cause isoimmunization is only 0.01 mL to 0.03 mL.  As such, it is not a test to determine if there is FMH, but rather, to better estimate the amount of FMH.  If positive, it is used to determine additional RhoGAM dosing over the standard 150 or 300 mcg dosage that should be administered.

Clinical Significance

Calculating the RhIg Dosage

The following calculations should be used to determine an additional number of vials of RhoGAM needed if the KB test is positive, indicating a large amount of FMH.  1 vial contains 300mcg and will protect against 30 mL of fetal blood.

Volume (mL) of Fetal Blood = % Fetal Cells x 50

Number of Vials of 300 mcg RhIG Required= Volume of Fetal Blood/30mL

Combining Equations:  # of Vials* = % Fetal Cells x 50 / 30

*  If the number to the right of the decimal point is <5, round down and add one vial

* If the number to the right of the decimal point is ≥5, round up and add one vial  Example 1:  If 2.9% fetal cells are reported on KB, what dose of RhIG would be required?

  • Volume of Fetal Blood:  2.9% x 50 = 145 mL
  • Number of Vials = 145 mL/30 = 4.83
  • The decimal is >5, therefore round up and add 1 vial (5+1).
  • Answer: 6 Vials

Example 2:  If 1.1% fetal cells are reported on KB, what dose of RhIG would be required?

  • Volume of Fetal Blood: 2.6% x 50 = 130 mL
  • Number of Vials = 130 mL/30 = 4.33
  • The decimal is <5, therefore round down and add 1 vial (4+1).
  • Answer: 5 Vials


[1] Homicide: a leading cause of injury deaths among pregnant and postpartum women in the United States, 1991-1999., Chang J,Berg CJ,Saltzman LE,Herndon J,, American journal of public health, 2005 Mar     [PubMed PMID: 15727979]
[2] [Demonstration of fetal hemoglobin in erythrocytes of a blood smear]., KLEIHAUER E,BRAUN H,BETKE K,, Klinische Wochenschrift, 1957 Jun 15     [PubMed PMID: 13450287]
[3] Improvement of the Kleihauer-Betke test by automated detection of fetal erythrocytes in maternal blood., Pelikan DM,Mesker WE,Scherjon SA,Kanhai HH,Tanke HJ,, Cytometry. Part B, Clinical cytometry, 2003 Jul     [PubMed PMID: 12827662]
[4] Persistent fetal hemoglobin in maternal circulation complicating the diagnosis of fetomaternal hemorrhage., Kush ML,Muench MV,Harman CR,Baschat AA,, Obstetrics and gynecology, 2005 Apr     [PubMed PMID: 15802419]
[5] Kleihauer-betke testing is important in all cases of maternal trauma., Muench MV,Baschat AA,Reddy UM,Mighty HE,Weiner CP,Scalea TM,Harman CR,, The Journal of trauma, 2004 Nov     [PubMed PMID: 15580038]