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Is the Kleihauer-Betke test a reliable indicator of foetomaternal haemmorrhage?

Three Part Question

In [pregnant patients who have sufferered an abdominal trauma] is [the Kleihaur-Betke test a reliable indicator ] of [the presence of small numbers of foetal cells in the maternal circulation]?

Clinical Scenario

A pregnant patient is brought in by ambulance following a motor vehicle accident she is Rhesus D negative and the physician treating her takes blood to send off for analysis to determine if there is the presence of foetomaternal haemmorrhage. The test came back negative but just recently the doctor had read an article that cast doubt on the accuracy of the Kleihauer-Betke test in the presence of very small haemorrhages and was wondering just how accurate the test was in the presence of very small numbers of foetal cells in the maternal circulation.

Search Strategy

Medline 1950 to June Week 1 2008 using Ovid Interface
EMBASE 1980-2008 Week 1 using Ovid interface
The Cochrane Library

[{exp Hematologic Tests/} AND {kleihauer}]LIMIT to human and English language

Search Outcome

29 papers were found of which 3 were relevant, another paper was located from the reference of one that was not relevant and 2 additional papers were located from the search strategy for a BET on the prophylactic use of Rh anti-D, and one from a BET on uterine tenderness as an indicator for foetomaternal haemorrhage.

Relevant Paper(s)

Author, date and country Patient group Study type (level of evidence) Outcomes Key results Study Weaknesses
Pelikan et al.
The Netherlands
Blood samples from 44 patients admitted to the obstetric department at high risk of foetomaternal haemorrhage. Study to compare automated Kleihauer-Betke tests (KBT) against the manually counted test and blood flow cytometry to assess accuracy.FMH <0.1% were detected by manual and automated KBTsManual and automated KBTs are more accurate than blood flow cytometry in detecting FMH<0.1%No sample size calculation and relatively small sample size
Salama et al
420 unselected post-partum samples from Rh D negative women who had delivered a Rh D positive infant.Diagnostic - To compare the efficacy of a gel agglutination technique (GAT) with the KBT.All 406 samples tested negative in both GAT and KBT. Only 3 women tested positive for FMH and they were relatively large haemorrhages. The rest of the samples yielded false positives with the KBT but negative with the GAT. Gel agglutination technique is a more accurate test than KBT.The sample size only yielded positive results for FMH in 3 women so a very small sample size to assess accuracy of GAT and KBT in determining foetomaternal haemorrhage.
Towery R, English TP, Wisner D,
125 pregnant patients attending the ED due to blunt trauma and of these the KBT was performed in 70% of them.Retrospective review of cases.The KBT may be useful as an ongoing indicator of FMH There is reference made to the assertion that a FMH of below 0.1% may not be picked up by the KBT. The review covered all patients and the actual number of patients who were Rh D positive was only 5. There was no quantitative information in assessing accuracy of test given in the results.
Goodwin TM, Breen MT
205 gravid women following noncatastrophic trauma over an 18 month period from Jan 87 – Sep 88Consecutive case studyThe amount of FMH sufficient to sensitize RH-negative mothers (0.1ml or 1 foetal cell in 50000 maternal cells) is far below the detection limit of the clinical laboratory.Study recommendation: Full-dose Rho (D) immune globulin is given to all RH-negative mothers. It should not be withheld because a Kleihaur-Betke test has not been obtained or is called negative.Study only focuses on patients after non-catastrophic trauma.
Ochsenbein-Imhof N et al,
384 samples of D+ cord blood post delivery both parents D+ and mixed with D- blood from a transfusion centre to in various dilutions of 1, 0.5,0.3,0.1% to represent a FMHA study to compare KBT, fluorescence microscopy and flow cytometry.KBT failed to detect one case of 0.1% dilution, but generally it over-estimated the amount of foetal blood.There is the likelihood that the KBT will not detect a FMH of less than 0.1%No sample size estimates performed
Corsetti JP, Cox C, Leary JF et al.
Artificial FMH using Rh negative blood from an adult volunteer and umbilical cord Rh positive red cells Diagnostic studyThe accuracy and precision of the test is unacceptable at the 0.1% level The KBT may not detect small FMH Artificial FMH


To detect foetomaternal haemorrhage the Kleinhauer Betke Test is routinely use to detect the presence of foetal cells in the maternal circulation this is only a problem if the mother is Rhesus D negative as should there be a leak she may produce antibodies to the foetal cells which can produce harmful consequences. Tests to detect the presence of foetal cells in the maternal circulation may be inaccurate if only a few cells are present but that does not mean that the risk of alloimmunization does not exist.

Clinical Bottom Line

It is possible that the KBT will not detect small haemorrhages that may still be clinically significant, therefore prophylactic anti-d should still be given even with a negative result and a follow up test using another method such as flow cytometry should be used for accuracy


  1. Pelikan DM, et al Quantification of fetomaternal hemorrhage: A comparative study of the manual and automated microscopic Kleihauer-Betke tests and flow cytometry in clinical samples. Am Jour Obst Gynecol 2004:191;551-7
  2. Salama A et al. Use of the gel agglutination technique for determination of fetomaternal hemorrhage. Transfusion 1998:38;177-89
  3. Towery R, English TP, Wisner D. Evaluation of pregnant women after blunt injury, Journal of Trauma. 1993:35(5):731-6
  4. Goodwin TM, Breen MT. Pregnancy outcome and foetomaternal hemorrhage after noncatastrophic trauma. Am J Obstet Gynecol 1990:162;665-71
  5. Ochsenbein-Imhof N, Ochsenbein AF, Seifert B, Huch A, Huch R, Zimmermann R. Quantification of fetomaternal hemorrhage by fluorescence microscopy is equivalent to flow cytometry, Transfusion 2002:42(7);947–53
  6. Corsetti JP, Cox C, Leary JF et al. Comparison of quantitative acid-elution technique and flow cytometry for detecting fetomaternal hemorrhage. Ann Clin Lab Sci 1987;17(3):197-206