Export

Indication - Haemolytic disease of fetus or newborn
Kleihauer-Betke acid-elution test
Facility level:
2. Laboratory
Assay formats
Microscopic examination of slides which may use different types of microscopes and stains
Status history
First added in 2022
Purpose type
Aid to diagnosis
Purpose
To aid in the diagnosis and treatment of fetomaternal haemorrhage (FMH)
Specimen types
Venous whole blood
WHO prequalified or recommended products
None
WHO supporting documents
None
Technical specifications for procurements
None
Codes
ICD11 code: KA84.Z

Summary of evidence evaluation

Considering the fact that flow cytometry is not always available, this very easy, less accurate test can save lives. While flow cytometry may be more accurate, it is not readily available in all clinical situations and treatment may be delayed or inadequate without access to the KB test. The test kit is not too expensive.

Summary of SAGE IVD deliberations

The KB acid-elution test measures the presence of fetal blood in the maternal circulation. The test is extremely valuable in LMICs where FMH is suspected and the gold standard (flow cytometry) is scarcely available. The KB test is also used to determine the appropriate dose of anti-D immunoglobulin to give a mother to prevent problems related to blood incompatibility when there has been mixing of fetal and maternal blood. The KB test is easy to use. But it is microscopy based, which means that it requires a high standard of laboratory training. The test also only picks up more than 5 mL of bleed. Flow cytometry provides a more accurate quantification of the amount of haemorrhage, but it is expensive and is usually done in a tertiary institution. SAGE IVD members emphasized that this characteristic is true for high-income countries as well as LMICs. One SAGE IVD member noted that a colleague in Australia reports doing 14 000 KB tests per year for FMH vs 30 flow cytometry tests. One expert raised a concern about availability of anti-D immunoglobulin in LMICs and whether it makes sense to include the KB test in the EDL if the therapy is actually difficult to get. Experts working in India and Mexico reported that supplies of immunoglobulins in their countries could be unreliable. But it was pointed out that anti-D immunoglobulin has consistently appeared on essential medicine lists across the globe for years and is likely to be generally available. SAGE IVD noted that the submission did lack a clear algorithm. Group members also questioned the quality of the evidence provided in the application, and specifically the lack of a direct comparison with flow cytometry. However, these concerns were not judged substantial enough to affect the decision to list. Ultimately, SAGE IVD members agreed that the KB test is critical to have, especially to save the lives of babies and mothers in LMICs where access to flow cytometry is uncertain or nonexistent. In the statement of test purpose, SAGE IVD recommended replacing “prognosis” with “treatment” to avoid giving the impression that the test predicts outcome. Literature cited in the discussion: Thorpe SJ, Sands D, Fox B, Behr-Gross ME, Schaffner G, Yu MW. 2nd WHO international standard for anti-D immunoglobulin. Geneva: World Health Organization; 2003 (WHO/Bs03.1962; https://apps.who.int/iris/handle/10665/ 67735, accessed 14 December 2022). WHO recommendations on antenatal care for a positive pregnancy experience. Geneva: World Health Organization; 2016 (https://apps.who.int/iris/handle/ 10665/250796, accessed 14 December 2022).

SAGE IVD recommendation

SAGE IVD recommended including the Kleihauer-Betke acid-elution test category in EDL 4 ■ as a general IVD for use in clinical laboratories (EDL 4, Section II.a); ■ using microscopic examination of slides which may use different types of microscopes and stains; ■ using whole blood as specimen type; ■ to aid in the diagnosis and treatment of fetomaternal haemorrhage (FMH).

Details of submission from 2022

Background

Disease condition and impact on patients Haemolytic disease of the fetus and newborn can be life-threatening if not diagnosed or prevented. The disease affects an Rh-positive fetus of a sensitized Rh-negative mother. Sensitization, which can occur following childbirth, fetal loss or blunt trauma to the abdomen, can be prevented via the use of prophylactic anti-D immunoglobulin. When FMH occurs, fetal haemoglobin is mixed with maternal blood. In response to this exposure the maternal immune system is activated, and isoimmunization (formation of anti-Rh(D) antibodies) may occur if the mother is Rh(D) negative and the blood type of the fetus is Rh(D) positive. It takes only 0.01–0.03 mL of FMH for isoimmunization of the mother. Future pregnancies may be at risk for Rh(D) disease if the fetus is Rh(D) positive. The maternal antibodies bind to fetal Rh(D)-positive erythrocytes, leading to haemolysis, anaemia, hydrops fetalis and possibly fetal death. Identification and quantification of a fetomaternal bleed can be used to direct prophylactic therapy with anti-D immunoglobulin (also known as Rho(D) immune globulin). While quantification of the bleed can be undertaken using sophisticated methods such as flow cytometry, this is not readily available in all health care settings where maternity care is offered. The KB test is a simple examination requiring minimal equipment. It is, however, dependent on the skill of the laboratory scientist performing the analysis. The test remains useful for demonstrating haemoglobin of fetal origin in maternal circulation, and it is also recommended as part of the investigation of stillbirth. Does the test meet a medical need? The KB test is utilized to determine the presence of fetal blood in maternal circulation and to better estimate the amount of FMH with a threshold of 5 mL. KB testing has obstetrical implications in the diagnosis and prognosis of preterm labour, fetal demise and other conditions. The test can be used to guide anti-D immunoglobulin prophylaxis. How the test is used This test remains the gold standard for detecting FMH. For bleeds estimated to be less than 10 mL, the standard dose of anti-D prophylaxis can be used to prevent sensitization. Where the estimated bleed is > 5 mL, samples can be sent for confirmative flow cytometry where available.

Public health relevance

Prevalence Not provided. Socioeconomic impact Not provided.

WHO or other clinical guidelines relevant to the test

Not provided.

Evidence for diagnostic accuracy

The KB test is not as precise or accurate as flow cytometry and tends to overestimate the volume of fetal blood in maternal circulation.

Evidence for clinical usefulness and impact

Alloimmunization from FMH remains an ongoing cause of haemolytic disease of the fetus and newborn. Though failure rates of postnatal prophylaxis have decreased to less than 1–2%, it is critical to minimize laboratory errors when calculating the Rh immunoglobulin dose to prevent maternal alloimmunization. Flow cytometry and the KB method are two means of calculating the necessary Rh immunoglobulin dose. The advantages of using the KB test include its ease of access, inexpensive cost and lack of special equipment needed. Inherent limitations to using the test include a lack of standardization, associated labour to perform it and its relative imprecision, with a coefficient of variation of 30–80%. Although flow cytometry can analyse a larger batch of samples (resulting in greater quantitative accuracy and reproducibility; is more precise, with a coefficient of variation less than 20%; and can distinguish between adult F-cells and fetal cells), it is relatively more expensive, time-consuming and inaccessible. The KB test can also be used as a test to explain fetal anaemia.

Evidence for economic impact and/or cost–effectiveness

Not provided.

Ethical issues, equity and human rights issues

There is a 72-hour window from the sensitizing event within which to administer prophylactic anti-D immunoglobulin to prevent alloimmunization. Women may not present until 48 hours have elapsed. Where flow cytometry cannot be made available, a KB test is an acceptable alternative and its absence could lead to ethical issues in addition to inequal access to affordable health care.
Akorsu EE, Acquaye JK, Benneh AA, Oppong SA, Olayemi E. Fetomaternal hemorrhage among pregnant women in Accra, Ghana. Int J Gynaecol Obstet. 2019 Sep;146(3):333–8. doi:10.1002/ijgo.12890. Bataille P, Petit L, Winer N (2022) Performance of the Kleihauer Betke test in the prediction of neonatal anemia. J Matern Fetal Neonatal Med. 2022;35:19:3670–6. doi:10.1080/14767058.2020.1837768. Gereg C, Fung MK. Assessment of flow cytometry and Kleihauer-Betke method when calculating fetomaternal hemorrhage and Rh immunoglobulin dose. Arch Pathol Lab Med. 2022;146(3):271 (https://doi.org/10.5858/arpa.2021-0432-LE, accessed 31 July 2023). Krywko DM, Yarrarapu SNS, Shunkwiler SM. Kleihauer Betke test. [Updated 2022 Aug 8]. In: StatPearls [internet]. Treasure Island (FL): StatPearls Publishing; 2023 (https://www.ncbi.nlm.nih.gov/books/ NBK430876/, accessed 6 April 2023). Tsakiridis I, Giouleka S, Mamopoulos A, Athanasiadis A, Dagklis T. Investigation and management of stillbirth: a descriptive review of major guidelines. J Perinat Med. 2022;50(6):796–813 (https://doi. org/10.1515/jpm-2021-0403, accessed 31 July 2023).