Haematology

The Detection of Foetal Red Blood Cells in Maternal Blood Samples by Dual Flow Cytometry
a report by

L i e s b e t h B a k k e r - J o n g e s 1 , D G r o o t f a a m 2 and J o h a n n e s D u v e k o t 2
1. Department of Immunology; 2. Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Centre, Rotterdam

Foetomaternal Haemorrhage Foetomaternal haemorrhage (FMH) occurs as a result of the loss of integrity of the normal physiological barrier between the foetal and maternal circulation, allowing foetal red blood cells (RBCs) to enter the maternal circulation. This may be an acute or a chronic process. The pressure gradient between the arterial foetal circulation and the venous maternal circulation leads to foetal RBCs entering the maternal circulation. Quantification of the amount of foetal RBCs derived from FMH in the maternal circulation may be of importance in obstetrical management.1 Furthermore, the amount of foetal RBCs in the maternal circulation can be used as a measure of the severity of the underlying cause. FMH may occur during pregnancy but also during delivery (see Table 1). During pregnancy, abdominal trauma, intra-uterine infection and (partial) abruption may result in FMH. However, in many cases of FMH during pregnancy a direct cause cannot be found. During delivery the mechanical forces used in the expulsion of the placenta are supposed to initiate most cases of FMH. Two important complications may arise from FMH (see Table 1). The first is the direct effect of blood loss from the foetal circulation. The resulting foetal anaemia may lead to foetal cardiac decompensation, which leads to foetal hydrops and, consequently, foetal intra-uterine death. One of the first clinical signs of foetal anaemia is the disappearance of foetal movements. It is estimated that 314% of stillbirths are caused by FMH.2,3 The second complication results from (transient) maternal chimerism, which may lead to immunisation of the mother against foetal alloantigens and the formation of RBC alloantibodies. Maternal RBC alloantibodies of the immunoglobulin G (IgG) type cross the placenta easily and may induce haemolytic disease in the foetus. In this way, a limited or chronic FMH during pregnancy may indirectly lead to severe haemolytic consequences for the foetus and newborn. FMH during delivery may lead to similar problems in subsequent pregnancies. The development of haemolytic complications in the foetus and newborn are strongly dependent on the type of RBC alloantibodies. The most severe reactions are seen with RBC alloantibodies

of the rhesus D (RhD) type. Exact quantification of FMH during delivery is required to calculate the dosage of anti-D immunoglobulin needed for immune prophylaxis.4,5 Clinical Indications Acute determination of FMH is warranted with the following events. In cases of decreased foetal body movements and non-reactive foetal heart rate tracings, the detection of FMH leads to a strong suspicion of foetal anaemia.57 In these cases, foetal Doppler velocimetry may complete the diagnosis. Determination of FMH is also indicated in cases of vaginal bleeding due to (partial) placental abruption. In cases of maternal trauma, especially abdominal trauma, signs of FMH are often described. The presence of FMH in the latter indication is not clearly correlated with clinical severity and prognosis of the trauma,8 although others find a strong correlation with subsequent delivery.9 After external cephalic version (ECV), FMH is only seldom observed,10,11 and for this indication determination of FMH seems unreasonable. In all cases of sudden and unexplained intrauterine demise, determination of FMH is an important diagnostic tool.6 The amount of FMH is important in RhD-negative mothers who have given birth to an RhD-positive child.1 Especially after Caesarean delivery, delivery of twins or manual removal of the placenta, FMH is larger than after spontaneous and uncomplicated deliveries.12 Anti-D immunoglobulin should be administered within 48 hours after delivery, which makes it necessary to perform the determination of FMH within this period. Furthermore, the test can also be used to confirm the effect of the administered anti-D immunoglobulin. Another post-partum indication for the determination of FMH may be an unexplainable anaemia in the newborn. Importance of the Detection of Small Amounts of Foetomaternal Haemorrhage Detection of small amounts of foetal RBCs in the maternal circulation may be of great importance because a relatively small FMH may reflect a relatively large proportion of the total volume of foetal blood, and because a small amount of foetal RBCs may have caused the formation of RBC alloantibodies. As little as 0.5ml of foetal blood passed transplacentally may result in maternal isoimmunisation.12

Liesbeth Bakker-Jonges is a Medical Immunologist in the Department of Immunology at the Erasmus MC, University Medical Centre in Rotterdam, The Netherlands. She is involved in the diagnostics of immunological diseases and the development and validation of (auto)antibody assays. She also participates in research projects, especially on autoimmune diseases. E: l.e.bakker@erasmusmc.nl Johannes Duvekot is a GynaecologistPerinatologist in the Department of Obstetrics and Gynaecology at the Erasmus MC, University Medical Centre in Rotterdam, The Netherlands. This department is the largest tertiary referral centre in The Netherlands. He is involved in research in clinical obstetrics, and is the author or co-author of more than 30 scientific papers on this subject. E: j.j.duvekot@erasmusmc.nl

A simple calculation to estimate the amount of blood transferred from the foetus to the mother can be made with the following assumptions: a nonpregnant woman has 70ml of blood per kilogram bodyweight, which increases during pregnancy to 100ml of blood per kilogram bodyweight. The foetus has approximately 80ml blood per kilogram bodyweight. This means that when an FMH of 1% is detected in a blood sample of an 80kg pregnant women, there is 80ml of foetal RBC in the maternal circulation. This is about one-third of the total amount of the circulating volume of a foetus of 3,000g. An FMH of 0.1% equates to a leakage of 8ml in this situation.

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The Detection of Foetal Red Blood Cells in Maternal Blood Samples by Dual Flow Cytometry

An acute bleeding of 20% of the foetal blood volume has been associated with a 50% mortality rate, and an acute bleeding exceeding 40% has been found to result in foetal death.13,14 In many countries, an RhD-negative mother is routinely administered 1,000mg IE anti-D immunoglobulin within 48 hours post-partum if she has delivered an RhD-positive child. This amount of anti-D immunoglobulin is sufficient to neutralise up to 20ml of foetal blood, thus preventing the formation of RhD-alloantibodies, which may have devastating effects in subsequent pregnancies. On the basis of the calculated volume of FMH, extra anti-D immunoglobulin may be administered. Methods of Foetomaternal Haemorrhage Detection Role of Haemoglobin A method that is independent of the blood group typing of the mother and child is preferred to detect foetal RBCs in the blood of the newborn is the type of haemoglobin. Foetal RBCs contain mostly foetal haemoglobin (HbF), while in maternal RBCs this is expressed only in a minority of the RBCs (see Figure 1). Thus, HbF molecules can be used to discriminate between foetal and maternal red blood cells. Kleihauer-Betke Test The Kleihauer-Betke method is applied to blood smears, and the principle is based on the difference of the haemoglobin type in maternal and foetal RBCs (see Figure 2).1517 Until now, the Kleihauer-Betke acid elution test was the gold standard for the quantification of FMH; however, this test is rather time-consuming, is not objective and requires considerable experience. The test shows a large inter-observer and inter-hospital variation.18 A great variability between observers was shown in an external quality control round in Ontario. This resulted in poor intra-laboratory reproducibility and a large error of calculated mean (>20%).19 The Kleihauer-Betke test is stained and evaluated manually by counting in a number of microscopic fields using a x40 objective. It is possible to automate this microscopic analysis. However, only a moderate agreement has been observed between the manual and automated Kleihauer-Betke test.20 Agreement between the results of the manual Kleihauer-Betke test and single-parameter flow cytometry was fair. The detection limit of the Kleihauer-Betke test lies at about 0.02%, but depends on the experience of the observer.20 The amount of maternal HbF-positive RBCs (F cells) is normally small: 0.50.7%.21 The percentage of F cells increases during pregnancy and reaches a peak at 1822 weeks.22,23 The classic Kleihauer-Betke test cannot discriminate between maternal F cells and foetal HbF-positive RBCs, which may lead to overestimation of the amount of FMH. This may even lead to false-positive results in the classic Kleihauer-Betke test.24 Flow Cytometric Test The first use of a flow cytometric test to detect foetal cells in the maternal circulation made use of the expression of RhD on the foetal cells in case of RhD antagonism. This test was used to calculate the amount of anti-D immunoglobulin to be administered. It may be obvious that this method
25

Table 1: Causes and Consequences of Foetomaternal Haemmorhage

Causes of Foetomaternal Haemorrhage Abdominal trauma Infection Placental abruption Delivery Unknown Consequences of Foetomaternal Haemorrhage Anaemia of the foetus Intra-uterine death Formation of maternal red blood cell alloantibodies

Figure 1: Types of Haemoglobin During Gestation and Early Life

100

Polypeptide present (%)

mother. The major difference between the RBCs of the mother and her

80

60

40

20

0 3 Gestation (months) -chain (embryonic) -chain 6 Birth HbF -chain (fetal) 3 Age (months) HbA -chain (adult) HbA2 -chain 6

The haemoglobin molecule, a transporter protein for oxygen, is a heterotetramer. It consists of four polypeptide chains that are two to two identical with heme groups inside and a ferritin atom in the centre. The foetal haemoglobin (HbF) consist of two -chains and two -chains. These -chains are replaced shortly after birth. Adult Hb mostly consists of two -chains and two -chains forming Hb A (HbA) (>95%), but there is also a HbA2 type (3%), which has a chain. Fewer than 1% of adult red blood cells contain HbF.

Kleihauer-Betke test.20,25,26 However, as in the classic Kleihauer-Betke test, this may lead to overestimation of FMH because of the presence of F cells. However, this will occur to a lesser extent because of the higher intensity of expression of HbF in the foetal RBCs than in the maternal RBCs. Therefore, because of its objectivity flow cytometry may be a good alternative for the Kleihauer-Betke test, and shows a good correlation with the Kleihauer-Betke test.25,27,28 The additional value of this method came when an extra marker was introduced in addition to HbF. With this marker, arbitrary settings for HbF expression and false-positivity by F cells could be prevented. The Foetal Cell Count Kit (IQ Products, Groningen, The Netherlands) is such a dual flow cytometric test for detection of FMH using a murine monoclonal antibody for the detection of HbF and a polyclonal antibody to detect carbonic anhydrase II (CA II) in RBCs. The CA isoenzymes are mainly represented by CA I and CA II. These enzymes only start to be expressed in RBCs of newborns, reaching adult levels at six months of age.29 Adult RBCs have a 100-fold higher expression of these CO2-converting enzymes than foetal RBCs. This makes it possible to identify both foetal and maternal RBCs (see Figure 3). Discrimination between true foetal RBCs and possible false-positive F is achieved because the latter cells will stain with both anti-HbF and anti-CA II (see Figure 3).

cannot be used for other combinations of blood group types. In the following development step, detection of FMH by flow cytometry was again based on a single parameter: the presence of HbF in foetal cells. This showed a good correlation with the results obtained by the

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Haematology
used in the patient group, 50 of the 455 samples were positive with the Kleihauer-Betke test, but only 44 were positive with the dual flow cytometric method. The discrepancy between the two techniques could be explained by staining of false-positive F cells with the Kleihauer-Betke test.30 The Dual Flow Cytometric Test in Erasmus MC, University Medical Centre, Rotterdam Over the last two years Foetal Cell Count Kit dual flow cytometric test was performed in 777 patients with different obstetric problems. Indications to perform the test were maternal abdominal trauma (55), stillbirth (21), ECV (58), vaginal bleeding before delivery (139), a decreased or absent foetal movement pattern (FMP) (379), Rh-negative patients post-partum (109) and a miscellaneous group (16). An FMH of 0.1% was chosen as the cut-off for a positive test. FMH was calculated according to the previously described assumptions and maternal weight. Only four patients (0.5%) tested positive. Three patients were in the group of absent or reduced FMP. In all of these cases, delivery occurred soon after testing because of foetal distress or because delivery started
The official result in this patient was 12%. Upon acid elution, foetal haemoglobin (HbF) remains in the lysed red blood cells (RBCs), whereas other haemoglobins are eluted. After staining, the HbF-positive RBCs have a pink-red colour and can be distinguished microscopically from RBCs without HbF, of which only the empty membranes are visible.

Figure 2: Foetal Haemoblobin-positive Cells in a Blood Smear Using the Kleihauer-Betke Test

spontaneously. Two of the newborns were clearly anaemic and received RBC transfusion. One Rh-negative patient showed a positive test result post-partum. She delivered by Caesarean section and during this procedure the placenta was removed manually. The dosage of anti-D immunoglobulin was adapted. To our surprise, only a few samples showed positive results. The avoidance

Figure 3: Flow Cytometric Analysis Based on Dual Staining with Foetal Haemoblobin and Carbonic Anhydrase II

A
Side scatter BhF-PE

of confusion with maternal F cells and the more exact determination of the volume of transferred foetal cells may have led to this small percentage. A positive dual flow cytometric test was found in 9.7% of the patients in the study by Porra.30 Unfortunately, no details were documented about this group of patients, which makes this study difficult to compare with our results. Furthermore, the detection threshold in this

Forward scatter

CA- FITC

study was 0.01%, versus 0.1% in our study. Summary FMH may be a serious complication during pregnancy, leading to acute foetal anaemia and resulting in reduced foetal movements, foetal cardiac decompensation and foetal death. The presence of such a clinically relevant FMH is not often encountered. In cases of acute absent or decreased foetal movements, a diagnosis of FMH should be anticipated. New detection methods such as the dual flow cytometric method which is sensitive and avoids confusion with maternal F cells are superior to the classic Kleihauer-Betke test. Using the assumption of a blood volume of 100ml per kilogram bodyweight in pregnant women, the exact amount of transferred foetal blood can be calculated. With a detection threshold of 0.1%, clinically relevant cases of FMH may be detected. Post-partum, the new dual flow cytometric test allows accurate dosing of anti-D immunoglobulin in RhD-negative women.

The erythrocytes (red) are gated based on their forward and side scatter (A). Within the erythrocyte gate (200,000 events) a foetal haemoglobin (HbF)-positive carbonic anhydrase (CA)-negative cell population (foetal erythrocytes, red population) is detected, while the maternal erythrocytes are depicted in grey. This results in this case in 0.03% foetomaternal haemorrhage (B). Maternal HbF positive cells, the F cells, are defined as HbF-positive/ CA-positive cells and are depicted in green (C).

Evaluation of blood samples of 455 pregnant or just delivered women and 124 artificial mixture experiments in French laboratories showed that the Foetal Cell Count kit allowed clear discrimination and accurate quantification of foetal cells.30 This study showed a detection limit for the dual flow cytometric method of 0.03% in 100,000 events and 0.02% in 200,000 events, which is in line with our own results. The expected values in healthy female blood donors was below even this detection limit (0.008%).
30

This study also showed that there was a good correlation

between the results obtained with the Kleihauer-Betke test and the Foetal Cell Count kit (ratio [r] = 0.94; p<0.01) in the spiking experiments. When

Dziegiel MH, et al., Curr Opin Hematol, 2006;13:49095. Laube DW, Schauberger CW, Obstet Gynecol, 1982;60:64951. Owen J, et al., Am J Obstet Gynecol, 1989;161:6636. Augustson BM, et al., Med J Aust, 2006;184:61113. David M, et al., J Perinat Med, 2004;32:2547. Silver RM, et al., Am J Obstet Gynecol, 2007;196:43344. Stanchev H, et al., Thromb Haemost, 2006;95:1958. Dahmus MA, Sibai BM, Am J Obstet Gynecol, 1993;169: 10549. 9. Muench MV, et al., J Trauma, 2004;57:10948. 10. Karantis E, et al., Aust N Z J Obstet Gynaecol Suppl, 2001; 41:3957. 11. Nord E, et al., Acta Obstet Gynaecol Scand, 1989;68:558.

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Leavitt BG, et al., Obstet Gynecol, 2007;110:60811. Giacoia GP, Obstet Gynecol Surv, 1997;52:37280. Hartwell EA, Am J Clin Pathol, 1998;110:28192. Mollison P, et al., Acid elution method for the detection of fetal red cells. Blood transfusion in clinical medicine, Vol. 9, Oxford: Blackwell, 1993;798800. Kleihauer E, et al., Klin Wochenschr, 1957;35:6378. Betke K, Nierhaus K, Munch Med Wochenschr, 1968;110: 50916. Duckett JR, Constantine G, Br J Obstet Gynaecol, 1997;104: 8456. Lafferty JD, et al., Am J Clin Pathol, 2003;119:727. Pelikan DM, et al., Am J Obstet Gynecol, 2004;191:5517.

21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

Wood WG, et al., Blood, 1975;46:67182. Pembrey ME, et al., Lancet, 1973;1:135054. Popat N, et al., Lancet, 1977;2:3779. Leers MP, et al., Eur J Obstet Gynecol Reprod Biol, 2007;134: 1279. Janssen WC, Hoffmann JJ, Clin Lab Haematol, 2002;24:8992. Italia KY, et al., Int J Lab Hematol, 2007;29:40914. Gomez-Arbones X, et al., Clin Lab Haematol, 2002;24:4753. Greiss MA, et al., Transfusion, 2002;42:106778. Aliakbar S, Brown PR, Clin Biochem, 1996;29:15764. Porra V, et al., Transfusion, 2007;47:12819.

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Feto-maternal hemorrhage
Fetal Cell Count Kit
Application areas
Cases with possible RhD incompatibilities Abdominal trauma Preeclampsia Blood loss during pregnancy Intra-Uterine Transfusion (IUT) Thallasemia

Diagnosis of

Special features
Clear separation of fetal and adult red blood cells Quantitation of Feto-maternal hemorrhage Intracellular detection of fetal hemoglobin and carbonic anhydrase Detection of as low as 0.02% fetal cells in maternal blood

bright fluorescence
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