View of Treatment of pregnant women by RhD type

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Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia

Correspondence/

Korespondenca:

Janja Mrak, e: janja.jerina@

gmail.com Key words:

variants D; weak D; partial D; serological techniques;

molecular-biological methods

Ključne besede:

variante D; šibki D; parcialni D; serološke tehnike;

molekularnobiološke metode

Received: 9. 1. 2018 Accepted: 6. 8. 2019

9.1.2018 date-received

6.8.2019 date-accepted

Cardiovascular system Srce in ožilje discipline

Professional article Strokovni članek article-type

Treatment of pregnant women by RhD type Obravnava nosečnic glede na tip RhD article-title Treatment of pregnant women by RhD type Obravnava nosečnic glede na tip RhD alt-title variants D, weak D, partial D, serological tech-

niques, molecular-biological methods variante D, šibki D, parcialni D, serološke tehnike, molekularnobiološke metode

kwd-group The authors declare that there are no conflicts

of interest present. Avtorji so izjavili, da ne obstajajo nobeni

konkurenčni interesi. conflict

year volume first month last month first page last page

2019 88 11 12 582 592

name surname aff email

Janja Mrak 1 janja.jerina@gmail.com

name surname aff

Klara Železnik 1

Tadeja Dovč-Drnovšek 1

Irena Bricl 1

eng slo aff-id

Blood Transfusion Centre of

Slovenia, Ljubljana, Slovenia Zavod RS za transfuzijsko

medicino, Ljubljana, Slovenija 1

Treatment of pregnant women by RhD type

Obravnava nosečnic glede na tip RhD

Janja Mrak, Klara Železnik, Tadeja Dovč-Drnovšek, Irena Bricl

Abstract

An accurate determination of RhD (D) antigen (Ag) is important for pregnant women, transfusion recipients and blood donors. The decision to receive the pre- and postnatal prophylaxis with anti-D immunoglobulin (RhIG) is based on the D-type. Individuals can be classified as D-positive (D-pos) or D-negative (D-neg) based on the determination of Ag D but there are numerous other versions of Ag D as well (variants D, D-var). Allele variants of the RhD (D) gene can encode different forms of protein D. They are divided into weak, partial and D_el. Ag D are determined on the basis of serological techniques. If in doubt, we use the molecular-biological methods for determining the D gene. The definitive definition of D-var is given on the basis of the molecular-biological methods.

From the peripheral venous blood samples taken from pregnant women, we roughly determined Ag D on plates (the Seraclone anti-D reagent) and on gel cards (the DiaClon ABO / D + Reverse Grouping test system (monoclonal antibodies) (Bio-Rad, Germany), which we do not detect D category VI blood group). For 49 pregnant women we performed extended serological tests of D blood group (commercial ID-Partial RhD Typing Set (Bio-Rad, Germany)). We proceeded with molecular-biological methods: DNA isolation (utilisation of the BioRobot EZ1 and the commercial set EZ1 DNA Blood 350 μl Kit (Qiagen, Germany)), determination of the genotype D with the PCR-SSP method (commercial RBC-Ready Gene CDE, RBC -Ready Gene D weak and RBC-Ready Gene D AddOn (Inno-Train; Germany)), amplification (Veriti apparatus (Apllied BioSystems, USA)), product separation (electrophoretic system (BioRad, USA) on agarose gel (Sigma, Germany).

Our results showed that weak D forms were found in 41 cases (83.7%) and partial forms 8 in cases (16.3%) of all D-var. The most common D-var in pregnant women was weak D type 1 with 17 cases (34.7%), followed by weak D type 3 with 13 cases (26.5%) and weak D type 2 with 10 cases (20.4%). The most common partial D form was D category VII with 5 cases (10.2%).

Pregnant women and transfusion recipients who are carriers of weak D types 1, 2 or 3 can be safely treated as D-pos, while carriers of all other D-var can be treated as D-neg. Blood donors with D-var are treated as D-pos. By means of this algorithm, approximately 172 unnecessary RhIG applications can be prevented annually and adequate supplies of D-neg erythrocyte blood components can be maintained.

Izvleček

Natančna opredelitev antigena (Ag) RhD (D) je pomembna pri nosečnicah, prejemnikih transfuzij in krvodajalcih. Glede na tip D se pri nosečnicah odločamo o pred- in poporodni zaščiti z imunoglobulinom anti-D (Ig anti-D). Glede na določitev Ag D lahko posameznike razvrstimo med D-pozitivne (D-poz) ali D-negativne (D-neg), obstajajo pa tudi številne druge različice Ag D (variante D, D-var). Variantni aleli gena RHD (D) lahko nosijo zapis za različne oblike proteina D, razdelimo jih lahko na šibke, parcialne in D_el. Ag D določamo s serološkimi metodami, v prime- ru nejasnosti si pomagamo z molekularnobiološkimi metodami za določitev gena D. Dokončno opredelitev D-var podamo na podlagi molekularnobioloških metod.

Slovenian Medical

Journal

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1 Introduction

An accurate determination of blood type (BT) RhD (D) is important for pregnant women, transfusion recipients and blood donors. The pregnant woman’s BT D type is important for deciding on the need for pre- and postnatal preventive prophylaxis with anti-D immunoglobulin (Ig), while with transfusion recipients it supports the decision whether they should receive D-positive (D-pos) or D-negative (D-neg) blood components, and with blood donors we can correctly decide whether their components will be safe for D-pos or D-neg recipients. Most people are carriers of the D antigen (Ag); however, in a minority the D Ag is absent, and some are carriers of other variants or versions of D Ag (D-var). So far, more than 200 types of D-var have been identified, and they are divided into weak D, partial D and D_el (1,2). It has been established that carriers

Nosečnicam smo iz vzorcev periferne venske krvi določili Ag D na ploščici (reagent Seraclone anti-D) in na gelu (testni sistem z gelskimi karticami DiaClon ABO/D + Reverse Grouping, monoclonal antibodies, Bio-Rad, Nemčija, s katerimi ne zaznamo krvne skupine D kategorija VI). 49 nosečnicam smo opravili razširjeno serološko testiranje KS D (komercialni set ID-Partial RhD Typ- ing Set, Bio-Rad, Nemčija). Nadaljevali smo z molekularnobiološkimi metodami: osamitev DNA (uporaba aparata BioRobot EZ1 in komercialnega set EZ1 DNA Blood 350 μl Kit, Qiagen, Nemčija), določitev genotipa D z metodo PCR-SSP (komercialni seti RBC-Ready Gene CDE, RBC-Ready Gene D weak in RBC-Ready Gene D AddOn, Inno-Train; Nemčija), pomnoževanje (aparat Veriti, Apllied BioSystems, ZDA), ločevanje produktov (elektroforezni system, BioRad, ZDA) na agaroznem gelu (Sigma, Nemčija).

Ugotovili smo, da so šibke oblike D predstavljale 41 primerov (83,7 %), parcialne oblike D pa 8 primerov (16,3 %) vseh D-var. Najpogostejša oblika D-var med nosečnicami je bila šibki D tip 1 s 17 primeri (34,7 %), sledil je šibki D tip 3 s 13 primeri (26,5 %) in šibki D tip 2 z 10 primeri (20,4 %).

Najpogostejša parcialna oblika D je bila D kategorija VII s 5 primeri (10,2 %).

Nosečnice in prejemnike transfuzij, ki so nosilci šibkih D tipa 1, 2 ali 3, lahko varno obravnavamo kot D-poz, nosilce vseh ostalih D-var pa kot D-neg. Krvodajalce z D-var obravnavamo kot D-poz. S tem algoritmom lahko preprečimo približno 172 nepotrebnih aplikacij Ig anti-D letno in ohranja- mo tudi primerne zaloge D-neg eritrocitnih komponent krvi.

Cite as/Citirajte kot: Mrak J, Železnik K, Dovč-Drnovšek T, Bricl I. Treatment of pregnant women by RhD type.

Zdrav Vestn. 2019;88(11–12):582–92.

DOI: https://doi.org/10.6016/ZdravVestn.2687

of weak D types 1, 2 and 3 do not form anti-D antibodies when coming into contact with D-pos red blood cells, while the carriers of other types of D-var do, even though there is not yet any data in the literature on the formation of antibodies. D Ag is rou- tinely determined using serological testing methods; however, these cannot be reli- ably enough differentiated between to determine individual types of D-var. In such cases, we support typing BT D using molecular-biological methods to determine the most frequent D-var. Anti-D alloantibodies may cause a haemolytic transfusion reaction (HTR) or a haemolytic disease of the foetus and newborn (HDFN) (3). The objective of this article is to show the algorithm for determining BT D type with pregnant women, management of pregnant women according to their BT D type, determining the frequency of

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individual D-var among pregnant women in Slovenia and comparing the data with other European countries.

2 The history of haemolytic disease of the foetus and newborn (HDFN)

Antibodies against the Ag of the Rh system can form during exposure of an Ag-negative individual with Ag-positive red blood cells when receiving the blood components or during pregnancy; in rare cases, these are naturally present antibodies. With HDFN, antibodies to only one of the numerous Ag of the Rh system, namely D Ag, are most frequently present (4,5). Because of transfusions of blood components consistent with D Ag, and preventive prophylaxis of D-neg pregnant women with Ig anti-D, newly discovered sensitizations to Ag D are rare in Slovenia.

If we discover anti-D with a serologically D-pos person, we first consider D-var, anti-D autoantibodies, or passively obtained antibodies. With D-neg persons, the anti-D antibodies may have been obtained passively through prophylaxis with anti-D Ig, they may result from immunisation after contact with D-pos or D-var red blood cells, and very rarely anti-D antibodies may be naturally present (6,7).

The Rhesus blood system (Rh) was discovered by Landsteiner and Wiener in 1940, when they established that when mixing antibodies against the Rh system with samples of white people, the antibodies agglutinated with the serum in 85% of the cases, while 15% of the cases did not agglutinate. They called the first group Rh- positive, and the second Rh-negative. This was the basis for establishing HDFN aetiol- ogy. Many years later, it was discovered that the sensitization of D-neg pregnant women can be effectively blocked by injecting anti-D Ig after the birth of a D-pos baby.

In 1968, antenatal prophylaxis of D-neg postpartum women with anti-D Ig was established in the US and several countries

in Europe (4,8), along with the guidelines for preventive injection of anti-D Ig at ter- mination of pregnancy, invasive diagnostic procedures, and other potential events that may cause sensitization during pregnancy (9). This has reduced the risk for sensitization of D-neg pregnant women carrying a D-pos foetus from 13.2% to 1.6% (4,10).

In the mid-90s, prenatal injections of anti-D Ig were introduced to prevent sensitization from undetected fetomaternal haemorrhages (FMH) in the last trimester of pregnancy. This measure resulted in the number of cases of sensitization addition- ally declining to 0.1–0.3% (9). The basis for performing prophylactic injection of D-neg pregnant women with anti-D Ig in Slovenia is the Rules on transfusion examinations and procedures during transfusion (Rules) (11). In 1970, we introduced antenatal prophylaxis, followed by prenatal prophylaxis, which has been mandatory since 1994. In 1997, cell-free foetal DNA (cffDNA) was discovered in the mother’s blood, leading to the development of tests for determining the foetus’s RHD (D) genotype from the mother’s blood and the introduction of targeted prophylaxis with anti-D Ig. Only those pregnant women with a D-pos foetus receive anti-D Ig. Targeted prophylaxis was introduced as a routine test in 2010 in Denmark, in 2011 in the Netherlands, and in 2014 in Finland, with individual regions in France, England, Sweden and Belgium following suit (10). Since the changes to the Rules in May 2018, targeted immunoprophylaxis is also practised in Slovenia.

We determine the foetus’s D type between the 25th and 26th week of pregnancy, and then decide whether to inject anti-D Ig.

The Rules also define a full introduction of targeted immunoprophylaxis within two years (11-14).

3 The Rh system

The Rh blood group system is a complex group of red blood cell antigens, encoded by two genes, the D and RHCE (CE) genes, located on the first chromosome. D encodes

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D Ag, CE determines C, c, E, and e Ag (5).

The D and CE genes consist of 10 exons and introns. The products of these genes are polypeptides that cross a red blood cell’s membrane twelve times, are already fully developed at birth, and can be already detected in the eighth week of pregnancy (5).

With regard to the D gene, an individual’s phenotype can be either D-pos, D-neg or D-var. If a D-neg or D-pos person who is not a weak D type 1, 2 or 3 is exposed to D Ag, anti-D antibodies may develop.

In the Caucasian population, 85% are D-pos, in the Sub-Saharan Africa 95% are positive, and in the Asian population more than 99.5% are D-pos. Persons who are D-neg either lack the D gene (deletion), or it is not expressed because of various changes in the gene. In the Caucasian population, the deletion of the D gene is characteristic, while in the black population it is characteristic for the D-neg forms to be the result of the D gene deletion, mutations in the D gene or hybrid genes D-CE-D (5,15).

4 Variants of the D gene

The first D-var variant was established in 1946 and called D^u. D-var is divided into weak D, partial D and D_el. The molecular reasons or changes to the D gene that lead to the onset of D-var may be single nucle- otide polymorphisms (SNP) that cause the replacement of one or several amino acids in the cytoplasm, transmembrane, or extracellular part of the D protein, a recom- bination that is the cause of the onset of hybrid D-CE genes, deletion and insertion.

It is characteristic of weak types of D to express D Ag on the surface of their cells, which has all the epitopes expressed, ex- cept that their number is smaller on the surface of red blood cells. Normal density of D Ag on the surface of red blood cells is between 13,000 and 24,000 D Ag/red blood cell (15,18), and the weak D is expressed with between 70 and 4,000 D Ag, with 95%

above 400 D Ag. Replacements of amino acids in the transmembrane or cytoplasm part of the D protein is characteristic of

weak types of D. The serologically weak D type may also be the result of the sup- pressive effect of Cde in the trans position.

Sometimes, these Ag can also be qualita- tively changed, which is then referred to as weak partial D Ag (5,7,18).

Changes in the amino acid sequence that changes the extracellular part of the D protein are characteristic for partial D forms, and consequently persons with these D-var can form anti-D antibodies when exposed to normal D Ag (5). Partial D can have normal or lowered density of D Ag on the surface of red blood cells. It is characteristic for them that the Ag are changed in a qualitative way (15,18).

D_el is a D-var type characteristic for populations of Asian origin (19). The D_el red blood cells react as D-neg with routine serology tests, and can only be detected using the adsorption and elution technique.

The number of D Ag on D_el red blood cells is below 22–36. In the literature, there are cases of anti-D antibodies forming after a D-neg person receives a transfusion with D_el red blood cells (15,18,20,21).

The D-var frequency depends on the ethnicity, and in different populations, it occurs with 0.3–1.7%. It is characteristic for Caucasians that between 0.2% and 1% of the population have D allele, which encode the serologically weak forms of D Ag. 95% of serologically weak types in Caucasians are weak D types 1, 2, 3, which in contact with D-pos red blood cells do not create anti-D antibodies (17,22,23,25).

The frequency of occurrence of individual forms of weak D is different between individual European countries. Considering all types of weak D, the frequency of weak D type 1 in Germany is 60–65%, in Spain it is 49%, in France %, in Tyrol 33%, and in Portugal 16%. The frequency of weak D type 2 in Portugal is 64%, in France and Spain it is 32–22%, in Germany 17–

27%, and in Tyrol 8%. Weak D type 3 has the frequency of 50% in Tyrol, 4–17% in Germany, 14% in Portugal, 9% in Spain, and 5% in France (24-27). In the Slovenian population weak D types occur in 0,4 %

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and represent 75-84 % of all D-var. Of all types of weak D, the most common is type 1 with a frequency of 43–47.2%, followed by weak D types 2 and 3 with a frequency of 22.5–26.3% (25,31). Partial forms of D are more frequent among the black population, among which the most frequent categories are IIIa, IIIb, IVa and Va. With Caucasians the most frequent partial D form is category VI, which occurs in 0.02–

0.05% of cases (28-29). In Germany, D category VI type II occurs with a frequency of 1:6200 (28-30), in the United Kingdom with a frequency of 1:4000 (4,20,30-32).

Partial D in the Slovenian population occurs with 0.05% of the total popul–ation, including the most frequent D category VI type II with a frequency of 1:5058, followed by D category V, VII, DFR (4,25,31).

D_el types are characteristic for the Asian population. They occur in 10–33% of serologically D-neg individuals (24). With Caucasians, the D_el type frequency is ex- ceptionally rare (19), in one research it stood at only 0.03% (32). In Slovenia, we have so far found two D_el allele (4,33).

5 Determining the D antigen

D Ag can be determined using serologic typing or its presence can be found using molecular-biological methods. With serological tests, commercial reagents with different antibody characteristics are used.

There are monoclonal (specific to a certain epitope) or polyclonal (specific to several epitopes) reagents (5,22). Different monoclonal reagents show reactions of varying degrees with D-var red blood cells; therefore, typing D-var based on serological tests alone is not possible (1,22). If the red blood cells do not produce a strong positive result with the anti-D reagent, they are serologically weak D Ag or D-var types. Since the most immunogenic and the most frequent partial type of D-var in the Caucasian population is the D category VI, the D Ag of pregnant women and transfusion recipients in Slovenia and in other European countries is determined using monoclonal reagents that do not detect the D category VI blood group. Such patients are defined as D-neg. D-var can

had weaker positive results, we conduct- ed expanded serological testing of D BT using the commercial set ID-Partial RhD Typing Set (Bio-Rad, Germany). For fi- nal determination of D BT, we then used molecular-biological methods. We isolat- ed DNA from full blood in accordance with the instructions of the manufacturer of the BioRobot EZ1 device and the EZ1 DNA Blood 350 μl Kit commercial set (Qiagen, Germany). We determined the D genotype according to the manufac- turer’s instructions applying the PCR-SSP method and using the RBC-Ready Gene CDE, RBC-Ready Gene D weak and RBC- Ready Gene D AddOn commercial sets (Inno-Train; Germany). After completing amplification using the Veriti (Applied BioSystems, USA) device we performed product separation of PCR reactions by length using an electrophoresis system (BioRad, USA) on agarose gel.

7 Results

According to the test results of the 49 samples of peripheral blood of pregnant women for the risk for alloantibody formation, 41 (83.7%) of the participating pregnant women who are carriers of the weak D type 1, 2, 3 do not require pre- or postnatal prophylaxis with anti-D Ig. According to the data from the literature, Figure 1: The algorithm for determining D blood group among pregnant women and their

continued management according to RhD type.

Serological determination D BT D-pos or D-neg

D genotyping*

*PCR-SSP for detecting partial and weak D types

Weak D type 1, 2, 3

is possible

D-var, which is not weak D type 1,2 or 3

PREGNANT WOMAN:

does not require prophylaxis with anti-D Ig, may receive

D-pos blood components

PREGNANT WOMAN:

requires prophylaxis with anti-D Ig, must receive

D-neg blood components is not possible

Table 1: Results of D genotyping among pregnant women.

Week D

type 1 Week D

type 3 Week D type 1 and type 3

D category VII

D category VI type 2

Partial

RHD DNB Unknow D-var Total

No. 17 10 13 1 5 1 1 1 49

% 34.7 20.4 26.5 2.0 10.2 2.0 2.0 2.0 100.0

Risks for forming anti-D and

candidate (pregnant woman) for receiving anti-D Ig

no yes

Transfusion of red

blood cell can be D-pos

83.7%41

D-neg 16.3%8

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be typed serologically more exactly or par- tially using monoclonal antibodies with a known specificity for individual epitopes.

From the combinations of results, we can determine whether the D Ag type is weak or partial. Since we cannot use serological methods to determine whether an individual has weak D type 1, 2, 3, which are treated as D-pos, or for any other type of serologically weak D, which should be treated as D-neg, additional tests are re- quired using molecular-biological methods. The most frequent method used is Polimerase Chain Reaction - Sequence Specific Priming (PCR-SSP) (34-35).

6 Samples and method

Samples of peripheral venous blood were taken from pregnant women in stan- dard vials with Ethylenediaminetetraacetic acid (EDTA) as an anticoagulant during routine determination of D BT at the beginning of pregnancy or for immunoprophylaxis with anti-D Ig. D BT of all pregnant women was determined on the plate according to the manufacturer’s instructions using the commercial reagent Seraclone anti-D and on DiaClon ABO/D + Reverse Grouping (monoclonal antibodies) gel cards (Bio-Rad, Germany), which do not detect the D category VI blood type. With 49 pregnant women who

had weaker positive results, we conduct- ed expanded serological testing of D BT using the commercial set ID-Partial RhD Typing Set (Bio-Rad, Germany). For fi- nal determination of D BT, we then used molecular-biological methods. We isolat- ed DNA from full blood in accordance with the instructions of the manufacturer of the BioRobot EZ1 device and the EZ1 DNA Blood 350 μl Kit commercial set (Qiagen, Germany). We determined the D genotype according to the manufac- turer’s instructions applying the PCR-SSP method and using the RBC-Ready Gene CDE, RBC-Ready Gene D weak and RBC- Ready Gene D AddOn commercial sets (Inno-Train; Germany). After completing amplification using the Veriti (Applied BioSystems, USA) device we performed product separation of PCR reactions by length using an electrophoresis system (BioRad, USA) on agarose gel.

7 Results

According to the test results of the 49 samples of peripheral blood of pregnant women for the risk for alloantibody formation, 41 (83.7%) of the participating pregnant women who are carriers of the weak D type 1, 2, 3 do not require pre- or postnatal prophylaxis with anti-D Ig.

According to the data from the literature, Figure 1: The algorithm for determining D blood group among pregnant women and their

continued management according to RhD type.

Serological determination D BT D-pos or D-neg

D genotyping*

*PCR-SSP for detecting partial and weak D types

Weak D type 1, 2, 3

is possible

D-var, which is not weak D type 1,2 or 3

PREGNANT WOMAN:

does not require prophylaxis with anti-D Ig, may receive

D-pos blood components

PREGNANT WOMAN:

requires prophylaxis with anti-D Ig, must receive

D-neg blood components is not possible

Table 1: Results of D genotyping among pregnant women.

Week D

type 3 Week D type 1 and type 3

D category VII

D category VI type 2

Partial

RHD DNB Unknow D-var Total

No. 17 10 13 1 5 1 1 1 49

% 34.7 20.4 26.5 2.0 10.2 2.0 2.0 2.0 100.0

Risks for forming anti-D and

candidate (pregnant woman) for receiving anti-D Ig

no yes

Transfusion of red

blood cell can be D-pos

83.7%41

D-neg 16.3%8

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it is also safe for them to receive D-pos blood components. Among the carriers of the other D-var, 8 cases (16.3%) needed appropriate prophylaxis with anti-D Ig during pregnancy and potentially also after the birth (if the newborn was D-pos or D-var) because of the risk of alloimmuni- sation, and in case of a blood transfusion, needed D-neg blood units. The algorithm of determining D BT with pregnant women and their continued management according to their D BT type is depicted in Figure 1 (1,34-36). The results of tests for the 49 pregnant women are listed in Table 1. We did not discover any cases of D-var, category VI, as we use monoclonal antibodies for serological determination of D Ag, which do not detect the D category VI, and consequently, such pregnant women are designated as D-neg.

8 Discussion

D Ag is highly immunogenic, therefore making an exact determination of D BT very important for the further treatment of pregnant women, blood donors and patients requiring a blood transfusion. The basic method for determining D Ag is serological. Due to the exceptional complexity of the D system, we most of- ten cannot differentiate between different types of D-var with serological tests alone, even when using different anti-D reagents.

In the event of an inconclusive serological D determination, we move on to molecular-biology methods.

In our research, we established that among Slovenian pregnant women the most frequent D-var type is the weak D, type 1, followed by weak D, types 3 and 2.

The frequency matches previous data on the occurrence of weak types of D in the Slovenian population (4,25,31). Compared to neighbouring European countries, we are most similar to the population of Tyrol (4). Partial D types are characteristic for the black population; however, they are also significant with Caucasians because of immunogenicity. Those who are carriers

of the partial D category VI can begin to produce anti-D after coming into contact with D-pos red blood cells, while D-pos newborns, whose mothers are carriers of D category IV blood type and form anti-D alloantibodies may develop a severe form of HDFN. Therefore, in Slovenia, as in other European countries, pregnant women and recipients of blood transfusions have their serological D type determined using monoclonal antibodies which do not detect the D category VI blood type.

Consequently, we treat pregnant women who are carriers of D category VI as D-neg, and blood transfusion recipients receive D-neg transfusions. On the other hand, we treat blood donors who are carriers of D category VI as D-pos. Only D-pos persons may receive such blood (4). This explains why the most frequent partial D type among the pregnant women in our results is D category VII and not D category VI, which we detect as D-neg.

The discovery of anti-D Ig in the mid- 1960s significantly contributed to the de- cline of HDFN cases and fatalities result- ing from them. Even though anti-D Ig is a safe medication, it is still a product of human origin, obtained from the plasma do- nations of actively immunised donors. As this medication is made from blood, there is a possibility, albeit a very low one, of risk of transferring infections with known or yet unknown agents. In some parts of the world, there are also issues with the supply of a satisfactory amount of plasma with a high titer of anti-D antibodies (37), and the European Medicines Agency (EMEA) already requires limited immunization of D-neg volunteers, meaning that sufficient future supply of the solution is not certain (38). Therefore, it is important to determine a suitable algorithm which can, on the one hand, be used to prevent alloimmunization of pregnant women and the onset of HDFN, and on the other hand, stop unnecessary administration of anti-D Ig.In the past, determination of D Ag and the management of pregnant women

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according to their D type has changed.

At first, pregnant women with serological weak D types were treated as D-pos and did not receive protection with anti-D Ig (recommendation of the American Association of Blood Banks, AABB) of 1981 and the American College of Obstetricians and Gynecologists, ACOG)) (20). With increasingly detailed knowl- edge of genetic information on locus D and CE and the cases of the formation of anti-D antibodies in some types of serological weak D, there was a change in the recommendations for determining D Ag and management of pregnant women. In 2013, Daniels recommended the following algorithm: pregnant women who are serologically determined as D-neg, require prophylaxis with anti-D Ig, and those who are D-pos do not need it. If the serological determination of D is inconclusive, it is recommended to genotype the D gene. If the pregnant woman is a carrier of weak D type 1, 2, or 3, she can be treated as D-pos, and D-neg in all other cases. Sandler recommended the same in 2015 (15,20).

Sandler established that, considering the frequency of D-var in the racial- ly mixed population of the US, a precise determination of serological weak D types could prevent the administration of 24,700 anti-D Ig injections per year (20). It is characteristic for the Slovenian population, where there have been on av- erage 21,020 births per year for the past five years (39), that weak D types have a frequency of 0.4%, and represent 80% of all D-var. Consequently, we discover approximately 105 (0.5%) pregnant women who are carriers of D-var. Among those, approximately 80 (80%) of them are carriers of weak D type 1, 2, and 3, who do not require perinatal prophylaxis with anti-D Ig. Each of these pregnant women would receive anti-D Ig in the 28th week of pregnancy, even though there is no need for this. As the Caucasian population has between 80–85% of D-pos people, and therefore 15–20% of D-neg people (40,41), and there are 44% of homozygotes (D/D) and

56% of heterozygotes (D/d) among D-pos population (42), we can calculate that approximately 67 pregnant women (80%) with D-var BT (and therefore also mostly the D/d genotype) would receive perinatal prophylaxis with anti-D Ig unnecessar- ily, even after the birth of a D-pos child, and approximately 21 (25%) of them also because of potential sensitization events during pregnancy (bleeding, amniocen- tesis, etc.) (20). Based on this calculation, we would spare about 84 pregnant women approximately 172 unnecessary injections of anti-D Ig per year.

9 Conclusion

Exact determination of D BT is very important for the further management of pregnant women, blood donors and patients requiring a blood transfusion. The basic method for determining D Ag is still serological. The Rh system is highly polymorphic and leads to different phenotypes, D-pos, D-neg or D-var. The latter is divided into weak D, partial D and D_el. Today, we know from experience that people who are carriers of alleles for weak D types 1, 2, or 3, will not form anti-D alloantibodies when coming into contact with D-pos red blood cells. Therefore, selective integration of molecular-biological methods for determining the D genotype into laboratory practice represents a support method for determining D BT, and has significantly contributed to the advance- ment of obstetric and blood transfusion protocols. This way, we can limit the unnecessary injections of anti-D Ig for certain D-var pregnant women and unnecessary transfusions of D-neg blood for those patients with D-var who can safely receive D-pos blood (carriers of weak D type 1, 2, 3). This allows us to conserve supplies of anti-D Ig and D-neg blood units. By in- troducing targeted immunoprophylaxis of D-neg pregnant women with anti-D Ig, we can expect to take an additional step in the optimum use of anti-D Ig medication (43-46).

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References

1. Investigation strategy for RhD typing discrepancies using a combination of PCR-SSP and serological technicques. Bethesda: AABB; 2017 [cited 2017 Sep 03]. Available from: http://www.aabb.org/

development/scholarships/Documents/11er.pdf.

2. Sandler SG, Queenan JT. A Guide to Terminology for Rh Immunoprophylaxis. Obstet Gynecol.

2017;130(3):633-5. DOI: 10.1097/AOG.0000000000002190 PMID: 28796682

3. Koelewijn JM, Vrijkotte TG, van der Schoot CE, Bonsel GJ, de Haas M. Effect of screening for red cell antibodies, other than anti-D, to detect hemolytic disease of the fetus and newborn: a population study in Neetherlands. Trasfusion. 2008;48(5):941-52. DOI: 10.1111/j.1537-2995.2007.01625.x

4. Dovč T. Določanje plodovega genotipa RHD iz plazme RhD-negativnih nosečnic z metodo PCR v realnem času. [PhD Thesis]. Ljubljana: T. Dovč; 2013.

5. Westhoff CM. The Rh System. In: Roback JD, Combs MR, Grossman BJ, Hillyer CD, eds. AABB Technical Manual. 16th ed. Richmond, TX, USA: Ergodebooks; 2005. pp. 315-32.

6. Flegl WA, Castilho SL, Keller MA, Klapper EB, Moulds JM, Noizat-Pirenne F, et al. Molecular

immunohaematology round table discussions. In: AABB Annual Meeting. Philadelphia. 2014. Blood Trasfusion 2015;1-9.

7. Yousuf R, Mustafa AN, Ho SL, Tang YL, Leong CF. Anti-G with concomitant anti-C and anti-D: A case report in a pregnant woman. Asian J Transfus Sci. 2017;11(1):62-4. DOI: 10.4103/0973-6247.200770 PMID: 28316444 8. Bowman J. Thirty-five years of Rh prophylaxis. Transfusion. 2003;43(12):1661-6. DOI: 10.1111/j.0041-

1132.2003.00632.x PMID: 14641860

9. Koelewijn JM, de Haas M, Vrijkotte TG, van der Schoot CE, Bonsel GJ. Risk factors for RhD immunisation despite antenatal and postnatal anti-D prophylaxis. BJOG. 2009;116(10):1307-14. DOI: 10.1111/j.1471- 0528.2009.02244.x PMID: 19538414

10. van der Schoot C, deHaas M, Clausen FB. Genotyping to prevent Rh disease: has the time come? Curr Opin Hematol. 2017;24(6):544-50. DOI: https://doi.org/10.1097/MOH.0000000000000379

11. Pravilnik o transfuzijskih preiskavah in postopkih ob transfuziji. UL RS. 2007;(9).

12. Pravilnik o spremembah Pravilnika o transfuzijskih preiskavah in postopkih ob transfuziji. Ur l RS. 2018;(32).

13. Bricl I. Ciljana zaščita z IgG anti-D. Neinvazivna določitev plodovega genotipa RhD iz periferne krvi RhD- negativnih nosečnic. In: Stezinar SL, ed. Zbornik predavanj strokovnih srečanj Združenja za transfuzijsko medicino Slovenije. In: Srečanje Združenja za transfuzijsko medicino Slovenije. Zreče, Slovenija. Ljubljana:

Slovensko zdravniško društvo; 2017. p.31.

14. Koelewijn JM, de Haas M, Vrijkotte TG, Bonsel GJ, van der Schoot CE. One single dose of 200 microg of antenatal RhIG halves the risk of anti-D immunization and hemolytic disease of the fetus and newborn in the next pregnancy. Transfusion. 2008;48(8):1721-9. DOI: 10.1111/j.1537-2995.2008.01742.x PMID: 18507749 15. Daniels G. Variants of RhD—current testing and clinical consequences. Br J Haematol. 2013;161(4):461-70.

DOI: 10.1111/bjh.12275 PMID: 23432139

16. Wagner FF, Gassner C, Müller TH, Schönitzer D, Schunter F, Flegel WA. Molecular basis of weak D phenotypes. Blood. 1999;93(1):385-93. DOI: 10.1182/blood.V93.1.385 PMID: 9864185

17. Wagner FF, Flegel WA. The Rhesus site. Transfus Med Hemother. 2014;41(5):357-63. DOI: 10.1159/000366176 PMID: 25538538

18. Wagner FF, Frohmajer A, Ladewig B, Eicher NI, Lonicer CB, Müller TH, et al. Weak D alleles express distinct phenotypes. Blood. 2000;95(8):2699-708. DOI: 10.1182/blood.V95.8.2699 PMID: 10753853

19. Li Q, Hou L, Guo ZH, Ye LY, Yue DQ, Zhu ZY. Molecular basis of the RHD gene in blood donors with DEL phenotypes in Shanghai. Vox Sang. 2009;97(2):139-46. DOI: 10.1111/j.1423-0410.2009.01181.x PMID:

19490579

20. Sandler SG, Flegel WA, Westhoff CM, Denomme GA, Delaney M, Keller MA, et al.; College of American Pathologists Transfusion Medicine Resource Committee Work Group. It’s time to phase in RHD genotyping for patients with a serologic weak D phenotype. Transfusion. 2015;55(3):680-9. DOI: 10.1111/trf.12941 PMID:

25438646

21. Horn T. Use of RHD Genotyping for Serologic Weak D Phenotypes. American Red cross. MclEAn: American Society for Clinical Laboratory Science, 2018 [cited 2018 Jun 08]. Available from: http://www.ascls-nj.

org/Documents/Speaker%20Handouts%202016%20SS/Use%20of%20RHD%20Genotyping%20for%20 Serologic%20Weak%20D%20Phenotypes%20-%20ASCLS%20NJ%20TH%202016.pdf.

22. Haspel RL, Westhoff CM. How do I manage Rh typing in obstetric patients? Transfusion. 2015;55(3):470-4.

DOI: 10.1111/trf.12995 PMID: 25647404

(10)

23. Sandler SG, Roseff SD, Domen RE, Shaz B, Gottschall JL. Policies and procedures related to testing for weak D phenotypes and administration of Rh immune globulin: results and recommendations related to supplemental questions in the Comprehensive Transfusion Medicine survey of the College of American Pathologists. Arch Pathol Lab Med. 2014;138(5):620-5. DOI: 10.5858/arpa.2013-0141-CP PMID: 24786120 24. Müller TH, Wagner FF, Trockenbacher A, Eicher NI, Flegel WA, Schönitzer D, et al. PCR screening for

common weak D types shows different distributions in three Central European populations. Transfusion.

2001;41(1):45-52. DOI: 10.1046/j.1537-2995.2001.41010045.x PMID: https://pubmed.ncbi.nlm.nih.

gov/11161244

25. Dovc Drnovsek T, Rozman P. The distribution of different RHD variants in Slovenian population. Transfus Clin Biol. 2006;13:174-5.

26. Ansart-Pirenne H, Asso-Bonnet M, Le Pennec PY, Roussel M, Patereau C, Noizat-Pirenne F. RhD variants in Caucasians: consequences for checking clinically relevant alleles. Transfusion. 2004;44(9):1282-6. DOI:

10.1111/j.1537-2995.2004.04063.x PMID: 15318849

27. Araújo F, Rodrigues MJ, Monteiro F, Chabert T, Tavares G, Sousa G, et al. Weak D type 2 is the most prevalent weak D type in Portugal. Transfus Med. 2006;16(1):63-7. DOI: 10.1111/j.1365-3148.2005.00638.x PMID: 16480441

28. Wagner FF, Kasulke D, Kerowgan M, Flegel WA. Frequencies of the blood groups ABO, Rhesus, D category VI, Kell, and of clinically relevant high-frequency antigens in south-western Germany. Infusionsther Transfusionsmed. 1995;22(5):285-90. PMID: 8924742

29. Leader KA, Kumpel BM, Poole GD, Kirkwood JT, Merry AH, Bradley BA. Human monoclonal anti-D with reactivity against category DVI cells used in blood grouping and determination of the incidence of the category DVI phenotype in the DU population. Vox Sang. 1990;58(2):106-11. PMID: 2111059

30. van Rhenen DJ, Thijssen PM, Overbeeke MA. Serological characteristics of partial D antigen category VI in 8 unrelated blood donors. Vox Sang. 1994;66(2):133-6. DOI: 10.1159/000462492 PMID: 8184595

31. Rupreht RR, Pretnar HK, Galvani V, et al. Weak D and partial D in Slovenian population through serology and genotyping. Pflugers Arch. 2000;440:R195-6. DOI: 10.1007/s004240000062 PMID: 28008538

32. Wagner FF, Frohmajer A, Flegel WA. RHD positive haplotypes in D negative Europeans. BMC Genet.

2001;2(1):10. DOI: 10.1186/1471-2156-2-10 PMID: 11495631

33. Wagner FF, Gassner C, Muller TH, Schonitzer D, Schunter F, Flegel WA. Three molecular structures cause rhesus D category VI phenotypes with distinct immunohematologic features. Blood. 1998;91(6):2157-68.

DOI: 10.1182/blood.V91.6.2157.2157_2157_2168 PMID: 9490704

34. Avent ND. RHD genotyping from maternal plasma: guidelines and technical challenges. Methods Mol Biol.

2008;444:185-201. DOI: 10.1007/978-1-59745-066-9_14 PMID: 18425481

35. Clarke G, Hannon J, Berardi P, Barr G, Cote J, Fallis R, et al. Resolving variable maternal D typing using serology and genotyping in selected prenatal patients. Transfusion. 2016;56(12):2980-5. DOI: 10.1111/

trf.13798 PMID: 27611891

36. Garratty G. Do we need to be more concerned about weak D antigens? Transfusion. 2005;45(10):1547-51.

DOI: 10.1111/j.1537-2995.2005.00625.x PMID: 16181202

37. Kumpel BM. Efficacy of RhD monoclonal antibodies in clinical trials as replacement therapy for prophylactic anti-D immunoglobulin: more questions than answers. Vox Sang. 2007;93(2):99-111. DOI:

10.1111/j.1423-0410.2007.00945.x PMID: 17683353

38. Overview of comments received on ‘Draft Guideline on the core SmPC for human Anti-D immunoglobulin for intravenous use’ (EMA/CHMP/BPWP/319619). Amsterdam: European Medicines Agency; 2016

[cited 2018 Jun 08]. Available from: https://www.ema.europa.eu/en/documents/comments/overview- comments-received-draft-guideline-core-smpc-human-anti-d-immunoglobulin-intravenous-use-ema/

chmp/bpwp/319619/2005-rev-2_en.pdf.

39. Statistični urad republike Slovenije, SURS. Ljubljana: Statistični urad RS; 2018. Available from: http://www.

stat.si/StatWeb/Field/Index/17.

40. Daniels G. Human Blood Groups. Oxford: Blackwell Science Ltd; 2002. pp. 195-274. DOI:

10.1002/9780470987018.ch5

41. Dovč-Drnovšek T, Klemenc P, Toplak N, Blejec T, Bricl I, Rožman P. Reliable Determination of Fetal RhD Status by RHD Genotyping from Maternal Plasma. Transfus Med Hemother. 2013;40(1):37-43. DOI:

10.1159/000345682 PMID: 23637648

42. Avent ND, Reid ME. The Rh blood group system: a review. Blood. 2000;95(2):375-87. DOI: 10.1182/blood.

V95.2.375 PMID: 10627438

43. Kacker S, Vassallo R, Keller MA, Westhoff CM, Frick KD, Sandler SG, et al. Financial implications of RHD genotyping of pregnant women with a serologic weak D phenotype. Transfusion. 2015;55(9):2095-103. DOI:

10.1111/trf.13074 PMID: 25808011

(11)

44. Flegel WA, Roseff SD, Tholpady A. Phasing-in RHD genotyping. Arch Pathol Lab Med. 2014;138(5):585-8.

DOI: 10.5858/2013-0509-ED PMID: 24786114

45. Denomme GA, Wagner FF, Fernandes BJ, Li W, Flegel WA. Partial D, weak D types, and novel RHD alleles among 33,864 multiethnic patients: implications for anti-D alloimmunization and prevention. Transfusion.

2005;45(10):1554-60. DOI: 10.1111/j.1537-2995.2005.00586.x PMID: 16181204

46. Drnovšek DT. Molekularno-biološke preiskave za določanje krvnih skupin na Zavodu RS za transfuzijsko medicino. In: Stezinar SL, ed. Zbornik predavanj strokovnih srečanj Združenja za transfuzijsko medicino Slovenije. 2017 Apr 7-8; Zreče, Slovenija. V Ljubljani: Slovensko zdravniško društvo; 2017. p. 28-30.