EVALUATING ABNORMAL HAEMOGLOBIN VARIANTS AND RHESUS BLOOD GROUP AMONG PREGNANT WOMEN

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EVALUATING ABNORMAL HAEMOGLOBIN VARIANTS AND RHESUS BLOOD GROUP AMONG PREGNANT WOMEN

ABSTRACT

The rhesus (Rh) blood group system is one of the most polymorphic and immunogenic blood group systems known in humans. The common potent Rh antigens reside on the RH polypeptides.  The antigenic expression is controlled by the RHD and RHCE genes, linked in tandem on chromosome 1. The D antigen is clinically the most important because of its high immunogenicity and polymorphism. HDFN is perpetuated by foeto-maternal rhesus antigens incompatibility. The aim of this study was to identify and classify rhesus D (RH1) variants among pregnant women, that were at a risk of haemolytic disease of the foetus and newborn (HDFN). This was a cross sectional study involving 110 pregnant women.  The RHD status of participants was confirmed using monoclonal antibodies and weak D expression was confirmed by indirect antiglobulin test.  Antibody screening was performed using coombs gel cards. RH primers were used to amplify exon 7 which is identical to both RHCcEe and RHD cDNA, and exon 10 that is specific to the D sequence. PCR multiplex assay using six RHD specific primer sets designed to amplify RHD exons 3, 4, 5, 6, 7 and 9 was used for RHD genotyping. All the 110 samples were confirmed as RHD negative by serology. Eight (7.3%) out of the 110 RHD negative samples showed the presence of red cell antibodies in the serum. Thirty four (30%) samples had both exon 7 and 10, depicting an RHD positive phenotype, 67 (60%) samples had only exon 7 amplified, denoting RHD negative phenotype and 9 (8.2%) samples, had neither exon 7 nor 10 denoting indeterminate status. None of the six RHD specific exons were amplified from the 34 D+ and 67 D- individuals.

The rate of alloimmunisation was 7.3% that require RHD immunoglobulin prophylaxis. Thirty percent (30%) of the serological RHD negative samples were molecularly RHD positive hence need to be classified as such. This study shows that not all RHD negative pregnant women require anti-D immunoglobulin prophylaxis.

 

CHAPTER ONE

INTRODUCTION  

1.1 BACKGROUND

The Rhesus blood group system is one of the most polymorphic and immunogenic systems of blood group antigens known in humans. This blood group system is comprise of numerous antigens, principally among them is ‘’RHD’’, ‘’RhC’’, “Rhc”,

“RhE” and “Rhe” antigens (Flegel, 2007; Sell et al., 2013).

 

The Rh factor is a term used to describe the presence of the D antigen, the first antigen that was identified belonging to the Rh blood group system (Huang, 2013), on the surface of erythrocytes. The common potent Rh antigens reside on the RH polypeptides, whose expression is controlled by the RHD and RHCE genes, linked in tandem on chromosome one. The RHD produces the D antigen and RHCE produces the alleles Ce, ce, cE and CE compound antigens. There are about 300 allelic forms of the RHD and RHCE genes named at the molecular level. This polymorphism is due to various pattern of genomic diversification at the RH locus. The predominant mechanism of diversification at the RH locus is coding nucleotide changes and genomic rearrangement through homologous recombination.

 

The D antigen is clinically the most important because of its high immunogenicity and polymorphism. It has a prevalence rate of about 85% in Caucasians and about 95% in blacks (Daniels et al., 2007; Poole and Daniels, 2007). The RHD negative phenotype is characterised by high molecular diversity between serologic and molecular methods. It accounts for the incidence of alloimmunization due to pregnancy or blood transfusion, despite the numerous red cell antigens (Delaney et al., 2016; Egbor, Fellow and Knott, 2012). Most red cell alloimmunization is attributed to  antigens “D” “C”, “c”, “E”, “K”, and “Jk”a (Evers et al., 2016; Flegel et al., 2016). Alloimmunization in mothers is caused by pregnancy, blood transfusion, abortion, ectopic pregnancy, amniocentesis

(iatrogenic) and foeto-maternal haemorrhage (Nour, ARamy and Ali,  2011).

 

The absence of the D antigen designates RH negative status on human erythrocytes; its molecular basis is divided into deletion and non-deletion types. Vast majority of Caucasians with the phenotype have a total deletion of the RHD gene. The non-deletion type occurs commonly in Africans, Japanese and other Asians (Huang, 2013).  There are genomic alterations of three types that silence the D antigen expression in RH negative subjects who carry either a partial or completely intact RHD (Avent et al., 2006; Flegel, 2011). The weak D (DU) phenotype is associated with various forms of mutations that affect the quantity and quality of D antigen (Daniels, 2013; Singleton et al., 2016). Most of these mutations are novel missense mutation that results in a single amino acid substitution that resides on either transmembrane domains or cytoplasmic portion of the protein. Partial D phenotype typifies a loss and/ or an alteration of one or more of D epitopes within the context of entire D protein. They often show weakened D expression making it difficult to differentiate between partial D and weak D (Denomme et al., 2005).

 

Due to the density and epitope expression of the D antigen on the red cell surface, the phenotypes commonly observed are normal D positive, weak D, partial D, DEL and D negative (Ye et al., 2014). Expression of weak D and partial D is attributed to variants of antigens and a large number of alleles of RHD gene (Rizzo et al., 2012). The weak D phenotype is due to quantitative expression of the D antigen epitopes whiles that of the partial D is due to qualitative expression of antigenic epitopes of the D antigen.

 

The discovery of the Rh blood group system was an insight into the instances of the haemolytic disease of the foetus and new-born (HDFN). HDFN is perpetuated by foetomaternal rhesus antigens incompatibility (Rouillac-Le Sciellour et al., 2007). HDFN occurs as result of the immune-mediated destruction of red cells antigens of a foetus that are not usually found on maternal red cells ( (Fasano, 2016). The common antibody produced is immunoglobulin G (IgG). The trans placental transfer of IgG antibodies attacks foetal RHD antigens leading to their destruction (Fasano, 2016).  Other RH blood group antigens that can cause alloimmunization include antigen C, E, c and e (Shao et al., 2010). Antibodies directed against epitopes of these antigens can cause alloimmunization through transfusion or pregnancy. The antibodies often encountered in Africa are anti-c, anti-C, anti-e and anti-D (Ngoma et al., 2016).

 

1.2 PROBLEM STATEMENT

The highly immunogenic nature of the RHD antigen and the consequent risk of alloimmunization of RHD negative individuals, when exposed to RHD positive foetus or transfused with RHD positive blood is well documented (Ngoma et al., 2016). Approximately 80% of RHD- negative individuals develop anti-D when exposed to Dpositive blood or foetus (Wang et al., 2010).

 

RHD antigen incompatibility has led to haemolytic disease of the foetus and new-born

(HDFN) and haemolytic transfusion reaction. About 40% RHD negative women carry

RHD negative children (Daniels et al., 2007) and hence are not at risk of HDFN. The common variants of RHD antigens are difficult to detect serologically and are often mistyped as RHD negative by conventional serological assays (Gowland et al., 2014). Some of these RHD variants have caused anti-D alloimmunization in transfused patients. D variants prevalence in Nigeria has not be been established.

 

Though the inception of anti-D immunoglobulin prophylaxis has reduced the frequency

HDFN, there is still alloimmunization to RHD antigens (Egbor, Fellow and Knott, 2012), especially in developing countries where there is, the lack of the anti-D immunoglobulin prophylaxis(Pal and Williams, 2015). Antenatal service provision remains an avenue for promoting maternal and neonatal health. It has being reported that low prenatal and maternal health service utilisation are among the factors contributing to adverse birth outcomes (Asundep et al., 2013). Though there is relatively high patronage of antenatal services in Nigeria (Gyebi, 2015), antenatal screening for alloantibodies in pregnant women, even if carried out, is at the tertiary hospitals. This approach has left out many potentially alloimmunised pregnant women (Egbor, Fellow and Knott, 2012), seeking antenatal at other levels of the health care system in Nigeria.

 

Currently, data in Nigeria shows  that 48% of all under-five mortality occur within the first month of life (Gyebi, 2015)). RHD variants need to be investigated because of the risk of alloimmunization in patients especially pregnant women to prevent HDFN (Abdelkefi et al., 2014; Sankaralingam et al., 2016). Furthermore, anti-D immunoglobulin administration has theoretically exposed pregnant women to prions and other blood transmissible pathogens (Neovius et al., 2016).

 

1.3 SIGNIFICANCE OF STUDY

RHD negative women often require anti-D immunoglobulin prophylaxis especially during the third trimester of pregnancy or within 72 hours after delivery depending on the local policy. The use of anti-D immunoglobulin prophylaxis to prevent alloimmunization in RHD negative women has significantly decreased HDFN (Mcbain, Crowther and Middleton, 2015). In sub-Saharan Africa, management of RHD negative women has inherent challenges, which  include the absence of policy on universal access to anti-D immunoglobulin, poor health infrastructure, lack of foeto-maternal testing facilities, unaffordability of anti-D immunoglobulin and suboptimal management and poor data on potentially sensitising events (Erhabor et al., 2013). The majority of the potentially sensitising events of D antigen are silent (Mcbain, Crowther and Middleton, 2015).  Routine anti-D immunoglobulin prophylaxis is only effective in nonsensitised RHD negative pregnant women. Approximately 40% of RHD negative women who carry RHD negative foetus will be given anti-D immunoglobin

unnecessarily (Mcbain, Crowther and Middleton, 2015)).  Furthermore, this prophylaxis cannot prevent all cases of sensitisation; some are either prior to or despite the administration of anti-D immunoglobulin.

 

Determining pregnant women with partial D or weak D phenotype may help in identifying pregnancies at risk of HDFN. Some weak D types (type 1, 2, 3 and 4.1) do not cause alloimmunization and pregnant women with such D types often do not require anti-D immunoglobulin prophylaxis (Hussein & Teruya, 2014). Those with weak D type 4.2, 11, 15 and 21 are the ones at risk of HDFN (Rizzo et al., 2012). Pregnant women with common weak D types should be manage as RHD positive as this is clinically beneficial without increasing overall cost of unnecessary testing and routine antenatal care (Kacker et al., 2015). Identifying pregnant women at risk of HDFN, that is the true RHD negative pregnant women, will eliminate the need for unnecessary administration of anti-D immunoglobulin prophylaxis. Screening for weak D and partial D will eliminate unnecessary administration of anti-D immunoglobulin prophylaxis in routine antenatal and postnatal care and enhances a cost-effective management of RHD negative mothers.

 

Molecular typing of pregnant women with weak D and partial D phenotypes may help prevent unnecessary anti-D immunoglobulin prophylaxis for individuals with weak D type 1, 2, 3, and 4. It will also prevent mistyping of partial D and weak D blood as D negative, thus conserving blood for the true RHD negatives (Shao et al., 2010), with particular regards to transfusion cross-matching. RH incompatibility due to variant forms of the D and other RH antigens is still crucial for pregnancies and patients with chronic diseases who require frequent blood transfusion or organ transplant (Huang .,

2013).

 

1.4 AIM AND SPECIFIC OBJECTIVES

1.4.1 Aim

The aim of this study was to identify and classify rhesus D (RH1) variants among pregnant women, that are at risk of haemolytic disease of the foetus and newborn (HDFN).

 

1.4.2 Specific Objectives

  1. To screen for anti –D antibody in RHD negative pregnant women in order to determine those who are already sensitised against antigen D (RHD) and are at risk of HDFN.
  2. To screen for molecular RHD status omitted by conventional serology which has the potential of causing alloimmunization in the pregnant women.
  3. To determine the prevalence of RHD variants that can cause alloimmunization in the RHD negative pregnant women.

EVALUATING ABNORMAL HAEMOGLOBIN VARIANTS AND RHESUS BLOOD GROUP AMONG PREGNANT WOMEN

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