Monday, 24 October 2016

Seminar Report On Haemolytic Disease Of The New-Born

HAEMOLYTIC DISEASE OF THE NEW-BORN

BY

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MLS/11/###

A SEMINAR WORK PRESENTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF BACHELOR OF
MEDICAL LABORATORY SCIENCE
(B.MLS)

DEPARTMENT OF MEDICAL LABORATORY SCIENCE

HAEMATOLOGY UNIT
FACULTY OF HEALTH SCIENCE
MADONNA UNIVERSITY
ELELE RIVERS STATE

SUPERVISOR: MR. EZE
CO-ORDINATOR: DR. ADEGOKE ADEBAYO

                                                               
                                                                       MARCH 2016


CERTIFICATION
This is to certify that this seminar work title “Haemolytic disease of the new-born” was done by Udoutun Rosemary Patrick (MLS/11/140) met the regulation governing the award of Medical Laboratory Science, Madonna University, Nigeria and is hereby approved for its contribution to knowledge.


MR. EZE                                                        …………………………                 ………………………..
Supervisor                                                     Signature                                    Date


DR. ADEGOKE ADEBAYO                       ………………………....                 ………………………..
Co-ordinator                                               Signature                                   Date


ASSOC. PROF. NNATUANYA .I.             …………………………..               ………………………..
H.O.D                                                              Signature                                    Date




                                          DEDICATION
This work is dedicated to God Almighty and Blessed Virgin Mary
                            



ACKNOWLEGEMENT
My special thanks goes to God Almighty, Jesus Christ the son and Blessed Virgin Mary for the graces bestowed upon me. My acknowledgement goes to my supervisor Mr. Eze in a very special way for his wonderful advice and knowledge he impacted in me to make sure that am successful in studies and also for his supervision. My heartfelt gratitude goes to my parents, Mr/Mrs Patrick .M. Udoutun and also my siblings who were always there to support me morally, spiritually, financially and otherwise. I also want to acknowledge my HOD. ASSOC. PROF. Isaac Nnatuanya, my lecturers and friends for their moral and academic support.



  


SUMMARY
Haemolytic Disease of the New-born (HDN) also known as Erythroblastosis fetalis is an immune haemolytic anaemia that is characterized by the presence of immunoglobulin G (IgG) antibodies in the maternal circulation, directed against paternally derived antigen present in the foetus causing destruction of baby's red blood cells. Symptoms such as; Oedema, new-born jaundice, enlarged liver and spleen, anaemia, still birth may occur in severe cases. The vast majority of HDN is caused by Rhesus “D” antigen; it may be caused by ABO incompatibility, and also due to other blood group incompatibility. IgG class antibody is mostly implicated in HDN because of its ability to cross the placenta. The mother usually develops antibodies following an early stimulus before affecting the foetus; this exposure can be during first pregnancy. During antenatal visit, screening tests are done to predict pregnancies at risk of HDN. The administration of RhIg (Rhogam) a human anti-D globulin to all unsensitised Rh-negative mothers after abortion, miscarriage and deliver of an Rh-Positive infant prevents sensitization to Rhesus factor. Treatment can be with intrauterine transfusion, induction of early pregnancy, phototherapy, exchange transfusion, and depending on the form of HDN.


CHAPTER ONE

1.0      INTRODUCTION
Haemolytic Disease of the New-born(HDN) also known as Erythroblastosis fetalis or haemolytic disease of the foetus is an immune haemolytic anaemia that is characterized by the presence of immunoglobulin G (IgG) antibodies in the maternal circulation, directed against a paternally derived antigen present in the foetal/neonatal red cells that cause haemolysis in the fetus by crossing the placenta and sensitizing red cells for destruction by the macrophages in the fetal spleen thereby destroying the new-born baby's blood cells very quickly and causing symptoms such as; Oedema(swelling under the surface of the skin),New-born  jaundice, enlarged liver and spleen, anaemia, still birth may occur in severe case(Hadley AG and Vox Sang,1998).                       
The vast majority of HDN is caused by Rhesus “D” antigen. Several foetal rhesus antigen may cause alloimmunization (c, C, D, e, E) and this occurs with the Kell, Duffy, ABO, MNS and other blood group systems. Pregnancies at risk of HND are those in which an Rh D-negative mother becomes pregnant with an Rh D-positive child (the child having inherited the D antigen from the father). The mother's immune response to the fetal D antigen is to form antibodies against it (anti-D). These antibodies are usually of the IgG type, the type that can cross the placenta into the foetal circulation. HDN can also be caused by an incompatibility of the ABO blood group. It arises when a mother with blood type O becomes pregnant with a fetus with a different blood type (type A, B, or AB). The mother's serum contains naturally occurring anti-A and anti-B, which tend to be of the IgG class and can therefore cross the placenta and haemolyses fetal RBCs. HDN due to ABO incompatibility is usually less severe than Rh incompatibility. (Urbaniak, et al., 2000, Garratty, et al., 2004).
Antenatally, the presence of anti-D antibodies in the mother is detected by indirect coomb’s test which is usually carried out on all Rhesus negative mothers during their subsequent antenatal visit. Once the presence of maternal anti-D has been confirmed, the next step is to determine whether the fetal RBCs are a target, i.e., confirm the Rh status of the fetus. If the father is homozygous for the D allele (D/D), the fetus will be D positive. If however the father is heterozygous (D/d), there is a 50:50 chance that the foetus maybe D positive or negative, and the only way to know the blood type for sure is to test a sample of fetal cells taken from the amniotic fluid or umbilical cord. If the fetus is Rh D-positive, the pregnancy is carefully monitored for signs of HDN. Monitoring includes regular ultrasound scans of the fetus and monitoring of the amount of anti-D in the mother's serum (Urbaniak, et al., 2000, Garratty, et al., 2004). If anti-D is not found in the mother's serum, it is likely that she has not been sensitized to the Rh D antigen. The risk of future sensitization can be greatly reduced by prophylactic administration of anti-D Ig (RhoGAM) at 28weeks and again at 32-34weeks of pregnancy and if needed within 72hours of delivery to "mops up" any fetal RBCs that may have leaked into the maternal circulation.
Incidence of HDN is dependent on the population who are Rh-D negative. In most tropical countries like Africa and Asia, HDN is likely to be caused by ABO incompatibility than Rhesus incompatibility due to low number of Rhesus negative women (Garratty et al., 2004).

.1 HISTORY

Haemolytic disease of the new-born used to be a major cause of fetal loss and death among new-born babies. The first description of HDN is thought to be in 1609 by a French midwife; Louise Bourgeois (Bowman JM, 1998). In 1609, Bourgeois assisted at a twin birth: the first twin was markedly hydropic and practically dead at delivery, whereas the second developed rapidly worsening jaundice within a few hours and died three days after birth. For the next 300 years, many similar cases were described in which new-borns failed to survive (Gruslin, et al., 2011, Cohen and Waltham, 2009).
In 1932 the three most characteristic clinical signs of Rh HDN, that is, hydrops fetalis, severe neonatal jaundice and delayed anaemia of the new-born, were recognised as expressions (with different incidences and severities) of a single pathological process, confirming the hypothesis proposed by Diamond and his colleagues, they believed that the pathogenesis of the disease involved a defect of the erythron (Bowman, 1999). It was not until the 1950s that the underlying cause of HDN was clarified that the new-born’s red blood cells (RBCs) are being attacked by antibodies from the mother. The attack begins while the baby is still in the womb and is caused by an incompatibility between the mother's and baby's blood (Gruslin, et al., 2011, Cohen and Waltham, 2009)
In the 1960s, trials in the United States and the United Kingdom tested the use of therapeutic antibodies that could remove the antibodies that cause HDN from the mother's circulation. The trials showed that giving therapeutic antibodies to women during their pregnancy largely prevented HDN from developing (Urbaniak, et al., 2000). In the 1970s, routine antenatal care included screening of all expectant mothers to find those whose pregnancy may be at risk of HDN, and giving preventative treatment accordingly. This has led to a dramatic decrease in the incidence of HDN, particularly severe cases that were responsible for stillbirth and neonatal death. (Garratty, et al., 2004).

1.2 GENETICS

Although the Rh antibody is the most common cause of severe haemolytic disease of the new-born, other alloimmune antibodies belonging to Kell (K and k), Duffy (Fya), Kidd (Jka and Jkb), and MNSs (M, N, S, and s) systems do cause severe haemolytic disease of the new-born(Van der Schoot CE, et al.,2003). The Rh blood group system uses Fisher-Race nomenclature, and the Rh gene complex consists of 3 genetic loci each with 2 major alleles. They code for 5 major antigens denoted by letters, C, c, E, e, and D. Rh blood group antigens are inherited as determined by at least 2 homologous but distinct membrane-associated proteins. Two separate genes: RhCE and RhD, located on the short arm of chromosome 1, encode Rh proteins. Production of 2 distinct proteins from the RHCE gene is due to alternative splicing of messenger RNA. Rh gene complex is described by 3 loci, and, therefore, 8 gene complexes are possible.
These complexes are as follows; CDe, cde, CDE, cDe, Cde, cdE, cDE, and CdE. Expression is limited to RBCs, with an increasing density during their maturation, unlike the ABH system, which exists in a wide variety of tissues. Rh antigen is not expressed on RBC progenitors. Of individuals who are Rh positive, 45% are homozygous (CDe/CDe), and 55% are heterozygous (CDe/cde) for the RhD gene (Singleton BK, et al., 2000). The Rh-negative phenotype represents absence of D protein on RBCs and most commonly results from deletion of the RHD gene on both chromosomes. However, the RHD gene has significant heterogeneity, and several inherited mutations and rearrangements in its structure can result in a lack of expressions of the RhD phenotype as well. Important examples of such mutations include the RHD pseudogene and RHD-CE-D hybrid gene (Flegel WA, 2011)
Beyond the 5 major antigens, more than 100 antigenic variants of Rh group system have been identified. Individuals with these weak-D phenotypes comprise of 2 populations: first group (90%) that expresses normal but reduced quantities of D antigen on the RBC surface and most cannot be sensitized to produce anti-D. However, the second group (remaining 10%) known as partial-D (e.g., Cw, Du) that express partial D epitopes on RBC surface and can make anti-D and rarely experience foetal haemolytic disease of the new-born. The partial D phenotype results from amino acid substitution in the active RhD epitope (Moise KJ, 2008). Most women with partial-D phenotype are classified as Rh negative on routine testing and are candidates for Rhesus immune globulin (RhIG). However, Rh-negative infants born to Rh-negative women should undergo testing to detect the partial-D phenotype so that RhIG can be administered in the event of weak expression.

1.3   PATHOPHYSIOLOGY

Occurrence of HDN as a result of red blood cell alloimmunization secondary to pregnancy involves three key stages. First, a paternally derived red blood cell antigen foreign to the mother must be inherited by the foetus. Second, the volume of foetal red cells that gain access to the maternal circulation must be sufficient to stimulate an immune response in the particular individual. Finally, maternal antibodies to foetal red cells must gain transplacental access and cause immune destruction of sensitized red cells by Fc receptor-bearing effector cells in the foetus and neonate(Bidyut, et al .,2010).

illustration showing the pathophysiology of HDN
Diagram showing the pathophysiology of HDN
Figure 1: Diagram showing the pathophysiology of HDN. Source:McGraw-Hill Companies, Inc.


Rhesus alloimmunisation begins with red blood cells from a rhesus positive foetus crossing the placental barrier during pregnancy and delivery, and entering the maternal blood circulation. A rhesus positive father and a rhesus negative mother are required for this situation to develop. The incompatible antigens introduced result in a primary immune response and stimulate the production of maternal antibodies (Kumar and Regan; 2005).
In a first pregnancy, Rh sensitization is not likely to cause problems; it becomes a problem in a future pregnancy with another Rh positive baby. When the next pregnancy occur, the mother’s antibodies cross the placenta then reacts with the corresponding antigen to fight the Rh positive cells in the baby’s body that the baby has inherited from the father which is foreign to the mother. Hence, antigen antibody interaction occurs. Sensitization of baby’s red blood cell (RBC) by mother’s IgG antibody causes the baby’s RBC to be destroyed, thereby causing haemolysis. These antibody-coated RBCs are then removed from fetal circulation by the macrophages of the spleen and liver. This haemolysis results in anaemia, hyperbilirubinaemia and the production of excessive erythroid tissue in the liver, spleen, bone marrow, skin and placenta. In severe cases, multi-organ dysfunction and hypoproteinaemia can develop. The severity of anaemia depends on the amount of mother’s antibody, its specificity, its avidity, and other characteristics. Anaemia will stimulate bone marrow to produce more RBC including immature RBC, which is then released to fetus circulation. This is also known as Erythroblastosis fetalis.
HDN is often classified into three categories, on the basis of the specificity of the causative IgG antibody: D haemolytic disease caused by anti-D alone or, less often, in combination with anti-C or anti-E, other haemolytic disease caused by antibodies against other antigens in the Rhesus system or against antigens in other systems; anti-c and anti-K are most often implicated, and ABO HDN caused by anti-A or anti-B (Brecher M.E. 2005).


CHAPTER TWO
LITERATURE REVIEW

2.1 EPIDEMIOLOGY

2.1.1 FREQUENCY

The incidence of haemolytic disease of the new-born depends on the proportion of the population who are RhD negative. This varies within ethnic minorities but, in the UK, it is highest in the Caucasian population (approximately 16%). Before immunoprophylaxis was available, HDN affected 1% of all new-borns and was responsible for the death of one baby in every 2,200 births but incidence of Rh sensitization has declined from 45 cases per 10,000 births to 10.2 cases per 10,000 total births, with less than 10% requiring intrauterine transfusion (Chavez et al., 1991). Currently, anti-D is still one of the most common antibodies found in pregnant women, followed by anti-K, anti-c, and anti-E. Of those foetus that require intrauterine transfusions, 85%, 10%, and 3.5% were due to anti-D, anti-K, and anti-c, respectively (Lindenburg et al., 2012). ABO incompatibility frequently occurs during the first pregnancy and is present in approximately 12% of pregnancies, with evidence of foetal sensitization in 3% of live births. Less than 1% of births are associated with significant haemolysis.

2.1.2 MORTALITY AND MORBIDITY

Only 3 antibodies are associated with severe foetal disease: anti-Rh-D, anti-Rh-c, and anti-Kell (K1). Nearly 50% of the affected new-borns do not require treatment, have mild anaemia and hyperbilirubinemia at birth, and survive and develop normally. Approximately 25% are born near term but become extremely jaundiced without treatment and either die or become severely affected by kernicterus. The remaining 25% of affected new-borns are severely affected in utero and become hydropic (Bowman JM, Creasy RK et al., 1999).

2.1.3 RACE
Incompatibility involving Rh antigens (anti-D or anti-c) occurs in about 10% of all pregnancies among whites and blacks; in contrast, it is very rare in Asian and African women because of the low number of Rh negative women.

2.1.4 SEX

Fetal sex plays a significant role in the degree of response to maternal antibodies. An apparent 13-fold increase is observed in fetal hydrops in RhD-positive male foetus compared with female foetus in similarly sensitized pregnancies (Kaplan M, Na'amad M, et al., 2009)

2.2 PROGNOSIS 

The severity of this condition can vary. Some babies have no symptoms. In other cases, problems such as hydrops can cause the baby to die before, or shortly after birth. Severe HDN may be treated before birth by intrauterine blood transfusion (Gruslin AM et al., 2011; Cohen and Waltham; 2009).

2.3 SIGNS AND SYMPTOMS

HDN can destroy the new-born baby's blood cells very quickly, which can cause symptoms and each infant may experience symptoms differently. Complications can range from mild to severe.

Before birth:
·        With amniocentesis (process of withdrawal of amniotic fluid with a needle for the purpose of analysis) the amniotic fluid may have a yellow colouring and contain bilirubin.
·        Hydrops Fetalis: This will occur as the baby's organs are unable to handle the anaemia. The heart begins to fail and large amounts of fluid build-up in the baby's tissues and organs. In severe forms this can include petechiae and purpura. The infant may be stillborn or die shortly after birth.
·        Ultrasound of the foetus shows enlarged liver, spleen, or heart and fluid build-up in the foetus' abdomen.

After birth:

·        Jaundice: Due to the destruction of red blood cells, there’s a usually elevated bilirubin level. After delivery bilirubin is no longer cleared (via the placenta) from the neonate's blood, Infants unable to get rid of the bilirubin so bilirubin builds up in the blood(hyperbilirubinemia) and other tissues and fluids of the infant's body resulting in JAUNDICE. Symptoms of jaundice (yellowish skin and yellow discoloration of the whites of the eyes) increase within 24hours after birth; there is the possibility of acute or chronic kernicterus (damage to Brain centres).
·        Anaemia: Anaemia limits the ability of the blood to carry oxygen to the infant's organs and tissues and lead to breathing difficulties to the infant. Profound anaemia can cause high-output heart failure, with pallor, and respiratory distress.
·        Babies with hydrops fetalis have severe oedema of the entire body and are extremely pale. They often have breathing problems.

2.4 CAUSES

HDN may develop when a mother and her unborn baby have different blood types called "incompatibility"(Gruslin, et al., 2011; Cohen and Waltham,2009). The mother produces substances called antibodies that attack the developing baby's red blood cells. Antibodies produced by the mother potentially causes HDN and is antibody of IgG class, this antibody is known to cause various degree of complications to the foetus if the foetus has RBCs containing the corresponding antigen. IgG class antibody is important in HDN because their ability to cross placenta. The 2 main common cause of HDN are:

Ø Rhesus incompatibility
Ø ABO incompatibility
Ø HDN can also be caused by other alloantibodies and this is uncommon. Most frequently involved antibodies of Kell, Duffy, Kidd and MNS blood group system.

2.5: HAEMOLYTIC DISEASE OF THE NEWBORN CAUSED BY RHESUS INCOMPATIBILITY (RHESUS ISSOIMUNIZATION)


In 1941, Levine and co-workers first described the involvement of the Rhesus factor in erythroblastosis fetalis (Levine et al 1941). Rhesus incompatibility is the most severe form of HDN and sometimes fatal. This form of HDN happens when mother who is rh-D negative is bearing an Rh-D positive foetus. D positivity of the foetus is due to genetically inheritance of D from father. Anti-D is the commonest antibody in Rh system that causes HDN although other Rh antigens such as c, C, E, and e, can also cause problems. If the father is heterozygous for the Rh D deletion, there is a 50% chance of the foetus being D-negative. If the father is homozygous for the Rh D gene, the foetus will definitely inherit the D antigen. Rh D negative mother lacks a functional Rh D gene and so does not produce the D antigen, and may be immunized by D-positive foetal blood.

2.5.1: PATHOGENESIS

Isoimmune haemolytic disease from D antigen is approximately three times more frequent among white persons than among black persons. When small quantities of    Rh-D foetal blood containing D antigen inherited from an Rh-positive father enter the maternal circulation during pregnancy, with spontaneous or induced abortion, or at delivery, antibody formation against D antigen may be induced in the unsensitised Rh-negative recipient mother. Once sensitization has taken place, considerably smaller dose of antigen can stimulate an increase in antibody titre.
Haemolytic disease rarely occurs during a first pregnancy because transfusion of Rh-Positive foetal blood into the Rh-Negative mother occurs near the time of delivery, too late for the mother to become sensitized and transmit antibody to her baby before delivery. The fact that 55% of Rh-Positive fathers are heterozygous (D/d) and may have Rh-negative offspring and that foetal-to-maternal transfusion occurs in only 50% of pregnancies and has reduced the chance of sensitization. But once a mother has been sensitized, in subsequent pregnancies with Rh-positive baby, the baby is likely to have haemolytic disease; the severity of Rh illness worsens with successive pregnancies. The injection of anti-D gamma globulin (RhoGAM) into the mother immediately after the delivery of each Rh-Positive infant has been a successful strategy to reduce Rh haemolytic disease.

2.5.2: CLINICAL MANIFESTATIONS

A wide spectrum of haemolytic disease occurs in affected infants born to sensitized mothers, depending on the nature of the individual's immune response. The severity of the disease may range from only laboratory evidence of mild haemolysis (15% of cases) to severe anaemia with compensatory hyperplasia of erythropoietic tissue leading to massive enlargement of the liver and spleen. When the compensatory capacity of the haematopoietic system is exceeded, profound anaemia occurs and results in pallor, signs of cardiac decompensation, massive anasarca, and circulatory collapse.

Ø HYDROPS FETALIS: This clinical picture of excessive abnormal fluid in two or more foetal compartment (skin, pleura, pericardium, placenta, peritoneum, amniotic fluid) termed hydrops fetalis which frequently results in death in utero or shortly after birth due to profound anaemia and circulatory failure.

diagram of a neonate suffering from hydrops fetalis.
Image of a neonate suffering from hydrops fetalis.
Figure 2: Image of a neonate suffering from hydrops fetalis. Source: Adamimages.com


The severity of hydrops fetalis is related to the level of anaemia and degree of reduction in serum albumin which is due in part to hepatic dysfunction. Alternatively heart failure may increase right heart pressure, with subsequent development of oedema and ascites, failure to initiate spontaneous effective ventilation becomes pulmonary oedema, after a successful resuscitation, severe respiratory distress may develop. Petechiae, purpura and thrombocytopenia may also be present in severe cases as a result of decreased platelet production.

Ø JAUNDICE: Jaundice may be absent at birth because of placental clearance of lipid soluble conjugated bilirubin, but in severe cases, bilirubin pigments can stain the amniotic fluid, cord, and Vernix caseosa yellow. Jaundice is generally evident on the first day of life because the infant's bilirubin conjugating and excretory systems are unable to cope with the load resulting from massive haemolysis. Indirectly reacting bilirubin accumulates postnatal and may reach extremely high levels and present a significant risk of bilirubin encephalopathy and the risk of development of kernicterus is very high.

illustration of a neonate suffering from Jaundice and Kernicterus.
Diagram of a neonate suffering from Jaundice and Kernicterus.
Figure 3: A diagram of a neonate suffering from Jaundice and Kernicterus.source:Adamimages.com
Infants born after intrauterine transfusion for prenatally diagnosed Erythroblastosis may be severely affected because the indications for transfusion are evidence for already severe diseases utero (hydrops, foetal anaemia).such infants usually have very high cord level of bilirubin reflecting severity of haemolysis. Infants treated with intraumbilical vein transfusion in utero also have benign. Anaemia from continuing haemolysis may be masked by the previous intrauterine transfusion and the clinical manifestations of erythroblastosis may be superimposed on various degrees of immaturity resulting from induced premature delivery.

2.5.3: LABORATORY TEST

Maternal blood

·        ABO and Rh grouping:  ABO and Rh grouping are ordered the first time a pregnant woman sees a physician. The father may also be tested at this time. If the woman is Rh negative and the father is heterozygous for the D antigen, the possibility of the infant being D positive is 50%.
·        Antibody screen and identification: Antibody screen is done to detect whether sensitization to D antigen has developed, if the initial antibody screen is positive, the antibody is identified and a titre is determined. Antibody titration is done to help the obstetrician determine the severity of HDN and the need for foetal monitoring (ultrasound, amniocentesis, and cordocentesis).
·        The Kleihauer–Betke test or flow cytometry on a postnatal maternal blood sample can confirm that fetal blood has passed into the maternal circulation and can also be used to estimate the amount of foetal blood that has passed into the maternal circulation.
·        The indirect Coombs test is used to screen blood from antenatal women for IgG antibodies that may pass through the placenta and cause haemolytic disease of the new-born.

Foetal blood (or umbilical cord blood)

·        Cord blood: Perform ABO and Rh grouping.
·        If HDN is suspected, do direct antiglobulin (DAT) test. If the infant’s cells are heavily coated with maternal antibody, there may be a reaction in the Rh control as well as the Rh test. It may not be possible to get a proper Rh type or even ABO without first performing heat elution of the antibody.
·        If the DAT is positive, or if the DAT is negative but the infant shows symptoms, elute the antibody from the cord blood cells and run a panel to determine the identity of the antibody. Compare to the mother’s antibody.
·        If the mother has anti-A, anti-B or anti-AB, and the infant is Group A or B, test the elute against screening cells, A1 and B cells. In ABO-HDN the cord cells may be DAT negative but the cord serum may have anti-A, anti-B or anti-A,B. Test the serum against A1, B, and O red cells by indirect antiglobulin method.
·        Full blood count.
·        Bilirubin (total and indirect).

2.5.4: LABORATORY FINDINGS

·        Before treatment, direct coombs test result is usually positive and anaemia is usually present.
·        The cord blood haemoglobin content varies and is usually proportional to the severity of the disease; with hydrops fetalis it may be as low as 3-4g/dL. Alternatively, despite haemolysis, it may be within the normal range because compensatory bone marrow and extra medullary haematopoiesis.
·        Blood smear typically shows polychromsia and a marked increase in nucleated red blood cells.
·        The reticulocyte count is increased.
·        The white blood cell count is usually normal but may be elevated in some cases; thrombocytopenia may develop in severe cases.
·        Biochemical test: Cord bilirubin is generally between 3-5 mg/dL, the direct reacting bilirubin content is usually elevated, especially if there was an intrauterine transfusion. Indirect reacting bilirubin content rises rapidly to high levels in the first 6hrs of life.
·        After intrauterine transfusions, cord blood may show a normal haemoglobin concentration, negative direct coombs test result, predominantly type O Rh-negative adult red blood cells and relatively normal smear findings.

2.5.5: DIAGNOSIS

Definitive diagnosis of Erythroblastosis fetalis requires demonstration of blood group incompatibility and corresponding antibody bound to the infant's red blood cells and can be done through prenatal and postnatal test.

Antenatal diagnosis

In Rh-negative women with history of previous transfusions, abortions, miscarriages and pregnancy should suggest the possibility of sensitization. Expectant parent's blood types should be tested for potential incompatibility and the maternal titre of IgG antibodies to D antigen should be assayed at 12-16, 28-32 and 36weeks of gestation. The presence of elevated antibody titres at the beginning of pregnancy, a rapid rise in titre suggests significant haemolytic disease, although the exact titre correlates poorly with the severity of the disease. If the mother is found to have antibody against D antigen at a titre of 1:16 or greater at any time during subsequent pregnancy, the severity of foetal disease is monitored by Doppler ultrasonography of the middle cerebral artery and also by percutaneous umbilical blood sampling (PUBS) if indicated.
Information obtained from ultrasonography and PUBS is required for the assessment of the foetus. Ultrasonographic signs of hydrops include organomegaly (liver, spleen, and heart), double bowel wall sign and placenta thickening. Real time ultrasonography predicts fetal well-being by means of the biophysical profile which includes; fetal breathing movement, gross body movement, fetal tone, reactive fetal heart rate, qualitative amniotic fluid volume, whereas Doppler ultrasonography assesses fetal distress by demonstrating increased vascular resistance in fetal arteries (middle cerebral). PUBS is performed to determine the fetal haemoglobin levels and to transfuse packed red blood cells in those with anaemia (Hct 25-30%).
Amniocentesis is used for detection of bilirubin level, fetal lung maturity profile, biophysical profiles and middle cerebral artery peak systolic velocity. It allows spectrophotometric measurement of deviation in optical density (OD) at 450nm due to bilirubin level in amniotic fluid, which reflects fetal RBC haemolysis. Ultrasonographically guided, transabdominal aspiration of amniotic fluid may be performed at 18-20 weeks of gestation. Amniocentesis procedure should be carried out only after careful ultrasound placental localization. If it must be carried out without the use of ultrasound, a suprapubic approach may be less likely to encounter the placenta. Amniocentesis is an invasive procedure with risk to both the foetus and mother, including fetal death, bleeding, bradycardia, worsening of alloimmunization, premature rupture of membranes, preterm Labour and chorioamnionitis.

Postnatal diagnosis

Immediately after the birth of an infant to an Rh-negative mother, blood from the umbilical cord or from the infant should be examined for ABO blood group, Rh type, Hct and haemoglobin levels, and reaction to the direct coombs test. If the direct coombs test result is positive, a baseline serum bilirubin level should be examined and a commercially available RBC panel should be used to identify antibodies present in the mother's serum, both tests being performed not only to establish the diagnosis but also to ensure selection of the most compatible blood for exchange transfusion, if necessary. The direct coombs test result is usually positive in clinically affected infants and may remain so for few days up to several months.

2.5.6: TREATMENT    

Before delivery of an unborn infant: If HDN is detected during pregnancy (before delivery) that is Rh HDN cases, it will be treated using;

·        Intrauterine transfusion (IUT),
·        Induction of early delivery if the foetus develops complication.

Intrauterine Transfusion: Intrauterine transfusion is done if the foetus if affected severely, transfusions will be done every 1 to 4 weeks until the foetus is mature enough to be delivered safely. Amniocentesis may be done to determine the maturity of the foetus’s lungs before delivery is scheduled. It is given to the fetus to prevent hydrops fetalis and fetal death. This method can be done as early as 17 weeks, although preferable to wait until 20 weeks. After multiple IUTs, most of the baby’s blood will be D negative donor blood, therefore, the direct antiglobulin test will be negative, but the indirect antiglobulin test will be positive.
                      
diagram of a neonate receiving Intrauterine Transfusion.
Image of a neonate receiving Intrauterine Transfusion.
Figure 4: Image of a neonate receiving Intrauterine Transfusion. Source: Adamimages.com


After IUTs, the cord bilirubin is not an accurate indicator of rate of haemolysis or of the likelihood of the need for post-natal exchange transfusion. Intrauterine transfusion procedure is done firstly by maternal and fetal sedation with diazepam and by fetal paralysis with pancuronium.
 After the mother's abdomen is cleaned with an antiseptic solution, she is given a local anaesthetics injection to numb the abdominal area where the transfusion needle will be inserted. Medication is also given to the foetus to temporarily stop foetal movement. An ultrasound image is obtained to determine the position of the fetus and placenta. Ultrasound is used to guide the needle through the mother's abdomen into the foetus’s abdomen or an umbilical cord vein. A compatible blood type (usually type O, Rh-negative) is delivered into the foetus’s abdominal cavity or into an umbilical cord blood vessel. The mother is usually given antibiotics to prevent infection. She may also be given oncolytic medication to prevent labour from beginning, though this is unusual. The risk of these procedures is now largely dependent on the prior condition of the fetus and the gestational age at which transfusion is commenced.
Transfusion should achieve a post transfusion Hct of 45-55% and can be repeated every 3-5weeks. Indications for delivery include: pulmonary maturity, fetal distress, complications of PUBS. The survival rate for intrauterine transfusion is 89%. Complications include rupture of the membranes, preterm delivery, infection, fetal distress requiring emergency caesarean section and perinatal death.

Induction of early pregnancy: Early delivery is recommended if the foetus develops complications. If the fetus has mature lungs, labour and delivery may be induced to prevent worsening of HDN.

AFTER DELIVERY OF A LIVE-BORN INFANT: The birth should be attended to by a physician skilled in neonatal resuscitation. Fresh, low titre, group O, leuko-reduced and irradiated Rh-negative blood cross matched with mother’s serum should be immediately available. If clinical signs of severe haemolytic anaemia are seen at birth, immediate resuscitation and supportive therapy, temperature stabilization, and monitoring before proceeding with exchange transfusion may save some severely affected infants. Supportive therapies like;

·        Correction of acidosis with 1-2 mEq/kg of sodium bicarbonate
·        A small transfusion of compatible packed red blood cells to correct anaemia
·        Volume expansion for hypotension, especially in those with hydrops
·        Provision of assisted ventilation for respiratory failure.

EXCHANGE TRANSFUSION

Exchange transfusion in which the infant’s blood is removed in small amounts usually 5 to 10 ml at a time and is replaced with a compatible blood (Rh-negative blood), is a standard mode of therapy for treatment of severe hyperbilrubinemia that is unresponsive to phototherapy, and it is the treatment of choice for severe hyperbilirubinemia and hydrops fetalis caused by Rh incompatibility. Exchange transfusion removes the sensitized erythrocytes, lowers the serum bilirubin levels to prevent bilirubin encephalopathy, corrects the anaemia, prevent cardiac failure. Indications for exchange transfusion include rapidly increasing serum bilirubin levels and haemolysis despite aggressive phototherapy. The criteria for exchange transfusion in preterm infants vary according to associated illness factor.
For exchange transfusion, fresh whole blood is typed and cross matched to the mother's serum. Heparin or citrate-phosphate-dextrose-adenine solution may be used as an anticoagulant. If the blood obtained before delivery, it should be taken from a type O, Rh-negative donor with a low titre of anti-A and anti-B antibodies and it should be compatible with the mother's serum by the indirect coombs test. After delivery, blood should be obtained from an Rh-Negative donor whose cells are compatible with the infant's and the mother's sera; when possible, type O donor cells are generally used. The amount of donors blood used is usually double the infant’s blood volume, which is approximately 85 ml/kg body weight. The double-volume exchange transfusion replaces approximately 85% of the neonate’s blood.
 An exchange transfusion is a sterile surgical procedure, with aseptic techniques a polyvinyl catheter is inserted into the umbilical vein and threaded into the inferior vena cava. Depending on the infant's weight, 5 to 10 ml of blood is withdrawn within 15 to 20 minutes and the same volume of donor's blood is infused until the targeted volume (double the estimated blood volume) is reached. If he donor's blood has been citrate, calcium gluconate may be given after the infusion of each 100ml of donor's blood to prevent hypocalcaemia. During exchange transfusion, blood is gradually warmed and maintained at a temperature between 35 and 37°c.i It is kept well mixed by gentle squeezing or agitation of the bag to avoid sedimentation; otherwise the use of supernatant serum with low Rbc count at the end of the infusion will leave the infant anaemia. The infant's stomach should be emptied before transfusion to prevent aspiration, and body temperature should be maintained and vital signs monitored during the process. Acute complications noted in 5-10% of infants include; transient vasospasm, thrombosis, apnea with bradycardia requiring resuscitation and death. Infection risks include CMV, HIV, and hepatitis.
After exchange transfusion, the bilirubin level must be determined at frequent intervals (4-8hours) because bilirubin may rebound 40-50% within hours. Repeated exchange transfusion should be carried out to keep the indirect fraction from exceeding the levels.

Benefits of exchange transfusion

·        Removal of bilirubin
·        Removal of sensitized RBCs
·        Removal of incompatible antibody
·        Replacement with compatible RBCs
·        Suppression of erythropoiesis: reduced production of incompatible RBCs

2.5.7: LATE COMPLICATIONS

Infants who have haemolytic disease or who have had an exchange or an intrauterine transfusion must be observed carefully for the development of anaemia and cholestasis. Late anaemia may be haemolytic or hypo regenerative. Treatment with supplemental iron, blood transfusion or erythropoietin may be indicated. A mild GVH reaction may manifest as diarrhoea, rash, hepatitis or eosinophilia.
Insipissated bile syndrome refers to the rare occurrence of persistence citrus in association with significant elevations in direct and indirect bilirubin levels in infants with haemolytic disease. The cause is still unclear but the jaundice clears spontaneously within a few weeks or months.
Portal vein thrombosis and portal hypotension may occur in children who have been subjected to exchange transfusion as new-born infants. It is probably associated with prolonged, traumatic, or septic umbilical vein catheterization.

2.5.8: PREVENTION OF RHESUS ISSOIMMUNIZATION

Immunization of the D antigen can be prevented by the administration of Rh immunoglobulin either before or shortly after exposure to Rh-positive cells. This dose of immunoglobulin has three mechanisms of action: antigen blocking (i.e. competitive inhibition) by attaching to or covering antigenic sites on the Rh-positive red cells; clearance and antigen deviation; central inhibition by the generation of antigen-specific suppressor cells (Schott J.R, et al 1999). Despite this; some investigators believed that the precise mechanism is still unclear (Koelewijn JM, et al 2009). The percentage of anti-D immunization decreased to 0.7-2.5% in the various countries after the introduction of anti-D immunoprophylaxis (Engelfriet CP et al 2003). Initial studies proved that the postpartum administration of a single dose of anti-D immune globulin to susceptible RhD-negative women within 72 h of delivery reduced the alloimmunization rate by 90% (Bidyut K, et al 2010).
Rh immunoglobulin (RhIg) is a concentrate of predominantly IgG anti-D derived from pools of human plasma. A full dose of anti-D (300-µg-1500 IU) is sufficient to counteract the immunizing effects of 15 ml of D-positive red cells; this corresponds to approximately 30 ml of foetal whole blood (Brecher M.E. 2005). RhIg prophylaxis is administrated to unimmunized Rh-negative women following events that might allow foetal red cells to enter the maternal circulations, i.e. delivery, spontaneous or therapeutic abortion, ectopic pregnancy, amniocentesis, chorionic villus sampling, cordocentesis, antepartum haemorrhage, blunt abdominal trauma, and foetal death (Engelfriet CP, et al 2003). Antepartum RhIg at or after 28 weeks is also recommended (Brecher M.E, 2005; Bidyut K, et al 2010; Engelfriet CP, et al 2003). Massive FMH can lead to immunization as the standard dose of RhIg fail to cover this excess of amount. A screening test such as the rosette technique should be used, and, if positive, quantification of the haemorrhage must be done by Kleihauer-Betke test or by flow cytometry (Brecher, 2005; Engelfriet, et al 2003; Harmening, 2005).
The failure of anti-D Ig prophylaxis related to increased FMH and /or insufficient anti-D Ig levels (Koelewijn, et al 2009)

2.6 HAEMOLYTIC DISEASE OF THE NEWBORN CAUSED BY ABO INCOMPATIBILITY

Haemolytic disease can also occur when the major blood groups antigen of the foetus are different from those of the mother. The major blood groups are A, B, AB, and O. It is the most common cause of HDN with most cases being mild. Significant problems with ABO incompatibility occur mostly with babies whose mothers have O blood type and where the baby is either A or B blood type. The mother’s history of prior transfusions or pregnancies seems unrelated to the occurrence and severity of the disease, thus ABO HDN may occur in the first pregnancy and in any subsequent pregnancies (Harmening D.M; 2005).
Antibodies in the plasma of one blood group (except the AB group, which contains no antibodies) will produce agglutination when mixed with antigens of a different blood group. The agglutinated donor cells become trapped in peripheral blood vessels, where they haemolyse, releasing large amount of bilirubin into the circulation. The most common blood group incompatibility in the neonate is between a mother with O blood group and an infant with A and B blood group as seen below;

MARTENAL BLOOD GROUP
INCOMPATIBLE FETAL BLOOD GROUP
O
A or B
B
A or AB
A
B or AB

Figure 5: Maternal-foetal ABO incompatibilities. Source: expertconsult.com


Naturally occurring anti-A or anti-B antibodies already present in the maternal circulation cross the placenta and attach to the foetal red blood cells causing haemolysis. Usually the haemolytic reaction is less severe than in Rh incompatibility. Unlike Rh reaction, ABO incompatibility may occur in the first pregnancy. The risk significant haemolysis in subsequent pregnancies is thought to be unchanged from the first (Luchtman-Jones, et al 1997).
There seem to be two main reasons for low incidence and severity of ABO HDN despite considerable foetal-maternal ABO incompatibility; first, the A and B antigens are not fully developed at birth, and second, A and B substances are not confined to the red cells so that only a small fraction of IgG anti-A and anti-B which cross the placenta combines with the infants red cells (Anstee, 2005). Prenatal screening for maternal ABO antibodies can demonstrate the presence of IgG antibody but do not correlate well with the extent of foetal RBCS destruction. Therefore, detection of ABO HDN is best done after birth (Duguid, 1997; Harmening, 2005).

2.6.1: CLINICAL MANIFESTATIONS

Most cases are mild, with jaundice being the only clinical manifestation. The infant is not generally affected at birth; pallor is not present, and hydrops fetalis is extremely rare. The liver and the spleen are not generally enlarged. Jaundice usually appeared during the first 24 hrs. Rarely, it may become severe and symptoms and signs of kernicterus develop rapidly.

2.6.2: DIAGNOSIS

A presumptive diagnosis is based on the presence of ABO incompatibility, a weakly to moderately positive direct coombs test result, and spherocytes in the blood smear, which may at times suggest the presence of hereditary spherocytosis. Hyperbilirubinemia is often the only other laboratory abnormality. The haemoglobin level is usually normal but may be as low as 10-22 g/dL. Reticulocytes may be increased to 10-15%, with extensive polychromasia and increased numbers of nucleated Rbcs. In 10-20% of affected infants, the unconjugated serum bilirubin level may reach 20 mg/dL or motor unless phototherapy is administered.

2.6.3: SEROLOGICAL DIAGNOSIS

·        Direct coomb test  on cord blood is usually positive in the first 24hours of life
·        Direct on blood drawn after the first 24hours of life is usually negative
·        Elution: the presence of ABO antibody in elute from the baby’s blood usually suggest ABO HDN
·        Haemoglobin level is usually normal or near normal.

2.6.4: TREATMENT

Phototherapy may be effective in lowering serum bilirubin levels. In severe cases, IVIG administration can reduce the rate of haemolysis and the need for exchange transfusion. Exchange transfusion with type O blood of the same Rh type as the infant may be needed in some cases to correct dangerous degrees of anaemia or hyperbilirubinemia. Indications for this procedure are similar to those previously described for haemolytic disease may require transfusion of packed Rbcs at several weeks of age because of slowly progressive anaemia. Post discharge monitoring of haemoglobin is essential in new-born with ABO haemolytic disease.
For phototherapy at the hospital, a light box and/or fiber-optic blanket directs fluorescent light onto the jaundiced baby. The baby lies in a bassinet or an enclosed crib (incubator) while light is absorbed into the skin.

phototherapyy in an incubator.
phototherapy in an incubator.
Figure 6: phototherapy in an incubator.


The blue wave length of natural light is absorbed by the bilirubin chemical in his skin when it is exposed to the light. The light wave changes the shape of the bilirubin molecules (the process is called photo-isomerization). This different– shaped bilirubin molecule is more easily taken out of the baby's body by his kidneys. The baby’s urine will turn a dark, dusty colour during phototherapy. The light treatment is safe and effective. The baby's eyes are usually covered for comfort because if he stares at that bright blue light long enough he get headache. The light is not dangerous to his eyes, or ours. Also, babies under phototherapy treatment need to sleep, and they seem to sleep better if their eyes are covered. The more a baby's skin is exposed to the blue light, the better it works. The light treatment does not work through clothes or diapers. After a few hours of light exposure, you can tell that the yellow is disappearing from his skin, but is still there under the diaper and the eye-shades.
Feeding your baby is an essential part of treating yellow jaundice. Milk going through your baby's digestive system is the signal to his liver that tells it to turn on. So, your baby must be taken out from under the phototherapy lights to get a good feeding at least every three hours. If your milk hasn't come in yet, and the baby needs phototherapy, you may be asked to supplement your baby's feedings with your pumped milk or formula until the baby is nursing well at the breast. If the baby isn't under the lights, the bilirubin keeps going up. If the baby doesn't get fed, the liver isn't turning on. So there must be a balance between the two important parts of treatment, phototherapy and feedings.
Natural sunlight contains all the wavelengths of light, so it has plenty of the blue wavelengths that convert the bilirubin molecule into the easier-to-pee- out form. But it must be light through a window, not outside. The window glass filters out the ultraviolet light that causes sunburn, so that the baby will not have sunburn. Two or three times a day, fifteen to twenty minutes at a time of bright sunlight shining on your baby's bare-naked skin really will help keep yellow jaundice down. It sort of depends on what kind of windows people have at your home.

2.7: OTHER FORMS OF HAEMOLYTIC DISEASE

Blood group incompatibilities other than Rh or ABO account for <5% of haemolytic disease of the new-born.
·        Haemolytic disease of the new-born due to anti-Kell alloimmunization
·        Other blood group antibodies (Kidd, Lewis, Duffy, MN, P and others) but the occurrence is very rare.

2.9: SENSITIZATION DURING PREGNANCY

Sensitization to an antigen occurs when the immune system encounters an antigen for the first time and mounts an immune response. The three most common models in which a woman becomes sensitized toward (i.e., produces IgG antibodies against) a particular antigen are; Fetal-maternal haemorrhage, Blood transfusion, Immune response to antigen A and B.

2.9.1: Foetal-maternal haemorrhage

Pregnancy presents special immunohematology problems for the transfusion service. The mother may exhibit alloimmunization to antigens on foetal cells, and the foetus may be affected by maternal antibodies provoked by transplacental passage, are produced. Most women who become alloimmunized do so as a result of FMH of less than 0.1 ml (Scott J.R. et al 1999, Bidyut K. 2010). Several studies suggested that abnormalities of pregnancy, methods of delivery, and therapeutic procedures are possible causes of FMH. The American College of Obstetrics and Gynaecology (ACOG) has defined pregnancies with one of the following circumstances as being at high risk for FMH of 30 ml or more: antepartum foetal death, antepartum bleeding, intrauterine manipulation, placenta prevail, abruption placenta, and caesarean section. Manual removal of the placenta (Sebring E.S and Polesky H.F 1990), amniocentesis, threatened abortion, abdominal trauma, anaemic infant (Schott J.R. etal 1999), therapeutic and spontaneous abortion, ectopic pregnancy (Bidyut K. etal 2010) represent additional causes of FMH.
The risk of sensitization to the Rh D antigen is decreased if the foetus is ABO incompatible. This is because any foetal cells that leak into the maternal circulation are rapidly destroyed by potent maternal anti-A and/or anti-B, reducing the likelihood of maternal exposure to the D antigen.

2.9.2: Blood Transfusion

ABO blood group system and the D antigen of the Rhesus (Rh) blood group system typing are routine prior to transfusion. Suggestions have been made that women of child bearing age or young girls should not be given a transfusion with Rhc-positive blood or Kell1- positive blood to avoid possible sensitization, but this would strain the resources of blood transfusion services, and it is currently considered uneconomical to screen for these blood groups. HDN can also be caused by antibodies to a variety of other blood group system antigens, but Kell and Rh are the most frequently encountered.

2.9.3: Immune response to antigen A and B

Another sensitization model can occur in women of blood type O. The immune response to A and B antigens, that are widespread in the environment, usually leads to the production of IgM or IgG anti-A and anti-B antibodies early in life. Women of blood type O are more prone than women of types A and B to making IgG anti-A and anti-B antibodies, and these IgG antibodies are able to cross the placenta. For unknown reasons, the incidence of maternal antibodies against type A and B antigens of the IgG type that could potentially cause haemolytic disease of the new-born is greater than the observed incidence of "ABO disease." About 15% of pregnancies involve a type O mother and a type A, B, or AB child; only 3% of these pregnancies result in haemolytic disease due to A/B/O incompatibility. In contrast to antibodies to A and B antigens, Rhesus antibodies are generally not produced from exposure to environmental antigens.

3.1: HEAMOLYTIC DISEASES IN SUBSEQUENT PREGNANCIES

Initially if a woman has been sensitized in the past, it is very important to be closely monitored by an experienced Obstetrics/Gynaecologist during any future pregnancy with an Rh-positive partner. The maternal anti-D that is formed at the time of sensitization is of the IgM type, which cannot cross the placenta. In subsequent pregnancies, a repeat encounter with the Rh D antigen stimulates the rapid production of type IgG anti-D, which can be transported across the placenta and enter the fetal circulation. Once in the fetal circulation, anti-D attaches to the Rh D antigens found on the fetal RBCs, marking them to be destroyed. The rate of haemolysis determines whether the nature of HDN is mild, moderate, or severe.

·        In Severe cases: Fetal hydrops refers to the widespread and serious destruction fetal red blood cells. This condition leads to the fetus developing severe anaemia which can lead to heart failure, total body swelling, respiratory distress, liver and/or spleen enlargement, jaundice and highly elevated bilirubin levels. One or more fetal blood transfusions may be needed before birth. In very severe cases of Rh disease the fetus may need a blood exchange, which replaces the majority of the new-born’s blood with donor blood, usually type O-Negative. Other treatment procedures that may be implemented include use of phenobarbital before delivery and infusions of albumin after delivery to help reduce bilirubin levels, as well as phototherapy treatments.
·        In Moderate cases: there is destruction of a larger numbers of fetal red blood cells, moderate anaemia, possible development of an enlarged liver and jaundice is looked for closely. Preterm delivery may be necessary to remove the fetus form the hostile environment and they may require a blood transfusion before and/or after birth.
·        In Mild cases: it may involve limited destruction of the fetal red blood cells and could result in mild fetal anaemia. Usually the fetus can be carried to term and requires little or no special treatment. However, some jaundice may occur after delivery. This lighter level of Rh-Disease is usually seen in a first pregnancy after sensitization has occurred.
·        After delivery: infants who have suffered a severe Rh incompatibility and are experiencing extreme cases of jaundice are at the greatest risk for developing Kernicterus; which is a neurological syndrome caused by deposits of bilirubin in the tissue of the brain. This syndrome is characterized by symptoms which may include the loss of the new-born’s startle reflex, a decreased amount of activity and poor feeding. Additional more serious symptoms include a shrill sounding cry, a bulging fontanel and possibly seizures. If the infant survives Kernicterus, they may later experience poor muscle tone, seizures, tone specific hearing loss and sometimes decreased mental ability. Kernicterus is a very serious condition that demands a physician or specialist who is experienced in Rh incompatible pregnancies and their possible complications.

4.1: PREDICTION OF HAEMOLYIC DISEASE

Prediction of haemolytic is a process of forecasting the possibility of haemolytic disease occurring under specific conditions.
·        During the first antenatal visit, all pregnant women should have samples collected, ideally at 10-16 weeks gestation for ABO and Rhesus typing and for the presence of red cell alloantibodies. Whether the woman is rhesus positive or negative, further blood samples should be taken at 28weeks for rechecking the ABO and Rhesus group and further red cell alloantibodies (Thompson et al, 2003).
·        Rhesus negative mothers with antibodies are screened for rise in antibody titre at intervals of 20, 28, 36, and 38 weeks of gestation.
·        When red cell antibodies are detected in Rhesus negative mothers, further testing of maternal blood should be undertaken to determine the specificity, concentration, origin and level of antibody or antibodies, and the likelihood of HDN. Anti-D, anti-c and anti-K are the antibodies most often implicated in causing haemolytic disease severe enough to warrant antenatal intervention. The antibody titre is checked at 2weeks interval, if constant it is disregarded but if it keeps rising to a critical point, induction maybe recommended at 38weeks, if the titre is indicative or if the amniotic fluid bilirubin is high.
·        For those in first pregnancy who are Rhesus negative, where a clinically significant antibody capable of causing HDN, particularly anti-D, anti-c or anti-K, is present in a maternal sample, determining the father’s phenotype provides useful information to predict the likelihood of a fetus carrying the relevant red cell antigen. The complexities of paternal testing and the potential for misidentification of the father need to be acknowledge [National Collaborating Centre for Women’s and Children’s Health, 2003] the husbands are screened for ABO and Rhesus group, if the husband is negative, then the pregnancy is not at risk but if the husband is Rhesus positive, the pregnancy might be at risk and there might be problems in subsequent pregnancies and may qualify her for RhoGAM injection.

5.1: CAUSATIVE ANTIBODIES

Ø ANTI D:  Despite the use of Rh immunoglobulin, anti-D is still a common antibody identified in alloimmunized women (O.Geifman-Holtzman et al 1995, Bidyut K. etal 2010, Howard H etal 1998). Anti-D shows a wide spectrum of severity. Not all D positive infants born to mothers with anti-D in their serum are affected by HDN. About 50% of Rh positive new-born infants with Rh (D) HDN are so mildly affected that they require no treatment. Anti-D is the most important after anti-A and B. It is mostly IgG with IgG1 and IgG3 as predominant subclasses. Naturally occurring IgG anti-D that were only detectable in an auto