HAEMOLYTIC DISEASE OF THE NEWBORN

GTB 313 19/9/2010 BT 5

OBJECTIVES
1. Define hemolytic disease of the new born (HDN)

2. Briefly explain the mechanisms of sensitization of ABO and RH haemolytic disease of the newborn.
3. Compare and contrast ABO-HDN with HDN associated with other blood group alloantibodies. 4. Describe the use of RhIG in the prevention of HDN due to anti-D. 5. List and explain the procedures used in immunohaematology laboratory in prenatal postnatal testing of potential haemolytic disease of newborn:1 Mother 2 Baby 6. Describe methods for screening and quantitation of fetal-maternal bleeds 7. Explain the criteria for selection of blood for exchange transfusion

Hemolytic disease of the new born (HDN)

HDN is the destruction of the red blood cells of the fetus

and the neonate by antibodies produced by the mother. Red cell antibodies are produced as a result of sensitisation due to a previous pregnancy, previous blood transfusion and occasionally transplantation. The current pregnancy will be affected only if the fetus has inherited the blood group antigen against which the mother has formed a clinically significant antibody.

 Many red cell antibodies have the potential to cause

HDN. It is essential, therefore, for ALL pregnant women found to have clinically significant red cell antibodies to referred to a hospital obstetric unit for management of their pregnancy.

Classification of HDN

According to the specificity of the antibody, haemolytic disease can be classified as :

1. HDN due to Rh(D) antibodies 2. HDN due to other Rh antibodies ie. anti-c, anti-E, anti-e. 3. HDN due to ABO IgG antibodies - ABO - anti-A, anti-B

 HDN due to other blood group system antibodies

Kell - anti-K, Cellano - anti-k Duffy - anti-Fya, Kidd - anti-JKa, anti-Jkb , MNS - anti-S, anti-s, anti-M

 Maternal anti-D, anti-K and anti-Fya antibodies are

amongst the important causes of severe HDN.

Anti-c is sometimes associated with severe HDN, while

HDN associated with anti-E and anti-c is usually not severe.

 In addition, microbes encountered by way of the

digestive tract and other mucosal surfaces regularly (eg, anti-A, -B, -P, -Pk) or sometimes (eg, anti-M, -P1, -Lea, Leb) result in production of so-called naturally occurring antibodies. These antibody specificities are the most common antibodies present in children and nontransfused male patients.

 Antibodies considered clinically insignificant unless the

antibody reacts in tests performed strictly at 37C) include those to A1, P1, M, N, Lua, Lea, Leb, and Sda antigens.

Hemolytic disease of the new born (HDN)

Hemolytic disease of the newborn (HDN) used to be a

major cause of fetal loss and death among newborn babies. During pregnancy, some of the mother's antibodies are transported across the placenta and enter the fetal circulation. This is necessary because by the time of birth, newborns have only a primitive immune system, and the continuing presence of maternal antibodies helps ensure that they survive while their immune system matures.

 Haemolytic disease of the newborn (HDN) occurs when the

mother has anti-red-cell IgG antibodies in her plasma that cross the placenta and bind to fetal red cells bearing the corresponding antigen. Fetal red cells binding sufficient maternally derived antibody are destroyed in the fetal reticuloendothelial system, producing extravascular haemolysis and a variable degree of fetal anaemia. In severe cases the fetus may die in utero of heart failure (hydrops fetalis). If the fetus survives birth, the neonate rapidly develops jaundice and is at risk of neurological damage due to the high bilirubin level.

Mechanisms of sensitization of ABO and RH haemolytic disease of the newborn.

A major cause of HDN is an incompatibility of the Rh blood

group between the mother and fetus. Most commonly, hemolytic disease is triggered by the D antigen, although other Rh antigens, such as c, C, E, and e, can also cause problems. Pregnancies at risk of HDN are those in which an Rh Dnegative mother becomes pregnant with an RhD-positive child (the child having inherited the D antigen from the father).

 Development of red cell antibodies in the mother may occur

either as a result of previous pregnancies (because fetal blood displaying paternal red cell antigens frequently enters the mothers circulation during pregnancy) or as a result of a previous blood transfusion. 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 is transported across the placenta and hence delivered to the fetal 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 antiA and anti-B, which tend to be of the IgG class and can therefore cross the placenta and hemolyse fetal RBCs.

 HDN due to ABO incompatibility is usually less severe

than Rh incompatibility. One reason is that fetal RBCs express less of the ABO blood group antigens compared with adult levels. In addition, in contrast to the Rh antigens, the ABO blood group antigens are expressed by a variety of fetal (and adult) tissues, reducing the chances of antiA and anti-B binding their target antigens on the fetal RBCs.

 Less common causes of HDN include antibodies directed

against antigens of the Kell blood group (e.g., anti-K and anti-k), Kidd blood group (e.g., anti-Jka and antiJkb), Duffy blood group (e.g., anti-Fya), and MNS and s blood group antibodies. To date, antibodies directed against the P and Lewis blood groups have not been associated with HDN.

Sensitization occurs during the first pregnancy

Sensitization to an antigen occurs when the immune

system encounters an antigen for the first time and mounts an immune response. In the case of HDN caused by Rh incompatibility, an Rh D-negative mother may first encounter the D antigen while being pregnant with an Rh D-positive child, or by receiving a blood transfusion of Rh D-positive blood. Once a mother has been sensitized to the D antigen, her serum will contain anti-D. The direct Coombs test confirms the presence of anti-D and hence that the mother has been sensitized.

 Only a small amount of fetal blood need enter the

mother's circulation for sensitization to occur. Typically, this occurs during the delivery of the first-born Rh D-positive child. Fetal-maternal hemorrhage is common during labor and is increased during a prolonged or complicated labor, which in turn increases the risk of sensitization.

 Sensitization can also occur earlier in the pregnancy, for

example during a prenatal bleed or a miscarriage. It may also occur during medical procedures, such as a termination of pregnancy or chorionic villus sampling. The risk of sensitization to the Rh D antigen is decreased if the fetus is ABO incompatible. This is because any fetal 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.

 Procedures used in immunohaematology laboratory in

prenatal and postnatal testing of potential haemolytic disease of newborn:1 2 Mother Baby

Mother lab. Testings for every pregnant women

ABO and D typing Antibody screening(anti-D, etc.) Antibody identification Antibody titration

Pregnant women Rh neg. mother and no anti-D Repeat screening for anti-D every 2-4 weeks until delivery Rh neg. mother with anti-D perform anti-D titer repeat titer every 2-4 week

Post-natal
Baby depends on the serum bilirubin level Exchange Transfusion
To replace normal red blood cells(correction

of anemia. To remove bilirubin. To remove free antibody. To remove fetal red blood cells coated with maternal antibody

The use of RhIG in the prevention of HDN due to anti-D.

Prevention of Hemolytic Disease of the Newborn Due to Anti-D
Routine testing and appropriate use of Rh Immune Globulin

(RhIG) during pregnancy and immediately after delivery or termination of pregnancy can successfully prevent most cases of hemolytic disease of the newborn (HDN) caused by alloimmunization to the D antigen. RhIg timing and dosage depend on the gestational date and on whether any events that increase the risk of fetomaternal hemorrhage (FMH) have occurred.

 Most women who receive antepartum RhIG will develop a

positive antibody screen due to passively acquired anti-D. The half-life of RhIG , in the absence of significant fetomaternal hemorrhage, is 21 to 30 days. Therefore, when a patient has received a standard 300 ug dose of RhIG, anti-D is often detectable up to 12 weeks later. In some reported cases, anti-D has remained detectable for as long as 6 months. For this reason, it is important to notify the laboratory that a patient has received RhIG, whenever an antibody screen is ordered. If the laboratory is not aware of this pertinent history, passively acquired anti-D may be misinterpreted as Rh sensitization.

 This misinterpretation could result in further unnecessary

serological testing or in failure to give RhIG postpartum. Once an antibody has been identified, it need not be reidentified. A selected cell panel should be run to exclude the presence of other clinically significant alloantibodies. Identification of any new alloantibodies is indicated. Periodic repeat titration of clinically significant alloantibodies may be appropriate during the continuation of pregnancy. Each new sample should be tested in parallel with the immediately preceding sample or with the original sample.

 The antibody screen should include an antiglobulin phase

using anti-IgG following 37oC incubation. If the antibody screen is positive, antibody identification must be performed. Once an antibody has been identified, repeat identification is not necessary. A selected cell panel should be performed to exclude the presence of other antibodies. Titration of clinically significant alloantibodies found early in pregnancy might be appropriate to establish a baseline for comparison to subsequent samples. Samples should be retained for repeat subsequent testing.

At delivery - Mother
If the delivering facility has a verified record of a

negative antibody screen during the current pregnancy, repeat maternal testing is not required unless a question of HDN arises. If the delivering facility has a verified record of immunization to D, RhIg should not be given. If the mother is known to be D-negative and not immunized to D and the cord blood types as D negative, RhIg should not be given and no further testing is necessary.

 If the mother is known to be D-negative and not

immunized to D and the cord blood types as D positive (including weak D), a test for excessive FMH should be performed to determine RhIg dosage. An appropriate dose of RhIg should be given. If the mother is known to be D-negative and not immunized to D and the cord blood is not tested, a test for excessive FMH should be performed to determine RhIg dosage. An appropriate dose of RhIg should be administered.

Infant: No cord blood testing is required except to establish candidacy of mother to receive RhIg or to investigate suspected HDN. If a question of HDN arises when the mother has clinically significant alloantibodies, ABO and Rh typing and direct antiglobulin testing should be performed on infants cord blood. In the absence of a maternal sample, eluate testing may be useful to confirm an antibody implicated in HDN.

Assessment of feto-maternal haemorrhage (Kleihauer testing)

In over 99% of cases less than 4mL of fetal blood enters the

mothers circulation at delivery and 500 IU anti-D immunoglobulin is sufficient to eliminate this volume and thereby prevent anti-D formation by the mother.
It is important to identify the small minority of women (less

than 1%) who have more than 4mL of fetal blood enter their circulation at the time of delivery.

Acid Elution Test

The acid-elution technique has been used for many

years as the principal method for assessment of FMH.

original test described by Kleihauer et al differentiates red cells containing fetal haemoglobin (HbF) from those containing adult haemoglobin by the relative resistance of HbF to elution at a low pH. After counter-staining, the fetal red cells stand out as brightly red stained cells in a field of ghost red blood cells. 2000 cells counted and percentage of fetal cells were determined.
The

The advantages of these tests are that they are: ! not dependent on the presence or absence of the Rh D antigen; ! they require only basic laboratory equipment, and; ! are inexpensive to perform.

CALCULATION

RhIg Dose for Fetal Maternal Hemorrhage

The amount of fetal maternal hemorrhage is calculated

by multiplying the percent fetal cells by 50 (maternal blood volume is typically 5 liters or 50 deciliters). This product is then divided by 30, which is the volume of fetal blood neutralized by a single vial of RhIg .

Fetal bleed (ml) = % fetal cells x maternal blood volume Doses/ Vials of RhIg = fetal bleed (ml)/30 ml For example, if the percent of fetal hemorrhage is 2%, then the volume of fetal hemorrhage is 100 mL. Dividing 100 mL by 30 mL/vial yields 3.3 vials.

 RhIg is very safe and has a very low risk of viral

transmission, especially of enveloped viruses. A potential risk of anaphylaxis exists in IgA deficient patients. No more than 5 vials should be injected into each buttock at one time. Large doses should be given at 12-hour intervals over a 72-hour period.

 For this reason, a blood sample taken from all D negative

mothers within 2 hours of delivery is used for a Kleihauer test to estimate the volume of fetal blood that has entered the maternal circulation and so identify those women who require additional anti-D immunoglobulin.

When the Kleihauer test indicates that there has been a

fetal bleed of more than 4mL we recommend that a more accurate measurement of fetal cells is undertaken by flow cytometry to determine how much additional anti-D immunoglobulin is required.

There are however significant disadvantages to these tests. This variation is due to a combination of factors including:
There are however significant disadvantages to these tests..

This variation is due to a combination of factors including: ! Technique sensitivity. The Haemoglobin elution step is sensitive to pH, time and temperature; ! Subjective interpretation of the stained blood film; ! Experience of the scientist/technician performing the test; ! Assumptions in the calculation of results; ! Increased levels of HbF in maternal red cells during pregnancy. It is known that in about 25% of pregnant women, the level of maternal HbF rises above the upper limit of normal. ! Hereditary persistence of fetal haemoglobin.

Flow Cytometric Testing

Flow cytometers are designed to quantitate small numbers of cells

present in a larger cell population. Differentiation and quantitation of the populations is most often achieved using fluorescent labelled antibodies. Flow cytometry allows large numbers of cells (eg. 50,000 100,000) to be readily counted. The test should then be more sensitive and accurate; and produce objective, quantitative results. However flow cytometers are expensive and generally available only in major centres and require staff with specific expertise to operate the analyser. Assessment of FMH can be achieved with flow cytometry using commercially available antibodies to the Rh D antigen or to Haemoglobin F [HbF].

 In situations where the mother is known to be Rh D negative

and the fetus is known to be Rh D positive the use of anti-D provides sensitive and accurate assessment of FMH, and determination of Rh D Immunoglobulin requirements. However where the mother and the fetus are of the same Rh D type or unknown anti-D cannot be used to determine whether FMH has occurred. Similarly it is of no value when the mother is Rh D positive. In these circumstances determination of FMH by flow cytometry should use anti-HbF.

 In common with the Kleihauer test, use of HbF as a marker of

FMH can result in false positive results due to hereditary persistence of fetal haemoglobin, increased levels of HbF in maternal red cells during pregnancy and in some disease states such as thalassaemia. Further, since all adult red cells contain small amounts of HbF (resulting in a background level), discrimination of fetal and adult cells can be difficult and at times uncertain. Commercial manufacturers have recently combined antibody tests for other unique properties of fetal red cells (eg. quantitation of carbonic anhydrase, Ii antigens) with HbF testing in an attempt to increase test specificity.

Other Techniques
Other techniques are available and have been used but are known to

produce a significant false negative rate. They are included for completeness only and are NOT recommended. Rosette Test

a)

The rosette test utilises an Indirect Antiglobulin Test with increased

sensitivity achieved by the addition of Rh D positive indicator red cells. These indicator cells adhere to the anti-D coating the minor population of Rh D positive fetal red cells. This results in clusters or rosettes that can be counted microscopically. The rosette test is only applicable in situations where the fetus is Rh D positive and mother is Rh D negative. It is not therefore not a replacement for the Kleihauer test.

b)Enzyme linked antiglobulin test (ELAT) The enzyme linked antiglobulin test is a quantitative test for FMH based on the indirect antiglobulin test. c) Surrogate Tests Elevation of maternal serum alpha-fetoprotein (AFP) has been reported as a consequence of FMH. However, many other fetal conditions are associated with raised levels of AFP. Hence this technique is not recommended as a sole indicator of FMH. Other tests have also been used as surrogate markers of FMH. These include placental alkaline phosphatase (PLAP), polymerase chain reaction [PCR], fluorescence in situ hybridisation [FISH].

Criteria for selection of blood for exchange transfusion

Neonatal transfusion - Exchange transfusion Indication and aims
Exchange transfusion may be used to manage severe anaemia at birth,

particularly in the presence of heart failure, and to treat severe hyperbilirubinaemia, usually caused by HDN.
In the treatment of HDN, the aim is to remove both the antibody-coated

red cells and the excess bilirubin.

Component and procedure specifications

.
Red cells for ET should be group O or ABO compatible with

maternal and neonatal plasma, RhD negative (or RhD identical with neonate); be negative for any red cell antigens to which the mother has antibodies; be IAT-cross-match compatible with maternal plasma; be 5 d old or less (to ensure optimal red cell function and low supernatant potassium levels); be collected into CPD anticoagulant; be CMV seronegative; be irradiated and transfused within 24 h of irradiation. Irradiation is essential if the infant has had a previous IUT (intrauterine transfusion) and is recommended for all ETs .

 Irradiation for ET in absence of IUT is not essential if this

would lead to clinically significant delay; have a haematocrit of 50%60%; not be transfused straight from 4oC storage. volume transfused is usually 80160 ml/kg for a term infant and 100200 ml/kg for a preterm infant depending on the clinical indication.

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