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Hemolytic Disease of the Fetus Hemolytic Disease of the Fetus and Newborn (HDFNand Newborn (HDFN((

DR. RAFIQ AHMAD

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DefinitionDefinition

» Fetal red cells become coated with IgG alloantibody of maternal origin, directed against a paternally inherited antigen present on the fetal cells that is absent from maternal cells.

» The IgG coated cells may undergo accelerated destruction both before and after birth, but the severity of the disease can vary from serologic abnormalities detected in an asymptomatic infant to intrauterine death

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PathophysiologyPathophysiology

» Erythroblastosis Fetalis

» Ictrus gravis neonatorum

» Hydrops Fetalis

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Mechanisms of Maternal ImmunizationHDFN is often classified into three categories,

1. D hemolytic disease caused by anti-D alone or, less often, in combination with anti-C or anti-E.

2. “Other” hemolytic disease caused by antibodies against other antigens in the Rh system or against antigens in other systems; anti-c and anti-K1 are most often implicated.

3. ABO HDFN caused by anti-A,B in a group O woman or by isolated anti-A or anti-B.

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Immunization

» Previous Transfusion» Previous Pregnancy - at the time of delivery –FMH - amniocentesis. - spontaneous or induced abortion. - chorionic villus sampling. - cordocentesis. - rupture of an ectopic pregnancy. - blunt trauma to the abdomen.

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Immunogenic Specificities

The antigen that most frequently induces immunization is D (only 0.1ml of fetal blood can immunize)

but any red cell antigen present on fetal cells and absent from the mother can stimulate antibody production

One retrospective study determined that there was a 0.24% prevalence of production of clinically significant antibodies other than anti-D during pregnancy (needs large volume of cells e.g., transfusion)

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Frequency of Immunization

Depends upon volume of cellsD sensitization- 16% 1.5% to 2% become sensitized at the time of

their first delivery, an additional 7% become sensitized within 6 months of the delivery, and the final 7% become sensitized during the second affected

pregnancy

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Frequency of Immunization

In susceptible women not immunized after two D-positive pregnancies, later pregnancies may be affected but with diminished frequency.

Once immunization has occurred, successive

D-positive pregnancies often manifest

HDFN of increasing severity, particularly

between the first and second affected

pregnancies.

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Effect of ABO Incompatibility

ABO incompatibility between mother and fetus has a substantial but not absolute protective effect against maternal immunization by virtue

The rate of immunization is decreased

from16% to between 1.5% and 2%.

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Transfusion as the Immunizing Stimulus It is extremely important to avoid transfusing D-

positive whole blood or red cells to D-negative females of childbearing potential because anti-D stimulated by transfusion characteristically causes severe HDFN in subsequent pregnancies with a D-positive fetus

The risk of immunization to a red cell antigen other than D after an allogeneic red cell transfusion has been estimated to be

1% to 2.5% in the general hospital population

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““Other” hemolytic disease Other” hemolytic disease antibodiesantibodies

Experience with other alloantibodies has not been as extensive as with anti-D; in some series, anti-c and anti-K1 were by far the most common causes of severe HDFN, other than anti-D.

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ABO antibodies

The IgG antibodies that cause ABO HDFN nearly always occur in the mother’s circulation without a history of prior exposure to human red cells.

It can occur in any pregnancy, including the first.

It is restricted almost entirely to group A or B infants born to group O mothers because group O individuals make the IgG antibody, anti-A,B.

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Prenatal Evaluation

Maternal History

Information about previous pregnancies or blood transfusions.

For a woman with a history of an infant with hydrops fetalis due to anti-D, there is a 90% or more chance of a subsequent fetus being similarly affected.

In contrast, during the first sensitized pregnancy, the risk of a hydropic fetus is 8% to 10%.

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Serologic Studies

Should be performed on all pregnant women as early

in pregnancy as possible.

Tests for:

» Blood typing for ABO and D,

» red cell antibody screening.

weak D test is not required.

partial D phenotypes, such as DVI, will also most

likely type as D negative

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Follow upFollow up

The test can be repeated at 28 weeks’ gestation before administration of RhIG to detect immunization that might have occurred before 28 weeks, in accordance with AABB recommendations

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Antibody Specificity

All positive screens for red cell antibodies

require identification of the antibody

Typing the FetusThe fetal D type can be established by using the polymerase chain reaction (PCR) to amplify DNA obtained from amniotic fluid, chorionic villus samples, or by serologic typing of fetal blood obtained by cordocentesis/amniocentesis

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Molecular MethodsMolecular Methods

Fetal DNA typing is also available for Jka/Jkb,

K1/K2, c, and E/e antigens.

A more recent development in fetal RhD typing

involves the isolation of free fetal DNA in the

maternal serum. Although not routinely available in

at this time, this will likely replace amniocentesis for

Fetal genotyping in the near future

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Maternal Antibody Titer

The antibody titer should be established in the first trimester to serve as a baseline, and the specimen should be frozen for future comparisons

Critical titer for anti-D - 1:16-32 for anti- K1 1- 8 For antibodies other than anti-D, and ant-K1 critical titers have not been identified

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Amniotic Fluid Analysis

A good index of intrauterine hemolysisand fetal well-being is the level of bilirubinpigment found in amniotic fluid obtainedby amniocentesisAmniotic fluid is obtained by inserting a long needle through the mother’s abdominal wall and uterusinto the uterine cavity under continuous ultrasound guidance.

wavelengths of 350 to 700 nm. Peak absorbance of bilirubin is at 450nm.

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Liley’s GraphLiley’s Graph

This method is

applicable to

pregnancies from

27 weeks through

term.

The ΔOD450 value is plotted on a graph against the estimated length of gestationThe ΔOD450 value is plotted on a graph against the estimated length of gestation

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Queenan’s graphQueenan’s graph

This method is

applicable to

pregnancies from

14 weeks through

term.

when amniocentesis or cordocentesis is performed for any reason on aD-negative woman who does not have anti-D, Rh immunoprophylaxis should be given

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Percutaneous Umbilical Blood Sampling (PUBS) or Cordocentesis» A needle into an umbilical blood vessel, preferably the vein at its insertion into the placenta, and obtain a fetal blood sample.» It allows direct measurement of Hematologic and biochemical variables. The fetal mortality of intrauterine fetal blood sampling has been reported to be 1%to 2%, and the procedure carries a high FMH

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Doppler Flow Studies

» Because fetal anemia results in increased cardiac output, several investigators have measured various blood velocities in fetal vessels using Doppler ultrasonography to determine the clinical status of the fetus in a noninvasive manner» There is good correlation between middle cerebral artery (MCA) peak velocity, fetal hemoglobin, and ΔOD450 reading » MCA Doppler is associated with a high false-positive rate for the diagnosis of fetal anemia after 35 weeks gestation

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Suppression of MaternalAlloimmunization

» Intensive plasma exchange and the administration of intravenous immunoglobulin (IGIV) is being used to reduce the maternal antibody titer.» Plasma exchange can reduce antibody levels by as much as 75%. Unfortunately, rebound usually follows because the IgG antibody is mostly extravascular and antigen exposure may be ongoing.» AABB and the American Society for Apheresis (ASFA) categorize plasma exchange as treatment Category III because its efficacy and safety

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Intrauterine Transfusion

IT can be performed by the intraperitoneal

route (IPT) or the direct intravascular

approach (IVT) by the umbilical vein. In

many instances, IVT is the procedure of

choice, but there may be problems of access

that make IPT preferable

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It is usually done after 20th week of gestation;

Once initiated, transfusions are usually

administered periodically until delivery.

It carries a 1%to 2% risk of perinatal loss, it

should be performed only after careful clinical

evaluation

Intrauterine Transfusion

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Techniques

IPT is performed through a needlepassed, with ultrasonographic monitoring, through the mother’s abdominal wall into the abdominal cavity of the fetusTransfused red cells enter the fetalcirculation through lymphatic channels that drain the

peritonealcavity.In IVT, the umbilical vein is penetrated under ultrasoundguidance and a blood sample is taken to verify positioningin the fetal vasculature.

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Selection of Red Cells

» The red cells used should be group O, D-negative, or negative for the antigen corresponding to the mother’s antibody if the specificity is not anti-D.

» Blood for intrauterine transfusion should be as fresh as possible, Irradiated, CMV reduced, and HbS negative » Red cells of mother, washed in saline, with final Hct of 75%-85% or deglycerolized, and irradiated to prevent

graft vs host disease have also been used.

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Volume Administered

For IPT, a volume calculated by the formulaV = (gestation in weeks – 20) x 10 mL appears to be well tolerated by the fetus.

For IVTFetoplacental volume (mL) = ultrasound estimated fetal weight (g) X 0.14Volume to transfuse (mL) = Fetoplacental volume X (Hct after IVT – Hct before IVT) Hct of donor cells where Hct = hematocrit

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Postpartum Evaluation

A D negative mother who is not immunized should receive an appropriate dose of RhIG, if delivered Rh positive infant.

cord blood should be tested immediately after delivery

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Assessment of FMHAssessment of FMH

» The Rosette Test: A qualitative test

» Kleihauer Betke Test: A quantitative test

» Flow Cytometry

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Kleihauer Betke TestKleihauer Betke Test

Positive ControlNegative Control

KB Test

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Indication (Indicated Dose a )

approximately

Postpartum (if the newborn is Rh-positive) 300 µg b

Antepartum: Prophylaxis at 26 to 28 weeks' gestation c 300 µg

Antepartum: Amniocentesis, chorionic villus sampling (CVS) and percutaneous umbilical blood sampling (PUBS(

300 µg

Antepartum: Abdominal trauma or obstetrical manipulation 300 µg

Antepartum: Ectopic pregnancy d 300 µg

Antepartum: Abortion or threatened abortion at any stage of gestation with continuation of pregnancy d 300 µg

Transfusion of Rh-incompatible blood or blood products d 300 µg

a Additional doses of RhoGAM are indicated when the patient has been exposed to > 15 mL of Rh-positive red blood cells. This may be determined by use of qualitative or quantitative tests for FMH (see below).

b See DESCRIPTION section.

c If antepartum prophylaxis is indicated, it is essential that the mother receive a postpartum dose if the infant is Rh-positive.

d If abortion or termination of pregnancy occurs up to and including 12 weeks' gestation, or less than 2.5 mL of Rh-incompatible red blood cells were administered, a single dose of (MICRhoGAM) Rh0D Immune Globulin (Human) (approximately 50 µg)* may be used instead of RhoGAM.

RhoGAM

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Dose CalculationDose CalculationFetal and maternal cells are counted separately

for a total of 2000 cells

The following formula can be used for fetal

bleeding calculation:

Fetal cells x maternal blood volume (ml) = fetal hemorrhage (ml)

total cells counted

Example:

6cells/2000 cells X 5000 ml = 15 ml fetal whole blood

300 µg of RhIG is required for 30 ml of fetal blood

= ( 0.5 of the vial), but needs 2 vials

if the calculated dose is right of the decimal point ≥ 0.5 add one more vial

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Flow Cytometry: FMH estimationFlow Cytometry: FMH estimation

Sample negative for fetal HbF. Positive fetal control. Fetal HbF positive cells show increased fluorescent intensity staining with anti-HbF-FITC conjugated antibodies compared to adult HbF and HbF negative cells.

Patient sample with 0.53 per cent fetal HbF positive cells, equating to an 11.0 ml FMH.

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RhIG dose for FMHRhIG dose for FMH

fetal cells percentage Vials to inject µg(mcg) IU

6-8 0.3-0.8 2 600 3000

9-14 0.9-1.4 3 900 4500

15-26 1.5-2.0 4 1200 6000

21-26 2.1-2.6 5 1500 7500

DoseDose

Note:Note:1. Based on maternal blood volume of 5000 ml.1. Based on maternal blood volume of 5000 ml.2. 1 vial of 300 µg (1500 IU) is needed for each 15 ml of fetal red cells or2. 1 vial of 300 µg (1500 IU) is needed for each 15 ml of fetal red cells or 30 ml of fetal whole blood30 ml of fetal whole blood

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ABO HDFNABO HDFN

» ABO incompatibilities are the most common cause of HDFN but are less severe– About 1 in 5 pregnancies are ABO-incompatible– 65% of HDFN are due to ABO incompatibility

» Usually, the mother is type O and the child has the A or B antigen…Why?– Group O individuals have a high titer of IgG anti-A,B

in addition to having IgM anti-A and anti-B

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ABO HDFNABO HDFN

» ABO HDFN can occur during the FIRST pregnancy because prior sensitization is not necessary

» ABO HDFN is less severe than Rh HDFN because there is less RBC destruction– Fetal RBCs are less developed at birth, so there is less

destruction by maternal antibodies– When delivered, infants may present with mild anemia

or normal hemoglobin levels– Most infants will have hyperbilirubinemia and jaundice

within 12 to 48 hours after birth

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Diagnosis of ABO HDNDiagnosis of ABO HDN

» Infant presents with jaundice 12-48 hrs after birth

» Testing done after birth on cord blood samples:– Sample is washed 3x to remove Wharton’s jelly– Anticoagulated EDTA tube (purple or pink)

– ABO, Rh and DAT performed– Most cases will have a positive DAT

• If DAT positive, perform elution to ID antibody

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Treatment of ABO HDFNTreatment of ABO HDFN

» Only about 10% require therapy

» Phototherapy is sufficient

» Rarely is exchange transfusion needed

» Phototherapy is exposure to artificial or sunlight to reduce jaundice

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PhototherapyPhototherapy

Fluorescent blue light in the 420-475 nm range

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Exchange transfusionExchange transfusion

Exchange transfusion involves removing newborn’s RBCs and replacing them with normal fresh donor cells

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What type of blood to be given:What type of blood to be given:

» Exchange transfusion: Fresh Whole Blood (to avoid Ca++), less than 7 days old

» Irradiated» SCT negative» Group O, or ABO compatible with mother’s serum

with D-negative in case of Rh-D HDFN» Group O red cells reconstituted with AB plasma in case of ABO HDFN» Leukoreduced» CMV negative

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The end!!The end!!

Thank you