Rh-ISOIMMUNIZATION

DR.SURENDRA NATH BERADR. MITALI DASH

M K C G MEDICAL COLLEGE , ORISSA

ISOIMMUNIZATION: A process by which immune

antibodies are produced in a person by the entry of an antigen of another individual of same species, the former lacking the antigen.

Rh- Iso imunizationDefinitionknown as:Rhesus incompatibility, Rhesus disease

RhD Hemolytic Disease of the Newborn.-When Rh– mother gets pregnant to Rh+ fetus

—she may be sensitized to Rh antigen and develop antibodies. These will cross the placenta and cause hemolysis of fetal red blood cells.

HISTORY 1609-description of hydrops fetalis. 1939-Levine and Stetson discovered atypical aglutinin. 1940-Landsteiner and wiener Rh-antigen. 1941-Levine discover Rh-antidody.

Rhesus factor (1940):Agglutinogen (C,D,E) - mainly DC,D,E - dominant antigenc,e - recessive antigen Person lack D-antigen called Rh-ve

- Rh positive (85%) - homozygous (DD) (35%), or heterozygous (Dd) (50%)

- Rh negative (15%)- Incidence of Rh-ve in far east is about 1%

Examples of Rh factor: (CDe=R1) , (Cde=r) (cDE=R2)

Other systems: Kell. Lweis, Deigo, luther, Duffy, MNS,

Kell is most common of minor gr. Responsible for 10% of cases of

severe antibody-mediated anemia Only anti-Fy(a) antibody associated

with HDFN- may range from mild to sever.

INCIDENCE OF Rh-ve

Chinese and Japanese 1% North American Indian 1—2% Indo-Eurasian 2% india 5% African American 4 - 8% Caucasian 15 - 16% Basque 30 - 35%

Rh-isoimmunization occurs

A. Mismatched blood transfusion.B. Rh-negative women bearing Rh-

positive fetous with feto-maternal hemorrhage.

CAUSE OF FMHFeto-maternal haemorrhage: during pregnancy

leakage of fetal cells in the maternal circulation (Rh+ fetal cells in Rh- maternal circulation)

Examples: 1.Abortion 2.APH3.E.C.V. 4.Cordocentesis,5.CVS,6.Amniocentesis

CAUSE OF FMH

7.Severe preeclampsia

8.Ectopic pregnancy

9.Caesarean section

10.Manual removal of placenta

11.Silent feto-maternal hage

Pathophysiology

Rh-isoimmunization is due to D antigen in more than 90% of cases.

Occasionally result of other than Rh group like anti- Kell and anti- Duffy

Pathophysiology

Following feto-maternal hemorrhage. Initial response is forming IgM antibodies for

the short period (6wks-6month)o Followed by production of IgG on subsequent

pregnancy which crosses placenta. IgG antibodies adhere to the antigen site on

the surface of erythrocytes causing hemolysis. The excessive removal of circulatory RBCs

leads to severe anemia and hypoxia.

IGM antibodies

1. Cleared by Macrophage

2. Plasma stem cells

Fetal Anaemia

Mother

Placental

Primary Response

• 6 wks to 6 M.

• IGM.

Anti - D

Macroph. antigenPresenting cell

T- helper cell

B cell

Fetal Anaemia

Mother

Placental

Secondary Response

• Small amount• Rapid • IgG

IgG

Rh Antibodies

Antibodies Coated Red Cells

Destruction of Fetal Cells by Fetal RES

Fetal Anemia

Fetal Hypoxia and Stimulate of Erythropoitin

Extra Medullary red Cells Synthesis

Hepato spleenomegally

Hepatic Cell Failure

Hypoproteinemia, Increased Intrahepatic Pressure, Portal hypertension

Ascetic, Edema, hypoxia, Placental Thickness, Polyhydramnios, Pericardial

effusion

Development of Rhesus antibodies:

depends on : 1- Inborn inability to respond to Rh-antigenic

stimulus.2- Protection if ABO incompatible 1\103- Strength of Rh antigen stimumlus (CDe=R1)4- Volume of leaking feta blood (0.1ml)5- Immunological nonresponders found in 30%

of Rh-negative women.

1- If ABO is incompatible: Red blood cells is easily destroyed, so

not reaching enough immunological component to cause antibody response and reaction.

The risk of sensitization after ABO incompatible pregnancy is only 2%.

- If ABO is compatible:

Rh+ fetal cells remain in circulation (life span) until removed by (R.E.S) destroyed liberating antigen (D) isoimmunization

Macroph. AntigenPresenting Cell

T-Hellper

B-cell

Anti-D

Anti - A Anti - B

Mother

InfantA Rh positive B Rh Positive

“O” Rh positive

Group “O” Rh Negative

Placenta

Fetomaternal hemorrhage as a reason of Rh –isoimmunization has been documented in:

6.7% in the first trimester.

13.9% in the second trimester

29% in the third trimester.

Amount needed for sensitisation 0.1ml

FMH/ML SENSITIZATION%

0.1 1

0.5 - 1 25

>5 65

Complications Hydrops fetalis And Stillbirth. Icterus gravis neonatorum. Neonatal Jaundice. Compilations Of Neonatal Kernicterus

(Lethargy, Hypertonicity, Hearing Loss, Cerebral Palsy And Learning Disability)

Congenital Anemia of newborn.

ICTERUS GRAVIS NEONATORUM

Less severe form of hemolytic disease.

Baby born alive without jaundice but soon develops within 24 hrs of birth.

Kernicterus

Concentration of bilirubin in the newborn blood exceeds 20mg/100ml or

in-term fetus – > 342 mmoll/L

in pre-term fetus – >205 mmoll/L,

Billirubin crosses the BBB and damage the basal nuclei of brain.

Screening and diagnosis

Maternal blood grouping, Rh-typing and antibody screening at their 1st pre-natal visit.

Presence of anti-D antibodies in serum is diagnostic of maternal Rh-alloimmunization.

Antibody screening in mother

Should be done in….I. Rh-negative women who have

received anti-D immune globulin.II. Rh-positive mother who have… --blood transfusion. --unexplained fetal loss. --infant with unexplained jaundice.

Gold Standard Test Indirect Coombs:-mix Rh(D)+ cells with maternal serum-anti-Rh(D) Ab will adhere-RBC’s then washed & suspended in

Coombs serum (antihuman globulin)-RBC’s coated with Ab will be agglutinated

Direct Coombs:-mix infant’s RBC’s with Coombs serum-maternal Ab present if cells agglutinate

If indirect coombs test is positive, the father’s Rh should be tested.

Serial maternal Anti D titers should be done every 2- 4 weeks.

If titer is less than 1/16 the fetus is not at risk.

If titer is more than 1/16 then severity of condition should be evaluated.

USG : detection of hydropic change. CVS Amniocentasis Cordocentesis MCA-PSV

USG

Confirmation of gestational age. Early detection of hydrops when finding one or

more of the following: Ascites, pleural effusion, pericardial effusion, skin edema. Increase placental size, Hepato-spleenomegally.

USG FINDINGS

Fetal Ascites

Rh- Iso imunizationBody wall

edema hydropic fetus

MCA-PSV

Non-invasive Mother not put at risk for worsening

alloimmunization Can be used with alloantibodies

other than RhD, including anti-Kell antibodies

Biophysical surveillance Middle cerebral artery peak velocity

Biophysical surveillanceMiddle Cerebral Artery peak systolic velocity

B = moderate-severe anaemiaA = mild anaemiaC = no anaemia

C

CVS

To know fetal Rh genotype. Advantage: early detection. Disadvantage: --more complicated. --increase severity of alloimmunization if baby Rh +

AMNOCENTESIS Methode of choice for detection of Rh factor of

fetous and amniotic fluid bilirubin. Critical titre/previous affected infant. Bilirubin correlates with fetal hemolysis. Spectophotometric analysis optical density of

amniotic fluid @ ▲OD 450nm. Data plotted on Liley curve.

CORDOCENTESIS

More complicated procedure.. Fetal Rh phenotype can be known

rapidly by blood bank serology. Gold standard for detection of fetal

anemia Greater morbidity and mortality 2.7% total risk of fetal loss

Diagram of cordocentesis procedure Cordocentesis -

MANAGEMENT

Rh-negative women categorized in two groups.

I. Rh-negative non-immunized women.II. Rh-negative immunized women. immunized against -D-antigens -non D-Rh antigens -other blood group system

The aim of antenatal management

To predict which pregnancy is at risk

To predict whether or not the fetus is severely affected.

To correct anemia and reverse hydrops by intrauterine transfusion.

To deliver the baby at the appropriate time, weighing the risks of prematurity against these of intrauterine transfusion.

OBJECTIVES

A. Non immunized women– prevention of allo-immunization.

B. Immunized women— a. early detection. b. adequet treatment of fetal anemia.

Rh-negative non immunized

Phenotype of father

Rh-positive

Ante-partum sensitization of Ab screening at

20,24,28 wks GA.

Rh-negative

Baby Rh-negative

Rh-positive Manage as normal pregnancy

Cont…

Antibody screening at 20,24& 28wks ga

No anti-Ab detected

Pt should received anti-D-immunoglobin at 28wks of GA.

If anti-D antibodies detected

Manage as Rh-negative immunised

women

At times of delivery to determine the mother’s eligibility for a second dose of

anti-D immunoglobin

RH-NEGATIVE IMMUNIZED WOMEN

Management based on---

A. First affected prenancy.

B. Previous affected pregnancy.

FIRST AFFECTED PREGNANCY

Should have antibody triter every 4 wks.A. If triter≥critical level - amniocntasis - MCA-PSV. B. If triter<critical level upto 36 wks of

gestation,should deliverd between 38-40wks.

Sudden elevation of Ab-triter when GA>34WKS <37WKS

amniocentasis

Lung imaturityContineu

pregnancy if

bilirubin<0.05 with serial

amniocentasis

weekly

Lung maturity

Lung maturity

delivery

PREVIOUS AFFECTED PREGNANCY

a. Maternal anti-D triter not predict the fetal anemia

b. MCA-PSV to determine the anemia.c. Serial amniocentasis.

PREVIOUS AFFECTED PREGNANCY

a. Maternal anti-D triter not predict the fetal anemia

b. MCA-PSV to determine the anemia.

c. Serial amniocentasis.

TRITER LEVEL PAST OBSTETRIC HISTORY

% OF IUD

<64 NEGATIVE 4%

<64 POSITIVE 32%

>64 NEGATIVE 17.2%

>64 POSITIVE 68.7%

Suggested management after amniocentesis for ΔOD 450

Serial Amniocentesis

Lily zone ILower Zone II

Upper Zone II Zone IIIHydramnios & Hydrops

Repeat Amniocentesis every

2-4 weeks

Delivery at OR near term

Repeat Amniocentesis in 7 days or FBS

Hct < 30% Hct > 30%

Intrauterine Transfusion

Repeat Sampling7 to 14 days

Fetal hematocrit<30%

Fetal hematocrit>30%

Intrauterine Transfusion

Follow with fetalBlood sampling& USG

DELIVERY WHEN LUNG

MATURE

Fetal blood sampling

LILEY’S CHART

WHITEFIELD’ ACTION LINE

QUEENAN CHART

Transfusion therapy Intraperitoneal First done in 1963 Instill blood through needle or epidural catheter Volume to transfuse = (G.A.-20) x 10ml. Generally, repeat in ~ 10 days, then every

4wks. Risk of death about 4% per procedure. Not effective in hydropic fetus. Some advocate combined approach (IPT and

IVT)

Transfusion therapy

Intravascular Goal is to have post-transfusion Hct 40-45% Can infuse about 10 ml/min Goal:keep Hct>25% Estimate requirement based on EFW and pre-

transfusion Hct Repeat in 1 wk., then about every 3 wkly. Fetal loss about 1.5% per procedure

Direct fetal intravascular transfusion

Other therapies:A. Plasmapheresis : tried to remove

several liters of maternal plasma with anti-D antibodies.

B. High dose i.v immunoglobulin : 1000mg/kg weekly.

MANAGEMENT DURING DELIVERY

Pregnant women undergo cesarean section in isoimunization:

Severe form of hemolytic infant disease in the term or 34-35 weeks after previous antenatal prevention of fetal hyaline membranes syndrome;

Measures to be taken Use abdominal packs in the sides of the

uterus before opening the lower segment to prevent spilled blood from the placenta to inter the peritoneal cavity.

Let the placenta to be delivered spontaneous using control cord traction without squeezing the uterus.

… A void avulsions of the cord.

undergo delivery in the term of 37-38 weeks of gestation.

Induction of labor is performen by prostaglandin (in the case of “unripe” uterine cervix) or by intravenous oxytocin infusion administration (in the case of “ripe” uterine cervix).

Vaginal delivery in Rh-isoimmunization

Vaginal delivery in Rh-isoimmunization During labor: No fundal pushing in 1st or 2nd stage of labor. With hold inj methergin after ant. shoulder delivery. Early cord clamping and no milking. No uterine massage or squeeze in 3rd stage. Let the placenta to be delivered spontaneous to

avoid avulsions of the cord. Protect the vaginal and perineal wounds and

laceration from being exposed to the fetal blood spilled from cord

At birth Maternal blooda. antibodies by indirect Comb's test ( ICT )..b. fetal red blood cells in maternal circulation Cord blood sample ( Neonatal blood sample ) fora. antibodies by Direct Comb's test ( DCT )b. Infant blood group and rh-typing.c. Infant bilirubin leveld. Infant Hb & Hct level

Rh- Iso imunization Prevention- Screening of all pregnant mothers to Rh D

antigen and antibody screening for Rh D negative mother.

Word of Vincent Freda the rule of thumb should be to administer

anti-D immunoglobin when in doubt rather than to withhold it.

Prophylactic anti D immunoglobulinTo be given All Rh – mothers after delivery if the fetus

is Rh+ At 28, 32 weeks of pregnancy or after

40wks if pregnancy contineued After abortion, amniocentesis, abruption,

ectopic pregnancy.

Rh- Iso imunizationPrevention The standard dose of anti D is

0.3 mg —will eradicate 15 ml of fetal red blood cells (routine for all Rh –ve pregnancies) within 3 days of delivery.

-If more feto-maternal bleeding is suspected as in abruption or ante partum hemorrhage-Do Kleihauer –Betke test to estimate the amount of fetal red cells in maternal circulation and re-calculate the dose of the anti-D.

Kleihauer-Betke test measure amount of feto-maternal haemorrhage. PRINCIPLE: Adult hemoglobin, but not fetal hemoglobin, is soluble in a

citrate buffer with pH 3.2 and will elute out of the red cell.

(critical volume) isoimmunization represented by 5 fetal cells in 50 low power microscopic field of peripheral maternal blood.

So 1 ml is represented by 20 fetal cells

method

Prepare patient blood smears Fix smears in 80% ethanol Incubate slides in citrate buffer Stain smears with erythrosine Count fetal cells on patient slid Red cells containing Hgb F stain bright pink due to erythrosin stain ,Negative

staining, adult red cells that contained Hgb A,appear as pale, ghost cells.

Kleihauer Calculations

%Fetal red cells = fetal cell counted in total slids x 100 total maternal cell

%fetal red cell x 5000ml(MBV) volume of FMH = 100MBV – maternal blood volume (usually 5000ml) volume of FMH VIAL REQUIRS = 30

positive cells on the Kleihauer-Betke stain

Post-partum mothers following a transplacental hemorrhage

• Newborns/infants less than 6 months old

• Hereditary persistence of hemoglobin F • Disorders that compensate with

hemoglobin F such as beta thal major or sickle cell disease

Other test: to detect FMH Flow cytometry Rosette test Enzyme linked antiglobulin test Surrogate test

Immunoglobulin (RhoGAM) prophylaxis (RhIgG)

Schedules Spon.abortionBefore 12wks – RCOG,UK- no dose require Austalian soc.- 50 μg RhIgG Canadian soc.- 120 μg RhIgG 2nd trimester abortion- 50 μg RhIgG Threatened abortion- 1st trimester - not requir 2nd trimester- 50 μg RhIgG (should be repeated 6wkly if heavy bleeding)

A minimum dose of 50 μg RhIgG is recomendate upto 19wks+6days GA.

After 20wks minimum dose 125 μg RhIgG .

Antepartum (RAADP)regimen. Two dose 500IU at 28 and 34wks GA. Single dose 1500IU at 28 wks GA. Postpartum <72 hr - 300 μg RhIgG;

IT can be given upto 28 days of delivery 0.3% require > 300 μg RhIgG

prophylaxis

Hydatidiform mole complete mole-controversial. patrial mole-anti-D to be given.

Following BTL-controversial. given…. women wants new partner desire

for IVF. In future if major accident occurs and Rh- blood not available at emergency.

300 μg anti-D neutralizes 30 mL fetal Rh-positive blood (15 mL packed fetal RBC)

Management of sensitized newborn

Mild anemia (Hb <14gm/dl, cord bilirubin>4 mg/dl)---Phototherapy

-Moderate to severe----Exchange transfusion.

-Mild Hydrops improves in 88% of cases

-Severe hydrops—Mortality is 39%