1. Rh Factor ISOIMMUNIZATION Associate Professor Iolanda Blidaru, MD, PhD

2. INTRODUCTION ANTIGEN >400 Agglutinogens on the cell membrane R.B.C.Plasma ANTIBODY Natural & Immune Agglutinins/ Isoantibodies Antigen-Antobody reaction on the cell surface  Hemolysis

3. INTRODUCTION controlled by genes at chromosomal loci appeared by 40 days of i.u. life unchanged till death present in tissues and tissue fluids Blood group system - a group of antigens controlled by a locus having a variable no. of allele genes. Antigens

4. INTRODUCTION > 15 recognised blood group systems ABO, Rh, Kell, Duffy, MNS, P, Lewis, Lutheran, Xg, Li, Yt, Public antigens & Private antigens. Blood-type means individual antigen phenotype which is the serological expression of the inherited genes. Most of these blood group antigens have been found to be associated with hemolytic disease. ABO & Rh account for 98%. Antigens

5. INTRODUCTION Alloantibodies / Agglutinins Pre-existent IgM Iso-antibodies IgG Formed in response to foreign R.B.C. or soluble blood group substances Antibodies

6. INTRODUCTION Formed against most of the major group antigens and present in individuals. IgM type. Do not cross placenta. React poorly at body temperature (except anti-A and anti-B), but agglutinate erythrocytes at 5-20°C Pre-existent (natural) Antibodies

7. INTRODUCTION The isoantibodies are IgG. Best react at body temperature Cross the placenta readily. Most Ab are complement binding. Iso-Antibodies

8. Immunology of Rhesus Blood Group System First demonstrated by testing human blood with rabit anti sera against RBC of Rhesus monkey → classifying into Rh negative / Rh positive. The genotype is determined by the inheritance of 5 closely linked allelic genes situated on the short arm of chromosome 1, named as D,C,c,E,e (Fisher-Race). No ”d” → absence of D The foetus inherits one gene from each group as a haplotype such as sets of cDe, CDE, etc from each parent.

9. Immunology of Rhesus Blood Group System 12 sets of combinations and 78 genotypes are possible. For clinical and all practical purposes it is enough to know whether one is Rh POSITIVE or NEGATIVE against anti D sera. The antigenic expressions of these genes are the coresponding antigens, consisting of C/c, D/d, E/e. The antigenic determinants form an intrinsic part of the red cell membrane protein structure.

10. Immunology of Rhesus Blood Group System C/c and E/e are weak antigens. ‘D’ is the most immunogenic in the Rh system. There is a rare type of Rh negative called Rh null who lack all known Rh antigens. ‘D’ antigen has no natural antibody. A single transfusion of Rh+ blood to a Rh– person has a 50% chance of forming anti Rh D antibodies.

11. Incidence of Rhesus Blood Group System Incidence of Rh negative varies in different races and ethnic groups Mongoloids = nil Chinese, Japanese = 1-2% Indians = 5% Africans = 5-8% Causcasians = 15-17% Basques = 30-35%

12. Pathogenesis Of Rh Iso-immunization  Rh Negative Women Man Rh positive (Homo/Hetero)    Fetus   Rh Neg Fetus No problem Rh positive Fetus   Rh+ RBCs enter maternal circulation  Mother previously sensitized Secondary immune response  Iso-antibody (IgG) (   Non sensitized mother Primary immune response  Fetus  unaffected 1 st baby usually escapes. Mother gets sensitised?   Fetus Haemolysis 

13. Pathogenesis Of Rh Iso- immunization Chances of feto-maternal hemorrhage / passage are only 5% in 1st trimester but 47% in 3rd trimester; many conditions can increase the risk. Chances of primary sensitization during 1st pregnancy is only 1-2%, but 10-15% of patients may become sensitized after delivery. ABO incompatibility and Rh non-responder status may protect. Amount of antibodies that enter the fetal circulation will determine the degree of haemolysis.

14. Pathology Of Erythroblastosis fetalis HAEMOLYSIS  IN UTEROAFTER BIRTH  BILLIRUBIN  ANAEMIA   MAT. LIV → NO EFFECT   HEPATIC ERYTHROPOIESIS & DYSFUNCTION  PORTAL & UMBILICAL VEIN HYPERTENSION, HEART FAILURE, HYDROPS FETALIS       BIRTH OF AN AFFECTED INFANT - Wide spectrum of presentations. Rapid deterioration of the infant after birth. May continue for few days to few months.  Jaundice Kernicterus Hepatic Failure  DEATH ERYTHROBLASTOSIS FETALIS    INTRAUTERINE DEATH     

15. Antenatal Diagnosis Of Rh Iso- immunization the history and physical exam. the maternal and paternal Rh blood type anti-Rh Ab screen (monthly after 20-th week) – by indirect Coombs test; if positive as 1/8-1/32, there is needed further assessment ultrasound exams fetal anemia and hyperbilirubinemia – by ultrasound guided amniocentesis → spectrophotometry of amnionic fluid (Liley chart) → CORDOCENTESIS – fetal blood tests

16. Antenatal Diagnosis Of Rh Iso- immunization - ultrasound exams

17. Antenatal Diagnosis Of Rh Iso-immunization Liley chart

18. Antenatal Diagnosis Of Rh Iso-immunization

20. Postnatal Diagnosis Of Rh Iso- immunization Hemolytic disease of the newborn Hemolytic anemia (Hb = 13-14g%; bilirubin < 3.5mg%) Icterus gravis neonatorum (Hb = 7-12g%; bilirubin > 10mg%) Kernicterus (bilirubin > 18mg%) Hydrops fetalis (Hb 10mg%)

21. Postnatal Diagnosis Of Rh Iso-immunization

22. Hydrops fetalis

24. Postnatal Diagnosis Of Rh Iso-immunization Laboratory tests (newborn) ABO and Rh blood group test Hb and Ht Reticulocyte count Bilirubin level Direct Coombs test

25. Prevention of Rh Iso- immunization Premarital counseling. Blood grouping for every woman, before 1st pregnancy. Proper management of unsensitised Rh negative pregnancies.

26. Prevention of Rh Iso- immunization  Blood typing at 1-st visit, If negative → husband’s typing.  Anti-Rh Ab screen (indirect Coomb’s test) of Rh–negative mother.  At about 28 weeks – negative → 300μg anti D immunoglobulin.  In abortion, ectopic pregnancy, abruption of placenta, placenta praevia, molar pregnancy, abdominal trauma, chorionic villi sampling, amniocentesis = foetal-maternal hemorrhage → 150-300μg anti D  At birth- cord blood for ABO and Rh typing → Baby Rh positive → 300μg anti D within 72 hours of delivery

27. Prevention of Rh Iso- immunization In case of large fetal-maternal hemorrhage: 1.the Kleihauer-Betke test estimates the amount of fetal blood in circulation 2.the indirect Coombs test 3.an additional dose of anti-D, if needed

28. The Kleihauer-Betke test

29. Prevention of Rh Iso- immunization Errors - Causes of sensitization Misinterpretation of maternal Rh type Rh+ blood transfusion Unprotected pregnancy and labour Inadequate dose / improper use of IgG on previous occasions Immunization to cross-reacting antigen

30. Management of Rh Iso- immunized Pregnancy ANTEPARTUM Careful planning during antepartum, intrapartum and neonatal period Known repeated maternal anti-D Ab titer Intrauterine fetal monitoring with repeated US examinations, cordocentesis & fetal blood sampling / amniocentesis, the measurement of the fetal middle cerebral artery peak velocity (Doppler)

31. Management of Iso- immunized Pregnancy Fetus Rh Positive + anemia Intrauterine transfusion of Rh- negative blood in selected cases Planned preterm delivery any time after 34 weeks or as soon as the lung maturity is documented by inducing the labor or cesarean section (for the severely affected fetuses)

32. Management of Iso- immunized Pregnancy POSTPARTUM Management of the infant Monitoring up to 8 weeks Exchange-transfusion in the newborn in the umbilical vein Phototherapy In cases of severely sensitized women, consider medical termination of pregnancy and sterilization.