ALLOIMMUNIZATION IN PREGNANCY Brooke Grizzell, M.D. PGY-2 OBGYN Department, UKSM September 28 th, 2005

Objectives 1. Understand history of HDFN 1. Understand history of HDFN 2. Learn correct terminology 2. Learn correct terminology 3. Outline ABO and CDE blood groups “Major blood group antigens” 3. Outline ABO and CDE blood groups “Major blood group antigens” 4. Discuss Minor blood group antigens, including Kell, Lewis, Duffy, and various others 4. Discuss Minor blood group antigens, including Kell, Lewis, Duffy, and various others 5. Review clinical management 5. Review clinical management 6. Compare Delta OD 450 and MCA-PSV 6. Compare Delta OD 450 and MCA-PSV 7. Provide Rh alloimmunization management summary 7. Provide Rh alloimmunization management summary 8. Learn prevention strategies 8. Learn prevention strategies

History of HDFN 1609: First case of HDFN described 1939: Levine & Stetson described antibody 1941: Levine demonstrated causal relationship between anti-D antibodies and HDFN 1945: Neonatal exchange transfusion began 1956: Bevis proposed amniotic fluid assessment 1961: Liley proposed amniotic fluid assessment 1963: Liley introduced intraperitoneal fetal transfusion 1968: Rh immune globulin (RhoGAM) introduced 1980’s: Real-time ultrasound 1990’s: Genetic techniques to perform fetal RBC typing

“erythroblastosis fetalis” vs. “hemolytic disease of the fetus and newborn” (HDFN) “erythroblastosis fetalis” vs. “hemolytic disease of the fetus and newborn” (HDFN) “alloimmunization” vs. isoimmuniation” “alloimmunization” vs. isoimmuniation” Correct Terminology

Pathophysiology After 1 st antigenic exposure, memory B lymphocytes recognize appearance of RBC’s containing the antigen in subsequent pregnancies After 1 st antigenic exposure, memory B lymphocytes recognize appearance of RBC’s containing the antigen in subsequent pregnancies B lymphocytes - plasma cells - IgG B lymphocytes - plasma cells - IgG Initial IgM response changes to IgG Initial IgM response changes to IgG

Pathophysiology- Continued Maternal antibodies cross placenta- attach to fetal RBC’s- lead to RBC destruction Maternal antibodies cross placenta- attach to fetal RBC’s- lead to RBC destruction Sequestration by macrophages in fetal spleen-extravascular hemolysis-produces fetal anemia Sequestration by macrophages in fetal spleen-extravascular hemolysis-produces fetal anemia

ABO Blood Group Invariably causes only mild disease Invariably causes only mild disease Treatment generally limited to phototherapy Treatment generally limited to phototherapy No need for antenatal detection No need for antenatal detection

CDE (Rhesus) System Very important!!! Very important!!! Includes c, C, D, e, E Includes c, C, D, e, E D negativity defined as absence of D antigen D negativity defined as absence of D antigen Only 87% of Caucasians carry the D antigen Only 87% of Caucasians carry the D antigen

Various Other Antibodies Antigens such as A, P, Le (a), M, I, IH, and Sd (a) are innocuous Antigens such as A, P, Le (a), M, I, IH, and Sd (a) are innocuous Most are IgM Most are IgM Lewis antibodies and cold agglutinins of I are prevalent but not clinically significant Lewis antibodies and cold agglutinins of I are prevalent but not clinically significant

Antibodies Associated with HDFN Anti-c, Anti-D, Anti-E, and Anti- Kell Anti-c, Anti-D, Anti-E, and Anti- Kell RhoGAM has decreased HDFN caused by anti-D, but Anti-D antibody is still MOST COMMON CAUSE of red cell alloimmunization RhoGAM has decreased HDFN caused by anti-D, but Anti-D antibody is still MOST COMMON CAUSE of red cell alloimmunization

Minor RBC Antigens Kell is most common of minor Kell is most common of minor Responsible for 10% of cases of severe antibody-mediated anemia Responsible for 10% of cases of severe antibody-mediated anemia Mechanism of anemia two-fold Mechanism of anemia two-fold 1. Hemolysis 2. Suppression of erythropoiesis ** Transfuse women with Kell(-) blood **

Minor RBC Antigens ( continued ) Lewis antigen also a common minor RBC antigen Lewis antigen also a common minor RBC antigen Most anti-Lewis antibodies are IgM antibodies Most anti-Lewis antibodies are IgM antibodies Mothers with anti-Le (a) antibodies who require transfusion, need blood negative for the Le(a) antigen Mothers with anti-Le (a) antibodies who require transfusion, need blood negative for the Le(a) antigen

Minor RBC Antigens ( continued ) Duffy antigens Fy(a) and Fy(b) Duffy antigens Fy(a) and Fy(b) Only anti-Fy(a) antibody associated with HDFN- may range from mild to severe Only anti-Fy(a) antibody associated with HDFN- may range from mild to severe ACOG: treat sensitization to minor RBC antigens similar to those with Rh alloimmunization ACOG: treat sensitization to minor RBC antigens similar to those with Rh alloimmunization

Minor Antigens ( continued ) MNS system = M, N, S, s, U antigens MNS system = M, N, S, s, U antigens Anti-M and anti-N naturally occurring- no clinical significance Anti-M and anti-N naturally occurring- no clinical significance Anti-S, anti-s, and anti-U antibodies ~ mild to severe HDFN Anti-S, anti-s, and anti-U antibodies ~ mild to severe HDFN

Clinical Management 1. Routine blood type &AB screen 1. Routine blood type &AB screen 2. Repeat AB screen at wga for Rh (-) women prior to receiving RhoGAM (some recommend repeat screens for Rh (+) women also) 2. Repeat AB screen at wga for Rh (-) women prior to receiving RhoGAM (some recommend repeat screens for Rh (+) women also) 3. If AB screen is (+), identify antibody and potential for HDFN 3. If AB screen is (+), identify antibody and potential for HDFN

Clinical Management (cont) 4. Elicit risk factors for alloimmunization (past pregnancies, transfusions, shared needles) 4. Elicit risk factors for alloimmunization (past pregnancies, transfusions, shared needles) 5. Determine father’s RBC antigen status and zygosity 5. Determine father’s RBC antigen status and zygosity 6. If paternity unknown or father is (+) for antigen, fetus is at RISK 6. If paternity unknown or father is (+) for antigen, fetus is at RISK

Clinical Management (cont) 6. (cont) Obtain antibody titer~ the reciprocal of the highest dilution still giving a positive reaction 6. (cont) Obtain antibody titer~ the reciprocal of the highest dilution still giving a positive reaction ACOG: Consider invasive testing at titer of 1:32 or greater by indirect Coombs (1:16 most often used) **Titers less reliable after a sensitized pregnancy**

Clinical Management (cont) 7. If AB titer remains below critical titer- invasive testing can be deferred and pt. evaluated by serial AB titers 7. If AB titer remains below critical titer- invasive testing can be deferred and pt. evaluated by serial AB titers Serial titers before wga not necessary Serial titers before wga not necessary If critical titer noted at first visit, amnio for delta OD450 at wga If critical titer noted at first visit, amnio for delta OD450 at wga

Clinical Management (cont) 8. Obtain amniocytes to determine fetal blood type if father is heterozygous for the antigen responsible for alloimmunization 8. Obtain amniocytes to determine fetal blood type if father is heterozygous for the antigen responsible for alloimmunization 9. MCA-PSV can be used as early as 18 wga ~ if greater than 1.5 MoM, consider fetal blood sampling 9. MCA-PSV can be used as early as 18 wga ~ if greater than 1.5 MoM, consider fetal blood sampling

Clinical Management (cont) 10. Serial amnio to measure delta OD450 and plot values on Liley or Queenan graph 10. Serial amnio to measure delta OD450 and plot values on Liley or Queenan graph Delta OD450 vs. Delta OD450 vs. MCA-PSV MCA-PSV

Delta OD450 Spectral analysis of amniotic fluid at 450 nm proposed in 1961 by Liley- measures change in OD Spectral analysis of amniotic fluid at 450 nm proposed in 1961 by Liley- measures change in OD Measures the level of bilirubin and predicts severity of hemolytic disease after 27 wga Measures the level of bilirubin and predicts severity of hemolytic disease after 27 wga Delivery or intrauterine transfusion if delta OD450 falls into zone III or upper zone II Delivery or intrauterine transfusion if delta OD450 falls into zone III or upper zone II

Queenan Method Proposed another method of using delta OD450 Proposed another method of using delta OD450 Suggested four zones in his graph Suggested four zones in his graph

Limitations of spectral analysis Kell-antigen sensitized pregnancies Kell-antigen sensitized pregnancies Erythroid suppression and hemolysis Erythroid suppression and hemolysis Not an accurate prediction of fetal anemia Not an accurate prediction of fetal anemia

MCA-PSV Velocity of blood flow in brain increased with anemia b/c of Velocity of blood flow in brain increased with anemia b/c of 1. Increased cardiac output 1. Increased cardiac output 2. Vasodilation in the brain 3. Decreased blood viscosity 3. Decreased blood viscosity Prospective studies Prospective studies 111 fetuses~ 100% sensitivity 125 fetuses~ 88% sens. and 98% NPV

Correct Technique for MCA Doppler Fetus resting Fetus resting Circle of Willis imaged in axial image using color doppler Circle of Willis imaged in axial image using color doppler Entire length of MCA Entire length of MCA Close to origin of internal carotid artery Close to origin of internal carotid artery

Limitations of Each Method Delta OD450 falsely elevated in presence of mec or blood Delta OD450 falsely elevated in presence of mec or blood Delta OD450 misleadingly low after inadvertent exposure to light or in Kell alloimmunization Delta OD450 misleadingly low after inadvertent exposure to light or in Kell alloimmunization MCA accuracy diminishes after 35 wga and after multiple transfusions MCA accuracy diminishes after 35 wga and after multiple transfusions

Comparison Studies? Few have been completed Few have been completed Small retrospective studies Small retrospective studies 28 pregnancies~ 100% sens. using Doppler MCA-PSV vs. 80% sens. using amnio and delta OD450 Another study with 28 pregnancies~ 75% sens and 60% PPV using MCA-PSV vs. 75% and 53% respectively for delta OD450

Cordocentesis Gold standard for detection of fetal anemia Gold standard for detection of fetal anemia Complications! Complications! 2.7% total risk of fetal loss 2.7% total risk of fetal loss Reserved for patients with increased MCA-PSV or delta OD450 Reserved for patients with increased MCA-PSV or delta OD450

Advantages of MCA-PSV Non-invasive Non-invasive Mother not put at risk for worsening alloimmunization Mother not put at risk for worsening alloimmunization Can be used with alloantibodies other than RhD, including anti-Kell antibodies Can be used with alloantibodies other than RhD, including anti-Kell antibodies

Summary of Management for Rh Alloimmunization Monthly indirect coombs titer (in first sensitized pregnancy) Monthly indirect coombs titer (in first sensitized pregnancy) If critical titer reached, determine paternal and fetal antigen status If critical titer reached, determine paternal and fetal antigen status Amniocentesis and delta OD450 OR MCA-PSV Amniocentesis and delta OD450 OR MCA-PSV ** For 2 nd or greater sensitized pregnancy, initiate amnio or MCA at wga**

Rh Alloimmunization management (cont) If using MCA-PSV, and initial is less than 1.5 MoM, do weekly testing x 3 wks If using MCA-PSV, and initial is less than 1.5 MoM, do weekly testing x 3 wks Regression line/slope Regression line/slope Repeat testing Q 1-4 wks Repeat testing Q 1-4 wks Cordocentesis or delivery once MCA-PSV reaches 1.5 MoM Cordocentesis or delivery once MCA-PSV reaches 1.5 MoM

Prevention of Alloimmunization ACOG recommends RhoGAM for Rh (-) pts after 1 st trimester loss ACOG recommends RhoGAM for Rh (-) pts after 1 st trimester loss RhoGAM for threatened abortion controversial- no evidence based recomm. RhoGAM for threatened abortion controversial- no evidence based recomm. 50 mcg dose protects against 2.5 ml of Rh (+) RBC’s 50 mcg dose protects against 2.5 ml of Rh (+) RBC’s 300 mcg dose protects against 15 ml of RBC’s or 30 ml of Rh (+) blood 300 mcg dose protects against 15 ml of RBC’s or 30 ml of Rh (+) blood

Prevention (cont) Give 300 mcg dose within 72 hrs of delivery to unsensitized Rh (-) women (Rh positive infant) Give 300 mcg dose within 72 hrs of delivery to unsensitized Rh (-) women (Rh positive infant) ACOG: 300 mcg at 28 wga UNLESS father known to be Rh (-) ACOG: 300 mcg at 28 wga UNLESS father known to be Rh (-) Repeat Antibody Screen ? Repeat Antibody Screen ?

Prevention (cont) Test for excessive fetal-maternal hemorrhage after blunt trauma, abruption, cordocentesis, and bleeding assoc. with previa Test for excessive fetal-maternal hemorrhage after blunt trauma, abruption, cordocentesis, and bleeding assoc. with previa Kleihauer Betke or rosette test Kleihauer Betke or rosette test Give RhoGAM for partial molar pregnancy, SAB, TAB, ectopic, chorionic villus sampling, external version Give RhoGAM for partial molar pregnancy, SAB, TAB, ectopic, chorionic villus sampling, external version

Conclusions Remember the instances in which to consider RhoGAM Remember the instances in which to consider RhoGAM -SAB, TAB, threatened AB (controversial), ectopic, previa/bleeding, abruption, partial molar, CVS, blunt trauma, cordocentesis -SAB, TAB, threatened AB (controversial), ectopic, previa/bleeding, abruption, partial molar, CVS, blunt trauma, cordocentesis Clinically important antibodies: Anti-c, Anti-D, Anti-E, and Anti-Kell, Rarely Anti- Duffy Fy(a) Clinically important antibodies: Anti-c, Anti-D, Anti-E, and Anti-Kell, Rarely Anti- Duffy Fy(a) Usually not associated with severe HDFN: Anti-Lewis, ABO incompatibilities, Anti- Duffy(Fy-b) antibodies, (Duffy Fy-a causes mild to severe HDFN), Anti-A, Anti-P, Anti-M, Anti-I, Anti-IH, Anti-Sd(a) Usually not associated with severe HDFN: Anti-Lewis, ABO incompatibilities, Anti- Duffy(Fy-b) antibodies, (Duffy Fy-a causes mild to severe HDFN), Anti-A, Anti-P, Anti-M, Anti-I, Anti-IH, Anti-Sd(a)

Conclusions (cont) Anti-D still most common cause of red cell alloimmunization, despite RhoGAM Anti-D still most common cause of red cell alloimmunization, despite RhoGAM Kell = most common minor antigen *anemia mechanism 2-fold Kell = most common minor antigen *anemia mechanism 2-fold Critical titer most often used is 1:16 by indirect coombs Critical titer most often used is 1:16 by indirect coombs Amnio with delta OD450 vs. MCA-PSV Amnio with delta OD450 vs. MCA-PSV Liley or Queenan graphs Liley or Queenan graphs Remember AB screens and indications for RhoGAM!! Remember AB screens and indications for RhoGAM!!

References 1. Gabbe Obstetrics – Normal and Problem Pregnancies, 4 th edition. 2. Creasy R., Resnik R., Iams J., Maternal Fetal Medicine Principles and Practice, 5 th edition. 3. ACOG Compendium Harkness U., Spinnato J., Prevention and Management of RhD isoimmunization. Clinics in Perinatology, Dec :4. 5. Pereira L., Jenkins T., Conventional management of maternal red cell alloimmunization compared with management by Doppler assessment of MCA-PSV. American Journal of Obstetrics and Gynecology, Oct :4.

References (cont) 6. Cohen D., Hemolytic disease of the newborn: RBC alloantibodies in pregnancy and associated serologic issues. Up to Date, Oct Barss V., Moise K., Significance of minor red blood cell antibodies during pregnancy. Up to Date, Apr 2005.