ALLOIMMUNIZATION ALLOIMMUNIZATION IN PREGNANCYIN PREGNANCY

Brooke Grizzell, M.D.Brooke Grizzell, M.D.PGY-2PGY-2

OBGYN Department, UKSMOBGYN Department, UKSM

September 28September 28thth, 2005, 2005

ObjectivesObjectives 1. Understand history of HDFN 1. Understand history of HDFN 2. Learn correct terminology2. Learn correct terminology 3. Outline ABO and CDE blood groups “Major blood 3. Outline ABO and CDE blood groups “Major blood

group antigens”group antigens” 4. Discuss Minor blood group antigens, including Kell, 4. Discuss Minor blood group antigens, including Kell,

Lewis, Duffy, and various othersLewis, Duffy, and various others 5. Review clinical management5. Review clinical management 6. Compare Delta OD 450 and MCA-PSV6. Compare Delta OD 450 and MCA-PSV 7. Provide Rh alloimmunization management summary7. Provide Rh alloimmunization management summary 8. Learn prevention strategies8. Learn prevention strategies

History of HDFNHistory of HDFN1609: 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 assessment1961: Liley proposed amniotic fluid assessment

1963: Liley introduced intraperitoneal fetal transfusion 1968: Rh immune globulin (RhoGAM) introduced1980’s: Real-time ultrasound 1990’s: Genetic techniques to perform fetal RBC typing

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

“ “alloimmunization” alloimmunization” vs.vs. isoimmuniation”isoimmuniation”

Correct TerminologyCorrect Terminology

PathophysiologyPathophysiology After 1After 1stst antigenic exposure, memory antigenic exposure, memory

B lymphocytes recognize appearance B lymphocytes recognize appearance of RBC’s containing the antigen in of RBC’s containing the antigen in subsequent pregnanciessubsequent pregnancies

B lymphocytes - plasma cells - IgGB lymphocytes - plasma cells - IgG Initial IgM response changes to IgG Initial IgM response changes to IgG

Pathophysiology- Pathophysiology- ContinuedContinued

Maternal antibodies cross placenta- Maternal antibodies cross placenta- attach to fetal RBC’s- lead to RBC attach to fetal RBC’s- lead to RBC destructiondestruction

Sequestration by macrophages in Sequestration by macrophages in fetal spleen-extravascular fetal spleen-extravascular hemolysis-produces fetal anemiahemolysis-produces fetal anemia

ABO Blood Group ABO Blood Group Invariably causes only mild diseaseInvariably causes only mild disease Treatment generally limited to Treatment generally limited to

phototherapyphototherapy No need for antenatal detectionNo need for antenatal detection

CDE (Rhesus) SystemCDE (Rhesus) System

Very important!!!Very important!!! Includes Includes c, C, D, e, Ec, C, D, e, E D negativity defined as absence of D negativity defined as absence of

D antigenD antigen Only 87% of Caucasians carry the Only 87% of Caucasians carry the

D antigenD antigen

Various Other AntibodiesVarious Other Antibodies

Antigens such as A, P, Le (a), M, I, Antigens such as A, P, Le (a), M, I, IH, and Sd (a) are innocuousIH, and Sd (a) are innocuous

Most are IgMMost are IgM Lewis antibodies and cold Lewis antibodies and cold

agglutinins of I are prevalent but agglutinins of I are prevalent but not clinically significantnot clinically significant

Antibodies Associated Antibodies Associated with HDFNwith HDFN

Anti-c, Anti-D, Anti-E, and Anti-Anti-c, Anti-D, Anti-E, and Anti-KellKell

RhoGAM has decreased HDFN RhoGAM has decreased HDFN caused by anti-D, but Anti-D caused by anti-D, but Anti-D antibody is still antibody is still MOST MOST COMMON CAUSE COMMON CAUSE of red cell of red cell alloimmunizationalloimmunization

Minor RBC AntigensMinor RBC Antigens

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

severe antibody-mediated anemiasevere antibody-mediated anemia Mechanism of anemia two-foldMechanism of anemia two-fold

1. Hemolysis1. Hemolysis

2. Suppression of erythropoiesis2. Suppression of erythropoiesis

****Transfuse women with Kell(-) bloodTransfuse women with Kell(-) blood****

Minor RBC Antigens Minor RBC Antigens ((continuedcontinued))

Lewis antigen also a common minor Lewis antigen also a common minor RBC antigenRBC antigen

Most anti-Lewis antibodies are IgM Most anti-Lewis antibodies are IgM antibodiesantibodies

Mothers with anti-Le (a) antibodies Mothers with anti-Le (a) antibodies who require transfusion, need blood who require transfusion, need blood negative for the Le(a) antigennegative for the Le(a) antigen

Minor RBC Antigens Minor RBC Antigens ((continuedcontinued))

Duffy antigens Fy(a) and Fy(b)Duffy antigens Fy(a) and Fy(b) Only anti-Fy(a) antibody Only anti-Fy(a) antibody

associated with HDFN- may range associated with HDFN- may range from mild to severefrom mild to severe

ACOGACOG:: treat sensitization to treat sensitization to minor RBC antigens similar to minor RBC antigens similar to those with Rh alloimmunizationthose with Rh alloimmunization

Minor Antigens (Minor Antigens (continuedcontinued))

MNS system = MNS system = M, N, S, s, U M, N, S, s, U antigensantigens

Anti-M and anti-N naturally Anti-M and anti-N naturally occurring- no clinical significanceoccurring- no clinical significance

Anti-S, anti-s, and anti-U Anti-S, anti-s, and anti-U antibodies ~ mild to severe HDFNantibodies ~ mild to severe HDFN

Clinical ManagementClinical Management 1. Routine blood type &AB screen1. Routine blood type &AB screen 2. Repeat AB screen at 24-28 wga 2. Repeat AB screen at 24-28 wga

for Rh (-) women prior to receiving for Rh (-) women prior to receiving RhoGAM RhoGAM (some recommend repeat (some recommend repeat screens for Rh (+) women also)screens for Rh (+) women also)

3. If AB screen is (+), identify 3. If AB screen is (+), identify antibody and potential for HDFNantibody and potential for HDFN

Clinical Management Clinical Management (cont)(cont)

4. Elicit risk factors for 4. Elicit risk factors for alloimmunizationalloimmunization (past pregnancies, (past pregnancies, transfusions, shared needles)transfusions, shared needles)

5. Determine father’s RBC antigen 5. Determine father’s RBC antigen status and zygositystatus and zygosity

6. If paternity unknown or father is 6. If paternity unknown or father is (+) for antigen, fetus is at (+) for antigen, fetus is at RISKRISK

Clinical Management Clinical Management (cont)(cont)

6. 6. (cont) (cont) Obtain antibody titer~ the Obtain antibody titer~ the reciprocal of the highest dilution reciprocal of the highest dilution still giving a positive reactionstill giving a positive reaction

ACOGACOG: : Consider invasive testing at Consider invasive testing at titer of 1:32 or greater by indirect titer of 1:32 or greater by indirect Coombs Coombs (1:16 most often used)(1:16 most often used)

**Titers less reliable after a sensitized **Titers less reliable after a sensitized pregnancy**pregnancy**

Clinical Management Clinical Management (cont)(cont) 7. If AB titer remains below 7. If AB titer remains below

critical titer- invasive testing can critical titer- invasive testing can be deferred and pt. evaluated by be deferred and pt. evaluated by serial AB titersserial AB titers

Serial titers before 18-20 wga not Serial titers before 18-20 wga not necessarynecessary

If critical titer noted at first visit, If critical titer noted at first visit, amnio for delta OD450 at 22-24 amnio for delta OD450 at 22-24 wgawga

Clinical Management Clinical Management (cont)(cont)

8. Obtain amniocytes to determine 8. Obtain amniocytes to determine fetal blood type if father is fetal blood type if father is heterozygous for the antigen heterozygous for the antigen responsible for alloimmunizationresponsible for alloimmunization

9. MCA-PSV can be used as early 9. MCA-PSV can be used as early as 18 wga ~ if greater than 1.5 as 18 wga ~ if greater than 1.5 MoM, consider fetal blood MoM, consider fetal blood samplingsampling

Clinical Management Clinical Management (cont)(cont)

10. Serial amnio to measure delta 10. Serial amnio to measure delta OD450 and plot values on Liley or OD450 and plot values on Liley or Queenan graphQueenan graph

Delta OD450 vs. Delta OD450 vs.

MCA-PSVMCA-PSV

Delta OD450Delta OD450 Spectral analysis of amniotic fluid Spectral analysis of amniotic fluid

at 450 nm proposed in 1961 by at 450 nm proposed in 1961 by Liley- measures change in ODLiley- measures change in OD

Measures the level of bilirubin and Measures the level of bilirubin and predicts severity of hemolytic predicts severity of hemolytic disease disease after 27 wgaafter 27 wga

Delivery or intrauterine transfusion Delivery or intrauterine transfusion if delta OD450 falls into if delta OD450 falls into zone III zone III or upper zone IIor upper zone II

Queenan MethodQueenan Method Proposed another method of using Proposed another method of using

delta OD450delta OD450

Suggested four zones in his graphSuggested four zones in his graph

Limitations of spectral Limitations of spectral analysisanalysis

Kell-antigen sensitized pregnanciesKell-antigen sensitized pregnancies Erythroid suppression Erythroid suppression and and

hemolysishemolysis Not an accurate prediction of fetal Not an accurate prediction of fetal

anemiaanemia

MCA-PSVMCA-PSV Velocity of blood flow in brain Velocity of blood flow in brain

increased with anemia b/c of increased with anemia b/c of 1. Increased cardiac output1. Increased cardiac output

2. Vasodilation in the brain2. Vasodilation in the brain 3. Decreased blood viscosity3. Decreased blood viscosity

Prospective studiesProspective studies111 fetuses~ 100% sensitivity111 fetuses~ 100% sensitivity

125 fetuses~ 88% sens. and 98% NPV125 fetuses~ 88% sens. and 98% NPV

Correct Technique for Correct Technique for MCA DopplerMCA Doppler

Fetus restingFetus resting Circle of Willis imaged in axial Circle of Willis imaged in axial

image using color dopplerimage using color doppler Entire length of MCAEntire length of MCA Close to origin of internal carotid Close to origin of internal carotid

arteryartery

Limitations of Each Limitations of Each MethodMethod

Delta OD450 falsely elevated in Delta OD450 falsely elevated in presence of mec or bloodpresence of mec or blood

Delta OD450 misleadingly low Delta OD450 misleadingly low after inadvertent exposure to light after inadvertent exposure to light or in Kell alloimmunizationor in Kell alloimmunization

MCA accuracy diminishes after 35 MCA accuracy diminishes after 35 wga and after multiple transfusionswga and after multiple transfusions

Comparison Studies?Comparison Studies? Few have been completedFew have been completed Small retrospective studiesSmall retrospective studies

28 pregnancies~ 100% sens. using 28 pregnancies~ 100% sens. using Doppler MCA-PSV vs. 80% sens. using Doppler MCA-PSV vs. 80% sens. using amnio and delta OD450amnio and delta OD450

Another study with 28 pregnancies~ Another study with 28 pregnancies~ 75% sens and 60% PPV using MCA-PSV 75% sens and 60% PPV using MCA-PSV vs. 75% and 53% respectively for delta vs. 75% and 53% respectively for delta OD450OD450

CordocentesisCordocentesis Gold standard for detection of fetal Gold standard for detection of fetal

anemiaanemia Complications!Complications! 2.7% total risk of fetal loss2.7% total risk of fetal loss Reserved for patients with Reserved for patients with

increased MCA-PSV or delta increased MCA-PSV or delta OD450OD450

Advantages of MCA-PSVAdvantages of MCA-PSV

Non-invasiveNon-invasive Mother not put at risk for Mother not put at risk for

worsening alloimmunizationworsening alloimmunization Can be used with alloantibodies Can be used with alloantibodies

other than RhD, including anti-Kell other than RhD, including anti-Kell antibodiesantibodies

Summary of Management for Summary of Management for Rh AlloimmunizationRh Alloimmunization

Monthly indirect coombs titer (in first Monthly indirect coombs titer (in first sensitized pregnancy)sensitized pregnancy)

If critical titer reached, determine If critical titer reached, determine paternal and fetal antigen statuspaternal and fetal antigen status

Amniocentesis and delta OD450 OR Amniocentesis and delta OD450 OR MCA-PSVMCA-PSV

** For 2** For 2ndnd or greater sensitized pregnancy, or greater sensitized pregnancy, initiate amnio or MCA at 18-20 wga**initiate amnio or MCA at 18-20 wga**

Rh Alloimmunization Rh Alloimmunization management management (cont)(cont)

If using MCA-PSV, and initial is If using MCA-PSV, and initial is less than 1.5 MoM, do weekly less than 1.5 MoM, do weekly testing x 3 wkstesting x 3 wks

Regression line/slopeRegression line/slope Repeat testing Q 1-4 wksRepeat testing Q 1-4 wks Cordocentesis or delivery once Cordocentesis or delivery once

MCA-PSV reaches 1.5 MoMMCA-PSV reaches 1.5 MoM

Prevention of Prevention of AlloimmunizationAlloimmunization

ACOGACOG recommends RhoGAM for recommends RhoGAM for Rh (-) pts after 1Rh (-) pts after 1stst trimester loss trimester loss

RhoGAM for threatened abortion RhoGAM for threatened abortion controversial- controversial- no evidence based recomm.no evidence based recomm.

50 mcg dose protects against 2.5 ml of 50 mcg dose protects against 2.5 ml of Rh (+) RBC’sRh (+) RBC’s

300 mcg dose protects against 15 ml of 300 mcg dose protects against 15 ml of RBC’s or 30 ml of Rh (+) bloodRBC’s or 30 ml of Rh (+) blood

Prevention (cont)Prevention (cont)

Give 300 mcg dose within 72 hrs Give 300 mcg dose within 72 hrs of delivery to unsensitized Rh (-) of delivery to unsensitized Rh (-) women (Rh positive infant)women (Rh positive infant)

ACOGACOG:: 300 mcg at 28 wga 300 mcg at 28 wga UNLESS father known to be Rh (-)UNLESS father known to be Rh (-)

Repeat Antibody Screen ?Repeat Antibody Screen ?

Prevention (cont)Prevention (cont) Test for excessive fetal-maternal Test for excessive fetal-maternal

hemorrhage after blunt trauma, hemorrhage after blunt trauma, abruption, cordocentesis, and bleeding abruption, cordocentesis, and bleeding assoc. with previaassoc. with previa

Kleihauer Betke or rosette testKleihauer Betke or rosette test Give RhoGAM for partial molar Give RhoGAM for partial molar

pregnancy, SAB, TAB, ectopic, pregnancy, SAB, TAB, ectopic, chorionic villus sampling, external chorionic villus sampling, external versionversion

ConclusionsConclusions Remember the instances in which to consider RhoGAMRemember the instances in which to consider RhoGAM -SAB, TAB, threatened AB (controversial), ectopic, previa/bleeding, abruption, -SAB, TAB, threatened AB (controversial), ectopic, previa/bleeding, abruption,

partial molar, CVS, blunt trauma, cordocentesispartial molar, CVS, blunt trauma, cordocentesis Clinically important antibodies: Anti-c, Anti-D, Anti-E, and Anti-Clinically important antibodies: Anti-c, Anti-D, Anti-E, and Anti-

Kell, Rarely Anti-Duffy Fy(a)Kell, Rarely Anti-Duffy Fy(a) Usually not associated with severe HDFN:Usually not associated with severe HDFN: Anti-Lewis, ABO Anti-Lewis, ABO

incompatibilities, Anti-Duffy(Fy-b) antibodies, (Duffy Fy-a causes mild to 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)severe HDFN), Anti-A, Anti-P, Anti-M, Anti-I, Anti-IH, Anti-Sd(a)

Conclusions Conclusions (cont)(cont)

Anti-D still most common cause of red cell Anti-D still most common cause of red cell alloimmunization, despite RhoGAMalloimmunization, despite RhoGAM

Kell = most common Kell = most common minorminor antigen antigen *anemia mechanism 2-fold*anemia mechanism 2-fold

Critical titer most often used is 1:16 by Critical titer most often used is 1:16 by indirect coombsindirect coombs

Amnio with delta OD450 vs. MCA-PSVAmnio with delta OD450 vs. MCA-PSV Liley or Queenan graphsLiley or Queenan graphs Remember AB screens and indications for Remember AB screens and indications for

RhoGAM!!RhoGAM!!

ReferencesReferences1. Gabbe Obstetrics – Normal and Problem 1. Gabbe Obstetrics – Normal and Problem Pregnancies, 4Pregnancies, 4thth edition. edition.2. Creasy R., Resnik R., Iams J., Maternal Fetal 2. Creasy R., Resnik R., Iams J., Maternal Fetal Medicine Principles and Practice, 5Medicine Principles and Practice, 5 thth edition. edition.3. ACOG Compendium 20053. ACOG Compendium 20054. Harkness U., Spinnato J., 4. Harkness U., Spinnato J., Prevention and Prevention and Management of RhD isoimmunization. Management of RhD isoimmunization. Clinics in Clinics in

Perinatology, Dec 2004 31:4.Perinatology, Dec 2004 31:4.5. Pereira L., Jenkins T., 5. Pereira L., Jenkins T., Conventional management of Conventional management of

maternal red cell alloimmunization compared maternal red cell alloimmunization compared with management by Doppler assessment of with management by Doppler assessment of MCA-PSV. MCA-PSV. American Journal of Obstetrics and American Journal of Obstetrics and Gynecology, Oct 2003 189:4.Gynecology, Oct 2003 189:4.

References References (cont)(cont)

6. Cohen D., 6. Cohen D., Hemolytic disease of Hemolytic disease of the newborn: the newborn: RBC alloantibodies in pregnancy and RBC alloantibodies in pregnancy and

associated serologic issues. associated serologic issues. Up to Date, Oct Up to Date, Oct 2004.2004.

7. 7. Barss V., Moise K., Barss V., Moise K., Significance of minor red Significance of minor red blood cell antibodies during pregnancy. blood cell antibodies during pregnancy. Up Up to to Date, Apr 2005.Date, Apr 2005.