Lecture 11: Hemolytic Disease of Fetus and Newborn

Hemolytic disease of the newborn

• blood problem found in newborns

  • fast rate of red cell breakdown due to mother having clinically significant antibody

• mother forms IgG antibodies from previous transfusion or pregnancy

• IgG antibodies in mother circulation can cross placenta during pregnancy

  • Corresponding antigen present on baby RBCs

  • antibody in fetal circulation binds to antigens on baby RBCs

• baby must get antigen from father

  • mother has to lack the antigen to form antibody

Severity HDN varies

• antibody ID, titer

• greatest danger to fetus while still in utero in anemia

• greatest danger to newborn is build up of unconjugated bilirubin

Antibodies causing HDN

• most severe: D, K, c

• other: E, C, e, Fy^a, Fy^b, Jk^a, Jk^b, S

• mild HDN can be caused by ABO antibodies

  • most commonly IgG anti-A,B

Prenatal studies

• ABO grouping and Rh typing

• Antibody screen

  • antibodies to low incidence antigens typically not detected with antibody screens but can still cause HDN

Presence of antibodies doesn’t mean HDN will occur

• baby needs to have antigen to corresponding antibody

• phenotype of father for likelihood of babying being +

  • father -: 0%

  • father heterozygous: 50%

  • father homozygous: 100%

Antibody titer

• done for any clinically significant antibodies detected

• can help clinicians decide to do more invasive procedure

  • amniotic fluid analysis

  • intrauterine transfusions

• serum is serial diluted to determine titer of antibody

  • titer is highest dilution at which agglutination occurs

  • current titer result run in parallel with previous frozen sample

  • an increase in titer could indicate HDN

Amniocentesis

• can help in prediction of severity of fetal anemia

  • measures the level of bilirubin pigment in amniotic fluid

    • does spectrophotometrically

    • concentration of bilirubin correlates with degree of fetal anemia

Cordocentesis

• also known as percutaneous umbilical blood sampling

• fetal blood sample is withdrawn for testing

  • direct indicator for severity of HDN

  • HCT

  • phenotyping

Assessment of values obtained

• HCT and bilirubin values given an idea of how much RBC destruction is occurring in fetus

• clinical team determines if medical intervention is needed to reduce fetal anemia

  • intrauterine transfusion

Intrauterine transfusion

• used in cases of severe fetal anemia

• provides fetus with RBCs that will survive normally

  • blood is transfused through fetus’s abdomen or an umbilical cord vein

  • transfused cells must be compatible with maternal antibodies

Characteristics of blood used

• freshest unit available

  • usually no older than 5 days after collection 

• group O RBCs

• typically Rh - RBCs

• negative for antigens corresponding to maternal antibodies 

• CMV - 

• hemoglobin S negative

• irradiated

• crossmatch compatible at AHG with maternal serum

  • infant eluate or serum can be used if maternal sample unavail

Plasma reduced blood

• citrate phosphate dextrose (CPD) anticoagulant

  • no additive solutions

  • if unit had additive solutions, must be washed

• crossmatch compatible at AHG with maternal serum

Postpartum testing

• cord blood should be contained for newborns with:

  • Type O mothers

    • could have IgG anti-A,B

  • mothers with allo-antibodies

  • Rh negative mothers

Cord blood testing - ABO group

• only forward type can be performed

  • serum testing not performed

• newborns don’t develop antibodies until 4-6 months old

  • any ABO antibodies present in cord blood sample are from mother 

Cord blood testing - Rh type

• immediate spin test and weak D test performed on newborns

• especially important to do weak D for newborns of Rh negative mothers

  • used to assess the mother’s Rh immune globulin candidacy

Cord blood testing - DAT

• determines presence of antibodies on surface of RBCs in vivo

• antibodies present on newborn RBCs will have + result and are from mother

  • these RBCs will be destroyed

• strength of DAT doesn’t correlate to severity of HDN

• HDN due to ABO incompatibilities typically have weak DAT reactions

• stronger + DAT found in HDN due to anti-D, anti-K, anti-c and other non-ABO blood group antibodies

cord blood testing - Elution

• if DAT is +, elution can remove maternal antibodies bound to newborn RBCs

• a panel can be performed with eluate to determine specificity of antibodies

• only necessary if mother has multiple antibodies 

Mom: O +, - antibody screen/Newborn: A pos, weak positive DAT

• ABO HDN

  • can occur in any pregnancy 

  • most commonly seen in O mother and AB or B newborn

  • DAT is weak

    • anti A or anti B or anti A,B can be eluated 

Mom: O +, - antibody screen Newborn: O +, 2+ positive DAT

• HDN due to low incidence antibody

  • antigen to maternal antibody is not present on screening cells

    • example: Kp^a, Js^a

  • eluate will not react with A cells, B cells, or screening cells

  • eluate would react against newborn cells and paternal RBCs

Mom: O +, + antibody Newborn: A +, 3+ positive DAT

• HDN due to other blood group antibodies 

  • eluated antibody will react with screening cells

Wharton’s Jelly

• gelatinous substance within umbilical cord

• causes false positive reactions in cord blood testing

  • avoid by washing forward type several times before testing RBCs

Intrauterine transfusion

• following an intrauterine transfusion, recipient circulating blood could contain up to 90% of donor cells

  • cause weak or mixed field reactions

  • could type exactly as donor cells (O-)

  • will have to wait until donor cells are out of patient’s system to know true blood type 

Newborn’s cells heavily coated with IgG antibodies

• maternal antibodies cross placenta and bind a significant portion of newborn antigen binding sites

  • strong positive DAT

• RBCs tested with antisera

  • antisera has reduced or no place to bind

  • false negative

Suspect fetus in Rh + but types as Rh -

• maternal anti-D: coating the Rh + newborn cells

• reagent anti-D has no place to bind

• known as blocked D

Resolution of blocked D

• a gentle heat elution will remove the maternal anti-D

  • cord RBCs can now be re-tested with reagent anti-D 

  • Rh + binding sites are available for reagent anti-D to react and agglutinate 

Used in cases of severe HDN

• remove aliquots of neonatal blood and replaces it with donor blood in order to

  • reduce bilirubin levels

  • remove antibody coated RBCs that will be destroyed

  • lower concentration of maternal antibodies in newborn circulation

  • maintain adequate blood in circulation to deliver O₂ to tissues

Rh immune Globulin 

• common brand RhoGAM 

• RhD immune contains mostly IgG anti-D from pools of human plasma 

• used to prevent Rh - mothers from developing anti-D 

  • thought to block immune system from recognizing foreign D antigen (ie from baby)

• important in preventing HDN in future pregnancies due to anti-D

When is RhoGAM given

• Rh negative woman may come into contact with Rh positive RBCs

• When Rh - woman is pregnant:

  • must be given within 72 hours of bleeding event

    • abortion

    • miscarriage

    • amniocentesis

    • antepartum hemorrhage

  • Rh negative women at 28th week of gestation

  • must be given 72 hours of giving birth to Rh + newborn

    • immediate spin or weak D +

When mother received RhIG during pregnancy

• neonate may have + DAT

  • weak, doesn’t cause hemolysis

• RhIG can still be present and cause + antibody screen at time of delivery

  • can sty detectable at 3-4 months

What testing is performed prior to administration of RhIg

• ABORh typing

• Antibody screen and ID

ABORh type

• given to Rh - women

• given to women with partial D mutations

  • can develop anti-D to missing epitopes

Antibody screen and identification

• women who already developed anti-D don’t need Rh immune globulin

  • careful about presence of anti-D from dose at 28th week still + at delivery

  • don’t want to miss-ID anti-D RhIG as real anti-D

  • women with all other antibodies are a candidate 

Rh immune globulin given circumstances

• Rh - women pregnant and experiences:

  • abortion

  • miscarriage

  • amniocentesis

• Rh negative women at 28th week of gestation

• Rh negative women gives birth to Rh positive baby

• women with partial D mutations

Rh immune globulin not given circumstances

• Rh positive women

• Rh negative women with anti-D

• Rh negative women gives birth to Rh negative baby

Determining RhIG dose

• full dose of Rh immune globulin protects against 15 mL of Rh + packed RBCs

  • equates to 30 mL of whole blood

• antepartum administration

  • usually 1 dose given

• postpartum administration

  • # of doses given is determined by presence and volume of fetal blood cells in maternal circulation

Determining RhIG dose - fetal blood screen

• determines if there is any Rh + fetal blood in Rh - mother

  • Anti-D is added to post-partum maternal sample to coat any Rh positive fetal RBCs

  • enzyme treated Rh + RBCs are added

    • clumps will form around the anti-D coated, Rh positive fetal cells

  • - result: 1 dose of RhIG

  • + result: detects FMH> ~10mL; needs a quantitative test to determine dose

Kleihauer-Betke test used to quantify FMH

• maternal blood is treated with acid

  • fetal cells have higher HGB F and are more resistance to acid elution

  • fetal cells remain intact and are red

  • maternal cells lose hgb and appear as ghost cells

KB test calculation

• [% Fetal cells x 50mL] / 30mL

• KB test gives % of fetal cells

• calculation gives volume of fetal cells in maternal circulation

• Round up if >.5 Round down if <.5

• an extra vial is given for safety

  • if KB is 0.3%, volume of fetal cells in circulation is 15%mL, which divided by 30mL is 0.5. Rounded up to 1 and give an extra to make 2 vials of RhIg