Red cell alloimmunization, also known as Rh disease, occurs when a woman develops antibodies against the Rh factor in her baby's blood. This can cause hemolytic disease of the fetus and newborn if she has a subsequent Rh-positive pregnancy. Screening involves testing the mother's blood type and performing an antibody screen and Kleihauer-Betke test if needed. Diagnosis may involve ultrasound to check the fetus for signs of anemia like increased blood flow in the middle cerebral artery. Treatment depends on the severity and includes monitoring the pregnancy, intrauterine fetal transfusions if indicated, or early delivery. Noninvasive prenatal testing using cell-free fetal DNA in the mother's blood can also determine the baby's

1. Red Cell Alloimmunization
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2. Contents
Introduction
The Rhesus System
Prevalence
Pathogenesis
Screening Vs Diagnosis
Treatment & Management
– Unsensitized Mother
– First affected pregnancy
– Previously affected fetus/infant
Intrauterine fetal transfusion
Other Treatment Options
Special Issues
Hemolytic Disease due to non
RhD Antibodies

3. Introduction
• Rh (Rhesus) factor
– named after the monkeys in which it was first discovered
– is a RBC surface antigen
• Terminology
– red cell alloimmunization / sensitization (formerly - isoimmunization) : production of anti
RBC antibodies for foreign RBC antigens
• Hemolytic disease of fetus & newborn (HDFB)
– more appropriate to describe this disorder
– Also = erythroblastosis fetalis
• Because the peripheral blood smear of these infants demonstrated a large percentage of
circulating immature red cells known as erythroblasts
– mechanism of fetal anemia
• Sequestration & destruction of antibody-coated red cells in fetal liver and spleen
3

4. Historic Perspectives
• The first case of HDFN was probably
described by a midwife in 1609 in
the French literature
• Sir Albert William Liley
– a New Zealand medical practitioner
– committed suicide in 1983
– major contribution to the story of
rhesus disease was the introduction of
the fetal intraperitoneal transfusion (IPT)
4
12 March 1929 – 15 June 1983

5. The Rhesus System
• The International Society of BloodTransfusion currently recognizes
– 33 different blood group systems & 339 red cell antigens
• Any individual who lacks a specific red cell antigen may produce an antibody when exposed to that antigen
• Two important antigens are common as a cause of transfusion reaction
• OAB
– most important blood group system
– immediate spontaneous agglutinin response
• Rh system (6 Rh antigens : C,c,D,d,E,e)
– Second most important blood group system
– No immediate spontaneous agglutinin response from serum
– Two separate genes for the Rh system are found on short arm of chromosome 1
• RHD gene - encodes for D antigen
– Rh(D) status described - positive or negative [no d antigen]
– Rh Positive—95% (black American), 85% (whites), 100% (in Africans)
• RHCE gene, encodes for a combination of CE or ce antigens
– Severe fetal anemia requiring antenatal transfusion are due to anti-D, anti-Kell, anti-c, or anti-E
alloimmunization
5

6. • Which blood group is Universal Donor??
– Type O- Blood only
– Not Type O+ Blood
• Incidence: O – 47%;A – 41%; B – 9%;AB – 3%
• Du phenotype
– Du antigen is manifested by weak expression of the D gene on the RBC
envelope
– not considered candidates for Rh immunoprophylaxis
– Rh Du (+) is considered Rh (D) positive for transfusion
6
• Type A agglutinates
only in anti-A
• Type B agglutinates
only in anti-B
• Type AB agglutinates in
both
• Type O agglutinates in
neither of them
Determining B/G & Rh

7. • Alloimmunization is uncommon for the following reasons
– low prevalence of incompatible red cell antigens;
– insufficient transplacental passage of fetal antigens or maternal
antibodies;
– maternal-fetal ABO incompatibility, which leads to rapid clearance of
fetal erythrocytes before they elicit an immune response;
– variable antigenicity; and
– variable maternal immune response to the antigen
7

8. Prevalence
• Incidence of D positive
– varies according to racial and ethnic origin
– 85% of non-Hispanic white Americans
– 90% for Native Americans
– 93% for African Americans and Hispanic Americans
– 99% for Asians
• Frequency of "Rh-negative" individuals (Medscape)
– 6% of blacks; <1% of Asians
• With no apparent predisposing factors, fetal RBCs - detected in maternal blood in (ACOG 2018)
– 7% of women during 1st trimester,
– 16% during 2nd trimester, and
– 29% during 3rd trimester
• Prevalence of D alloimmunization complicating pregnancy: 0.5 to 0.9%
– In population-based screening studies: prevalence of red cell alloimmunization in pregnancy ~ 1 percent
8

9. Incidence of Rh incompatibility (ACOG 2018)
• varies by race and ethnicity
• Rh negative
– 15% of whites
• Rh-negative woman has an approximate 85% chance of mating with an Rh-
positive man,
– 60% of whom are heterozygous and
– 40% of whom are homozygous at the D locus
– 5–8% of African Americans
– 1–2% of Asians and Native Americans
9

10. D sensitization
• Without Anti D + ABO-compatible newborn
– W 25th: D alloimmunization risk = 16%
• 2% will become sensitized by the time of delivery,
• 7% by 6 months postpartum, and
• 7% will be “sensibilized”—producing detectable antibodies only in a subsequent
pregnancy
– G 7th: 13%
• Without Anti D + ABO-incompatible newborn
– D alloimmunization risk = 2% due to erythrocyte destruction of ABO-
incompatible cells, which thereby limits sensitizing opportunities
• Reduction with prophylactic Anti D
– Antepartum (from 16% to 2%) and post partum (from 2% to 0.1%)
10

11. Pathogenesis
• Rh incompatibility can occur by 2 main mechanisms
1. Exposure to Rh-positive fetal RBCs
– The most common type
– FMH: abortion (spontaneous/induced), trauma, invasive obstetric
procedures, or normal delivery
– Grandmother Effect /Theory
• D negative female fetus – sensitized by her Rh+ve blood ➔ produce
anti-D antibodies even before or early in her first pregnancy ➔ fetus in
the 1st pregnancy is jeopardized
• As many as ¼ of RhD-negative babies are immunized in early life (G 7th)
2. Rh-positive blood transfusion
– blood banks prefer using blood type "O negative" as universal
donor in emergency situations
• Once sensitization has occurred, it is irreversible
– This sensitization may produce immunological memory in the
mother for future pregnancies
11

12. • Amount of fetal blood necessary to produce Rh incompatibility
– as little as 0.1 mL of fetal erythrocytes (ACOG, 2018)
• Risk of sensitization depends on
– Volume of transplacental hemorrhage
– Extent of the maternal immune response
– Concurrent presence of ABO incompatibility
• Fetal sex may also play a significant role in the fetal response to maternal
antibodies
– RhD-positive male fetuses (G 7th)
• 13 times more likely than their female counterparts to become hydropic
• 3 times more likely to die of their disease.
• In most cases of red cell alloimmunization, a FMH occurs in the antenatal
period or, more commonly, at the time of delivery
12

13. Maternal antibodies
• After a sensitizing event, the human antiglobulin anti-D titer can usually be detected after 5
to 16 weeks
– Once sensitized, it takes ~ 4 weks for Rh antibodies in the maternal circulation to equilibrate in
the fetal circulation
– approximately 50% of alloimmunized patients are sensibilized
• In this scenario, an antibody screen will be negative,
• but memory B lymphocytes are present that can create an anti-D antibody response
– With subsequent pregnancy involving an RhD-positive fetus, the antiD titer becomes detectable
• In 90% of cases, sensitization occurs during delivery
(initial response mostly) can cross placenta
– Mostly IgG1
– a non agglutinating antibody that does not bind complement
• So no intravascular hemolysis;
• Rather sequestration and subsequent destruction of antibody-coated red cells in the fetal liver and spleen →
fetal anemia
– hemolytic anemia → hyperbilirubinemia → ultimately hydrops fetalis
– can’t cross placenta
13

14. • Associated physiologic changes (G 7th)
– Reticulocytosis from the bone marrow (when Hb ↓ by > 2 g/dL)
– Erythroblasts are released from the fetal liver (when Hb ↓ by > 7
g/dL)
– 2-3 diphosphatidylglycerol (DPG) levels ↑
• In effort to increase oxygen delivery to peripheral tissues
– Increased umbilical artery lactate level (when Hb↓ < 8 g/dL)
– Increased venous lactate (when Hb 4 g/dL)
– Hydrops fetalis - in at least two body compartments (late finding)
14

15. • Immune response to D antigen - three groups (G 7th)
: 60% to 70%
• develop an antibody to relatively small volumes of red cells
• the probability of immunization increases with escalating volumes of cells
• A small percentage of responders can be called hyperresponders in that they will be
immunized by very small quantities of red cells
: 10% to 20%
• immunized only by exposure to very large volumes of cells
: 10% to 20%
• Totally nonresponders
• Sensitization to C, c, E, and e antigens
– have lower immunogenicity than the D antigen
– complicates ~ 0.3% of pregnancies in screening studies and accounts for about
30% of red cell alloimmunization cases
– Anti-E alloimmunization
• most common, but the need for fetal or neonatal transfusions is greater with anti-c
alloimmunization than with anti-E or anti-C
15

16. Alloimmunization to Minor Antigens
• Kell antigens - the most frequent
– 90% of non-Hispanic white Americans and up to 98% of African Americans are Kell
negative
– nearly 90% result from transfusion with Kell-positive blood (so transfusion history is
important)
– may develop more rapidly and may be more severe than with sensitization to D
– mechanism of fetal anemia - differs somewhat from that in Rh-D
• anti-K antibodies cause suppression of fetal erythropoiesis rather than due to hemolysis
– In one study, a critical anti-K titer of 1:32 support as a predictor of disease severity
• Most cases of sensitization to minor antigens result from incompatible blood
transfusions
• few blood group antigens pose no fetal risk (does not cause fetal hemolysis)
– Lewis antibodies - Lea and Leb, as well as I antibodies, are cold agglutinins
• They are predominantly IgM and are not expressed on fetal red cells
– Duffy group B - Fyb
16

17. Determining Fetal Risk
• Up to 40% of D-negative pregnant women carry a D-negative fetus
• presence of anti-D antibodies reflects maternal sensitization
– but does not indicate whether the fetus is D positive
– If a prior sensitization → higher titer despite fetal D status
• Chorionic villus sampling (CVS) is not recommended
– Due to greater risk for FMH & subsequent worsening of alloimmunization
• Fetal testing for other antigens
– E/e, C/c, Duffy, Kell, Kidd, and M/N - is also available
Fetal BloodTyping
• Ultrasound-directed cordocentesis
– disruption of the chorion villi during the procedure can result in FMH and a rise in maternal titer,
thereby worsening the fetal disease
• Amniocentesis for DNA testing – Invasive
• ccffDNA: Circulating cell-free fetal DNA
17

18. Rh positive father
• Homozygous father (45%)
– all of his children will be
Rh positive
• Heterozygous father
(55%)
– Children: 50% chance of
being Rh positive
• By way of contrast the
Rh-negative individual is
always homozygous
18
Circulating free DNA (cfDNA)

19. 19

20. ccffDNA: Circulating cell-free fetal DNA
• Early studies in pregnant women carrying a male fetus indicated that
– 3% of ccffDNA in the maternal circulation in the first trimester is fetal in origin;
– this increases to 6% by the third trimester
• Source of this DNA appears to be
• Noninvasive assessment: sample from maternal plasma after 10 weeks for PCR
• can be performed with reliable results at as early as 10 weeks’ gestation
• Sensitivity > 99, specificity > 95%, and P/N PPV - very high
• Indications
– ACOG: does not recommend routinely (Since it is costly)
– To identify fetuses that are D Negative and do not require anemia surveillance, and
– To withheld anti-D immune globulin if the fetus is D negative
• Fetal DNA is rapidly cleared from the maternal circulation with a mean half-life
of 16 minutes after cesarean delivery; after vaginal delivery,
– fetal free DNA is cleared by 100 hours
20

21. Fetomaternal Hemorrhage
• a large blood loss & true fetomaternal hemorrhage is rare
– In one series of > 30,000 pregnancies,
• FMH ≥150 mL occurred in 1 per 2800 births
• Prevalence of FMH of
– at least 30 mL of fetal blood (which can be covered by a standard
300-μg dose of anti-D immune globulin)
• is estimated to occur - 3 per 1000 pregnancies
– in > 80 percent of cases, no cause is identified
21

22. ABO blood group incompatibility
• Incompatibility for antigens A and B - most common cause
of hemolytic disease in newborns,
– but it does not cause appreciable hemolysis in the fetus
• ~ 20% of newborns have ABO blood group incompatibility
– yet only 5 percent are affected clinically (mild anemia)
• often seen in firstborn neonates
• Rarely becomes more severe in successive pregnancies
• considered a pediatric disease - rarely of obstetrical concern
– because most anti-A and anti-B antibodies are IgM and do not
cross the placenta.
– Fetal red cells also have fewer A and B antigenic sites than adult
cells and are thus less immunogenic
• No need fetal surveillance and early delivery
• neonatal observation for hyperbilirubinemia - ?
phototherapy
CDE incompatibility
• Seen in successive
newborns
• More severe in successive
pregnancies
• obstetrical concern
• fetal surveillance and early
delivery is mandatory if
mother is sensitized
22
Differences in ABO & CDE incompatibility

23. Screening Vs Diagnosis
• Screening
– Blood type: ± D antigen?
– Antibody screen:
• Coombs test (  maternal blood &  fetal/neonatal blood)
– Rosette Test (Fetal RBC Screen [Qualitative])
– Kleihauer-Betke test
• Diagnostic
– Sonography:
• MCA peak systolic velocity (most accurate predictor)
• Hydrops (ominous finding)
– CTG: sinusoidal FHR pattern
23

24. Coombs test
Indirect
• For prenatal detection of Rh Antibodies
• Take maternal blood → serum → then
• In a test tube:
– Maternal serum + Animal/human Rh+ve RBCs +
coombs reagent (antihuman globulin) → if
– Agglutination: mother has produced antibodies against
fetal Rh +ve RBCs
• -ve (No Agglutination): Give Anti D
• +ve (Agglutination): No need to give Anti D
– determine antibody titers
– critical titer:The level at which significant fetal anemia
could potentially develop
• Usually ranges between 1:8 and 1:32
• If the critical titer for anti-D antibodies is 1:16, a titer ≥1:16
indicates the possibility of severe hemolytic disease
• Exception → Kell sensitization
• Kell sensitization
– since there is less hemolysis, and severe
anemia may not be predicted by the
maternal Kell antibody titer
• Ancillary tests that should follow titer
– Paternal Genotype
– Fetal Genotype:Amniocentesis is the
primary modality (PCR)
• 2 mL of amniotic fluid.
• sensitivity and specificity - 98.7% and 100
• Positive and negative predictive values of 100%
and 96.9%
• in 2nd trimester with > 99% accuracy
Direct
• To detect anemia 20 to Hemolytic
disease of the newborn
• Take fetal blood → serum → then
• In a test tube mix this serum with
coombs reagent → if
– Agglutination: neonate’s blood has Abs from
mother destroying its blood
24

25. The RosetteTest
• A Qualitative (not quantitative) test
• the initial test of choice, is highly sensitive in qualitatively
detecting 10 mL of fetal whole blood in the maternal
circulation
• To identify quantity of fetal RBC in maternal circulation
Technique
– Take maternal blood → add anti D (binds to D positive
RBCs, but doesn’t agglutinate them) → add indicator RBC
bearing D-antigen to form rosette ➔ count number of
rosette ➔
• Thus, if rosettes are visualized, there are fetal D-
positive cells in that sample
• Results interpretation
– Negative: result warrants administration of a standard
300 µg dose of RhIG
– Positive →Kleihauer-Betke acid-elution test - for
quantifying FMH
25

26. Quantitative Tests
Kleihauer-Betke acid-elution test
• the most widely used confirmatory test for quantifying FMH
• inexpensive and requires no special equipment
– it lacks standardization and precision, and may not be accurate in conditions with elevated F-cells
• Principle: fetal hemoglobin (HbF) is resistant to acid-elution whereas adult hemoglobin is acid-
sensitive
• Technique
– smears are prepared from maternal capillary / venous blood → Ehrlich's acid hematoxylin → examined
under light microscopy
– fetal erythrocytes appear red and adult erythrocytes appear as “ghosts”
– fetal cells are then counted and expressed as a percentage of adult cells
FMH (ml) = KB % X Estimated maternal blood volume (usually 5000 ml)
RhIG vial = (Calculated FMH (ml) ÷ 30 ml)
• used for calculating qty o FMH & determining dose o anti D
• two scenarios in which it may not be accurate:
– Maternal hemoglobinopathies such as β- thalassemia in which the fetal hemoglobin level is elevated and
– pregnancies at or near term, when the fetus has already started to produce hemoglobin A
Anti-HbF flow cytometry
• is a promising alternative, although its use is limited by equipment and staffing costs
26

27. MCA Doppler Velocimetry (PSV)
• for detection of fetal anemia
• anemic fetus shunts blood preferentially to the brain to maintain adequate oxygenation
– So velocity rises because of increased cardiac output and decreased blood viscosity
• MCA peak systolic velocity
– ≤ 1.5 MoM ➔ begin antenatal testing at 32 weeks
• A value > 1.5 MoM for the corresponding GA predicts moderate to severe fetal anemia with a sensitivity of
88% and a negative predictive rate of 89%
• Deliver at 37 – 38 weeks
– > 1.5 MoM ➔ check fetal Hct by fetal blood sampling
• sensitivity of 100% and a false-positive rate of 12% (ACOG 2018)
• < 30%
 < 35 wk → IUT and for ≥ 35 wk → Deliver
• ≥ 30% → begin antenatal testing at 32 weeks → Deliver at 37 – 38 weeks
• false positive rate of MCA PSV increases significantly beyond 34 - 35 weeks (ACOG 2018)
– due to the normal augmentation in cardiac output that develops at this gestational age
27

28. 28
• Circle of Willis begins to form when the right & left internal carotid artery enters the cranial cavity and each
one divides into two main branches: the anterior cerebral artery (ACA) and middle cerebral artery (MCA)
• Importance: to provide collateral blood flow bn anterior & posterior circulations of the brain, protecting
against ischemia in the event of vessel disease or damage in one or more areas
Anterior circulation: From the bilateral internal carotid arteries
• supplies most of the cerebral hemispheres (frontal lobes, parietal lobes,
lateral temporal lobes and anterior part of deep cerebral hemispheres)
Posterior circulation: from bilateral vertebral arteries
• Supplies brainstem, cerebellum, occipital lobes, medial temporal lobes
and posterior part of the deep hemisphere, mainly the thalamus

29. • The Doppler gate is then placed in the
proximal MCA where the vessel arises from
the carotid siphon
– Measurements in the more distal aspect of
the vessel will be inaccurate because reduced
peak velocities will be obtained
• fetus should be in a quiescent state
– because accelerations of FHR can result in a
decrease in the PSV, especially late in 3rd
trimester
• administration of antenatal steroids
– decreases in the peak MCA velocity
– effect usually lasts for 24 to 48 hours after
the last dose
• MCA measurements can be obtained reliably
as early as 18 weeks’ gestation
• Values should be converted to MoM using
Internet based calculators (e.g.,
www.perinatology.com)
29
Correct determination of the fetal peak
middle cerebral artery Doppler velocity
Techniques
– First locate anterior wing of sphenoid bone at the
base of the skull → using Color or power Doppler,
locate MCA
– Angle of insonation is maintained as close to zero

30. Amnionic Fluid Spectral Analysis
• This test is included for historical interest –
demonstrated by Liley (1961)
• Amnionic fluid bilirubin concentration
– indirect indication of the degree of fetal hemolysis
– measured by a spectrophotometer (represented as
change in optical density absorbance at 450 nm =
ΔOD450)
• likelihood of fetal anemia – 3 zone (by plotting the
ΔOD450 value)
– zones roughly correlated with fetal hemoglobin
concentration (anemia severity)
– The original Liley graph
• valid from 27 to 42 weeks’ gestation
– Modified by Queenan (1993)
• include gestational ages as early as 14 weeks
• NB: amnionic fluid bilirubin level is normally high in mid pregnancy,
limiting the reliability of this technique
• Currently replace with MCA PSV
– Due to its less accuracy & invasiveness
30
• Liley Curve
– Zone I – fetus very low risk of severe fetal anemia
– Zone II – mild to moderate fetal hemolysis
– Zone III – severe fetal anemia with high
probability of fetal death 7-10 days
• Liley good after 27 weeks
• 98% sensitive for detecting anemia in upper zone
2/ zone 3

31. Percutaneous umbilical blood sampling (PUBS)
• also known as
– cordocentesis or funipuncture
• associated with a > 4-fold ↑ in perinatal loss
compared with amniocentesis
• fetal blood type, hematocrit, direct Coombs
test, reticulocyte count, and total bilirubin
• Although serial PUBS was once proposed as
a primary method of fetal surveillance after
a maternal critical titer is reached, it has
been associated with
– a 1% to 2% rate of fetal loss and
– up to a 50% risk for FMH with subsequent
worsening of the alloimmunization
• For these reasons, FBS is reserved for
patients with elevated peak systolic MCA
Doppler velocities
31

32. Hydrops fetalis
• is defined as the presence of extracellular fluid in at least
two fetal compartments
• When hydrops is present, fetal hemoglobin deficits of 7 to 10
g/dL from the mean hemoglobin value for the corresponding
gestational age can be expected
• the early second-trimester fetus can be severely anemic
without signs of hydrops
32

33. Treatment & Management
• Issue to consider
– Status of sensitization
– GA
– Severity
33

34. Unsensitized Mother
• Criteria to provide prophylaxis
– Rh-ve Mother
– no evidence of anti-D alloimmunization : Indirect – Negative;
RosetteTest - Negative
• Omit prophylaxis & screening at 28 weeks
– if biologic father: certainly D-negative
– if cell-free DNA suggest D-negative fetus
• Mini dose vial (50 µg) - Covers fetal 5 ml of fetal blood (2.5 ml
of fetal RBC
34

35. 1st Dose: at approximately 28 weeks’ gestation: 300 µg
• 1 full dose = 300 µg (1500 IU): Covers fetal 30 ml of fetal blood (15 ml of
fetal RBC, since Hct is 50%)
• Reduces risk from 16% (ABO compatible newborn) to 2% or from 2%
(ABO incompatible newborn) to 0.1%
• If both antepartum & postpartum: risk of alloimmunization is reduced to
0.1% [Current 12th]
• repeat antibody screening is recommended to identify individuals who have
become alloimmunized (AAP, 2017).
• Duration: Suppression of Rh isoimmunization: ~ 12 weeks
• If > 12 weeks have elapsed since anti-D immunoglobulin administration,
consideration should be given to administering 300 µg of anti-D
immunoglobulin at 40 weeks of gestation
35

36. 2nd Dose: after delivery if the newborn is D-positive
• given within 72 hours (some protection up to 28 days postpartum )
• If delivery occurs < 3 weeks from the administration of RhIG used
for antenatal indications such as external cephalic version, a repeat
dose is unnecessary unless a large FMH is detected at the time of
delivery
• Failed prophylaxis after the appropriate dose of RhIG is rare
• anti-D antibody screen may remain positive for up to 6 months.
– may produce a weekly positive 1:1 to 1:4 indirect Coombs titer in the
mother
– Persistence after 6 months is likely to be the result of sensitization
36

37. Anti D Immunoglobulin (Pregnancy risk factor: C)
• used to prevent hemolytic disease of the fetus and newborn
• Anti-D is a polyclonal IgG product purified from the plasma of D alloimmunized individuals
• Half life of RhIG is approximately 16 days
• Mechanism of action - Unproven
– Possibilities
• rapid macrophage-mediated clearance of anti-D-coated red cells and/or
• down-regulation of antigen-specific B cells before an immune response occurs
• Its MoA in Rx of ITP
– Not completely characterized
– Rho(D) immune globulin is thought to form anti-D-coated red blood cell complexes which
bind to macrophage Fc receptors within the spleen → blocking or saturating the spleens
ability to clear antibody-coated cells, including platelets
– In this manner, platelets are spared from destruction
• Risks
– Infection: hepatitis C virus, human immunodeficiency viruses, hepatitis B virus, and parvovirus B19
– Allergic reaction
– Serious adverse reactions are rare
37

38. Formulations prepared by
• Cold ethanol fractionation and ultrafiltration
– must be administered IM
– because they contain plasma proteins that could result in anaphylaxis if given
intravenously
– Two products: RhoGAM & HyperRho
• Using ion exchange chromatography
– may be administered either IV or IM
– This is important for treatment of significant fetomaternal hemorrhage
– two products:WinRho-SDF & Rhophlac
• Both preparation methods effectively remove viral particles, including
hepatitis and human immunodeficiency viruses
38

39. 39
Special Fetomaternal Risk States
Condition & anti D dose Risk of sensitization Remark
Abortion
o 1st trimester = 50 µg
o 300-µg (usually)
• 2% - spontaneous
• 4-5% - induced
o A dose of 50 µg of RhIG is effective until 13 wk due to small volume of
RBCs in the fetoplacental circulation
Amniocentesis, chorionic villus
sampling: 300 µg
• 11% o procedures are performed in the unsensitized patient
Antepartum bleeding: 300 µg
o If there is evidence of a subchorionic hematoma or placenta abruption
o If the pregnancy is carried > 12 weeks from the time of RhlgG
administration, a repeat prophylactic dose is recommended
External cephalic version: 300 µg o Fetomaternal hemorrhage occurs in 2 - 6% of patients
Delivery With extensive FMH
o Quantify dose based on KB
test estimation
o occurs in ~ 0.4% of patients
o in 2 – 3 per 1000 pregnancies, the volume of FMH exceeds 30 mL of whole
blood (ACOG, 2017)
o No > 5 vial RhIG should be administered by the intramuscular route in one
24-hour period
o Doses of up to 600 µg (3000 IU) can be given 8 hourly till total dose
has been achieved
o The rule of thumb should be to administer RhIG when in doubt, rather
than to withhold it

40. Classification of Indications for RhIG
A = high
 Spontaneous miscarriage
 Elective abortion
 Ectopic pregnancy
 Genetic amniocentesis
 Chorion villus biopsy
 Fetal blood sampling (FBS)
 At 28 weeks’ gestation unless father of
fetus is RhD negative
 Amniocentesis for fetal lung maturity
 Within 72 hours of delivery of an RhD
positive infant
B = moderate
 Hydatidiform mole
C = low
 Threatened miscarriage
 Placenta previa with bleeding
 Suspected abruption
 Intrauterine fetal demise
 Blunt trauma to the abdomen
 External cephalic version
 After administration of RhD-positive
blood component
40
LEVEL OF EVIDENCE: A, B, C

41. • Volume of RhoGAM injected (ml)
• (ml of whole blood transfused) (HCT of donor blood)
• Volume (ml) of packed RBCs each ml of RhoGAM will
“neutralize”, based on the “rule of thumb” ratio of 20 µg of
anti-Rho(D)/ml of Rh-positive red blood cells
41

42. First affected pregnancy
• ~ 25 to 30% of fetuses from D-
alloimmunized pregnancies will have
mild-to-moderate hemolytic anemia
– And without treatment, up to 25 percent
will develop hydrops
• Management of 1st alloimmunized
pregnancy is individualized and may
consist of
 Determine antibody titers at 20 weeks
– titer < critical value → titer is repeated
every 4 weeks till 24th wk → every 2 wk
– titer ≥ critical value → no benefit to
repeating it ➔ US (hydrops), MCA PSV & ?
IUT
• Doppler @ circle ofWillis & blood flow in
the proximal third of MCA
• PSV > 1.5 multiples of the median - severe
fetal anemia
 MCA peak systolic velocity: 1.5 MoM
– MCA-PSVs <1.5 MoMs → induce labor at
37 to 38 weeks of gestation
– MCA-PSV ≥1.5 MoM → severe anemia →
management depends on gestational age
• < 35 weeks: fetal blood sampling → if fetal
hematocrit <30 percent (< 2 SD) IUT
• 35 to 37 weeks: amniocentesis to assess fetal
lung maturity →
– If matured induce labor (CD for obstetrical
indication)
– If immature & reassuring BPP → continue to
monitor fetal well-being and delay delivery
until fetal lung maturity is likely
 Fetal blood sampling (Fetal HCT)
 Gestational age
 Amnionic fluid bilirubin (Currently not
used)
42

43. 43

44. • No need of serial fetal surveillance
– In cases of a heterozygous paternal phenotype or questionable paternity →
ccffDNA testing should be sent to a DNA → RhD negative paternal blood or
fetal RHD negative genotype
• serial fetal surveillance indicated
– If - RHD positive fetus, homozygous paternal phenotype or RHD positive fetus
by DNA testing
• When is the best time to deliver the infant of an alloimmunized patient?
– Mild fetal hemolysis
• induction of labor at 37–38 weeks of gestation
– Severely sensitized pregnancies requiring multiple invasive procedures
• Weigh risk of continued cord blood sampling and transfusionsVs neonatal risks associated
with early delivery
• delivery at 32–34 weeks of gestation after maternal steroid administration to enhance fetal
pulmonary maturity
44

45. Previously affected fetus/infant
• The management of the second (or more) affected pregnancy is similar,
except maternal antibody titers are not helpful in following the degree of
fetal anemia
– Because maternal titers are not predictive of the degree of fetal anemia
– after a first affected pregnancy, future pregnancies tend to manifest with more
severe disease and at an earlier gestational age
• No need of antibody titers
– the pregnancy is assumed to be at risk regardless of titer
– because fetal surveillance is indicated by the history of prior affected fetus
• a prior pregnancy in which fetal transfusions were required,
• a prior stillbirth related to alloirnmunization, or
• a prior neonate who required exchange transfusion after delivery
45

46. • measure MCA-PSV weekly, beginning at 16 to 18 weeks gestation
– Serial every 1 to 2 weeks
• In the case of a heterozygous paternal phenotype or questionable paternity,
ccffDNA analysis to determine the fetal RHD status is indicated.
• Amniocentesis
– to determine fetal genotype when father is heterozygous for D ➔
• Fetus with D antigen - at risk of hemolytic disease and severe anemia regardless of maternal
antibody titers
– MCA PSV initiated at 18 weeks → repeated every 1-2 weeks
• Fetus with no D antigen: No risk of hemolytic disease
– Amniocentesis can be used after 15 weeks’ gestation to determine the status of
the fetal red cell antigen in cases of other maternal antibodies such as anti-Kell
46

47. • Ultrasound parameters that could predict the early onset of anemia
– enlargement of fetal liver and spleen
– Since these are sites of extramedullary hematopoiesis and the destruction
and sequestration of sensitized red cells in cases of severe HDFN
• Both splenic perimeter and hepatic length correlate with the degree of fetal anemia
– increased cardiac output
• in an effort to enhance oxygen delivery to peripheral tissues
– Lower blood viscosity
• ➔ produces fewer shearing forces in blood vessels increased blood velocities
47

48. • Results of MCA Dopplers will place the fetus into 1 of 3 categories:
– normal MCA Doppler
– Repeat doppler study every 2 weeks
– Delivery - at term or near term (after fetus has achieved pulmonary maturity.
– MCA PSV nearing 1.5 MoM
– Repeat every 1-2 weeks
– Delivery - may be before term & as soon as pulmonary maturity is reached.
– Sometimes enhancement of pulmonary maturity by use of corticosteroids may be
necessary.
– MCA PSV > 1.55 MoM or frank evidence of hydrops {ascites, pleural or pericardia!
effusion, subcutaneous edema)
– Delivery – when fetus reach a GA at which delivery and neonatal risks are fewer than
the risks of in utero therapy
48

49. • If the fetus is preterm
– Determine fetal HCT
• cordocentesis or percutaneous umbilical cord blood sampling (PUBS)
• Intrauterine transfusions (IUT)
– between 18 and 35 weeks
– Before 18 weeks, access to the umbilical vein is limited due to the small caliber of the vessel
– After 35 weeks, the risk/benefit ratio favors delivery of a fetus with evidence of severe anemia
– After transfusion,
• repeat transfusions or delivery usually will be necessary, as production of fetal blood
markedly decreases or ceases
• Timing of these transfusions may be assisted by MCA Doppler studies
• Delivery should take place when the fetus has documented pulmonary maturity
49

50. Intrauterine fetal transfusion
• Indication - fetal anemia
– Peak MCA velocity ≥1.5 MoM and Fetal hematocrit ≤30%
• Begin antenatal testing at 32 weeks
• Deliver by 37-38 weeks
• performed prior to 34 to 35 weeks
• women who have undergone several fetal transfusions are given
oral phenobarbital for 7 days prior to delivery to enhance fetal
hepatic maturity
– Phenobarbital: enhance the capability of the neonatal liver to conjugate
and eliminate bilirubin
– Oral phenobarbital (30 mg TID) for 10 days
• decrease the need for neonatal exchange transfusions for hyperbilirubinemia by 75%
50

51. • Target hematocrit
– In a nonhydropic fetus - 40 to 50%
• Severely anemic fetuses
– in the early second trimester do not tolerate the acute correction of their hematocrit
to normal values
– less blood is transfused initially, and another transfusion may be planned for
approximately 2 days later.
• initial hematocrit should not be increased by > 4X at the time of the first procedure
• A repeat IUT is then performed within 48 hours to correct the fetal hematocrit into the normal
range
• Before transfusion, a paralytic agent may be given to the fetus
– Vecuronium - 0.01 mg/kg administered into the umbilical vein
– Fentanyl (2 to 3 µg/kg EFW) can also be used and can be mixed with the vecuronium
– Paralysis is almost immediate and lasts 2 to 3 hours
• Subsequent transfusions usually take place every 2 to 4 weeks, depending on the
hematocrit
51

52. • Route
– Intravascular transfusion
• into the umbilical vein under sonographic guidance
– Puncture UV in the portion of umbilical cord near its insertion into the placenta
• preferred method of fetal transfusion
– Intraperitoneal transfusion
• may be necessary with severe, early-onset hemolytic disease in the early second trimester, a
time when the umbilical vein is too narrow to readily permit needle entry
• With hydrops, although peritoneal absorption is impaired, some prefer to transfuse into both
the fetal peritoneal cavity and the umbilical vein
• In the case of
– Anterior placenta
• needle is passed through the placental mass into the cord root
– Posterior placentation
• the cord insertion into the placenta is preferred because this represents a site of immobility
compared with a “floating” loop of cord
– A sample of fetal blood is obtained for an initial hematocrit
52

53. • Red cells transfused are
o Type O
o D negative
o Cytomegalovirus-negative
o Washed and packed to a final hematocrit of approximately 75% to 80% to prevent
volume overload in the fetus
o Irradiated to prevent fetal graft-versus-host reaction
o Leukocyte poor
• Leukoreduced PRBCs should be used in all neonatal transfusions because it reduces febrile non-
hemolytic transfusion reactions, prevents alloimmunization, and reduces the transmission of certain
infections (notably CMV) (Grade 2C)
• irradiated RBCs - prevent transfusion-associated-graft-versus-host disease
• The patient is admitted to the labor and delivery unit as an
• skin is prepped with hexachlorophene, and sterile drapes are applied
• A 20 gauge procedure needle (a 22-gauge needle is used for gestations <22
weeks) is introduced into the amniotic cavity and then into the umbilical vein
under continuous ultrasound guidance
53

54. Methods to EstimateVolume
transfused
• Method 1
– (Transfusion coefficient) X EFW
• Method 2
– The fetoplacental volume (mL) is
calculated from the ultrasound estimate of
the fetal weight according to the formula
= (1.046 + EFW in grams x 0.14)
54
Target - beginning HCT =
Desired increment in HCT
Transfusion
coefficient
10 0.02
15 0.03
20 0.04
25 0.05
30 0.06
Transfusion coefficient for
calculating transfusion volume
for fetal transfusion

55. • MCA peak systolic velocity threshold for severe anemia
– is higher following an initial transfusion
• 1.70 MoM rather than 1.50 MoM
• It has been shown to be useful in timing the second IUT
• After the second procedure, the MCA Doppler loses its validity in predicting fetal anemia
– Why?
• It is hypothesized that the change in threshold compensates for the contribution of donor cells in
the initial transfusion, because donor cells (from adults) have a smaller mean corpuscular volume
• Alternately, the timing of subsequent transfusions is based on anemia severity
and posttransfusion hematocrit
• Following transfusion, the fetal hematocrit generally drops by approximately 1%
per day
– A more rapid initial decline may be encountered in the setting of fetal hydrops
55

56. Outcomes
• Complications from IUT are uncommon
• overall survival rates exceeding 95 percent
– G 7th: overall survival rate of 91%
• Procedure-related complications
– Fetal death: 2 percent,
• stillbirth rate exceeds 15% if transfusion is required before 20 weeks
• Considering that fetal transfusion is potentially lifesaving in severely compromised fetuses, these risks should
not dissuade therapy.
– need for emergent cesarean delivery in 1 percent, and
– infection and preterm rupture of membranes in 0.3 percent
• Survival rate approached 75 to 80 percent
– in nearly two thirds with resolution of hydrops following transfusion, more than 95 percent
survived
– survival rate was <40 percent if hydrops persisted
• Long-term outcomes
– more than half - required exchange transfusion in the neonatal period
– Few (4.8%) - severe impairments: developmental delay, cerebral palsy & deafness
56

57. NeonatalTransfusions
• prolonging the gestation of the treated fetus with HDFN until near term has
resulted in a virtual absence of the need for neonatal exchange transfusions
• if cord blood at delivery is submitted for neonatal red cell typing
– O-ve reflecting antigen status of the donor blood used for the IUTs
• Top up transfusions (simple transfusions)
– Most blood transfusions involve adding blood or blood products without removing any
blood
– Due to:
• Elevated levels of circulating maternal antibodies in the neonatal circulation and
• Conjunction with suppression of the fetal bone marrow production of red cells
– this occurs in approximately 50% of infants near 1 month of age
• Supplemental iron therapy in these infants is unnecessary
– because there is excess levels of stored iron due to previous hemolysis in utero and
lysis of red cells from the IUTs
– Supplemental folate therapy (0.5 mg/day) should be considered
57

58. Other Treatment Options
• Maternal plasmapheresis (Gabbe 7th )
– Perinatal survival rate of 69%
– Plasmapheresis is started at 12 weeks’ gestation and repeated three times in that week.
– The maternal titer should be expected to be reduced by 50%.
• Intravenous immune globulin (IVIG) (Gabbe 7th )
– has also been used effectively as the sole antenatal treatment for HDFN
– given to replace the globulin fraction removed by plasmapheresis in the form of a 2 g/kg loading dose after
the 3rd plasmapheresis;
• this is followed by 1 g/kg/week of IVIG until 20 weeks’ gestation
FutureTherapeutic Options
• Patients with
– High anti red cell titers and recurrent perinatal loss in the second trimester have few options
• Artificial insemination with red cell antigen – negative donor semen
• Surrogate pregnancy, or
• Preimplantation diagnosis (if the father is heterozygous)
• Proteasome inhibitors
58

59. Special Issues
59
Special Issues
Condition Remark
Positive titer at delivery
• After dose at 28th week, low titer at term (≤4) may exist, still give anti D if infant is D-positive
• But, if IgM antibody is present or IgG titer >1:4 - alloimmunization is proven
D positive mother at
delivery
• Large FMH ? → KB test / flow cytometry and to determine the appropriate dose of anti-D
immunoglobulin
• See prior D antigen typing
Inadvertently omitted
Anti-D
• Give as soon as possible after recognition of the omission
• Partial protection is afforded with administration within 13 days of the birth or potentially
sensitizing event, and some experts recommend giving it as late as 28 days
anti-D given shortly
before delivery
• Women who deliver < three weeks from the administration of anti-D immunoglobulin for the
usual indications do not require a post partum dose (unless a large FMH )
Postpartum sterilization
procedures
• Controversial
• ACOG considers women undergoing postpartum sterilization procedures candidates for anti-D
immune globulin

60. 60
Special Issues
Condition Remark
Women undergoing
intraoperative cell salvage
• maternal blood sample should be obtained 30 to 45 minutes after reinfusion to estimate the
volume of fetal red cells in the maternal circulation in case more anti-D immunoglobulin is
indicated
False-positive anti-D
antibody screen
• a typical D-sensitized patient, the anti-D antibody titer exceeds that of anti-C antibody
• If the anti-C titer equals or exceeds the anti-D titer, the clinician should consider the
possibility that the anti-D antibody is actually anti-G antibody
• G antigen is present on almost all D+ or C+ cells and absent from virtually all red cells which
lack D and C antigens.Anti-G antibody serologically mimics a combination of Anti-C and Anti
D.The challenge of anti G in the antenatal setting is to identify whether Anti D is present or
not
Weak / discordant D typing
• treated as negative & provide antenatal and postpartum anti – D
• For the purposes of
• blood donation, they are categorized as D positive
• transfusion recipients with weak D are considered D negative
Inadvertent administration
of anti-D to a +ve woman
• not harmful

61. 61
Special Issues
Condition Remark
pregnancies with
severe fetal anemia
before 20 wk
• intrauterine transfusion - mainstay of treatment,
• Plasma exchange and administration of IVIG
women with
multiple antibodies
• no specific guidelines
• warrant close observation
Prevention of an
affected fetus in
future pregnancies
• Avoid conception of an Rh(D)- positive fetus – with following
methods
1. In vitro fertilization (IVF) with preimplantation genetic diagnosis
2. Use of a gestational surrogate
3. Use of donor sperm from an Rh(D)-negative donor
IVF/donor
egg/surrogacy
• Management of pregnancies involving a donor gamete or a
gestational surrogate is based on
• the predicted antigen status of the fetus and
• the known antibody status of the gestational carrier

62. Hemolytic Disease due to non RhD Antibodies
• antibodies to minor antigens occur in 1.5–2.5% of obstetric patients
• Most cases of alloimmunization due to these minor antigens are caused by
incompatible blood transfusion
• antibodies to > 50 other red cell antigens have been reported to be
associated with HDFN
– only three antibodies
• anti-RhD, antiRhc, and anti-Kell (K1) ➔ cause significant enough fetal hemolysis that
treatment with IUT is considered necessary
• In one series from a tertiary care center for IUT in the Netherlands,
– 85% of cases involved anti-D;
– 10%, anti-K1; and
– 3.5%, anti-c
62

63. • care of the pregnant patient with antibodies to one of the clinically
significant minor antigens is similar to care of Rh-D alloimmunized
pregnant women
• Exception
– alloimmunization to the K or K1 antigens of the Kell blood group system
– Kell alloimmunization appears to be less predictable and often results in more
severe fetal anemia than alloimmunization due to other erythrocyte antigens.
– mechanism of anemia due to Kell alloimmunization to be different than with Rh-
D alloimmunization
• So maternal Kell antibody titers and amniotic fluid ΔOD450 values are not as predictive of
the degree of fetal anemia as with Rh-D sensitization
• Doppler measurements, however, appear to be accurate in predicting severe fetal anemia
63

64. 64