Hemolytic Diseases of the Newborn (HDN )

Nurlina Sirait
Noormartany

Selasa, 6 April 2010

Bagian Patologi Klinik
Fakultas Kedokteran Universitas Padjadjaran
RSUP Dr Hasan Sadikin Bandung
 URAIAN KASUS

• Nyonya C golongan darah O, Rhesus negatif

• Tn. C. memiliki golongan darah A, Rhesus positif.
• Mereka memiliki 4 anak, 2 diantaranya menderita
hemolytic diseases of the newborn (HDN).
• Kehamilan kedua, ketiga dan keempat Ny. C. telah
mendapat terapi antibodi Rhesus-D.
 Ny. C Tn. C
Gol O, Rh - Gol A, Rh +

Anak 1 Anak 2 Anak 3 Anak 4

Gol O Rh + Gol B Rh + Gol A Rh + Gol A Rh -
 Anak 1
Gol O Rh +
Anti-D pada ibu baru terbentuk
setelah anak pertama lahir

Ny.C hanya mengalami sensitisasi,

karena masuknya eritrosit janin
keperedaran darah ibu

Normal
 Anak 2
Gol B Rh +
Janin dengan Rh +  IgG anti-D
ibu yang telah terbentuk 
melewati sawar darah plasenta
masuk ke janin  HDN.
HDN
ringan
 Anak 3
Reaksi hemolisis >> anak kedua
Gol A Rh + sampai memerlukan transfusi
intrauterin.

Anti-D ibu menjadi lebih sensitif

terhadap eritrosit janin

Respon yang lebih kuat  reaksi

HDN Berat hemolisis yang lebih berat.
 Anak 4
Gol A Rh -
Pada anak keempat Rhesus janin
negatif = Rhesus ibu

Tidak menderita HDN.

Normal
Kejadian HDN paling sering disebabkan :
- inkompatibilitas Rhesus
- inkompatibilitas ABO.

Inkompatibilitas Rhesus memiliki perbedaan sensitisasi dengan inkompatibilitas ABO

Sistem ●

●
Tidak ada bahan-bahan alamiah yang secara kimiawi
sama dengan antigen D
Individu memiliki anti-D melalui :
Masuknya eritrosit janin Rh positif ke ibu Rh negatif.

Rh
●
Transfusi darah dari donor Rh positif kepada resipien Rh
negatif.

Sistem ●
Mirip dengan antigen bakteri atau
tumbuh-tumbuhan
●
Individu akan memiliki antibodi

ABO terhadap anti-A dan B

 Anak kedua dan ketiga
mengalami HDN, dan
anak ketiga reaksi >>
inkompatibilitas
HDN akibat

Rhesus

Anak ke empat
 rhesus (-)
tidak HDN
dan Ibu Rh -
Ayah Rh+
Pada kehamilan anak ke-2 sampai ke-4 ibu mendapat antibodi Rhesus D, namun
HDN masih tetap terjadi.

Seharusnya pemberian antibodi Rhesus D telah diberikan pada kehamilan pertama

sebelum anti-D pada ibu terbentuk
 Ibu
gol O
(OO)
Rh (-)
dd

Anak
50%
golongan
darah A,
dan 50%
golongan
darah O
50% Rhesus
positif dan
50% Rhesus
negatif
Ayah
Gol A
(Ao)
Rh(+)
Dd

Anak Ke-2 golongan darah B  bukan anak dari Tn.C

 PEMBAHASAN

Antibodi maternal sirkulasi janin HDN

 PEMBAHASAN
• HDN  reaksi hipersentivitas tipe II.
• Reaksi sitotoksik atau sitolitik
• Antibodi Ig G dan Ig M  antigen permukaan
sel atau jaringan yang berinteraksi komplemen
dan berbagai jenis sel efektor
 Pemeriksaan Diagnostik
• Pemeriksaan gol. Darah ABO dan Rhesus
Pemerikasaan gol. Darah forward grouping
dan reverse grouping .
Forward grouping berdasarkan antigen pada
eritrosit.
Reverse grouping berdasarkan ada tidaknya
antibodi dalam serum.
 Tes skrining HDN
• Tes Coombs : - Coombs direk
- Coombs indirek
Coombs direk mendeteksi adanya antibodi
IgG ibu yang mengikat antigen pada sel darah
merah janin
• Prosedur :
– Bagikan 1 tetes suspensi Eritrosit ke masing2
tabung
– Cuci tabung dengan saline selama 3 sampai 4
menit
– Tambahkan antisera, campur lalu sentrifugasi
– Lihat agglutinasi dari sel-sel darah merah
– Jika menggunakan AHG, inkubasi selama 5 menit,
kemudian di sentrifugasi kembali & baca kembali
• Interpretasi :
– Hasil positif ketika terjadi agglutinasi
– Hasil negatif jika tidak terjadi agglutinasi
 Coombs indirek
• Tujuan menentukan ada tidaknya antibodi
anti-D dalam serum ibu dengan Rh negatif
atau yang melahirkan anak dengan Rh positif.
• Serum ibu diinkubasi selama 15 menit pada
suhu 37 °C dengan sel darah merah yang Rh
positif.
 Penatalaksanaan
• Pencegahan
– Imunoprofilaksis Rh merupakan imunisasi
pencegahan pada wanita Rh-negatif.
– Waktu paruh RhIG adalah 24 hari  15-20% ibu
yang mendapat suntikan RhIG pada usia
kehamilan 28 minggu akan memiliki titer anti-D
yang rendah saat melahirkan
 Terapi
• Sebelum melahirkan, pada
janin dapat dilakukan:
– Terapi transfusi intrauterin
dapat diberikan pada kasus
HDN yang berat.
– Transfusi dapat diberikan
secara intraperitoneal atau
intravaskuler.
 Terapi
• Terapi pada bayi baru lahir
– Fototerapi
– Transfusi tukar : Indikasi transfusi tukar adalah
adanya penigkatan bilirubin yang cepat dan
anemia.
Fototerapi Transfusi tukar
KESIMPULAN
• Kasus HDN  Ibu Gol O Rh (-) ; ayah gol A Rh
(+)
• HDN ini akibat inkompatibilitas Rhesus
• Anak ketiga terjadi reaksi yang lebih berat
dibanding anak kedua  Antibodi ibu lebih
sensitif
• Pemberian antibodi Rhesus sebagai
pencengahan tidak efektif  kehamilan kedua
 KESIMPULAN
• Pemeriksaan diagnostik pada kasus HDN yaitu
pemeriksaan awal meliputi pemeriksaan golongan
darah ABO dan Rh(D).
• Pasien Rh negatif diskrining pada usia kehamilan 28-
30 minggu, sebelum mendapat RhIG.
Terimakasih
 (From Robbins Basic Pathology ,2003 ）
Slide 7.10
Tabel 1 Grup Antigen Eritrosit

Erythrocyte surface glycoprotein Blood groups expressed Number of epitopes per cell

Anion transport protein ABO, Li 106

Glycoporin A MN 106

Glucose Transporter ABO, Li 5 x 105

MR 45.000-100.000 ABO

MR 30.000 ABO, Rh 1.2 x 105

Glycoporin B N,Ss 2.5 x 105

Glycoporins C and D Gerbich (Ge) 105

DAF Cromer < 105

CD 44 (80 kDa) Ina/Inh 3000-6000

Zinc Endopeptidase Kell 3000-6000

DARC Fy 12.000
Laminin-binding glycoprotein Lutheran 1500-4000
Type II Hypersensitivity:
Cytotoxic
 Examples
Examples of of drug-induced
drug-induced
type
type IIII hypersensitivity
hypersensitivity
 Red
Redcells:
cells:
Penicillin,
Penicillin,chloropromazine,
chloropromazine,phenacetin
phenacetin

 Granulocytes:
Granulocytes:
Quinidine,
Quinidine,amidopyridine
amidopyridine

 Platelets:
Platelets:
sulphonamides,
sulphonamides,thiazides
thiazides
 Blood Group Ags
Blood Group Ag Ab
A A anti-B

B B anti-A

AB A&B None

O ---- anti-A&
anti-B
Abs against blood group Ags are naturally
present and are IgM type.
 Hemolytic Disease of the New Born

RhD-ve mother

Anti-RhD Abs

RhD +ve fetus RhD +ve fetus

If mother and fetus have different blood
groups, hemolytic disease does not occur.

‘A’ blood group

mother

Anti-B Abs

‘B’ blood group fetus

 Child with Hemolytic Disease

 Mother is Rh- and fetus is Rh+.

 Fetal RBC entering mother, will trigger anti- Rh Abs.
 Such Abs cross placenta and can destroy fetal RBC.
 Prophylaxis (RhoGAM)

Anti-RhD Abs
RhD-ve mother

RhD +ve fetus RhD +ve fetus

Mid-term injection of RhoGAM and a second

injection within a few days of delivery
Prophylaxis

Inject Anti-RhD Abs mothers

immediately after delivery

Prevents sensitization
 Intrauterine Transfusion (IUT)
• Given to the fetus to prevent hydrops fetalis and fetal death.
• Can be done as early as 17 weeks, although preferable to wait until 20
weeks
• Severely affected fetus, transfusions done every 1 to 4 weeks until the
fetus is mature enough to be delivered safely. Amniocentesis may be done
to determine the maturity of the fetus's lungs before delivery is
scheduled.
• After multiple IUTs, most of the baby’s blood will be D negative donor
blood, therefore, the Direct Antiglobulin test will be negative, but the
Indirect Antiglobulin Test will be positive.
• After IUTs, the cord bilirubin is not an accurate indicator of rate of
hemolysis or of the likelihood of the need for post-natal exchange
transfusion.
 Intrauterine Transfusion
• An intrauterine fetal blood transfusion is done in the hospital. The mother may
have to stay overnight after the procedure.
• The mother is sedated, and an ultrasound image is obtained to determine the
position of the fetus and placenta.
• After the mother's abdomen is cleaned with an antiseptic solution, she is given a
local anesthetic injection to numb the abdominal area where the transfusion
needle will be inserted.
• Medication may be given to the fetus to temporarily stop fetal movement.
• Ultrasound is used to guide the needle through the mother's abdomen into the
fetus's abdomen or an umbilical cord vein.
• A compatible blood type (usually type O, Rh-negative) is delivered into the fetus's
abdominal cavity or into an umbilical cord blood vessel.
• The mother is usually given antibiotics to prevent infection. She may also be given
tocolytic medication to prevent labor from beginning, though this is unusual.
 Intrauterine Transfusion
• Increasingly common and relatively safe procedure since the development
of high resolution ultrasound particularly with colour Doppler capability.
• MCA Doppler velocity as a reliable non-invasive screening tool to detect
fetal anemia.
– The vessel can be easily visualized with color flow Doppler as early as 18
weeks’ gestation.
– In cases of fetal anemia, an increase in the fetal cardiac output and a decrease
in blood viscosity contribute to an increased blood flow velocity
 Intrauterine Transfusion

• The risk of these

procedures is now
largely dependent on
the prior condition of
the fetus and the
gestational age at which
transfusion is
commenced.
 Intrauterine Transfusion
• Titer greater than 32 for anti-D and 8 for anti-K OR four fold increase in titer
indicates need for analysis of amniotic fluid.
• Amniocentesis
– Perform at 28 wks if HDN in previous child
– Perform at 22 wks if previous child severely affected
– Perform if maternal antibody increases before 34th wk.
• High values of bilirubin in amniotic fluid analyses by the Liley method or a
hemoglobin concentration of cord blood below 10.0 g/mL.
• Type fetus -recent development in fetal RhD typing involves the isolation of free
fetal DNA in maternal serum. In the United Kingdom, this technique has virtually
replaced amniocentesis for fetal RhD determination in the case of a heterozygous
paternal phenotype
• Maternal plasma exchange may be instituted if the fetus is too young for
intrauterine transfusion.
Liley Graph
 Selection of Blood
• CPD, as fresh as possible, preferably <5 days old.
• A hematocrit of 80% or greater is desirable to minimize the
chance of volume overload in the fetus.
• The volume transfused ranges from 75-175 mL depending on
the fetal size and age.
• CMV negative
• Hemoglobin S negative
• IRRADIATED
• O negative, lack all antigens to which mom has antibodies and
Coomb’s compatible.
 Treatment of Mild HDN
• Phototherapy is the treatment of choice.
• Phototherapy process slowly
decomposes/converts bilirubin into a nontoxic
isomer, photobilirubin, which is transported in
the plasma to the liver.
• HDN is judged to be clinically significant
(phototherapy treatment) if the peak bilirubin
level reaches 12 mg/dL or more.
Bilirubin Degradation by Phototherapy
 Phototherapy

• The therapy uses a blue light (420-470 nm) that converts

bilirubin so that it can be excreted in the urine and feces.
• Soft eye shields are placed on the baby to protect their eyes
from damage that may lead to retinopathy due to the bili
lights.
 Phototherapy

• Lightweight, fiberoptic pad delivers up to 45 microwatts of

therapeutic light for the treatment of jaundice while allowing
the infant to be swaddled, held and cared for by parents and
hospital staff.
• Compact unit is ideal for hospital and homecare.
Exchange Transfusion
 Exchange Transfusion

• Full-term infants rarely require an exchange transfusion if intense

phototherapy is initiated in a timely manner.
• It should be considered if the total serum bilirubin level is approaching 20
mg/dL and continues to rise despite intense in-hospital phototherapy.
• The procedure carries a mortality rate of approximately 1% and there may
be substantial morbidity
 Goals of Exchange Transfusion
• Remove sensitized cells.
• Reduce level of maternal antibody.
• Removes about 60 percent of bilirubin from the plasma,
resulting in a clearance of about 30 percent to 40 percent of
the total bilirubin.
• Correct anemia by providing blood that will have normal
survival.
• Replacement with donor plasma restores albumin and any
needed coagulation factors.
• Rebound – usually a 2 volume exchange is needed as bilirubin
in tissues will return to blood stream.
 Testing Baby
• Antibody elution testing from cord red blood cells.
• ABO/D typing
– If baby received intrauterine transfusions will type as O negative
– If baby’s Direct Antiglobulin Test is strongly positive due to anti-D may
get FALSE NEGATIVE immediate spin reaction with reagent anti-D
(blocking phenomenon), weak D (Du) test will be STRONGLY positive
• Antibody screen
• Coomb’s crossmatch antigen negative donor.
 Testing Mom
• Type and screen on mom.
• Identification of unexpected antibodies.
• More than 40 antigens have been identified as
causing HDN.
• Select blood that lacks antigens to which mom has
antibodies.
• Perform coomb’s crossmatch with Mom and baby’s
blood.
 Phototherapy

Fluorescent blue light in the 420-

475 nm range
Exchange transfusion
 What type of blood to give fetus:
• CMV negative
• Irradiated
• Fresh Whole Blood (to avoid Ca++)
• Maternal blood if possible
• Leukoreduced
 FetoMaternal Hemorrhage
• Sensitization occurs as a result of seepage of
fetal cells into maternal circulation as a result
of a fetomaternal hemorrhage
– Placental membrane rupture (7%)
– Trauma to abdomen
– Delivery (>50%)
– Amniocentesis
– Abortion
 Risk
• Rh-negative women can be exposed to Rh-
Positive cells through transfusion or pregnancy
• Each individual varies in their immune
response (depends on amount exposed to)
– 85%* transfused with 200 mL Rh-positive cells will develop
anti-D
– There is only about a 9%* chance that Rh-neg mothers
pregnant with an Rh-positive child will be stimulated to
produce anti-D (without RhIg)

*Mollison, PL, Engelfriet, CP & Contreras, M. (1997). Blood Transfusion in Clinical

Medicine (ed. 10). London: Blackwell Scientific, p 395.
 Pathogenesis
• Maternal IgG attaches to antigens on fetal cells
– Sensitized cells are removed by macrophages in
spleen
– Destruction depends on antibody titer and number
of antigen sites
– IgG has half-life of 25 days, so the condition can
range from days to weeks
• RBC destruction and anemia cause bone
marrow to release erythroblasts, hence the
name “erythroblastosis fetalis”)
 Pathogenesis
• When erythroblasts are
used up in the bone
marrow, erythropoiesis in
the spleen and liver are
increased
– Hepatosplenomegaly
(enlarged liver & spleen)
– Hypoproteinemia (from
decreased liver function)
leads to cardiac failure
edema, etc called
“Hydrops fetalis”
• Gambaran klinis HDF/N dapat dibedakan :
- HDN berat : kematian intrauterin akibat hidrops
fetalis
- HDN sedang : bayi lahir dengan anemia dan
ikterus
- HDN ringan : terdapat anemia ringan
dengan/atau tanpa disertai ikterus
Tes Rosette
-Digunakan untuk menentukkan jumlah FMH pada
♀ Rh (-) yang telah melahirkan bayi Rh (+)
-Tes Rosette (+) → tes kuantitatif untuk
menentukan volume FMH
Prinsip : eri ibu Rh (-) yang sudah melahirkan bayi
Rh (+) diinkubasi dengan anti-D maka selama
inkubasi anti-D akan mensensitisasi sel-sel janin
Rh (+) dalam suspensi eri
-Sampel: darah postpartum ibu dalam
antikoagulan EDTA yang belum mendapat RhIG
(1 jam postpartum) dan dibuat suspensi eritrosit
3-5%
Hasil : bila jumlah total rosette dalam 10 LpB
adalah >3, hasil dilaporkan sebagai “positif”, dan
harus dilanjutkan dengan tes KB. Bila jumlah
total rosette dalam 10 LpB adalah <3, hasil
dilaporkan sebagai “negatif”.
Tes Kleihauer-Betke (Kleihauer-Betke stain; tes
KB)
-Prinsip: Hemoglobin F resisten terhadap asam
dan akan berwarna merah pada pemberian
eosin. Hemoglobin A larut pada suasana asam
sehingga eritrosit akan tidak berwarna (ghost
cells). Jumlah HbF dihitung secara mikroskopis.
Prosedur :
-Buat 2 apusan darah tepi pasien, biarkan kering,
fiksasi dengan ethyl alkohol 80 % selama 5
menit dan cuci menggunakan air suling.
Diamkan apusan sampai kering.
-Letakkan apusan pada larutan bufer sitrat selama
10 menit (bufer ini akan melarutkan HbA),
kemudian bilas menggunakan air suling.
-Warnai apusan menggunakan larutan Ehrlich
hematoxylin selama 3 menit
kemudian dalam larutan Erythrosine 0,1 % selama
3 menit kemudian dibilas menggunakan air
suling.
-Periksa dengan pembesaran 40X
-Menghitung banyaknya eritrosit janin dalam 1.000
eritrosit dewasa dalam tiap apusan (total
dihitung pada 2.000 eritrosit).
Perhitungan volume FMH dilakukan
menggunakan rumus:

Interpretasi :
-HbF (+) → merah terang
 Comparison of ABO and Rh HDN
ABO Rh
Severity Mild Severe
Child Affected First born (40-50%) Usually second or
subsequent births
Blood groups Mother O,child A or B Mother Rh-,child Rh+
Anemia Uncommon, mild Severe
Hydrops fetalis Rare Frequent
Jaundice Mild Severe
Spherocytes on peripheral Usually present None
blood smear
Direct Coombs test Negatif or weakly positive Positive
Maternal antibodies Inconsistent,inconclusive Always present
Antenatal diagnosis Unnecessary Necessary
Treatment Phototherapy Exchange transfusion
Exchange transfusion Intrauterine
Types of antibody IgG (immune) IgG (immune)
Prophylaxis None RhIG, antenatal RhIG
 DAT / IAT

False positive result False negative result

Overcentrifugation Failure to add antiglobulin
reagent

Inappropiate blood sample Inadequate washing

Sample contamination Improper technique
Spontaneous aggregration Undercentrifugation
Improper technique pH of isotonic saline
 FetoMaternal Hemorrhage
• Sensitization occurs as a result of seepage of
fetal cells into maternal circulation as a result
of a fetomaternal hemorrhage
– Placental membrane rupture (7%)
– Trauma to abdomen
– Delivery (>50%)
– Amniocentesis
– Abortion
 Risk
• Rh-negative women can be exposed to Rh-
Positive cells through transfusion or pregnancy
• Each individual varies in their immune
response (depends on amount exposed to)
– 85%* transfused with 200 mL Rh-positive cells will develop
anti-D
– There is only about a 9%* chance that Rh-neg mothers
pregnant with an Rh-positive child will be stimulated to
produce anti-D (without RhIg)

*Mollison, PL, Engelfriet, CP & Contreras, M. (1997). Blood Transfusion in Clinical

Medicine (ed. 10). London: Blackwell Scientific, p 395.
 Pathogenesis
• Maternal IgG attaches to antigens on fetal cells
– Sensitized cells are removed by macrophages in
spleen
– Destruction depends on antibody titer and number
of antigen sites
– IgG has half-life of 25 days, so the condition can
range from days to weeks
• RBC destruction and anemia cause bone
marrow to release erythroblasts, hence the
name “erythroblastosis fetalis”)
Increased immature RBCs
 Pathogenesis
• When erythroblasts are
used up in the bone
marrow, erythropoiesis in
the spleen and liver are
increased
– Hepatosplenomegaly
(enlarged liver & spleen)
– Hypoproteinemia (from
decreased liver function)
leads to cardiac failure
edema, etc called
“Hydrops fetalis”
 Bilirubin
• Hemoglobin is metabolized to bilirubin
– Before birth, “indirect” bilirubin is transported
across placenta and conjugated in maternal liver
(“direct”) where it is excreted
– After birth, the newborn liver is unable to
conjugate the bilirubin
• Unconjugated (“indirect”) bilirubin can reach toxic
levels (18-20 mg/dL)
• This is called kernicterus and can lead to permanent
brain damage
 Diagnosis & Management
• Serologic Testing (mother & newborn)
• Amniocentesis and Cordocentesis
• Intrauterine Transfusion
• Early Delivery
• Phototherapy & Newborn Transfusions
 Serologic testing on mother
• ABO and Rh testing
– Test for D antigen (test for weak D if initially negative)

• Antibody Screen
– To test detect for IgG alloantibodies that react at 37°C
– If negative, repeat before RhIg therapy and/or if patient is transfused
or has history of antibodies (3rd trimester)

• Antibody ID
– Weakly reacting anti-D may be due to FMH or passively administered
anti-G (RhIg)
– If antibody is IgG, anti-D is most common followed by anti-K and other
Rh antibodies
 Serologic Tests (cont’d)
• Paternal phenotype
• Amniocyte testing
– If mother has anti-D, then father probably is heterozygous for D
antigen
– Amniocytes can be tested as early as 10-12 weeks gestation to detect
the gene for the D antigen and any other antigens
 Serologic testing (cont’d)
• Antibody titration
– Antibody concentration is determined by antibody titration
– Mother’s serum is diluted to determine the highest dilution that reacts
with reagent RBCs at 37°C (60 min) and AHG phase
– First sample is frozen and run with later specimens
– Testing is repeated at 16 and 22 weeks and 1- to 4- week intervals
after
– A Difference of >2 dilutions; or a score change of more than 10 is
considered a significant change in titer (Marsh score)
• A titer of 16-32 is significant
• >16 should be repeated at 18-20 weeks’ gestation
• >32 indicates a need for amniocentesis or cordocentesis between 18-
24 weeks’ gestation
• <32 is repeated every 4 weeks (18-20 weeks) and every 2-4 weeks
(third trimester)
 Marsh score
• The agglutination reactions for each dilution are given a corresponding
score; scores are added:
• 4+ 12
• 3+ 10
• 2+ 8
• 1+ 5
• w+ 3

1:1 1:2 1:4 1:8 1:16 1:32 1:64

Example:

3+ +3 +3 +2 +2 +2 1+
10 + 10 + 10 + 8 + 8 + 8 + 5
= 59
Amniocentesis & Cordocentesis
• About 18-20 weeks’ gestation
• Cordocentesis takes a sample of
umbilical vessel to obtain blood
sample
• Amniocentesis assesses the
status of the fetus using
amniotic fluid
– Fluid is read on a
spectrophotometer (350-700
nm)
– Change in optical density (ΔOD)
above the baseline of 450 nm
is the bilirubin measurement
Analysis of amniotic fluid (example)

ΔOD
 Liley graph
• The ΔOD is plotted on the Liley graph
according to gestational age
• Three zones estimate the severity of HDN
– Lower: mildly or unaffected fetus (Zone 1)
– Midzone: moderate HDN, repeat testing (Zone 2)
– Upper: severe HDN and fetal death (Zone 3)
Liley graph

a ΔOD of .206 nm at
35 weeks correlates
* with severe HDN
 What to do?
• Intrauterine transfusion is
done if:
– Amniotic fluid ΔOD is in high
zone II or zone III
– Cordocentesis has hemoblobin
<10 g/dL
– Hydrops is noticed on ultrasound
• Removes bilirubin
• Removes sensitized RBCs
• Removes antibody
 Other treatments
• Early Delivery
– If labor is induced, fetal lung maturity must be determined using
the lecithin/sphingomyelon (L/S) ratio (thin layer
chromatography) to avoid respiratory distress syndrome
• Phototherapy (after birth)
– Change unconjugated bilirubin to biliverdin
– May avoid the need for exchange transfusion
• Newborn transfusion
– Small aliquots of blood (PediPak)
– Corrects anemia
 Postpartum testing
• ABO – forward only
• Rh grouping – including weak D
• DAT
• Elution
– Done when a DAT is positive and HDN is
questionable
– Removes antibody from RBC to identify
– Treatment does not change
 Prevention
• RhIg (RhoGAM®) is given to the mother to
prevent immunization to the D antigen
– “Fools” mom into thinking she has the antibody
– RhIg (1 dose) is given at 28 weeks’ gestation
– RhIg attaches to fetal RBCs in maternal circulation
and are removed in maternal spleen; this prevents
alloimmunization by mother
– May cause a positive DAT in newborn (check
history)
 Postpartum administration of RhIg

• Another dose of RhIg should be given to the

mother within 72 hours of delivery (even if
stillborn)
– Mother should be D negative
– Newborn should be D positive or weak D
– About 10% of the original dose will be present at
birth, so it’s important to give another dose to
prevent immunization
 Dose
• Each vial of RhIg contains enough anti-D to
protect against a FMH of 30 mL
– One vial contains 300 μg of anti-D
– Given intramuscularly of intravenously
– Massive fetomaternal hemorrhage (>30 mL)
requires more than one vial
– To assess a FMH, a maternal sample is screened
within 1 hour of delivery (rosette test)
 Rosette Test
• A qualitative measure of fetomaternal
hemorrhage

Fetomaternal Hemorrhage:
<1 rosette per 3 lpf = 1 dose of RhIg
>1 rosette per 3 lpf = Quantitate bleed
 Kleihauer-Betke acid elution
• Quantitates the number of fetal cells in
circulation
– Fetal hemoglobin is resistant to acid and retain
their hemoglobin (appear bright pink)
– Adult hemoglobin is susceptible to acid and
leaches hemoglobin into buffer (“ghost” cells)
 Calculating KB test

Step 1) stain and count the amount of fetal

cells out of 1000 total cells counted
Step 2) calculate the amount of fetal blood in # fetal cells
 % of fetal cells
cirulation by multiplying %fetal cells by 50 mL total cells (2000)
Step 3) divide mL of fetal blood by 30 (each
vial protects against a 30 mL bleed
Step 4) Round the calculated dose up and
add one more vial for safety

% fetal cells x 50
Required dose of RhIg 
30
 Considerations
• RhIg is of no benefit once a person has formed
anti-D
• It is VERY important to distinguish the
presence of anti-D as:
– Residual RhIg from a previous dose OR
– True immunization from exposure to D+ RBCs
• RhIg is not given to the mother if the infant is
D negative (and not given to the infant)
 Maternal Specimen

D Positive D negative

* Make sure presence of anti-D is not

due to antenatal administration of Rh
immune globulin