Title: CLS 3311 Advanced Clinical Immunohematology
1CLS 3311 Advanced Clinical Immunohematology
- Hemolytic Disease of
- the Newborn
- HDN
2Hemolytic Disease of the Newborn
- HDN occurs when the Mother has an antibody
capable of crossing the placental barrier that is
specific to an antigen present on the red blood
cells of the fetus. - Fetal red cells become coated with the IgG
alloantibody and undergo accelerated destruction
both before and after birth. - Where does the baby get an antigen that is
foreign to the Mom? - Its the Dads fault!
3Mechanism of HDN
- Immunization and Production of Antibody
- Fetomaternal Bleed Fetal RBCs enter moms
circulation - During birth, trauma to abdomen, etc.
- Maternal antibodies are formed against foreign
fetal red cell antigens - During subsequent pregnancies unexpected IgG
antibody crosses placenta and attaches to fetal
red cell antigens causing hemolysis.
4Categories of HDN
- Most severe form of HDN.
- Anti-D is 1.
- Less common due to RhIg
- Anti-K, -Fya, -s, etc. Page 424, Table 20-1
- Least severe. Group O mom with A or B fetus. Most
common form of HDN.
- Rh System Antibodies
- Other Blood Group Antibodies
- ABO Antibodies
5ABO vs. Rh HDN
Rh ABO
Mother Negative Group O
Infant Positive A or B (AB)
Occurrence in first born 5 40-50
Stillbirth and or hydrops Frequent Rare
Severe Anemia Frequent Rare
DAT Positive Pos or Negative
Spherocytes None Present
Exchange Transfusion Frequent Infrequent
Phototherapy Adjunct to exchange Often only treatment
6Pathophysiology of HDN
- Accelerated red cell destruction leads fetus to
increase production of RBCs therefore there are
increased numbers of nucleated RBCs. Also called
Erythroblastosis fetalis. - Severe cases of HDN can result in
- Generalized edema of the fetus Hydrops fetalis
- Severe anemia that can lead to cardiovascular
failure and tissue hypoxia, both of which can
lead to fetal death.
7Pathophysiology of HDN
- Bilirubinemia
- Results from increased RBC destruction
- Fetus in utero Not a problem because Moms liver
conjugates the bilirubin - Newborn Problem
- Newborn liver not yet able to conjugate the
bilirubin. Can build up to toxic levels and cause
Kernicterus.
8Prenatal Testing
- Patient History
- Which pregnancy is this?
- First? Second? Does it make a difference?
- Has she ever been transfused?
- May indicate allo antibody.
- Is she Rh negative? Has she had antenatal RhIg?
- May have anti-D. May have RhIg anti-D present in
serum NOW. - Does she have a previously identified unexpected
antibody?
9Prenatal Testing
- Test Mom for ABO, Rh (Weak D), and Antibody
Screen - Group O Mom
- Not a problem until baby is born OR is also Group
O. - Rh Negative Mom
- If she is Rh negative, has she been administered
antenatal RhIg? Is this her first or second
pregnancy?
10Prenatal Testing
- Positive Antibody Screen?
- Identify antibody and perform Titration if
antibody is clinically significant (anti-D, -K,
etc.). FREEZE the serum sample. If a subsequent
titer is requested you need to compare the first
titer results with the second titer. Run both
titers in parallel and compare endpoints. - Has the titer increased? Two tube increase is
clinically significant. May lead to more
sensitive testing (Amniocentesis, etc.) to
determine severity of disease.
11Prenatal Serological Studies
- Amniocentesis
- A good indicator of intrauterine hemolysis and
fetal well-being is the level of bilirubin
pigment found in the amniotic fluid. - Usually performed on women with allo antibody or
have an antibody titer at or greater than
critical level. - A change in optical density (?OD450) value of
the amniotic fluid in the upper mid zone of a
Liley graph indicates the need for fetal blood
sampling.
12Liley Graph
- The amniotic fluid is subjected to a
spectrophoto-metric scan at steadily increasing
wavelengths so that the change in the optical
density at 450 nm (?OD450) can be calculated. - Liley graph plots the change in OD at 450nm
versus gestational age in weeks. - Zone 1 - Observe fetus for stress-repeat 2-4
weeks - Zone 2 - Moderate disease May require treatment
- Zone 3 - Severe problems - Deliver/treat
13Zone 3
Zone 2
Zone 1
14Percutaneous Umbilical Blood Sampling
- Insertion of a needle into umbilicus vein and
withdrawal of fetal blood. - Allows direct measurement of Fetal hemoglobin and
hematocrit which gives a better assessment of
fetal anemia. - How do you know if you have, indeed, collected
fetal blood? - Can test with anti-I. How does this help?
15Intrauterine Transfusion
- Indications
- Correct fetal anemia lt10 gm/dl Hemoglobin
- 24-26 week gestation
- Blood component
- Frozen, deglycerolized blood Normal
electrolytes, no anticoagulant or plasma (washed
out during deglycerolization), and low
platelets/WBCs. - Group O Negative, 75 to 80 Hematocrit,
Hemoglobin S negative, CMV negative
(leukoreduce), Irradiated red blood cells
16Intrauterine Transfusion (IUT)
- Methods
- Intraperitoneal Red cells are infused into fetal
abdomen and absorbed into circulation. - Intravascular Red cells are infused directly
into umbilical vein using ultrasound guidance.
Quicker resolution of anemia. - A combination of methods may be used to avoid
peaks and troughs of fetal hematocrit. - Once began, IUT are administered periodically
until delivery of the baby. Such as every two
weeks.
17Rh Immune Globulin
- What is it?
- Its a concentrate of predominantly IgG anti-D
developed from pools of human plasma. (Trade name
is RhoGam) - How does it work?
- Prevents mom from making immune anti-D by
suppression of immune response. RhIg attaches to
Rh positive fetal red cells activating suppressor
T- cells. At least that is the current theory.
18Rh Immune Globulin
- Full Dose 300 micrograms of anti-D
- Sufficient to counteract 15 mls of D positive
packed red cells (30 mls whole blood) - Mini dose 50 ?g
- Sufficient for 2.5 mls D positive blood - for
first trimester abortion or miscarriage. NOT used
much. Why?
19When to give RhIg
- Antenatal administration
- Given at 28 (to 32) weeks gestation to Rh
negative pregnant women as long as the antibody
screen is negative for anti-D. - Amniocentesis
- When an amniocentesis is preformed (16 to 18
weeks gestation) should receive full dose.
20When to Give RhIg
- Postpartum Administration
- When Mother is Rh negative (and is negative for
allo anti-D) and Baby is Rh positive. It is that
simple. - How much? Need to determine Fetal Bleed. How much
fetal blood transferred into the mothers
circulation can be determined.
21Rosette Test
- Purpose Screening test to detect the presence of
Rh positive RBCs in the circulation of Rh
negative person. - Qualitative Tells us that there are Rh Positive
cells in an Rh Negative person. Nothing more.
22Rosette Test - Principle
- Add chemically modified anti-D to Mothers washed
Post Partum (EDTA) red cells and incubate at
37oC. Anti-D will attach to Rh Positive cells
present. - Wash cells and add R2R2 indicator cells.
Indicator cells will rosette around anti-D that
has attached to the Rh positive cells. - Centrifuge and resuspend the suspension and read
microscopically looking for Rosettes. - Rosettes present? Rh positive cells are present,
but we dont know how many.
23Kliehauer Betke (Acid Elution)
- Purpose To detect the presence of Hemoglobin F.
If a fetomaternal bleed has occurred then fetal
red cells will be present in the maternal
circulation. - Quantitative Can determine the extent of the
fetomaternal bleed. How much fetal blood entered
the maternal circulation. (And thus how much
RhIg to administer!)
24Kliehauer Betke (Acid Elution)
- Principle Draw a Post Partum EDTA sample from
the mother and make and fix a blood smear on a
glass slide. Flood the smear with an acid
solution. The Hemoglobin of adult red cells is
washed out by the acid solution while red cells
with Hgb F are not. Rinse slide and counter stain
(Safranin) the smear. Cells with Hgb F stain red
while the adult red cells remain transparent. - Count number of stained Hgb F red cells within
2000 adult (Hgb A) red cells.
25Kliehauer Betke StainCalculations
- Fetal cells / 2000 adult cells x 100 of
Fetal cells present in the maternal circulation. - of Fetal cells X 50 number of mls of Fetal
bleed - of mls of fetal bleed / 30 vials of RhIg
required Plus 1. - We always add one additional dose of RhIg to
insure adequate suppression of immune production
of allo anti-D.
26Practice Calculation of RhIg Dose
- Count 26 Fetal Cells in 2000 adult cells.
- (26 / 2000) x 100 1.3 Fetal Red Cells
- 1.3 x 50 65 mL fetal blood. Another way to
calculate the same result is - (1.3/100) x 5000 65 The 5000 represents the
total blood volume of Mother. - 65 mL / 30 2.2 doses of RhIg, Plus 1.
- So this Mom would receive 3 vials of RhIg to
counteract the fetal bleed.
27Cord Blood Studies
- Required testing on the Cord Blood of Newborns
with Rh Negative Moms (suggested on Group O
Moms) - ABO group If Mom is Group O and Baby is Group A
or B baby may have ABO HDN. - Rh typing If baby is Rh Negative Mom is NOT a
candidate for RhIg. If baby is Rh Positive then
she is a candidate for RhIg. - Direct Antiglobulin Test If DAT is positive
perform eluate to identify antibody that is
coating the babies red blood cells.
28Exchange Transfusion
- Exchange transfusion may be definitive therapy
for newborns with severe HDN. - A process where you exchange baby red cells with
transfused red cells. Accomplishes the
following - Remove antibody coated RBCs Not all but many.
- Removal of maternal antibody. Remember this
antibody is passively transferred so the more we
remove the better. - Removal of bilirubin reduce bilirubin in
newborn. - Replacement of RBCs Treating the anemia
29Compatibility testing for Exchange Transfusion
- Crossmatch blood for exchange transfusion with
Mothers serum. Why? - Can crossmatch with baby serum or eluate if mom
is not available, but best indication of red cell
survival is to crossmatch with the Mothers serum.
Remember the source of the antibody is MOM.
30Exchange Transfusion
- Selected red cells need to be compatible with
Moms ABO antibodies in addition to any other
antibodies. - Group O red cells (lt5 days old) suspended in
Group AB plasma are commonly used. - If Mom and Baby are ABO identical, group specific
red cells or whole blood may be used. - The blood should also be Irradiated.
- Typically, a volume of twice the infants blood
volume is used.
31Which mothers are candidates for RhIg?
- Mother Rh positive with anti-K
- Baby Rh positive with negative DAT
-
- Mother O negative with anti-C
- Baby A negative with positive DAT
-
- Mother A negative with negative IAT
- Baby O positive with negative DAT
32Which mothers are candidates for RhIg?
- Mother A negative with anti-D, C, K
- Baby B positive, DAT eluate showed D, C
- Mother A negative with negative IAT
- Baby O positive with positive DAT, eluate neg
- Mother AB negative with anti-D
- Baby A negative with negative DAT