CLS 3311 Advanced Clinical Immunohematology

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CLS 3311 Advanced Clinical Immunohematology

• Hemolytic Disease of
• the Newborn
• HDN

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Hemolytic 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!

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Mechanism 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.

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Categories 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.

1. Rh System Antibodies
2. Other Blood Group Antibodies
3. ABO Antibodies

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ABO 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
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Pathophysiology 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.

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Pathophysiology 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.

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Prenatal 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?

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Prenatal 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?

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Prenatal 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.

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Prenatal 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.

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Liley 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

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Zone 3
Zone 2
Zone 1
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Percutaneous 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?

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Intrauterine 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

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Intrauterine 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.

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Rh 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.

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Rh 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?

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When 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.

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When 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.

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Rosette 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.

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Rosette 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.

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Kliehauer 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!)

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Kliehauer 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.

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Kliehauer 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.

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Practice 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.

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Cord 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.

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Exchange 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

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Compatibility 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.

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Exchange 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.

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Which 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

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Which 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