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CLS 3311 Advanced Clinical Immunohematology

CLS 3311 Advanced Clinical Immunohematology. Hemolytic Disease of the Newborn HDN. 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.

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CLS 3311 Advanced Clinical Immunohematology

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  1. CLS 3311 Advanced Clinical Immunohematology Hemolytic Disease of the Newborn HDN

  2. 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? • It’s the Dads fault!

  3. 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.

  4. RhSystem Antibodies Other Blood Group Antibodies ABO Antibodies 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. Categoriesof HDN

  5. ABO vs. Rh HDN

  6. 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.

  7. Pathophysiology of HDN Bilirubinemia • Results from increased RBC destruction • Fetus in utero:Not a problem because Mom’s liver conjugates the bilirubin • Newborn:Problem • Newborn liver not yet able to conjugate the bilirubin. Can build up to toxic levels and cause Kernicterus.

  8. 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?

  9. 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?

  10. Prenatal Testing Positive Antibody Screen? • Identify antibody and perform Titration if antibody is clinically significant (anti-D, -K, etc.). FREEZEthe 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.

  11. 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.

  12. 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

  13. Zone 3 Zone 2 Zone 1

  14. 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?

  15. Intrauterine Transfusion Indications • Correct fetal anemia: <10 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

  16. 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.

  17. Rh Immune Globulin What is it? • It’s 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.

  18. 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?

  19. Whento 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.

  20. When to Give RhIg Postpartum Administration • WhenMother is Rh negative(and is negative for allo anti-D)andBaby 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.

  21. 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.

  22. 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 don’t know how many.

  23. 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!)

  24. 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.

  25. 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.

  26. 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.

  27. Cord Blood Studies Required testing on the Cord Blood of Newborn’s 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.

  28. 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

  29. 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.

  30. Exchange Transfusion • Selected red cells need to be compatible with Mom’s ABO antibodies in addition to any other antibodies. • Group O red cells (<5 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.

  31. 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

  32. 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

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