1. Unit 11 Neonatal and Obstetrical Transfusion Practice – Part 3
Terry Kotrla, MS, MT(ASCP)BB
The clinical laboratory continues to play a major role in the diagnosis of Hemolytic Disease of the Newborn (HDN). The results of laboratory tests will assist the physician in deciding upon treatment options. Moderate to aggressive therapy may be needed, including transfusion support both in-utero and/or after birth. The transfusion service must be aware of the laboratory results which indicate the severity of the disease as well as the unique transfusion requirements needed for these situations.

2. Exchange Transfusion
Used most often for HDFN but has recently been used to treat RDS, DIC and sepsis.
Mortality rate 1%,may be substantial morbidity.
Quantified (i.e. 2 volume, single volume) to reflect affect on infant’s total blood volume.
Depends on bilirubin levels, amount and rate of increase.

3. Exchange Transfusion
Immediate effectiveness of a two-volume exchange transfusion is 45-50%.
Plasma bilirubin tends to rise or rebound after an exchange transfusion because:
entry of bilirubin from the extravascular spaces and tissues
continued production of bilirubin from residual maternal antibody coating newly released RBCs.
Additional exchanges are necessary if the bilirubin level threatens to exceed 20 mg/dL in a full term infant.
Phototherapy is used as an adjunct

4. Exchange Transfusion
Infant severely affected with HDFN will require exchange transfusion.
Goals – MEMORIZE
Reduce the load of accumulated bilirubin
Reduce the amount of unbound maternal antibody.
Remove antibody coated cells, whose destruction would further raise the bilirubin load.
Replace infant cells with RBCs compatible with the maternal antibody, which will have a normal survival rate.
Replacement with donor plasma restores albumin and any needed coagulation factors.

5. Compatibility Testing for Exchange
Donor blood
Usually O negative,
lacks all antigens to which mom has antibodies
Coomb’s crossmatch compatible.
Serum or plasma of infant or mother may be used for crossmatch, mother best as
larger quantity available and
has highest concentration of antibody.
Blood must be compatible with baby AND mom.

6. Compatibility Testing for Exchange
Mom’s blood not available:
Use eluate from cells which concentrates antibody, serum may have lower concentration.
Use of infant’s serum or eluate not ideal but preferable to delaying the transfusion.

7. Selection and Preparation of Blood
CPD, as fresh as possible, preferably <5 days old.
Hematocrit of 80% or greater to minimize volume overload.
Volume transfused ranges from mL depending on the fetal size and age.
CMV negative
Hemoglobin S negative
IRRADIATED
Preparation
Physician will specify a hematocrit.
Reconstitute donor unit with plasma.
Most facilities prefer to use group O red cells and AB plasma.

8. Exchange Transfusion Cannulate umbilical vein. “Pull” baby blood. Wait
“Push” donor blood.
Repeat

9. Rh Immune Globulin
Concentrated solution of IgG anti-D from human plasma.
A 1 mL FULL dose contains 300 ug of anti-D to counteract immunizing effects of:
15 mLs D pos packed RBCs
30 mLs D pos whole blood
Does NOT transmit hepatitis or HIV

10. Rh Immune Globulin
Used to protect D negative individuals exposed to D positive rbcs.
Prenatally to D neg moms
Postnatally when D neg gives birth to D pos
After transfusion of D neg with blood products with D pos rbcs.
The IgG anti-D coats D pos rbcs causing their destruction before being recognized.

11. HDFN

12. Rh Immune Globulin Became available in 1968. Major breakthrough.
Immunization to D antigen from 8% during first pregnancy and additional 8% during second pregnancy to 1-2&

13. Antepartum Rh Immune Globulin
Discovered that D neg women given RhIg after birth of D pos child still developing anti-D – termed “RhIg failures”
Discovered that immunization decreased from 1% to less than 0.1% if dose given at 28 weeks.

14. Rh Immune Globulin Testing at delivery:
ABO/D type to identify D negative mothers.
Antibody screen to detect immune antibodies.
ABO/D type of cord blood.

15. Women Who are NOT RhIg Candidates
D negative women with D negative babies.
D positive women
D negative women immunized to the D antigen.

16. Evaluation of Anti-D in Postpartum Specimen
Women who receive antenatal RhIg may have positive antibody screen.
History IMPORTANT
RhIg given at 28 weeks WEAKLY reactive if delivery is full term.
Do NOT assume antibody is immune.
When in doubt give it out.

17. Evaluation of Anti-D in Postpartum Specimen
Clues to distinguish origin of antibody
RhIg is IgG, if anti-D is saline reactive probably represents active immunization.
RhIg rarely achieves titer above 4, high titer anti-D is immune.
Obtain confirmation from physician records.
Administer RhIg if there is any doubt.

18. Administration of RhIg
Antepartum dose given at weeks gestation, 92% of women who develop anti-D do so at 28 weeks or later.
Obtain blood sample BEFORE administering.
Perform T&S
Must be D and weak D negative
Identify antibodies if screen positive, if antibody other than anti-D give RhIg

19. Administration of RhIg
RhIg anti-D may be detectable for as long as 6 months.
Half live is 23 days, 300 ug given at 28 weeks, ug present at delivery 12 weeks later.
Inject within 72 hours of delivery.
Do not delay or omit injection due to problems with antibody screen interpretation.
If RhIg NOT given within 72 hours give as soon as possible, do not withhold!!!

20. RhIg Utilization Gap
Administration of RhIG is indicated, but sometimes inadvertently omitted, after several common events (MEMORIZE):
abortion/miscarriage
ectopic pregnancy
antepartum hemorrhage
fetal death
amniocentesis
chorionic villus sampling
cordocentesis
After an external cephalic version of a breech fetus
blunt trauma to the abdomen

21. Detection of Fetomaternal Hemorrhage
Immunization to D can occur if amount of fetal rbcs entering maternal circulation exceeds 30 mLs of whole blood, the amount covered by a single 300 ug dose of RhIg.
Risk of hemorrhage exceeding 30 mLs estimated to be 0.3%.
Standards requires that post-partum maternal sample be evaluated for excessive bleed.

22. Detection of Fetomaternal Hemorrhage
Weak D test was used for screening for excessive fetomaternal hemorrhage (FMH).
Fetal D pos rbcs would be coated by the reagent anti-D
Microscopic evaluation of the weak D reveals small, tight clumps of rbcs, MF agglutination
Very insensitive test and requires skill.
CAP survey 12% of labs failed to detect D pos cells in sample simulating 30 mL bleed.

23. Rosette Screening Test
Similar to weak D, drop of maternal rbcs incubated with anti-D which coats D antigens of fetal rbcs present.
Wash off anti-D.
Add D pos indicator rbcs which will bind to second Fab of the anti-D coating the cells.
Forms rosettes around the cells.
Detects FHM of approximately 10 mLs
Qualitative test only, if positive must perform Kleihauer Betke acid elution test to quantitate.
IMPORTANT: If baby types weak D positive a Kleihauer Betke MUST be done instead of the Rosette.

24. Kleihauer-Betke Acid Elution
Hemoglobin F is resistant to acid elution, adult hemoglobin is not.
Expose thin smear of blood to acid buffer, adult hemoglobin eluted out.
Smears are stained with hematoxylin and erythrosin B.

25. Kleihauer-Betke Acid Elution
Adult rbcs appear as ghost cells, stroma only, while fetal cells appear bright pink and refractile.
Count number of fetal cells in 2000 adult cells.
Precision of test poor even in experienced hands.

26. Kleihauer-Betke Acid Elution
To calculate volume of fetomaternal hemorrhage:
Determine percentage of fetal rbcs.
Multiply x 50 (represents 5000 mL, arbitrary blood volume of mother).
Divide by 30, volume of whole blood covered by 1 vial of RhIg, to determine number of vials required.
Round up or down and ADD 1 VIAL for safety.

27. Kleihauer-Betke Acid Elution
EXAMPLE
26 fetal cells counted in 2000 total.
26/2000= 1.3%
1.3 x 50 = 65 mL of fetal whole blood
65/30 = 2.2 doses of RhIg
2.2+ 1(safety dose) = 3 vials
You will be required to calculate for the next exam.

28. Giving RhIg Given intramuscularly.
No more than 5 doses should be injected at one time.
If more than 5 doses needed space over 72 hours.

29. Kleihauer-Betke Acid Elution
False positive may occur if patient has persistent presence of hemoglobin F due to certain diseases: sickle cell anemia, thalassemia, acquired aplastic anemia, and several other hemoglobinopathies.
The amount of hemoglobin F in these conditions varies in the cell so the staining is not uniform.

30. Flow Cytometry
Uses anti-hemoglobin F antibodies to bind, followed by incubation with labeled anti-antibody.
Counted in flow cytometer.

31. End of Unit 11Part 3