1. Haemolytic Disease of the Fetus and Newborn
Chapter 11
Mollison 11th Edition- notes and embellishments
Dr Debra Lane
Medical Director
Canadian Blood Services Winnipeg

2. First Case
Levine and Stetson (1939) described a woman who bore a macerated fetus and then developed a transfusion reaction after a transfusion from her husband
POSTULATED IT WAS PRODUCTS FROM THE FETUS

3. HDN-Definition
Haemolytic Disease of Newborn is a condition in which the lifespan of the infants red cells is shortened by the action of mother’s antibodies which have crossed the placenta
The disease starts in the fetus and should be renamed Hemolytic Disease of the Fetus

4. Sensitization
RH (D) negative fetus may develop a positive DAT by the 8th week
Severe death and anemia can occur by the 18th week
The hemolytic process is maximal at birth and decreases with the diminution of the maternal antibody
Jaundice increases after birth

5. +DAT≠ HDN
Many infants with a positive DAT do not have increased red cell destruction
Serum bilirubins rise in most infants first two to three days of life
Hemoglobins fall for the first two months of life

6. Transfer of Antibodies
Only occurs via the placenta
IgG only bound to FC receptor
Specialized neonatal FC receptor (FcRn)
Function depends on dimerization with β2 microglobulin
Only occurs between mother and fetus
It is an active process- only small amounts transferred early in pregnancy

7. IgG transfer This is slow initially
At term infants IgG may be higher than mothers
Half life two to three weeks in the newborn
DAT may remain positive for three months in non-treated infants

8. Most common Anti-A and Anti-B are the most common antibodies
ABO hemolytic disease is common, but not severe
Anti-D used to be the most common antibody causing HDN
Still the most common causing Fatal HDN

9. In England Wales fatal HDN:
Fatal anti-c and Kell
1977 4
1990 4

10. Immunogenicity D>c>K Most antibodies can cause HDN
Severe ones include RH, anti-K, -k, -Kpa, -Ku, -Jsa, -Jsb, -Jka, -Fya, -M, -U, -PP1Pk, -Dib, -LAN, LW, -FAR, -Good,
-WRA, -Zd

11. Quantification of Fetal Cells
Number of fetal cells
Number of Adult cells

12. Detection of Fetal Cells by Acid Elution
Described by Kleihauer and Betke
Blood films are treated with acid buffer
Counter-stained the fetal cells appear as red cells on a sea of white ghosts
Normally 1 to 2% red cells are fetal
In 25% of females the fetal hemoglobin rises during pregnancy and may reach 7%

13. Must remember Fetal cells are larger than adult cells
Not all fetal cells stain darkly by acid-elution
Arbitrary figure for maternal cell volume has to be assumed

14. Assumptions Fetal red cells 22% bigger
92% of the red cell stain darkly
Average red cell volume of recently delivered woman is 1800 mls
The volume of fetal red cells in the maternal circulation is 2400/ ratio of darkly staining cells to regular cells

15. Estimating Transplacental Hemmorhage-Slides
Using slides is inaccurate
Depends on the thickness of slides
Skill of technologists

16. Rosetting Tests Used as a screen
Anti-D is added to red cells from the D neg mom which coats any D pos cells
Red cells are washed
D positive (enzyme treated) detector cells are added which form rosettes around the D positive cells in the original sample

17. Flow Cytometry
Maternal sample treated with anti-D and then fluorescein-labelled anti-IgG
Can detect 1/1000
Lack of consensus in studies comparing flow to BK may be a reflection of technical differences.
Need standardization of flow
Gating of red cells
Not universally accessible

18. Fetal Cell detection in Third Trimester
Authors
28-30 weeks
30-39 weeks
Bowman and Pollock
0.40%
1.84%
Huchet et al
5.8%
7.0%

19. Trans Placental Hemorrhage (TPH) with Amniocentesis
1981 to 1984 of 1000 women with amniocentesis for genetic disorders,
2.6% had TPH >0.1 ml
1.6% > 1.0 ml

20. TPH with Amniocentesis
Bowman and Pollock
1200 having amniocentesis for HDN
2.3% TPH >0.1 ml
1.8% TPH > 1.0 ml

21. Chorionic Villus Biopsy
Done at 7 to 10 weeks
No fetal cells were detected
But αfetoprotein increases- so Rh Immune globulin is recommended

22. Normal Delivery 1.0% have 3.0 ml or more 0.3% have 10 mls or more
When there is ABO incompatibility between the fetus and mom, less fetal cells are found in circulation
Caesarean section and manual removal of placenta give a considerable increase in fetal cells

23. Clinical manifestations of HDN
Rapid jaundice
If not treated may develop kernicterus with:
Lethargy
Spasticity
Opisthotonos

24. Kernicterus Rare under 306μmol/L Non-immunologic hydrops 1/3500 births
Cardiac
Genetic
Twin to twin transfusion

25. Anti-D Develops late in first pregnancies- 28 weeks
First child rarely affected
Stillbirth rate
6% with second pregnancy
29% with third

26. Prenatal testing
All D negative women should be tested at the first antenatal visit and at 28 weeks
Anti-D at first trimester due to previous transfusion or undiagnosed or undisclosed abortion

27. New anti-D in Second Pregnancy
Due to missed anti-D
Anti-D given too late or insufficient dose

28. PCR for Prenatal Determination of “D” Status
PCR amplification of fetal DNA for RHD
Uses fetal DNA present in the maternal sample
No risk of TPH
No possibility of sensitizing the mother
RHD is absent in D negatives- in whites
Pseudo RHD in blacks

29. Real time PCR
Read Mollison

30. Antenatal Assessment
Used to include fetal blood sampling

31. Amniocentesis
Estimating the amount of bile pigment in amniotic fluid is performed by measuring the optical density at 450 nm between the observed density and an extrapolated baseline
Amount falls with pregnancy so the age of the fetus must be considered

32. Liley (1961)
Developed charts to determine the severity from 27 weeks onward
They cannot be used prior to 27 weeks
Liley had three zones indicating the approximate severity of HDN

33. Comparison of Liley {Delta}A450 values with bilirubin concentrations measured with the standard (A) and modified (B) iterative methods, and log-linear chart (C), extrapolated to 17 weeks of gestation, showing the three risk zones described by Liley
Egberts, J. et al. Clin Chem 2002;48:
Copyright ©2002 American Association for Clinical Chemistry

34. Examination of the Fetus by Ultrasongraphy and fetal Doppler blood flow
Can be used to diagnose hydrops fetalis
Ascites
Pleural effusions
Pericardial effusions
Skin edema (Bowman 1983)

35. Ultrasonography Can detect small pericardial effusions
Dilated cardiac chambers

36. Fetal Doppler Reported 2006 NEJM Oepkes et all 355(2) 156
Prospective International Trial of woman with anti-D, anti-c, anti-E,and anti-FYa
With a titre of 1/64
165 fetus
74 with anemia

38. Comparison Fetal Doppler versus Amniotic Fluid
Sensitivity
88 %
76%
Specificity
82%
77%
Accuracy
85%

39. Fetal Blood Sampling
Ultrasound guidance with a needle into the fetal umbilical or hepatic vein
394 woman with 606 samplings the fetal loss was 0.8%
Fetal MCV 118 to 135
Betke- Kleihauer Fetal hemoglobin determined prior to fetal transfusion

40. Assessment of Severity of HDN in Newborn
Cord blood gives the most reliable hemoglobin
13.6 to 19.6
Range is wider for first day of life

41. Methods of Treatment Plasma exchange in the mother IVIG to mom
IVIG to fetus
Transfusion of the fetus in-utero
Post –natal exchange transfusion
Phototherapy to reduce serum bilirubin

42. IVIG to mom/baby IVIG 2g/kg over 5 days
Weekly injections of 1g/kg +/- plasma exchange
Might act by saturating the fetal FcRn and inhibiting placental transfer
A small series was done transfusing fetuses with IVIG mean dose 85.7mg/kg/body weight
No benefit- Dooren et al 1994

43. Transfusion of the Fetus in Utero
1963 Liley used intra-peritoneal transfusion
Taken into the bloodstream via the sub-diaphragmatic lacunae and right lymphatic duct
Uptake depends on diagphragmatic movements
Easy to perform
Can be done in conjunction with intravascular transfusion
Cannot be done in infants without diaphragm movement

44. Intravascular Transfusion
Needle into the umbilical vein with ultra sonic guidance
Excellent results by Bowman 1990
16/22 hydropic fetuses survived
3/6 deaths were between weeks in fetuses of iv drug abusers
Rodeck 1984 infants weeks
25/29 with RH D HDN survived ( 10 hydropic)

45. Requirements for Exchange Transfusion blood
UK uses blood less than 72 hours
In Manitoba less than 5 days or we wash the units
The supranatant is removed and replaced with AB plasma
St Justines uses albumin- reduces donor exposures
O negtive, K negative and compatible with mothers antibody ( c negative if required)
PCV 0.70 to 0.85
Irradiated
CMV negative

46. Transfusion volume 50/ml /kg estimated non-hydropic weight
30-40ml/kg estimated non-hydropic weight for hydropic fetuses (Bowman 1994)
Red cells suspended in non-protein solutions such as SAG-M or Adsol should not be used for transfusion to the fetus in-utero. ( In Mollison no reference)

47. Alloimummization of the Mother
May occur with fetal transfusion
Fetal cells may be shed into the peritoneum
May enter maternal vessels on placenta
I had one mother develop three new antibodies
Donor red cells are less frequently the source of immunization than fetal cells

48. Exchange Transfusion- Postnatal
Diamond 1947 introduced the method
Blood is withdrawn and injected, intermittently through a catheter
Passed up umbilical vein
Primary objective- remove D pos rbcs
Secondary- remove bilirubin
Clinicians like to exchange relatively large amounts of blood( 200 ml/kg)

49. Blood requirements Plasma reduced Less than 7 days
Rh and Kell neg/ compatible with mom
PCV 0.60
Screened for anti-HBS
CMV neg
Mollison does not say irradiated- we irradiate.

50. Indications for Exchange Transfusion
When fetus is exchange prenatally they usually do not need exchanges just booster transfusions
80% okay with phototherapy
Exchange if bilirubin threatens to exceed 340umol/L
Hg should be checked every 10 days
if Hg, 70g/L 20ml/kg given- Bowman

51. Exchange continued
Not transfused prenatally- cord Hg, 110g/L is an indication for exchange

52. Phototherapy Light at 420-480nm bilirubin converts to biliverdin
First reported Cremer et al 1958
Tabb 1972 (controlled trial)
Some have combined phototherapy with IVIG and have avoided exchange
11/16 only phototherapy needed exchange
2/16 needed exchange with both (Rubo and Wahn 1991)

53. Kell HDN Very sever at any titre 1/1000 pregnancies
History of transfusion in 88%
1/40,000 Bowman 1994
Fetal anemia is due to the suppression of erythropoiesis
Kell antigens are present on early erythroblasts

54. Kell HDN
Poor correlation with severity of the disease and the mother’s titre
Women with anti-K should be sent to Fetal assessment immediately and not wait for a rise in titre
Bowman (1989) saw a hydrops at 23 with a anti-Kell of 8

55. Anti-k HDN Very rare May also be sever despite low titre
Anti-k (cellano) anti-k 16
The hemoglobin was 60g/L at 31 weeks Bowman 1989

56. Anti-c HDN Titre of 1:32 or greater identified all affected fetuses
32/49 deaths from HDN to 1990 in Wales
0.7/1000 pregnancies
40 to 50% immunized by transfusion
Fetus relatively often c negative
Antibody is of low titre

57. Anti-c Only 20% of c positive infants required exchange transfusion
(Astrup and Kornstad 1977)
C typing of fetal DNS
Polymorphism at nucleotide 307 of RHCE
Quantitative real time PCR can be used for c typing of cell-free DNS from maternal blood in pregnancies at risk (Finning 2004)

58. Anti-E Most common after anti-D Often naturally occurring
Seldom causes HDN
See more with Galileo
Review 1959 to 2004 one perinatal death due to anti-E alone (Joy)

59. Anti-e
Very rare
Disease usually mild

60. Anti-Fya Mild hemolytic disease
At titres of .1/64 they should be closely monitored

61. Anti-Jkb
Rarely causes sever HDN
Single case of hydrops

62. Anti-M
May fail to affect the fetus
Rare cases of hydrops-two cases

63. ABO hemolyti disease Only two cases of hydrops due to ABO
Main need for exchange is to treat hyperbilirubinemia
Moderate bilirubinemia controlled by phototherapy

64. I could go on forever- but this is it for today.
Questions?