Hemolytisk sjukdom

1
Hemolytisk sjukdom hos foster og nyfødd
Svein Magne Skulstad
Seksjon for Fostermedisin/Obstetrisk seksjon,
Kvinneklinikken Blodbanken Haukeland
Universitetssjukehus Nettundervisning for
Immunologi og transfusjonsmedisin 15.06.06
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Background Hydrops foetalis due to hemolytic
disease
Clinical findings Mild forms - intrauterine
death Anemia Reticulocytosis Hydrops
foetalis Hb5g/dl Heart failure Hepatosplenomegaly
Jaundice Hyperbilirubinemia Kernicterus
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Background Hydrops foetalis due to hemolytic
disease
Enlarged heart Hydrothorax with pericardial
effusion
Enlarged liver Ascites
4
Background Hydrops foetalis due to hemolytic
disease
Kernicterus Unconjugated bilirubin
deposits Cerebellum Basal ganglia Chorioathetosi
s, cerebral paresis, mental retardation
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Post-partum anti-D prophylaxisEffect on
morbidity and mortality
From 1969 250-300mg anti-D i.m. within 72
hours Major impact on mortality down from
12000 to165.000
Hussey Clarke. BMJ 1991 303 445-6
The frequency of primary anti-D formation per
10.000 births, and perinatal deaths from anti-D
hemolytic disease in Rogaland county from
1965-1994
Hervik et al. Abstract in Concensus conference on
anti-D prophylaxis, Edinburgh, UK, 1997
6
Blood Groups on the RBC
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7
Risk of fetal anemiaRelation to type of maternal
antibody
Anti-K Suppresses erythropoiesis at the
progenitor-cell level in addition to hemolysis.
Rapid evolvement of fetal anemia.
Vaughan et al. N Engl J Med 1998 338 798-803
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Model of topology for RhAG, RhCE and RhD
Exofacial
Intracellular
Exofacial
The RhAG-RhCE-RhD complex in the red blood cell
membrane 3 protein complex 11 helices
Intracellular
Exofacial
Intracellular
Avent Reid. Blood 2000 95 375-87
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RhD protein function
3D structure resolved
AmtB Ammonia transporter of E. coli Homology
to the Rh proteins Alternative Gas channels
for CO2? pH regulation?
Soupene et al. PNAS 2001 99 7769-73 Soupene et
al. PNAS 2004 101 7787-7792
Khademi et al. Science 2004 305
1587-94 Transfus Med Hemother 2004 31 (suppl
3) Kustu Inwood. Transfus Clin Biol 2006 in
press Nedjma et al. Biochem. J. 2005) 391 3340
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Three types of D neg
D
D
D
D
Wagner et al. Blood 2000, 95_3662-8 Transfus Med
Hemother 2004 31 (suppl 3) http//www.uni-ulm.de/
wflegel/RH/SympDGTI2004/
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Anti-D alloimmunization Epidemology
Rh-system is complex 52 different antigens Rh(D)
is highly immunogenic 85 Rh(D) Homozygote DD
(42) Heterozygote Dd (43) 15
Rh(D)- Homozygote dd
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Anti-D alloimmunization Epidemology
In 10 of all pregnancies Rh(D)- mother
Rh(D) fetus Rh(D)- women 60 of newborn
Rh(D) Transplacental hemorrhage - sensitisation
of the mother Dose ABO-compatible RhD
positive fetus increased risk Non-responders
15 Sensitisation of the mother 1.5 with post
partum prophylaxis
Contreras. Br J Obstet Gynaecol 1998 105 Suppl
18 7-10
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Anti-D alloimmunizationEtiology
Fetomaternal hemorrhage Spontaneous 3 At
birth gt0.2ml in 50 lt1 of fetomaternal
bleeds gt 2.5mL of fetal cells 0.1 ml
transferred sensitizes 30 of primigravida
at risk Sensitisation possible from week 6
Southcott et al. Blood 1999 93 4425-4435
Mollison et al. 1993 Blood transfusion in
clinical medicine, Blackwell, Oxford
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Fetomaternal hemorrhage Risk of sensitisation
Spontaneous abortions 1-2 Provoked abortions
4-5 In the third trimester 1-2 At
term 13-14 Amnioscentesis 1-2 Ectopic
pregnancy lt1 Caecarean section gt20 Transfusi
on of D pos blood 20-90
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Diagnosis
Maternal anti-D antibodies in blood samples
IAT-titer and titer increase Positive DAT
(direct antiglobulin test) in cord
blood Erytrocytes from the newborn covered by
maternal antibodies
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Diagnosis
Fetal ultrasound Blood flow velocities Edema -
hydrops Heart failure Establish RhD type of the
fetus/infant During pregnancy - in blood sample
from mother In cord (at birth or during
pregnancy)
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RhD immunization and prophylaxis
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Kumpel Elson. Trends Immunol 2001 22 26 31
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Anti-D prophylaxis modes of action
(a)

• Key process Inhibiton of B cells by
  crosslinking heterologous receptors
• (co-inhibition)
• (a) Crosslinking BCR and Fc?RIIb on the B cell,
  with D antigen and anti-D on fetal RBC
• (b) Interaction of anti-D coated RBC with Fc?RI
  and
• Fc?RIIIa on macrophage leads to rapid
  phagocytosis and destruction of D antigen
• Fetal RBCs are rapidly cleared from circulation

(b)
Modified from Kumpel Elson. Trends Immunol
2001 22 26 31
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When during pregnancy does immunization occur?
Silent sensitisation 55-80 of all
sensitisation due to transplacental hemorrhage
Robson et al. Br J Obstet Gynaecol 1998 105
129-34 Hughes et al. Br J Obstet Gynaecol 1998
105 38
Mortality underreported 35 of fetal losses due
to the condition occur before week 24
Whitfield et al. BMJ 1997 315 1504-5
Sensitisation often during first
pregnancy/delivery 20 infants born to
primigravidae with antibodies before or at
delivery 2 moderately affected (requiring
exchange transfusions) 16 mildly affected
Tovey et al. Lancet 1983 2 2446
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When during pregnancy does immunization occur?
42 (1.2) sentisised of 3487 Rh(D) neg (12.0 of
29 115 women) Few multigravida sentisised after
36 weeks 16/42 primigravida (38)
Tuohy et al. 2004 Aust N Z J Obstet Gynaecol 44
458-9
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Systematic reviewUK National Institute for
Clinical Excellence (NICE)
11 studies 31262 patients included 19793 as
controls Outcome Alloimmunization in
pregnancy Non-RCA studies Many studies poor in
design and performance
Jones et al. BJOG 2004 111 892-902 Chilcott et
al. Health Technol Assess 2003 7 iii-62
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Clinically important outcome measure
The number of mothers having delivered a
RhD-positive baby who are found to be sensitised
during a subsequent RhD-positive pregnancy Only
two studies had this as their primary end-point
Mayne et al. BMJ 1997 315 1588 MacKenzie et al.
Br J Obstet Gynaecol 1999106492 497 Chilcott
et al. Health Technol Assess 2003 7 iii-62.
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Women sensitised in a subsequent pregnancy as
result of a previous pregnancy
n number of RhD negative women in the trial
group undergoing subsequent pregnancy with a
RhD-positive infant
Mayne et al. BMJ 1997 315 1588 MacKenzie et al.
Br J Obstet Gynaecol 1999106492 497 Chilcott
et al. Health Technol Assess 2003 7 iii-62.
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Meta-analysis
Sensitisation rate of antenatal prophylaxis group
using meta-analysis OR of sensitisation, (95 CI)
in
Chilcott et al. Health Technol Assess 2003 7
iii-62
25
Results
OR for the risk of sensation 0.37 (two UK
non-randomised community-based studies)
Jones et al. BJOG 2004 111 892-902 Chilcott et
al. Health Technol Assess 2003 7 iii-62
26
NNTnumbers needed to treat
Reduction in risk of sensitisation 0.6 in
RhD-negative mothers at risk (i.e. carrying a
RhD-positive child) If the fetus is
RhD-positive NNT 1/0.006 166 to avoid a
sensitisation NNT 166/0.048 3458 to avoid a
fetal/neonatal death If the RhD status of the
fetus is unknown NNT 166 x 10/6 278 to avoid
a sensitisation NNT 278/0.048 5792 to avoid a
fetal/neonatal death
Knowing the RhD status of the fetus is important!
Jones et al. BJOG 2004 111 892-902 Chilcott et
al. Health Technol Assess 2003 7 iii-62
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Fetal RhD DNA detection in maternal plasma
To restrict antenatal immuno-prophylaxis to
RhD-negative women carrying a RhD positive
fetus Non-invasive fetal bloodgroup detection in
mothers at risk for alloimmunization
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Fetal DNA in maternal plasma
Present in very small amounts in plasma of normal
individuals Cell free, impossible to spin
down Increased in patients with cancer,
tumor-associated DNA mutations are present in
plasma DNA Placenta can be seen as a
pseudomalignant tissue gt Lo and co-workers
hypothesized that placental derived fetal DNA is
present in plasma
Lo et al. Lancet 1993 341 1147-8 Lo et al.
Lancet 1997 350 485-487
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Fetal RhD DNA concentration
16th week n 120, Mean 149 pg/ml (range 23-952)
gt 23 geq/ml 30th week n 299, Mean 522 pg/ml
(range 20-4640) gt 79 geq/ml
Geq genome-equivalent/ml
Range of Fetal DNA
numbers
DNA concentration (Pg/ml)
http//www.uni-ulm.de/wflegel/RH/SympDGTI2004/
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Rapid clearance of fetal DNA from maternal plasma
T1/2 10 100 hours after vaginal
delivery T1/2 16 minutes (range 4-30) after
CS
Nelson et al. Vox Sang 2001 80 112116
Lo et al. Am J Hum Gen 1999 64 218-24
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Concordant results in 99.1 of the tested samples
(n1257)
van der Schoot et al. Transfus Med Hemother 2004
31 (suppl 3) 50
32
Conclusions - fetal RhD genotyping
At least as reliable as cord blood serology
(gt99 diagnostic accuracy) Can restrict
antenatal prophylaxis to D-negative women
pregnant of a D-positive child Postnatal cord
blood typing can be omitted, at least in all
women with a fetal D PCR result. Postnatal
prophylaxis directly after delivery, with
increased effectiveness Assay costs for reagents
and equipment are below 15 Euro/assay

Finning et al. Ann N Y Acad Sci 2004 1022
119-23 van der Schoot et al. Transfus Med
Hemother 2004 31 (suppl 3) 50
33
Fetal D blood grouping - trends in UK
Finning et al. Ann N Y Acad Sci 2004 1022 119-23
Reduction in the numbers of referreals to IBGRL
for fetal D blood grouping using invasively
derived fetal material, due to the introduction
(in 2001) of a test using free fetal DNA in
maternal blood
International Blood Group Reference Laboratory,
National Blood Service, Bristol, UK
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Antenatal anti-D prophylaxisSuggestion
Who? All RhD negative women with no anti-D
antibodies carrying a RhD positive
fetus When? Gestational week 17-20 and
32 Dose? 100?g each time Another 100?g at
birth Additional dose in patients at risk for
FMH Abdominal trauma, invasive procedures,
late abortion, vaginal bleeding during last
half of pregnancy, external version,
intrauterine death FMB estimated (flow
cytometry), anti-D prophylaxis in accordance
with volume
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Incremental cost effectiveness of providing RAADP
for RhD-negative primigravidae and for all
RhD-negative pregnant women
RAADP Routine antenatal anti-D prophylaxis
Adapted from UK NATIONAL INSTITUTE FOR CLINICAL
EXCELLENCE 2005, Technology Appraisal Guidance
No. 41
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Next part follows..
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Fetal surveillance in red blood cell
alloimmunization
Incidence stable at around 1 per 1000 live
births Fetal loss rates 4-15 (when fetal
transfusion required) Aims of surveillance Early
detection of fetal hemolytic anemia Timely
intervention with intrauterine blood
transfusion before hydrops occur Early
delivery when needed followed by neonatal
treatment
Oepkes. Eur J Obstet Gynecol Reprod Biol 2000
92 83-9 Ryan Morrow. Clin Perinatol
199421573-89 Schumacher et al. Obstet Gynecol
1996 88 137-50 Weiner et al. Am J Obstet
Gynecol 1991 165 1302-7
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When should ultrasound be done?
Antibody screening and titration identifies the
groups at risk anti-D, anti-Rh(c) Titer 64,
titer increase x 4 anti-K First examination
at time of screening or before When titer
8 regular examinations at 1-2 weeks intervals
Titer increase x 4 Other antibodies Only
examination when titers are high or a pronounced
titer increase is demonstrated
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Fetal anemia - hyperdynamic circulation
Anemia ?? viscosity ? ?? cathecolamines
? heart rate ? cardiac output ? Increase
in blood flow velocities in veins and
arteries Most important vessel Middle cerebral
artery
Kirkinen et al. Lancet 198111004-5 Kirkinen et
al. Br J Obstet Gynaecol 1983 90 640-3
Rightmire et al. Obstet Gynecol 1986 68 233236
Yyas et al. Am J Obstet Gynecol 1990 162
10661068 Mari et al. N Engl J Med 2000 342 9-14
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Modes of surveillance
Invasive testing Amniocentesis - Bilirubin -
Liley chart/Queenan's chart Cordocentesis -
Direct measurement of Hb/Hct Non-invasive
testing Ultrasound 2D Doppler
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Tests for fetal hemolysis?OD 450 spectrometry
Queenan's chart
Consept Hemolysis gives hyperbilirubinemia to be
detected in the amniotic fluid Unreliable
method Repeated amniocentesises with risk of
immunization! Should be discontinued!
Zone IV
Zone III
Zone II
Zone I
10
35
30
25
20
15
40
Nicolaides et al. AJOG 1986155 90-4 Vaughan et
al. AJOG 1994 171 247-52 Nicolaides et al. BMJ
1992 304 1155-6 Rahman et al. Acta Obstet
Gynecol Scand 1998 8 804-7
Spong et al. Am J Obstet Gynecol 2001 185 481-
4)
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2D ultrasound In surveillance of fetal anemia
Pericardial fluid
Edema in placenta
Acites
Dilated intrahepatic UV
2D ultrasound should not be the only
tool Hydrops and placenta edema is
visualized Spleen and liver enlargement
indicators for anemia In early second trimester,
hydrops before Doppler changes Doppler
examination in addition
Iskaros et al. Ultrasound Obstet Gynecol 1998
11 432-7 Dukler et al. Am J Obstet Gynecol 2003
188 1310-4
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MCA DopplerIn surveillance of fetal anemia
MCA Doppler predicts moderate to severe anemia
(5SD) Cutoff MCA Vmax gt 1.5 MoM
Specificity 88 (sensitivity 100) to predict Hb
concentrations of 0.65 MoM or lower (equivalent
to a Hb deficiency of 4.5 and 4.8g/dL at 30 and
40 weeks)
Mari et al. N Engl J Med 2000 342
9-14 Zimmermann et al. BJOG 2002 109
746752 Haugen et al. Acta Obstet Gynecol Scand
2002 81 227233
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MCA DopplerIn surveillance of fetal anemia
Middle cerebral artery Doppler Fetal anemia,
weeks 26.4
Before transfusion Vmax 0.56 m/s
After transfusion Vmax 0.49 m/s
MCA Doppler visualizes immediate effect of fetal
transfusion
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Surveillance in RhD immunization
Early gestation Maternal blood typing and
screening of antibodies
Mother RhD negative RhD antibodies Titration
Mother RhD negative No antibodies
Week 17-20 Fetal RhD genotyping in maternal plasma
Fetus RhD negative
Fetus RhD negative
Fetus RhD positive
Week 32 RhD antibodies Titration
Fetus RhD positive First ultrasound at 17-20
weeks (Doppler, MCA, UV, DV)
No further control in pregnancy
Anti-D prophylaxis Week 17-20 100?g
Fetal RhD serotyping at birth (to be discontinued)
Fetal RhD serotyping at birth (to be discontinued)
Anti-D prophylaxis Week 32 100?g
Ultrasound every 2nd week (Doppler, MCA, UV,
DV) Titration every 4th week
From week 28 onwards Titration every 2nd week
Delivery at 37 weeks
UL indicators of anemia (MCA Vmax 1.5
MoM) (And/or Titer increase 4)
Fetal RhD serotyping at birth (to be discontinued)
From week 32 onwards Titration every 2nd
week Ultrasound every week (Doppler, MCA, UV, DV)
Cordocentesis Transfusion Control weekly
thereafter
Anti-D prophylaxis At birth 100?g
Delivery at 37 weeks
Delivery at 34 weeks
46
Surveillance in immunization (K, Rh(c))
Early gestation Maternal blood typing and
screening of antibodies
Mother anti-K negative
Mother anti-c negative
Mother anti-c positive Titration
Mother anti-K positive Titration
No further control in pregnancy
Fetus RhD positive
No further control in pregnancy
First ultrasound at 18 weeks (Doppler, MCA, UV,
DV)
First ultrasound at 17-20 weeks (Doppler, MCA,
UV, DV)
Ultrasound every 2nd week (Doppler, MCA, UV,
DV) Titration every 4th week
From week 24 onwards Ultrasound every
week (Doppler, MCA, UV, DV) Titration every 2nd
week
UL indicators of anemia (MCA Vmax 1.5
MoM) (And/or Titer increase 4)
Cordocentesis Transfusion Control weekly
thereafter
From week 32 onwards Titration every week
Delivery at 37 weeks
Delivery at 34 weeks
47
Takk!