2004 Fetal blood group genotyping from DNA from maternal plasma: an important advance in the management and prevention of haemolytic disease of the fetus and newborn.. Haematologica 53Su
Trang 1Borst-Eilers E (1972) Rhesusimmunisatie: onstaan en
pre-ventie MD Thesis, University of Amsterdam, Amsterdam
Bowell PJ, Wainscot JS, Peto TEA et al (1982) Maternal
anti-D concentrations and outcome in rhesus haemolytic
disease of the newborn BMJ 285: 327–329
Bowell PJ, Brown SE, Dike AE et al (1986) The significance
of anti-c alloimmunization in pregnancy Br J Obstet
Gynaecol 93: 144 –1048
Bowell PJ, Inskip MJ, Jones MN (1988) The significance of
anti-C sensitization in pregnancy Clin Lab Haematol 10:
251–255
Bowley CC, Dunsford I (1949) The agglutinin anti-M
asso-ciated with pregnancy BMJ ii: 681
Bowman JM (1983) Blood group immunization in obstetric
practice Curr Probl Obstet Gynecol 7: 4 – 61
Bowman JM (1984) Controversies in Rh prophylaxis In:
Hemolytic Disease of the Newborn G Garratty (ed.),
pp 67–85 Arlington, VA: Am Assoc Blood Banks
Bowman JM (1990) Treatment options for the fetus with
allo-immune hemolytic disease Transfus Med Rev 4: 191–207
Bowman JM (1994) Intrauterine and neonatal transfusion.
In: Scientific Basis of Transfusion Medicine KC Anderson,
PM Ness (eds) Philadelphia, PA: WB Saunders
Bowman JM, Pollock JM (1965) Amniotic fluid
spectro-photometry and early delivery in the management of
erythroblastosis fetalis Pediatrics 35: 815
Bowman JM, Pollock JM (1985) Transplacental fetal
hemor-rhage after amniocentesis Obstet Gynecol 66: 749–754
Bowman JM, Pollock JM (1987) Failures of intravenous Rh
immune globulin prophylaxis; an analysis of the reasons
for such failures Transfus Med Rev 1: 101–112
Bowman JM, Pollock J (1993) Maternal C w
alloimmuniza-tion Vox Sang 64: 226–230
Bowman JM, Chown B, Lewis M et al (1978)
Rh-immunization during pregnancy, antenatal prophylaxis.
Can Med Assoc J 118: 623
Bowman JM, Friesen AD, Pollack JM et al (1980) WinRho:
Rh immune globulin prepared by ion exchange for
intra-venous use Can Med Assoc J 123: 1121–1125
Bowman JM, Lewis M, De Sa DJ et al (1984) Hydrops fetalis
caused by massive maternofetal transplacental haemorrhage.
J Pediatr 104: 769–772
Bowman JM, Harman FA, Manning CR et al (1989)
Erythroblastosis fetalis produced by anti-k Vox Sang 56:
187–189
Brossard Y, Pons JC, Jrad I et al (1996) Maternal-fetal
hemorrhage: a reappraisal Vox Sang 71: 103–107
Brouwers HAA, Overbeeke MAM, van Ertbruggen I et al.
(1988a) What is the best predictor of the severity of ABO
haemolytic disease of the newborn? Lancet ii: 641– 644
Brouwers HAA, Overbeeke MAM, Huiskes E et al (1988b)
Complement is not activated in ABO-haemolytic disease of
the newborn Br J Haematol 68: 363–366
Bruce M, Watt AH, Gabra GS et al (1985) Rh null with
anti-Rh29 complicating pregnancy: the first example in the United Kingdom Commun Br Blood Transfus Soc, Oxford
Brumit MC, Carnahan GE, Stubbs JR et al (2002) Moderate
hemolytic disease of the newborn (HDN) due to anti-Rh17 produced by a black female with an e variant phenotype Immunohematology 18: 40 – 42
Budin P (1875) A quel moment doit-on pratiquer la ligature
du cordon ombilical? Progr Méd (Paris) 3: 750 (see also (1876) 4: 2 and 36)
Bürki U, Degnan TJ, Rosenfield RE (1964) Stillbirth due to anti-U Vox Sang 9: 209
Bystryn J-C, Graf MW, Uhr JW (1970) Regulation of body formation by serum antibody II Removal of specific antibody by means of exchange transfusion J Exp Med 132: 1279
anti-Caballero C, Vekemans M, Lopez del Campo JG et al (1977)
Serum alpha-fetoprotein in adults, in women during nancy, in children at birth, and during the first week of life:
preg-a sex difference Am J Obstet Gynecol 127: 384 Caine ME, Mueller-Heubach E (1986) Kell sensitization in pregnancy Am J Obstet Gynecol 154: 85–90
Cariani L, Romano EL, Martinez N et al (1995)
ABO-haemolytic disease of the newborn (ABO-HDN): factors influencing its severity and incidence in Venezuela J Trop Pediatr 41: 14 –21
Carpentier M, Meersseman F (1956) Considération sur le traitement, à propos d’un cas d’isoimmunisation gra- vidique vis-à-vis du facteur A Bull Soc Roy Belge Gynécol Obstet 26: 374
Chilcott J, Lloyd Jones M, Wight J et al (2003) A review of
the clinical effectiveness and cost-effectiveness of routine anti-D prophylaxis for pregnant women who are rhesus- negative Health Technol Assess 7(4)
Chilcott J, Tappenden P, Lloyd Jones M et al (2004) The
economics of routine antenatal anti-D prophylaxis for pregnant women who are rhesus negative Br J Gynaecol 111: 903–7
Chitkara U, Bussel J, Alvarez M et al (1990) High dose
intra-venous gamma globulin: does it have a role in the treatment
of severe erythroblastosis fetalis Obstet Gynecol 76: 703–708
Chiu RW, Murphy MF, Fidler C et al (2001) Determination
of RhD zygosity: Comparison of a double amplification refractory mutation system approach and a multiplex real- time quantitative PCR approach Clin Chem 47: 667–672 Chown B (1955) On a search for rhesus antibodies in very young foetuses Arch Dis Child 30: 237
Clarke CA, Mollison PL (1989) Deaths from Rh haemolytic disease of the fetus and newborn, 1977–87 J R Coll Phys 23: 181–184
Clarke SC, Whitfield AGW (1979) Deaths from rhesus haemolytic disease in England and Wales in 1977: accuracy
Trang 2of records and assessment of anti-D prophylaxis BMJ i:
1665–1669
Cohen F, Zuelzer WW (1967) Mechanisms of
isoimmuniza-tion II Transplacental passage and postnatal survival of fetal
erythrocytes in heterospecific pregnancies Blood 30: 796
Cohen F, Zuelzer WW, Gustafson DC et al (1964)
Mechanisms of iso-immunisation I The transplacental
passage of fetal erythrocytes in homospecific pregnancies.
Blood 23: 621
Contreras M, Gordon H, Tidmarsh E (1983) A proven case
of maternal alloimmunization due to Duffy antigens in
donor blood used for intrauterine transfusion (Letter) Br J
Haematol 53: 355–356
Costa FH, Cardim WH, Mellone O (1960) Fototerapia.
Novo recurso terapeutico na hiperbilirubinemia do
recem-nascido J Pediatr 25: 347–391
Crawford DH, Barlow MJ, Harrison JF et al (1983)
Production of human monoclonal antibody to rhesus
D antigen Lancet 1(8321): 386 –388
Crawford H, Cutbush M, Mollison PL (1953) Hemolytic
dis-ease of the newborn due to anti-A Blood 8: 620
Cremer RJ, Perryman P, Richards DH (1958) Influence
of light on the hyperbilirubinaemia of infants Lancet i:
1094
Dacus JV, Spinnato JA (1984) Severe erythroblastosis
sec-ondary to anti-Kp b sensitization Am J Obstet Gynecol
150: 888–889
Daffos F, Capella-Pavlovsky M, Forrestier F (1985) Fetal
blood sampling during pregnancy with use of a needle
guided by ultrasound: a study of 606 consecutive cases Am
Daniels GL, Hadley AG, Green CA (1999) Fetal anaemia due
to anti-Kell may result from immune destruction of early
erythroid progenitors Transfus Med 9(Suppl.): 16
Daniels GL, Finning K, Martin P et al (2004) Fetal blood
group genotyping from DNA from maternal plasma: an
important advance in the management and prevention of
haemolytic disease of the fetus and newborn Vox Sang 87:
225–232
Davey MG (1976a) Antenatal administration of anti-Rh:
Australia 1969–1975 In: Proceedings of Symposium on
Rh Antibody Mediated Immunosuppression Raritan, NJ:
Ortho Research Institute
Davey MG (1976b) Epidemiology of failures of Rh immune
globulin and ABO protection In: Proceedings of
Sym-posium on Rh Antibody Mediated Immunosuppression.
Raritan, NJ: Ortho Research Institute
Davidson LT, Merritt KT, Weech AA (1941)
Hyper-bilirubinemia in the newborn Am J Dis Child 61: 958
Davis BH, Olsen S, Bigelow NC et al (1998) Detection of
fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry Transfusion 38: 749–756
DeMarsh QB, Windle WF, Alt HL (1942) Blood volume of newborn infants in relation to early and late clamping of the umbilical cord Am J Dis Child 63: 1123
Denomme GA, Ryan G, Seaward PG et al (2004) Maternal
ABO-mismatched blood for intrauterine transfusion of severe hemolytic disease of the newborn due to anti-Rh17 Transfusion 44: 1357–1360
Desjardins L, Blajchman MA, Chintu C et al (1979) The
spectrum of ABO hemolytic disease of the new born infant.
J Pediatr 95: 447– 449
De Young-Owens A, Kennedy M, Rose RL et al (1997)
Anti-M isoimmunization: management and outcome at the Ohio State University from 1969–1995 Obstet Gynecol 90: 962–996
Diamond LK (1947) Erythroblastosis foetalis or haemolytic disease of the newborn Proc R Soc Med 40: 546
van Dijk BA, de Man AJM, Kunst VAJM (1994) tion of fetomaternal hemorrhage by a fluorescent micro- sphere method (Abstract) Vox Sang 67 (Suppl 2): 34
Quantita-Divakaran TG, Waugh J, Clark TJ et al (2001) Noninvasive
techniques to detect fetal anemia due to red cell ization: a systematic review Obstet Gynecol 98: 509–517 Dooren MC, Engelfriet CP (1993) Protection against Rh D- haemolytic disease of the newborn by a diminished trans- port of maternal IgG to the fetus Vox Sang 65: 59–61
alloimmun-Dooren MC, Kuijpers RWAM, Joekes EC et al (1992)
Protection against immune haemolytic disease of newborn infants by maternal monocyte-reactive IgG alloantibodies (anti-HLA-DR) Lancet i: 1067–1070
Dooren MC, van Kamp IL, Kanhai HHH et al (1993)
Evidence for the protective effect of maternal Fc-R blocking IgG alloantibodies HLA-DR in Rh-D haemolytic disease of the newborn Vox Sang 65: 55–58
Dooren MC, Kamp IL, Scherpenisse JW et al (1994) No
beneficial effect of low-dose fetal intravenous globulin administration in combination with intravascular transfusions in severe RhD haemolytic disease Vox Sang 66: 253–257
gamma-Dukler D, Oepkes D, Seaward G et al (2003) Noninvasive
tests to predict fetal anemia: a study comparing Doppler and ultrasound parameters Am J Obstet Gynecol 188: 1310–1314
DuPan RM, Wenger P, Koechli S et al (1959) Étude du
passage de la γ-globuline marquée à travers le placenta humain Clin Chim Acta 4: 110
Dziegiel MH, Koldkjaer O, Berkowicz A (2005) Massive antenatal fetomaternal hemorrhage: evidence for long- term survival of fetal red blood cells Transfusion 45: 539–544
Trang 3Edelman L, Margaritte C, Chaabihi H et al (1997) Obtaining
a functional recombinant anti-rhesus (D) antibody using
the baculovirus-insect cell expression system Immunology
91: 13–19
Einhorn MS, Granoff DM, Nahm MH et al (1987)
Concentration of antibodies in paired maternal and infant
sera: relationship to IgG subclass J Pediatr 111: 783–788
Eklund J (1978) Prevention of Rh immunization in Finland
A national study, 1969–1977 Acta Paediatr Scand 274
(Suppl.): 1–57
Eklund J, Nevanlinna HR (1971) Immuno-suppression
therapy in Rh-incompatible transfusion BMJ iii: 623
Eklund J, Nevanlinna HR (1973) Rh prevention: a report and
analysis of a national programme J Med Genet 10: 1
Eklund J, Hermann M, Kjellman H et al (1982) Turnover rate
of anti-D IgG injected during pregnancy BMJ 284: 854–855
Ellis MI, Hey EN, Walker W (1979) Neonatal death in babies
with Rhesus isoimmunization Q J Med (NS) 48: 211–225
Engelfriet CF, Ouwehand WH (1990) ADCC and other
cellular bioassays for predicting the clinical significance of
red cell alloantibodies In: Blood Transfusion: the Impact
of New Technologies M Contreras (ed.) Baillière’s Clinical
Haematology 3: 321–337
Falterman CG, Richardson J (1980) Transfusion reaction due
to unrecognized ABO hemolytic disease of the newborn
infant J Pediatr 97: 812–814
Finn R, Harper DT, Stallings SA et al (1963) Transplacental
hemorrhage Transfusion 3: 114
Finning KM, Martin PG, Soothill PW et al (2002) Prediction
of fetal D status from maternal plasma: introduction of
a new noninvasive fetal RHD genotyping service
Trans-fusion 42: 1079–1085
Finning K, Martin P, Daniels G (2004) A clinical service in the
UK to predict fetal Rh (Rhesus) D blood group using free
fetal DNA in maternal plasma Ann NY Acad Sci 1022:
119–23
Firan M, Bawdon R, Radu C et al (2001) The MHC class
I-related receptor, FcRn, plays an essential role in the
mater-nofetal transfer of gammaglobulin in humans Int Immunol
13: 993–1002
Fischer K (1961) Morbus Haemolyticus Neonatorum im
ABO-System Stuttgart: Georg Thieme Verlag
Fleetwood P, De Silva PM, Knight RC (1996) Clinical
significance of red cell antibody concentration in
preg-nancy (Abstract) Br J Haematol 93 (Suppl 1.): 13
Forestier F, Daffos F, Galacteaos F et al (1986)
Hematological values of 163 normal fetuses between 18
and 30 weeks gestation Pediatr Res 20: 342–346
Fraser ID, Tovey GH (1976) Observations on Rh
isoimmun-isation: past, present and future Clin Haematol 5: 149
Freiesleben E, Jensen KG (1961) Haemolytic disease of the
newborn caused by anti-M The value of the direct
con-glutination test Vox Sang 6: 328
Frigoletto FD, Greene MF, Benaceraff BR et al (1986)
Ultrasonographic fetal surveillance in the management of the isoimmunized pregnancy N Engl J Med 315: 430 – 432
Garner SF, Gorick BD, Lai WYY et al (1995) Prediction
of the severity of haemolytic disease of the newborn Vox Sang 68: 169–176
Gemke RJ, Kanhai HH, Overbecke MA et al (1986) ABO
and Rhesus phenotyping of fetal erythrocytes in the first trimester of pregnancy Br J Haematol 64: 689 – 697
Gerlini G, Ottaviano S, Sbraccia C et al (1968) Reattività
dell’antigene A1e suoi rapporti con la malattia emolitica del neonato da incompatibilità ABO Haematologica 53(Suppl.): 1019
Giblett ER (1964) Blood group antibodies causing hemolytic disease of the newborn Clin Obset Gynecol 7: 1044 Gilja BK, Shah VP (1988) Hydrops fetalis due to ABO incom- patibility Clin Pediatr (Phila) 27: 210 –212
Gitlin D, Kumate J, Urrusti J et al (1964) The selectivity of
the human placenta in the transfer of plasma proteins from mother to fetus J Clin Invest 43: 1938
Goodrick MJ, Hadley AG, Poole G (1997) Haemolytic ease of the fetus and newborn due to anti-Fy(a) and the potential clinical value of Duffy genotyping in pregnancies
dis-at risk Transfus Med 7: 301– 304
Gordon MC, Kennedy MS, O’Shaughnessy RW et al (1995)
Severe hemolytic disease of the newborn due to anti-Js b Vox Sang 69: 140 –141
Gorlin JB, Kelly L (1994) Alloimmunization via previous transfusion places female Kp b -negative recipients at risk for having children with clinically significant hemolytic disease of the newborn Vox Sang 66: 46 – 48
Gottstein R, Cooke RW (2003) Systematic review of venous immunoglobulin in haemolytic disease of the new- born Arch Dis Child Fetal Neonatal Ed 88: F6 –F10 Grannum PAT, Copel JA (1988) Prevention of Rh isoimmun- ization and treatment of the compromised fetus Semin Perinatol 12: 324 –335
intra-Grant CJ, Hamblin TJ, Smith DS et al (1983) Plasmapheresis
in Rh hemolytic disease: the danger of amniocentesis Int
J Artif Organs 6: 83–86 Greenwalt TJ, Sasaki T, Gajewski M (1959) Further exam- ples of haemolytic disease of the newborn due to anti-Duffy (anti-Fy a ) Vox Sang 4: 138
Greiss MA, Armstrong-Fisher SS, Perera WS et al (2002)
Semiautomated data analysis of flow cytometric estimation
of fetomaternal hemorhagge in D-women Transfusion 42: 1067–1078
Grozdea J, Alie-Daram S, Vergnes H et al (1995) About
diminished transport of maternal IgG (RhD tion) to the fetus Vox Sang 68: 134 –135
alloimmuniza-Grumbach A, Gasser C (1948) ABO-inkompatibilitäten und Morbus haemolyticus neonatorum Helv Paediatr Acta 3: 447
Trang 4Hackney DN, Knudtson EJ, Rossi KQ et al (2004)
Manage-ment of pregnancies complicated by anti-c isoimmunisation.
Obstet Gynecol 103: 24 –30
Hadley AG, Garner SF, Taverner JM (1993) AutoAnalyzer
quantification, monocyte-mediated cytotoxicity and
chemilu-minescence assays for predicting the severity of haemolytic
disease of the newborn Transfus Med 3: 195–200
Halbrecht I (1951) Icterus precox: further studies on its
fre-quency, etiology, prognosis and the blood chemistry of the
cord blood J Pediatr 39: 185
Hall AM, Cairns LS, Altmann DM et al (2005) Immune
responses and tolerance to the RhD blood group protein
in HL-A transgenic mice Blood 105: 2175–2179
Hammerström L, Smith CIE (1986) Placental transfer of
intravenous immunoglobulin (Letter) Lancet i: 681
Hardy J, Napier JAF (1981) Red cell antibodies detected in
antenatal tests on Rhesus positive women in south and mid
Wales 1948–1978 Br J Obstet Gynaecol 88: 91–100
Hartmann O, Brendemoen OJ (1953) Incidence of Rh antibody
formation in first pregnancies Acta Paediatr (Uppsala) 42: 20
Hay FC, Hull MGR, Torrigiani G (1971) The transfer of
human IgG subclasses from mother to foetus Clin Exp
Immunol 9: 355–358
Hayashida Y, Watanabe A (1968) A case of a blood group p
Taiwanese woman delivered of an infant with hemolytic
disease of the newborn Jap J Legal Med 22: 10
Helderweirt G (1963) Le passage de globules foetaux et
maternels à travers le placenta Ann Soc Belge Méd Trop
43: 575
Herron B, Reynolds W, Northcott M et al (1996) Data from
two patients providing evidence that the placenta may act
as a barrier to the maternal fetal transfer of anti-Lutheran
antibodies (Abstract) Transfus Med 6(Suppl 2): 24
Hindemann P (1966) Experimentelle und klinische
Unter-suchungen über eine transabdominale
Späteinsch-wemmung fetaler Erythrozyten in den mütterlichen
Kreislauf Gerburtsh Frauenheilk 26: 1359
Ho cevar M, Glonar L (1974) Rhesus factor immunization
In: The Ljubljana Abortion Study 1971–1973 L Ardolsek
(ed.) Maryland: National Institute of Health Center for
Population Research
Holtrop PC, Ruedisueli K, Regan R et al (1991) Double vs.
single phototherapy in low-birth-weight infants Pediatr
Res 29: 218A
Holzgreve W, Holzgreve B, Curry CJ et al (1985)
Nonimmune hydrops fetalis: diagnosis and management.
Semin Perinatol 9: 52– 67
Hopkins DF (1970) Maternal anti-Rh(D) and the
D-negative fetus Am J Obstet Gynecol 108: 268
Hoppe HH, Mester T, Hennig et al (1973) Prevention of Rh
immunisation Modified production of IgG anti-Rh for
intravenous application by ion exchange chromatography
(IEC) Vox Sang 25: 308
Howarth DJ, Robinson FM, Williams M et al (2002) A
modified Kleihauer technique for the quantitation of maternal haemorrhage Transfus Med 12: 373–378 Hsu TCS, Rosenfield RE, Rubinstein P (1974) Instrumented PVP-augmented antiglobulin tests III IgG-coated cells in ABO incompatible babies: depressed hemoglobin levels in type A babies of type O mothers Vox Sang 26: 326
feto-Huchet J, Crégut R, Pinon F et al (1975) Les hémorragies
foeto-maternelles et leur importance dans la pathologie perinatale Rev Fr Transfus Immunohématol 18: 361 Huchet J, Defossez Y, Brossard Y (1988) Detection of transplacental haemorrhage during the last trimester of pregnancy (Letter) Transfusion 28: 506
Hughes-Jones NC, Hughes MIJ, Walker W (1967) The amount of anti-D on red cells in haemolytic disease of the newborn Vox Sang 12: 279
Hughes-Jones NC, Ellis M, Ivona J et al (1971) Anti-D
concentration in mother and child in haemolytic disease
of the newborn Vox Sang 21: 135
Huntley CC, Lyerley AD, Littlejohn MP et al (1976) ABO
hemolytic disease in Puerto Rico and North Carolina Pediatrics 57: 875
Hussey R, Clarke CA (1992) Deaths from haemolytic disease
of the newborn in 1990 (Letter) BMJ 304: 444
Hutchinson AA, Drew JH, Yu VYH et al (1982)
Nonimmunologic hydrops fetalis: a review of 61 cases Obstet Gynecol 59: 347–352
Issitt PD (1981) The MN Blood Group System Cincinnati, OH: Montgomery Scientific Publications
Johnson PRE, Tait RC, Austin EB et al (1995) Flow
cyto-metry in diagnosis and management of large fetomaternal haemorrhage J Clin Pathol 48: 1005–1008
Jørgensen J (1975) Foeto-maternal blødning MD Thesis, University of Copenhagen, Copenhagen
Joy SD, Rossi KQ, Krigh D et al (2005) Management of
preg-nancies complicated by anti-E alloimmunisation Obstet Gynecol 105: 24 –28
Katz J (1969) Transplacental passage of fetal red cells in abortion; increased incidence after curettage and effect of oxytoxic drugs BMJ 4: 84
Kennedy GA, Shaw R, Just S et al (2003) Quantification
of feto-maternal haemorrhage (FMH) by flow cytometry: anti-fetal haemoglobin labelling potentially underestimates massive FMH in comparison to labelling with anti-D Transfus Med 13: 25–33
King JC, Sacher RA (1989) Percutaneous umbilical blood sampling In: Contemporary Issues in Pediatric Trans- fusion Medicine RA Sacher, RG Strauss (eds), pp 33–53 Arlington, VA: Am Assoc Blood Banks
Kirkman NN (1977) Further evidence for a racial ence in frequency of ABO hemolytic disease J Pediatr 90: 717
differ-Kleihauer E, Betke K (1960) Praktische Anwendung des
Trang 5Nachweises von Hb F-haltigen Zellen in fixierten
Blutaus-strichen Internist 1: 292
Kochwa S, Rosenfield RE, Tallal L et al (1961) Isoagglutinins
associated with erythroblastosis J Clin Invest 40: 874
Kohler PF, Farr RS (1966) Elevation of cord over maternal
IgG immunoglobulin: evidence for an active placental IgG
transport Nature (Lond) 210: 1070
Konugres AA, Fontain DK, Thiet M-P et al (1994)
Intrauterine transfusion to prevent fetal demise in a case of
fetal-maternal hemorrhage Vox Sang 67(Suppl 2): 145
Kornstad L (1983) New cases of irregular blood group
antibodies other than anti-D in pregnancy: frequency
and clinical significance Acta Obstet Gynecol Scand 62:
431– 436
Krevans JR, Woodrow JC, Nosenzo C et al (1964) Patterns
of Rh-immunization Commun 10th Congr Int Soc
Haematol, Stockholm
Kumpel BM, Goodrick J, Pamphilon DH et al (1995)
Human Rh D monoclonal antibodies (BRAD-3 and
BRAD-5) cause accelerated clearance of Rh D+ red blood
cells and suppression of Rh D immunization in Rh D–
volunteers Blood 86: 1701–1709
Kumpel BM, van de Winkel JGJ, Westerdaal NAC et al.
(1996) Antigen topography is critical for interaction of
IgG2 anti-red-cell antibodies with Fc γ receptors Br J
Haematol 94: 175–183
Lacey PA, Caskey CR, Werner OJ et al (1983) Fatal
hemolytic disease of a newborn due to anti-D in an
Rh-positive Du variant mother Transfusion 23: 91–94
Ladipo OA (1972) Management of third stage of labour, with
particular reference to reduction of fetomaternal
transfu-sion BMJ i: 721
Lakatos L, Csathy L, Nemes E (1999) ‘Bloodless’ treatment
of a Jehovah’s Witness infant with ABO hemolytic disease.
J Perinatol 19: 530–2
Lam GK, Subramanyam L, Orton S et al (2003) Minimizing
red blood cell contamination while isolating mononuclear
cells from whole blood: the next step for the treatment of
severe hemolytic disease of the fetus/newborn Am J Obstet
Gynecol 189: 1012–6
Lambin P, Debbia M, Puillandre P et al (2002) IgG1 and
IgG3 anti-D in maternal serum and on the RBCs of infants
suffering from HDN: relationship with severity of the
dis-ease Transfusion 42: 1537–1546
Lawler SD, Vanloghem JJ (1947) The rhesus antigen C w
caus-ing haemolytic disease of the newborn Lancet ii: 545
Lee SI, Heiner DC, Wara D (1986) Development of serum
IgG subclass levels in children Monogr Allergy 19: 108–121
Lee S, Bennett PR, Overton T et al (1996) Prenatal diagnosis
of Kell blood group (genotypes: KEL1 and KEL2) Am J
Obstet Gynecol 175: 455– 459
Legler TJ, Maas JH, Kohler M et al (2001) RHD sequencing:
A new tool for decision making on transfusion therapy
and provision of Rh prophylaxis Transfus Med 11: 383–388
Lenkiewicz B, Zupanska B (2000) Moderate hemolytic ease of the newborn due to anti-Hr0 in a mother with the D–/D-phenotype Immunohematology 16: 109–111
dis-Levene C, Sela R, Rudolphson Y et al (1977) Hemolytic
disease of the newborn due to anti-PP1P k (anti-Tj a ) fusion 17: 569
Trans-Levine P (1943) Serological factors as possible causes in taneous abortions J Hered 34: 71
spon-Levine P (1958) The influence of the ABO system on lytic disease Hum Biol 30: 14
hemo-Levine P, Stetson R (1939) An unusual case of intragroup agglutination JAMA 113: 126
Levine P, Burnham L, Katzin EM et al (1941) The role of
iso-immunization in the pathogenesis of erythroblastosis fetalis Am J Obstet Gynecol 42: 925
Levine P, Ferraro LR, Koch E (1952) Hemolytic disease of the newborn due to anti-S Blood 7: 1030
Levine P, Vogel P, Rosenfield RE (1953) Hemolytic disease of the newborn Adv Pediatr 6: 97
Lewi S, Clarke TK (1960) A hitherto undescribed enon in ABO haemolytic disease of the newborn Lancet, ii: 456
phenom-Li TC, Bromham DR, Balmer BM (1988) Fetomaternal macrotransfusion in the Yorkshire region 1 Prevalence and obstetric factors Br J Obstet Gynaecol 95: 1144 – 1151
Liedholm P (1971) Feto maternal haemorrhage in ectopic pregnancy Acta Obstet Gynecol Scand 50: 367
Liley AW (1961) Liquor amnii analysis in management
of pregnancy complicated by rhesus sensitization Am
J Obstet Gynecol 82: 1359 Liley AW (1963) Intrauterine transfusion of foetus in haemolytic disease BMJ ii: 1107
Lloyd-Evans P, Kumpel BM, Bromelow I et al (1996) Use of a
directly conjugated monoclonal anti-D (BRAD-3) for quantification of foetomaternal haemorrhage by flow cytometry Transfusion 36: 432– 437
Lloyd-Evans P, Guest AG, Austin EB et al (2000) Use of a
phycoerythrin-conjugated anti-glycophorin A monoclonal antibody as a double label to improve the accuracy of FMH quantification by flow cytometry Transfus Med 9: 155– 160
Lo YM, Hjelm NM, Fidler C et al (1998) Prenatal diagnosis
of fetal RhD status by molecular analysis of maternal plasma N Engl J Med 339: 1734 –1738
Longster GH, Robinson EAE (1981) Four further examples
of anti-In b detected during pregnancy Clin Lab Haematol 3: 351–356
Lubenko A, Contreras M (1989) A review: low-frequency red cell antigens Immunohematology 5: 7–14
Lubenko A, Contreras M, Habash J (1989) Should anti-Rh
Trang 6immunoglobulin be given to D variant women? Br J
Haematol 72: 429 – 433
Lubenko A, Contreras M, Portugal CL (1992) Severe
haemolytic disease in an infant born to an Rhnullproposita.
Vox Sang 63: 43 – 47
Maaskant-van Wijk PA, Faas BH, de Ruijter JA et al (1998)
Genotyping of RHD by multiplex polymerase chain
reac-tion analysis of six RHD-specific exons Transfusion 38:
1015–1021
MacAfee CAJ, Fortune DW, Beischer NA (1970)
Non-immunological hydrops fetalis J Obstet Gynaecol Br
Cwlth 77: 226
MacKenzie IZ, Bichler J, Mason GC et al (2004) Efficacy and
safety of a new, chromatographically purified rhesus (D)
immunoglobulin Eur J Obstet Gynecol Reprod Biol 117:
154–161
McNabb T, Koh T-Y, Dorrington KJ et al (1976) Structure
and function of immunoglobulin domains V Binding of
immunoglobulin G and fragments to placental membrane
preparations J Immunol 117: 882
Margulies M, Voto LS (1991) High dose intravenous gamma
globulin: does it have a role in the treatment of severe
ery-throblastosis fetalis Obstet Gynecol 77: 804–806
Mari G, Deter RL, Carpenter RL et al (2000) Noninvasive
diagnosis by Doppler ultrasonography of fetal anemia due
to maternal red cell alloimmunisation N Engl J Med 342:
9–14
Marsh GW, Stirling Y, Mollison PL (1970) Accidental
injec-tion of anti-D immunoglobulin to an infant Vox Sang 19:
468
Marsh WL, Redman CM (1990) The Kell blood group
system: a review Transfusion 30: 158–167
Martin WL, West AP Jr, Gan L et al (2001) Crystal structure
at 2.8A of an FcRn/heterodimeric Fc complex: mechanism
of pH-dependent binding Mol Cell 7: 867–877
Matson GA, Swanson J, Tobin JD (1959) Severe
haem-olytic disease of the newborn due to anti-Jk a Vox Sang 4:
144
Matsumoto H, Tamaki Y, Sato S et al (1981) A case of
hemolytic disease of the newborn caused by anti-M:
sero-logical study of maternal blood Acta Obstet Gynaecol
Japan 33: 525–528
Matthews CD, Matthews AEB (1969) Transplacental
haem-orrhage: spontaneous and induced abortion Lancet i:
694
Mayne KM, Bowell PJ, Pratt GA (1990) The significance of
anti-Kell sensitization in pregnancy Clin Lab Haematol
12: 379–385
Menticoglou SM, Harman CR, Manning FA et al (1987)
Intraperitoneal fetal transfusion: paralysis inhibits red cell
absorption Fetal Therapy 2: 154
Miescher S, Zahn-Zabal M, De Jesus M et al (2000) CHO
expression of a novel human recombinant IgG1 anti-RhD
antibody isolated by phage display Br J Haematol 111: 157–166
Miescher S, Spycher MO, Amstutz H et al (2004) A single
recombinant anti-RhD IgG prevents RhD immunization: association of RhD-positive red blood cell clearance rate with polymorphisms in the Fc γRIIA and FcγIIIA genes Blood 103: 4028–4035
Miles RM, Maurer HM, Valdes OS (1971) Iron-deficiency anaemia at birth Two examples secondary to chronic fetal-maternal hemorrhage Clin Pediatr 10: 223
Millar DS, Davis LR, Rodeck CH et al (1985) Normal blood
cell values in the early mid-trimester fetus Prenatal Diagn 5: 367–373
Miqdad AM, Abdelbasit OB, Shaheed MM et al (2004)
Intravenous immunoglobulin G (IVIG) therapy for significant hyperbilirubinemia in ABO hemolytic disease of the newborn J Matern Fetal Neonatal Med 16: 163–166
Miyoshi K, Kaneto Y, Lawai H et al (1988) X-linked
domin-ant control of F-cells in normal adult life: characterization
of the Swiss type as hereditary persistence of fetal globin regulated dominantly by gene(s) on X chromosome Blood 72: 1854 –1860
hemo-Mollison PL (1943) The survival of transfused red cells in haemolytic disease of the newborn Arch Dis Child 18: 161 Mollison PL (1951) Blood Transfusion in Clinical Medicine Oxford: Blackwell Scientific Publications
Mollison PL (1972) Quantitation of transplacental orrhage BMJ iii: 31 and 115
haem-Mollison PL (1983) Blood Transfusion in Clinical Medicine, 7th edn Oxford: Blackwell Scientific Publications Mollison PL, Cutbush M (1949a) Haemolytic disease of the newborn: criteria of severity BMJ i: 123
Mollison PL, Cutbush M (1949b) Haemolytic disease of the newborn due to anti-A antibodies Lancet ii: 173
Mollison PL, Cutbush M (1951) A method of measuring the severity of a series of cases of hemolytic disease of the new- born Blood 6: 777
Mollison PL, Cutbush M (1954) Haemolytic disease of the newborn In: Recent Advances in Paediatrics D Gairdner (ed.) London: J & A Churchill
Mollison PL, Walker W (1952) Controlled trials of the treatment of haemolytic disease of the newborn Lancet i: 429
Mollison PL, Engelfriet CP, Contreras M (1987) Blood Transfusion in Clinical Medicine, 8th edn Oxford: Blackwell Scientific Publications
Moncharmont P, Juron-Dupraz F, Rigal M et al (1990)
Haemolytic disease of two newborns in a rhesus anti-e alloimmunised woman Review of literature Haemato- logia 23: 97–100
Moores PP, Smart E, Gabriel B (1994) Hemolytic disease of the newborn in infants of an Ohmother (Letter) Trans- fusion 34: 1015–1016
Trang 7Morell A, Skvaril F, van Loghem E et al (1971) Human IgG
subclasses in maternal and fetal serum Vox Sang 21: 481
Morgan CL, Cannell GR, Addison RS et al (1991) The effect
of intravenous immunoglobulin on placental transfer of
a platelet-specific antibody: anti-Pl A1 Transfus Med 1:
209–216
MRC (1978) An assessment of the hazards of amniocentesis
(Report of a Working Party) Br J Obstet Gynaecol 85
(Suppl 2): 1
Munk-Andersen G (1958) Excess of group O-mothers in
ABO-haemolytic disease Acta Pathol Microbiol Scand 42:
43
Murray S (1957) The effect of Rh genotypes on severity in
haemolytic disease of the newborn Br J Haematol 3: 143
Murray S, Knox EG, Walker W (1965) Rhesus haemolytic
disease of the newborn and the ABO groups Vox Sang 10: 6
Nance SJ, Nelson JM, Arndt et al (1989a) Quantitation
of fetal-maternal hemorrhage by flow cytometry A simple
and accurate method Am J Clin Pathol 91: 288–292
Nance SJ, Nelson JM, Horenstein J et al (1989b) Monocyte
monolayer assay: an efficient noninvasive technique for
predicting the severity of haemolytic disease of the
new-born Am J Clin Pathol 92: 89–92
Ness PM, Salamon JL (1986) The failure of postinjection Rh
immune globulin titers to detect large fetalmaternal
hemor-rhages Am J Clin Pathol 85: 604 – 606
Nevanlinna HR (1953) Factors affecting maternal Rh
immun-ization Ann Med Exp Fenn 31(Suppl 2): 1–80
Nevanlinna HR (1965) ABO protection in Rh immunization.
Commun 10th Congr Eur Soc Haematol, Strasbourg
Nicolaides KH, Rodeck C (1992) Maternal serum anti-D
antibody concentration and assessment of rhesus
isoimmun-ization BMJ 304: 1155–1156
Nicolaides KH, Rodeck CH, Millar DS et al (1985a) Fetal
haematology in rhesus isoimmunization BMJ 290:
661–663
Nicolaides KH, Warenski JC, Rodeck CH (1985b) The
relationship of fetal plasma protein concentration and
hemoglobin level to the development of hydrops in rhesus
isoimmunization Am J Obstet Gynaecol 152: 341–344
Nicolaides KH, Rodeck CH, Mibashan RS et al (1986) Have
Liley charts outlived their usefulness? Am J Obstet Gynecol
155: 90–94
Nicolaides KH, Fontanarosa M, Gabbe SG et al (1988a)
Failure of ultrasonographic parameters to predict the
severity of fetal anaemia in rhesus isoimmunisation Am J
Obstet Gynecol 158: 920 –6
Nicolaides KH, Soothill PW, Clewell WH et al (1988b) Fetal
haemoglobin measurement in the assessment of red cell
immunisation Lancet i: 1073 –1075
Nicolini U, Rodeck CH (1988) A proposed scheme for
plan-ning intrauterine transfusion in patient with severe
Rh-immunisation J Obstet Gynecol 9: 162–163
Osborn LM, Lenarsky C, Oakes RC et al (1984)
Phototherapy in full-term infants with hemolytic ease secondary to ABO incompatibility Pediatrics 74: 371–374
dis-Owen RD, Woon HR, Foord AG et al (1954) Evidence for
actively acquired tolerance to Rh antigens Proc Natl Acad Sci USA 40: 420
Parinaud J, Blanc M, Grandjean H et al (1985) IgG
sub-classes and Gm allotypes of anti-D antibodies during nancy: correlation with the gravity of the fetal disease Am
preg-J Obstet Gynecol 151: 1111–1115 Pearson HA, Diamond LK (1959) Fetomaternal transfusion.
Am J Dis Child 97: 267 Peevy KJ, Wiseman HJ (1978) ABO hemolytic disease of the newborn: evaluation of management and identification of racial and antigenic factors Pediatrics 61: 475
Pembrey ME, Weatherall DJ, Clegg JB (1973) Maternal thesis of haemoglobin F in pregnancy Lancet i: 1350 Pepperell RJ, Barrie JU, Fliegner JR (1977) Significance of red-cell irregular antibodies in the obstetric patient Med
syn-J Aust ii: 453 Pereira L, Jenkins TM, Berghella V (2003) Conventional management of maternal red cell alloimmunization com- pared with management by Doppler assessment of middle cerebral artery peak systolic velocity Am J Obstet Gynecol 189: 1002–1006
Petz LD, Garratty G (2004) Immune Hemolytic Anemias, 2nd edn New York: Churchill Livingstone
Phibbs RH, Johnson P, Tooley WH (1974) Cardiorespiratory status of erythroblastic newborn infants II Blood volume, hematocrit, and serum albumin concentration in relation
to hydrops fetalis Pediatrics 53: 13
Polley MJ, Mollison PL, Rose J et al (1965) A simple
sero-logical test for antibodies causing ABO-haemolytic disease
of the newborn Lancet i: 291 Pollock A (1968) Transplacental haemorrhage after external cephalic version Lancet i: 612
Pollock JM, Bowman JM (1990) Anti-Rh(D) subclasses and severity of Rh hemolytic disease of the newborn Vox Sang 59: 176 –179
Popat N, Wood WG, Weatherall DJ (1977) Pattern of nal F-cell production during pregnancy Lancet ii: 377 Poulain M, Huchet J (1971) Appréciation de l’hémorragie foeto-maternelle après l’accouchement en vue de la préven- tion de l’immunisation anti-D (Bilan de 5.488 tests de Kleihauer) Rev Fr Transfus 14: 219
mater-Pratt GA, Bowell PJ, MacKenzie IZ et al (1989) Production
of additional atypical antibodies in Rh(D)-sensitised nancies managed by intrauterine investigation method Clin Lab Haematol 11: 241–248
preg-Proulx C, Boyer L, St-Amour I et al (2002) Higher affinity
human D MoAB prepared by light-chain shuffling and selected phage display Transfusion 42: 59–65
Trang 8Ranasinghe E, Goodyear E, Burgess G (2003) Anti-Ce
complicating two consecutive pregnancies with increasing
severity of haemolytic disease of the newborn Transfus
Med 13: 53–55.
Rawson AJ, Abelson NM (1960) Studies of blood group
antibodies IV Physico-chemical differences between
iso-anti-A,B and iso-anti-A or iso-anti-B J Immunol 85: 640
Reid ME, Chandrasekaran V, Sausais L et al (1996)
Dis-appearance of antibodies to Cromer blood group system
antigens during mid pregnancy Vox Sang 71: 48–50
Renaer M, van de Putte I, Vermylen C (1976) Massive
feto-maternal hemorrhage as a cause of perinatal mortality and
morbidity Eur J Obstet Gynecol Reprod Biol 6: 125
Renaer M, van der Putte I, Vermylen C (1976) Massive
feto-maternal hemorrhage as a cause of perinatal mortality and
morbidity Eur J Obstet Gynecol Reprod Biol 6: 125
Renkonen KO, Seppälä M (1962) The sex of the sensitizing
Rh-positive child Ann Med Exp Fenn 40: 108
Renkonen KO, Timonen S (1967) Factors influencing the
immunization of Rh-negative mothers J Med Genet 4: 166
Report from 9 Collaborating Laboratories (1991) Results of
tests with different cellular bioassays in relation to severity
of Rh D haemolytic disease Vox Sang 60: 225–229
Revill JA, Emblin KF, Hutchinson RM (1979) Failure of
anti-D immunoglobulin to remove fetal red cells from maternal
circulation Vox Sang 36: 93–96
Robinson AE (1981) Unsuccessful use of absorbed
autolo-gous plasma in Rh-incompatible pregnancy (Letter) N
Engl J Med 305: 1346
Robinson AE (1984) Principles and practice of plasma
exchange in the management of Rh hemolytic disease of the
newborn Plasma Therapy 5: 7–14
Rodeck CH, Letsky E (1989) How the management of
erythroblastosis fetalis has changed (Commentary) Br J
Obstet Gynaecol 96: 759–763
Rodeck CH, Nicolaides KH, Warshof SL et al (1984) The
management of severe rhesus isoimmunization by
feto-scopic intravascular transfusions Am J Obstet Gynecol
150: 769–774
Romano EL, Mollison PL (1975) Red cell destruction in vivo
by low concentrations of IgG anti-A Br J Haematol 29:
121
Romano EL, Hughes-Jones NC, Mollison PL (1973) Direct
antiglobulin reaction in ABO-haemolytic disease of the
newborn BMJ i: 524
Romano EL, Linares J, Suarez G (1982) Plasma fibrinogen
concentration in ABO-hemolytic disease of the newborn.
Int Arch Allergy Appl Immunol 67: 74 –77
Romano EL, Zabner-Oziel P, Soyano A et al (1983) Studies
on the binding of IgG and F(ab) anti-A to adult and
new-born group A red cells Vox Sang 45: 378–383
Romano EL, Rossi DML, Hagel I et al (1988) Infestacion por
Ascaris: una posible explicacion para los altos niveles de
IgG anti-A observados en la poblacion Venezolana Acta Cient Venezol 39: 75–78
Romano EL, Soyano A, Montano RF et al (1994) Treatment
of ABO hemolytic disease with synthetic blood group trisaccharides Vox Sang 66: 194 –199
Romans DG, Tilley CA, Dorrington KJ (1980) Monogamous bivalency of IgG antibodies I Deficiency of branched ABHI-active oligosaccharide chains on red cells of infants causes the weak antiglobulin reactions in hemolytic disease
of the newborn due to ABO incompatibility J Immunol 124: 2807–2811
Roopenian DC, Christianson GJ, Sproule TJ et al (2003) The
MHC Class I-like IgG receptor controls perinatal IgG transport, IgG homeostasis and fate of IgG-Fc-coupled drugs J Immunol 170: 3528–3533
Rosenfield RE (1955) A-B hemolytic disease of the newborn Analysis of 1480 cord blood specimens, with special refer- ence to the direct anti-globulin test and to the group O mother Blood 10: 17
Rosenfield RE, Ohno G (1955) A-B hemolytic disease of the newborn Rev Hématol 10: 231
Rubo J, Wahn V (1990) A trial with high-dose gamma lin therapy in 3 children with hyperbilirubinemia in rhesus incompatibility Monatsschr Kinderheilkd 138: 216 Rubo J, Wahn V (1991) High-dose intravenous gammag- lobulin in rhesus-haemolytic disease (Letter) Lancet 337: 914
globu-Sampietro M, Thein SL, Contreras M et al (1992) Variation
of HbF and F-cell number with the G-gamma Xmn l (C-T) polymorphism in normal individuals (Letter) Blood 79: 832–833
Schellong G (1964) Über den Einfluss mütterlicher Antikörper des ABO-systems auf Reticulocyten-zahl und Serumbilirubin bei Frühgeborenen Commun 10th Congr Int Soc Blood Transfus, Stockholm
Schneider J (1969) Prophylaxe der Rh-Sensibiliserung durch Anti-D-Applikation, in Ergebnisse der Bluttransfusions- forschung Bibl Haematol (Basel) 32: 113
Schneider J (1976) German Trials In: Proceedings of Symposium on Rh Antibody Mediated Immunosuppres- sion Raritan, NJ: Ortho Research Institute
Schur PH, Alpert E, Alper C (1973) Gamma G subgroups in human fetal, cord, and maternal sera Clin Immunol Immunopathol 2: 62
Scott JR (1976) Immunologic risks to fetuses from maternal
to fetal transfer of erythrocytes In: Proceedings of posium on Rh antibody Mediated Immunosuppression Raritan, NJ: Ortho Research Institute
Sym-Sebring ES (1984) Fetomaternal hemorrhage – incidence and methods of detection and quantitation In: Hemolytic Disease of the Newborn G Garratty (ed.), pp 87–117 Arlington, VA: Am Assoc Blood Banks
Sebring ES, Polesky HF (1990) Fetomaternal hemorrhage:
Trang 9incidence, risk factors, time of occurrence, and clinical
effects Transfusion 30: 344 –357
Sender A, Maigret P, Poulain M et al (1971) La règle de
com-patibilité transfusionnelle A.B.C à Ia période néonatale.
Bull Feb Soc Gynecol Obstet 23: 560
Seppola M, Ruoslahti E (1972) Alpha fetoprotein in amniotic
fluid: an index of gestational age Am J Obstet Gynecol
114: 595–598
Sherer DM, Abramowicz JS, Ryan RM et al (1991) Severe
fetal hydrops resulting from ABO incompatibility Obstet
Gynecol 78: 897–899
Simonovits I, Timár I, Bajtai G (1980) Rate of Rh
immuniza-tion after induced aborimmuniza-tion Vox Sang 38: 161–164
Sims DG, Barron SL, Wadehra V et al (1976) Massive
chronic feto-maternal bleeding associated with placental
chorioangiomas Acta Paediatr Scand 65: 271
Singleton BK, Green CA, Avent ND et al (2000) The
pres-ence of an RHD pseudogene containing a 37 base pair
duplication and a nonsense mutation in Africans with the
RhD-negative blood group phenotype Blood 95: 12–18
Smith G, Knott P, Rissik J et al (1998) Anti-U and haemolytic
disease of the fetus and newborn Br J Obstet Gynaecol
105: 1318–1321
Smith NA, Ala FA, Lee D et al (2000) A multi-centre trial
of monoclonal anti-D in the prevention of
Rh-immunisa-tion of RhD negative male volunteers by RhD + red cells
Transfus Med 10 (Suppl 1): 8
Southcott MJ, Tanner MJ, Anstee DJ (1999) The expression
of human blood group antigens during erythropoiesis in a
cell culture system Blood 93: 4425– 4435
Stone B, Marsh WL (1959) Haemolytic disease of the
new-born caused by anti-M Br J Haematol 5: 344
Tabb PA, Inglis J, Savage DCL et al (1972) Controlled trial of
phototherapy of limited duration in the treatment of
physio-logical hyperbilirubinaemia in low-birth-weight infants.
Lancet 2: 1211
Tannirandorn Y, Rodeck CH (1990) New approaches in the
treatment of haemolytic disease of the fetus In: Blood
Transfusion: the Impact of New Technologies M
Contreras (ed.) Baillière’s Clinical Haematology 3: 289–
320
Terry MF (1970) A management of the third stage to reduce
feto-maternal transfusion J Obstet Gynaecol Br Cwlth 77:
129
Tovey GH (1945) A study of the protective factors in
hetero-specific group pregnancy and their role in the prevention
of haemolytic disease of the newborn J Pathol Bact 57:
295
Tovey LAD (1986) Haemolytic disease of the newborn: the
changing scene Br J Obstet Gynaecol 93: 960–966
Tovey LAD, Townley A Stevenson BJ et al (1983) The
Yorkshire antenatal anti-D immunoglobulin trial in
primi-gravidae Lancet 2: 244 –246
Urbaniak SJ, Greiss MA, Crawford RJ et al (1984)
Prediction of the outcome of rhesus haemolytic disease of the newborn: additional information using an ADCC assay Vox Sang 46: 323–329
Urbaniak SJ, Duncan JI, Armstrong-Fisher SS et al (1999)
Variable inhibition of placental IgG transfer in vitro with commercial IvgG preparations Br J Haematol 107: 815–817
Valentine GH (1958) ABO incompatibility and haemolytic disease of the newborn Arch Dis Child 33: 185
Van der Schoot CE, Tax GHM, Rijnders RJP et al (2003)
Prenatal typing of Rh and Kell blood group system antigens: the edge of a watershed Transfus Med Rev 17: 31– 44
Van der Schoot CE, Soussan AA, Dee R et al (2004) Screening
for fetal RHD-genotype by plasma PCR in all D-negative pregnant women is feasible Vox Sang 87(Suppl 3): 9
Van Kamp IL, Klumper FJ, Meerman RH et al (2004)
Treatment of fetal anemia due to red cell alloimmunization with intrauterine transfusions in the Netherlands, 1988–
1999 Acta Obstet Gynecol Scand 83: 731–737
Vaughan JI, Warwick R, Letsky E et al (1994) Erythropoietic
suppression in fetal anemia because of Kell tion Am J Obstet Gynecol 171: 247–252
alloimmuniza-Vaughan JI, Manning M, Warwick RM et al (1998)
Inhibition of erythroid progenitor cells by Kell bodies in fetal alloimmune anemia N Engl J Med 338: 798–803
anti-Veall N, Mollison PL (1950) The rate of red cell exchange in replacement transfusion Lancet ii: 792
Viëtor HE, Kanhai HHH, Brand A (1994) Induction of tional red cell antibodies after intrauterine transfusions Transfusion 34: 970–974
addi-Voak D (1969) The pathogenesis of ABO haemolytic disease
of the newborn Vox Sang 17: 481 Voak D, Bowley CC (1969) A detailed serological study on the prediction and diagnosis of ABO haemolytic disease of the newborn (ABO HD) Vox Sang 17: 321
Voak D, Williams MA (1971) An explanation of the failure of the direct antiglobulin test to detect erythrocyte sensitiza- tion in ABO haemolytic disease of the newborn and observa- tions on pinocytosis of IgG anti-A antibodies in infant (cord) red cells Br J Haematol 20: 9
Wagner FF, Flegel WA (2000) RHD gene deletion occurred in the Rhesus box Blood 95: 3662–3668
Wagner T, Berer A, Lanzer G et al (2000a) Kell is not
restricted to erythropoietic lineage but is also expressed
on myeloid progenitor cells Br J Haematol 110: 409 – 411
Wagner T, Bernaschek G, Geissler K (2000b) Inhibition of megakaryopoiesis by Kell-related antibodies N Engl J Med 343: 72
Walker RH (1984) Relevance in the selection of serologic tests for the obstetric patient In: Hemolytic Disease of the
Trang 10Newborn G Garratty (ed.), pp 173–200 Arlington, VA:
Am Assoc Blood Banks
Walker RH, Hartrick MB (1991) Non-ABO clinically
signi-ficant erythrocyte alloantibodies in Caucasian obstetric
patients (Abstract) Transfusion 31(Suppl.): 52S
Walker RH, Batten DG, Morrison MM (1993) The current
rarity of Rh D hemolytic disease of the newborn in a
com-munity hospital Am J Clin Pathol 100: 340–341
Walker W (1958) The changing pattern of haemolytic disease
of the newborn (1948 –1957) Vox Sang 3: 225, 236
Walker W, Murray S, Russell JK (1957) Stillbirth due to
haemolytic disease of the newborn J Obstet Gynaecol Br
Emp 44: 573
Wang AC, Faulk WP, Stuckey MA et al (1970) Chemical
dif-ferences of adult, fetal and hypogammaglobulinemic IgG
immunoglobulins Immunochemistry 7: 703
Ward HK, Walsh RJ, Kooptzoff O (1957) Rh antigens and
immunological tolerance Nature (Lond) 179: 1352
Warren RC, Butler J, Morsman JM et al (1985) Does
chori-onic villus sampling cause fetomaternal haemorrhage?
Lancet i: 691
Weisert O, Marstrander J (1960) Severe anemia in a newborn
caused by protracted feto-maternal ‘transfusion’ Acta
Paediatr 49: 426
Wenk RE, Goldstein P, Felix JK (1985) Kell
alloimmun-ization, hemolytic disease of the newborn, and perinatal
management Obstet Gynecol 66: 473 – 476
WHO (1971) Prevention of Rh sensitization Technical
Report Series 468
Wiener AS, Wexler LB, Hurst JG (1949) The use of exchange
transfusion for the treatment of severe erythroblastosis due
to A-B sensitization with observations on the pathogenesis
of the disease Blood 4: 1014
Williamson LM, Warwick RM (1995)
Transfusion-associated graft-versus-host disease and its prevention.
Blood Rev 9: 251–261
Walport MJ, Lachmann PJ (1993) Complement In: Clinical
Aspects of Immunology, 5th edn PJ Lachmann, K Peters,
FS Rosen et al (eds) Oxford: Blackwell Scientific
Publications, pp 347–375
Wolter LC, Hyland CA, Saul A (1993) Rhesus D genotyping using polymerase chain reaction Blood 82: 1682–1683 Woodrow JC (1970) Rh immunization and its prevention Ser Haematol 3: no 3
Woodrow JC, Donohoe WTA (1968) Rh-immunization by pregnancy: results of a survey and their relevance to pro- phylactic therapy BMJ iv: 139
Woodrow JC, Finn R (1966) Transplacental haemorrhage.
Br J Haematol 12: 297
Woodrow JC, Clarke CA, Donohoe WTA et al (1965)
Prevention of Rh-haemolytic disease: a third report BMJ i: 279
Worlledge S, Ogiemudia SE, Thomas CO et al (1974) Blood
group antigens and antibodies in Nigeria Ann Trop Med Parasitol 68: 249
Yao AC, Moinian M, Lind J (1969) Distribution of blood between infant and placenta after birth Lancet ii: 871 Yeung CY, Hobbs JR (1968) Serum-γG-globulin levels in normal, premature, post-mature, and ‘small-for-dates’ newborn babies Lancet ii: 1167
Yoshida Y, Yoshida H, Tatsumi K et al (1981) Successful
antibody elimination in severe M-incompatible pregnancy.
N Engl J Med 305: 460 – 461
Zhu X, Meng G, Dickinson BL et al (2001) MHC class
I-related neonatal Fc receptor for IgG is functionally expressed in monocytes, intestinal macrophages and dendritic cells J Immunol 166: 3266–3276
Zipursky A (1971) The universal prevention of Rh ization Clin Obstet Gynecol 14: 869
immun-Zipursky A, Pollock J, Neelands P et al (1963a) The
transplacental passage of foetal red blood-cells and the pathogenesis of Rh immunization during pregnancy Lancet ii: 489
Zipursky A, Pollock J, Chown B et al (1963b) Transplacental
foetal hemorrhage after placental injury during delivery or amniocentesis Lancet ii: 493
Zuelzer WW, Kaplan E (1954) ABO heterospecific pregnancy and hemolytic disease IV Pathologic variants Am J Dis Child 88: 319
Trang 11contained mixtures of several antibodies This problemwas partly overcome by applying extensive statisticalanalysis to the results (van Rood and van Leeuwen1963) in order to define single specificities Progressbecame more rapid with (1) the replacement of leuco-agglutination by lymphocytotoxicity tests and (2) therealization that HLA antibodies are frequently formed
in pregnancy, particularly as these antibodies, in trast with those formed after blood transfusion aredirected against a limited number of HLA antigens(Payne and Rolfs 1958; van Rood 1958) and (3) theorganization of international histocompatibility work-shops in which different laboratories were able to com-pare results by sharing reagents and typing commonpanels of cells These workshops, which continue on aregular basis, have been instrumental in the orderlydevelopment of the HLA system and its nomenclature(Bodmer 1997) The HLA antigens first detected werefound to be encoded by three closely linked genes:
con-HLA-A, -B and C, subsequently named class I genes.
The observation that lymphocytes from two related individuals can stimulate each other to blast formation when cultured together (mixed lymphocyteculture (MLC) assay), and that the antigens respons-ible for this stimulation are inherited together withHLA antigens, led to the discovery of HLA-D antigens
un-(Bach and Hirschhorn 1964; Bain et al 1964), which
were later detected serologically on B cells and named
DR antigens (van Rood et al 1975, 1976; van Rood
and van Leeuwen 1976) HLA-DR molecules togetherwith HLA-DQ and HLA-DP constitute the classicalclass II molecules
The ability to study HLA genes and their alleles
at the molecular level has enormously advanced the
and plasma components
13
Antigens expressed on leucocytes and platelets include
HLA class I and II molecules as well as those that are
specific for particular cells and those that are shared
with red cells In this chapter, these antigens, their
structure, function and corresponding antibodies
are described, together with methods for detecting
them The chapter includes an account of the clinical
relevance of these systems and effects owing to related
incompatibilities
The human leucocyte antigen (HLA)
system
The human leucocyte antigens (HLAs), coded by genes
of the major histocompatibility complex (MHC) are
cell surface glycoproteins that are critical in
deter-mining the compatibility of tissue grafts, in selecting
donor–recipient pairs for transfusion and
transplanta-tion and in designing epitope-specific cellular
immuno-therapy HLA antibodies are commonly formed after
blood transfusion and pregnancy The name is
mis-leading HLA molecules are expressed on a wide range
of cells in addition to leucocytes, and although these
molecules prove ‘alloantigenic’ to gravid women and
to transfusion and graft recipients, they function not
as antigens but as peptide chaperones crucial to the
process of adaptive immunity (Paul 2003; Wang et al.
2005) HLA molecules play a key role in host defences
by presenting foreign antigens to the immune
The first HLA antigen to be clearly defined was
HLA-A2, first named MAC (Dausset 1958) Early
work on the definition of HLA antigens was
complic-ated by the poor reproducibility of the
leucoagglutina-tion assay and by the fact that the early seroreagents
Trang 12knowledge of the HLA genes Not only is the region
now known ‘nucleotide by nucleotide’ at the genome
level, but also hundreds of alleles of the different loci
have been sequenced in the population Most HLA
typing is now done at the DNA level
Human leucocyte antigen: the human major
histocompatibility complex
The name MHC refers to the ability of the genes of this
genomic region to determine graft rejection between
individuals of the same species In the 1960s, the HLA
genes, first discovered through leucocyte
agglutina-tion, were established as the genes responsible for graft
rejection in man The physiologic function of these
molecules was determined during the following decade:
presentation of antigens to T cells (Zinkernagel and
Doherty 1974) The mechanism of this ‘HLA
restric-tion’ was first explained by Townsend and co-workers
(1986), who showed that synthetic peptides could be
presented to the T cell The final explanation of how
this peptide could be presented to the T cell awaited
the discovery of the structure of the HLA molecules in
1987 and the crystallization of a T-cell receptor bound
to an MHC molecule
The extremely polymorphic, closely linked genes of
the HLA system are located in a region that spans
about 4000 kilobases (kb) on the short arm of
chromo-some 6 (Breuning et al 1977) Moving from
centro-mere to telocentro-mere, the class II genes are separated
from the class I genes by a number of functionally
unrelated genes (and pseudogenes) that encode the
complement factors C2, C4a and C4b heat shock
proteins, cytokines and enzymes (class III genes)
(Fig 13.1)
After three decades of maps of ever increasing
elaboration, the complete sequence of the human
MHC was published in 1999 by the MHC Sequencing
Consortium (1999)
Class I region
HLA-A, -B and -C code for the heavy chain of the
MHC class I molecules expressed on most cells
HLA-F, -G and -E code for the heavy chain of non-classical
class I molecules, with highly specialized functions
The MIC genes or human MHC class I chain-related
genes encode stress-inducible proteins implicated in
the regulation of NK cell activity HFE is a class I-like
gene located approximately 4 Mb telomeric of HLA-Fand responsible for most hereditary haemochromatosis
Class III region
TNFB and -A code for tumour necrosis factors, HSPgenes for heat shock proteins, C2, Bf and C genes forproteins of the complement system, and P450-C21Bfor a steroid 21-hydroxylase
Class II region
The HLA-DRBA1, -DQA1 and -DPA1 genes code foralpha chains of the DR, DQ and DP class II molecules.HLA-DRB1, -DQB1 and -DPB1 code for the betachain of the DR, DQ and DP class II molecules Inaddition to HLA-DRB1, which codes for the primaryHLA specificities such as DR1, DR2, DR4, etc., otherDRB genes code for the beta chain of the specificitiesDR52 (DRB3), DR53 (DRB4) and DR51 (DRB5) notpresent in all haplotypes The DO and DM mole-cules regulate the loading of exogenous peptides intoclass II molecules LMP2 and LMP7, which encode the subunits of the proteasome, and TAP1 and TAP2,which encode a peptide transporter, are involved in the processing and presentation of antigens by class Imolecules
HLA class I and II molecules: structure and function
HLA molecules engage two distinct arms of the mediated immune response MHC class I moleculespresent antigen to cytotoxic T cells (CTLs), whereasMHC class II present to helper T cells Antigens are notpresented by HLA molecules as intact proteins Theantigen must first be degraded to peptide fragmentsand presented in the context of the HLA molecule tothe T-cell receptor
T-cell-HLA class I molecules
HLA-A, -B and -C genes produce a transmembraneglycosylated polypeptide of molecular weight 4300(the α or heavy chain) linked non-covalently to β2-microglobulin, a non-polymorphic and non-glycosylated polypeptide of molecular weight 1200(the β or light chain), which is encoded by a gene on
chromosome 15 (Snary et al 1977a; Barnstable et al.
Trang 13100- 200- 300- 400- 500- 600- 700- 800- 900- 1000- 1100- 1200- 1300- 1400- 1500- 1600- 1700- 1800- 1900- 2000- 2100- 2200- 2300- 2400- 2500- 2600- 2700- 2800- 2900- 3000- 3100- 3200- 3300- 3400-
0-HFE
C L A S S I
C L A S S I I I
C L A S S I I
HLA-F MICE
HLA-DPA2 HLA-DPA3 HLA-DPB2
HLA-A
HLA-E
HLA-C HLA-B
MICA
TNFA
HSPA1L
HLA-DOB TAP2 LMP7 TAP1 LMP2 HLA-DMB HLA-DMA HLA-DOA
HSPA1A HSPA1B C2 C4B P450-C21B
HLA-DRA HLA-DRB3 HLA-DRB1 HLA-DQA1 HLA-DQB1
HLA-DPA1 HLA-DPB1
BF
MICB TNFB
Fig 13.1 The segment of the small arm of chromosome 6
that contains the HLA complex is shown in detail The first
bar shows the division of the complex into class I, II and III
regions The ruler indicates the number of kilobases The
genomic map shows the approximate positions of the gene
loci mentioned in the text Bars to the right show expressed
genes and to the left, pseudogenes (not expressed) In bold
are shown the genes coding for the heavy chain of the classical class I molecules and the alpha and beta chains of
the class II molecules Class I region: HLA-A, B and C code
for the heavy chain of the MHC class I molecules expressed
on most cells HLA-F, G and E code for the heavy chain of non-classical class I molecules, with highly specialized functions The MIC genes or human MHC class I
Trang 141978) The extracellular part of the heavy chain
con-sists of three domains: α1, α2 and α3 (Fig 13.2)
The three-dimensional structure of class I molecules
has been revealed by X-ray crystallographic analysis,
first of A2 (Fig 13.3) and subsequently of
HLA-A68 and HLA-B27 (Bjorkman et al 1987; Garretti
et al 1989; Madden et al 1991) The α3 and β2m
domains have tertiary structures similar to domains in
the constant region of the immunoglobulins The top
of the molecule is formed by pairing the α1 and α3
domains, which together form the antigen
peptide-binding groove The majority of the polymorphic
determinants in class I molecules occur on the floor of
this cleft (Bjorkman et al 1987) (see Fig 13.3) The
class I molecules specifically bind peptides of defined
length, usually 6–10 residues (Falk et al 1991) All
peptides bind similarly with their N- and C-terminisequestered in the binding groove by a network ofhydrogen bonds to residues conserved in all class I glycoproteins (Madden and Wiley 1992) In addition,there are allele-specific binding pockets with a strongpreference for a few side-chains at some positions ofthe peptide This explains the correlation betweenclass I polymorphism and the affinity of peptide bind-
ing (Falk et al 1991) Generally, these peptides derive
from self-proteins, but in virus-infected cells the tides from the pathogen may be processed in thecytosol and migrate with the HLA molecules to the cellsurface (Pamer and Cresswell 1998) Tumour antigenscan be detected in the same way Class I molecules are expressed on most cells and they inform the scan-ning CTL about the status of potential target cells for
pep-Fig 13.1 (cont.)
chain-related genes encode stress-inducible proteins
implicated in the regulation of NK cell activity HFE is a
class I-like gene located approximately 4 Mb telomeric of
HLA-F and responsible for hereditary haemochromatosis.
Class III region: TNFB and A code for tumour necrosis
factors, HSP genes for heat shock proteins, C2, Bf and
C genes for proteins of the complement system, and
P450-C21B for a steroid 21-hydroxylase Class II region:
The HLA-DRBA1, -DQA1 and -DPA1 genes code for
alpha chains of the DR, DQ and DP class II molecules
HLA-DRB1, -DQB1 and -DPB1 code for the beta chain of the DR, DQ and DP class II molecules In addition to HLA- DRB1, which codes for the primary HLA specificities such as DR1, DR2, DR4, etc., other DRB genes code for the beta chain of the specificities DR52 (DRB3), DR53 (DRB4) and DR51 (DRB5) not present in all haplotypes The DO and
DM molecules regulate the loading of exogenous peptides into class II molecules LMP2 and LMP7, which encode the subunits of the proteasome, and TAP1 and TAP2, which, encode a peptide transporter, are involved in the processing and presentation of antigens by class I molecules.
Fig 13.2 Structure of class I and class
II HLA molecules showing domains
and transmembrane segments From
Roitt and Delves (2001).
Trang 15destruction Peptide epitopes presented by them can
only be recognized by CTL if these (1) have a specific
receptor for the antigen and (2) the same HLA class
antigens as the target cell This phenomenon, known
as HLA restriction, was first described in the mouse
(Zinkernagel and Doherty 1974) The HLA-A, -B and
-C antigens are expressed on all nucleated cells except
spermatozoa and placental trophoblast The antigens
are also found on platelets and some class I antigens
have been detected on red cells The number of class I
molecules on various cells differs and, particularly on
platelets, some of the antigens are weakly expressed
HLA class II molecules
All typical class II molecules consist of two
transmem-brane glycoprotein chains of molecular weight 33 kDa
(the heavy or α-chain) and 28 kDa (the light or
β-chain) respectively (Snary et al 1977b) The
extracel-lular component of both chains consists of two distinctdomains: α1, α2 and β1, β2 The domains distal to thecell surface carry most of the polymorphic determinants.The constant domain near the cell surface is very similar
to the constant domain of the immunoglobulin heavy
chains (Shackelford et al 1982; see also Fig 13.2).
The crystal structure of class II molecules (DR1) issimilar to that of class I molecules; polymorphic deter-minants of class II molecules are also clustered in the
antigen peptide-binding groove (Brown et al 1993) In
contrast with class I molecules, class II molecules bindlonger peptides with no apparent restriction on pep-
tide length (Rudensky et al 1991) The peptides bind
to the groove as a straight extended chain with a singlepronounced twist Hydrogen bonds along the main
(b)
(a)
Peptide-binding cleft
N N
Fig 13.3 Schematic representations of the crystallized
structure of the HLA-A2 molecule (a) The four domains,
with the α 1 and α 2 domains forming a putative
peptide-binding region (b) Top surface of the molecule The putative antigen-binding groove is shown, made up of a β-pleated sheet flanked by two α-helices (Bjorkman et al 1987).
Trang 16chain of all peptides interact with residues from the
α-helical regions and the β-sheet in the peptide-binding
groove and thus provide a binding component that
is independent of the sequence of the peptide Twelve
hydrogen bonds on the peptide bind to determinants
encoded by residues conserved in most class II alleles
and this suggests that peptides bind to class II
molecules by a universal mode However, particular
side-chains of the peptide are accommodated in
poly-morphic pockets in the binding groove that determine
specific binding of peptides and thus the affinity of
the peptide class II molecule bond (Stern et al 1994).
The expression of class II antigen is restricted to B cells
and to antigen-presenting cells such as macrophages,
dendritic cells and Langerhans cells Class II antigens
are also present on activated T lymphocytes and some
tumour cells (Winchester and Kunkel 1979)
The cells that express class II molecules, specialized
antigen-presenting cells (APCs), such as dendritic cells,
mononuclear phagocytes and B cells, bind
exogen-ously derived peptides of 9–22 residues In the case of
macrophages and B cells, the HLA molecule–antigen
complex is assembled within intracellular organelles
With all APCs the antigen peptides are held in a groove
in HLA class II molecules, and this plasma
membrane-bound compound antigen is recognized by helper T
lymphocytes via their T-cell receptors during
immuno-surveillance The polymorphic HLA determinants in
the peptide-binding groove of the class II molecule
strongly influence peptide binding
Dendritic cells, which express HLA class II antigens
particularly well, are the APCs that present antigen to
helper T cells to induce a primary immune response
Memory T cells can be stimulated by macrophages, B
cells and even by free antigens (Berg et al 1994) Class
II antigen complexes instruct the helper T-cell system
to initiate the humoral immune response and assist
in the cellular immune process; for this reason, class II
genes are often referred to as ‘immune response genes’
HLA genes and antigens
The linkage between the HLA genes is so strong that
crossing over between them is rare; therefore the alleles
of the HLA genes present on one chromosome usually
segregate together within a family The two alleles of
each individual gene are expressed co-dominantly (e.g
HLA-A1, -A11) The set of HLA alleles present on a
single chromosome is known as a haplotype Siblings
who inherit the same haplotypes from their parents are thus HLA identical, unless crossing over betweenHLA genes has occurred
As crossing over within the HLA region is rare, withrandom assortment equilibrium should be reached in apopulation over a long period of time; particular com-
binations of alleles at, for example, the A and B loci
or at the loci of the D region should not be more
com-mon than predicted from the product of their relative frequencies in the population However, in any givenpopulation, certain combinations of alleles or haplo-types are more frequent than expected, a phenomenonknown as ‘linkage disequilibrium’ For example, thefrequency of HLA-A1-B8 in European white people is8.8%, whereas the expected frequency of this haplo-type, based on the individual frequencies of A1 and B8, is 1.6% Selective pressures that affect survival orreproductive capacity usually drive linkage disequilib-rium Patterns of linkage disequilibrium vary in differ-ent populations
Nomenclature
A history of the development of HLA system nomenclature has been compiled by Boolmer (1997).Nomenclature for the HLA genes and antigens is regu-larly updated by the WHO Nomenclature Committeefor factors of the HLA system (Marsh 2003) For thenon-aficionado, nomenclature remains a challenge,because two systems remain in general use The olderserological nomenclature relies on identification ofantigens on the leucocyte surface (HLA antigens)
(Tiercy et al 2002) The following terms are used
for the ‘classical’ HLA antigens: for class I, the capital
letters -A, -B -C are appended to identify the locus; for class II, the prefix D followed by a letter (-DR, -DQ, -DP) for the subregion and by the letters A or B
to indicate whether the gene codes for the α- or chain, for example DRA, DQB, etc The letters are followed by a number that identifies epitopes deter-mined by alloantibodies or less often alloreactive cytotoxic T cells
β-Sequence-based nomenclature separates the HLAlocus with an asterisk (*) followed by four digits used
to designate the alleles of a particular gene; the firsttwo digits describe the serologically defined antigenwith which the allele is (or alleles are) most closelyassociated, and the last two or three digits completethe number of the allele as defined by molecular
Trang 17techniques (DNA typing, oligonucleotide typing,
nucleotide and amino acid sequencing, cloning), for
example HLA-A*0101 for the allele that encodes the
A1 antigen and A*0201, A*0202, etc for the alleles
associated with the antigen A2; the serologically
defined antigens encoded by alleles of each gene are
also numbered: A1, A2 etc.; w (‘workshop’) was used
to indicate that the specificity was provisional, but
in the future all serological specificities will be named
on the basis of correlation with an identified sequence
The letter ‘w’ can therefore be dropped with three sets
of exceptions: (1) Bw4 and Bw6 to distinguish them
as epitopes from those encoded by other alleles of
the HLA-B gene; (2) the C antigens for which the w
is retained throughout to avoid confusion with the
nomenclature of the complement system; and (3) the
Dw specificities defined by the MLC assay; and the
DP specificities defined by a secondary response of T
lymphocytes that had been primed by a first step in
the MLC (primed lymphocyte typing) (Bodmer et al.
1992)
One of the non-classical HLA class I genes, the
HLA-G gene, encodes a non-polymorphic α-chain
with a shortened cytoplasmic segment The HLA-G
molecule is expressed only on the trophoblast, which
suggests that it may have a role in embryonic
develop-ment or fetal–maternal immune interactions, or both
(Geraghty et al 1987; Kovats et al 1991) Class I
anti-gens are detected in a lymphocytotoxicity test, using
either alloantibodies or human or murine monoclonal
antibodies
Some 250 alleles of the class I A gene, about twice
this number of B alleles and 119 C alleles are
recog-nized by the WHO Nomenclature Committee The
numbers have been increasing rapidly For a list of
these and of class I antigens, see Marsh (2003)
Class II genes and antigens
The polymorphism of the class II genes is much greater
than detected on their products by serological typing
and by the MLC assay Studies at the DNA level have
shown that, in addition to the classical DR, DQ and
DP series of genes, there are several other non-classical
class II genes in the D region: DOA, DOB, DNA and
the DMA and DMB genes In addition, in the class II
chromosomal region, there are four genes, TAP1 and
TAP2, and LMP2 and LMP7, which encode molecules
involved in antigen processing (see below)
In the DR subregion there is a single α-chain gene
(DRA) with two alleles that, however, do not encode a
polymorphism on the α-chain There are nine DRB genes, five of which are pseudogenes (DRB2, DRB6, DRB7, DRB8 and DRB9) Seven of the DRB genes (except DRB1 and DRB9) are restricted to certain
DR haplotypes The genomic organization of the DR
region is shown in Figure 13.4 The DRB1, DRB3, DRB4 and DRB5 genes encode four separate β-
chains DRB1 encodes the major DR antigens, whereas the DRB3 gene codes for DR52, and the DRB5 gene for DR51.
In the DQ subregion there are two α genes: DQA1,
which encodes an α-chain and DQA2, which is not known to be expressed There are three B genes: DQB1, which encodes a β-chain and DQB2 and DQB3, not known to be expressed DQA1 and DQB1 are polymorphic but only the products of the DQB1
alleles have been serologically recognized (DQ antigens)
In the DP subregion there are two α and two β genes:
DPA1, DPA2, DPB1 and DPB2 DPA1 and DPB1
encode an α- and a β-chain respectively, whereas DPA2 and DPB2 are pseudogenes.
Additional HLA class II genes
The genes DOB and DNA located between the DP and
DQ subregions encode a β- and α-chain, respectively,
whose function is as yet unknown The genes DMA and DMB encode an α- and a β-chain, which associate
to form a class II molecule that contains a binding groove involved in antigen presentation (Kelly
peptide-et al 1991) The DM genes are polymorphic, but the
polymorphism is limited and the resulting antigenicdeterminants occur only on the area of the extracellu-lar portion of the protein that is proximal to the cellmembrane They therefore do not occur in the peptide-binding groove and thus do not affect antigen pre-sentation, in which the DM molecule probably has a
specialized function (Sanderson et al 1994).
Non-HLA genes involved in antigen processing
The TAP1 and TAP2 genes, located between DBO and DNA, do not encode typical class II proteins
but instead encode an important peptide transportermolecule involved in the endogenous processing of
antigen (Spies et al 1991) The TAP genes are morphic (Colonna et al 1992; Powis et al 1992a,b,
Trang 18poly-1993) This polymorphism may affect antigen
process-ing and thus the immune response, but at present this
is speculation The LMP2 and LMP7 genes, located
near the TAP genes, encode proteasomes that affect
the degradation of antigen molecules to peptides
(Cerundolo et al 1995).
The class II antigens are detected by alloantibodies
and, in some cases, also by monoclonal antibodies, using
a complement-dependent cytotoxicity test on isolated
B lymphocytes or a two-colour immunofluorescence
test on unseparated cells (van Rood et al 1976).
As mentioned above, a polymorphism (Dw) encoded
by the D region has been defined by using homozygous
typing cells in the mixed lymphocyte culture (MLC)
The exact relationship between Dw determinants and
the polymorphic determinants encoded by the DR, DP
genes is not known However, a strong correlation has
been observed between matching and mismatching for
DR polymorphic DNA sequences and reactivity in the
MLC The lymphocytes of all DR mismatched pairs
were reactive and 37% of the matched pairs were
non-reactive in the MLC The MLC reactivity of 63% of
the matched pairs may be due to unrecognized DR
alleles (Baxter-Lowe et al 1992), but incompatibility
for HLA-DP specificities has also been found to inducesignificant proliferation in the primary MLC in HLA-
A, -B, -DR and -DQ identical subjects (Olerup et al 1990) HLA-DQA1 or -DQB1 allele differences are not
important in the primary MLC among otherwise HLA
identical, unrelated subjects (Termijtelen et al 1991).
For a list of the very numerous class II alleles, antigens and determinants agreed by the WHONomenclature Committee, see Marsh (2003)
Other genes in the HLA region
As the products of these genes are not leucocyte gens and are not involved in antigen presentation byHLA molecules, they will not be discussed further
anti-Crossreactions in the HLA system
Sera from subjects alloimmunized against HLA gens are frequently crossreactive, as shown by the following example: a given serum may react with twodifferent serologically defined antigens, for example
Fig 13.4 Genomic organization of the HLA-DR region and
encoded products (specificities) (Bodmer et al 1992).
Pseudogenes are indicated by shaded boxes, expressed genes
by open boxes The serological specificity encoded by a gene
is shown underneath in italics *, rarely observed haplotypes;
#, DR51 and DR53 may not be expressed on certain haplotypes; §, the presence of DRB9 in these haplotypes needs confirmation.
Trang 19HLA-B51 and HLA-B52, but antibodies recognizing
these two specificities individually cannot be separated
from the serum The antibodies in such a serum are
in fact directed against a different antigen, HLA-B5
in this example, which occurs together with B51 and
B52 Thus the antibodies (anti-B5) crossreact with
B51- and B52-positive cells
This kind of crossreactivity is explained by the
mul-tiple mutations within the HLA genes A single allele of
a gene may code for different, separate polymorphisms
on the single HLA molecule it produces The
frequen-cies of the epitopes encoded by these different
poly-morphisms within a single allele differ greatly Some
have a very high frequency, for example HLA-B4 and
HLA-B6, and are called supratypic or ‘public’
anti-gens At the other end of the scale are antigens with a
very low frequency (1–2%) that are called ‘private’
antigens Thus, epitopes with different frequencies in
the population, and against which separate
alloanti-bodies can be made, occur on a single HLA molecule
and are the basis of the crossreactivity: antibodies
against public antigens (also called crossreactive
gens) react with cells carrying different private
anti-gens Particular public antigens occur together with
particular private antigens that are said to form
cross-reactive groups (CREGs) of (private) antigens The
higher the frequency of the public antigen in the
popu-lation, the more important are antibodies against the
antigen for crossreactivity Thus, anti-HLA-B4 and
-B6 are responsible for much of the crossreactivity
among the HLA-B antigens
The occurrence of crossreactive antigens is also
responsible for what are called ‘splits’ of HLA
anti-gens Frequently, a crossreactive antigen, for example
the antigen B5 in the above example, was defined
before the two private antigens B51 and B52, together
with which it occurs Later, when antibodies
recogniz-ing B51 and B52 were found, the B5 antigen was ‘split’
into B51 and B52 (see Table 13.2)
Soluble HLA class I antigens in plasma
Using monoclonal antibodies coated on to
immuno-beads and one-dimensional isoelectric focusing,
fol-lowed by immunoblotting using specific class I antisera,
all antigens defined to date have been detected in
plasma (Doxiadis and Grosse-Wilde 1989)
Soluble HLA class I antigens (sHLA) may have
important immunological effects sHLA have been
shown to inhibit alloreactive cytotoxic T cells
(Zavazava et al 1991) and to block specifically the
induction of HLA alloantibody formation (Grumet
et al 1994) Allogeneic sHLA alone or complexed with
antibody induces prolongation of allograft survival
(Sumimoto and Kamada 1990; Wang et al 1993a).
When a graft is rejected, sHLA is shed from it and thus graft rejection episodes can be identified by serial
measurements of donor-specific sHLA (Claas et al 1993; Puppo et al 1994).
HLA antibodies
Mechanisms of alloimmunization
The high density of HLA molecules on the cell surfacerenders allogeneic leucocytes highly immunogenic fol-lowing transfusion or pregnancy Sensitization dependsupon both donor and recipient factors Two recipientT-cell recognition mechanisms have been shown to becritical for the initiation of alloimmunity (Semple andFreedman 2002) The direct pathway occurs whenrecipient helper T cells interact with MHC molecules
on donor APCs The indirect pathway is more ous to the normal immune response Indirect recogni-tion involves processing of allogeneic donor molecules
analog-by recipient APCs and presentation to recipient helper
T cells With indirect allorecognition, interactions tween donor antigen and recipient APCs are essentialfor T-cell activation and subsequent antibody forma-tion For both pathways, the MHC molecules areexpressed on the surface of the APC and are availablefor presentation to circulating T cells If a T cell has areceptor with sufficient affinity for the peptide–MHCcombination (first signal) and various co-stimulatory(second signal) events occur, the T cell will be activatedand differentiate into an effector cell Cytokines such
be-as interleukin 2 (IL-2), IL-4 and alpha-interferon α) secreted from the activated helper cells stimulatedonor MHC class I-primed B cells to differentiate intothe plasma cells that secrete IgG antibodies and helper
(IFN-T cells (Weiss and Samuelsen 2003)
Development of HLA antibodies after transfusion
Unless measures are taken to reduce the number oftransfused leucocytes (see below), a high incidence ofHLA antibodies will be encountered in patients who
Trang 20receive multiple transfusions from different donors.
However, even in subjects exposed to the blood of a
single donor, the incidence of HLA antibodies is high
In a series in which patients awaiting renal grafting
were given three transfusions at 2-weekly intervals
from a potential donor, who in each case had a
haplo-type identical with one of the recipient’s haplohaplo-types,
HLA antibodies developed in some 30% of recipients
(Salvatierra et al 1980).
After the massive blood transfusion that used to be
associated with open heart surgery, lymphocytotoxic
antibodies and/or leucoagglutinins could be found in
almost all subjects, provided that repeated tests are
made, as some of the antibodies can be detected only
transiently In a series in which patients were tested at
1 week and usually also at 2, 4 and 12 weeks after open
heart surgery, 52 out of 54 developed leucocyte
anti-bodies; 12 weeks after transfusion antibodies were
present in only 62.5% of the subjects (Gleichmann
and Breininger 1975) The majority of HLA antibodies
formed after blood transfusion are directed against
class I antigens HLA antibodies are the most
import-ant cause of import-antibody-mediated refractoriness to
platelet transfusions (see later), of febrile transfusion
reactions prior to leucoreduction and of
transfusion-associated acute lung injury (see Chapter 15)
Some patients never become immunized despite
repeated transfusions of blood or of platelets Such
subjects are considered to be non-responders to HLA
Development of HLA antibodies in pregnancy
In primiparous women, lymphocytotoxic class I
anti-bodies may be found as early as the twenty-fourth
week of pregnancy and are present by the last trimester
in 10% of women (Overweg and Engelfriet 1969)
Estimates of the incidence of lymphocytotoxic
anti-bodies after a first pregnancy vary widely: 4.3%
(Ahrons 1971), 9.1% (Nymand 1974), 13% (Overweg
and Engelfriet 1969) and 25% (Goodman and Masaitis
1967) The discrepancies may well be due to the
vary-ing extent of the panels of lymphocytes with which the
sera were tested and the sensitivity of the techniques
applied The majority of HLA antibodies developed in
pregnancy are directed against class I antigens
Women tend to make antibodies against only
cer-tain of the HLA antigens to which they are exposed
during pregnancy In multiparous women who had
had at least four pregnancies, and were therefore likely
to have been exposed to antigens encoded by both oftheir partner’s haplotypes, the frequency of womenwith antibodies against only a single paternal antigenwas the same as that in primiparous women (Tongio
et al 1985) Both maternal and paternal (fetal) HLA
antigens play a role in the class I differential
immuno-genicity (Dankers et al 2003).
Although HLA antibodies are usually IgG, they produce no obvious damage to the fetus, presumablybecause they are absorbed by fetal cells in the placenta
Monoclonal HLA antibodies
Most murine monoclonal HLA antibodies are directedagainst non-polymorphic determinants of the HLA
molecules (Brodsky et al 1979; Trucco et al 1980;
1979); some antibodies detect a polymorphism that isdifferent from those detected by alloantisera (Quaranta1980) However, many murine monoclonals whichrecognize HLA antigens as defined by alloantiserahave been described, particularly anti-DR and anti-
DQ (Marsh and Bodmer 1989)
In addition, many human monoclonal HLA bodies against both class I and class II antigens havenow been described
anti-Some features of HLA antibodies
HLA antibodies formed after blood transfusion orpregnancy are characteristically IgG They are comple-ment activating and have cytotoxic properties and,like most granulocyte-reactive IgG antibodies, are leucoagglutinins (see below) HLA antibodies may benaturally occurring Using very sensitive techniques,weak HLA antibodies, particularly anti-B8, have beendemonstrated in the serum of about 1% of normaldonors who had had no known pregnancies or trans-
fusions (Tongio et al 1985) These antibodies are
usu-ally IgM and, in the cytotoxicity test, react only with
B cells, on which class I antigens are more stronglyexpressed than on T cells
HLA and haematopoietic progenitor cell grafts
Graft-versus-host-disease and graft-versus-tumour or -leukaemia
Haematopoietic cell (marrow, umbilical cord blood
or mobilized peripheral blood) transplantation is
Trang 21performed to replace inadequate or defective blood
cell production, for example in aplastic anaemia, sickle
cell disease and thalassaemia (Walters et al 2000; La
Nasa et al 2002; Ades et al 2003; Atkins and Walters
2003), for adoptive immunotherapy of malignancy
(Landsteiner and Levine 1929; Landsteiner 1931;
Barrett 2003; Chakrabarti and Childs 2003) and for
reconstitution of ‘normal’ immune function as in
treat-ment of severe combined immunodeficiency (SCID) and
Wiskott–Aldrich syndrome (Filipovich et al 2001).
The role of HLA ‘compatibility’ falls into four different
areas: (1) sufficient compatibility to permit engraftment
and prevent late rejection (with appropriate preparative
and immunosuppressive regimens); (2) enough
com-patibility to minimize graft-versus-host-disease (GvHD);
(3) ample immune reconstitution to permit
immuno-surveillance; and (4) sufficient immune potency to effect
adoptive immune therapy of neoplasia HLA identity is
neither necessary nor sufficient to ensure these effects,
but serological and molecular similarities are the best
available surrogate assays to guide related and unrelated
transplants Both GvHD and graft-versus-tumour (GvL)
occur in the presence of a full HLA match, suggesting
that the classical HLA molecules themselves are not
targets of allosensitization, but rather present
poly-morphic molecules expressed by recipient cells that are
recognized by the grafted immune cells
Graft-versus-host disease: the dark cloud of
haematopoietic cell grafts
Donor lymphocytes engraft, replicate and react against
the normal tissues of the recipient, resulting in a
syn-drome known as GvHD Myeloablative conditioning
administered before transplant effectively minimizes
graft rejection The art of post-transplant
immunosup-pression consists of achieving a balance between graft
immunocompetence and GvHD without allowing
rejection of the graft The risk of GvHD increases with
genetic disparity between donor and recipient HLA
identical twins have the least chance of developing
GvHD, followed by HLA identical siblings, minor
degrees of mismatching among siblings, and unrelated
donors of differing degrees of similarity at the MHC
locus (Longster and Major 1975; Hansen et al 1999).
However, while the genetic homogeneity between donor
and recipient generally decreases the risk of GvHD, it
lessens the therapeutic benefit and increases the chance
of tumour relapse as well (Weiden et al 1981).
Graft-versus-tumor effect: a silver lining of graft-versus-host disease?
One possibly beneficial effect of GvHD, or perhaps
an immunological activity difficult to separate fromGvHD, is rejection of recipient tumour cells by thedonor immune system (graft-versus-leukaemia (GvL)
effect) (Mavroudis and Barrett 1996; Mavroudis et al.
1998) The GvL effect has long been recognized to play
a powerful therapeutic role in the treatment of chronicmyelocytic leukaemia and more recently recognized
as treatment of refractory malignant disorders ing some solid tumours (graft-versus-tumour, GVT)(Childs and Barrett 2002) GVT may be the mostpotent form of tumour immunotherapy currently inclinical use, but its mechanism(s) of action is stillpoorly understood Allogeneic T cells clearly play afundamental role in the initiation and maintenance
includ-of the effect on neoplastic cells (Kolb et al 1990) The
risk of relapse increases markedly for patients withchronic myelogenous leukaemia who received a T-cell-depleted graft compared with a subset of patients whohad received a T-cell-replete one, although the former
patients avoided significant GvHD (Leak et al 1990; Champlin et al 2000) These results suggest that GvHD
is a biological entity different from the GvL effect
In addition, upon leukaemia relapse, administration
of donor lymphocyte infusion can induce clinical and molecular remission Donor T cells may target notonly tumour-specific antigens but also allelic variants
of these antigens, minor histocompatibility antigensand, in the case of HLA-mismatched transplants, HLAantigens disparate from the donor but expressed by thetumour cells (Leddy and Bakemeier 1967; Lederman
et al 1983; Marijt et al 2003) Although several
theories about the mechanisms of the GvL effect havebeen proposed, the reasons that allogeneic T cells seemsuperior to native tumour immunity for some leuk-aemias and solid tumours remain to be clarified
Effect of previous transfusion on success
of bone marrow grafting
Previous transfusions, particularly from close relatives,prejudice the success of subsequent bone marrow
grafting (Storb et al 1980) The chance of rejection of
the graft increases with the number of transfusions
If future recipients of a bone marrow graft need to
be transfused, they should receive leucocyte-depleted
Trang 22blood or blood components from random donors
and not from relatives The discrepancy between the
effect of transfusion of blood containing white cells
on grafted bone marrow and the apparent mitigating
effect on renal grafts has not been explained The
development of GvHD after the transfusion of
allo-geneic leucocytes is described in Chapter 15
HLA and organ grafting
Renal grafts
Significance of HLA antibodies When HLA
allo-antibodies directed against antigens expressed on the
donor kidney are present in the serum of a renal graft
recipient, acute or hyperacute rejection of the graft will
occur It is therefore necessary to perform a
cross-match between the patient’s serum and the B and T
lymphocytes of the donor Not all antibodies detected
in the crossmatch are harmful Cold-reacting IgM
autoantibodies directed against B and T cells may be
present in the serum of dialysis patients and do not
appear to be harmful (Ting 1983)
Significance of matching for HLA The extent to
which matching for HLA improves renal graft survival
remains controversial In many studies, matching has
had no obvious benefit due, perhaps, to the small
num-bers of cases studied, interference of the many factors
influencing graft survival and incomplete tissue typing
Furthermore, the survival of mismatched kidneys
has improved greatly following the discovery of the
beneficial effect of previous blood transfusion and of
the value of ciclosporin A as an immunosuppressive
drug Nevertheless, in some large studies, a significant
beneficial effect of HLA matching on long-term graft
survival has been observed
In a study in which 240 laboratories participated,
the results of 30 000 first cadaver kidney transplants
were analysed Cases in which the donor and recipient
were typed for all known ‘splits’ of HLA-A and -B
antigens and those in which typing was restricted to
the broad antigens were analysed separately At 3 years,
an 18% difference in survival rates between grafts with
zero and four mismatches typed for A and B antigen
splits was found, but only a 2% difference when typing
was restricted to broad antigens When A, B and DR
antigens were considered together, the differences in
rates of survival were 31% and 6%, respectively, in the
two groups It was concluded that typing for antigensplits is important (Opelz 1992) Molecular typing of
DR alleles revealed an error rate in serological typing
of about 25% (Mytelineos et al 1990) The impact of
DR matching is particularly significant if patients and
donors are typed at the DNA level (Opelz et al 1993).
In a recent study, complete matching for serologicallydetermined HLA-A, B and DR antigens was found tohave a significant and clinically important impact on
short- and long-term graft survival (Opelz et al 1999).
On the other hand, partial matching provided little
benefit (Held et al 1994) The advantage of complete
matching was diminished by the negative influence oflonger periods of organ preservation and by the factthat in practice only 50% of perfectly matched kidneyswere actually transplanted into the identified recipient
An analysis of more than 150 000 renal transplantsbetween 1987 and 1997 in the Collaborative TransplantStudy indicates that a first cadaver graft with a 6-locusmismatch has a 17% lower 10-year survival than a graft
with no mismatch at these sites (Opelz et al 1999) In
the latter study, the matching effect is even more ing in patients with highly reactive preformed lympho-cytotoxic antibodies Among first cadaver transplantrecipients with antibody reactivity against > 50% ofthe test panel, the difference in graft survival at 5 yearsbetween patients with zero or six mismatches reached30% Once again, correction of serological HLA typ-ing errors by more accurate DNA typing resulted in
strik-a significstrik-antly improved HLA mstrik-atching effect, strik-andmatching for the class II locus HLA-DP, a locus thatcan be typed reliably only by DNA methods, showed
a significant effect for cadaver kidney re-transplants.Non-HLA transplant immunity may be more import-
ant for long-term graft survival (Opelz et al 2005).
For brief discussions of the importance of ABO as amajor histocompatibility system and of the possibleeffects of Lewis groups on renal transplantation, seeChapter 4
Liver grafts and heart–lung grafts
The survival of liver grafts is reportedly improved withHLA matching and is worse when the T-cell cross-
match is positive (Nikaein et al 1994) However, this
finding could not be confirmed in the Collaborative
Transplant analysis (Opelz et al 1999) Matching for
HLA-DR diminished the frequency of rejection episodesafter heart transplantation from 34% to 16%, and at
Trang 233 months there was an additional beneficial effect of
HLA-B matching (Sheldon et al 1994) An independent
study of heart transplants showed a highly significant
impact of HLA compatibility on graft outcome (P<
0.0001) (Opelz et al 1999) In practice, matching for
HLA is much more difficult in the transplantation of
liver and heart than of kidney, mainly because there is
no large pool of HLA-typed recipients to choose from
Immunomodulatory effect(s) of transfusion
As knowledge about the mechanisms of immune
responsiveness and tolerance evolves, and as tools
to measure alterations in immunity become available,
additional immunological consequences of blood
transfusion are being detected Numerous variationsin
circulating blood cells have been reported in patients
transfused with allogeneic blood (see below) Some of
these changes persist for months or even longer after
transfusion The lingering question has been whether
these observations represent no more than laboratory
curiosities, or whether they reflect some clinically
relevant alteration in the recipient’s immune status,the
so-called ‘immunomodulatory effects’ of blood
trans-fusion Based on the sum of clinical evidence (see below)
immunomodulation seems likely to be added to the list
of unintended consequences of allogeneic blood
trans-fusion The magnitude and importance of these effects,
the causative agents, the biological mechanisms and
the patients or patient groups that are at particular risk
have yet to be defined (Klein 1999)
Dzik (2003) has suggested that there may be two
categories of immunosuppressive transfusion effect:
one that is HLA dependent and directed against
adap-tive immunity and a second that is mild, non-specific
and directed against innate immunity The
non-specific effect might result from the infusion of blood
cells that undergo apoptosis during refrigerated
storage The infusion of apoptotic cells has been
shown to be immunosuppressive in animal models
Immunosuppression resulting from the infusion of
apoptotic cells may be linked to transforming growth
factor beta (TGF-β) (Dzik 2003)
Changes in recipient’s lymphocytes after
blood transfusion
Following the transfusion of large amounts of fresh
or stored blood, changes develop in the recipient’s
lymphocytes after an interval of about 1 week Atypicallymphocytes increase by a factor of five or more and lymphocytes may incorporate 3H-thymidine in vitro
at an increased rate Values return to the sion level by about 3 weeks Changes are not seen aftertransfusion of frozen and washed (leucocyte-depleted)
pretransfu-red cells (Schechter et al 1972) Confirmatory
observa-tions were published by Hutchinson and co-workers(1976) The changes are interpreted as a response todonor HLA antigens (presumably of the Dw series)
and may be regarded as those of an MLC in vivo A
number of other alterations of immune cells including
a decrease in NK function and delayed hypersensitivity
have been published (Tartter et al 1986, 1989; Jensen
et al 1992) Blood transfusion alters immune cell
anti-gen expression in premature neonates and may initially
be immunostimulatory and later immunosuppressive
(Wang-Rodriguez et al 2000) Donor lymphocytes
may circulate for prolonged periods in some patientgroups, such as trauma victims, whereas in others such as patients infected with HIV, microchimerism
appears to be transient (Kruskall et al 2001; Lee et al.
1995, 1999, 2001) The immunomodulatory role ofpersistent microchimerism post transfusion and itsrelationship to the HLA system are areas of activeinvestigation For the relationship of microchimerismand transfusion-associated GVHD, see Chapter 15
Effect of previous transfusion on success
of renal graft
Patients who have antibodies against HLA antigens ofthe donor undergo acute rejection of renal allografts
On the other hand, blood transfusion has been shown
to have a striking effect in improving the survival
of subsequent renal grafts in subjects who have notdeveloped cytotoxic antibodies, or who have done soand have received renal grafts from HLA-compatible
donors (Opelz et al 1973; van Hooff et al 1976).
Leucocytes in the donor blood have been found to beessential for the beneficial effect (Persijn 1984).After the introduction of more potent immunosup-pression with ciclosporin, transfusions before cadavergrafting were found to confer little additional benefit
(Kaban et al 1983; Lundgren et al 1986; Opelz 1987),
although a single-centre randomized study attributedpretransplant transfusion benefit to reduced mortality
related to immunosuppression (Vanrenterghem et al.
1994) However, transfusions improved the 1-year
Trang 24graft survival rate by 8% (P< 0.01) in recipients of a
one-DR mismatched graft and by 10% (P< 0.01) in
recipients of a two-DR mismatched graft (Iwaki et al.
1990) Two to four transfusions from random donors
were sufficient to obtain this effect This study
con-cluded that despite the use of ciclosporin, the
prac-tice of giving deliberate transfusions before grafting
should not be abandoned In a randomized, controlled
multicentre trial, cadaver graft survival rate was
significantly higher in the 205 recipients who
under-went three pretransplant transfusions than in the 218
patients who did not receive transfusions (Opelz et al.
1997)
When a kidney from a live donor is used, it is
pos-sible to give both transfusion and graft from the same
donor Donor-specific blood transfusions (DSTs) lead
to increased graft survival rates (Salvatierra et al.
1981, 1986; Kaplan 1984) A disadvantage of DST
is that the patient may develop lymphocytotoxic
antibodies against donor HLA antigens In animal
models it was found that heat treatment of the donor
blood (Martinelli et al 1987), or pre-treatment of the
recipient with donor leucocytes coated with
anti-lymphocyte antibody, diminished the chance of such
immunization (Susal et al 1990) Treatment of the
patient with azathioprine also had this effect
(Anderson et al 1982).
Several mechanisms have been suggested to explain
the beneficial effect of previous blood transfusions
on renal graft survival: (1) the induction of increased
suppressor cell activity (Marquet and Heystek 1981;
Quigley et al 1989); (2) decreased natural killer cell
activity (Gascon et al 1984); (3) specific
unresponsive-ness due to idiotype antibodies, which inactivate T-cell
clones (Woodruff and van Rood 1983; Kawamura
et al 1989); (4) impairment of the function of the
mononuclear phagocyte system (MPS) by iron loading
(Akbar et al 1986; de Sousa 1989); (5) deletion of
clones of cells, which are first activated by blood
transfusion and then killed or inactivated by
high-dose immunosuppressive therapy during the anamnestic
response after transplantation (Terasaki 1984); and
(6) the production of non-cytotoxic, Fc
receptor-blocking antibodies (MacLeod et al 1985; Petranyi
et al 1988).
Sharing of MHC antigens between donor and
re-cipient has been found to determine the extent of the
blood transfusion effect The survival of kidney grafts
in recipients who were given transfusions, and who
shared one HLA-DR antigen with the donors, wassignificantly better (81% at 5 years) than in recipientswho were given transfusions from donors mismatchedfor both DR antigens (57% at 5 years), or in recipientswho were not transfused (45%) Immunizationoccurred less frequently in the recipients who shared
one DR antigen with the donor (Lagaaij et al 1989)
In another study, sharing of one HLA haplotype (or
at least one HLA B and DR antigens) between donorand recipients had a mitigating effect because it led to
a specific suppression of the formation of cytotoxic Tlymphocytes (CTLs), i.e to CTL non-responsiveness;recipients of blood from fully identical donors
remained CTL responders (van Twuyver et al 1991).
Furthermore, transfusion of blood from HLA-identicaldonors induces the generation of suppressor cell-independent, high-affinity CTL against donor antigens
(van Twuyver et al 1994) These mitigating and
im-munizing effects are donor specific, but the beneficialeffect of blood transfusion on kidney survival is alsodue to a non-specific effect Transfusion of blood fromdonors who share one HLA haplotype induces a general decrease in the usage of T-cell Vβ families
(Munson et al 1995) These effects are probably due
to the survival of donor lymphocytes in the recipient.Studies in mice have shown that sharing of H2 anti-gens between donor and recipient of a blood trans-fusion facilitates the persistence of donor lymphoid cells in the recipient, which is associated with tolerancefor donor alloantigens Donor lymphocytes can bedetected 10 –20 years after transplantation in patients
in whom the graft survives for long periods of time.Such chimerism may be important in modulating the
immune response (Starzl et al 1992).
Effect of transfusion on tumour growth and recurrence of cancer
A retrospective analysis of the recurrence rate of cinoma of the colon after surgical resection first suggested that the 5-year disease-free survival rate was reduced by blood transfusion given at the time ofsurgery (Burrows and Tartter 1982) However, despitenumerous subsequent reports, including more than
car-100 observational studies and three controlled trials,and several meta-analyses, the relationship betweenblood transfusion, cancer growth and cancer-free sur-vival remains murky and contradictory (Klein 2001).The numerous variables including different tumours,
Trang 25locations, extent of disease, histological grade and
modes of treatment make this a particularly difficult
area to evaluate
Prospective randomized clinical studies have been
conducted with patients undergoing surgery for
colo-rectal carcinoma In one large multicentre
random-ized trial, patients who were operated on because of
colorectal cancer and who needed blood transfusion
were randomized to an autologous or allogeneic
trans-fusion regimen At study conclusion, patients received
allogeneic blood only (133), autologous and
allo-geneic blood (66), autologous blood only (112) or no
blood at all (164) There were no significant
differ-ences between the groups receiving allogeneic blood
or autologous blood only: at 4 years, survival rates
were 67% and 62%, respectively, and in survivors, no
recurrence of cancer in 63% and 66% respectively
On the other hand, many patients did not receive the
‘treatment’ specified by their prospective treatment
assignment, and cancer recurred significantly more
fre-quently in the transfused than in the non-transfused
patients This difference may have been associated
with the circumstances that necessitated transfusion
(Klein 1999) The red cells transfused in the allogeneic
arm were buffy coat depleted as was the routine in the
Netherlands at the time In a second controlled study
of colorectal cancer in the Netherlands, leucoreduced
red cells were compared with buffy coat-reduced red
cells No significant differences were found between
the two trial transfusions in survival, disease-free
sur-vival or cancer recurrence rate after an average
follow-up of 36 months Patients who had a curative resection
and who received blood of any sort had a lower 3-year
survival than non-transfused patients (69% vs 81%,
P= 0.001) These observations confirm an association
between blood transfusion and poor patient survival,
but suggest that the relation is not due to promotion of
cancer (Houbiers et al 1994) The third prospective
study of colorectal cancer from a single centre in
Germany found that blood transfusion was an
inde-pendent factor associated with tumour recurrence,
and that survival of transfused patients tended to be
shorter although the difference was not statistically
significant (Heiss et al 1994) There may well be some
subset of patients, perhaps defined by immune status
or tumour subtype, that is particularly susceptible to
the effects of allogeneic transfusion Demonstration of
such a difference will probably require a large,
care-fully controlled, prospective study
Effect of transfusion on postoperative infections
As is the case with cancer, a large number of tional studies find an association between allogeneicblood transfusion and postoperative bacterial infection,while a few do not (Vamvakas and Blachman 2001) Forexample, Carson and co-workers (1999) conducted aretrospective cohort study of 9598 consecutive patientswith hip fracture who underwentsurgical repair between
observa-1983 and 1993 at 20 hospitals across the USA Bacterialinfection, defined as bacteraemia, pneumonia, deepwound infection or septic arthritis/osteomyelitis wasthe primary endpoint and numerous variables wereincluded in the statistical model; a highly significantassociation was found between serious postoperativeinfection and transfusion Chang and co-workers (2000)analysed a database of 1349 patients undergoing elective colorectal surgery for any disease of the colon
or rectum at 11 university hospitals across Canada Toadjust for confounding effects associated with remoteinfections such as pneumonia and urinary tract infec-tions, the study limited the analysis to postoperativewound infection Allogeneic blood transfusion was ahighly significant independent predictor of postoperat-ive wound infection Vamvakas and Carven (1998)reported a retrospective cohort study of 416 coronaryartery bypass graft patients admitted to one hospital.The endpoints were limited to postoperative woundinfection or pneumonia, and adjustment was made forthe effects of chronic systemic illness and specific riskfactors for wound infection or pneumonia The risk ofpostoperative wound infection or pneumonia increased
by 6% per unit of allogeneic red blood cells (RBCs) and/
or platelets transfused, or by 43% for a patient receivingthe mean transfusion dose of 7.2 units of either compon-ent Nevertheless, these analyses are inevitably flawed,despite meticulous multivariate testing, by the numerousvariables that predispose to postoperative infection(comorbidities, catheters, respirator time, impairedconsciousness, etc.), not to mention factors related tothe blood components such as storage time and method
of preparation (Vamvakas and Carven 1998)
Seven randomized controlled trials compare theincidence of postoperative infection between recipi-
ents of buffy coat-reduced (Heiss et al 1993; Busch
et al 1994; Houbiers et al 1994; Jensen et al 1996; van de Watering et al 1998) or standard allogeneic red cells (Tartter et al 1998) or whole blood (Jensen et al.
1992) and recipients of autologous or WBC-reduced,
Trang 26buffy coat-reduced allogeneic red cells or whole blood.
Two studies (Jensen et al 1992, 1996) reported a
significant effect, two studies (Heiss et al 1993; van
de Watering et al 1998) reported a marginal effect,
and three studies (Busch et al 1994; Houbiers et al.
1994; Tartter et al 1998) did not detect an effect
The strengths and weakness of these studies have
been analysed exhaustively (Vamvakas and Blachman
2001) However, insufficient data are available to
perform the kind of meta-analysis that might help
draw conclusions from these studies
Postoperative mortality
In addition to the possible association between
allo-geneic transfusion and postoperative infection, van de
Watering and co-workers (1998) detected an
unex-pected association between WBC-containing allogeneic
blood transfusion and postoperative mortality from
causes other than postoperative infection In total,
24 out of 306 patients (7.8%) transfused with buffy
coat-reduced red cells died, compared with 11 out of
305 patients (3.6%) receiving buffy coat-reduced red
cells that were leucoreduced before storage, and 10 out
of 303 patients (3.3%) receiving buffy coat-reduced
red cells that were leucoreduced after storage (P= 015)
The overall difference in 60-day mortality was due to a
highly significant difference among the three
random-ization arms The number of RBC units transfused
was the most significant predictor of postoperative
mor-tality The association between leucocyte-containing
allogeneic blood and increased mortality may be
lim-ited to cardiac surgery and should not be extended to
other clinical settings At the very least, the finding
requires confirmation by a study designed with
mor-tality as the primary endpoint
Possible role of HLA in habitual abortion
Parental sharing of HLA antigens has been thought to
be a cause of habitual abortion In such cases,
non-cytotoxic antibodies that are normally produced in the
mother and that protect the fetus are absent (Adinolfi
1986; Scott et al 1987).
Immunization of women with leucocytes has been
employed with the object of correcting the
immuno-logical unresponsiveness (Taylor and Falk 1981; Beer
et al 1985) One problem in assessing the benefit of
such immunization is that the definition of habitual
abortion varies Furthermore the chance of a successfulpregnancy after three abortions is about 60% (Regan1991) One prospective randomized trial (Mowbray
et al 1987) has shown an apparent benefit; in another
trial, no clear advantage of leucocyte injection wasobserved, and the authors expressed their concernabout severe growth retardation seen in some fetuses
(Beer et al 1985) The Recurrent Miscarriage Study
enrolled women who had had three or more eous abortions of unknown cause in a double-blind,
spontan-multicentre, randomized clinical trial (Ober et al.
1999) In total, 91 women were assigned to tion with paternal mononuclear cells (treatment) and
immuniza-92 to immunization with sterile saline (control) Theprimary endpoints were the inability to achieve preg-nancy within 12 months of randomization, or a preg-nancy that terminated before 28 weeks of gestation(failure); and pregnancy of 28 or more weeks of gesta-tion (success) Immunization with paternal mono-nuclear cells did not improve pregnancy outcome
in women with unexplained recurrent miscarriage.However, it is possible that a subset of respondersmight be identified by using some as yet unrecognizedlaboratory determination or susceptibility factor.Until this is possible, immune therapy in women withhabitual abortion should be restricted to clinical trials
(Moloney et al 1989).
Tests for HLA alleles, antigens and antibodies
HLA allelesHLA alleles can be determined directly at the DNAlevel The resolution of DNA-based typing is limitedonly by the available allele-specific probes The relev-ant techniques are based on several different prin-ciples (see Chapter 3) While the heterogeneity of theMHC has made high-resolution typing problematicfor matching donor and recipient for transplantation
(Petersdorf et al 2001), the stringency of the HLA role
in antigen presentation has made high resolutionincreasingly desirable for immunotherapy trials.Anthony Nolan HLA informatics group publishes up-to-date online HLA Class I and II Sequence Alignments(http://www.anthonynolan.com/HIG/data.html)
Sequence-specific oligonucleotides DNA is amplified
in the polymerase chain reaction (PCR) and a set of
Trang 27sequence-specific oligonucleotides (SSOs) is used in a
dot-blot or reverse dot-blot hybridization technique
to detect allelic sequences (Saiki et al 1986, 1989; Ng
et al 1993) Several modifications of this technique
have been described (Bidwell 1994) In one, a strand of
heat-denatured, amplified DNA is ligated with SSOs
by an added ligase Ligation only occurs when the
sequences of the DNA and SSO are identical The ligated
product is detected by enzyme-linked immunosorbent
assay (ELISA) (Fischer et al 1995) The techniques
permit the identification of alleles, even of those
differ-ing from each other by a sdiffer-ingle nucleotide
Sequence-specific primers PCR is performed with a
set of sequence-specific primers (SSPs) that will only
amplify DNA with sequences complementary to the
primers (Olerup et al 1993; Olerup 1994; Bunce et al.
1995) A simple and quick SSP test in microplates has
been described (Chia et al 1994).
The limitations of both SSO and SSP are
require-ments for a large number of PCRs to include the known
alleles, and the inability to identify polymorphisms
unless the variation happens to lie within the region
spanned by the assay These limitations are addressed
by nucleotide sequencing of PCR-amplified DNA, the
method of choice for ‘high-resolution’ typing that is
required in the selection of an unrelated stem cell donor
(Spurkland et al 1993) High-throughput robotic
sequence-based typing allows daily sequencing of
hundreds of genomic fragments, and high-density
array technology promises to permit extensive typing
of polymorphisms, both known and unknown, on
microchips (Adams et al 2001; Wang et al 2003).
HLA antigens and antibodies
Class I antigens are determined using the
lymphocyto-toxicity test Crossreactivity and the lack of specific
antisera led to difficulties in HLA typing; several antisera
must be used in typing for a particular antigen In
sero-logical typing for DR and DQ antigens, the two-colour
fluorescence test or the lymphocytotoxicity test on B
cells is applied (see below) These same techniques are
used for the detection of class I and class II antibodies
respectively Lymphocytotoxicity is declining in interest
in the USA as most laboratories switch to easier, higher
resolution molecular methods However,
immunolo-gical methods remain valuable to characterize
func-tional aspects of HLA as molecular methods cannot
define whether an HLA allele is expressed or how asequence correlates with empirically determined anti-gen importance
Lymphocytotoxicity test
Complement-dependent cytotoxicity remains the standard test for determining HLA class I antigens.Lymphocytes are incubated with antibody and rabbitcomplement, and a dye (Trypan blue or eosin) is thenadded If the lymphocytes carry an antigen corres-ponding to the antibody, complement is fixed, the cell membrane is damaged and dye enters and stainsthe cell (blue or red) The percentage of stained cells iscounted Live cells are unstained, smaller and refrac-tile It is essential to use a pure lymphocyte suspension,
as platelets carry A, B and C antigens and granulocytesare always killed in the cytotoxic assay and stain non-specifically Details of the NIH-recommendedlymphocytotoxicity test, using microdroplets, werepublished by Terasaki and co-workers (1973)
For the determination of DR and DQ antigens bylymphocytotoxicity, B lymphocytes can be isolated:(1) by removing the T lymphocytes from a lymphocytesuspension by rosetting with Z-aminoethylisothiouro-nium bromide-treated sheep red cells and centrifuga-tion on Ficoll-hypaque (density 1.077) (Pellegrino
et al 1976); (2) by the use of nylon fibre columns (Wernet et al 1977); or (3) by the use of magnetic
beads coated with monoclonal antibodies specific for
class II epitopes (Vartdal et al 1986).
In the two-colour fluorescence tests the IgG on the Bcells is capped with FITC-labelled anti-IgG followed
by a cytotoxicity test The B cells can be distinguishedfrom the T cells by the green IgG cap on their surface.For a description of the technique, see van Rood andvan Leeuwen (1976)
The mixed lymphocyte culture
This test was described by Bain and co-workers(1964) The principle is to irradiate or add a substancesuch as mitomycin C to one sample, usually thedonor’s, and to mix these lymphocytes with thosefrom another subject such as a potential recipient.Irradiation, or treatment with mitomycin C, preventslymphocytes from transforming to blast cells but doesnot destroy their ability to stimulate other lymphocytes.Blast transformation of the untreated lymphocytes
Trang 28indicates that they have recognized a foreign antigen
on the treated lymphocytes (Bach and Voynow 1966),
and this transformation can be assessed by measuring
the incorporation of tritiated thymidine (one-way
MLC)
In the MLC, the cells that stimulate are B cells
and monocytes carrying Dw determinants and class II
antigens Those that respond are T cells (Potter and
Moore 1977)
If irradiated or mitomycin C-treated stimulator
cells, homozygous for a Dw determinant (homozygous
typing cells, or HT), are used they can only stimulate
untreated lymphocytes that do not carry the Dw
deter-minant for which they are homozygous Thus, panels
of HTC are used to identify Dw determinants (Bradley
et al 1972).
The two-way MLC, in which the lymphocytes in
both samples are able to respond by blast formation,
has been used as a final test for HLA identity of donors
and recipients of bone marrow who are serologically
identical
HLA antibody detection
Typed repository cell lines are used in a
complement-dependent cytotoxicity assay to identify alloantibodies
in sera of sensitized subjects The percentage of cell
lines killed by the sera is used as a rough measure of
the degree of sensitization or ‘panel reactive antibody’
(PRA) reactivity Some antibodies activate complement,
yet kill cells inefficiently, a phenomenon known as
‘cytotoxicity negative absorption positive’ (CYNAP)
(Lublin and Grumet 1982) The CYNAP phenomenon
may result in underestimation of sensitization
How-ever, augmentation of the assay to increase sensitivity
may implicate innocuous antibodies and thus
over-estimate clinically relevant sensitization Another
method of identifying alloantibodies uses (Le Pendu
et al 1986; Le Pont et al 1995) flow cytometry of
a variety of microbeads loaded with known HLA
alleles (Guertler et al 1984; Moses et al 2000) An
interlaboratory comparison of techniques suggests
that considerable inconsistencies in serum screening
and crossmatching exist among laboratories
particip-ating in the American Society for Histocompatibility
and Immunogenetics/College of American Pathologists
surveys (Duquesnoy and Marrari 2003) The lack of
uniformity in test results may limit the usefulness of
these methods in a clinical setting
Other antigens found on leucocytes
Some red cell antigens are also found on leucocytes; see Chapters 4 and 6
Antigens located on granulocytes (humanneutrophil antigens)
Nomenclature: confusion, controversial and evolving
Neutrophil antigens were first characterized using seracollected from neutropenic patients who formed clin-ically important allo- and autoantibodies Althoughthe presence of the first granulocyte-specific antigen,NA1, was inferred from the presence of an antibody in
a case of neonatal neutropenia in 1960, the antigen
was not identified until 1966 (Lalezari et al 1960;
Lalezari and Bernard 1966a) As new antigens werediscovered, nomenclature threatened to assume some
of the quirky randomness that characterized red cellblood group antigens A new nomenclature was pro-posed in 1998 by an International Society of BloodTransfusion (ISBT) Working Party to permit separatenotations for the phenotype associated with the glyco-protein location and for the alleles, according to theguidelines for human gene nomenclature (Bux 1999).Although the proposed nomenclature has been criti-cized for including antigens found on cells other thangranulocytes, the ISBT proposal represents a soundfirst attempt at standardization
In the ISBT nomenclature, antigen systems arereferred to as human neutrophil antigens (HNA) The antigen systems, the polymorphic forms of theimmunogenic proteins, are indicated by integers andspecific antigens within each system are designatedalphabetically by date of publication Alleles of thecoding genes are named according to the Guidelinesfor Human Gene Nomenclature Neutrophil antigensNA1 and NA2 became HNA-1a and HNA-1b in thenew nomenclature and the third antigen reported,NB1 became HNA-2a (Table 13.1)
The HNA-1 system
The neutrophil-specific antigens HNA-1a and -1b(NAl and NA2) are products of alleles that form a biallelic system confined to granulocytes (Lalezari
et al 1960; Lalezari and Radel 1974) and NK cells.
Trang 29Exceptions to the inheritance of the NA antigens first
suggested the possibility of a silent allele at the NA
locus (Lalezari et al 1975; Clay 1985) The HNA-1
antigens are located on the FcγRIIIb of neutrophils
(Huizinga et al 1990) FcγRIIIb on neutrophil
mem-branes is a phosphatidylinositol-linked glycoprotein
with a molecular weight of 50 –80 kDa (Huizinga et al.
1989, 1990)
FcγRIIIb and the HNA-1 antigens are encoded by
the FCGR3B gene located on chromosome 1q23–24
within a cluster of two families of the FcγR genes,
Fc γR2 and FcγR3 The FcγR3 family is made up of
FCGR3A and FCGR3B FCGR3B is highly
homo-logous to FCGR3A, which encodes FcRIIIa The most
important difference between the two genes is a C-to-T
change at 733 in FCGR3B, which creates a stop codon
in FcγRIIIb As a result, FCGR3A has 21 more amino
acids than FCGR3B, and FCGR3A is a
transmem-brane rather than a GPI-anchored glycoprotein (Ory
et al 1989; Ravetch and Perussia 1989; Huizinga et al.
1990; Trounstine et al 1990) FcγRIIIa is expressed
only by NK cells and FcRIIIb only by neutrophils
(Trounstine et al 1990).
HNA-1a, -1b and –1c polymorphisms The HNA-1
antigen system consists of the three alleles HNA-1a,
-1b and -1c (Bux et al 1997) The antigens are also
known as NA1, NA2 and SH (Table 13.1) The gene
frequencies of the three alleles vary widely among
dif-ferent racial groups (Hessner et al 1999; Matsuo et al.
2001) Among white people, the frequency of the gene
encoding HBA-1a, FCGR3B*1, is between 0.30 and
0.37, and the frequency of the gene encoding HNA-1b,FCGR3B*2, is from 0.63 to 0.70 In Japanese andChinese populations, the FCGR3B*1 gene frequency
is from 0.60 to 0.66, and the FCGR3B*2 gene quency is from 0.30 to 0.33 The gene frequency of the gene encoding HNA-1c, FCGR3B*3, also variesamong racial groups FCGR3B*3 is expressed by neutrophils from 4% to 5% of white people and 25
fre-to 38% of African Americans (Kissel et al 2000).
The FCGR3B*1 gene differs from the FCGR3B*2gene by only five nucleotides in the coding region at
positions 141, 147, 227, 277 and 349 (Ory et al 1989; Ravetch and Perussia 1989; Huizinga et al 1990; Trounstine et al 1990) Four of the nucleotide changes
result in changes in amino acid sequence between theHNA-1a and the HNA-1b forms of the glycoprotein.The fifth polymorphism at site 147 is silent The gly-cosylation pattern differs between the two antigensbecause of the two nucleotide changes at bases 227
and 277 The HNA-1a form of Fc γRIIIb has six
N-linked glycosylation sites and the HNA-1a form hasfour glycosylation sites
The gene encoding the HNA-1c form of F γcRIIIb,
FCGR3B*3, is identical to FCGR3B*2 except for
a C-to-A substitution at amino acid 78 of FcRIIIb (Bux et al 1997) In many cases, FCGR3B*3 exists
on the same chromosome with a second or duplicate
FCGR3B gene (Koene et al 1998).
Several other sequence variations in FCGR3B havebeen described These chimeric alleles have single-basesubstitutions involving one of the five SNPs that
distinguish FCGRB3B*1 and FCGR3B*2 FCGR3B
Antigen system Antigens Location Former name Alleles
CR3, C3bi receptor; gp, glycoprotein; HNA, human neutrophil antigen; ISBT,
International Society of Blood Transfusion; LFA-1, leukocyte function antigen-1.
From Wang E, Marincola FM, Stroncek D Human leukocyte antigen (HLA) and
human neutrophil antigen (HNA) systems In: Hematology: Basic Principles and
Practice (2005), Philadelphia, PA: Elsevier Churchill Livingstone.
Table 13.1 ISBT Human Neutrophil
Antigen (HNA) nomenclature.
Trang 30alleles that more closely resembled FCGR3B*2 were
found more often in African Americans than in white
people or Japanese people (Matsuo et al 2001).
Function of HNA-1 antigens The low-affinity
FcγRIIIb receptors link humoral and cellular immune
function The FcγRIIIb on effector cells bind cytotoxic
IgG molecules and immune complexes containing
IgG Polymorphisms in FcγRIIIb affect neutrophil
func-tion Neutrophils that are homozygous for HNA-1a
have a greater affinity for IgG3 than do those that
are homozygous for HNA-1b (Nagarajan et al 1995).
Neutrophils from subjects homozygous for HNA-1b
phagocytize erythrocytes sensitized with IgG1 and
IgG3 anti-Rh monoclonal antibodies and bacteria
opsonized with IgG1 less efficiently than do
granulo-cytes homozygous for HNA-1a (Bredius et al 1994).
Fc γRIIIb deficiency Blood cells from patients with
paroxysmal nocturnal haemoglobinuria (PNH) lack
the GPI-linked glycoproteins and their granulocytes
express reduced amounts of FcγRIIIb and the HNA-1
antigens (Huizinga et al 1990) Genetic deficiency of
granulocyte FcγRIIIb and the HNA-1 antigens has
been reported With inherited deficiency of FcγRIIIb,
the FCGR3B gene is deleted along with an adjacent
gene, FCGR2C (De Haas et al 1995) Despite the
prim-ary role of FcγRIIIb in neutrophil function, deletion
of the entire Fc γRIIIB gene results in no obvious
clin-ical abnormality Although most subjects who lack
FcγRIIIb appear healthy, too few have been studied
to ensure that no subtle alteration in immune
func-tion is present In a study of 21 FcγRIIIb subjects
with FcγRIIIb deficiency, two were found to have
autoimmune thyroiditis and four had sustained
mul-tiple episodes of bacterial infections (De Haas et al.
1995)
FCGR3B polymorphisms and disease
associations
Several studies suggest that FCGR3B polymorphisms
affect the incidence and outcome of some autoimmune
and inflammatory diseases Children with chronic
immune thrombocytopenic purpura were more likely
to be FCGR3B*1 homozygous than were control
sub-jects (Foster et al 2001), but Spanish patients with
systemic lupus erythematosus were more likely to be
FCGR3B*2 homozygous (Gonzalez-Escribano et al.
2002) Myasthenia gravis is more severe in FCGR3B*1
homozygous patients (Raknes et al 1998), but multiple
sclerosis is more benign in FCGR3B*1 homozygous
patients (Myhr et al 1999) Patients with chronic
granulomatous disease who are FCGR3B*1 zygous are less likely to develop major gastrointestinal
homo-or urinary tract infectious complications comparedwith those who are heterozygous or FCGR3B*2 homo-
zygous (Foster et al 1998) As FCGR3B is clustered
with FCGR3BA and FCGR2B on chromosome 1q22,some of these findings may reflect in part linkage dis-equilibrium among Fc receptors
The HNA-2 system
The HNA-2 system has one well-described allele,HNA-2a (NB1) expressed only on neutrophils, meta-
myelocytes and myelocytes (Stroncek et al 1998a).
The 58- to 64-kDa glycoprotein that carries HNA-2a(NB1 gp) is located on neutrophil plasma membranesand in secondary granules, and is linked to the plasmamembrane by a glycosylphosphatidylinositol (GPI)anchor HNA-2a is expressed on 45–65% of circulat-ing neutrophils; expression is greater on neutrophils
from women than from men (Stroncek et al 1996; Matsuo et al 2001) Pregnant women express HNA-
2a more strongly than do healthy female blood
donors (Caruccio et al 2003) Expression of HNA-2a
decreases with age in women, but remains constant
in men Administration of G-CSF to healthy subjects can increase the proportion of neutrophils express-
ing HNA-2a to near 90% (Stroncek et al 1998b).
Monoclonal antibodies specific to HNA-2a have beenclustered as CD177 The role of CD177 in neutrophilfunction is unknown Women who lack NB1 gp arehealthy Although the expression of HNA-2a is reduced
on neutrophils from patients with PNH and chronicmyelocytic leukaemia (CML), no clinical significancehas been attributed to this observation
HNA-2 polymorphisms HNA-2a is expressed on
neutrophils by approximately 97% of white people,95% of African Americans and 89–99% of Japanese
people (Matsuo et al 2000; Taniguchi et al 2002).
HNA-2a has been reported to have an allele, NB2, but the product of this gene cannot be identified reli-ably with alloantisera, and no monoclonal antibody
specificity for NB2 has been identified (Stroncek et al.
1993a) The HNS-2a-negative neutrophil phenotype is
Trang 31due to a CD177 transcription defect Kissel HNA-2a
genes from two women with HNA-2a-negative
neu-trophils, who produced HNA-2a-specific
alloanti-bodies have been studied and CD177 cDNA sequences
were present in both women Sequencing of cDNA
prepared from neutrophil mRNA demonstrated
acces-sory sequences in the lengths coding CD177
The HNA-3 system
The HNA-3 antigen system has one antigen HNA-3a
that was previously known as 5b HNA-3a is
expressed by neutrophils, lymphocytes, platelets,
endothelial cells, kidney, spleen and placental cells
(van Rood and Ernisse 1968) and weakly expressed on
red cells (Rosenfield et al 1967) The gene encoding
HNA-3a is located on chromosome 4 (van Kessel et al.
1983), but has not yet been cloned The nature and
function of the 70 –95 kDa gp is unknown Potent
anti-HNA-3a agglutinins in transfused plasma can cause
transfusion-related acute lung injury (TRALI) (see
Chapter 15)
HNA-4 and HNA-5 systems
The HNA-4 and HNA-5 antigens are located on the β2
integrins Each system contains only a single antigen,
HNA-4a and HNA-5a The HNA-4a antigen,
previ-ously known as Marta, has a phenotype frequency of
99.1% in white people (Kline et al 1982) HNA-4a is
expressed on granulocytes, monocytes and
lympho-cytes, but not on platelets or red blood cells HNA-4a
has been located on the αM chain (CD11b) of the
receptor CR3 Neonatal alloimmune neutropenia has
been caused by antibody to HNA-4a, but this is the
exception rather than the rule (Fung et al 2003) A
second polymorphism of the β2 integrins, HNA-5a,
previously Ondawith a frequency of 95%, is located
on the α-chain of the leucocyte function-associated
antigen (LFA-1, CD11a) molecule (Simsek et al.
1996)
Other granulocyte-specific antigens
Other granulocyte-specific antigens are ND1, NE1
and LAN (Lalezari and Radel 1974; Verheugt et al.
1978; Claas et al 1979; Rodwell et al 1991) Like the
NA antigens, LAN is located on the FcγRIIIb (Metcalfe
and Waters 1992) Another high-frequency antigen,
also located on the FcγRIIIb, was described by Bux and colleagues (1994) The antigen NC1 (Lalezari
and Radel 1974) is identical with HNA-1b (Bux et al.
1995a) Most granulocyte-specific antigens have beendefined by alloantibodies, but ND1 and NE1 weredefined by autoantibodies These granulocyte-specificantigens appear to be true differentiation antigens, asthey appear at the myelocyte or metamyelocyte stage
or even later (Lalezari 1977; Evans and Mage 1978)
Antigens on granulocytes and monocytes
The following antigens have been shown to be present
on granulocytes and monocytes: HGA-1 (Thompson
et al 1980) and the HMA-1 and HMA-2 antigens, products of a biallelic gene (Jager et al 1986) The
AYD antigen is shared by granulocytes, monocytesand endothelial cells (Thompson and Severson 1980).The 9a antigen, which was first thought to be granulo-cyte specific, is also expressed on monocytes (Jager
et al 1986).
Antibodies to neutrophil antigens
Antibodies to neutrophil antigens may be responsiblefor five different clinical syndromes: (1) neonatal allo-and isoimmune neutropenia; (2) autoimmune neutro-penia; (3) transfusion-related alloimmune neutropenia;(4) pulmonary infiltrates following transfusion (TRALI);and (5) febrile reactions following transfusion Thelast two conditions are discussed in detail in Chapter 15.Transfusion-related alloimmune neutropenia is prob-ably a variant of TRALI and appears to be rare (Wallis
et al 2002).
Neonatal alloimmune neutropenia
This syndrome, analogous to haemolytic disease of thenewborn (HDN), is usually recognized when infection
in a newborn infant is found to be accompanied bysevere neutropenia Neutrophil antigens in the fetusthat are inherited from the father but foreign to themother provoke formation of maternal IgG antibodiesthat cross the placenta and react with the neonate’sneutrophils (Lalezari and Bernard 1966b) Absoluteneutrophil counts typically range from 0.100 to 0.200
× 109/l Bone marrow examination reveals myeloidhyperplasia The syndrome is self-limited, but maypersist from days to weeks as passive antibody is
Trang 32cleared (Bux et al 1992) Treatment with IVIG or
recombinant cytokines such as granulocyte
colony-stimulating factor (G-CSF) has met with variable
success (Maheshwari et al 2002).
In reviewing the syndrome, Lalezari and Radel
(1974) described results in 19 infants from 10 families
The specificity of the antibody in three families was
HNA-1a; in two, HNA-1b; in four,
anti-HNA-2a; and in one, not determined In four of the
families the first-born infant was affected Antibodies
of other specificities have been implicated, but much
less frequently (Stroncek 2002)
A prospective survey of some 200 pregnant women,
either primiparae at term or multiparae, in which
the woman’s serum was tested against her partner’s
granulocytes and lymphocytes, indicated that the
incidence of neutrophil antibodies was about 3%
(Verheugt et al 1979) The incidence of diagnosed
cases of neonatal alloimmune neutropenia is much
lower
Neonatal isoimmune neutropenia
Subjects who lack a membrane glycoprotein due to
deletion of the gene encoding the glycoprotein may
form strong antibodies (named isoantibodies) against
non-polymorphic determinants on the glycoprotein
Severe neonatal neutropenia caused by maternal
isoantibodies may occur in infants from mothers with
a deletion of the FcγRIIIb gene (Huizinga et al 1990;
Stroncek et al 1991; Cartron et al 1992; Fromont
et al 1992).
Autoantibodies to granulocytes
The first convincing case that implicated
autoanti-bodies as the cause of neutropenia involved a female
infant who had severe infections and was found at the
age of 7 months to have a neutrophil count of 1.0 × 109/l
The peripheral blood contained fewer than 3% mature
neutrophils and the bone marrow revealed virtually no
mature granulocytes, although it did contain normal
granulocyte precursors The patient’s serum contained
the neutrophil-specific autoantibody anti-HNA-1b
with a titre of 16–256 The antibody was mainly IgG
and the patient was HNA-1b positive After steroid
therapy, the granulocyte count rose to 310 × 109/l and
the leucoagglutinin titre fell to 2, but the patient
relapsed when steroids were discontinued (Lalezari
et al 1975) The syndrome is now well established (McCullough 1988; Bux et al 1998).
Autoimmune neutropenia (AIN) in children is tionally divided into two forms In so-called ‘primary’AIN, neutropenia is the sole abnormality and,although neutrophil counts may fall below 0.500/l,bacterial infections, when they occur, are generallybenign Primary AIN is commonly diagnosed betweenthe ages of 5 and 15 months, but has been observed as
tradi-early as day 33 of life (Bux et al 1998) Spontaneous
remission occurs in 95% of the patients within 7–24months A high percentage of autoantibodies (35–86%)bind preferentially to granulocytes from HNA-1a andHNA-1b homozygous donors, but other specificities
have been found (Bux et al 1998; Bruin et al 1999).
The bone marrow is typically normocellular or hypercellular, with a variably diminished number ofsegmented granulocytes For severe infections or prior to surgery, G-CSF, corticosteroids and IVIG (19 mg/kg per day) can effect neutrophil increases of50–100% and each has been used successfully in many
cases (Pollack et al 1982; Bussel and Lalezari 1983; Bux et al 1998).
Secondary AIN occurs in association with otherautoimmune diseases In contrast with primary AIN,infections are usually more severe and the autoanti-body is more commonly directed against FcγRIIIb
(Shastri and Logue 1993; Bruin et al 1999).
Drug-induced immune granulocytopenia
Mechanisms responsible for drug-induced immuneneutropenia are similar to those involving red cells (see Chapter 7) The classic case of pyramidon-induced granulocytopenia described by Moeschlin and Wagner (1952) is an example of the mechanism
in which the drug does not bind firmly to the cells, but in which drug, antibody and a determinant on the cell membrane form a trimolecular complex.Although quinine is usually implicated in drug-induced thrombocytopenia, it may be involved rarely
in cases of drug-induced neutropenia in which the quinine antibodies react with the same glycoprotein
as anti-HNA-2a, and/or an 85-kDa glycoprotein
(Stroncek et al 1994) Drug-induced neutrophil
anti-bodies may be directed against a metabolite of the
drug (Salama et al 1989) Recombinant G-CSF has
reportedly shortened the period of granulocytopenia
in some of these cases
Trang 33Reactions to granulocyte transfusions
Granulocyte transfusion recipients sometimes produce
antibodies specific to HNA-1a, HNA-1b and HNA-2a
(see also Chapters 14 and 15) Further transfusion of
granulocytes to patients with these antibodies may
lead to severe febrile and pulmonary transfusion
reac-tions (Stroncek 1996) Haematopoietic progenitor cell
transplant recipients who produce HNA-2a antibodies
as a result of granulocyte transfusions have
experi-enced marrow graft failure (Stroncek et al 1993b).
Tests for granulocyte antibodies and antigens
The following techniques are used in detecting
granulocyte-specific antibodies: (1) granulocyte
agglutination; (2) immunofluorescence; (3)
chemilu-minescence; and (4) monoclonal antibody-specific
immobilization of granulocyte antigen assays
The granulocyte agglutination technique
Pure granulocyte suspensions are prepared by dextran
sedimentation followed by centrifugation of the
super-natant on Ficoll-hypaque (density 1.077) The
con-taminating red cells in the granulocyte pellet at the
bottom of the tube are lysed with ammonium chloride
or distilled water Alternatively, granulocytes can
be isolated by double-density gradient
centrifuga-tion Agglutination techniques are carried out in
microplates
Granulocytes, in contrast to red cells and platelets,
are agglutinated by two different mechanisms:
1 Like red cells and platelets granulocytes are
agglu-tinated by crosslinking of cells by IgM antibodies
2 An entirely different mechanism is responsible for
agglutination of granulocytes by IgG antibodies In
this case, agglutination results from a response to
sensitization by an antibody that requires active cell
participation Sensitization does not lead to immediate
agglutination but to the formation of pseudopods
The granulocytes migrate towards each other until
membrane contact is established (Lalezari and Radel
1974) This process is time and temperature (37°C)
dependent Agglutination may be due to changes in
membrane-bound molecules that cause granulocytes
to adhere to each other, or to IgG antibodies on one
granulocyte that adhere to Fc receptors on other
gran-ulocytes In any case, both IgM and IgG antibodies can
be detected by the granulocyte agglutination test Bothgranulocyte-specific and HLA-A, -B and -C antibodiesare detected, but HLA antibodies are better detected
by the lymphocytotoxicity test
Granulocyte immunofluorescence technique
Purified suspensions of granulocytes are prepared
as described above The granulocytes are fixed withparaformaldehyde, incubated with the serum to
be tested, then washed and finally incubated withfluorescein isothiocyanate-labelled anti-Ig serum TheFab or F(ab′)2fragments of the IgG fraction of anti-human Ig are used because whole IgG anti-Ig tends
to bind to the Fc receptor on granulocytes With theabove modifications, the fluorescein-labelled anti-globulin test is more sensitive than granulocyte agglu-tination for the detection of IgG antibodies (Verheugt
et al 1977).
Using flow cytometry for the granulocyteimmunofluorescence technique (GIFT) instead ofmicroscopy, there is no need for isolating granulo-cytes, as granulocytes can be identified according
to light scatter patterns Furthermore, granulocytes,platelets and lymphocytes can be tested simultane-
ously (Robinson et al 1987) Flow cytometry has been
found to be slightly more sensitive for the detection
of granulocyte antibodies than the GIFT (Sintnicolaas
et al 1991).
Both granulocyte-specific and HLA-A, -B and -Cantibodies are detected in the GIFT The lymphocyto-toxicity test (LCT) and the immunofluorescence testare more sensitive for the detection of HLA class I antibodies If a serum is negative in these tests but pos-itive in the GIFT, the serum is very likely to containgranulocyte-specific antibodies If the tests on lympho-cytes are positive and if it is necessary to ascertainwhether granulocyte-specific antibodies are present,the serum should be absorbed with pooled platelets toremove any HLA class I antibodies or the serum must
be tested with a granulocyte panel typed for specific antigens Unfortunately, positive reactions withlymphocytes particularly in the immunofluorescencetest may be due to lymphocyte-specific antibodies,
granulocyte-in which case it may be difficult to ascertagranulocyte-in the ence of granulocyte-specific antibodies, unless a knownspecificity is detected
pres-With the GIFT, not only antibodies but also formed immune complexes cause positive reactions,
Trang 34pre-due to adherence to Fc and complement receptors
(Camussi et al 1979; Engelfriet et al 1984) There are
three possible ways of distinguishing between
anti-bodies and fixed immune complexes:
1 Preparation of an eluate from positively reacting
granulocytes Eluted antibodies will again react with
granulocytes while immune complexes are usually
dis-sociated by the elution procedure (Helmerhorst et al.
1982)
2 Testing the serum under investigation in an ADCC
assay on granulocytes
3 Blocking Fc receptors on target granulocytes with
monoclonal antibodies (Engelfriet et al 1984) In
practice, it is difficult to distinguish between
autoanti-bodies and bound immune complexes, because there
are seldom enough cells to prepare an eluate and
because the results of the ADCC assay on patients’
granulocytes are difficult to interpret
Chemiluminescence test
To prepare suspensions of mononuclear cells and
granulocytes, fresh EDTA blood is centrifuged on
Ficoll-hypaque (density 1.077) The mononuclear
cell fraction is washed three times The red cell/
granulocyte fraction is resuspended in PBS and
mixed 1:4 with dextran solution After sedimentation
(30 min) the granulocytes in the supernatant are
packed and then washed twice in PBS The
granulo-cytes are resuspended in PBS and transferred to a
glass tube pre-located in a waterbath at 52°C and
after 2 min are allowed to cool in another tube at
room temperature The purpose of the heat treatment
is to render the granulocytes incapable of responding
to immunoglobulin aggregates in the sera to be
tested which would lead to non-specific generation of
chemiluminescence
For the assay, granulocytes are incubated with
serum and then washed in phosphate-buffered
solu-tion (PBS) and resuspended in Hanks’ BSS The
gran-ulocytes are then incubated with freshly prepared
mononuclear cells from the peripheral blood and with
luminol Chemiluminescence is generated as a result of
phagocytosis of sensitized granulocytes, which leads
to the formation of oxygen radicals and oxidation of
luminol Chemiluminescence is measured in a
lumino-meter (Hadley and Holburn 1984) The sensitivity of
the chemiluminescence (CL) test is similar to that of
the GIFT (Lucas 1994)
Phenotyping and genotyping of neutrophil antigens
Phenotyping By tradition, neutrophil antigen typing
has been performed using human alloantibodies in thegranulocyte agglutination or GIFT assay However,alloantisera are difficult to obtain Monoclonal anti-bodies specific to HNA-1a, HNA-1b and HNA-2ahave been described and are available commercially.These reagents have been used to phenotype neutro-phils by flow cytometry This method is faster and easier than manual methods with alloantibodies, aswhole blood rather than isolated neutrophils can be used
Genotyping As alloantibodies are difficult to obtain
and monoclonal antibodies are not available for allneutrophil antigens, genotyping assays have gainedimportance Genotyping assays are performed withDNA isolated from whole blood, thus eliminating theneed to isolate granulocytes Furthermore, leucocyteDNA can be stored for months before testing is per-formed The characterization of the genes encodingHNA-1 antigens has led to the development of assays
for these antigens (Bux et al 1995b; Hessner et al.
1996) Genotyping of HNA-1 antigens is particularlyvaluable given the rarity of alloantibodies to HNA-1cand the absence of monoclonal antibodies Geno-typing for FCGR3B alleles is complicated by the high degree of homology between FCGR3B andFCGR3A Among the five nucleotides that differbetween FCGR3A*1 and FCGR3A*2, FCGR3A is thesame as FCGR3A*1 at three nucleotides and the same
as FCGR3A*2 at two nucleotides As a result, mostlaboratories use PCR and sequence-specific primers todistinguish FCGR3A alleles A unique set of primers isused to amplify each of the three alleles
Unfortunately, HNA-2a genotyping reagents are notavailable The HNA-2a-negative phenotype is caused
by CD177 mRNA splicing defects (Kissel et al 2002).
However, no mutations have been detected in theCD177 genomic DNA from subjects with HNA-2a-negative neutrophils It may be possible to distinguishpositive and negative phenotypes by analysing CD177mRNA for accessory sequences, but this is a highlysophisticated methodology
Antigens found only on lymphocytes
In addition to HLA antigens (see above) to some red
Trang 35cell antigens (see Chapter 5), 5a and HNA-3a antigens,
and antigens also present on granulocytes and
mono-cytes, lymphocytes carry antigens that do not occur on
other cells
Two biallelic systems, one on Tγ cells with the
antigens TCA1 and TCA2, and one on Tµ cells with
the antigens TCB1 and TCB2 have been defined (van
Leeuwen et al 1982a) Non-HLA antigens only
expressed on activated T cells have been described
(Gerbase et al 1981; Wollman et al 1984) The
clin-ical significance of alloantibodies against
lymphocyte-specific antigens is uncertain, but a case of alloimmune
lymphocytopenia of the newborn, due to maternal
alloantibodies and resulting in severe combined
immune deficiency has been reported (Bastian et al.
1984)
Cold autoantibodies to lymphocytes
Anti-I and anti-i See Chapter 4.
Lymphocyte autoantibodies reactive
at 37°C in vitro
Single cases of (1) hypogammaglobulinaemia with
cytotoxic autoantibodies against B cells reactive at
37°C and (2) acquired hypogammaglobulinaemia
with autoantibodies specific for T-helper cells, leading
to increased activity of T-suppressor cells, have been
described (Tursz et al 1977).
Antibodies against various subsets of lymphocytes
have been described in patients with AIDS and may
contribute to the decline in the CD4+T-cell count
Antigens found only on monocytes
In addition to HLA class I and class II antigens, and
the antigens shared by monocytes and granulocytes
mentioned above, monocytes carry alloantigens that
do not occur on other blood cells Some of these
anti-gens (EM antianti-gens) are also present on endothelial
cells (Moraes and Stastny 1977; Claas et al 1980;
Cerilli et al 1981; Stastny and Nunez 1981); others
are monocyte specific (Cerilli et al 1981; Baldwin et al.
1983; Paul 1984) Antibodies against EM antigens
are detrimental to transplanted kidneys and may be
involved in GvHD EM antibodies and antibodies
react-ing with monocytes, tubular endothelium and kidney
cells in the cortex can be eluted from rejected kidneys
(Joyce et al 1988) The significance of
monocyte-specific alloantibodies needs further evaluation
mole-platelets are absorbed from the plasma (Santoso et al.
1993a) The number of some of the class I antigens onplatelets varies greatly in different subjects Class IIantigens are not detectable on platelets but HLA-DRantigens can be induced at the platelet surface by stimu-lation with cytokines, for example gamma-interferon,
both in vitro and in vivo (Boshkov et al 1992).
Red cell antigens also found on platelets
ABH, Lewis, I, i and P antigens on platelets aredescribed in Chapter 4 Using a sensitive two-stageradioimmunoassay the major antigens of the Rh, Duffy,Kell, Kidd and Lutheran systems have been shown to
be absent from platelets (Dunstan et al 1984).
Antigens found only on platelets (platelet-specific antigens)Several systems have been defined whose antigens arefound on glycoproteins of platelets Some of these anti-gens are found on other cells such as endothelial cells
as well The human platelet antigen (HPA) ture system was adopted in 1990 (von dem Borne andDecary 1990) The HPA nomenclature categorizes allalloantigens expressed on the platelet membrane, exceptthose encoded by genes of the major histocompatibil-ity complex A platelet-specific alloantigen is called aHPA when its molecular basis has been defined Thedifferent HPAs are grouped in systems based on hav-ing alloantibodies defining a given alloantigen and its
nomencla-‘antithetical’ alloantigen A large number of antigenshave been described and the molecular basis of many has been resolved To date, 24 platelet-specificalloantigens have been defined by immune sera, ofwhich 12 are grouped into six biallelic systems (HPA-1,
-2, -3, -4, -5, -15) (Table 13.2 and Metcalfe et al.
Trang 362003) For the remaining 12 antibodies, alloantibodies
against the antithetical antigen have yet to be
discov-ered The molecular basis of 22 out of the 24
serologic-ally defined antigens has been resolved In all but one,
the difference involves a single amino acid substitution
generally caused by a single nucleotide polymorphism
(SNP) in the gene encoding the relevant membrane
gly-coprotein The systems are numbered in the order of
the date of publication and the antigens are designated
alphabetically in the order of their frequency in the
population This nomenclature has been criticized, the
main objection being that HPA-1a, HPA-4a, HPA-6a,
HPA-7a and HPA-8a are five different names for
ident-ical GPIIIa molecules carrying these high-frequency
antigens Only the GPIIIa molecules that carry the
low-frequency antigens of these systems differ from
each other due to amino acid substitutions at different
positions of the molecule (Newman 1994)
HPA-1 system (Zw, Pl A )
The first system to be described was recognized by
van Loghem and co-workers (1959) when a serum was found that agglutinated some samples of plateletsbut not others; the antigen was named Zwawhen anantithetical antigen (Zwb) was recognized (van der
Weerdt et al 1962, 1963) Anti-PlA1(Shulman et al.
1961) was subsequently shown to have the samespecificity as anti-Zwa The system is now namedHPA-1 and the antigens, HPA-1a and HPA-1b.Ninety-eight per cent of white people are HPA-1(a+)and 27% HPA-1 (b+)
The 1 gene has two alleles, 1a and 1b Anti-HPA-1a is associated with most cases of post-transfusion purpura and neonatal alloimmunethrombocytopenia (NATP)
HPA-HPA-1a and -1b antigen sites are situated on themembrane glycoprotein IIIa (Kunicki and Aster 1979;van der Schoot and von dem Borne 1986) The HPA-1polymorphism results from the substitution of a singlebasepair in the coding DNA at position 33, coding for leucine in HPA-1a and for proline in HPA-1b
(Newman et al 1989) Patients with Glanzmann’s
thrombocytopenia type I have no detectable membrane
System Antigen Original names Glycoprotein CD
HPA-4b Yuk a , Pen b
From Metcalfe et al (2003).
Table 13.2 Human platelet antigens
(HPAs).
Trang 37glycoprotein IIIa (or IIb) on their platelets and are
therefore unable to express the HPA-1 antigens
(Kunicki et al 1981; van Leeuwen et al 1981).
The HPA-1 polymorphism is not found in Japanese
people (Shibata et al 1986b).
HPA-2 system (Ko)
A second biallelic system, Ko (HPA-2), was described
by van der Weerdt and co-workers (1962) In total,
16% of subjects were found to be HPA-2(b+) (Ko(a+)
and 99% were HPA-2(a+) (Ko(b+) Like anti-HPA-1a,
anti-HPA-2a and -2b were detected by platelet
agglut-ination The HPA-2 antigens are situated on GPIb/IX
(Kuijpers et al 1989) The polymorphism involves
substitution of a single nucleotide in the DNA at
posi-tion 434, which codes for the β-chain of GPIb, to
give methionine in HPA-2b and threonine in HPA-2a
at position 145 (Kuijpers et al 1992a) A platelet
anti-gen Siba, described by Saji and co-workers (1989), was
shown to be identical to Koa(Kuijpers et al 1989).
HPA-3 system (Bak, Lek)
The platelet antigen, Baka(HPA-3a) is present in about
90% of the Dutch population (von dem Borne et al.
1980) The first example of anti-HPA-3a was
respons-ible for NATP An antigen, Leka, at first found to be
closely associated serologically with Baka (Boizard
1984) was subsequently shown to be identical (von
dem Borne and van der Plas-van Dalen CM 1985)
HPA-3a is present on glycoprotein IIb (Kieffer et al.
1984; van der Schoot and von dem Borne 1986) The
antigen HPA-3b, antithetical to HPA-3a, was described
independently by Kickler and co-workers (1988a) and
Kiefel and co-workers (1989a) In both cases,
anti-HPA-3b was responsible for post-transfusion purpura
The HPA-3 polymorphism is also due to the
substitu-tion of a single basepair in the coding DNA, to give
isoleucine at amino acid residue 843 in HPA-3a and
serine in HPA-3b (Lyman and Aster 1990)
HPA-4 system (Pen, Yuk)
Another biallelic system, Yuk (HPA-4) was described
by Shibata and co-workers (1986a,b) Both the
low-frequency antigen Yuka(HPA-4b) and the
high-frequency antigen Yukb(HPA-4a) were detected with
antibodies that caused NATP
The antigen, Pena, that had been described byFriedman and Aster (1985), proved to be identical toYukb(RH Aster and Y Shibata, unpublished observa-
tion) HPA-4a is present on GPIIIa (Furihata et al 1987; Santoso et al 1987) The HPA-4 polymorphism
has not been found in white people (Friedman and
Aster 1985; Kiefel et al 1988) The Yuk
polymor-phism involves substitution of a single nucleotide inthe DNA which encodes the GPIIIa protein, coding forarginine at position 526 in HPA-4a and for glutamine
in HPA-4b (Wang et al 1991).
HPA-5 system (Br, He, Zav, Tu a , Ca a Mo a , Sr a , Max a , La a , Gro a , ly a , Sit a , Oe a )
The antigens Bra (HPA-5b) and Brb (HPA-5a) weredescribed by Kiefel and co-workers (1988, 1989a).The HPA-5 antigens are present on glycoprotein la
(Kiefel et al 1989a; Santoso et al 1989) Anti-HPA-5a
and -5b have been responsible for NATP
Most HPA-5 antibodies are non-reactive in theimmunofluorescence test because of the low number of
antigenic sites (Kiefel et al 1989b) HPA-5 antibodies
can best be detected by the MAIPA, a specific assay (see below) The polymorphism involvessubstitution of a single nucleotide in the cDNA at posi-tion 1648, to give glutamine in HPA-5a and lysine
glycoprotein-in HPA-5b at position 505 (Santoso et al 1993b)
The biallelic Zav system described by Smith and co-workers (1989) is identical with the HPA-5 systemand the antigen Hca is the same as HPA-5b (Woods
et al 1989) Tu/Ca (HPA-6bw), a low-frequency
antigen located on GPIIIa and involved in NATP was
at first named Tua(HPA-6b) (Kekomaki et al 1993)
It is identical to Caadescribed by McFarland and workers (1993) The polymorphism involves a singlenucleotide substitution at position 1564 to give 489glutamine in HPA-6a and 489 arginine in HPA-6b
co-(Wang et al 1993b) The antigen Mo (HPA-7bw),
involved in NATP, is located on GPIIIa and due to
a C–G substitution at position 1267 in the cDNA,resulting in a substitution of proline by alanine at
position 407 (Kuijpers et al 1993) Sra(HPA-8b) has
so far been detected in only one family, in which it wasinvolved in NATP The antigen is located on GPIIIaand is due to the substitution at position 636 of cys-teine (in HPA-8b) for arginine (normally present in
GPAIIIa) (Kroll et al 1990; Santoso et al 1994) Maxa
(HPA-9bw), a low-frequency alloantigen responsible
Trang 38for NATP, is located on GPIIb and the polymorphism
is due to a single nucleotide substitution G→A at
posi-tion 2603 (Noris et al 1995) HPA-11bw (Groa) is
located on GPIIIa and involved in NATP It has so
far been found in only a single family A guanine
for adenine mutation was found, predicting an
arginine→histidine substitution at position 633 of the
mature glycoprotein (Simsek et al 1997) The antigen
ly (HPA-12 bw) is a low-frequency antigen located on
the glycoprotein Ib/IX complex Anti-Iyawas the cause
of severe NATP (Kiefel et al 1995) Sita(HPA-13bw),
a low-frequency antigen in the German population,
was identified in a severe case of neonatal alloimmune
thrombocytopenia Sit(a) epitopes reside on platelet
GPIa A threonine→methionine substitution at the
799 position is responsible for formation of the Sit(a)
alloantigen, and diminished platelet aggregation
responses of Sit(a)(+) individuals indicate that the
Thr(799)Met mutation affects the function of the
GPIa–IIa complex (Santoso et al 1999) Oea
(HPA-14bw), a low-frequency alloantigen responsible for a
case of neonatal NATP, has been assigned to platelet
GPIIIa Molecular studies suggest that Oeaarose as a
result of a mutational event from an already mutated
GPIIIa allele (Santoso et al 2002).
HPA-15 system (Gov, Duv)
A biallelic system with the alleles Gova(HPA-15b)
and Govb (HPA-15a) was reported by Kelton and
co-workers (1990) Anti-Govawas found in a patient
with post-transfusion purpura The Gov antigens are
expressed on the CDw 109 protein (Smith et al 1995).
Anti-Duv(a+), directed against an antigen HPA-16bw
(Duva) on glycoprotein GPIIIa has been implicated in
a case of neonatal thrombocytopenia Sequencing of
the exons 2–15 of GPIIIa revealed a single base
sub-stitution 517C→T (complementary DNA) present in a
heterozygous state in DNA from the father leading
to amino acid substitution of threonine for isoleucine
at position within the Arg-Gly-Asp binding domain of
GPIIIa (Jallu et al 2002).
Obsolete systems and systems not yet included
in the HPA nomenclature
DUZO Moulinier (1957), using the antiglobulin
con-sumption technique, demonstrated a platelet antibody
in the serum of a woman whose four children had died
from neonatal purpura The corresponding antigenwas termed ‘DUZO’ However, no second example ofanti-DUZO has been found and this antigen has there-fore become obsolete
PlE system The two alleles of this system (PlE1and
PlE2) were defined by Shulman and co-workers (1964).This system has not been included in the HPA nomen-clature because anti-PlE1was probably an isoantibodyfrom a patient with Bernard–Soulier syndrome andanti-PlE2is no longer available (Shulman 1987)
PlT antigen An antigen, PlT, with a very high quency was described by Beardsley and co-workers(1987) It is present on glycoprotein V
fre-Nak a The antigen Naka is absent in 3 –11% of
Japanese people and is present on GPIV (Ikeda et al.
1989) However, the Nak antigen appears to be a polymorphic determinant of GPIV, Nak(a–) subjectsbeing deficient for GPIV Anti-Nak therefore is not an
non-alloantibody, but an isoantibody (Yamamoto et al.
1990)
Va a The low-frequency antigen Vaa, involved in
NATP, is located on GPIIIa (Kekomaki et al 1992).
Presence of platelet-‘specific’ antigens
on other cells
The antigens of the HPA-1 system are present on
endothelial cells (Leeksma et al 1987; Giltay et al.
1988a) HPA-1a has also been detected on vascular
smooth muscle cells and fibroblasts (Giltay et al.
1988) The HPA-5 antigens are probably also present
on endothelial cells, which express VLA-2
Alloimmunization to platelet antigens
Role of HLA class I antibodies in refractoriness
to platelet transfusions
When no measures are taken to reduce the number
Trang 39of leucocytes in red cell or platelet concentrates,
80–100% of patients, depending upon the disease,
may develop HLA antibodies; only 40 –70% patients
treated with immunosuppressive regimens become
immunized (Howard and Perkins 1978; Dutcher et al.
1980, 1981) In the great majority of immunized
patients, the alloantibodies are directed against HLA
class I antigens (Schiffer et al 1976) Primary
immun-ization occurs as early as 10 days after transfusion,
although 3– 4 weeks is more usual; reappearance of
lymphocytotoxic antibodies appeared as early as 4
days in previously sensitized subjects In total, 62% of
women with acute myelocytic leukaemia and previous
pregnancies became alloimmunized following
trans-fusion during cytoreductive therapy (Trial to Reduce
Alloimmunization to Platelets Study Group 1997)
HLA alloimmunization does not necessarily correlate
with the number or schedule of transfusions, at least
when multiple transfusions are administered When
the number of leucocytes in the transfused cell
concen-trate is reduced, the percentage of immunized patients
decreases, and at levels of 1–5 × 106 or fewer, primary
immunization against HLA class I antigens is
pre-vented (see below)
The presence of HLA class I antibodies in a patient’s
serum does not equate with refractoriness to platelet
transfusions The frequency of most HLA antigens
is low and antibodies against them may not react
with the platelets of any randomly chosen donors
or may react with only a few of them Furthermore,
some class I antigens may be expressed so weakly
on the donor platelets that they survive normally,
or nearly normally, in a patient with antibodies
against them Furthermore, patients with
allobodies against HLA class I antiallobodies may form
anti-idiotype antibodies that react with and inactivate
the class I antibodies For this reason refractoriness
to platelet transfusions may be overcome in spite
of continued transfusions of incompatible platelets
(Atlas et al 1993) In a significant percentage of
alloimmunized patients, evidence of alloimmunization
declines or disappears with time (Lee and Schiffer
1987; Murphy et al 1987) Nevertheless, 10 –20%
of patients in large prospective and retrospective
series of thrombocytopenic patients treated for
malignancy become alloimmune refractory
follow-ing transfusion therapy (Trial to Reduce
Alloimmu-nization to Platelets Study Group 1997; Seftel et al.
2004)
Role of platelet-specific antibodies
Even when HLA-matched platelets, either from closerelatives or from random donors, are transfused, 19% of recipients became refractory (Schiffer 1987)
In most cases refractoriness was probably related to HLA incompatibilities that went undetected ratherthan to antibodies to platelet-specific antigens, becausethe latter occur almost exclusively in patients who are strongly immunized to HLA class I antigens Usingthe MAIPA, platelet-specific antibodies were found
in 25% (9 out of 36) of patients with high levels of
HLA immunization (Schnaidt et al 1996) This figure
corresponds well with the observation that fusions of HLA-compatible platelets are unsuccessful
trans-in about 20% of HLA-immunized patients (Saji et al.
antigens (Welsh et al 1977; Claas et al 1981; van Marwijk et al 1991) Formation of such antibodies
occurs if the platelets are contaminated with cytes, but platelets alone are capable of inducing a
leuco-secondary immune response (Gouttefangeas et al.
2000) Although foreign antigen is presented to helper
T cells by the subject’s own HLA class II-positive gen-presenting cells (APCs), the induction of a primaryimmune response to foreign class I antigens must be presented by class II-positive APCs of the donor
anti-(Lechler and Batchelor 1982; Sherwood et al 1986).
Dendritic cells are probably the class II-positive donor
cells responsible for antigen presentation (Deeg et al.
1988) Thus alloimmunization against class I antigensshould be prevented when class II-positive cells havebeen either removed from red cell or platelet concen-trates or inactivated
Removal of leucocytes
An early study showed that patients transfused withplatelet concentrates from which most of the leuco-cytes have been removed, for example by passagethrough cotton wool filters, were substantially lesslikely to become refractory to transfusion of platelets
Trang 40(Eernisse and Brand 1981) This observation has
been frequently confirmed (Murphy et al 1986; van
Marwijk et al 1991) A multi-institutional,
random-ized, blinded trial was conducted to determine
whether transfusion of platelets from which leucocytes
had been removed by a filter before storage would
prevent the formation of platelet alloantibodies and
refractoriness to platelet transfusions Patients who
were receiving induction chemotherapy for acute
myeloid leukaemia were randomly assigned to receive
one of four types of platelet transfusions: unmodified,
pooled platelet concentrates from random donors
(control); filtered, pooled platelet concentrates from
random donors (F-PC); ultraviolet B-irradiated,
pooled platelet concentrates from random donors
(UVB-PC); or filtered platelets obtained by apheresis
from single random donors (F-AP) All patients
received transfusions of filtered, leucocyte-reduced red
cells Of 530 patients with no alloantibodies at the trial
initiation, 13% of those in the control group produced
lymphocytotoxic antibodies and their
thrombocytope-nia became refractory to platelet transfusions,
com-pared with 3% in the F-PC group, 5% in the UVB-PC
group and 4% in the F-AP group (P= 0.03 for each
treated group compared with the control subjects)
Lymphocytotoxic antibodies were found in 45% of
the controls, compared with 17–21% in the treated
groups (P< 0.001 for each treated group compared
with the control subjects) Reduction of leucocytes by
filtration and ultraviolet B irradiation of platelets was
equally effective in preventing alloantibody-mediated
refractoriness to platelets during chemotherapy for
acute myeloid leukaemia Platelets obtained by
apheresis from single random donors provided no
additional benefit compared with pooled platelet
concentrates from random donors (Trial to Reduce
Alloimmunization to Platelets Study Group 1997)
Universal pre-storage leucoreduction (ULR) of red
cell and platelet products has been performed in
Canada since August 1999 In a retrospective analysis
of 13 902 platelet transfusions in 617 patients
under-going chemotherapy for acute leukaemia or stem cell
transplantation before (n = 315) and after (n = 302)
the introduction of ULR, alloimmunization was
significantly reduced (19–7%) in the post-ULR group
Alloimmune platelet refractoriness was similarly
reduced (14 – 4%) Fewer patients in the post-ULR
group received HLA-matched platelets (14% vs
5%) Thus leucocyte reduction (see below) reduces
alloimmunization, refractoriness and requirements forHLA-matched platelets when applied as routine trans-fusion practice to patients receiving chemotherapy or
stem cell transplant (Seftel et al 2004) While the total
number of platelet transfusions was also reduced afterULR, this was probably related to other factors such as
a reduction in the platelet transfusion trigger
As might be expected, following the transfusion ofleucocyte-poor platelet concentrates, the development
of platelet refractoriness is much more common inpatients who have been transfused previously or havebeen pregnant, which confirms the suspicion that secondary immune responses to HLA class I antigens
cannot be prevented (Brand et al 1988; Novotny
et al 1995; Sintnicolaas et al 1995) However, in the
two large studies, alloimmunization and alloimmunerefractoriness in patients who were previously preg-nant or transfused were also reduced after ULR (Trial
to Reduce Alloimmunization to Platelets Study Group
1997; Seftel et al 2004).
Counting small numbers of leucocytes
The small numbers of residual leucocytes in filteredconcentrates (1–3 cells/µl) can be determined accur-ately either by flow cytometry, after staining the
nucleated cells with propidium iodide (Wenz et al.
1991), or by using a large-volume counting chamber,
for example the Nageotte chamber (Masse et al.
1991) In trials in 20 laboratories, the detection limitsfor the flow cytometry and NC techniques were 0.1and 1 leucocyte/µl respectively Both methods are suitable for assessing the adequacy of leucodeple-tion filters Sampling error and instrument precisionremain hurdles for all proposed methods Issuesinvolving counting technique and guidelines for pro-cess control of leucoreduced blood components have
been published (Dumont et al 1996; Dzik 2000).
Filters for whole blood and red cell concentrates
There is ample evidence that primary HLA tion does not occur, or occurs only rarely, when fewerthan 5 × 106leucocytes are transfused (Sirchia et al 1982; Saarinen et al 1990; Novotny et al 1995) To
immuniza-avoid primary immunization, the total number of leucocytes transfused in a red cell or platelet concentr-ate must therefore be less than this number For redcells and whole blood collections, the only practical