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Typing of fetal platelet alloantigens when platelets are not available
1319
cytogenetic
hallmarks of
secondary leukaemia.2 Chromosomal
abnormalities are of prognostic importance since patients with normal karyotype have a substantially longer median survival (10 months) than do those with an abnormal karyotype (4 months).3 a higher percentage of FAB types M1 and M2 AML in patients exposed to environmental agents than in patients not so exposed.1
There is
Infante et al report two patients with chronic myeloid leukaemia (CML) among the five with leukaemia. In a previous report, one out of eight Ohio plioform workers who died of leukaemia had CML; the other seven had AML.4 The Philadelphia chromosome is rarely seen in acute leukaemia from environmental toxins.’ At least one of Infante and colleagues’ patients was diagnosed several years after being removed from exposure to petrol, indicating that former workers should continue to be screened.5 The association of non-random chromosomal changes with leukaemia that is possibly induced by benzene or other environmental agents might prove to have medicolegal
implications. Department of Haematology, Selly Oak Hospital, Birmingham B29 6JD, UK
typing of fetus by RFLP analysis on amplified DNA. PCR amplification products digested with Ncil (Bethesda Research Laboratories) and analysed on a 12% polyacrylamide gel by ethidium bromide staining and ultraviolet visualisation. Lane 1 b= H PA-(1 a +,b ),lane 2b=HPA-1(a+,b+), lane 3b=HPA-1(a-,b+), lane 4=fetus, lane 5 = 146 bp marker, lane 6 = negative control (primers only)In lanes 1 a, 2a, and 3a undigested PCR products are shown (Details of primers and the PCR cycles are available from R W. A. M. K.). HPA-1
M. LUMLEY H. BARKER J. A. MURRAY
1. Mitelman
F, Brandt L, Nilsson PG. Relation among occupational exposure to potential mitogenic/carcinogenic agents, clinical findings, and bone marrow chromosomes in acute non lymphocytic leukaemia. Blood 1978; 52: 1229-37. 2. Rowley JD. Cytogenetic studies in haematological disorders. Recent advances m haematology 3. Edinburgh: Churchill Livingstone, 1982: 233-52. 3. Golomb HM, Vardiman JW, Rowley JD, Testa JR, Mintz U. Correlation of clinical findings with quinacrine-banded chromosomes in 90 adults with acute nonlymphocytic leukaemia. N Engl J Med 1978, 299: 613-19. 4. Infante PF, Rinsky RA, Wagoner JK, Young RJ. Leukaemia in benzene workers. Lancet 1977; ii: 76-78. 5 Raffle PAB, ed. Hunter’s diseases of occupations London: Hodder & Stoughton, 1987: 298-300.
Typing of fetal platelet alloantigens when platelets are not available SIR,-Neonatal alloimmune thrombocytopenia carries the risk of haemorrhage, with intracranial bleeding in utero or during delivery. The alloantigen most frequently involved is HPA-la(Zwa or PIAl). When a mother is HPA-l(a-) and HLA DRw52a positive, the fetus is potentially at risk. The father determines the chance that the child will have the HPA-la phenotype and if the father is heterozygous it is important to find out whether the fetus is HPA-l(a+) because of the possibility of prophylactic high-dose intravenous immunoglobulin to the mother or other perinatal severe
care. 1,2 When
no platelets are available serological typing for platelet alloantigens is impossible. Genotyping for HPA-1 alleles became possible when Newman et aP showed that the polymorphism is due to a single base-pair substitution in DNA. By amplifying cDNA from platelet mRNA they showed that the human platelet alloantigens HPA-la and HPA-lb (Zwa and Zwb) are associated with a leucine and a proline, respectively, at aminoacid 33 of glycoprotein IIIa. The substitution at base-pair 196 created a unique restriction site for NciI on DNA from HPA-lb positive
donors. In the gene structure of glycoprotein II Ia4 there is a 10 kb intron adjacent to the polymorphic HPA-1 site which so far has made polymerase chain reaction (PCR) amplification directly on DNA impracticable. However, use of a primer complementary to a sequence of non-coding DNA directly upstream of the HPA-1 site yields a suitable 170 bp fragment. By this method we have genotyped a fetus and fourteen healthy individuals. The sister of an alloimmunised HPA-1(a-) female who had given birth to a thrombocytopenic child sought advice from the department of obstetrics in the 20th week of her first pregnancy. She was homozygous HPA-lb (HPA-1 [a - b + ]) and HLA DR3 and DRw52 positive. The father was HPA-1(a+,b+). Only weak platelet and lymphocyte reactive antibodies could be detected in her serum, too weak to establish specificity. In the 28th week of gestation 1 ml anticoagulated fetal blood was taken by ultrasound guided umbilical cord puncture. The platelet count was 149000/111 (normal 254 000s). DNA from the fetus and from six
HPA-l(a+b-) donors, six HPA-l(a-b+) donors, and two heterozygous donors was analysed. A 170 bp fragment was amplified with the primers. After digestion with AM amplified DNA from HPB-1 (a - b +) donors was cleaved with two fragments of 106 and 64 bp. From the DNA of HPA-1 (a + ,b - ) donors the 106 bp fragment only was obtained, and heterozygous HPAl(a + ,b +) donors yielded 106, 64, and 170 bp fragments. In this way the fetus was typed HPA-l(a+,b+) (figure). Serological typing confirmed that the platelets were HPA-la positive. Thus, restriction fragment length polymorphism analysis after NciI digestion is totally concordant with the phenotype evaluated
serologically. This method can now be used for typing for platelet HPA-1polymorphism, when no platelets are available, with DNA derived from lymphocytes or from chorion villous tissue or other fetal material. Department of Immunological Haematology, Central Laboratory of Blood Transfusion Service of Netherlands Red Cross, 1066 CX Amsterdam, Netherlands
ROBERT W. A. M. KUIJPER NANNE M. FABER
Department of Obstetrics, Academic University Hospital, Leiden
HUMPHREY H. H. KANHAI
Department of Haematology, Academic Medical Centre, University of Amsterdam
ALBERT E. G. KR. VON DEM BORNE
Kaplan C. Prenatal treatment in neonatal alloimmune thrombocytopenia. Curr Stud Hematol Blood Transf 1988; 55: 142-47. 2. Bussel JB, Berkowitz RL, McFarland JG, et al. Antenatal treatment of neonatal alloimmune thrombocytopenia. N Engl J Med 1988; 319: 1374-78. 3. Newman PJ, Derbes RS, Aster RH. The human platelet alloantigens PLA1 and PLA2 are associated with a leucin(33)/prolin(33) aminoacid polymorphism in membrane glycoprotein IIIa and are distinguishable by DNA typing. J Clin Invest 1989; 83: 1.
1778-81. 4. Zimrin AB, Gidwitz S, Lord S, et al. The genomic organizaton of platelet glycoprotein IIIa. J Biol Chem 1990; 265: 8590-95. 5. Forestier F, Daffos F, Galacteros F, Bardekjian J, Rainaut M. Haematological values of 163 normal fetuses between 18 and 30 weeks of gestation. Pediatr Res 1986; 20: 342-46
Genetics of
pre-eclampsia
SIR,-Dr Wilton and colleagues (Sept 15, p 653) are one of several groups examining the genetics of pre-eclampsia. Most family studies have suggested inheritance compatible with a single recessive gene acting in the mother, although a dominant gene with incomplete penetrance has been postulated. The apparent high heritability suggests high penetrance, and Wilton and colleagues’
cytogenetic
hallmarks of
secondary leukaemia.2 Chromosomal
abnormalities are of prognostic importance since patients with normal karyotype have a substantially longer median survival (10 months) than do those with an abnormal karyotype (4 months).3 a higher percentage of FAB types M1 and M2 AML in patients exposed to environmental agents than in patients not so exposed.1
There is
Infante et al report two patients with chronic myeloid leukaemia (CML) among the five with leukaemia. In a previous report, one out of eight Ohio plioform workers who died of leukaemia had CML; the other seven had AML.4 The Philadelphia chromosome is rarely seen in acute leukaemia from environmental toxins.’ At least one of Infante and colleagues’ patients was diagnosed several years after being removed from exposure to petrol, indicating that former workers should continue to be screened.5 The association of non-random chromosomal changes with leukaemia that is possibly induced by benzene or other environmental agents might prove to have medicolegal
implications. Department of Haematology, Selly Oak Hospital, Birmingham B29 6JD, UK
typing of fetus by RFLP analysis on amplified DNA. PCR amplification products digested with Ncil (Bethesda Research Laboratories) and analysed on a 12% polyacrylamide gel by ethidium bromide staining and ultraviolet visualisation. Lane 1 b= H PA-(1 a +,b ),lane 2b=HPA-1(a+,b+), lane 3b=HPA-1(a-,b+), lane 4=fetus, lane 5 = 146 bp marker, lane 6 = negative control (primers only)In lanes 1 a, 2a, and 3a undigested PCR products are shown (Details of primers and the PCR cycles are available from R W. A. M. K.). HPA-1
M. LUMLEY H. BARKER J. A. MURRAY
1. Mitelman
F, Brandt L, Nilsson PG. Relation among occupational exposure to potential mitogenic/carcinogenic agents, clinical findings, and bone marrow chromosomes in acute non lymphocytic leukaemia. Blood 1978; 52: 1229-37. 2. Rowley JD. Cytogenetic studies in haematological disorders. Recent advances m haematology 3. Edinburgh: Churchill Livingstone, 1982: 233-52. 3. Golomb HM, Vardiman JW, Rowley JD, Testa JR, Mintz U. Correlation of clinical findings with quinacrine-banded chromosomes in 90 adults with acute nonlymphocytic leukaemia. N Engl J Med 1978, 299: 613-19. 4. Infante PF, Rinsky RA, Wagoner JK, Young RJ. Leukaemia in benzene workers. Lancet 1977; ii: 76-78. 5 Raffle PAB, ed. Hunter’s diseases of occupations London: Hodder & Stoughton, 1987: 298-300.
Typing of fetal platelet alloantigens when platelets are not available SIR,-Neonatal alloimmune thrombocytopenia carries the risk of haemorrhage, with intracranial bleeding in utero or during delivery. The alloantigen most frequently involved is HPA-la(Zwa or PIAl). When a mother is HPA-l(a-) and HLA DRw52a positive, the fetus is potentially at risk. The father determines the chance that the child will have the HPA-la phenotype and if the father is heterozygous it is important to find out whether the fetus is HPA-l(a+) because of the possibility of prophylactic high-dose intravenous immunoglobulin to the mother or other perinatal severe
care. 1,2 When
no platelets are available serological typing for platelet alloantigens is impossible. Genotyping for HPA-1 alleles became possible when Newman et aP showed that the polymorphism is due to a single base-pair substitution in DNA. By amplifying cDNA from platelet mRNA they showed that the human platelet alloantigens HPA-la and HPA-lb (Zwa and Zwb) are associated with a leucine and a proline, respectively, at aminoacid 33 of glycoprotein IIIa. The substitution at base-pair 196 created a unique restriction site for NciI on DNA from HPA-lb positive
donors. In the gene structure of glycoprotein II Ia4 there is a 10 kb intron adjacent to the polymorphic HPA-1 site which so far has made polymerase chain reaction (PCR) amplification directly on DNA impracticable. However, use of a primer complementary to a sequence of non-coding DNA directly upstream of the HPA-1 site yields a suitable 170 bp fragment. By this method we have genotyped a fetus and fourteen healthy individuals. The sister of an alloimmunised HPA-1(a-) female who had given birth to a thrombocytopenic child sought advice from the department of obstetrics in the 20th week of her first pregnancy. She was homozygous HPA-lb (HPA-1 [a - b + ]) and HLA DR3 and DRw52 positive. The father was HPA-1(a+,b+). Only weak platelet and lymphocyte reactive antibodies could be detected in her serum, too weak to establish specificity. In the 28th week of gestation 1 ml anticoagulated fetal blood was taken by ultrasound guided umbilical cord puncture. The platelet count was 149000/111 (normal 254 000s). DNA from the fetus and from six
HPA-l(a+b-) donors, six HPA-l(a-b+) donors, and two heterozygous donors was analysed. A 170 bp fragment was amplified with the primers. After digestion with AM amplified DNA from HPB-1 (a - b +) donors was cleaved with two fragments of 106 and 64 bp. From the DNA of HPA-1 (a + ,b - ) donors the 106 bp fragment only was obtained, and heterozygous HPAl(a + ,b +) donors yielded 106, 64, and 170 bp fragments. In this way the fetus was typed HPA-l(a+,b+) (figure). Serological typing confirmed that the platelets were HPA-la positive. Thus, restriction fragment length polymorphism analysis after NciI digestion is totally concordant with the phenotype evaluated
serologically. This method can now be used for typing for platelet HPA-1polymorphism, when no platelets are available, with DNA derived from lymphocytes or from chorion villous tissue or other fetal material. Department of Immunological Haematology, Central Laboratory of Blood Transfusion Service of Netherlands Red Cross, 1066 CX Amsterdam, Netherlands
ROBERT W. A. M. KUIJPER NANNE M. FABER
Department of Obstetrics, Academic University Hospital, Leiden
HUMPHREY H. H. KANHAI
Department of Haematology, Academic Medical Centre, University of Amsterdam
ALBERT E. G. KR. VON DEM BORNE
Kaplan C. Prenatal treatment in neonatal alloimmune thrombocytopenia. Curr Stud Hematol Blood Transf 1988; 55: 142-47. 2. Bussel JB, Berkowitz RL, McFarland JG, et al. Antenatal treatment of neonatal alloimmune thrombocytopenia. N Engl J Med 1988; 319: 1374-78. 3. Newman PJ, Derbes RS, Aster RH. The human platelet alloantigens PLA1 and PLA2 are associated with a leucin(33)/prolin(33) aminoacid polymorphism in membrane glycoprotein IIIa and are distinguishable by DNA typing. J Clin Invest 1989; 83: 1.
1778-81. 4. Zimrin AB, Gidwitz S, Lord S, et al. The genomic organizaton of platelet glycoprotein IIIa. J Biol Chem 1990; 265: 8590-95. 5. Forestier F, Daffos F, Galacteros F, Bardekjian J, Rainaut M. Haematological values of 163 normal fetuses between 18 and 30 weeks of gestation. Pediatr Res 1986; 20: 342-46
Genetics of
pre-eclampsia
SIR,-Dr Wilton and colleagues (Sept 15, p 653) are one of several groups examining the genetics of pre-eclampsia. Most family studies have suggested inheritance compatible with a single recessive gene acting in the mother, although a dominant gene with incomplete penetrance has been postulated. The apparent high heritability suggests high penetrance, and Wilton and colleagues’