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Relationships between severe neonatal thrombocytopenia and maternal characteristics in pregnancies associated with autoimmune thrombocytopenia
240 heparin to achieve an activated clotting time greater than 400 seconds, as would be done during cardiopulmonary bypass in humans. A 25-pro filter was located on both the venous and arterial sides of the bypass device. At brain autopsy examination. the number of microvessel dilatations per square centimeter counted. The results showed a 10-fold higher average number of dilations among animals treated with cardiotomy suction t46/cm 2 v 4.5/cm 2 in group 3; 4.2/cm 2 in group 2: and 1.8/cm2 in group 1). Comparison of group 3 with group 4 animals showed that the dramatic increase in central nervous system lesions was attributable to the reinfusion of aspirated blood from the surgical field. The authors concluded that the data suggest that a major source of cerebral emboli is the suctioning of blood from the operative field. Whether the washing of aspirated shed blood would decrease the frequency of such microemboli could not be determined from the study. Readers interested in the potential side effects of intraoperative blood salvage may want to keep this article on file. (S.D. J
Human parvovirus B19: Prevalence of viral DNA in volunteer blood donors and clinical outcomes of transfusion recipients. Jordan J, 77angco B, Kiss J, et a/. Vo• Sang 75:97-102, 1998. Human parvovlrus B19 ts a nonenvelopped, single-stranded DNA virus, usually spread by aerosol droplets, that also may be transmitted by single-donor blood products and factor concentrates. Although most infections are mild, they can result in severe disease in pregnant women, immunodeficient patients, or patients with increased erythropoiesis. In this study, the authors performed polymerase chain reaction (PCR J-based screening of blood donors in the Pittsburgh area. and attempted to determine the clinical consequences in recipients of PCR-positive products. They also measured B19-specific tmmunoglobulin M (IgM) and immunoglobulin G (IgG) in donor samples positive for B19 DNA. PCR screening was done initially on pools of 50 donors. Of 9.568 donors screened during the spring and summer of 1995. 11 (0.1%) were PCR positive. Three of these donors lacked measurable levels of Bl9-specific antibodies. Chart review or interview with a treating physician was possible for 10 of 21 recipients of PCR-positive blood products. Only one recipient who received a PCR-positive. antibody-negative unit was traced. This patient received a red cell transfusion 2 days after liver transplantation, developed transient anemia and reticulocytopenia 4 months posttransfusion, and had evidence of recent parvovirus infection when tested 8 months posttransfusion. as demonstrated by the presence of B 19-specific IgM and IgG antibodies. This study provides interesting data on the prevalence of parvovirus DNA in a population of US blood donors. Reported prevalence rates have varied from 0.03% to 0.6% in different geographic locales and seasons. Because clinical follow-up was retrospective and occurred in only 48% of recipients for an uncertain duration of time. the clinical consequences of infection may have been underestimated. The imminent introduction of genomlc amplification testing for human immunodeficiency wrus (HIV) and hepatitis C virus (HCV) on all blood donations in many countries may make it feasible to perform parvovirus testing on selected donor pools, to provide PCR-negative products for patients at risk for severe infection. ( M . G . )
CURRENT LITERATURE
Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma: Lo YMD, /-Ije/m NM, Fid/erC, eta/. N Engl J Med 339:1734-1738, 1998. In RhD-a!loimmunized women with partners who are heterozygous for the RhD gene, there is a 50% chance that the fetus will be RhD-negative a n d therefore require no treatment or invasive monitoring. In this study, the authors show that fetal RhD genotyping can be accurately Performed on maternal plasma, beginning in the second trimester of Pregnancy. Fiftyseven pregnant women were studied. DNA was extracted from maternal plasma with the QIAmp Blood kit. A fluorescencebaSed polymerase chain reaction (PCR) assay wasperf0rmed Using primers and probes targeted to the 31 untranslated region (ex0n 10) of the Rh gene. The B-globin gene assay also was performed as a control for the amplifiability of the extracted DNA. Dilutional studies on DNA of an RhD-positive subject showed that positive signals were detected with the amount of DNA usuat!y contained in a single RhD-positive cell (7.8 pg). Among the 57 fetuses, 39 were RhD positive and 18 were RhD negative on serological analysis of cord blood or PCR testing of amniotic fluid. Among women studied in the second or third trimester of pregnancy, there was complete concordance between the results of PCR analysis of maternal plasma and serological analysis or PCR testing of amniotic fluid. Among the nine women in the first trimester of pregnancy with RhDpositive fetuses, two plasma samples yielded false-negative results, at gestational ages of 8 and 9 weeks, presumably because of the low concentration of fetal DNA in the plasma at that time. There were no false-positive results because of persistence of fetal cells from previous pregnancies in the 47 women who were multiparous. This method of determination of fetal Rh status was simpler, quicker, and more sensitive than other methods previously described by the authors and other groups, which require the isolation of fetal cells from maternal blood, or the detection of RhD messenger RNA in fetal nucleated red cellsl (M. G.)
Relationships between severe neonatal thrombocytopenia and maternal characteristics in pregnancies associated with autoimmunethrombocytopenia. Va/at AS, Caulier MT, Devos P, et al. Br J Haematol 103:397401, 1998. In the management of pregnancy and the delivery of women with autoimmune thrombocytopenia (AITP), it is difficult to identify the small number of infants at risk for severe neonatal thrombocytopenia and hemorrhage. In many studies most of the patients have mild pregnancy-associated thrombocytopenia, and not true AITE In this study from France, 57 women with chronic AITP diagnosed before pregnancy were followed-up for 64 pregnancies. The patients were treated with steroids or intravenous immune globulin during pregnancy as needed~Df the 64 newborns, 8 (12.5%) had severe thrombocyt0penia at birth, defined by the authors as a platelet count below 50 • 109]L, and four others developed severe thrombocytopenia within 4 days of delivery. These infants presented with petechiae or bruising, and 11 of 12 responded well to intravenous immune globulin therapy. In six women with multiple pregnancies, platelet counts were similar between siblings. The incidence of severe thrombocytopenia at birth varied according to the nadir of the maternal
CURRENT LITERATURE platelet count during the pregnancy, and the history of prior splenectomy. Severe thrombocytopenia was present in 57% (95% CI; 18% to 90%) of the infants born to mothers with both severe thrombocytopenia in the index pregnancy and prior splenectomy (n = 7), 30% (95% CI; 7% tO 65%) of the infants born to mothers with severe thrombocytopenia alone (n = 10), 4% (95% CI; 1 to 21%): Of the infants born to mothers with splenectomy alone (n - 2z~); and none of the infants born to mothers without these two variables (n 23, 95% ci; 0 to 15%); The confidence infervals are large, as the number of patients in each group is small. This study Confirms that the incidence of severe thrombocytopenia at birth is low in infants of mothers with AITR The authors suggest that severe maternal disease is a predictive factor for neonatal thi-ombocytopenia, and that invasive monitoring and cesarean section delivery be considered potentially useful only in this group. (M. G.)
Retrospective neuropathological review of prion disease in UK haemophilic patients; Lee CA, Ironside JW, Bell JE, et at. T h r o m b Haemost 80:909-911, 1998. New variant Creutzfeldt-Jacob disease (nvCJD) was first reported in the ~Unked Kingdom in 1996, and appears to be caused by a transmissible agent similar or identical to the agent of bovine spongiform encephalopathy (BSE). BSE was thought to enter the human food chain in the United Kingdom in the early 1980s, when an epidemic occurred in cattle. Although there have been no documented cases of transmission of nvCJD by blood- or fractionation products, the presence of the prion in lymphoid tissue and the recent description of the variant have raised concern. In November 1997, the National Blood Authority in the UK initiated recall of albumin and factor VIII made from a plasma pool containing a donation from each of two donors who had developed nvCJD. In this autopsy study, the brains of 33 hemophilia patients who were treated primarily with clotting factor concentrates manufactured from UK donor sonrce plasma from 1962 to 1995 were examined for evidence of CJD. Both conventional histology and immunocytochemistry for prion protein, using two monoclonal antibodies, were done. The patients were treated for a median of 9 years, and the median age at death was 42 years: No evidence of spongiform 9encephalopathy was found in any of the patients. The authors point out that this study would not necessarily detect preclinical disease. However, it reinforces other epidemiological data suggesting that the transmission of nvCJD by fractionation products, if it occurs at all, i's low. (M.G:)
Shift from Rh-positive to Rh-negative phenotype caused by a somatic mutation within the RHD gene in a patient with chronic myelocytic leukaemia. CherifZahar B, Ben)/ V, Steffensen R, et al. Br J Haematol 102:1263,1270, 1998. Altered expression of a blood group antigen has been described in association with changes in a chromosome as a result of diseases such as acute and chronic myetogenous leukemia (AML, CML) myeloid metaplasia, polycythemia, or myelofibrosis. This article describes a patient whose blood type changed from D-positive to D-negative as a consequence of a somatic mutation within RHD. Red blood cells (RBCs) from the patient, a 69-year-old white woman, had typed O, D + in 1979
241 and in 1991 when she was being treated for breast cancer. In 1994. when she was admitted to a hospital with a diagnosis of CML, her RBCs typed O. D C + E c + e + . A normal karyotype of 46.XX was detected in peripheral blood leukocytes. The Philadelphia chromosome abnormality, which was detected initially in 1994 by Southern blot and confirmed in 1997 by multiplex polymerase Chain reaction (PCR/analysis of mRNA transcripts, was consistent With the most common type of chromosome abnormality foUnd in CML patients. By flow cytometry, her RBCs were shown to express normal levels of C. c, and e anugens las compared to DCCee and dCcee controls), had no detectable E or D antigens, and a level of LW that was lower than D + controls but similar to D controls. Other typings appeared normal, and no mixed field agglutination was observed. By indirect hemagglutination, no D antigen was detected: however, a weak D antigen was detected by absorption and elution. Southern blot analysis after digestion with HindlII. BamH1. or EcoR1 was identical to DCe/DCe and DCe/ce controls. The results indicated that the patient had one chromosome carrying RHD and RHCE genes and the other chromo= some carrying only an RHCE gene. Thus. loss of the D antigen was not associated with either a gross deletion or a rearranged RHD gene. Sequence analysis of RHCE transcripts indicated the presence of normal RHCe and Rhce alleles and showed the presence of a single base deletion (G) from position 600 within exon 4 of the RHD cDNA. This deletion introduced a frameshifl resulting in a premature stop codon. The predicted size of this protein, }f translated, would consist of 227 amino acids, as compared with the normal RhD protein of 417 residues. A fragment of DNA (prepared from an Epstein-Barr virus (EBV)transformed lymphoblastoid cell line that was derived from the patient) encompassing the G600 deletion was amplified, cloned, and sequenced. Because there were no discordant results, the possibility of a somatic mutation in the myeloid compartment was addressed. To pursue this possibility, cDNA and mRNA were prepared from erythroid cells cultured in a two-phase liquid culture system. The G600 deletion was observed in most RHD transcripts. The authors also performed allele-specific PCR analysis, using a primer specific for the normal sequence; an amplicon of the expected size was obtained in patient's peripheral blood and the B-cell line. A very weak band was obtained on DNA from purified neutrophils. A band was also obtained in DNA prepared from cultured erythroblasts, which the authors conclude arose from the presence of nonerythroid ceils in the preparation or from nonmutated erythroid cells. They claim that if the latter is the case, this would correlate with the weak absorption and elution of anti-D from the patient's red blood cells. Using a primer specific for the sequence with the nucleotide deletion, an amplicon with the expected size was obtained using DNA from neutrophils and cultured erythroblasts but not from the B-ceil line. These results suggest that the G600 deletion is a somatic mutation restricted to the myeloid lineage. A single point mutation that changed codon 41 (Gln) to a stop codon has been described to silence the RHD gene (Avent et al, Blood 89:2568-2577, 1997) in a healthy person. However, analysis was restricted to cDNA from RNA obtained from the donor's reticulocytes, and studies were not performed to determine whether the mutation was inherited. Family members from the CML patient described here were not available for testing. (M.E.R.)
Human parvovirus B19: Prevalence of viral DNA in volunteer blood donors and clinical outcomes of transfusion recipients. Jordan J, 77angco B, Kiss J, et a/. Vo• Sang 75:97-102, 1998. Human parvovlrus B19 ts a nonenvelopped, single-stranded DNA virus, usually spread by aerosol droplets, that also may be transmitted by single-donor blood products and factor concentrates. Although most infections are mild, they can result in severe disease in pregnant women, immunodeficient patients, or patients with increased erythropoiesis. In this study, the authors performed polymerase chain reaction (PCR J-based screening of blood donors in the Pittsburgh area. and attempted to determine the clinical consequences in recipients of PCR-positive products. They also measured B19-specific tmmunoglobulin M (IgM) and immunoglobulin G (IgG) in donor samples positive for B19 DNA. PCR screening was done initially on pools of 50 donors. Of 9.568 donors screened during the spring and summer of 1995. 11 (0.1%) were PCR positive. Three of these donors lacked measurable levels of Bl9-specific antibodies. Chart review or interview with a treating physician was possible for 10 of 21 recipients of PCR-positive blood products. Only one recipient who received a PCR-positive. antibody-negative unit was traced. This patient received a red cell transfusion 2 days after liver transplantation, developed transient anemia and reticulocytopenia 4 months posttransfusion, and had evidence of recent parvovirus infection when tested 8 months posttransfusion. as demonstrated by the presence of B 19-specific IgM and IgG antibodies. This study provides interesting data on the prevalence of parvovirus DNA in a population of US blood donors. Reported prevalence rates have varied from 0.03% to 0.6% in different geographic locales and seasons. Because clinical follow-up was retrospective and occurred in only 48% of recipients for an uncertain duration of time. the clinical consequences of infection may have been underestimated. The imminent introduction of genomlc amplification testing for human immunodeficiency wrus (HIV) and hepatitis C virus (HCV) on all blood donations in many countries may make it feasible to perform parvovirus testing on selected donor pools, to provide PCR-negative products for patients at risk for severe infection. ( M . G . )
CURRENT LITERATURE
Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma: Lo YMD, /-Ije/m NM, Fid/erC, eta/. N Engl J Med 339:1734-1738, 1998. In RhD-a!loimmunized women with partners who are heterozygous for the RhD gene, there is a 50% chance that the fetus will be RhD-negative a n d therefore require no treatment or invasive monitoring. In this study, the authors show that fetal RhD genotyping can be accurately Performed on maternal plasma, beginning in the second trimester of Pregnancy. Fiftyseven pregnant women were studied. DNA was extracted from maternal plasma with the QIAmp Blood kit. A fluorescencebaSed polymerase chain reaction (PCR) assay wasperf0rmed Using primers and probes targeted to the 31 untranslated region (ex0n 10) of the Rh gene. The B-globin gene assay also was performed as a control for the amplifiability of the extracted DNA. Dilutional studies on DNA of an RhD-positive subject showed that positive signals were detected with the amount of DNA usuat!y contained in a single RhD-positive cell (7.8 pg). Among the 57 fetuses, 39 were RhD positive and 18 were RhD negative on serological analysis of cord blood or PCR testing of amniotic fluid. Among women studied in the second or third trimester of pregnancy, there was complete concordance between the results of PCR analysis of maternal plasma and serological analysis or PCR testing of amniotic fluid. Among the nine women in the first trimester of pregnancy with RhDpositive fetuses, two plasma samples yielded false-negative results, at gestational ages of 8 and 9 weeks, presumably because of the low concentration of fetal DNA in the plasma at that time. There were no false-positive results because of persistence of fetal cells from previous pregnancies in the 47 women who were multiparous. This method of determination of fetal Rh status was simpler, quicker, and more sensitive than other methods previously described by the authors and other groups, which require the isolation of fetal cells from maternal blood, or the detection of RhD messenger RNA in fetal nucleated red cellsl (M. G.)
Relationships between severe neonatal thrombocytopenia and maternal characteristics in pregnancies associated with autoimmunethrombocytopenia. Va/at AS, Caulier MT, Devos P, et al. Br J Haematol 103:397401, 1998. In the management of pregnancy and the delivery of women with autoimmune thrombocytopenia (AITP), it is difficult to identify the small number of infants at risk for severe neonatal thrombocytopenia and hemorrhage. In many studies most of the patients have mild pregnancy-associated thrombocytopenia, and not true AITE In this study from France, 57 women with chronic AITP diagnosed before pregnancy were followed-up for 64 pregnancies. The patients were treated with steroids or intravenous immune globulin during pregnancy as needed~Df the 64 newborns, 8 (12.5%) had severe thrombocyt0penia at birth, defined by the authors as a platelet count below 50 • 109]L, and four others developed severe thrombocytopenia within 4 days of delivery. These infants presented with petechiae or bruising, and 11 of 12 responded well to intravenous immune globulin therapy. In six women with multiple pregnancies, platelet counts were similar between siblings. The incidence of severe thrombocytopenia at birth varied according to the nadir of the maternal
CURRENT LITERATURE platelet count during the pregnancy, and the history of prior splenectomy. Severe thrombocytopenia was present in 57% (95% CI; 18% to 90%) of the infants born to mothers with both severe thrombocytopenia in the index pregnancy and prior splenectomy (n = 7), 30% (95% CI; 7% tO 65%) of the infants born to mothers with severe thrombocytopenia alone (n = 10), 4% (95% CI; 1 to 21%): Of the infants born to mothers with splenectomy alone (n - 2z~); and none of the infants born to mothers without these two variables (n 23, 95% ci; 0 to 15%); The confidence infervals are large, as the number of patients in each group is small. This study Confirms that the incidence of severe thrombocytopenia at birth is low in infants of mothers with AITR The authors suggest that severe maternal disease is a predictive factor for neonatal thi-ombocytopenia, and that invasive monitoring and cesarean section delivery be considered potentially useful only in this group. (M. G.)
Retrospective neuropathological review of prion disease in UK haemophilic patients; Lee CA, Ironside JW, Bell JE, et at. T h r o m b Haemost 80:909-911, 1998. New variant Creutzfeldt-Jacob disease (nvCJD) was first reported in the ~Unked Kingdom in 1996, and appears to be caused by a transmissible agent similar or identical to the agent of bovine spongiform encephalopathy (BSE). BSE was thought to enter the human food chain in the United Kingdom in the early 1980s, when an epidemic occurred in cattle. Although there have been no documented cases of transmission of nvCJD by blood- or fractionation products, the presence of the prion in lymphoid tissue and the recent description of the variant have raised concern. In November 1997, the National Blood Authority in the UK initiated recall of albumin and factor VIII made from a plasma pool containing a donation from each of two donors who had developed nvCJD. In this autopsy study, the brains of 33 hemophilia patients who were treated primarily with clotting factor concentrates manufactured from UK donor sonrce plasma from 1962 to 1995 were examined for evidence of CJD. Both conventional histology and immunocytochemistry for prion protein, using two monoclonal antibodies, were done. The patients were treated for a median of 9 years, and the median age at death was 42 years: No evidence of spongiform 9encephalopathy was found in any of the patients. The authors point out that this study would not necessarily detect preclinical disease. However, it reinforces other epidemiological data suggesting that the transmission of nvCJD by fractionation products, if it occurs at all, i's low. (M.G:)
Shift from Rh-positive to Rh-negative phenotype caused by a somatic mutation within the RHD gene in a patient with chronic myelocytic leukaemia. CherifZahar B, Ben)/ V, Steffensen R, et al. Br J Haematol 102:1263,1270, 1998. Altered expression of a blood group antigen has been described in association with changes in a chromosome as a result of diseases such as acute and chronic myetogenous leukemia (AML, CML) myeloid metaplasia, polycythemia, or myelofibrosis. This article describes a patient whose blood type changed from D-positive to D-negative as a consequence of a somatic mutation within RHD. Red blood cells (RBCs) from the patient, a 69-year-old white woman, had typed O, D + in 1979
241 and in 1991 when she was being treated for breast cancer. In 1994. when she was admitted to a hospital with a diagnosis of CML, her RBCs typed O. D C + E c + e + . A normal karyotype of 46.XX was detected in peripheral blood leukocytes. The Philadelphia chromosome abnormality, which was detected initially in 1994 by Southern blot and confirmed in 1997 by multiplex polymerase Chain reaction (PCR/analysis of mRNA transcripts, was consistent With the most common type of chromosome abnormality foUnd in CML patients. By flow cytometry, her RBCs were shown to express normal levels of C. c, and e anugens las compared to DCCee and dCcee controls), had no detectable E or D antigens, and a level of LW that was lower than D + controls but similar to D controls. Other typings appeared normal, and no mixed field agglutination was observed. By indirect hemagglutination, no D antigen was detected: however, a weak D antigen was detected by absorption and elution. Southern blot analysis after digestion with HindlII. BamH1. or EcoR1 was identical to DCe/DCe and DCe/ce controls. The results indicated that the patient had one chromosome carrying RHD and RHCE genes and the other chromo= some carrying only an RHCE gene. Thus. loss of the D antigen was not associated with either a gross deletion or a rearranged RHD gene. Sequence analysis of RHCE transcripts indicated the presence of normal RHCe and Rhce alleles and showed the presence of a single base deletion (G) from position 600 within exon 4 of the RHD cDNA. This deletion introduced a frameshifl resulting in a premature stop codon. The predicted size of this protein, }f translated, would consist of 227 amino acids, as compared with the normal RhD protein of 417 residues. A fragment of DNA (prepared from an Epstein-Barr virus (EBV)transformed lymphoblastoid cell line that was derived from the patient) encompassing the G600 deletion was amplified, cloned, and sequenced. Because there were no discordant results, the possibility of a somatic mutation in the myeloid compartment was addressed. To pursue this possibility, cDNA and mRNA were prepared from erythroid cells cultured in a two-phase liquid culture system. The G600 deletion was observed in most RHD transcripts. The authors also performed allele-specific PCR analysis, using a primer specific for the normal sequence; an amplicon of the expected size was obtained in patient's peripheral blood and the B-cell line. A very weak band was obtained on DNA from purified neutrophils. A band was also obtained in DNA prepared from cultured erythroblasts, which the authors conclude arose from the presence of nonerythroid ceils in the preparation or from nonmutated erythroid cells. They claim that if the latter is the case, this would correlate with the weak absorption and elution of anti-D from the patient's red blood cells. Using a primer specific for the sequence with the nucleotide deletion, an amplicon with the expected size was obtained using DNA from neutrophils and cultured erythroblasts but not from the B-ceil line. These results suggest that the G600 deletion is a somatic mutation restricted to the myeloid lineage. A single point mutation that changed codon 41 (Gln) to a stop codon has been described to silence the RHD gene (Avent et al, Blood 89:2568-2577, 1997) in a healthy person. However, analysis was restricted to cDNA from RNA obtained from the donor's reticulocytes, and studies were not performed to determine whether the mutation was inherited. Family members from the CML patient described here were not available for testing. (M.E.R.)