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1 DOENÇA HEMOLÍTICA PERINATAL Giovanni Fraga Lenza

2 1.Incompatibilidade sanguínea materno-fetal 2.Passagem do sangue fetal para circulação materna 3.Reconhecimento do Ag 4.Produção de Ac 5.Passagem dos Ac para circulação fetal 6.Ação dos Ac sobre as hemácias fetais 1.Incompatibilidade sanguínea materno-fetal 2.Passagem do sangue fetal para circulação materna 3.Reconhecimento do Ag 4.Produção de Ac 5.Passagem dos Ac para circulação fetal 6.Ação dos Ac sobre as hemácias fetais Doença Hemolítica Perinatal História natural

3 J Obstet Gynaecol Can.J Obstet Gynaecol Can May;34(5): J Obstet Gynaecol Can. Anti-d in rh(d)-negative pregnant women: are at-risk pregnancies and deliveries receiving appropriate prophylaxis ? Koby LKoby L, Grunbaum A, Benjamin A, Koby R, Abenhaim HA. Grunbaum ABenjamin AKoby RAbenhaim HA Koby LGrunbaum ABenjamin AKoby RAbenhaim HA Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Montreal QC. Abstract Objective: Although anti-D prophylaxis has greatly reduced the rate of Rh-immunization, there remain women who sensitize during or after pregnancy because of inadequate prophylaxis. The purpose of this study was to compare adherence to prophylaxis recommendations for antenatal and postnatal anti-D immunoglobulin administration. Methods: We conducted a retrospective cohort study of all pregnancies recorded at the Royal Victoria Hospital between 2001 and 2006 to determine the rates of antenatal and postnatal prophylaxis in Rh(D)-negative women. We compared adherence to anti-D prophylaxis recommendations between our institution's physician-dependent antenatal approach and the protocol-based postpartum approach. Logistic regression analysis was used to estimate the odds ratio and 95% confidence intervals of determinants of non-adherence to current recommendations for anti-D prophylaxis. Results: Antenatal administration was analyzed in 1868 pregnancies in eligible Rh-negative women. Among these women, 85.7% received appropriate antenatal prophylaxis and 98.5% of eligible women received appropriate postnatal prophylaxis. Factors independently associated with non- adherence to antepartum prophylaxis included first visit in the third trimester (P < 0.001), transfer from an outside hospital (P = 0.03), and physician licensing before 1980 (P = 0.04). Conclusion: Unlike hospital-based protocol-dependent systems, physician-dependent systems for antenatal anti-D prophylaxis remain subject to errors of omission. A more standardized system is needed to ensure effective antenatal prophylaxis. DOENÇA HEMOLÍTICA PERINATAL

4 História Natural História Natural Incompatibilidade sanguínea materno-fetal Passagem de sangue fetal para circulação materna Reconhecimento do AG e produção de AC pela mãe Reconhecimento do AG e produção de AC pela mãe Passagem de AC maternos para circulação fetal Passagem de AC maternos para circulação fetal Ação dos AC maternos sobre as hemácias fetais Ação dos AC maternos sobre as hemácias fetais Anemia Anemia Eritroblastose Eritroblastose Focos ectópicos de hematopoiese Focos ectópicos de hematopoiese Polidramnia e placentomegalia Polidramnia e placentomegalia Anasarca Anasarca Morte Morte DOENÇA HEMOLÍTICA PERINATAL

5 INCIDÊNCIA Sistema ABO e Rh – 98% 2/3 ABO 1/3 Rh 1/3 RhKell E 2% CellanoDuffyKiddoutros DOENÇA HEMOLÍTICA PERINATAL

6 Incompatibilidade sanguínea materno-fetal Passagem de SG fetal para circulação materna Reconhecimento do AG e produção de AC pela mãe Reconhecimento do AG e produção de AC pela mãe Passagem de AC para circulação fetal Passagem de AC para circulação fetal Ação dos AC maternos sobre as hemácias fetais Ação dos AC maternos sobre as hemácias fetais Anemia Anemia Eritroblastose Eritroblastose Focos ectópicos de hematopoiese Focos ectópicos de hematopoiese Polidramnia e placentomegalia Polidramnia e placentomegalia Anasarca Anasarca Morte Morte DOENÇA HEMOLÍTICA PERINATAL

7 Transfusion.Transfusion May;30(4): Transfusion. Fetomaternal hemorrhage: incidence, risk factors, time of occurrence, and clinical effects. Sebring ESSebring ES, Polesky HF. Polesky HF Sebring ESPolesky HF Memorial Blood Center of Minneapolis, Minnesota. Abstract Most women have only very small amounts of fetal blood in their circulations following pregnancy and delivery: the volume is less than 0.5 mL of whole blood in 93 percent of women, less than 1 mL in 96 percent, and less than 2 mL in 98 percent. FMH of 30 mL or more occurs in just 3 of 1000 women. When the FMH was 150 mL or more, 15 of 41 infants did not survive Rh-negative women with FMH of more than 30 mL of Rh-positive whole blood are at increased risk of Rh immunization, and thus the outcome of their future pregnancies also may be affected. ABO-compatible fetal red cells that have entered the maternal circulation have a life span similar to that of adult cells. ABO-incompatible fetal red cells may be cleared rapidly, but in some cases they circulate for weeks. Most FMHs of 30 mL or more occur before labor, delivery, or cesarean section. The majority occur with minimal clinical signs and symptoms in apparently normal pregnancies. The identification of postpartum Rh-negative women who have 30 mL or more of Rh-positive fetal blood in their circulation is important so that sufficient RhIG for immune suppression can be administered. It appears that more than one-half of women with FMH of 30 mL or more would not be identified if protocols were adopted to test only women in pregnancies considered to be at high risk. DOENÇA HEMOLÍTICA PERINATAL

8 Hemorragia feto-materna PARTO NORMAL X CESÁREO Hemorragias da primeira metade Abortamento espontâneo x provocado Abortamento espontâneo x provocado Prenhez ectópica Prenhez ectópica Doença trofoblástica Doença trofoblástica Hemorragias da segunda metade DPP DPP Rotura uterina Rotura uterina PP PP Dequitação manual Procedimentos invasivos Gemelidade Maior número de gstações Aumenta com o evoluir da gestação DOENÇA HEMOLÍTICA PERINATAL

9 Transfusion.Transfusion Feb 8. Transfusion. Fetomaternal hemorrhage in normal vaginal delivery and in delivery by cesarean section. Lubusky MLubusky M, Simetka O, Studnickova M, Prochazka M, Ordeltova M, Vomackova K. Simetka OStudnickova MProchazka MOrdeltova MVomackova K Lubusky MSimetka OStudnickova MProchazka MOrdeltova MVomackova K From the Department of Obstetrics and Gynecology, the Department of Medical Genetics and Fetal Medicine, the Department of Surgery I, and the Department of Immunology, University Hospital, Olomouc, Czech Republic; and the Department of Obstetrics and Gynecology, University Hospital, Ostrava, Czech Republic. AbstractBACKGROUND: The objective was to determine the incidence and volume of fetomaternal hemorrhage (FMH) in normal vaginal delivery and in delivery by cesarean section. Determination of these variables would enable optimalization of guidelines for D alloimmunization prophylaxis. STUDY DESIGN AND METHODS: In a prospective cohort study, a total of 3457 examinations were performed, 2413 after normal vaginal delivery and 1044 after cesarean delivery. FMH was assessed by flow cytometry. (FMH is fetal red blood cell [RBC] volume; fetal blood volume is double [expected fetal hematocrit is 50%].) RESULTS: The fetal RBC volume diagnosed in maternal circulation after delivery ranged from insignificant FMH of not more than 0.1 mL to excessive FMH of 65.9 mL (median, 0.7; mean, 0.78; SD, 1.48). FMH of more than 2.5 mL (immunoglobulin [Ig] G anti-D insufficient dose 50 µg) was observed in 1.4% (49/3457) and excessive volumes of FMH of more than 5 mL (insufficient dose, 100 µg) in 0.29% (10/3457). Delivery by cesarean section presented a higher risk of incidence of FMH of more than 2.5 mL (odds ratio, 2.2; p = 0.004) when compared with normal vaginal delivery. It did not, however, present a significant risk factor for the incidence of excessive volumes of FMH of more than 5 mL. CONCLUSION: During normal vaginal delivery as well as during delivery by cesarean section, FMH of less than 5 mL occurs in the great majority of cases, and thus for the prevention of D alloimmunization, an IgG anti-D dose of 100 µg should be sufficient. Contrarily, only rarely does greater FMH occur and delivery by cesarean section does not present a risk factor. DOENÇA HEMOLÍTICA PERINATAL

10 Hemorragia feto-materna Parto normal x cesáreo Hemorragias da primeira metade Abortamento espontâneo x provocado Abortamento espontâneo x provocado Prenhez ectópica Prenhez ectópica Doença trofoblástica Doença trofoblástica Hemorragias da segunda metade DPP DPP Rotura uterina Rotura uterina PP PP Dequitação manual Procedimentos invasivos Gemelidade Maior número de gstações Aumenta com o evoluir da gestação DOENÇA HEMOLÍTICA PERINATAL

11 Incompatibilidade sanguínea materno-fetal Passagem de SG fetal para circulação materna Reconhecimento do AG e produção de AC pela mãe Reconhecimento do AG e produção de AC pela mãe Passagem de AC para circulação fetal Passagem de AC para circulação fetal Ação dos AC maternos sobre as hemácias fetais Ação dos AC maternos sobre as hemácias fetais Anemia Anemia Eritroblastose Eritroblastose Focos ectópicos de hematopoiese Focos ectópicos de hematopoiese Polidramnia e placentomegalia Polidramnia e placentomegalia Anasarca Anasarca Morte Morte DOENÇA HEMOLÍTICA PERINATAL

12 Apenas 1 em cada 23 gestantes Rh negativo formam AC 0,01 à 0,03 ml de sangue fetal podem ser suficientes para sensibilizar gestante susceptível ( Mollison et.al., 1973 ) Incompatibilidade do sistema ABO Positivção do Coombs indireto DOENÇA HEMOLÍTICA PERINATAL

13 Incompatibilidade sanguínea materno-fetal Passagem de SG fetal para circulação materna Reconhecimento do AG e produção de AC pela mãe Reconhecimento do AG e produção de AC pela mãe Passagem de AC para circulação fetal Passagem de AC para circulação fetal Ação dos AC maternos sobre as hemácias fetais Ação dos AC maternos sobre as hemácias fetais Anemia Anemia Eritroblastose Eritroblastose Focos ectópicos de hematopoiese Focos ectópicos de hematopoiese Polidramnia e placentomegalia Polidramnia e placentomegalia Anasarca Anasarca Morte Morte DOENÇA HEMOLÍTICA PERINATAL

14 IgM ( AC completos ou precoces ) alto peso molecular Não atravessam a placenta IgG ( AC incompletos, bloquadores ou tardios ) tardios ) atravessam a placenta Hemólise RESPOSTA PRIMÁRIA RESPOSTA SECUNDÁRIA OU ANAMNÉSICA DOENÇA HEMOLÍTICA PERINATAL

15 Incompatibilidade sanguínea materno-fetal Passagem de SG fetal para circulação materna Reconhecimento do AG e produção de AC pela mãe Reconhecimento do AG e produção de AC pela mãe Passagem de AC para circulação fetal Passagem de AC para circulação fetal Ação dos AC maternos sobre as hemácias fetais Ação dos AC maternos sobre as hemácias fetais Anemia Anemia Eritroblastose Eritroblastose Focos ectópicos de hematopoiese Focos ectópicos de hematopoiese Polidramnia e placentomegalia Polidramnia e placentomegalia Anasarca Anasarca Morte Morte Coombs indireto positivo Coombs direto positivo Doppler USG CTG DOENÇA HEMOLÍTICA PERINATAL

16 Acompanhamento Gestante Rh negativo COOMBS INDIRETO TIPAGEM MARIDO Rh (-) Rh (+) heterozigoto PCR-DNA Tipagem fetal Sangue materno 1ª CONSULTA

17 Gestante Rh negativo COOMBS INDIRETO NEGATIVO NEGATIVO IMUNOGLOBULINA ANTI-D ou Repetir 28,32,36,parto Positivo ≤ 1/8 Mensal ate 28 semanas Quinzenal após ≥ 1/16 AmniocenteseCordocenteseDoppler sem Tipagem + fator Rh HgbHtc Coombs direto Contagem de reticulócitos Possível tratamento

18 Routine antenatal anti-D prophylaxis in women who are Rh(D) negative: meta- analyses adjusted for differences in study design and quality. Turner RMTurner RM, Lloyd-Jones M, Anumba DO, Smith GC, Spiegelhalter DJ, Squires H, Stevens JW, Sweeting MJ, Urbaniak SJ, Webster R, Thompson SG. 2012;7(2):e Lloyd-Jones MAnumba DOSmith GCSpiegelhalter DJSquires HStevens JWSweeting MJUrbaniak SJ Webster RThompson SG Turner RMLloyd-Jones MAnumba DOSmith GCSpiegelhalter DJSquires HStevens JWSweeting MJUrbaniak SJ Webster RThompson SG Medical Research Council Biostatistics Unit, Institute of Public Health, Cambridge, United Kingdom. bsu.cam.ac.uk AbstractBACKGROUND: To estimate the effectiveness of routine antenatal anti-D prophylaxis for preventing sensitisation in pregnant Rhesus negative women, and to explore whether this depends on the treatment regimen adopted. METHODS: Ten studies identified in a previous systematic literature search were included. Potential sources of bias were systematically identified using bias checklists, and their impact and uncertainty were quantified using expert opinion. Study results were adjusted for biases and combined, first in a random-effects meta-analysis and then in a random-effects meta-regression analysis. RESULTS: In a conventional meta-analysis, the pooled odds ratio for sensitisation was estimated as 0.25 (95% CI 0.18, 0.36), comparing routine antenatal anti-D prophylaxis to control, with some heterogeneity (I² = 19%). However, this naïve analysis ignores substantial differences in study quality and design. After adjusting for these, the pooled odds ratio for sensitisation was estimated as 0.31 (95% CI 0.17, 0.56), with no evidence of heterogeneity (I² = 0%). A meta-regression analysis was performed, which used the data available from the ten anti-D prophylaxis studies to inform us about the relative effectiveness of three licensed treatments. This gave an 83% probability that a dose of 1250 IU at 28 and 34 weeks is most effective and a 76% probability that a single dose of 1500 IU at weeks is least effective. CONCLUSION: There is strong evidence for the effectiveness of routine antenatal anti-D prophylaxis for prevention of sensitisation, in support of the policy of offering routine prophylaxis to all non-sensitised pregnant Rhesus negative women. All three licensed dose regimens are expected to be effective. DOENÇA HEMOLÍTICA PERINATAL

19 Gestante Rh negativo COOMBS INDIRETO NEGATIVO NEGATIVO IMUNOGLOBULINA ANTI-D ou Repetir 28,32,36,parto Positivo ≤ 1/8 Mensal ate 28 semanas Quinzenal após ≥ 1/16 AmniocenteseCordocenteseDoppler sem Tipagem + fator Rh HgbHtc Coombs direto Contagem de reticulócitos Possível tratamento

20 Gestante Rh negativo COOMBS INDIRETO NEGATIVO NEGATIVO IMUNOGLOBULINA ANTI-D ou Repetir 28,32,36,parto Positivo ≤ 1/8 Mensal ate 28 semanas Quinzenal após ≥ 1/16 AmniocenteseCordocenteseDoppler sem Tipagem + fator Rh HgbHtc Coombs direto Contagem de reticulócitos Possível tratamento

21 Doppler Obstétrico

22 Obstet Gynecol 2002;100:600 –11.

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24 Doença Hemolítica Perinatal TRATAMENTO TRATAMENTO

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26 PROFILAXIA IMUNOGLOBULINA ANTI-RH APÓS O PARTO IMUNOGLOBULINA ANTI-RH APÓS O PARTO SANGRAMENTO DURANTE A GRAVIDEZ SANGRAMENTO DURANTE A GRAVIDEZ PROCEDIENTOS INVASIVOS PROCEDIENTOS INVASIVOS ROTINA 28 SEMANAS ROTINA 28 SEMANAS

27 Acta Obstet Gynecol Scand May;91(5): Acta Obstet Gynecol Scand May;91(5): Acta Obstet Gynecol Scand. Acta Obstet Gynecol Scand. Pharmacokinetics of 250 μg anti-D IgG in the third trimester of pregnancy: An observational study. Pharmacokinetics of 250 μg anti-D IgG in the third trimester of pregnancy: An observational study. Tiblad E, Wikman A, Rane A, Jansson Y, Westgren M. Tiblad E, Wikman A, Rane A, Jansson Y, Westgren M. Tiblad EWikman ARane AJansson YWestgren M Tiblad EWikman ARane AJansson YWestgren M Department of Obstetrics and Gynecology, Karolinska University Hospital and Karolinska Institute, Stockholm, Sweden Department of Immunology and Transfusion Medicine, Karolinska University Hospital and Karolinska Institute, Stockholm, Sweden Department of Clinical Pharmacology, Karolinska University Hospital and Karolinska Institute, Stockholm, Sweden. Department of Obstetrics and Gynecology, Karolinska University Hospital and Karolinska Institute, Stockholm, Sweden Department of Immunology and Transfusion Medicine, Karolinska University Hospital and Karolinska Institute, Stockholm, Sweden Department of Clinical Pharmacology, Karolinska University Hospital and Karolinska Institute, Stockholm, Sweden. Abstract Abstract Objective. We present a pharmacokinetic study evaluating a single intramuscular dose of 250 μg anti-D immunoglobulin in the third trimester of pregnancy. The aim of the study was to determine the kinetic profile and duration of detectable levels of anti-D. Design. Prospective observational study. Setting. Antenatal outpatient clinic. Population. Healthy Rhesus D (RhD)-negative pregnant women with an RHD-positive fetus. Methods. Serial plasma anti-D quantitations following antenatal administration of anti-D immunoglobulin were performed using flow cytometry. Kinetic profiles for anti-D levels were generated from the concentration values at predetermined sampling time points. The half-lives were calculated by linear regression analysis. Main outcome measures. Time vs. concentration profile, half-life and anti-D concentration ≥1 ng/mL close to term. Results. The maximal plasma concentration of anti-D was usually seen at 3-10 days postinjection, with a median value of 25 ng/mL. The half-life varied between individuals, with a median of 23 days. We found detectable levels of anti-D IgG within two weeks of parturition in 11 of 12 women. Conclusions. The preparation of anti-D immunoglobulin used in the present study, if administrated in pregnancy week 28-30, is associated with detectable levels of anti-D in most women at the time of delivery. Although the half-time is 23 days, it is uncertain whether all mothers have adequate anti-D concentrations at term. Alternative strategies may be evaluated in the future, with repeated administration of antenatal prophylaxis at term rather than conventional postpartum administration of anti-D. Objective. We present a pharmacokinetic study evaluating a single intramuscular dose of 250 μg anti-D immunoglobulin in the third trimester of pregnancy. The aim of the study was to determine the kinetic profile and duration of detectable levels of anti-D. Design. Prospective observational study. Setting. Antenatal outpatient clinic. Population. Healthy Rhesus D (RhD)-negative pregnant women with an RHD-positive fetus. Methods. Serial plasma anti-D quantitations following antenatal administration of anti-D immunoglobulin were performed using flow cytometry. Kinetic profiles for anti-D levels were generated from the concentration values at predetermined sampling time points. The half-lives were calculated by linear regression analysis. Main outcome measures. Time vs. concentration profile, half-life and anti-D concentration ≥1 ng/mL close to term. Results. The maximal plasma concentration of anti-D was usually seen at 3-10 days postinjection, with a median value of 25 ng/mL. The half-life varied between individuals, with a median of 23 days. We found detectable levels of anti-D IgG within two weeks of parturition in 11 of 12 women. Conclusions. The preparation of anti-D immunoglobulin used in the present study, if administrated in pregnancy week 28-30, is associated with detectable levels of anti-D in most women at the time of delivery. Although the half-time is 23 days, it is uncertain whether all mothers have adequate anti-D concentrations at term. Alternative strategies may be evaluated in the future, with repeated administration of antenatal prophylaxis at term rather than conventional postpartum administration of anti-D. DOENÇA HEMOLÍTICA PERINATAL

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29 Best Pract Res Clin Obstet Gynaecol Feb;26(1): Epub 2011 Nov 26. Best Pract Res Clin Obstet Gynaecol Feb;26(1): Epub 2011 Nov 26. Best Pract Res Clin Obstet Gynaecol. Best Pract Res Clin Obstet Gynaecol. Red-cell and platelet alloimmunisation in pregnancy. Red-cell and platelet alloimmunisation in pregnancy. Egbor M, Knott P, Bhide A. Egbor M, Knott P, Bhide A. Egbor MKnott PBhide A Egbor MKnott PBhide A Source Source Department of Obstetrics and Gynaecology, and Fetal Medicine Unit, St Helier University Hospital, Surrey, UK. Department of Obstetrics and Gynaecology, and Fetal Medicine Unit, St Helier University Hospital, Surrey, UK. Abstract Abstract The management of red-cell alloimmunisation has been revolutionised by the widespread use of anti-D administration for mothers who are rhesus negative, and the availability of non-invasive, ultrasound- based techniques for reliable detection of moderate-to-severe fetal anaemia. With reduced frequency of alloimmunisation to the D antigen, antibodies to c and Kell antigen are increasingly responsible for red- cell alloimmunisation. Ultrasound-based, non-invasive diagnosis is now so reliable that invasive techniques are sparingly used to detect significant fetal anaemia. Treatment of fetal anaemia using ultrasound-guided intravascular transfusions is highly successful. Advances in molecular biology have led to the successful determination of fetal blood group using free fetal DNA from maternal blood. This development is highly likely to allow use of anti-D in only those pregnant women carrying rhesus- positive fetuses. Sensitisation to non-D group antibodies continues to occur owing to the lack of available prophylaxis for other blood-group antigens. The management of red-cell alloimmunisation has been revolutionised by the widespread use of anti-D administration for mothers who are rhesus negative, and the availability of non-invasive, ultrasound- based techniques for reliable detection of moderate-to-severe fetal anaemia. With reduced frequency of alloimmunisation to the D antigen, antibodies to c and Kell antigen are increasingly responsible for red- cell alloimmunisation. Ultrasound-based, non-invasive diagnosis is now so reliable that invasive techniques are sparingly used to detect significant fetal anaemia. Treatment of fetal anaemia using ultrasound-guided intravascular transfusions is highly successful. Advances in molecular biology have led to the successful determination of fetal blood group using free fetal DNA from maternal blood. This development is highly likely to allow use of anti-D in only those pregnant women carrying rhesus- positive fetuses. Sensitisation to non-D group antibodies continues to occur owing to the lack of available prophylaxis for other blood-group antigens. DOENÇA HEMOLÍTICA PERINATAL

30 Immunohematology. 2011;27(1):6-11. Immunohematology. 2011;27(1):6-11. Immunohematology. Serologic and molecular characterization of D variants in Brazilians: impact for typing and transfusion strategy. Serologic and molecular characterization of D variants in Brazilians: impact for typing and transfusion strategy. Credidio DC, Pellegrino J, Castilho L. Credidio DC, Pellegrino J, Castilho L. Credidio DCPellegrino JCastilho L Credidio DCPellegrino JCastilho L Source Source Instituto Nacional de Ciência e Tecnologia do Sangue, Hemocentro, UNICAMP, Rua Carlos Chagas, 480, Caixa Postal 6198, CEP Barão Geraldo, Campinas, SP, Brazil. Instituto Nacional de Ciência e Tecnologia do Sangue, Hemocentro, UNICAMP, Rua Carlos Chagas, 480, Caixa Postal 6198, CEP Barão Geraldo, Campinas, SP, Brazil. Abstract Abstract Rh discrepancies are a problem during routine testing because of partial D or weak D phenotypes. Panels of monoclonal antibodies (MoAb) are being developed to identify D variants such as partial D and weak D when there are anomalous D typing results; however, molecular characterization offers a more specific classification of weak and partial D. The weak D and partial D phenotypes are caused by many different RHD alleles encoding aberrant D proteins, resulting in distinct serologic phenotypes and the possibility of anti-D immunization. We evaluated currently used serologic methods and reagents to detect and identify D variants and correlated the results with molecular analyses. A total of 306 blood samples from Brazilian blood donors and patients with discrepant results in routine D typing were analyzed. In total, 166 (54.2%) weak D, 136 (44.4%) partial D, 3 (1%) DEL, and 1 (0.3%) DHAR variants were identified. Among weak D samples, 76 weak D type 1 (45.8%), 75 weak D type 2 (45.2%), 13 weak D type 3 (7.8%), and 2 weak D type 5 (1.2%) alleles were found. Among the partial D samples, 49 type 4.0 weak partial D (36%), 9 DAR (6.6%), 24 DFR (17.6%), 6 DBT (4.4%), 1 DHMi (0.73%), 26 DVI (19%), 14 DVa (10.3%), 5 DIVb (3.7%), and 2 DVII (1.5%) were observed. Two samples identified as DEL by adsorption-elution were characterized by molecular analyses as RHD(IVS5– 38DEL4) and one sample was characterized as RHD(K409K). One sample was characterized as DHAR, a CE variant positive with some monoclonal anti-D. Our results showed that the use of different methods and anti-D reagents in the serologic routine analysis revealed D variants that can be further investigated. Molecular methods can help to differentiate between partial D and weak D and to characterize the weak D types, providing additional information of value in the determination of D phenotypes. This distinction is important for optimized management of D– RBC units and for the prevention of anti-D–related hemolytic disease of the fetus and newborn. Rh discrepancies are a problem during routine testing because of partial D or weak D phenotypes. Panels of monoclonal antibodies (MoAb) are being developed to identify D variants such as partial D and weak D when there are anomalous D typing results; however, molecular characterization offers a more specific classification of weak and partial D. The weak D and partial D phenotypes are caused by many different RHD alleles encoding aberrant D proteins, resulting in distinct serologic phenotypes and the possibility of anti-D immunization. We evaluated currently used serologic methods and reagents to detect and identify D variants and correlated the results with molecular analyses. A total of 306 blood samples from Brazilian blood donors and patients with discrepant results in routine D typing were analyzed. In total, 166 (54.2%) weak D, 136 (44.4%) partial D, 3 (1%) DEL, and 1 (0.3%) DHAR variants were identified. Among weak D samples, 76 weak D type 1 (45.8%), 75 weak D type 2 (45.2%), 13 weak D type 3 (7.8%), and 2 weak D type 5 (1.2%) alleles were found. Among the partial D samples, 49 type 4.0 weak partial D (36%), 9 DAR (6.6%), 24 DFR (17.6%), 6 DBT (4.4%), 1 DHMi (0.73%), 26 DVI (19%), 14 DVa (10.3%), 5 DIVb (3.7%), and 2 DVII (1.5%) were observed. Two samples identified as DEL by adsorption-elution were characterized by molecular analyses as RHD(IVS5– 38DEL4) and one sample was characterized as RHD(K409K). One sample was characterized as DHAR, a CE variant positive with some monoclonal anti-D. Our results showed that the use of different methods and anti-D reagents in the serologic routine analysis revealed D variants that can be further investigated. Molecular methods can help to differentiate between partial D and weak D and to characterize the weak D types, providing additional information of value in the determination of D phenotypes. This distinction is important for optimized management of D– RBC units and for the prevention of anti-D–related hemolytic disease of the fetus and newborn.

31 Transfusion Feb;52(2): doi: /j x. Epub 2011 Jul 25. Transfusion Feb;52(2): doi: /j x. Epub 2011 Jul 25. Transfusion. Partial D phenotypes and genotypes in the Chinese population. Partial D phenotypes and genotypes in the Chinese population. Ye L, Wang P, Gao H, Zhang J, Wang C, Li Q, Han S, Guo Z, Yang Y, Zhu Z. Ye L, Wang P, Gao H, Zhang J, Wang C, Li Q, Han S, Guo Z, Yang Y, Zhu Z. Ye LWang PGao HZhang JWang CLi QHan SGuo ZYang YZhu Z Ye LWang PGao HZhang JWang CLi QHan SGuo ZYang YZhu Z Source Source Shanghai Institute of Transfusion Medicine, Shanghai Blood Center, and School of Life Science, East China Normal University, Shanghai, China. Shanghai Institute of Transfusion Medicine, Shanghai Blood Center, and School of Life Science, East China Normal University, Shanghai, China. Abstract Abstract BACKGROUND: BACKGROUND: The D variant phenotypes are often categorized into weak D types and partial D types. Although the molecular basis underlying the partial D phenotype has been investigated in several races, data from Chinese populations are rare. The D variant phenotypes are often categorized into weak D types and partial D types. Although the molecular basis underlying the partial D phenotype has been investigated in several races, data from Chinese populations are rare. STUDY DESIGN AND METHODS: STUDY DESIGN AND METHODS: We collected partial D samples from 1,274,540 blood donors, as well as from sporadic patients in the Chinese population, over a 4-year period. Samples with partial D phenotype were determined by commercial monoclonal anti-D panels and molecular methods. Blood samples with discrepant results of serologic and molecular methods were further investigated by polymerase chain reaction (PCR) with sequence-specific primers and nucleotide sequencing of RHD exons. The detection of antibodies was performed. We collected partial D samples from 1,274,540 blood donors, as well as from sporadic patients in the Chinese population, over a 4-year period. Samples with partial D phenotype were determined by commercial monoclonal anti-D panels and molecular methods. Blood samples with discrepant results of serologic and molecular methods were further investigated by polymerase chain reaction (PCR) with sequence-specific primers and nucleotide sequencing of RHD exons. The detection of antibodies was performed. RESULTS: RESULTS: A total of 44 samples with partial D phenotypes were confirmed. Molecular typing revealed five different known aberrant alleles as well as four new RHD alleles. As described previously, DVI represented the most frequent partial D type in China with a total of 36 samples. However, discrepant results were observed in four DVI samples with serotyping and genotyping (i.e., DVI category identified by D-screen test and grossly intact RHD gene identified by multiplex PCR). We also found four novel alleles, termed DFR-4, DCS-3, DCC, and DLX. A total of 44 samples with partial D phenotypes were confirmed. Molecular typing revealed five different known aberrant alleles as well as four new RHD alleles. As described previously, DVI represented the most frequent partial D type in China with a total of 36 samples. However, discrepant results were observed in four DVI samples with serotyping and genotyping (i.e., DVI category identified by D-screen test and grossly intact RHD gene identified by multiplex PCR). We also found four novel alleles, termed DFR-4, DCS-3, DCC, and DLX. CONCLUSION: CONCLUSION: To date, this study presents the most comprehensive report on partial D in China. The distribution of partial D types in China was found to be complicated and polymorphic, whereas RhD genotyping of DVI-variant samples might give inaccurate results due to a relatively high incidence of RHD(1227G>A) in the Chinese population. To date, this study presents the most comprehensive report on partial D in China. The distribution of partial D types in China was found to be complicated and polymorphic, whereas RhD genotyping of DVI-variant samples might give inaccurate results due to a relatively high incidence of RHD(1227G>A) in the Chinese population.

32 Obstet Gynecol Feb;119(2 Pt 2): Obstet Gynecol Feb;119(2 Pt 2): Obstet Gynecol. Obstet Gynecol. New laboratory procedures and Rh blood type changes in a pregnant woman. New laboratory procedures and Rh blood type changes in a pregnant woman. Sandler SG, Li W, Langeberg A, Landy HJ. Sandler SG, Li W, Langeberg A, Landy HJ. Sandler SGLi WLangeberg ALandy HJ Sandler SGLi WLangeberg ALandy HJ Source Source Georgetown University Hospital, Washington, DC 20007, USA. Georgetown University Hospital, Washington, DC 20007, USA. Abstract Abstract BACKGROUND: BACKGROUND: A woman's candidacy for Rh immune globulin depends on whether her blood type is Rh-positive (D antigen-positive) or Rh-negative (D antigen-negative). New molecular blood-typing methods have identified variant D antigens, which may be reported as Rh-positive or Rh-negative depending on the laboratory method. We describe a case illustrating the effect of the new laboratory methods on a woman's candidacy for Rh immune globulin and present recommendations for interpreting the new test results. A woman's candidacy for Rh immune globulin depends on whether her blood type is Rh-positive (D antigen-positive) or Rh-negative (D antigen-negative). New molecular blood-typing methods have identified variant D antigens, which may be reported as Rh-positive or Rh-negative depending on the laboratory method. We describe a case illustrating the effect of the new laboratory methods on a woman's candidacy for Rh immune globulin and present recommendations for interpreting the new test results. CASE: CASE: A 40-year-old woman presented for management of her third pregnancy. During her first pregnancy, she was typed as Rh-positive ("D") and did not receive Rh immune globulin. During her second pregnancy, she was typed as Rh-negative, in accordance with revised Rh-typing procedures. Anti-D antibody was detected. During her third pregnancy, she was genotyped as a partial D antigen, which was reported as Rh-negative. A 40-year-old woman presented for management of her third pregnancy. During her first pregnancy, she was typed as Rh-positive ("D") and did not receive Rh immune globulin. During her second pregnancy, she was typed as Rh-negative, in accordance with revised Rh-typing procedures. Anti-D antibody was detected. During her third pregnancy, she was genotyped as a partial D antigen, which was reported as Rh-negative. CONCLUSION: CONCLUSION: Revisions in laboratory procedures for Rh typing may present as a change in the Rh blood type of pregnant women-and as a change in their eligibility for Rh immune globulin. Revisions in laboratory procedures for Rh typing may present as a change in the Rh blood type of pregnant women-and as a change in their eligibility for Rh immune globulin.

33 Am J Hematol Apr;87(4): doi: /ajh Epub 2012 Jan 9. Am J Hematol Apr;87(4): doi: /ajh Epub 2012 Jan 9. Am J Hematol. Am J Hematol. Detection of fetomaternal hemorrhage. Detection of fetomaternal hemorrhage. Kim YA, Makar RS. Kim YA, Makar RS. Kim YAMakar RS Kim YAMakar RS Source Source Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. Abstract Abstract The prevention of Rhesus D alloimmunization through Rh immune globulin (RhIg) administration is the major indication for the accurate detection and quantification of fetomaternal hemorrhage (FMH). In the setting of D incompatibility, D-positive fetal cells can sensitize the D-negative mother, resulting in maternal anti-D alloantibody production. These anti-D alloantibodies may lead to undesirable sequelae such as hemolytic disease of the newborn (HDN). Since the widespread adoption of FMH screening and RhIg immunoprophylaxis, the overall risk of Rh alloimmunization and infant mortality from HDN has substantially decreased. The rosette screen, the initial test of choice, is highly sensitive in qualitatively detecting 10 mL of fetal whole blood in the maternal circulation. As the screen is reliant on the presence of the D antigen to distinguish fetal from maternal cells, it cannot be used to detect FMH in D-positive mothers or in D-negative mothers carrying a D-negative fetus. The Kleihauer-Betke acid-elution test, the most widely used confirmatory test for quantifying FMH, relies on the principle that fetal RBCs contain mostly fetal hemoglobin (HbF), which is resistant to acid-elution whereas adult hemoglobin is acid-sensitive. Although the Kleihauer-Betke test is inexpensive and requires no special equipment, it lacks standardization and precision, and may not be accurate in conditions with elevated F-cells. Anti-HbF flow cytometry is a promising alternative, although its use is limited by equipment and staffing costs. Hematology analyzers with flow cytometry capabilities may be adapted for fetal cell detection, thus giving clinical laboratories a potentially attractive automated alternative for quantifying FMH. The prevention of Rhesus D alloimmunization through Rh immune globulin (RhIg) administration is the major indication for the accurate detection and quantification of fetomaternal hemorrhage (FMH). In the setting of D incompatibility, D-positive fetal cells can sensitize the D-negative mother, resulting in maternal anti-D alloantibody production. These anti-D alloantibodies may lead to undesirable sequelae such as hemolytic disease of the newborn (HDN). Since the widespread adoption of FMH screening and RhIg immunoprophylaxis, the overall risk of Rh alloimmunization and infant mortality from HDN has substantially decreased. The rosette screen, the initial test of choice, is highly sensitive in qualitatively detecting 10 mL of fetal whole blood in the maternal circulation. As the screen is reliant on the presence of the D antigen to distinguish fetal from maternal cells, it cannot be used to detect FMH in D-positive mothers or in D-negative mothers carrying a D-negative fetus. The Kleihauer-Betke acid-elution test, the most widely used confirmatory test for quantifying FMH, relies on the principle that fetal RBCs contain mostly fetal hemoglobin (HbF), which is resistant to acid-elution whereas adult hemoglobin is acid-sensitive. Although the Kleihauer-Betke test is inexpensive and requires no special equipment, it lacks standardization and precision, and may not be accurate in conditions with elevated F-cells. Anti-HbF flow cytometry is a promising alternative, although its use is limited by equipment and staffing costs. Hematology analyzers with flow cytometry capabilities may be adapted for fetal cell detection, thus giving clinical laboratories a potentially attractive automated alternative for quantifying FMH.

34 Am J Obstet Gynecol Dec;193(6): Am J Obstet Gynecol Dec;193(6): Am J Obstet Gynecol. Am J Obstet Gynecol. Noninvasive prenatal RHD genotyping by real-time polymerase chain reaction using plasma from D- negative pregnant women. Noninvasive prenatal RHD genotyping by real-time polymerase chain reaction using plasma from D- negative pregnant women. Zhou L, Thorson JA, Nugent C, Davenport RD, Butch SH, Judd WJ. Zhou L, Thorson JA, Nugent C, Davenport RD, Butch SH, Judd WJ. Zhou LThorson JANugent CDavenport RDButch SHJudd WJ Zhou LThorson JANugent CDavenport RDButch SHJudd WJ Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA. Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA. Abstract Abstract OBJECTIVE: OBJECTIVE: Prenatal noninvasive determination of fetal Rh status is an important aid to the management of hemolytic disease of the fetus and newborn. We performed real-time polymerase chain reaction on fetal DNA derived from maternal plasma to determine fetal Rh status. Prenatal noninvasive determination of fetal Rh status is an important aid to the management of hemolytic disease of the fetus and newborn. We performed real-time polymerase chain reaction on fetal DNA derived from maternal plasma to determine fetal Rh status. STUDY DESIGN: STUDY DESIGN: Cell-free plasma DNA from 98 D-negative pregnant women was tested for the presence of exons 4, 5, and 10 of RHD. The presence of fetal DNA was confirmed by detection of SRY or biallelic insertion/deletion polymorphisms in the maternal plasma and buffy coat. Cell-free plasma DNA from 98 D-negative pregnant women was tested for the presence of exons 4, 5, and 10 of RHD. The presence of fetal DNA was confirmed by detection of SRY or biallelic insertion/deletion polymorphisms in the maternal plasma and buffy coat. RESULTS: RESULTS: Seventy-two D-positive infants and 26 D-negative infants were determined by serologic studies. All 3 RHD exon sequences were detected in 68 of 72 mothers of D-positive infants. The presence of fetal DNA in mothers of D-negative infants was confirmed in all 10 boys and in 14 of 16 girls. Seventy-two D-positive infants and 26 D-negative infants were determined by serologic studies. All 3 RHD exon sequences were detected in 68 of 72 mothers of D-positive infants. The presence of fetal DNA in mothers of D-negative infants was confirmed in all 10 boys and in 14 of 16 girls. CONCLUSION: CONCLUSION: Fetal RHD genotyping in this study correctly predicted fetal Rh status in 92 of 98 (94%) cases. Fetal RHD genotyping in this study correctly predicted fetal Rh status in 92 of 98 (94%) cases.


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