Genotipificación del gen RHD en ADN fetal libre en plasma de embarazadas RhD negativo / Genotyping of the RHD gene in free fetal DNA in plasma of RhD negative pregnant women

Objetivo: Determinar el grupo RhD fetal a través del estudio del gen RHD en ADN fetal que se encuentra libre en plasma de embarazadas RhD negativo. Método: Se analizó la presencia de los genes RHD, SRY y BGLO en ADNfl obtenido de plasma de 51 embarazadas RhD negativo no sensibilizadas, utilizando una qPCR. Los resultados del estudio genético del gen RHD se compararon con el estudio del grupo sanguíneo RhD realizado por método serológico en muestras de sangre de cordón, y los resultados del estudio del gen SRY fueron cotejados con el sexo fetal determinado por ecografía. Se calcularon la sensibilidad, la especificidad, los valores predictivos y la capacidad discriminativa del método estandarizado. Resultados: El gen RHD estaba presente en el 72,5% de las muestras y el gen SRY en el 55,5%, coincidiendo en un 100% con los resultados del grupo RhD detectado en sangre de cordón y con el sexo fetal confirmado por ecografía, respectivamente. Conclusiones: Fue posible deducir el grupo sanguíneo RhD del feto mediante el estudio del ADN fetal que se encuentra libre en el plasma de embarazadas con un método molecular no invasivo desarrollado y validado para este fin. Este test no invasivo puede ser utilizado para tomar la decisión de administrar inmunoglobulina anti-D solo a embarazadas RhD negativo que portan un feto RhD positivo.

Objective: To determine the fetal RhD group through the study of the RHD gene in fetal DNA found free in plasma of RhD negative pregnant women. Method: The presence of the RHD, SRY and BGLO genes in fetal DNA obtained from plasma of 51 non-sensitized RhD negative pregnant women was analyzed using qPCR. The results of the genetic study of the RHD gene were compared with the RhD blood group study performed by serological method in cord blood samples, and the results of the SRY gene study were compared with the fetal sex determined by ultrasound. Sensitivity, specificity, predictive values and discriminative capacity of the standardized method were calculated. Results: The RHD gene was present in 72.5% of the samples and the SRY gene in 55.5%, coinciding 100% with the results of the RhD group detected in cord blood, and with the fetal sex confirmed by ultrasound, respectively. Conclusions: It was possible to deduce the RhD blood group of the fetus through the study of fetal DNA found free in the plasma of pregnant women with a non-invasive molecular method developed and validated for this purpose. This non-invasive test can be used to make the decision to administer anti-D immunoglobulin only to RhD-negative pregnant women carrying an RhD-positive fetus.

Safety of RhD alloimmunization / 中国输血杂志

【Objective】 To explore the safety of RhD-positive red blood cells (RBCs) immunization schedules in RhD-negative volunteers, so as to facilitate the development of domestic anti-D immunoglobulin. 【Methods】 From January 2018 to April 2020, 23 RhD negative volunteers with informed consent were enrolled and divided into initial immunization group and booster immunization group. The initial immunization included first immunization, second immunization and third immunization. Four groups, i. e. 3 cases of 20 mL, 8 of 30 mL, 6 of 40 mL, and 6 of 50 mL, were involved in initial immunization. After the initial immunization response, booster immunizations were performed every 3 months. According to the anti-D titer before each immunization, the booster immunization doses were set to 0.5, 1 and 2 mL. Whole blood samples of 5mL/ person (time) were collected 24 h and 1 week after each infusion, and the blood routine, liver, kidney and blood coagulation function and anti-D titer were detected. The differences of detection (index) values at 24 h and 1 week after the first immunization and booster immunization in each (dose) group were compared. 【Results】 No statistically significant differences were observed in hemolysis index values (all within the range of medical reference values) 24 h or 1 week after initial immunization among RhD positive RBCs of 20, 30, 40 and 50mL(P>0.05). The differences between the hemolysis index values and the basic values before the immune response (all within the range of medical reference values) after 0.5 or 1 mL booster immunizations were also not statistically different (P>0.05). However, the differences (μmol/L)between total bilirubin levels and the basic values before the immune response (1.55±1.87, 6.29±2.66) were significantly different after 2 mL booster immunization (P<0.05). 【Conclusion】 No risks affecting the safety of RhD negative volunteers was found in the immunization schedule proposed in this study.

Fetomaternal hemorrhage in RhD-negative pregnant women in Tianjin / 国际生物医学工程杂志

Objective To study and monitor the situation of femomaternal hemorrhage (FMH) in RhD-negative pregnant women in Tianjin, obtain the FMH data of such population, and analyze the relationship between FMH and age, blood type, gestational age, hemolytic disease of postpartum neonates, etc. Methods The FMH level was detected by flow cytometry with FITC-anti-HbF monoclonal antibody. The blood type was detected by blood serum method. The irregular antibody was identified by saline method and indirect anti-human ball method. The hemolysis of postpartum neonates was detected by three tests of hemolysis. Results The FMH volume of 86 RhD negative pregnant women was between 0 and 11.48 ml, with an average of 1.82 ml. There were 63.95％of pregnant women showed a volume of FMH<2.0 ml, 23.26％between 2 and 4 ml, 11.63％between 4.0 and 10.0 ml, and 1.16％>10 ml. The proportion of lower FMH in pregnant women≤30 years old was>11.71％higher than that in the pregnant women>30 years old, but the difference was no statistical significant. There was no significant difference in FMH of pregnant women with O, A, B and AB types. The proportion of higher FMH in pregnant women with compatible ABO blood type with her husband was 12.46％ lower than that of the heterozygous cases, but the difference was no statistical significant. The proportion of higher FMH in the pregnant women with 28 to 32 weeks gestational age was 14.55％ higher than that of ≤28 weeks and was 35.32％ higher than that of >32 weeks, and the differences were statistical significant. Three samples in the 86 samples were positive for anti-D antibody, and their three hemolytic test results were strongly positive with the anti-D titer from 1:2 to 1:32 and the FMH volume from 1.50 to 6.93 ml. The proportion of lower FMH in the 10 pregnant women without postpartum hemolysis was 70％ higher than that in 5 pregnant women with postpartum hemolysis, but the differences were not statistical significant. Conclusions The results suggest that monitoring FMH content by flow cytometry can reflect FMH in Rh-negative pregnant women. The studies on the relationship between FMH and age, blood type, pregnant time and hemolytic disease of postpartum neonates can provide basically experimental data for standard use of anti-D immunoglobulin in pregnant women.

Anticuerpos anti-Fya con reacción anamnésica asociada con anti-D en transfusión intrauterina: reporte de un caso / Anti-Fya antibodies with anamnestic reaction associated to anti-D in intrauterine transfusion: A case report

Resumen ANTECEDENTES La enfermedad hemolítica perinatal ocurre después de una transfusión sanguínea, que sensibiliza con antígenos eritrocitarios, hemorragia materno-fetal durante el embarazo o al momento del parto. La incidencia de anticuerpos anti-D ha disminuido de 14 a 0.1% en las madres D-negativas. No existe una inmunoglobulina que evite o disminuya la aloinmunización por otros antígenos eritrocitarios durante el embarazo. La incompatibilidad del grupo sanguíneo Duffy es una causa común de enfermedad hemolítica perinatal. OBJETIVO Exponer el caso de una paciente con hijo con enfermedad hemolítica perinatal por anticuerpos anti-Fya y anti-D tratado con transfusión intrauterina. CASO CLÍNICO Paciente de 22 años, con antecedente de múltiples transfusiones sanguíneas y datos clínicos de síndrome anémico. En la semana 28 del embarazo fue valorada para aplicarle inmunoglobulina anti-D. Luego de aplicarle dos unidades de concentrado eritrocitario Rh negativo se observó incompatibilidad (++) en fase de antiglobulina humana (Coombs), por esto se realizó el escrutinio de anticuerpos irregulares en gel, que resultó positivo en células I y II (+++). Enseguida se inició el protocolo de identificación de anticuerpos irregulares con un panel de 11 células, que reportó aglutinación en las células 1, 2, 3, 5, 6, 7, 8 y 11, sin mostrar especificidad. El estudio de adsorción del anticuerpo anti-D mostró células de antígeno D+ con las que se estableció el diagnóstico de anticuerpos anti-Fya y anti-D. El embarazo finalizó mediante cesárea con el nacimiento de un varón con grupo y Rh O positivo, de 30.1 semanas de gestación (talla de 40 cm y peso de 2000 g) con hidrops fetal. Se le realizaron ciclos de reanimación e ingresó a la unidad de cuidados intensivos neonatales, sin tratamiento farmacológico, y después de una hora de vida extrauterina falleció. La madre se dio de alta del hospital 36 horas después del puerperio, sin complicaciones adicionales. CONCLUSIÓN Los anticuerpos antieritrocitarios anti-Fya, solos o en combinación con otros anticuerpos, provocan enfermedad hemolítica perinatal severa. El laboratorio de inmunohematología tiene participación importante en el diagnóstico, seguimiento y tratamiento de la enfermedad hemolítica perinatal.

Abstract BACKGROUND Hemolytic disease of the fetus and newborn occurs after alloimmunization with red blood cells antigens by blood transfusion, maternal-fetal hemorrhage during pregnancy or at delivery. Currently, the incidence of alloimmunization by anti-D antibody has been reduced from 14% to 0.1% of D-negative mothers, however, there is no immunoglobulin that prevents or decreases alloimmunization by other red blood cells antigens during pregnancy. The incompatibilities of the Duffy blood group are a common cause of hemolytic disease of the fetus and newborn. OBJECTIVE To present the case of a neonate with perinatal hemolytic disease secondary to anti-Fya and anti-D antibodies managed with intrauterine transfusion. CLINICAL CASE A 22-year-old patient with a history of multiple blood transfusions and clinical data of anemic syndrome. In the 28th week of pregnancy it was evaluated for the application of anti-D immunoglobulin. The blood bank was asked for two units of Rh negative erythrocyte concentrate. Incompatibility (++) in the human antiglobulin phase (Coombs) was observed, so the irregular antibody gel was screened, which was positive in cells I and II (+++). An identification protocol for irregular antibodies was initiated with a panel of 11 cells, which reported agglutination in cells 1, 2, 3, 5, 6, 7, 8 and 11, without specificity. The adsorption study of the anti-D antibody showed D + antigen cells. The diagnosis of anti-Fya and anti-D antibodies was established. The pregnant woman was terminated by caesarean section, from which a male with a group was born and Rh O positive, of 30.1 weeks of gestation (size of 40 cm and weight of 2000 g) with fetal hydrops. He underwent resuscitation cycles, entered the neonatal intensive care unit, without pharmacotherapy and died after one hour of extrauterine life. The mother withdrew 36 hours after the puerperium, without additional complications. CONCLUSION The antibodies anti-Fya alone or next to other alloantibodies produce severe hemolytic disease of the fetus and newborn. The laboratory of immunohematology in the blood bank is an essential tool in the diagnosis, monitoring and treatment of hemolytic disease of the fetus and newborn.

Comparison of anti-D immunoglobulin and high-dose intravenous immunoglobulin therapy in childhood acute idiopathic thrombocytopenic purpura:a systematic review and meta-analysis / 临床儿科杂志

Objectives To compare the effectiveness and safety of intravenous anti-D immunoglobulin (IV anti-D) with high-dose intravenous immunoglobulin (IVIG) as initial treatments of acute idiopathic thrombocytopenic purpura (ITP) in children. Methods Randomized controlled trials comparing anti-D with high-dose IVIG in the treatment of childhood acute ITP were systematically reviewed from PubMed, Embase and Cochrane Central Register of Controlled Trials and hand-searched reference lists. The number of patients with a platelet count>20 × 109/L at 72 hours after treatment initia-tion, and the decrease in hemoglobin were the primary outcomes. The meta-analysis was performed by RevMan 5.1. Results A total of 771 relevant articles were retrieved, and ifve studies were included. The RR (anti-D versus high dose IVIG) of achieving a platelet count>20×109/L at 72 hours was 0.90 (95%CI:0.82~0.98). However, subgroup analysis suggested no signiifcant difference between anti-D at a dose of 50μg/kg and high-dose IVIG (RR 0.98, 95% CI: 0.84~1.13), as well as between anti-D at a dose of 75μg/kg and high-dose IVIG (RR 0.88, 95%CI:0.75~1.03). Hemoglobin drop was greater in the anti-D group. No patients, however, required transfusions of erythrocyte suspensions. Conclusions IV anti-D may be as ef-fective as high-dose IVIG in the treatment of childhood acute ITP at 72 hours after therapy. The side effects of anti-D were tolerated and acceptable.

An Analysis of Neutropenia after the Administration of High-dose Intravenous Immunoglobulin or Anti-D Immunoglobulin on Acute Immune Thrombocytopenic Purpura Children: Age based Analysis / 임상소아혈액종양

BACKGROUND: The purpose of this study is to evaluate the decreased neutrophil count after the administration of high-dose intravenous immunoglobulin (IVIG) or anti-D immunoglobulin (anti-D Ig) to children with acute immune thrombocytopenic purpura (ITP). In addition, post-treatment changes in absolute neutrophil count (ANC) were also analyzed according to patient age. METHODS: We retrospectively performed a chart review of 83 patients who had received IVIG or anti-D Ig to manage the decreased platelet count due to acute ITP. Pre- and post-treatment ANC were analyzed in the IVIG treatment group and anti-D Ig treatment group, and ANC were compared between each group. In addition, all the subjects were divided into two groups according to age: infantile group and other group, and ANC of these two groups were compared. RESULTS: A decrease in ANC in just a day and recovering to its pre-treatment value on the 7th post-treatment day was observed for the IVIG treatment group. Especially, the decrease in ANC observed in the infantile group was found to recover in 14 days, where-as ANC recovery in the other group was seen within 2 days after the treatment. There was no statistically significant decrease in ANC in the anti-D Ig treatment group. CONCLUSION: IVIG used for childhood acute ITP caused a decrease in ANC more severely than with the administration of anti-D Ig. Subjects aged less than 12 months who were treated with IVIG for acute ITP showed more ANC decrease and needed longer time to recover normal ANC compared to those over 12 months of age.

An Analysis of Neutropenia after the Administration of High-dose Intravenous Immunoglobulin or Anti-D Immunoglobulin on Acute Immune Thrombocytopenic Purpura Children: Age based Analysis / 임상소아혈액종양

BACKGROUND: The purpose of this study is to evaluate the decreased neutrophil count after the administration of high-dose intravenous immunoglobulin (IVIG) or anti-D immunoglobulin (anti-D Ig) to children with acute immune thrombocytopenic purpura (ITP). In addition, post-treatment changes in absolute neutrophil count (ANC) were also analyzed according to patient age.METHODS: We retrospectively performed a chart review of 83 patients who had received IVIG or anti-D Ig to manage the decreased platelet count due to acute ITP. Pre- and post-treatment ANC were analyzed in the IVIG treatment group and anti-D Ig treatment group, and ANC were compared between each group. In addition, all the subjects were divided into two groups according to age: infantile group and other group, and ANC of these two groups were compared.RESULTS: A decrease in ANC in just a day and recovering to its pre-treatment value on the 7th post-treatment day was observed for the IVIG treatment group. Especially, the decrease in ANC observed in the infantile group was found to recover in 14 days, where-as ANC recovery in the other group was seen within 2 days after the treatment. There was no statistically significant decrease in ANC in the anti-D Ig treatment group.CONCLUSION: IVIG used for childhood acute ITP caused a decrease in ANC more severely than with the administration of anti-D Ig. Subjects aged less than 12 months who were treated with IVIG for acute ITP showed more ANC decrease and needed longer time to recover normal ANC compared to those over 12 months of age.

Development of Severe Hemolytic Anemia after Treatment with Anti-D Immunoglobulin in a Patient with Immune Thrombocytopenic Purpura / 대한수혈학회지

A 78-year-old female was admitted due to nasal bleeding and purpuric macules on both legs. The patient underwent renal biopsy, and a diagnosis of Henoch-Schonlein purpura nephritis was made. The patient's platelet count was 1.6x10(10)/L, and, based on results from bone marrow biopsy, the patient was diagnosed with immune thrombocytopenic purpura. Despite treatment with glucocorticoid and IV immunoglobulin, thrombocytopenia continued. The patient's blood group was Rhesus D positive and treatment with IV anti-D immunoglobulin followed. Thereafter, platelet count showed a rapid increase; however, occurrence of hemolytic anemia, hyperbilirubinemia, and hemoglobinuria consistent with intravascular hemolysis was observed.

Intravenous Anti-D immunoglobulin treatment of adult chronic immune thrombocytopenic purpura (ITP) / 대한내과학회지

BACKGROUND/AIMS: Immune thrombocytopenic purpura (ITP) is an autoimmune disease that is mediated by anti-platelet antibodies. Based on the pathogenesis of ITP we evaluated the efficacy of intravenous anti-D immunoglobulin for adult chronic ITP. METHODS: Fourteen patients (4 without splenectomy and 10 with splenectomy) with refractory chronic ITP were treated with 50-70 microgram/kg of intravenous anti-D immunoglobulin only once. Treatment effects were evaluated by measuring the platelet counts and hemoglobin levels. RESULTS: Five patients (36%) showed a response; improvement in the platelet count lasted for on average 7 days (range: 2~24 days). There were no serious adverse effects. CONCLUSION: Anti-D immunoglobulin, which is associated with an Fc receptor blockade, appeared to be safe and effective for the treatment of adults with chronic ITP. Further studies are needed to confirm these findings and define further potentially effective treatment protocols with intravenous anti-D immunoglobulin.

A Case of Idiopathic Thrombocytopenic Purpura with Anti-E Antibody Developed after Transfusion of Platelet Concentrates / 대한수혈학회지

Alloantibodies against RBCs after transfusion of only platelet products are rarely reported except for RhD mismatched platelet transfusion. We report a case of anti-E antibody developed after transfusion of platelet concentrates. The patient was a 27- month male infant with idiopathic thrombocytopenic purpura and was treated with IV immunoglobulin and IV Rh immunoglobulin. Anti-E antibody was detected after transfusion of 18 units of platelet concentrates for about 40 days. RBC alloimmunization was developed after transfusion of platelet products, which suggests platelet transfusion may be cause of RBC alloimmunization in childhood.