Noninvasive diagnostics of fetal KEL*01.01 allele from maternal plasma of immunized women using digital PCR protocols.

Allo-antibodies produced by K-negative pregnant women against a fetal K antigen from the Kell blood group system may cause hemolytic disease of the fetus and newborn (HDFN). Predicting the fetal K antigen using noninvasive prenatal testing (NIPT) is important for decisions concerning management of pregnancies. Digital and droplet digital PCR techniques permit the detection of fetal single nucleotide variant with a higher specificity and sensitivity than real-time polymerase chain reaction (PCR). AIM: The aim was to evaluate and compare protocols for fetal KEL*01.01 genotyping using different assays and digital PCR platforms. METHODS: DNA isolated from 59 pregnant women (9-39 weeks of gestation, 49 with anti-K) was tested using home-made and custom-ordered KEL*01.01/KEL*02 assays with Droplet Digital™ and QuantStudio™3D. The results were compared with fetal/neonatal genotypes/phenotypes. RESULTS: Fetal KEL*01.01 results using all tested protocols were concordant with fetal/neonatal KEL*01.01 genotypes/phenotypes. None of the tested combinations of assays or digital PCR platforms gave false KEL*01.01-negative results, but inconclusive KEL*01.01 reads were observed in all tested protocols. For 36 cases compared using two digital PCR platforms and assays, there were not statistically significant differences in a level of fetal KEL*01.01 fraction (p < .72). CONCLUSION: Independent of the applied dPCR and ddPCR platforms and KEL*01.01 assays, prediction of the fetal KEL*01.01 is highly reliable. Before implementation in routine practice further validation of the KEL*01.01 protocol with a larger group of pregnant women should be performed.

Prediction of fetal blood group and platelet antigens from maternal plasma using next-generation sequencing.

BACKGROUND: Fetuses whose mothers have produced antibodies to red blood cell (RBC) or platelet antigens are at risk of being affected by hemolytic disease or alloimmune thrombocytopenia, respectively, only if they inherit the incompatible antigen. Noninvasive diagnosis of the fetal antigen is employed for management of immunized pregnancies, but the specific detection of SNPs, encoding the majority of antigens, in maternal plasma is still a challenge. We applied targeted next-generation sequencing (NGS) to predict the fetal antigen based on the detection of fetomaternal chimerism. METHODS AND MATERIALS: The DNA of 13 pregnant women (with anti-K [3] anti-k [1], anti-Fya [1], anti-D + C + Jka [1], anti-D + E + K [1], anti-HPA-1a [1], anti-HPA-3b [1], anti-HPA-5b [1], and nonimmunized [3]) was sequenced using primers for regions encoding RhD, RhC, Rhc, RhE/e, K/k, Fya/b, Jka/b, MN, Ss, and HPA-1, 2, 3, 5, 15, 4 X-polymorphisms on the Ion Torrent Personal Genome Machine (PGM) System (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RESULTS: NGS results were in agreement with the phenotype/genotype of women and their neonates (except for the unsuccessful detection of MN and RhC). NGS determined fetal allele chimerism for K, k, Fya, Fyb, Jka, Jkb, S, RhE (from 0.42% to 6.08%); RhD, Rhc (100%); HPA-1a, -2b, -3a, 3b, -5b, -15a, 15b (from 0.23% to 4.11%). NGS revealed fetal chimerism for incompatible antigens (from 0.7% to 4.8%) in 7 immunized cases, excluded in 3 (with anti-K, anti-Fya , anti-HPA-3b). CONCLUSION: The designed NGS predicts the fetal RBC and platelet antigen status universally in cases with various clinically significant antibodies as well as providing confirmation of the presence of fetal DNA. However, some improvement of the unsuccessful primers is required.

[Standardization of the quantitative flow cytometric test with anti-D antibodies for fetomaternal hemorrhage in RhD negative women]. / Standaryzacja metody ilosciowej oceny przecieku plodowo-matczynego u kobiet RhD ujemnych przy pomocy cytometrii przeplywowej z uzyciem przeciwcial anty-D.

BACKGROUND: In order to determine the appropriate dose of anti-D immunoglobulin to be administered as a preventive measure against hemolytic disease of the fetus/newborn in the subsequent pregnancy it is necessary to assess the number of fetal red blood cells that infiltrate/penetrate into the maternal circulation as a result of fetomaternal hemorrhage (FMH). One of the quantitative methods of FMH analysis is based on flow cytometry (FACS) which makes use of monoclonal antibodies to RhD antigen (anti-D test). The aim of the study was to further develop the method, evaluate its sensitivity and reproducibility and to compare it with the test based on the detection of fetal hemoglobin (HbF). MATERIAL AND METHODS: The FACS study involved 20 RhD negative pregnant women and 80 RhD negative women after delivery. The following monoclonal antibodies were used: BRAD 3 FITC (anti-RhD antigen), CD45 PerCP (anti leukocyte antigen CD45), and anti-HbF PE. RESULTS: The fluorescence intensity of cells incubated with BRAD 3 FITC was demonstrated to depend on the RhD antigen expression, though the anti-D test also detects the weak D variant. The CD45 PerCP antibodies increased the sensitivity of anti-D test since they eliminated the leukocytes which non-specifically bind anti-D from the analysis. The presence of anti-D antibodies in maternal plasma does not affect the quantitative assessment of the fetal RhD positive fetal cells with BRAD 3 FITC. In case of FMH, the results of the anti-D test were similar to those with anti-HbF antibodies. CONCLUSIONS: The flow cytometric test with anti-D and anti-CD45 is useful in the assessment of the fetomaternal hemorrhage in RhD negative women. The sensitivity of the test is estimated at 0.05%.

[Maternal blood intrauterine transfusions in the therapy of red-cell alloimmunization performed in three difficult cases]. / Wykorzystanie krwi matczynej do transfuzji doplodowych w terapii konfliktu serologicznego na przykladzie trzech trudnych przypadków.

Maternal alloimmunization can lead to hemolytic anemia, hydrops fetalis and even fetal or neonatal death. Intrauterine treatment is possible and effective even though it is associated with some risk. We present a rare method of maternal blood intrauterine transfusions in the therapy of three difficult cases of erythroblastosis fetalis. The aim of this report was to present an alternative to volunteer donors. In severe cases, i.e. in the absence of matching blood types from the donor in the presence of multiple alloantibodies in the pregnant woman or if multiple transfusions are required, this can be the only therapeutic option. To the best of our knowledge, this has been the first publication on maternal blood donation for intrauterine transfusion in the Polish literature.

Identification and follow-up of pregnant women with platelet-type human platelet antigen (HPA)-1bb alloimmunized with fetal HPA-1a.

INTRODUCTION: Pregnant women negative for human platelet antigen 1a (HPA-1a) are at risk of alloimmunization with fetal HPA-1a antigen inherited from the father, and their offspring may develop fetal and neonatal alloimmune thrombocytopenia (FNAIT). The aim of this study was to analyze the frequency of HPA-1a alloimmunization in pregnant Polish women, the feasibility of using maternal platelets for intrauterine transfusions in women subjected to diagnostic fetal blood sampling (FBS) and to discuss potential consequences of alloimmunization. MATERIAL AND METHODS: Fifteen thousand two hundred and four pregnant women were typed for HPA-1a; HPA-1a negative were screened for anti-HPA-1a. Alloimmunized women received specialist perinatology care; some of them were subjected to FBS, followed by transfusion of HPA-1a negative platelet concentrates (PC) prepared from maternal blood. RESULTS: Three hundred seventy-three (2.5%) women were HPA-1a negative, and 32 (8.6%) tested positively for anti-HPA-1a. Antibodies were detected in 22 women during pregnancy. Diagnostic FBS followed by PC transfusion was performed in 14 woman, who were platelet donors for their 16 unborn babies. Blood donations were tolerated well by the patients, and also intrauterine platelet transfusions were uneventful. Pharmacotherapy with intravenous immunoglobulins was implemented in 11/22 patients. CONCLUSIONS: HPA-1a negative women (ca. 2.5% of all pregnant patients) are at risk of alloimmunization with HPA-1a antigen and developing FNAIT. Alloimmunized women can be donors of platelets for their offspring providing removal of antibodies from PC. Owing to potential complications, special care should be taken if an alloimmunized woman was qualified as a blood or stem cell recipient.

Recent advances in understanding the clinical relevance of antiplatelet alloantibodies.

Alloimmunization to human platelet antigens (HPAs) may occur either during pregnancy, when a HPA­negative mother gives birth to a newborn who inherits HPAs from the father, or following blood transfusion or stem cell transplantation. Antiplatelet alloantibodies do not cause thrombocytopenia in a patient, but their detection must always be recorded in medical records because they may induce fetal and neonatal alloimmune thrombocytopenia in present and all subsequent pregnancies, platelet refractoriness, posttransfusion purpura, or prolonged thrombocytopenia with engraftment failure after stem cell transplantation. Passive transfer of platelet alloantibodies through transfused blood components may trigger thrombocytopenia and severe posttransfusion reactions in the recipient. In a Caucasian population, such clinical outcome of platelet alloimmunization is mostly due to anti­HPA­1a antibodies, less frequently to anti­HPA­5b, anti­HPA­1b, and others. Information on anti­HPA alloantibodies is crucial for the prevention and treatment of their consequences.

[Bernard-Soulier syndrome during pregnancy: a case report]. / Przebieg ciazy, porodu i pologu u pacjentki z rozpoznaniem zespolu Bernarda-Soulier.

The Bernard-Soulier syndrome (B-S s.) is rare autosomal recessive bleeding disorder. It is characterized by prolonged bleeding time, fail to agglutinate with ristocetin and a normal to decreased number of unusually large platelets whose membrane lack glycoproteins complex GP Ib/IX/V. The manifestation of the disease may differ in consecutive pregnancies of the same patient. Presently we describe 24 years old white women diagnosed with B-S s. at the age of 22. Two years later she become pregnant. During the pregnancy platelet counts ranged from 26 x 10(9)/1 to 51 x 10(9)/1, without sings of bleeding. Because of the risk of immunization against GP Ib/IX, with might lead to severe isoimmune neonatal thrombocytopenia, 4 times during pregnancy she had done MAIPA-test (monoclonal antibody immobilization of platelet antigens). Specific antibodies were not detected. Subsequent ultrasounds with biophysical profile revealed normal fetus growth. Spontaneous labour in 38th week of gestation. The use of single-donor platelets, intravenous desmopressin and tranexamic acid was found to be useful in controlling postpartum vaginal bleeding in the patient.

[Hairy cell leukemia in pregnancy]. / Bialaczka wlochatokomórkowa u kobiety w ciazy.

Pregnancy complicated by HCL is an extremlely rare event: only 5 report of HCL in pregnancy have been previously described. A 36-year-old women was diagnosed of HCL presented in 14 week of her 5th pregnancy. Therapy was initiated in 18 wk of gestation with IFN-alpha at dose 6 x 106 U subcutaneous 3 times per week and prednisone 20 mg/d. IFN-alpha administration was well tolerated. After IFN-alpha therapy partial remission was achieved. The course of the pregnancy was normal. At week 39 of gestation a cesarean cection was performed and the patient delivered a healthy male infant weighing 3460 g with Apgar score of 10. The placenta was of normal size, no infiltration of hairy cell was founded. Post-partum course was uncomplicated. The patient elected to not breast feed her infant. 3 weeks after delivery patient had a 5-days course of 2CDA