Non-invasive Prenatal Diagnosis of Feto-Maternal Platelet Incompatibility by Cold High Resolution Melting Analysis.

Fetal and Neonatal alloimmune thrombocytopenia (FNAIT) is a condition which could occur when pregnant women develop an alloimmunization against paternally inherited antigens of the fetal platelets. Approximately 80 % of FNAIT cases are caused by anti-HPA-1a, about 15 % by anti-HPA-5b and 5 % by other HPA antibodies. Only 2 % of the total population is HPA-1a negative (HPA-1b1b). The HPA-1a allele differs by one single nucleotide from HPA-1b allele, yet it represents around 27 % of total severe thrombocytopenias. HPA-1 was studied in serum cDNA from 12 volunteer pregnant women to determine their HPA-1 genotype by HRM (high resolution melting) PCR. When an homozygous HPA-1 gene was detected in a mother, a COLD HRM was performed to determine whether or not the fetal genotype differs from the mother's.The differences in the melting curve shapes allow us to accurately distinguish the three pregnants genotypes. The fetal heterozygous genotype of homozygous pregnant women was correctly detected by COLD PCR HRM in maternal serum. HPA-1 genotyping by HRM may be a useful aproach for genotyping all pregnant women in inexpensively. Moreover, when HPA-1 homozygosis was detected in a pregnant woman, fetal heterozygosis may be diagnosed by COLD HRM to select pregnancies for preventive monitoring.

Intermediate doses of cytarabine plus granulocyte-colony-stimulating factor as an effective and safe regimen for hematopoietic stem cell collection in lymphoma patients with prior mobilization failure.

BACKGROUND: High-dose chemotherapy supported by autologous stem cell transplantation (ASCT) is an effective treatment for patients with lymphomas. However, failure to reach the minimum threshold of hematopoietic stem cells to proceed to ASCT may occur, even with the most effective strategies currently available. STUDY DESIGN AND METHODS: We report on 33 patients diagnosed with lymphoma who had at least one prior mobilization failure and received cytarabine at a dose of 400 mg/m(2) /day intravenously × 3 days plus granulocyte-colony-stimulating factor (G-CSF) 10 to 12 µg/kg/day as mobilization regimen. The median number of previous lines of chemotherapy was three. RESULTS: Thirty-two of 33 patients (96.8%) reached the target CD34+ cell dose (>2 × 10(6) /kg). The mean (range) number of apheresis procedures was 1.8 (1-3) with 4.69 × 10(6) (1.5 × 10(6) -6.8 × 10(6) )/kg CD34+ cells obtained. All but one patient received chemomobilization in the outpatient department. Severe infections or treatment-related mortality were not observed. All patients that received ASCT (31/33) engrafted without requiring G-CSF during the posttransplant period. CONCLUSION: This study shows that cytarabine at intermediate doses plus G-CSF in patients diagnosed with lymphoma who had a prior mobilization failure is a feasible and effective mobilization regimen.

Lenograstim compared to filgrastim for the mobilization of hematopoietic stem cells in healthy donors.

BACKGROUND: Recombinant human granulocyte-colony-stimulating factor (G-CSF) is used to mobilize hematopoietic stem cells for both autologous and allogeneic hematopoietic stem cell transplantation. The recombinant products clinically available are lenograstim and filgrastim, which differ from a biologic point of view as well as from their economical impact. In this regard, some studies have shown different in vitro activities although clinical studies comparing both drugs in the allogeneic transplant setting are scanty. STUDY DESIGN AND METHODS: In the current study we compare the efficacy of lenograstim and filgrastim in terms of number of circulating CD34+ cells/µL during the fifth day of G-CSF administration, the number of days of apheresis required to obtain the target cell dose, the median of CD34+ cells collected on the first day of apheresis, or the median number of total CD34+ cells collected at the end of the procedure, in a series of 146 healthy donors undergoing hematopoietic stem cell mobilization for allogeneic transplantation. RESULTS: We observed that, using a comparable dose for the two products, no significant differences were observed between the two groups. CONCLUSION: In conclusion, the current retrospective study shows that lenograstim and filgrastim are similar in terms of efficacy for the mobilization of hematopoietic stem cells in healthy donors.

Standardization non-invasive fetal RHD and SRY determination into clinical routine using a new multiplex RT-PCR assay for fetal cell-free DNA in pregnant women plasma: results in clinical benefits and cost saving.

INTRODUCTION: Among negative RhD mothers it is essential to know the fetal RhD status in order to avoid the possibility of hemolytic disease of the newborn. In this regard, the detection of fetal DNA in maternal plasma might become a new diagnostic tool. In the current study, we have evaluated the standardization of a Multiplex-PCR targeted towards two exons of the RHD and one SRY gene to monitor RhD negative women. The current study addresses questions concerning feasibility and applicability of this approach into the clinical practice. MATERIALS AND METHODS: Both single and multiplex real-time PCRs targeting RHD exons 5 and 7 and SRY were applied for the detection of fetal-specific RHD sequences and sex in maternal plasma. A large cohort of 2127 women was studied between 10 and 28 weeks of pregnancy. 134 of them were used for single TaqMan PCR studies and 1993 were evaluated using Multiplex TaqMan PCR studies. All of them were serologically typed as RhD negative according to Spanish guidelines. Single and multiplex real-time PCR results were compared with postnatal serology and sex identification. RESULTS: There was a 100% concordance between results obtained with single and multiplex real-time PCR assays. At present, 1012 of the 1993 pregnant women studied gave birth and the results of RHD status obtained with the multiplex TaqMan PCR assay were confirmed postpartum by serological methods showing that sensitivity, specificity, and accuracy of the multiplex assay were 100, 98.6, and 99.3%, respectively. This procedure improved the speed of the assay, avoided over-treatment among RhD negative pregnant women bearing RhD negative fetus, and reduced the requirements for clinical and biological monitoring, resulting in a clinical benefit and cost saving. CONCLUSIONS: The routine determination of fetal RHD status and SRY in maternal plasma, using multiplex real-time PCR, is feasible. The use of multiplex real-time PCR allows improving the response of the laboratory, saving time and reagent costs, opening the door to a complete automatization of the process.