METHODS::Fanconi anemia (FA) is a recessive disorder that predispose to bone marrow failure and multiple congenital anomalies in affected individuals worldwide. To date, 22 FA genes are known to harbor sequence variations in disease phenotype. Among these, mutations in the FANCA gene are associated with 60% to 70% of FA cases. The aim of the present study was to screen FA cases belonging to consanguineous Pakistani families for selected exons of FANCA gene which are known mutational hotspots for Asian populations. Blood samples were collected from 20 FA cases and 20 controls. RNA was extracted and cDNA was synthesized from blood samples of cases. DNA was extracted from blood samples of cases and ethnically matched healthy controls. Sanger's sequencing of the nine selected exons of FANCA gene in FA cases revealed 19 genetic alterations of which 15 were single nucleotide variants, three were insertions and one was microdeletion. Of the total 19 sequence changes, 13 were novel and six were previously reported. All identified variants were evaluated by computational programs including SIFT, PolyPhen-2 and Mutation taster. Seven out of 20 analyzed patients were carrying homozygous novel sequence variations, predicted to be associated with FA. These disease associated novel variants were not detected in ethnically matched controls and depict genetic heterogeneity of disease.

METHODS::In type 3 von Willebrand disease (VWD3), the most severe form with absent von Willebrand factor (VWF), the bleeding phenotype is variable. Platelet contribution to the hemostatic defect in VWD3 calls upon further studies. We investigated the contribution of platelets to in vitro thrombin generation (TG) and platelet procoagulant activity in VWD3. TG was assessed by calibrated automated thrombogram (CAT) in platelet-poor (PPP) and -rich plasma (PRP) from 9 patients before and in 6 patients also 30 min after receiving their regular VWF therapy. Responsiveness of PPP to FVIII and protein S was also investigated. TG data were compared with routine laboratory variables, rotational thromboelastometry (ROTEM) and platelet expression of P-selectin and phosphatidylserine in flow cytometry. Compared with healthy controls, TG was markedly decreased in VWD3 PPP (peak thrombin was 16% of normal median), but not in PRP (77% of normal median) (p = 0.002). Six out of nine patients (67%) were high responders in their platelet P-selectin, and 5/9 (56%) in phosphatidylserine expression. Replacement therapy improved TG in PPP, while in PRP TG only modestly increased or was unaffected. In PPP, FVIII levels associated with TG and in vitro FVIII-supplemented TG inclined up to threefold. Conversely, a FVIII inhibitory antibody reduced plasma TG in all, but especially in patients with remnant FVIII levels. Inhibition of protein S improved plasma TG, particularly at low FVIII levels. ROTEM failed to detect VWD3.In VWD3, TG is reduced in PPP and regulated by FVIII and protein S, but TG is close to normal in PRP. VWD3 platelets seem to compensate for the FVIII-associated reduction in TG by their exposure of P-selectin and phosphatidylserine.

METHODS:BACKGROUND:The optimal timing of initiating granulocyte-colony stimulating factor following chemotherapy in pediatric patients has not been clearly defined. This study aimed to compare the administration of granulocyte-colony stimulating factor on day 1 versus day 3 postchemotherapy in pediatric patients with Ewing sarcoma. METHOD:A retrospective study of pediatric patients with Ewing sarcoma who received granulocyte-colony stimulating factor following chemotherapy between January 2016 and September 2018 at a comprehensive cancer center. The institution's chemotherapy protocol for Ewing sarcoma was modified in April 2017 to include granulocyte-colony stimulating factor initiation on day 3 instead of day 1 post-chemotherapy. Febrile neutropenia requiring hospitalization, duration of hospital stay, and chemotherapy delay were compared for patients before and after the protocol change. RESULTS:Over the study period, 250 cycles were evaluated with day 1 granulocyte-colony stimulating factor and 221 cycles with day 3 granulocyte-colony stimulating factor. There were no differences between the day 1 and day 3 groups in the number of cycles associated with Febrile neutropenia requiring hospitalization (34 vs. 19, p = 0.086), and the length of Febrile neutropenia-related hospitalization (mean 4 ± 2.1 vs. 4.6 ± 1.8, p = 0.123). However, delay in chemotherapy due to neutropenia was reported in significantly more cycles in the day 1 group, compared to the day 3 group (37 vs. 16, p = 0.01). CONCLUSIONS:Febrile neutropenia resulting in hospital admission and the length of hospital stay was not different between pediatric patients with Ewing sarcoma who received granulocyte-colony stimulating factor on day 1 or day 3 post-chemotherapy. Chemotherapy delay due to neutropenia was higher in patients who received granulocyte-colony stimulating factor on day 1. Larger studies are required to fully determine the impact of delayed initiation of granulocyte-colony stimulating factor.