[A rare cause of anemia in a chronic dialyzed patient].

International audienceWe reported the case of a female in chronic dialysis since 3 years who developed a normocytic a regenerative anemia with no evident etiology. A bone marrow smear revealed a copper deficiency, a rare cause of anemia in general and dialyzed populations. Copper supplementation improved anemia with a decrease in transfusions rate and erythropoietin dosage. This case suggests that a copper deficiency has to be considered when a myelodysplastic syndrome is associated with neurologic symptoms and granulocytes precursors vacuolization identified in bone marrow smear. A blood titration of trace elements should be considered in front of a refractory anemia with no evident etiology

New methods for the quantification of mixed chimerism in transplantation

International audienceBackground Quantification of chimerism showing the proportion of the donor in a recipient is essential for the follow-up of hematopoietic stem cell transplantation but can also be useful to document an immune tolerance situation after solid organ transplantation. Historically, chimerism has been quantified from genomic DNA, but with technological advances, chimerism from donor-derived cell-free DNA seems particularly relevant in solid organ transplantation.Methods The reference method was until recently the short tandem repeat technique, but new innovative techniques as digital PCR (dPCR) and NGS, have revolutionized the quantification of chimerism, such as the so-called microchimerism analysis. After a short review of chimerism methods, a comparison of chimerism quantification data for two new digital PCR systems (QIAcuity™ dPCR (Qiagen ® ) and QuantStudio Absolute Q (ThermoFisher ® ) and two NGS-based chimerism quantification methods (AlloSeq HCT™ (CareDx ® ) and NGStrack™ (GenDX ® )) was performed.Results These new methods were correlated and concordant to routinely methods (r²=0.9978 and r²=0.9974 for dPCR methods, r²=0.9978 and r²=0.9988 for NGS methods), and had similar high performance (sensitivity, reproductibility, linearity).Conclusion Finally, the choice of the innovative method of chimerism within the laboratory does not depend on the analytical performances because they are similar but mainly on the amount of activity and the access to instruments and computer services

Evaluation of Next-Generation Sequencing and Crystal Digital PCR for Chimerism Monitoring of Post-Allogeneic Hematopoietic Stem Cell Transplantation.

International audienceHematopoietic stem cell transplantation (HSCT) is a curative treatment for most hematologic diseases. To evaluate the level of donor engraftment, chimerism must be carefully monitored after HSCT. Short tandem repeats, quantitative PCR (qPCR), and, more recently, digital PCR (dPCR) are widely used to determine the proportions of donor and recipient cells after HSCT. The screening and quantification of chimerism have been evaluated by 2 new methods: a ready-to-use next-generation sequencing (NGS)-based method using the Devyser ChimerismNGS kit and an original combination of the Stilla crystal digital PCR (cdPCR) platform with 3-color multiplexing capacity using GenDX KMRtrack reagents. The genotyping of 4 HSCT pairs by cdPCR using 11 triplex mixes of the GenDX KMRtype kit was consistent at 98.8% with qPCR. Informative samples (n = 20) from 6 donor-recipient pairs and 1 external proficiency test demonstrated the reliability of the results (0.1% to 50%) for the 2 methods. The methods are also highly sensitive (0.1%) and accurate. The chimerism values of the 2 methods are correlated and concordant with those of the reference methods. In addition, the ADVYSER software (Devyser) is user-friendly and well adapted to chimerism monitoring. In conclusion, these 2 innovative methods are easy to perform and user-friendly in all molecular, hematology, and immunogenetic laboratories and allow the genotyping and monitoring of chimerism with high performance and sensitivity

Clinical value of the quantitative expression of the three epitopes of CD34 in 300 cases of acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: The various epitopes of the CD34 molecule have been classified according to their different sensitivities to enzymatic cleavage by neuraminidase, chymopapain and a glycoprotease from Pasteurella haemolytica. Although monoclonal antibodies have been developed that specifically identify these epitopes, few studies have evaluated the distribution and quantitative expression of such epitopes on leukemic blasts. DESIGN AND METHODS: We report here a prospective multicenter study in which we examined and quantified the expression of the 3 classes of CD34 on fresh leukemic blast cells from 300 cases of acute myeloid leukemia (AML). The binding of monoclonal antibodies was studied by flow cytometry, allowing evaluation of blast cell positivity as well as their mean fluorescence intensity. These quantitative data were made comparable between centers by means of a calibration curve established with the same reagents in all laboratories. RESULTS: Quantitative expression of class I epitope was significantly higher than that of class II and class III epitopes (p<0.0001). The three classes were more frequently expressed in M0 and M1 and less in M3 and M5. The highest levels of CD34 expression were observed in M2, M0 and M1 and the lowest in M3, M5 and BAL for class II and III. CD34 expression was lower for all classes in cases with a normal karyotype, compared to in cases with structural or numerical abnormalities. INTERPRETATION AND CONCLUSIONS: In cases with a t(9;22) the expression of class I was significantly higher than that of class II and III and the opposite was observed in AML with t(15;17). Moreover, as a whole, a high intensity of class III CD34 appeared to be a marker of good prognosis