New methods for the quantification of mixed chimerism in transplantation.

Background: 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.

Hematopoietic 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.

Using EasyMatch® to anticipate the identification of an HLA identical unrelated donor: A validated efficient time and cost saving method.

In the absence of an HLA matched familial donor, a search for an unrelated donor or cord blood unit is initiated through worldwide registries. Although a first look-up on available HLA information of donors in the "book" at BMDW (Bone Marrow Donor Worldwide) can provide a good estimation of the number of compatible donors, the variety of resolution typing levels requires confirmatory typing (CT) which are expensive and time consuming. In order to help recipient centers in their work. The French donor registry (France Greffe de Moelle/Agence de la Biomedecine) has recently developed a software program called "EasyMatch®" that uses haplotype frequencies to compute the likelihood of phenotypic match in donors according to various typing resolution levels. The goal of our study is to report a single monocentric user-experience with EasyMatch®, demonstrating that its routine use reduced the cost and the delay of the donor search in our center, allowing the definition of a new strategy to search compatible unrelated donors. The strategy was first established on a retrospective cohort of 217 recipients (185 adults and 32 children=before score) and then validated on a prospective cohort of 171 recipients (160 adults and 11 children=after score). For all patients, we calculated the delay between the registration day and the donor identification day, and the number of CT requested to the donor centre. Considering both groups, we could observe a significant decrease of the number of CT from 8 to 2 (p<0,001), and a significant decrease of the median delay to identify a suitable donor from 43 to 31days (p<0.0001). EasyMatch® estimates the number of potentially identical donors, but doesn't foresee availability of the donors. It provides us an easy tracking of mismatches, an estimation of the number of potential donors, the selection of population following ethnic origin of patients and a high prediction when probability is high or low. It affords a new approach of donor search in our daily work and improves the efficiency in the great challenge of the compatible donor identification.

[A rare cause of anemia in a chronic dialyzed patient]. / Une cause rare d'anémie chez une patiente hémodialysée chronique.

We 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.

Confirmation of a novel recurrent association: BCR-ABL t(9;22) and t(19;21).

Association of a t(9;22)(q34;q11), BCR/ABL-positive, with a dic(19;21)(p13;p13) has been described in acute lymphoblastic leukemia in relapse, raising the question of whether this association is recurrent. Described here are two cases, one of myeloproliferative disease and one of acute lymphoblastic leukemia, both presenting a masked t(9;22) and t(19;21). Chromosomal rearrangements were ascertained by fluorescence in situ hybridization (FISH) using locus-specific probes, multicolor FISH, and bacterial artificial chromosome array. These additional observations suggest a nonrandom association.

[An unusual cause of pancytopenia: intravascular lymphoma]. / Etiologie inhabituelle d'une pancytopénie: le lymphome intravasculaire.

We report an observation of intravascular lymphoma occurring in a 69-year-old woman. This relatively rare disease presents polymorphic clinical features that render diagnosis difficult. Cutaneous and central nervous system signs and symptoms are frequently observed and should be recognized as suggestive of intravascular lymphoma. However, they are not always observed, in our case only pancytopenia was present. Pronostic is generally unfavorable but good response to chemotherapy has been described with early diagnosis. A large number of pathogenic hypotheses have been put forward: abnormality of cellular receptor, role of Epstein-Barr virus or transformation from lymphoma. Intravascular lymphoma should be included in the differential diagnosis of pancytopenia to increase chances of good response.

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.