Background: The definition of polycythemia, whether primary or secondary, is based on direct measurement of red cell mass (RCM) by isotope labelling method. Because of the lack of availability of ...isotopes, the use of hematocrit and hemoglobin values defined by the WHO recommendations has overtaken the use of the RCM, which can result in misdiagnosis of polycythemia.This determination is also useful for patients with myeloproliferative neoplasm (MPN) to separate two clinical entities: essential thrombocythemia and “masked” Polycythemia Vera, both of which result in high blood counts. These two conditions have different prognoses and therapeutic management. RCM measurement is also sometimes used in the follow-up of MPN with splanchnic thrombosis.The CO-rebreathing method is mainly used in sports to assess the RCM. It is minimally invasive, fast (<30 min) and its accuracy appears to be equivalent to the gold standard’s.To date, no study has compared RCM values obtained with CO-Rebreathing and with the state-of-the-art technique, i.e. isotopic labeling, in the diagnosis of polycythemia.Aims: Here, we present the result of a prospective bi-centric study comparing RCM obtained by CO-rebreathing and by isotopic measurement in a population referred to hematologists for suspicion of polycythemia.Methods: Forty-two patients were initially recruited for simultaneous RCM determination by Co-rebreathing and isotope labelling method. All patients signed an informed consent form. During the course of the study, two patients could not benefit from the Co-rebreathing measurement due to non-compliance with the inclusion criteria (smoking just before the examination) or due to the ergonomic complexity of using the spirometer. On the other hand, the isotope labelling method could not be performed for another patient. This prospective non-randomized study therefore included 39 patients (35 men and 4 women) with a median age of 57 years (range 19-91 years); three of them had Polycythemia Vera with a V617F JAK2 mutation.The isotope labelling method was performed according to the recommendations of the French Society of Radiopharmacy (SoFRa) in a nuclear medicine department using the labelling of the patient’s red blood cells (RBC) with technetium 99m.Co-rebreathing measurement was performed in a pneumology department immediately after the respiratory functional explorations and the determination of baseline HbCO. This method consists of labelling Hb with inspired carbon monoxide (CO), resulting in a temporary increase of COHb.Results: True polycythemia was defined by an increased red cell mass (RCM) above 125% of the theoretical one, for both techniques. All results were expressed as a percentage of the theoretical value.Comparing the RCM results obtained with these two methods, the two techniques were consistent for 31 patients (21 true polycythemia, 10 false polycythemia). For four patients, the CO-rebreathing measurement was underestimated, while for four others, the value obtained by CO-rebreathing was overestimated. Overall, the method yielded a sensitivity of 84% and a specificity of 71%.Summary/Conclusion: This study, carried out on a large series of patients, allows the validation of a simple, non-invasive and reliable tool for the measurement of RCM based on the use of CO-rebreathing, and which is well correlated with the reference isotopic method. It has the advantage of being more accessible to patients, for whom a hospital hosting a nuclear medicine department is too far.In addition, it will allow the measurement of RCM in countries where the isotopic method is no longer available, such as most European and North American countries.
The discovery in 2005 of the
V617F gain-of-function mutation in myeloproliferative neoplasms and more particularly in polycythemia vera has deeply changed the diagnostic and therapeutic approaches to ...polycythemia. More recently, the use of NGS in routine practice has revealed a large number of variants, although it is not always possible to classify them as pathogenic. This is notably the case for the
E846D variant for which for which questions remain unanswered. In a large French national cohort of 650 patients with well-characterized erythrocytosis, an isolated germline heterozygous
E846D substitution was observed in only two cases. For one of the patients, a family study could be performed, without segregation of the variant with the erythrocytosis phenotype. On the other hand, based on the large UK Biobank resource cohort including more than half a million UK participants, the
E846D variant was found in 760 individuals, associated with a moderate increase in hemoglobin and hematocrit values, but with no significant difference to the mean values of the rest of the studied population. Altogether, our data as well as UK Biobank cohort analyses suggest that the occurrence of an absolute polycythemia cannot be attributed to the sole demonstration of an isolated
E846D variant. However, it must be accompanied by other stimuli or favoring factors in order to generate absolute erythrocytosis.
A rapid, sensitive and specific method for ricinine identification and quantification in plasma has been developed by LC–HRMS. Deuterated ricinine was used as the internal standard. From 100 μL of ...plasma, ricinine was extracted using micro‐solid‐phase elution, which allows a reduced extraction time, by eliminating the evaporation step. Eluate is directly injected into the LC–HRMS system. Chromatographic separation was performed using a reverse‐phase C18 column with a 4.5 min gradient elution. The method was validated according to European Medicines Agency guidelines. Linearity was verified between 0.25 and 500.0 ng/mL; the maximum precision calculated was 19.9% for the lower limit of quantitation and 9.6% for quality control, and accuracy was within ± 5.6% of the nominal concentrations. Selectivity, carryover, matrix effect and stability were also verified according to European Medicines Agency guidelines. The method allows the rapid and reliable identification of ricin‐exposed victims in case of terrorist attacks or poisonings: three intoxication cases are reported.
Hereditary erythrocytosis (HE) is a rare hematological disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2160 ...patients with erythrocytosis sequenced in 10 different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of the Hypoxia-Inducible Factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: in silico study of localization, conservation, and deleterious effects; analysis of hematological parameters of carriers identified in the UK Biobank; functional studies of the protein activity and stability; and comprehensive study of PHD2 splicing. Altogether, this study allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extented to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences with the variants of unknown significance regarding the severity of the developed disease (hematological parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare pathology to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematological diseases.
Gain-of-function mutations in the EPAS1/HIF2A gene have been identified in patients with hereditary erythrocytosis that can be associated with the development of paraganglioma, pheochromocytoma and ...somatostatinoma. In the present study, we describe a unique European collection of 41 patients and 28 relatives diagnosed with an erythrocytosis associated with a germline genetic variant in EPAS1. In addition we identified two infants with severe erythrocytosis associated with a mosaic mutation present in less than 2% of the blood, one of whom later developed a paraganglioma. The aim of this study was to determine the causal role of these genetic variants, to establish pathogenicity, and to identify potential candidates eligible for the new hypoxia-inducible factor-2 α (HIF-2α) inhibitor treatment. Pathogenicity was predicted with in silico tools and the impact of 13 HIF-2b variants has been studied by using canonical and real-time reporter luciferase assays. These functional assays consisted of a novel edited vector containing an expanded region of the erythropoietin promoter combined with distal regulatory elements which substantially enhanced the HIF-2α-dependent induction. Altogether, our studies allowed the classification of 11 mutations as pathogenic in 17 patients and 23 relatives. We described four new mutations (D525G, L526F, G527K, A530S) close to the key proline P531, which broadens the spectrum of mutations involved in erythrocytosis. Notably, we identified patients with only erythrocytosis associated with germline mutations A530S and Y532C previously identified at somatic state in tumors, thereby raising the complexity of the genotype/phenotype correlations. Altogether, this study allows accurate clinical follow-up of patients and opens the possibility of benefiting from HIF-2α inhibitor treatment, so far the only targeted treatment in hypoxia-related erythrocytosis disease.
