An inter-laboratory comparison of PNH clone detection by high-sensitivity flow cytometry in a Russian cohort

Sipol, Alexandra; Babenko, Elena V.; Борисов, В. И.; Наумова, Е В; Boyakova, Elena; Yakunin, Dimitry I.; Глазанова, Т В; Чубукина, Ж В; Pronkina, Natalya V.; Popov, Alexander; Saveliev, Leonid; Lugovskaya, S. A.; Lisukov, Igor A.; Illingworth, Andrea

doi:10.1179/1607845414y.0000000162

Cited by 12 publications

(16 citation statements)

References 23 publications

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“…В ряде случаев болезнь одновременно про-является как интенсивным гемолизом, так и осложне-ниями цитопении. Внедрение современной хорошо стандартизо-ванной высокочувствительной проточной цитоме-трии сделало верификацию диагноза ПНГ быстрой и достоверной, что было показано в т. ч. в процессе валидизирующего исследования в России [16]. Од-нако осведомленность гематологов и врачей других специальностей об этом редком и гетерогенном забо-левании остается весьма слабой, что приводит к зна-чительным трудностям первичного распознавания ПНГ и, как правило, позднему диагнозу.…”

Section: Discussionunclassified

“…Наличие и размер клона ПНГ определялись ме-тодом проточной цитометрии на основе анализа экс-прессии GPI-якоря (FLAER) на гранулоцитах и моно-цитах, а также GPI-связанных белков на гранулоцитах (CD24), моноцитах (CD14) и эритроцитах (CD59), согласно подробно описанному протоколу [16].…”

Section: материалы и методыunclassified

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Clinical Manifestation and Errors in the Diagnosis of Classical Paroxysmal Nocturnal Hemoglobinuria:

Klimova

Ivanova

et al. 2017

Клиническая онкогематология

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Background. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal hematopoietic stem cell disorder, characterized by intravascular hemolysis, cytopenia and thrombosis. Diagnostic errors with delayed diagnosis of PNH are often due to the variety of the clinical presentation and the lack of awareness of the doctors of this rare disease. Aim. The aim of the study was to characterize the spectrum of clinical manifestations and the complexity of diagnosis of classical PNH. Materials & Methods. The study included 150 patients with classical PNH. The inclusion criteria were: 1) clinical and laboratory signs of intravascular hemolysis; 2) verification of the diagnosis using standard flow cytometry; 3) absence of aplastic anemia, myelodysplastic syndrome and primary myelofibrosis. Results. The study population consisted of 89 (59 %) women and 61 (41 %) men. Median age was 34 years (13-72 years). The time before the diagnosis ranged from 0 to 455 months (median 33 months). The median size of the PNH clone among granulocytes and erythrocytes was 95 % and 41 %, respectively. The median of the lactate dehydrogenase was 7.2 times the upper limit of normal (ULN). Cytopenia occurred in 65 % of patients, including a combination of thrombocytopenia and neutropenia in 29 % of cases. Weakness and fatigue (99 %), hemoglobinuria (57 %), pain (52 %), icterus (46 %), dysphagia (37 %) and infection/fever (23 %) were the most common symptoms on the onset of the disease. Before the diagnosis of PNH, thrombosis or acute kidney injury was found in 22 % and 12 % of patients, respectively. Only 22 % of patients were initially diagnosed with PNH. In the remaining patients, the primary diagnosis was incorrect. Conclusion. The clinical manifestation of PNH is characterised by the presence of hemoglobinuria, cytopenia and early thrombosis in 57 %, 65 % and 22 % of patients, respectively. Errors of the primary diagnosis reach 78 % and lead to inadequate treatment. The results of the study showed the need for multidisciplinary approach and strict adherence to diagnostic algorithms of PNH in the risk groups, according to current recommendations.

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Section: Discussionunclassified

Section: материалы и методыunclassified

Clinical Manifestation and Errors in the Diagnosis of Classical Paroxysmal Nocturnal Hemoglobinuria:

Klimova

Ivanova

et al. 2017

Клиническая онкогематология

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“…Sample 23 was originally labeled with panel 3, which included CD64-PECy7. Nonetheless our validation acceptance criteria was to as far as possible use reagents that minimize the need for re-drawing/ moving gates, and since CD64 (clone 10.1) has been previously used by several studies (e.g., (7,12,13) and CD64-BV421 clone 10.1 was used previously by one network laboratory for several years without any spurious results or artifacts; we elected to use CD64-BV421. S3).…”

Section: Wbc Assay: Issues Encounteredmentioning

confidence: 99%

“…Patients with bone marrow failure syndromes such as aplastic anaemia and myelodysplasia may present with low numbers of cells with a PNH phenotype; detecting these cells may aid diagnostic work-up of such cases (5). Since then, additional guidelines and studies that evaluate PNH testing protocols have been published (7)(8)(9)(10)(11)(12)(13)(14)(15). These guidelines provide a starting point for producing accurate data that are reproducible by different laboratories.…”

Section: Introductionmentioning

confidence: 99%

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Inter‐laboratory validation of a harmonized PNH flow cytometry assay

Payne

Johansson

Bloxham

et al. 2018

Cytometry Part B Clinical

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Background: A network comprising 11 laboratories aimed to consolidate PNH testing by developing and validating an assay guided by previous guidelines and studies. Network analyses of >20 native samples yielded key findings that were used to create and reshape the final protocol.Methods: Twenty-seven native samples were distributed to all participating laboratories for blind testing, local analysis, and subsequent re-analysis by a central laboratory. Inter-laboratory clone size precision (coefficient of variation [CV]) was monitored for each sample, and the findings used to refine the test protocol. Linearity and precision tests were performed, assay limits of blank and detection were calculated, and limits of quantification were determined.Results: When using the final protocol, enumeration of cells with a PNH phenotype by all participating laboratories was comparable, with no clinically significant discrepancies or false-positive or false-negative results reported. Of note, the biological characteristics of the sample affected precision. For example, CVs were higher when PNH and normal cells showed contiguous expression of GPI-linked antigens. A red cell gating strategy that eliminated non-specific type II PNH phenotypic events was devised, enabling reliable reporting of events in the type II red cell gate. The final protocol provided an assay with a Limit of Quantification of 0.01% for neutrophils and red cells, and 0.1% for monocytes.Conclusions: We describe a robust network-validated PNH assay that may aid other laboratories to set up and validate high resolution PNH analysis.

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Randomized multicenter noninferiority phase III clinical trial of the first biosimilar of eculizumab

et al. 2021

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Currently, eculizumab is the main effective treatment for paroxysmal nocturnal hemoglobinuria (PNH). The aim of this randomized multicenter noninferiority study was to evaluate the efficacy and safety of the Biosimilar (Elizaria) in comparison with the Originator (Soliris) in patients with PNH. Biosimilar and Originator were administered at a dose of 600 mg weekly for 4 weeks at the initial stage in naive patients, as well as for maintenance therapy at a dose of 900 mg every 2 weeks in all patients. The primary endpoint was a comparative assessment of hemolytic activity based on the area under the lactate dehydrogenase (LDH) concentration–time curve during the maintenance therapy. Thirty-two (32) patients were randomized for therapy with Biosimilar (n = 16) or Originator (n = 16). The mean values of LDH concentration–time curve were similar in both treatment groups without statistically significant differences (p > 0.05). Evaluation of secondary endpoints has shown no statistically significant differences between the groups. Safety values were comparable in both treatment groups. The data obtained confirm that the Biosimilar is not inferior to the Originator in terms of the main efficacy parameter, and is also comparable with it in terms of safety and additional efficacy parameters. Clinicaltrials.gov identifier: NCT04463056

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An inter-laboratory comparison of PNH clone detection by high-sensitivity flow cytometry in a Russian cohort

Abstract: The ICCS-recommended high-sensitivity flow cytometry protocol was effective for detecting major and minor PNH clones in Russian PNH patients, and showed high reproducibility between laboratories.

Cited by 12 publications

References 23 publications

Clinical Manifestation and Errors in the Diagnosis of Classical Paroxysmal Nocturnal Hemoglobinuria:

Clinical Manifestation and Errors in the Diagnosis of Classical Paroxysmal Nocturnal Hemoglobinuria:

Inter‐laboratory validation of a harmonized PNH flow cytometry assay

Randomized multicenter noninferiority phase III clinical trial of the first biosimilar of eculizumab

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