Clinical Cell Analysis Research Articles

Frequencies of glycosylphosphatidylinositol (GPI)-deficient cells using high-sensitivity flow cytometry as per the 2018 ICCS/ESCCA consensus guideline in patients with hematologic malignancy, aplastic anemia, or cytopenia

ABSTRACT Objectives We examined the frequencies and sizes of glycosylphosphatidylinositol (GPI)-deficient cells as per the International Clinical Cytometry Society/European Society for Clinical Cell Analysis (ICCS/ESCCA) consensus guidelines for the high-sensitivity detection of GPI-deficient cells. Methods In 2018, the ICCS/ESCCA guidelines for the high-sensitivity detection of GPI-deficient cells were published. We evaluated frequencies and sizes of GPI-deficient red blood cells (RBCs), neutrophils, and monocytes as determined using the ICCS/ESCCA guidelines and Clinical and Laboratory Standards Institute (CLSI) guidelines in patients with a hematologic malignancy, aplastic anemia, or cytopenia. Results A total of 106 (38.7%) patients exhibited GPI deficiency in at least one blood cell lineage. GPI-deficient cells of one or more lineages were found in 62.7% of patients with a hematologic malignancy, 51.1% of patients with aplastic anemia, and 23.4% of patients with cytopenia. GPI-deficient monocytes were most frequently detected in all three groups. By population size, GPI-deficient clones (>1%) in monocytes were mostly detected in patients with a hematologic malignancy or aplastic anemia. Rare cells with GPI deficiency (<0.1%) in monocytes were most common among patients with cytopenia. Conclusion High-sensitive flow cytometry analysis including monocytes may be necessary for patients with a hematologic disorder.

Issue Highlights - November 2020.

Issue Highlights - November 2020.

Monitoring anti-B cell immunotherapies in autoimmune diseases: Go with the flow. A Position Paper of the Italian Society for Clinical Cell Analysis (ISCCA)

During the past decades autoimmune diseases have been usually treated with immunosuppressive drugs mostly active on T-Cell mediated responses. Only in recent years, with our extended knowledge of the pathogenic mechanisms of autoreactive disorders and the tremendous development of new therapeutic monoclonal antibodies, anti-B-Cell therapies have emerged as a new option for treating autoimmune diseases. The rationale for this changeover from T-Cell to B-Cell targeted therapies resides in the recently accumulated evidence of the role of B-Cells in the pathogenesis of autoimmune diseases and in the generation of tissue damage. Targeting memory and effector BCells may then disrupt the production of pathogenic antibodies, counteract the role of B-Cells in sustaining antigen presentation to T-Cells and block the synthesis of B-Cell activation cytokines. The anti-CD20 monoclonal antibody Rituximab was first introduced more than 20 years ago for the treatment of CD20+ chronic B-lymphoproliferative disorders, and was then successfully experimented in the treatment of an ever-increasing spectrum of autoimmune diseases. Newer anti-CD20 monoclonal antibodies have been introduced more recently, which vary in their biological effects. The need for laboratory indicators that may help the rational usage and follow-up of anti-CD20 treatments has now emerged, due to the high variability of individual response, to the markedly different outcomes in the various diseases and to the controversial role of pathogenic autoantibodies as indicators of disease activity. Flow cytometric (FCM) analyses to identify and enumerate the B-cell functional subsets in the peripheral blood have been developed in recent years. They can be used to assess the degree and the persistence of memory B-Cell depletion, the quality and the timing of B-Cell reconstitution, along with the highly sensitive FCM counting technique needed for the detection of extremely low cell levels. The long-term aim of this innovative approach is to provide clinicians with a tool for a safer and more rational usage of anti-CD20 agents.

Acceptable "Real-Life" Variability for Lymphocyte Counts by Flow Cytometry.

As part of quality assurance in laboratories (labs) offering clinical cell analysis by flow cytometry (FCM), cell counting precision and robustness are evaluated but international desirable ranges are still missing. The aim of this study was to provide desirable interlaboratory variability reference values. A prospective survey on monthly quality assessment was proposed to all French laboratories routinely performing lymphocyte subpopulation quantification, over one arbitrarily selected month (June 2017), regardless of instrument, counting system and quality controls used. Relative variabilities of the commercially available internal quality control (IQC) used locally were collected. Robust mean, standard deviation and CV were calculated on relative and absolute counts. Sixty-two labs participated, providing 91 sets of data on 82 instruments. All but three were enrolled in external quality assessment (EQA) and 46 in externalized IQA. The mean CV of five repeats ranged from 1.00 ± 0.33 for T cells to 4.78 ± 1.92 for NK cells and from 2.88 ± 1.46 to 5.87 ± 1.83 for relative and absolute counts, respectively. The precision correlated directly to the concentration of cells rather than the phenotype. Negligible differences were observed between IQC material: Multicheck™ (3.36 ± 1.30, n = 11); Immunotrol™ (3.62 ± 3.24, n = 21) and Statusflow™ (3.63 ± 1.87, n = 24) on CD4+ T cell, for example. Little difference was observed between counting systems such as Flowcount™ (4.55 ± 3.45, n = 19), Trucount™ (3.17 ± 1.40, n = 30) and the fully automated Aquios™ system (1.87 ± 0.75, n = 5) on single platforms, while dual platform CV was at 2.00 ± 0.58 (n = 2) on CD4+ T cell, as example. Robustness was measured on 21 ± 11 consecutive analyses of the same IQC material, providing CVs ranging from 4.45 ± 1.74 for T cells to 7.57 ± 2.19 for NK absolute counts. Average residuals calculated from the different low count IQC samples for CD4 T cells (median value below 200 cells/μl) were below 10 cells/μl, demonstrating their robustness for medical decisions. Real life variabilities in cell counting are directly related to the cell concentration and not to their phenotype. Desirable ranges within three SD are proposed according to different cell levels, based on 62 labs, different IQC material and systems. © 2018 International Clinical Cytometry Society.

Abstracts for ESCCA 2018 Conference: At the Shore of Future Cytometry September 13-15, 2018, Valencia, Spain.

We are pleased to present abstracts from ESCCA 2018: At the Shore of Future Cytometry. This year, the European Society for Clinical Cell Analysis (ESCCA) organised its annual Conference over 3 days in Valencia, Spain. Four workshops for a limited number of participants - dedicated to Cytometry Analysis Tools, Cytometry of Microparticles, Cytometry in Veterinary and Animal Sciences and CLL MRD - as well as ESCCA certification exams took place before the Conference. The Conference itself included four Education Programmes from which the participants had to choose in advance: (i) Good Cytometry Practices; (ii) Advanced Hemato-Oncology; (iii) Clinical Diagnostic Cytometry and (v) Advanced Immunology. A group of experts in these fields coordinated, presented, and discussed these topics with the attendees. The Education Programme sessions were alternated with plenary keynote lectures and other sessions, such as an interactive voting session on interesting cases, a Round Table discussion and a session jointly organised with the Iberian Cytometry Society (SIC) on The future of flow cytometry in Europe. Finally, 125 accepted abstracts were scheduled as poster presentation. Three abstracts that have received the highest ranking from the abstract reviewer committee have been nominated for the ESCCA Best Oral Award, and were presented in one of the plenary sessions. In addition the next best 18 best abstracts were presented during 3 poster walks. The abstracts of the keynote lectures and the poster abstracts are published in this issue of Clinical Cytometry. The abstracts are available as Online Supporting Information.

Use FlowRepository to share your clinical data upon study publication.

A fundamental tenet of scientific research is that published results including underlying data should be open to independent validation and refutation. Data sharing encourages collaboration, facilitates quality and reduces redundancy in data production. Authors submitting manuscripts to several journals have already adopted the habit of sharing their underlying flow cytometry data by deposition to FlowRepository-a data repository that is jointly supported by the International Society for Advancement of Cytometry, the International Clinical Cytometry Society and the European Society for Clinical Cell Analysis. De-identification is required for publishing data from clinical studies and we discuss ways to satisfy data sharing requirements and patient privacy requirements simultaneously. Scientific communities in the fields of microarray, proteomics, and sequencing have been benefiting from reuse and re-exploration of data in public repositories for over decade. We believe it is time that clinicians follow suit and that de-identified clinical data also become routinely available along with published cytometry-based findings. © 2016 International Clinical Cytometry Society.

Paroxysmal Nocturnal Hemoglobinuria Testing by Flow Cytometry: Brief Overview for Clinicians

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare, acquired hematopoietic stem cell disorder, caused by a somatic mutation in the PIG-A gene. This mutation is responsible for the synthesis of glycophosphatidylinositol (GPI) anchor that attaches a number of proteins to the cell surface. The mutant gene leads to partial deficiency or absence of all proteins normally linked to the cell membrane by GPI anchor. The primary clinical manifestations of PNH are complement mediated hemolytic anemia, thrombosis in atypical locations and blood cytopenias. Flow cytometry has become the gold standard method for PNH clone detection due to its high sensitivity and specificity and due to the ability to examine multiple GPI-linked proteins on red and white blood cells surface. It is the method of choice for the detection of very small PNH clones in subclinical PNH that often accompanies aplastic anemia and other bone marrow disorders. PNH clone detection traditionally involves the analysis of CD55 and CD59 on red and white blood cells. Other markers such as CD14, CD16, CD24, CD66b and CD157 are suitable to detect GPI-linked proteins in the surface of granulocytes and monocytes. The most useful reagent to assess white blood cell PNH clones is Fluorescent Aerolysin (FLAER) which is a mutated form of proaerolysin conjugated with a fluorochrome. Its advantage in PNH clones detection is due to the ability to bind directly to the glycan portion of the GPI anchor. Flow cytometry is a sophisticated method and a useful tool for clinical cell analysis. However, PNH is a clinical diagnosis and flow cytometric results should always be interpreted with respect to clinical manifestations and other laboratory findings.

Toxicological Studies of 212Pb Intravenously or Intraperitoneally Injected into Mice for a Phase 1 Trial.

Faced with the novelty of a 212Pb-labeled monoclonal antibody (mAb) for clinical translation, concerns were expressed by the Food and Drug Administration (FDA) regarding 212Pb prematurely released from the mAb-chelate conjugate. The objective of this study was to simulate the worst case scenario of such a failure. Groups of Balb/c mice (n = 9–20) were administered 212Pb by intraperitoneal (0.0925–1.85 MBq) or intravenous (0.0925–1.11 MBq) injection and then euthanized at 7 or 90 days to assess acute or chronic effects. Weights were recorded prior to injection of the 212Pb and at the end of the observation periods. Blood samples were collected for clinical chemistry and blood cell analysis. Thirty tissues were harvested and formalin fixed for histopathological examination. Treatment related effects of the 212Pb were observed in the bone marrow, spleen, kidneys and the liver. Histological alterations in these organs were considered mild to moderate, indicating low grade toxicity, and not considered severe enough to affect function. This data was presented to the FDA and determined to be acceptable. The clinical trial with 212Pb-TCMC-trastuzumab was approved in January 2011 and the trial opened at the University of Alabama at Birmingham (UAB) in July.

Consensus guidelines for myeloma minimal residual disease sample staining and data acquisition.

Flow cytometric (FC) detection of minimal residual disease (MRD) in multiple myeloma (MM) is prognostic and predictive of response to therapy. Therefore, standardization of FC MM MRD testing is vital to ensure better and uniform assessment of response to therapy and clinical prognostication. The International Clinical Cytometry Society and European Society for Clinical Cell Analysis, recognizing the need for standardized FC approaches, organized a working group to develop consensus guidelines on good clinical practice in FC MM MRD. Consensus guidelines are presented for specimen quality, staining process, reagent combinations, and the data acquisition process, all key factors in achieving high quality FC MM MRD testing. A group of eight flow cytometrists currently performing FC testing in MM evaluated available literature on FC MM MRD testing. A document presenting best practice was developed and reviewed in successive rounds until consensus was reached. The consensus on best practice for detection of MRD in MM is that CD38, CD138, and CD45 are analyzed in combination with CD19, CD56, CD27, CD81, and CD117. Consensus guidelines on acceptable specimen quality, staining procedures, panel design, and data acquisition were developed.

Abstracts for ESCCA 2014 Conference: Exploring New Frontiers in Flow Cytometry September 14–17, 2014, Lisbon, Portugal

We are pleased to present abstracts from ESCCA 2014: Exploring New Frontiers in Flow Cytometry. The meeting, a joint production of the European Society for Clinical Cell Analysis (ESCCA) and the Iberian Society for Cytometry (SIC), has been of major importance for education and scientific exchange in basic, translational and clinical applications in cytometry; in particular flow cytometric applications in Hematology, Immunology, Cancer and Cell Biology. The meeting took place in Lisbon, Portugal on September 14–17, 2014, and was chaired by Dr. Maria Arroz (Lisbon, Portugal).The abstracts are available as Online Supporting Information.

Different distribution and activation degree of th17 cells in peripheral blood in patients with osteoarthritis, rheumatoid arthritis and healthy donors: preliminary results of the magenta clicao (clinical cell analysis in osteoarthritis) study

Background: Osteoarthritis (OA) as well as rheumatoid arthritis (RA) are chronic diseases associated with joint destruction and mobility impairment. Data about changes in immune system in RA and the significant role of T cells, primarily Th17 cells, are now well recognised. On the other hand data about immunological profile in OA are limited, because OA has long been regarded in the past as a no inflammatory disease. Our study aim to measure the distribution and the activation degree of CD4+ Th17 in peripheral blood of OA, RA and age-matched healthy controls. Methods: Patients with established diagnosis of RA according to ACR/EULAR 2010 criteria, knee or hip OA according to ACR criteria and volunteers healthy blood donors were eligible. Other inclusion criteria were a DAS28 between 3.2 and 5.1 or a WOMAC Likert score more than 50, respectively. Exclusion criteria were the presence of other autoimmune diseases, tumours or secondary osteoarthritis. Finally no changes in rheumatologic drugs were allowed from 3 months before enrolment. Multichannel flow cytometry was used for T cells subpopulation distinguishing and quantification using monoclonal antibodies against CD3, CD4, CD8, CCR6, CD38, CXCR3 and HLA DR. Participants were informed about the aim of the project and gave their written consent. The project was accepted by the Local Ethic Committee. Results: We analysed blood samples of 91 subjects (75 females, 16 males). 15 Patients with well-defined RA, 56 with hip or knee OA and 20 healthy controls. Mean age was 45 ± 2.7 years old (p > 0.05 between groups). Blood samples from the RA patients had significantly higher count of CD4+CD38+DR+ (activated CD4 T cells) and Th17 (CCR6+CXCR3-) cells as compared to OA patients and control group (Figure) (P < 0.01). Furthermore the samples from the OA patients had an higher percentage of activated CD4 T cells and Th17 cells as compared to control group (P < 0.05) Conclusion: According to the latest view of OA disease pathogenesis, our preliminary results give support to the hypothesis that OA may also (like RA) be a disease with an immunological/inflammatory involvement. In fact it seems that there is a quantitative but non qualitative difference in Th17 cells profile, including the expression of activation markers, between RA and OA.

Remote access technology offers tremendous possibilities to the cytometry community

THE opportunity for low cost, high quality training and supervision is an attractive one to most scientists in the cytometry field. Many have had little if any training, no expertise based locally, or may not have the funds to attend regional or international training courses. In addition, rather than general cytometry they may require assistance with a particular application, which could be accomplished in a number of hours through support from an expert. Separately, shared resource scientists, also known as core facility staff, are generally responsible for the performance and upkeep of multiple complex instruments, as well as monitoring, training etc. Some acquisitions or cell sorts may take hours for a single run, and with limited staff the scientist is usually multitasking on an hourly basis. Also, if users of the core facility are having problems and staff is unavailable, valuable samples or time may be lost. There is potential for error at many steps of flow cytometry, including sample preparation, experimental design and fluorochrome selection, instrument set up, compensation, gating, analysis, and interpretation. External/offsite tracking may improve monitoring, and perhaps limit such errors. Also interand intra-instrumentation variations can lead to different results for the same sample. A means of unifying performance, setup, and interpretation is vital, in particular with multicenter collaborations. Peer-review of results and analysis, as well as hierarchical approval systems, provides some solutions, but further stream-lining is clearly beneficial. Remote access technology for cytometry (or remote cytometry) may offer solutions to many such issues and can be performed at very low costs to the institution. There is potential for such systems to facilitate instrumentation monitoring, control equipment in containment areas, speed up vendor service, improve training courses, increase collaborations, and further promote cytometry globally.

Accreditation of flow cytometry in Europe

ISO 15189 has been introduced to enable any clinical laboratory, irrespective of geographic location, to be accredited against internationally recognized standards and therefore facilitate direct international comparison of laboratories. Together with increasing use of ISO 15189 for standardization and competition purposes, often triggered by demands of patients and clinicians, clinical flow cytometry laboratories are becoming increasingly challenged to introduce compliant quality management systems. Whilst in most countries, ISO 15189 accreditation is not yet compulsory, there is increasing evidence to suggest that the implementation of this standard is growing. As a result, the European Society of Clinical Cell Analysis (ESCCA) has analysed the impact of accreditation in clinical flow cytometry laboratories. It found, through a discussion forum, that staff qualification, adaptation of multicolour antibody panels, and implementation of a comprehensive quality system (including quality assessment) have been identified as major challenges.

Abstracts of the 12th European Society for Clinical Cell Analysis (ESCCA) Euroconference September 12–15, 2012 Budapest, Hungary

Abstracts of the 12th European Society for Clinical Cell Analysis (ESCCA) Euroconference September 12–15, 2012 Budapest, Hungary

Sensitive, robust and automated protein analysis of cell differentiation and of primary human blood cells by intact cell MALDI mass spectrometry biotyping

Intact cell mass spectrometry biotyping, a collection of methods for classification of cells based on mass spectrometric fingerprints, is an established method in clinical and environmental microbiology. It has recently also been applied to the investigation of mammalian cells including primary blood cells and cultured cells. However, few automated procedures suitable for higher throughput and little analytical standardization of mammalian biotyping approaches have been reported so far. Here, we present a novel automated method that robustly classifies as few as 250 cells per spot. Automatically acquired cell fingerprints from cultured and primary cells show high technical (R > 0.95) and biological reproducibility (R = 0.83-0.96), with a median peak variance below 12 %. Ion suppression is shown to be a major concern at higher cell numbers and needs to be carefully monitored. We demonstrate that intact cell mass spectrometric signatures of different cell lines start to resemble each other at higher trifluoroacetic acid (TFA) concentrations and that therefore low concentrations of TFA in the matrix solution are preferred. We show that in vitro differentiation of HL-60 cells into a neutrophil-like phenotype can be rapidly and robustly monitored. We utilize the method for global analysis of person-to-person differences in mass spectral signatures of intact polymorphonuclear neutrophils and monocytes obtained from healthy volunteers. Our data suggest that automated MALDI mass spectrometry cell biotyping could be a useful complementary approach in clinical cell analysis.

Affinity and enzyme-based biosensors: recent advances and emerging applications in cell analysis and point-of-care testing

The applications of biosensors range from environmental testing and biowarfare agent detection to clinical testing and cell analysis. In recent years, biosensors have become increasingly prevalent in clinical testing and point-of-care testing. This is driven in part by the desire to decrease the cost of health care, to shift some of the analytical tests from centralized facilities to "frontline" physicians and nurses, and to obtain more precise information more quickly about the health status of a patient. This article gives an overview of recent advances in the field of biosensors, focusing on biosensors based on enzymes, aptamers, antibodies, and phages. In addition, this article attempts to describe efforts to apply these biosensors to clinical testing and cell analysis.

Abstracts of the 11th Euroconference on Clinical Cell Analysis. Dublin, Ireland, 14–17 September 2011

Abstracts of the 11th Euroconference on Clinical Cell Analysis. Dublin, Ireland, 14–17 September 2011

Introduction to case reports

Clinical cases play a pivotal role in the daily routine of a clinical laboratory. The screening and diagnosis of the different types of samples sent to the flow cytometry laboratory represent a challenge to those in charge of processing, acquiring, and analyzing them. The fact that we are dealing with biological specimens make them unique and, as we all know, many times they do not replicate the phenotype of the entities described in the literature. According to these premises, the European Working Group for clinical cell analysis decided to promote a plenary session at their 9th Annual Conference in St. Etienne, France, September 2009, which I have chaired: Plenary 9, quiz in leukemia/lymphoma immunophenotyping. Four speakers from different parts of the world, France, Hungary, USA, and Australia, were invited to present five cases. The topics covered included two cases of nonhematological diseases, two concomitant hematological diseases affecting different lineages, an acute leukemia in a newborn, and a haemophagocytic syndrome in the presence of grossly abnormal T-cell population. The scope of this exercise was to highlight cases that are rare but can happen in everyone's routine. This was a participated session and as such we have decided to extend it to a larger audience by submitting some of the cases to this journal.

Clinical Cytometry in Europe, 2010

This year, the European Society for Clinical Cell Analysis (ESCCA) and the Iberian Society for Cytometry (SIC) jointly organized in Valencia (Spain) the 10th Euroconference on Clinical Cell Analysis in conjunction with the 6th European Clinical Cytometry Course. As of September 1, 385 paying participants from 45 countries had signed up for the 3-day conference and 220 for one of six 2-day courses, respectively. The 6th European Course offered lectures, debates, practical exercises, and data analysis sessions on the six themes that were decided this year: immunophenotyping of leukemias and lymphomas, immunodeficiency, integrated cytomics, cellular biomarkers of cancer, functional cytometry, and paroxysmal nocturnal hemoglobinuria. The scientific programme of the 10th Euroconference included interdisciplinary topics favored to confront and exchange common and specific approaches. Besides the keynote lecture and the presentation of the 2009–2010 best paper in Clinical Cytometry, an extensive program with plenary and parallel lectures was provided. Six proferred abstracts were selected for oral presentation. Morethan 100 posters were on display, whereas a further 10 were highlighted during two “poster walks.” All invited presentations, selected proferred abstracts, and abstracts that were shown as posters are shown, in abstract form, in this issue of Clinical Cytometry (see Table 1). From 2005 onward, the Euroconferences make use of a so-called interactive voting system (IVS) that enable the participants to cast their votes on statements and questions prepared by the moderators of specific sessions. This year, the IVS served to structure debates on the detection of circulating tumor cells (cell-based vs. molecular-based), and on the limits of flow cytometry in rare event detection, focused on leukemia/lymphoma immunophenotyping, and circulating endothelial cells. The 7th European Clinical Cytometry Course, the 11th Euroconference on Clinical Cell Analysis and the Joint Meeting of ISAC (International Society for Advancement of Cytometry), International Clinical Cytometry Society (iCCS), Irish Society for Cytometry (ICyS), and ESCCA will be held in Dublin, Ireland, from September 13–14, 2011 (Joint Meeting and European Course) and from September 14–17, 2011 (Euroconference), under the co-chairmanship of Alfonso Blanco and Jan Gratama. For details, see www.cytometry2011.eu. As scientific organizing committee, we are very pleased for the financial support of our 23 industrial partners, and for the financial and logistical support provided by the Generalitat Valenciana, the University of Valencia, the Centro de Investigación Principe Felipe, the Valencian Council of Culture, and the International Federation of Clinical Chemistry and Laboratory Medicine. In particular, we are very grateful to the International Clinical Cytometry Society (iCCS) and the Clinical Cytometry journal to publish the abstracts of the Conference.

Abstracts from the 10th Euroconference on Clinical Cell Analysis of the European Society for Clinical Cell Analysis (ESCCA) and the Iberian Society for Cytometry (SIC)

Abstracts from the 10th Euroconference on Clinical Cell Analysis of the European Society for Clinical Cell Analysis (ESCCA) and the Iberian Society for Cytometry (SIC)