Detection Of Paroxysmal Nocturnal Hemoglobinuria Clones Research Articles

Immunophenotypic assessment of PNH clones in major and minor cell lineages in the peripheral blood of patients with paroxysmal nocturnal hemoglobinuria.

Flow cytometric immunophenotyping is essential for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). Most cases have easy to interpret flow cytometry profiles with red cells, neutrophils and monocytes showing complete deficiency of glycophosphatidylinositol (GPI) linked antigen expression. Some cases are more challenging to interpret due to the presence of multiple populations of PNH cells and variable levels of GPI antigen expression. We studied 46 known PNH patients, many with complex immunophenotypic profiles using a novel, single tube, multi-parameter 7-color immunophenotyping assay that allowed simultaneous detection and assessment of PNH clones within multiple lineages of peripheral blood leucocytes. Red cell PNH clones were also assessed in total and immature (CD71+) components by CD59 expression. For individual patients, total PNH clones in each cell lineage were highly correlated. Monocytes, eosinophils and basophils showed the highest proportions of PNH cells. Red cell PNH clones were typically smaller than monocyte and neutrophil PNH clones. In most cases, PNH clones were detectable in minor leucocyte populations where multiple populations of PNH cells were present, variability in the proportions of type II and type III cells was seen across different cell lineages, even though total PNH clones remained similar. This study shows that PNH patients with multiple PNH clones do not always display the same abnormality across all cell lineages routinely tested. There is no simple explanation for this but is likely due to a combination of complex molecular, genetic and biochemical dysfunction in different blood cell types.

Frequency of Paroxysmal Nocturnal Hemoglobinuria in Patients with Lymphoma

Objective: To investigate the frequency of paroxysmal nocturnal hemoglobinuria (PNH), an acquired clonal hematopoietic stem cell disease resulting in complement-mediated hemolysis, in patients with lymphoma by flow cytometry.Methods: Fifty patients with lymphoma who were admitted to the hematology clinic, newly diagnosed and not yet treated were included in this study conducted in 2014. The presence of PNH clones was checked by FLAER flow cytometry method in peripheral blood samples. FLAER is a non-hemolytic fluorescently labeled inactive toxin aerolysin method that can detect up to 0.5% of PNH cells instead of bacterial toxin aerolysin, which binds to RBCs via the GPI anchor and initiates hemolysis for PNH screening or PNH clone detection. With this technique, PNH clones in all hematopoietic cell lines can be detected in an assay.Results: PNH clone was observed over 10% in two patients, one male and the other female. However, no hemolysis was found in patients with PNH clones. The lymphoma subtypes of the patients with positive PNH clone were B-cell small-cell lymphocytic lymphoma in the male patient and primary splenic lymphoma in the female patient.Conclusion: PNH or PNH-like disorders accompanying hematological malignancies, especially lymphomas, are not very common in the literature. There is a need to elucidate the relationship between hematological malignancies and PNH with the help of more advanced molecular techniques.

ANALYSIS OF BASELINE CHARACTERISTICS, DISEASE BURDEN AND LONG-TERM FOLLOW-UP OF 167 PATIENTS WITH BRAZILIAN PAROXYSMAL NOCTURNAL HEMOGLOBINURIA - ANOTHER NATURAL HISTORY

Paroxysmal nocturnal hemoglobinuria (PNH) may occur as classical hemolytic disease or a small PNH clone found in a patient with bone marrow failure. To describe clinical features and long-term follow-up of 167 patients with PNH and demonstrate differences between disease categories. Multiparametric flow cytometry performed on 1025 patients referred from Jan/2000 to Dec/2019 found 167 (16.3%) confirmed PNH clone, 87M/70F. Clinical characteristics at first visit, and laboratory results, incidence of thrombosis, treatment and outcome during follow-up visits were considered for statistical analysis. Most patients (89.2%) had hypocellular bone marrow at diagnosis; 55(32.9%) developed hemoglobinuria, and 22(13.2%) developed thrombosis during monitoring. Mean age at aplasia diagnosis 28.4 years (range 5.6 - 71.2), and mean age at PNH clone detection 31.0 years (7.2 - 71.3 years). Clonal evolution occurred in 77 (46.1%) patients at a median of 4 years (range 6-281 months) after the onset of cytopenia. The cohort was divided into 15 classic PNH, 55 hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 definitive bone marrow disease with minor PNH clones - subclinical PNH (sc-PNH). Subclinical patients had lower erythrocyte (2.0% vs 24.0% vs 57.8%) and granulocyte PNH clones (11.7% vs 58.8% vs 81.2%) than PNH/AA and classic PNH, respectively. LDH, reticulocytes, absolute neutrophils and bone marrow cellularity were all lower in sc-PNH than in the hemolytic group. Pearson's analysis showed a statistically significant correlation (p < 0.001) between PNH granulocyte clone and RBC clone (0.697), LDH (0.602), absolute neutrophils (0.310), platelet count (0.430), absolute reticulocytes (0.546), total bilirubin (0.330), and bone marrow biopsy (0.435). PNH granulocyte clone greater than 50% was found in 16/22 thrombotic patients. Mean granulocyte clone was 89.1% (3.4%-99.2%) versus 12.3%(0.1%-34.0%), p < 0.001, and erythrocytes 28.4% (3.4-99.8) versus 0.7% (0.1-14.0) in thrombotic versus the remaining 145 patients. Treatment was highly heterogeneous during this 20-year follow-up: immunosuppressive therapy with CSA and/or thymoglobulin was given to 72 patients in the subclinical group (73.1%) and 45 (64.3%) in the hemolytic group; 52 patients received HSCT (31.1%) of which 40 in sc-PNH and 11 in PNH/AA; a total of 19 patients received Eculizumab after 2008 and 3 patients never received treatment. Incidentally, there were 17 thrombotic events in hemolytic patients (38.6%) diagnosed by 2007, compared to only five events (19.2%) in patients diagnosed after 2008. The average follow-up period was 74 months (1 - 330 months), with 31 deaths in total (80.4% OS). Sepsis was the leading cause of sc-PNH mortality (16/18, 88.8%), and thrombosis in both hemolytic groups (11/13, 84.6%). Log rank test shows similar survival when comparing patients with diagnosis until and after 2007 (p = 0.04). this study suggests clonal evolution in the long-term follow-up of aplastic patients and confirms the observation that high levels of PNH and LDH clones are associated with life-threatening hemolysis and thrombosis, while small PNH clones and young age are associated with the subclinical form of the disease. Although without statistical significance, the cumulative incidence of thrombosis in the group diagnosed after 2008 appears to be lower than the previous period, perhaps due to the routine use of Eculizumab.

Analysis of baseline characteristics, disease burden and long-term follow-up of 167 patients with Paroxysmal Nocturnal Hemoglobinuria at a single center in Brazil

Paroxysmal nocturnal hemoglobinuria (PNH) can occur as a hemolytic form or small PNH clone found in a patient with bone marrow failure. MethodsDescribe Brazilian retrospective PNH cohort and identify the impact of disease burden on long-term follow-up. Results167 patients, mean age at diagnosis 28.4 (7.1–71.2 years), four years mean interval between onset of cytopenia/aplasia diagnosis and PNH clone detection. Patients were divided into 15 Classic PNH, 55 Hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 Subclinical PNH (sc-PNH). Hypocellular bone marrow was found in 89.2%; 55 had hemoglobinuria and 22 thrombosis during monitoring. WBC PNH clone correlated with RBC PNH clone, LDH and cytopenia. Subclinical patients had lower median lower RBC clone (2.0% vs 24.0% vs 57.8%) and WBC clone (11.7% vs 58.8% vs 81.2%) than PNH/AA and Classic PNH, respectively. PNH granulocyte clone was 89.1% in thrombotic patients. Ten-year overall survival 80.4% and mortality in transplanted patients 9.6%. Sepsis was mortality cause in subclinical PNH (16/18, 88.8%), and thrombosis in hemolytic PNH (11/13, 84.6%). ConclusionLarge PNH clones and LDH burden were associated with increased hemolysis and thrombosis risks, while young patients were associated with small PNH clones and subclinical form of the disease. Knowledge of the patient profile, the low risk associated with HSCT, and the use of long-term IST may be instrumental in the clinical management of PNH in restricted-resources countries.

Platelet counts on peripheral blood and Mean Platelet Volume as markers of clinical severity in Sickle Cell Disease

Paroxysmal nocturnal hemoglobinuria (PNH) can occur as a hemolytic form or small PNH clone found in a patient with bone marrow failure.Describe Brazilian retrospective PNH cohort and identify the impact of disease burden on long-term follow-up.167 patients, mean age at diagnosis 28.4 (7.1–71.2 years), four years mean interval between onset of cytopenia/aplasia diagnosis and PNH clone detection. Patients were divided into 15 Classic PNH, 55 Hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 Subclinical PNH (sc-PNH). Hypocellular bone marrow was found in 89.2%; 55 had hemoglobinuria and 22 thrombosis during monitoring. WBC PNH clone correlated with RBC PNH clone, LDH and cytopenia. Subclinical patients had lower median lower RBC clone (2.0% vs 24.0% vs 57.8%) and WBC clone (11.7% vs 58.8% vs 81.2%) than PNH/AA and Classic PNH, respectively. PNH granulocyte clone was 89.1% in thrombotic patients. Ten-year overall survival 80.4% and mortality in transplanted patients 9.6%. Sepsis was mortality cause in subclinical PNH (16/18, 88.8%), and thrombosis in hemolytic PNH (11/13, 84.6%).Large PNH clones and LDH burden were associated with increased hemolysis and thrombosis risks, while young patients were associated with small PNH clones and subclinical form of the disease. Knowledge of the patient profile, the low risk associated with HSCT, and the use of long-term IST may be instrumental in the clinical management of PNH in restricted-resources countries.

Persistence of PNH Clones over Time: Insights from the Mid-Term Analysis of the French Nation-Wide Multicenter Prospective Observational Study

Introduction: Since the publication of the International Guidelines (Borowitz, 2010; Illingworth, 2018), no study has assessed the long-term evolution of paroxysmal nocturnal hemoglobinuria (PNH) clones using high-resolution flow cytometry. The sole evaluation, performed by Sugimori et al, using a 2-color flow cytometry test, showed the disappearance of PNH clones in 24% of patients with bone marrow (BM) failure over 5 years (Sugimori, 2009). A diagnostic practice harmonization using high-resolution flow cytometry has spread in France since 2013 through an on-going inter-laboratory comparison program (Debliquis, 2015). Thus, our HPNAFC group has been able to initiate a French nation-wide multicenter prospective observational study. Objective: We aimed to assess the evolution of PNH clones over a long term period using mostly high sensitivity test, which is required for minor clone assessment, with validated flow cytometry data. Methods: All patients of any age with a PNH clone or GPI-deficient cells ≥0.01%, newly- or previously-diagnosed, detected in France from February 29th 2016, could be included in this Observatory, provided that the center had validated a PNH flow cytometry quality control. For each patient, the baseline assessment was always considered as the initial PNH clone detection, even if it occurred before the initiation of the Observatory. Thus, this strategy allowed the collection of cases with long-term follow-up. Referent cytometrist of each center included patients in the e-CRF available on the HPNAFC website providing clinical and biological information as well as flow cytometry raw data files. This study was approved by the national research ethics board. Results: As of July 15th 2019, 48 participating flow cytometry laboratories across France have enrolled 356 patients with a PNH clone or GPI-deficient cells ≥ 0.01%. All cases have been carefully reviewed by the 2 principal investigators, who both thoroughly re-examined flow cytometry data and the e-CRF filling that led to the update of roughly one third of the submitted files. This enabled the validation of 200 patients at diagnosis, the remaining 156 being ongoing. One hundred and three of the 200 validated patients displayed at least one follow-up point (more than 3 months apart from the diagnosis) with a clone size determined at diagnosis (see flow chart figure 1A). For 8/103 patients, exchanges with centers are still ongoing. Thus, we were able to assess the evolution of PNH clones of 95 patients with 2 [range: 1-8] follow-up points over a period of 4.1 [0.3-14.2] years, corresponding to 200 validated follow-up points. The patient median age at diagnosis was 40 years old [10-85] with 3 pediatric cases (&lt;18y) and a M/F sex ratio of 0.86. Diagnoses were made between 2003 and 2018 with clinical information available in 97% of cases: 19 patients (20%) had hemolytic anemia and most patients (n=73, 77%) displayed BM failure including aplastic anemia (n=62), myelodysplastic syndrome (n=7) and unexplained cytopenia(s) (n=4). No case of thrombosis was included. All patients with hemolytic anemia showed an increasing clone size over time including the two who were not treated with eculizumab (Figure 1B; median size at diagnosis on neutrophils: 81.3% vs median size over 5 years: 96.3%). The median clone size at diagnosis for patients with BM failure was 1.5% on neutrophils with a very wide range [0.01-97.87], almost half of them being less than 1%. When comparing the diagnosis point with the latest follow-up point, PNH clone size increased in 37 patients, decreased in 16 of them and remained stable in 20 cases (Figure 1C, D). Nine of the 37 patients reached a PNH clone size above 50% and 4 of them received a treatment by eculizumab in a median delay of 5 years [1.5-6.0]. Interestingly, no patient showed spontaneous disappearance of PNH clones, pending the use of a high-resolution flow cytometry test. The only five patients with undetectable PNH clones (Figure 1D, red lines) were those who underwent BM transplantation. Conclusion: This multicenter study based on robust flow cytometry analysis showed no disappearance of PNH clones, including minor ones, over a long period of time, regardless of the clinical manifestations, except for patients who underwent BM transplantation. Moreover, PNH clone size increased in half of patients with BM failure, justifying a long term PNH clone size monitoring, even in these patients. Figure 1 Disclosures Le Garff-Tavernier: Alexion: Consultancy, Honoraria. Pruvot Debliquis:Alexion: Honoraria; Takeda: Honoraria; Pfizer: Honoraria; Gilead: Honoraria; Genzyme: Honoraria. Socie:Alexion: Consultancy. Peffault de Latour:Alexion: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding. Drenou:Alexion: Consultancy, Honoraria. Wagner Ballon:Alexion: Consultancy, Honoraria.

FLAER Based Assay According to Newer Guidelines Increases Sensitivity of PNH Clone Detection.

Flow cytometry has become 'gold standard' for detecting abnormal clones in paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia (AA) and myelodysplastic syndrome (MDS). This pilot study was conducted in 2015 with a primary aim to evaluate the utility of single tube fluorescent aerolysin (FLAER) based testing and its comparison with two tubes non-FLAER based testing (CD55, CD59, CD24 and CD66b) in detecting abnormal PNH clones in these newly diagnosed cases. The secondary aim was an attempt to distinguish PNH from AA/MDS cases associated with PNH clones based on clinical, laboratory features and clone size at diagnosis. In this study, the abnormal PNH clones were detected using a single tube FLAER based testing and two tubes non-FLAER based testing in all cases of PNH (n = 12), healthy subjects (n = 18) and AA/MDS with PNH clone (n = 9) and compared with clinical and laboratory features at diagnosis. The receiver operator curve (ROC) analysis defined the optimal cut-offs for FLAER in granulocytes (> 0.7%) and monocytes (> 0.9%). There was significant positive correlation between FLAER and non-FLAER based testing in these cells (r > 0.3 and p < 0.05). FLAER based testing helped us in picking up smaller clones which were missed by latter technique in four patients thereby increasing its sensitivity and also technically proved to be cost-effective (Rs. 1800 vs. Rs. 2100). Even in PNH patients, the clone size was slightly higher by using FLAER when compared to non-FLAER based antibodies panel. The clone size of monocytes was always higher than granulocytes in both PNH and AA/MDS groups. Bone marrow cellularity and mean size of granulocytes and monocytes clone at diagnosis showed a striking statistically significant 'p' value of < 0.0001 between these groups. In this pilot study, a single tube FLAER based PNH testing had improved clone detection in all cases of PNH, AA/MDS with PNH clones. The clone size was > 30% in majority of PNH cases whereas in AA/MDS, it was usually < 10% at diagnosis. Hence this newer technique not only increased the sensitivity of PNH clone detection but also proved to be cost-effective.

Sensitive and accurate identification of PNH clones based on ICCS/ESCCA PNH Consensus Guidelines-A summary.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder resulting from the somatic mutation of the X-linked phosphatidyl-inositol glycan complementation Class A (PIG-A) gene. Depending on the severity of the mutation in the PIG-A gene, there is a partial or absolute inability to make glycosylphosphatidyl-inositol (GPI)-anchored proteins including complement-defense structures such as CD55 and CD59 on RBCs and WBCs. Flow cytometric detection of PNH clones has become the gold standard and has played an increasingly important role in the diagnosis, monitoring, and clinical management of patients with PNH. Recently, a 4-part set of Consensus Guidelines have been published by flow experts in the field to address the key assay-specific considerations for the identification of PNH clones in RBC and WBC, how to report such data and a full validation document for the assays described. Below, we have summarized the most significant aspects of this International effort.

Issue Highlight - July 2018.

Issue Highlight - July 2018.

The Monoclonal Anti-CD157 Antibody Clone SY11B5, Used for High Sensitivity Detection of PNH Clones on WBCs, Fails to Detect a Common Polymorphic Variant Encoded by BST-1.

CD157, encoded by BST-1, has been described as a useful flow cytometric marker for the analysis of paroxysmal nocturnal hemoglobinuria (PNH) as it is a glycosylphosphatidylinositol (GPI)-linked molecule highly expressed on normal monocytes and neutrophils. We and others observed isolated CD157 signal dropouts during intended PNH analysis. We hypothesize that these negative populations occur due to an antibody failure. To investigate the reason for this finding, we compared two different anti-CD157 antibody clones for PNH analysis. We sequenced BST-1 of CD157-negative probands that are not suffering from PNH and expressed wild type and a discovered variant form of CD157 in HEK293 cells. We compared the binding patterns of two different anti-CD157 antibody clones (SY11B5 and RF3) by flow cytometry and western blot analysis. When sequencing two CD157-negative probands we detected a common SNP (p.Arg145Gln) in exon 3 of BST-1. We found that only anti-CD157 antibody clone RF3 but not the more widely used clone SY11B5 was able to detect both, the wild type and the variant form of CD157 in flow cytometric experiments. The failure of anti-CD157 antibody clone SY11B5 to detect a common SNP can explain some CD157-negative cytometric data. This provides crucial knowledge for laboratories performing PNH analyses as such results can potentially lead to false-positive PNH interpretation. Our results confirm the importance of published PNH guidelines. © 2018 International Clinical Cytometry Society.

Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation

Paroxysmal nocturnal hemoglobinuria (PNH) is an extremely rare acquired disorder. The aim of this study was to investigate the demographics, clinical manifestations, and outcomes of PNH patients in southern Taiwan. Data on PNH patients diagnosed over a 30-year period (1985–2015) were retrospectively collected from four tertiary medical centers in southern Taiwan. Blood samples were collected for hematologic panel testing and flow cytometry detection of PNH clones. Radiologic studies were performed to assess the frequency of complications. Twenty-four patients were enrolled in this study. The median duration of disease in the study participants was 10.8 years. The median granulocyte PNH clone size was 92.5% (range, 1.3%–99.8%), and the median lactate dehydrogenase (LDH) level was 2920.2 ± 1462.0 IU/L. The incidence of thromboembolism and impaired renal function was 16.7% and 29.2%, respectively. The primary treatment strategies included steroids (79.2%), androgens (42.0%), eculizumab (33.3%), immunosuppressants (16.7%), and anticoagulants (4.2%). In eight patients treated with eculizumab, there was a marked reduction in the LDH levels of 14.89-fold–1.63-fold that of the upper limit of normal; seven patients exhibited decreased transfusion requirements. Twenty-one patients were alive with regular follow-up at the time of publication. Our study demonstrates that PNH patients in southern Taiwan may exhibit different clinical characteristics and outcomes relative to patients in other countries. There was a trend toward a greater PNH granulocyte clone size, which may lead to more hemolysis. In our study, the percentage of patients with impaired renal function, but not the percentage of patients with thrombotic events, was higher than values reported worldwide and in the observational cross-sectional International PNH Registry. More large-scale studies with comprehensive data on the clinical response to different treatments are needed.

Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria.

ABSTRACTObjective:To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols.Methods:A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria.Results:Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection.Conclusion:High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones.

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.

Frequency of Paroxysmal Nocturnal Hemoglobinuria Clones by Multiparametric Flow Cytometry in Pediatric Aplastic Anemia Patients of Indian Ethnic Origin.

The literature on paroxysmal nocturnal hemoglobinuria (PNH) in aplastic anemia (AA) is largely focused on adults with few studies in children. Moreover, large studies are conspicuously absent from developing countries. Knowledge of the prevalence and utility of their detection is required before widespread use of PNH screening in pediatric AA in resource-limited settings. We performed a retrospective audit over a period of 9 years to study the prevalence of PNH clones by flow cytometry (FCM) in children ≤12 years of age presenting with AA, and analyzed their response to immunosuppressant therapy. Nine (12.9%) out of 70 patients had PNH clones comprising >1% of the target cell population, including five patients (7.14%) with PNH clone size >10%. The clone size in monocytes ranged from 3.7% to 95.2% (median 21.1%) and in neutrophils from 1.6% to 87.6% (median 19.5%). Fluorescent aerolysin (FLAER)-based FCM screening significantly improved the detection of PNH clones compared to non-FLAER based screening techniques (18.4% vs. 6.25%). One child showed chronic intravascular hemolysis and another developed arterial stroke during the course of illness. None of our PNH-positive AA patients tested for chromosome breakage studies (n = 8) showed increased clastrogen-induced breakage. A lower frequency but moderate/large-sized PNH clones were seen in our pediatric AA population, compared to western data. FLAER-based FCM screening significantly improved the detection of PNH clones. We recommend routine FLAER-based screening of PNH in pediatric AA patients.

A New Approach of PNH Clone Detection

IntroductionThe presence of the CD157 GPI-protein on both monocytes and granulocytes only gives opportunity to use it as a target for paroxysmal nocturnal hemoglobinuria (PNH) phenotype cells detection instead of CD24 and CD14, thus improving the technique of PNH-cells evaluation. In present study we used standard technique (International Clinical Cytometry Society (ICCS) guideline proposal) together with 5 color combination of mAbs for detection of PNH-clone on the monocytes and granulocytes simultaneously in one test-tube.Objectives: improvement of PNH cells detection technique.Materials and methods: Blood samples were collected from 37 patients (pts) with bone marrow failure syndrome (aplastic anemia – 34 pts., PNH - 2 pts., myelodysplastic syndrome – 1 pt.; 23 female and 14 men; median age 24 (15 to 66). PNH clone was detected by flow cytometry in all 37 pts. Three healthy donors were in control group.The comparison of the standard 4-color technique of PNH clone size detection on monocytes (FLAER, CD14, CD64, CD45 reagents) and on granulocytes (FLAER, CD24, CD15, CD45 reagents) with the 5-color technique using CD157 GPI-protein antibodies for the both cells populations in one test-tube (FLAER, CD157, CD15, CD64, CD45) were performed.Results: Both methods showed the same high sensitivity for determining of PNH clone. The correlation coefficient was 0,9994 for granulocytes and 0,9924 for monocytes (Figure 1). [Display omitted] In group of patients with minor PNH-clone (range from 0,01% to 0,99%) the clone size was twice times bigger on monocytes compared to granulocytes using the standard method, whereas with antibodies against CD157 this difference disappeared. In patients with PNH-clone between 1% and 10% the clone was nearly three times less on granulocytes than monocytes using both methods; in the group with clone more than 10% there was no differences between cells populations.Table 1Average value of PNH clone on leukocytes in groups of patients both techniquesPNH cloneGr, % (mean)Mon, % (mean)CD24-/FLAER-CD157-/FLAER-CD14-/FLAER-CD157-/FLAER-0%-0,99%0,1780,3490,3230,3781%-9,99%3,983,769,549,98>10%71,5271,5271,8571,34Conclusion: The results of the PNH clone detection obtained with CD157 mAbs are comparable with the standard technique proposed by ISSC. However, the use of CD157 antibodies has important advantage: the PNH clone size detection in one test-tube with 5-color combination reduces time and expenses. Additionally, using of 5-color CD157 antibodies kit would be preferable for the monitoring and detection of minor PNH clone. DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometry Screening for Paroxysmal Nocturnal Hemoglobinuria Abnormal Clones in Saudi Arabia

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease with insidious process, chronic course and life-threatening condition. PNH is clinically defined by the deficiency of the endogenous glycosyl phosphatidylinositol (GPI)-anchored complement inhibitory protein. It has always aroused interest in the medical profession rendering screening and proper diagnosis by flow cytometry (FCM) technology a priorityWe reviewed all samples submitted for PNH/ FCM screening for the past 2 years (2012-2013) at hematology section, Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center (General Organization). We collected the positive cases and reviewed them for age, gender, indication for screening, sample type, size of the PNH clone and cell type affected. Immunophenotypic analysis was performed using gating antibodies CD45, CD15, CD33, CD235a GPI-linked antibodies, CD59, CD14, and CD24 as well as fluorescent Aerolysin (FLAER).In a total of 366 peripheral blood samples submitted for PNH/ FCM screening fifteen samples (4%) were positive for PNH clones but only 12 patients were available for analysis. The median age for our patients was 34 years with approximately equal male to female distribution.12 cases showed type II and III clones within the RBCs with clone size ranging between 0.04% and 56%. Analysis of granulocytes and monocytes revealed type III clone in 8 cases, type II and III clone in 3 cases and non in one case. The percentage of the clone varies between the granulocytes and monocytes and ranges from 1% up to 100%.Of 12 positive PNH cases, 8 (66.7%) patients were diagnosed as having aplastic anemia (AA), 1 (8.3%) patient with Budd-Chiari syndrome, 1 (8.3%) patient has chronic immune thrombocytopenia (ITP), and 2 (16.7%) patients presented with pancytopenia.This study confirms the rarity of the disease since only 4% of the submitted samples for analysis turned to be positive for PNH. The detection limit for a PNH clone by FCM in the RBC or WBC is 0.01%. Identification of small PNH clone is greater FCM sensitivity relative to old test used for the same purpose (Ham test). The use of the FLAER allowed us to detect granulocytic PNH clone, however, granulocytes PNH clone detection alone without RBCs clone detection is not recommended. This review confirms the previous percentage of positive cases (5.9%) reported from this center on a smaller number of cases during the past few years. DisclosuresNo relevant conflicts of interest to declare.

Multiparameter FLAER-based flow cytometry for screening of paroxysmal nocturnal hemoglobinuria enhances detection rates in patients with aplastic anemia.

Flow cytometry is the gold standard methodology for screening of paroxysmal nocturnal hemoglobinuria. In the last few years, proaerolysin conjugated with fluorescein (FLAER) has become an important component of antibody panel used for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clone. This study aimed to compare PNH clone detection by flow cytometry in the pre-FLAER era versus the FLAER era. This was a retrospective analysis of 4 years and included 1004 individuals screened for PNH clone, either presenting as hemolytic anemia or as aplastic anemia. In the pre-FLAER time period, the RBCs and neutrophils were screened with antibodies against CD55 and CD59. With the introduction of FLAER, neutrophils were screened with FLAER/CD24/CD15 and monocytes with FLAER/CD14/CD33 combination. A comparative analysis was done for detection of PNH clone in aplastic anemia patients versus non-aplastic anemia patients, as well as between pre-FLAER and FLAER era. Out of a total of 1004 individuals, 59 (5.8%) were detected to have PNH clone positivity. The frequency of PNH clone detected in aplastic anemia and non-aplastic anemia groups was 12.02 and 3.36%, respectively. The detection rate of PNH clone increased from 4.5% (32/711) in the pre-FLAER era to 9.2% (27/293) with the introduction of FLAER. However, this increase could be attributed to increased detection of PNH clone in the aplastic anemia group, which showed a significant increase from 8.3 to 18.2% after use of FLAER. In the non-aplastic group, PNH clone was detected with similar frequencies before and after use of FLAER (3.2 versus 3.8%, respectively). Mean PNH clone size was lower in the aplastic anemia group when compared with the non-aplastic group. RBCs always showed a lower clone size than neutrophils. PNH clone on neutrophils and monocytes was however similar. Inclusion of FLAER increases the sensitivity of the test which is especially useful in picking up small PNH clones in patients of aplastic anemia.

Standardizing leucocyte PNH clone detection: An international study

Consensus and Practical Guidelines for robust high-sensitivity detection of glycophosphatidylinostitol-deficient structures on red blood cells and white blood cells in paroxysmal nocturnal hemoglobinuria (PNH) were recently published. UK NEQAS LI issued three stabilized samples manufactured to contain no PNH cells (normal), approximately 0.1% and 8% PNH leucocyte populations, together with instrument-specific Standard Operating Procedures (SOPs) and pretitered antibody cocktails to 19 international laboratories experienced in PNH testing. Samples were tested using both standardized protocol/reagents and in-house protocols. Additionally, samples were issued to all participants in the full PNH External Quality Assessment (EQA) programs. Expert laboratory results showed no difference in PNH clone detection rates when using standardized and their "in-house" methods, though lower variation around the median was found for the standardized approach compared to in-house methods. Neutrophil analysis of the sample containing an 8% PNH population, for example, showed an interquartile range of 0.48% with the standardized approach compared with 1.29% for in-house methods. Results from the full EQA group showed the greatest variation with an interquartile range of 1.7% and this was demonstrated to be significantly different (P<0.001) to the standardized cohort. The results not only demonstrate that stabilized whole PNH blood samples are suitable for use with currently recommended high-sensitivity reagent cocktails/protocols but also highlight the importance of using carefully selected conjugates alongside the standardized protocols. While much more variation was seen among the full UK NEQAS LI EQA group, the standardized approach lead to reduced variation around the median even for the experienced laboratories.

Standardizing Leucocyte PNH clone detection: An international study.

Background: Consensus and Practical Guidelines for robust high-sensitivity detection of glycophosphatidylinostitol (GPI)-deficient structures on Red Blood Cells (RBCs) and White Blood Cells (WBCs) in Paroxysmal Nocturnal Hemoglobinuria (PNH) were recently published. Methods: UK NEQAS LI issued 3 stabilized samples manufactured to contain no PNH cells (normal), approximately 0.1% and 8% PNH leucocyte populations, together with instrument-specific SOPs and pre-titered antibody cocktails to 19 international laboratories experienced in PNH testing. Samples were tested using both standardized protocol/reagents and in-house protocols. Additionally, samples were issued to all participants in the full PNH EQA programmes. Results: Expert laboratory results showed no difference in PNH clone detection rates when using standardized and their 'in-house' methods though lower variation around the median was found for the standardized approach compared to in-house methods. Neutrophil analysis of the sample containing an 8% PNH population, for example, showed an interquartile range of 0.48% with the standardized approach compared with 1.29% for in-house methods. Results from the full EQA group showed the greatest variation with an inter-quartile range of 1.70 and this was demonstrated to be significantly different (P<0.001) to the standardized cohort. Conclusions: The results not only demonstrate that stabilized whole PNH blood samples are suitable for use with currently recommended high-sensitivity reagent cocktails/protocols but also highlight the importance of using carefully selected conjugates alongside the standardized protocols. While much more variation was seen amongst the full UK NEQAS LI EQA group, the standardized approach lead to reduced variation around the median even for the experienced laboratories. © 2014 Clinical Cytometry Society.

Evaluation and Comparison of Different Approaches for the Detection of PNH Clones by Flow Cytometry Following the ICCS Guidelines

Comparison between consensual approaches for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clones by flow cytometry (FCM) following the international clinical cytometry society (ICCS) guidelines has not been widely reported. We determined the performance characteristics of 4, 5, and 6-color protocols for white blood cell (WBC) and one and two-color protocols for red blood cell (RBC) evaluation for different PNH target clones and compared results from PNH patient analysis. Coefficient of variation (CV) for precision/reproducibility analysis ranged from 0.01%/0.12% to 2.56%/ 3.59% for granulocytes, from 0.07%/0.08% to 3.87%/11.61% for monocytes and from 0.4%/1.02% to 6.53%/ 5.1% for RBCs within different approaches and target PNH clones. Comparison of individual protocols revealed excellent correlation (r = 0.99), Wilcoxon rank tests found no statistically significant differences (p > 0.05), Bland-Altman analysis proved agreement for all PNH clones (mean bias ranging from 0.02 to 2.2). Our results confirm good intralaboratory characteristics for precision and reproducibility analysis, excellent correlation and agreement between approaches underlining the primary role of optimally selected glycophosphatidylinositol (GPI)-specific reagents and secondary role of number, type of gating reagents and gating strategy.