Prognostic Value of the Expression of Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR-1) in Chronic Lymphocytic Leukemia.

Background: The transmembrane receptor tyrosine kinase-like orphan receptor 1 (ROR1) has acted on the causation and sustentation of mature B-cell lymphomagenesis for chronic lymphocytic leukemia (CLL) cells. The study attempted to show whether there is a relationship between the level of ROR1 surface expression in CLL cells and disease findings. Materials and Methods: The level of ROR1 cell surface expression was determined in accordance with the flow cytometric analysis of CLL patients at the first diagnosis time.  Two groups were formed according to the high and low ROR1 levels. The cut-off point for the ROR1 level was calculated for advanced-stage disease using receiver operating characteristic (ROC) curves. A two-sided p-value <0,05 was considered statistically significant. Results: 108 CLL cases with a median age of 60 were enrolled. The median percentage of ROR1 cell surface marker positivity in the CD5/CD19 positive leukemic cell was 62%. The CLL cases with high ROR1 levels have thrombocytopenia (p=0.042), anemia (p=0.028), and high beta-2 microglobulin value ≥3 mg/dL (p=0.002) and the need for first-line treatment (p=0.043). Conclusion: The poor prognostic parameters such as splenomegaly, anemia, higher beta-2 microglobulin levels, intermediate/advanced RAI stage disease, and need for first-line treatment had associated high-level ROR 1 expression of our CLL patients. It needs to be investigated for its effect on predicting disease burden and aggressiveness with more comprehensive studies on ROR1 expression levels in CLL cases.

A novel differential diagnosis algorithm for chronic lymphocytic leukemia using immunophenotyping with flow cytometry.

INTRODUCTION: The availability of a clinical decision algorithm for diagnosis of chronic lymphocytic leukemia (CLL) may greatly contribute to the diagnosis of CLL, particularly in cases with ambiguous immunophenotypes. Herein we propose a novel differential diagnosis algorithm for the CLL diagnosis using immunophenotyping with flow cytometry. METHODS: The hierarchical logistic regression model (Backward LR) was used to build a predictive algorithm for the diagnosis of CLL, differentiated from other lymphoproliferative disorders (LPDs). RESULTS: A total of 302 patients, of whom 220 (72.8%) had CLL and 82 (27.2%), B-cell lymphoproliferative disorders other than CLL, were included in the study. The Backward LR model comprised the variables CD5, CD43, CD81, ROR1, CD23, CD79b, FMC7, sIg and CD200 in the model development process. The weak expression of CD81 and increased intensity of expression in markers CD5, CD23 and CD200 increased the probability of CLL diagnosis, (p < 0.05). The odd ratio for CD5, C23, CD200 and CD81 was 1.088 (1.050 - 1.126), 1.044 (1.012 - 1.077), 1.039 (1.007 - 1.072) and 0.946 (0.921 - 0.970) [95% C.I.], respectively. Our model provided a novel diagnostic algorithm with 95.27% of sensitivity and 91.46% of specificity. The model prediction for 97.3% (214) of 220 patients diagnosed with CLL, was CLL and for 91.5% (75) of 82 patients diagnosed with an LPD other than CLL, was others. The cases were correctly classified as CLL and others with a 95.7% correctness rate. CONCLUSIONS: Our model highlighting 4 markers (CD81, CD5, CD23 and CD200) provided high sensitivity and specificity in the CLL diagnosis and in distinguishing of CLL among other LPDs.

A novel differential diagnosis algorithm for chronic lymphocytic leukemia using immunophenotyping with flow cytometry

Abstract Introduction The availability of a clinical decision algorithm for diagnosis of chronic lymphocytic leukemia (CLL) may greatly contribute to the diagnosis of CLL, particularly in cases with ambiguous immunophenotypes. Herein we propose a novel differential diagnosis algorithm for the CLL diagnosis using immunophenotyping with flow cytometry. Methods The hierarchical logistic regression model (Backward LR) was used to build a predictive algorithm for the diagnosis of CLL, differentiated from other lymphoproliferative disorders (LPDs). Results A total of 302 patients, of whom 220 (72.8%) had CLL and 82 (27.2%), B-cell lymphoproliferative disorders other than CLL, were included in the study. The Backward LR model comprised the variables CD5, CD43, CD81, ROR1, CD23, CD79b, FMC7, sIg and CD200 in the model development process. The weak expression of CD81 and increased intensity of expression in markers CD5, CD23 and CD200 increased the probability of CLL diagnosis, (p < 0.05). The odd ratio for CD5, C23, CD200 and CD81 was 1.088 (1.050 - 1.126), 1.044 (1.012 - 1.077), 1.039 (1.007 - 1.072) and 0.946 (0.921 - 0.970) [95% C.I.], respectively. Our model provided a novel diagnostic algorithm with 95.27% of sensitivity and 91.46% of specificity. The model prediction for 97.3% (214) of 220 patients diagnosed with CLL, was CLL and for 91.5% (75) of 82 patients diagnosed with an LPD other than CLL, was others. The cases were correctly classified as CLL and others with a 95.7% correctness rate. Conclusions Our model highlighting 4 markers (CD81, CD5, CD23 and CD200) provided high sensitivity and specificity in the CLL diagnosis and in distinguishing of CLL among other LPDs.

Predictive Factors for Molecular Response in Chronic Myeloid Leukemia: Reduction Ratio and Halving Time of BCR::ABL1 IS Transcript Levels

Objective: Achieving an early molecular response (EMR) is crucial for improving the prognosis of patients with chronic myeloid leukemia (CML). The halving time (HT) and reduction ratio (RR) of BCR::ABL1 transcript levels have recently emerged as additional prognostic indexes besides the BCR::ABL1 International Scale (IS). We aimed to investigate the prognostic role of BCR::ABL1 transcript levels, HT, and RR on molecular response kinetics at 3 months in patients with newly diagnosed chronic-phase (CP)-CML. Materials and Methods: Forty patients with CP-CML who received first-line imatinib treatment were included in this study. BCR::ABL1 transcript levels and molecular responses at baseline and at 3, 6, 12, and 24 months of treatment were evaluated retrospectively. Major molecular response (MMR) at 12 months and event-free survival (EFS) were determined as primary endpoints and the effects of treatment kinetics on these parameters were examined. Results: Of the 40 patients, BCR::ABL1 IS was ≤10% at 3 months in 72.5%, representing EMR. The rate of event occurrence was 45.5% in patients with BCR::ABL1 IS of >10%, whereas it was 6.9% in those with BCR::ABL1 IS of ≤10% (p=0.004). MMR was detected in 62.1% of the patients with EMR and in 9.1% of those without EMR (p=0.003). The cut-off value for achieving MMR was 24 days for HT and 0.04 for RR. Deep molecular response (DMR) at 24 months was associated with HT of ≤24 days and RR of ≤0.04. EFS was found to be significantly better in the group with BCR::ABL1 IS of ≤10% and HT of ≤24 days (p=0.001) and in the group with BCR::ABL1 IS of ≤10% and RR of ≤0.04 (p=0.007) compared to others. Conclusion: Our findings revealed that MMR could be predicted via EMR as well as by HT and RR. Additionally, HT of ≤24 days and RR of ≤0.04 were more important than BCR::ABL1 IS of ≤10% in achieving DMR at 24 months, and the combination of BCR::ABL1 IS of ≤10% with both HT of ≤24 days and RR of ≤0.04 has the best predictive value for EFS.

Conditioning Regimens for Relapsed/Refractory Lymphoma Patients Undergoing Autologous Stem Cell Transplantation: BEAM Versus High Dose ICE.

There are different drug combinations and conditioning regimens in lymphoma transplants. However, no randomized data is available to demonstrate the superiority of any regimen and the optimal choice is unknown. In this analysis, we compared the efficacy, toxicity and the survival outcomes of the BEAM and the high dose ICE (hdICE) conditioning regimens in relapsed NHL and relapsed/refractory Hodgkin Lymphoma patients undergoing auto-SCT. 83 patients with relapsed/refractory HL or relapsed NHL who were treated with Auto-SCT between 2006 and 2016, were analyzed retrospectively. 52 patients (62.7%) received BEAM, while 31 patients (37.3%) received hdICE. Between two groups there is no significant difference in age, gender, diagnosis, disease stage, chemosensitivity, ECOG performance status, time from diagnosis to transplant, salvage regimens and previous lines of chemotherapy. After a median of 59-month follow-up, PFS and OS rates of both groups were similar (5-year PFS was 51.6% in BEAM group, 48.8% in hdICE group, p = 0.71; 5-year OS was 58% in BEAM group, 54.8% in hdICE group, p = 0.93). The median neutrophil (11 vs. 10 days, p = 0.06) and platelet engraftment (13 vs. 11 days, p = 0.01) was faster and demand of transfusions were lesser in hdICE group (p = 0.03). However, severe renal toxicity was significantly higher in hdICE group in our study (p = 0.01). hdICE conditioning regimen may be used as an alternative to BEAM, with similar survival outcomes and toxicity profile, especially transplant centers that experience some difficulties in the availability of the carmustine.

Synchronous detection of multiple myeloma and acute myeloid leukemia: A diagnostic and therapeutic challenge.

INTRODUCTION: Synchronous detection of multiple myeloma and acute myeloid leukemia in a single patient is a rare coincidence. Treatment of these patients is still unclear, mostly based on acute myeloid leukemia strategies combined with bortezomib. CASE REPORT: A 72-year-old male with no medical history was investigated for pancytopenia. On medical examination, he was complicated with a wide and severe skin infection on arm. On examination of bone marrow aspirate, 25% myeloblasts infiltration and additional 10% plasma cells were seen. Acute myeloid leukemia was diagnosed and plasma cell proliferation was attributed to reactive plasmacytosis due to skin infection. However, flowcytometric studies and immunohistochemical examination revealed two different cell populations with 30-40% atypical plasma cells and >20% myeloblasts. Serum M-protein detected by serum electrophoresis test and immunofixation test revealed a monoclonal IgG lambda band. He was diagnosed with concurrent acute myeloid leukemia and multiple myeloma without history of chemotherapy.Management and outcome: The patient was initially treated with bortezomib and dexamethasone for the myeloma. Subsequently, azacitidine was administered subcutaneously for the acute myeloid leukemia treatment. The tru-cut biopsy of the lesion on his arm revealed suppurative inflammatory findings and no malign cells detected. Antibiotherapy was started according to susceptibility. He expired after three months of survival. DISCUSSION: The synchronous occurrence of these two different clonal hematological malignancies is rare in hematology practice. Patient-based prospective studies and case series are needed to guide diagnosis and treatment strategies. Furthermore, this report highlights the importance of ruling out reactive plasmacytosis in patients with hematological malignancy who developed severe infections.

Comparative evaluation of memory T cells in COVID-19 patients and the predictive role of CD4+CD8+ double positive T lymphocytes as a new marker.

BACKGROUND: The COVID-19 pandemic has affected the entire world, posing a serious threat to human health. T cells play a critical role in the cellular immune response against viral infections. We aimed to reveal the relationship between T cell subsets and disease severity. METHODS: 40 COVID-19 patients were randomly recruited in this cross-sectional study. All cases were confirmed by quantitative RT-PCR. Patients were divided into two equivalent groups, one severe and one nonsevere. Clinical, laboratory and flow cytometric data were obtained from both clinical groups and compared. RESULTS: Lymphocyte subsets, CD4+ and CD8+ T cells, memory CD4+ T cells, memory CD8+ T cells, naive CD4+ T cells, effector memory CD4+ T cells, central memory CD4+ T cells, and CD3+CD4+ CD25+ T cells were significantly lower in severe patients. The naive T cell/CD4 + EM T cell ratio, which is an indicator of the differentiation from naive T cells to memory cells, was relatively reduced in severe disease. Peripheral CD4+CD8+ double-positive T cells were notably lower in severe presentations of the disease (median DP T cells 11.12 µL vs 1.95 µL; p< 0.001). CONCLUSIONS: As disease severity increases in COVID-19 infection, the number of T cell subsets decreases significantly. Suppression of differentiation from naive T cells to effector memory T cells is the result of severe impairment in adaptive immune functions. Peripheral CD4+CD8+ double-positive T cells were significantly reduced in severe disease presentations and may be a useful marker to predict disease severity.

Comparative evaluation of memory T cells in COVID-19 patients and the predictive role of CD4+CD8+ double positive T lymphocytes as a new marker

SUMMARY BACKGROUND: The COVID-19 pandemic has affected the entire world, posing a serious threat to human health. T cells play a critical role in the cellular immune response against viral infections. We aimed to reveal the relationship between T cell subsets and disease severity. METHODS: 40 COVID-19 patients were randomly recruited in this cross-sectional study. All cases were confirmed by quantitative RT-PCR. Patients were divided into two equivalent groups, one severe and one nonsevere. Clinical, laboratory and flow cytometric data were obtained from both clinical groups and compared. RESULTS: Lymphocyte subsets, CD4+ and CD8+ T cells, memory CD4+ T cells, memory CD8+ T cells, naive CD4+ T cells, effector memory CD4+ T cells, central memory CD4+ T cells, and CD3+CD4+ CD25+ T cells were significantly lower in severe patients. The naive T cell/CD4 + EM T cell ratio, which is an indicator of the differentiation from naive T cells to memory cells, was relatively reduced in severe disease. Peripheral CD4+CD8+ double-positive T cells were notably lower in severe presentations of the disease (median DP T cells 11.12 µL vs 1.95 µL; p< 0.001). CONCLUSIONS: As disease severity increases in COVID-19 infection, the number of T cell subsets decreases significantly. Suppression of differentiation from naive T cells to effector memory T cells is the result of severe impairment in adaptive immune functions. Peripheral CD4+CD8+ double-positive T cells were significantly reduced in severe disease presentations and may be a useful marker to predict disease severity.

RESUMO OBJETIVO: A pandemia de COVID-19 tem afetado o mundo todo, constituindo uma ameaça grave para a saúde humana. As células T desempenham um papel crítico na imunidade celular contra infecções virais. Procuramos desvendar a relação entre sub grupos de células T e a severidade da doença. MÉTODOS: Um total de 40 pacientes com COVID-19 foram aleatoriamente recrutados para o presente estudo transversal. Todos os casos foram confirmados por RT-PCR quantitativo. Os pacientes foram divididos em dois grupos equivalentes, um grave e um não-grave. Os dados da avaliação clínica, laboratorial e da citometria de fluxo foram obtidos para ambos os grupos e comparados. RESULTADOS: Os subconjuntos de linfócitos, células T CD4+ e CD8+, células T de memória CD4+, células T de memória CD8+, células T CD4+ virgens, células T efetoras CD4+, células T de memória central CD4+ e células T CD3+ CD4+ CD25+ estavam significativamente mais baixas nos pacientes graves. A razão células T virgens/células T efetoras TCD4+, que é um indicador da diferenciação entre células T virgens e células de memória, estava relativamente reduzida em casos graves da doença. As células T duplo-positivas CD4+CD8+ periféricas estavam notavelmente mais baixas em casos graves da doença (mediana das células T DP: 11,12 µL vs. 1,95 µL; p< 0,001). CONCLUSÃO: Conforme aumenta a gravidade da doença nos casos de COVID-19, o número de subconjuntos de células T diminui significativamente. A supressão da diferenciação de células T virgens para células T efetoras é o resultado do comprometimento grave das funções imunológicas adaptativas. As células T duplo-positivas CD4+CD8+ periféricas estavam notavelmente mais baixas em casos graves da doença e podem ser um marcador útil para predizer a severidade da doença.

Study for the Diagnostic Screening of Paroxysmal Nocturnal Hemoglobinuria in Older Patients with Unexplained Anemia and/or Cytopenia.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disease that may lead to weakness and death of patients, if unrecognized and untreated. Although consensus guidelines were reviewed recently for the diagnostic screening of PNH with multi-parameter flow cytometry (FCM), until now, no study has investigated the efficiency of such clinical indications in older patients. METHODS: Overall, 20 centers participated in the study and a total of 1,689 patients were included, 313 of whom were at geriatric age and 1,376 were aged 18 - 64 years. We evaluated the efficiency of consensus clinical indications for PNH testing using FCM in peripheral blood samples and compared the results of older patients and patients aged 18 - 64 years. RESULTS: PNH clones were detected positive in 7/313 (2.2%) of the older patients. Five (74.4%) of the patients with PNH clones had aplastic anemia, 1 had unexplained cytopenia, and 1 patient had myelodysplastic syndrome (MDS) with refractory anemia. PNH clones were not detected in any older patients who were screened for unexplained thrombosis, Coombs (-) hemolytic anemia, hemoglobinuria, and others (e.g., elevated lactate dehydrogenase (LDH), splenomegaly). We detected PNH clones in 55/1376 (4%) samples of the patients aged under 65 years. Forty-two (76.4%) patients with PNH clones had aplastic anemia, 2 patients had Coombs (-) hemolytic anemia, 3 patients had unexplained cytopenia, 1 patient had MDS with refractory anemia, 1 patient had hemoglobinuria, and 6 (10.9%) had others (e.g., elevated LDH, splenomegaly). PNH clones were not detected in any patients who were screened for unexplained thrombosis. There was no statistical difference between the geriatric population and patients aged 18 - 64 years in terms of clinical indications for PNH screening with FCM (p = 0.49). CONCLUSIONS: Our results showed that the current clinical indications for PNH screening with FCM were also efficient in older patients. We suggest that older patients with unexplained anemia, myelodysplastic syndrome with refractory anemia, and unexplained cytopenia should be screened for PNH with FCM to identify patients who would benefit from treatment.

Comparison of allogenic stem cell transplantations performed with frozen or fresh stem cell products with regard to GVHD and mortality.

PURPOSE: Stem cells are collected from donors and infused to the recipient in allogenic peripheral stem cell transplantations. The use of frozen stem cells can promote donor compatibility, and overcoming possible problems due to insufficient stem cell mobilization will also be easier. Nevertheless, studies about the use of frozen peripheral stem cells in allogenic transplantation are extremely rare. In this study, we aimed to compare the clinical outcomes of allogenic stem cell transplants from frozen or fresh stem cell products. MATERIALS AND METHODS: This retrospective analysis was conducted between April 2004 and September 2018 in the bone marrow transplantation unit of Ankara Numune Training and Research Hospital. Clinical data of patients who received allogenic peripheral stem cell transplantations from fully matched sibling donors were compared for 42 fresh and 30 frozen stem cell transplants. RESULTS: While the platelet engraftment period, febrile neutropenia period, hospitalization period, and 100-day mortality rates did not show any differences, the neutrophil engraftment period was longer in the frozen group (mean: 14 days vs. 16 days, p = 0.006). Acute and chronic graftversus-host disease (GVHD) rates were similar in both groups; however, the rate of grade 3 or4 chronic liver GVHD was slightly higher in transplants performed with fresh stem cells compared to the frozen group (p = 0.046). Overall survival was similar between the groups (p = 0.700). CONCLUSION: The use of frozen peripheral stem cells in allogenic stem cell transplantation may be a reasonable option that can be applied without causing a significant change in clinical results.

Which Markers Should the used for Diagnostic Chronic Lymphocytic Leukemia Immunophenotyping Scoring System by Flow Cytometry?

BACKGROUND: The scoring system used for chronic lymphocytic leukemia (CLL) cannot make an accurate diagnosis in some cases. Novel markers are available for the differential diagnosis of CLL, especially from MCL. However, these markers are still not incorporated into diagnostic algorithms. We investigated the role of CD43, CD81, CD200, and ROR1 in the differential diagnosis of CLL and their expression in non-CLL cases. METHODS: We investigated the role of CD43, CD81, CD20, and ROR1 in the differential diagnosis of CLL by incorporating them into the diagnostic panel after studying peripheral blood or bone marrow samples of 165 patients with 8-color flow cytometry. RESULTS: CD43 positivity was a sensitive marker but had a lower specificity for CLL. CD43 had high diagnostic value for CLL (sensitivity 100%, specificity 88.5%, AUC 98.0%). CD200 was a specific marker for CLL (sensitivity 98%, specificity 90%, AUC: 96%). CD81 expression was highest in the MCL cases, with a median expression rate of 68.5% (range: 54 - 82.5%). It was negative in all the CLL cases. For CLL, CD81 negativity had a sensitivity of 95%, a specificity of 82% and an AUC of 92%. ROR1 was positive in all CLL and MCL cases. CD79b, on the other hand, was a fairly sensitive and specific marker for MCL. CONCLUSIONS: CD43, CD81, CD200, and ROR1 should be incorporated into diagnostic algorithms for the differential diagnosis of CLL, especially from MCL.

Study for the diagnostic screening of paroxsymal nocturnal hemoglobinuria in Turkey: Prospective multicentric evaluation of suspected patients.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease presenting with variable and various clinical findings. PNH might be overlooked and diagnosis may be delayed due to low awareness about PNH. This is the first multicenter study in Turkey, investigating the efficiency of diagnostic screening of PNH by multiparameter flow cytometry (FCM) according to consensus guidelines. METHODS: We evaluate the efficiency of consensus clinical indications for PNH testing with FCM in 1689peripheral blood samples from 20 centers between January 2014 and December 2017. RESULTS: Overall, at the 20 centers contributing to this study, PNH clone were detected in 62/1689 samples (3.6%) by FCM test. 75.8% (n = 47) of patients with PNH clone had aplastic anemia, 3.2% (n = 2) had Coombs (-) hemolytic anemia, 6.5% (n = 4) had unexplained cytopenia, 3.2% (n = 2) had MDS with refractory anemia, 1.6% (n = 1) had hemoglobinuria and 9.7% (n = 6) had others (elevated LDH, splenomegaly, etc.). In contrast, we detect no PNH clone test in patients who were screened for unexplained thrombosis. CONCLUSIONS: Our study showed that current clinical indications for PNH testing are highly efficient and diagnostic screening of suspected patients for PNH with FCM is recommended. However, advanced screening algorithms are required for patients presenting with unexplained thrombosis and normal complete blood count.

The Importance of RET-He in the Diagnosis of Iron Deficiency and Iron Deficiency Anemia and the Evaluation of Response to Oral Iron Therapy.

BACKGROUND: The purpose of this study is to investigate whether or not reticulocyte hemoglobin equivalent (RET-He) is a superior indicator of blood count and other iron parameters in terms of diagnosing iron deficiency (ID) and iron deficiency anemia (IDA), and thus evaluating a patient's response to oral iron treatment. METHODS: The research population consisted of 217 participants in total: 54 control, 53 ID, 58 non-ID anemia, and 52 IDA patients. A hemoglobin (Hb) value of < 130.0 g/L was defined as indicating anemia for men, while an Hb value of < 120.0 g/L was defined as indicating anemia for women. All patients were administered 270 mg oral elemental iron sulphate daily. RESULTS: The RET-He was significantly lower in the IDA group, compared to other groups (IDA: 21.0 ± 4.1, ID: 26.0 ± 4.9, non-ID anemia: 32.1 ± 6.8, control: 36.6 ± 7.0; < 0.001). The ID group had a lower RET-He compared to the non-ID anemia group and the control group. On the 5th day of treatment, the ID and IDA group showed no significant differences in terms of Hb while the RET-He level demonstrated a significant increase. The increase in the RET-He level observed in the IDA group on the 5th day was significantly higher compared to the increase observed in the ID group. A RET-He value of 25.4 pg and below predicted ID diagnosis with 90.4% sensitivity and 49.1% specificity in IDA patients, compared to the ID group. CONCLUSIONS: The results of our study, therefore, suggest that RET-He may be a clinically useful marker in the diagnosis of ID and IDA.

Calculation of Biological Variation Components and Reference Change Value for Serum Copper and Zinc.

BACKGROUND: Zinc and copper are among the most important trace elements. Deficiencies of these trace elements cause a wide variety of disorders. The present study aims to report the definitive assessment of biological variation (BV) parameters for these elements as within-subject BV (CVI), between subject BV (CVG), index of individuality (II), and reference change value (RCV) in a Turkish cohort study group. METHODS: Ten blood specimens were collected weekly from 20 healthy volunteers (13 women, 7 men) for 10 weeks. Collected sera were stored at -80°C until the time of analysis. Serum zinc and copper levels were analyzed with atomic absorption spectrometry and ANOVA test was used to calculate the variations. RESULTS: The CVI and CVG for zinc were 6.26% and 23.27%, respectively. Analytical variation (CVA) was calculated as 4.24%. II and RCV for zinc were calculated as 0.26 and 21.51%, respectively. The CVI and CVG for copper were 6.05% and 19.64%, respectively. CVA was calculated as 4.24%. II and RCV for copper were calculated as 0.31 and 20.47%, respectively. CONCLUSIONS: Since II values were less than 0.6 for both analytes, the reference values will be of little use. RCV might be preferred for better evaluation instead.

Biological variation of peripheral blood T-lymphocytes.

BACKGROUND: Flow cytometric analysis of the lymphocyte subsets has become one of the most commonly used techniques in the routine clinical laboratory. It is frequently used in monitoring lymphocyte recovery after hematopoietic stem cell transplantation (HSCT), as well as diagnosis and treatment of acquired immunodeficiency syndrome (AIDS). Reliable biological variation (BV) data is needed for safe clinical application of these tests. In this study, similar preanalytical and analytical protocols to the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) checklist were followed and a stringent statistical approach was applied to define BV of T-lymphocytes. METHODS: During the 10 weeks study period, weekly blood samples were obtained from 30 healthy individuals (20 females, 10 males) and analyzed with Facs Canto (BD Biosciences, San Jose, CA, USA) analyzer using 4-colour BD Multitest CD3/CD8/CD45/CD4 reagents. Data were assessed in terms of normality, tendencies, outliers and variance homogeneity prior to applying coefficient of variance (CV)- analysis of variance (ANOVA) test. Sex-stratified within-individual (CVI) and between-individual (CVG) BV estimates of CD3+, CD3 + CD4+, CD3 + CD8+, and CD3 + CD4 + CD8+ T lymphocytes were calculated. RESULTS: No difference was found between males and females. Except for the CD3 + CD4 + CD8+ subset, stable BV was found for CD3+, CD3 + CD4+, and CD3 + CD8+ subsets. CONCLUSSION: Instead of using the conventional reference ranges of CD3+, CD3 + CD4+ and CD3 + CD8+ counts for monitoring HIV positive or post-HSCT patients, RCV should be used. Because individualityis characteristic of lymphocytes subsets RCVs should be used instead of RIs for patient monitoring.

CD81 Expression in the Differential Diagnosis of Chronic Lymphocytic Leukemia.

BACKGROUND: Immunophenotyping has a central role in CLL. However, CLL is a very heterogenous disease, both morphologically and immunophenotypically; thus, its diagnosis may prove a challenge. We investigated CD81 ex-pression in the differential diagnosis of CLL and MCL. METHODS: We retrospectively examined CD81 expression with 8 color Multiparameter Flow cytometry devices in 101 CLL and 19 MCL cases. RESULTS: We found negative CD81 expression in CLL cases whereas it was positive in MCL cases. CONCLUSIONS: Our results suggest that CD81 may be a valuable marker for the differential diagnosis of CLL. We are of the opinion that it should be definitely included in the diagnostic algorithm for CLL.

Biological variation estimates of prothrombin time, activated partial thromboplastin time, and fibrinogen in 28 healthy individuals.

BACKGROUND: Although tests of global hemostasis prothrombin time (PT) and activated partial thromboplastin time (aPTT) should not be used for prediction of bleeding risk, these tests are often used by many clinicians in daily practice particularly as a preoperative screening test. Robust biological variation (BV) data are needed for safe clinical applications of these tests. In this study, a stringent protocol was followed to estimate the BV's for PT, aPTT, and fibrinogen levels. METHODS: Weekly blood samples were obtained from 28 healthy individuals (18 females, 10 males) during 10 weeks study period. All measurements were performed with Stago STA-R coagulation analyzer. Prior to coefficient of variation (CV)-analysis of variance (ANOVA), the data were assessed for normality, trends, outliers, and variance homogeneity. Sex-stratified within-individual (CVI ) and between-individual (CVG ) BV estimates were determined for PT, aPTT, and fibrinogen tests. RESULTS: No difference was found between male and female estimates of BV. The observed CVI and CVG estimates were found to be lower than those previously published. Only for fibrinogen, CVI was higher than CVG . CONCLUSION: Following a meticulous protocol, our study results provide up-to-date and more stringent BV estimates of global hemostasis tests.

Both Granulocytic and Non-Granulocytic Blood Cells Are Affected in Patients with Severe Congenital Neutropenia and Their Non-Neutropenic Family Members: An Evaluation of Morphology, Function, and Cell Death

Objective: To examine granulocytic and non-granulocytic cells in children with severe congenital neutropenia (SCN) and their non-neutropenic parents. Materials and Methods: Fifteen patients with SCN and 21 non-neutropenic parents were evaluated for a) CD95, CD95 ligand, annexin V, propidium iodide, cell cycle, and lymphocyte subsets by flow cytometry; b) rapid cell senescence (of leukocytes) by senescence-associated ß-galactosidase stain; c) aggregation tests by aggregometer; d) in vitro bleeding time by PFA-100 instrument; e) mepacrine-labeled dense granule number of thrombocytes by fluorescence microscope; and f) hematomorphology by light and electron microscope. HAX1, ELANE, G6PC3, CSF3R, and JAGN1 mutations associated with SCN were studied in patients and several parents. Results: Significant increase in apoptosis and secondary necrosis in monocytes, lymphocytes, and granulocytes of the patients and parents was detected, irrespective of the mutation type. CD95 and CD95 ligand results implied that apoptosis was non-CD95-mediated. Leukocytes of 25%, 12.5%, and 0% of patients, parents, and controls showed rapid cell senescence. The cell cycle analysis testable in four cases showed G1 arrest and apoptosis in lymphocytes of three. The patients had HAX1 (n=6), ELANE (n=2), G6PC3 (n=2), and unidentified (n=5) mutations. The CD3, CD4, and NK lymphocytes were below normal levels in 16.6%, 8.3%, and 36.4% of the patients and in 0%, 0%, and 15.4% of the parents (controls: 0%, 0%, 5.6%). The thrombocytes aggregated at low rates, dense granule number/thrombocyte ratio was low, and in vitro bleeding time was prolonged in 37.5%-66.6% of patients and 33.3%-63.2% of parents (vs. 0% in controls). Under electron and/or light microscope, the neutrophils, monocytes, lymphocytes, and thrombocytes in the peripheral blood of both patients and parents were dysplastic and the bone marrow of patients revealed increased phagocytic activity, dysmegakaryopoiesis, and necrotic and apoptotic cells. Ultrastructurally, thrombocyte adhesion, aggregation, and release were inadequate. Conclusion: In cases of SCN, patients' pluripotent hematopoietic stem cells and their non-neutropenic parents are both affected irrespective of the genetic defect.

What should be the laboratory approach against isolated prolongation of a activated partial thromboplastin time?

BACKGROUND: This study is a retrospective evaluation of patients who were subject to mixing study in our laboratory due to prolonged APTT. The preliminary diagnoses, clinical manifestations, and results of additional ordered tests were reviewed. The study aims to investigate whether repeating APTT test with a different assay prior to performing mixed study in patients with prolonged APTT would be a better alternative algorithmic approach in order to save both time and costs. METHODS: We retrospectively evaluated 166 patients (65 females and 101 males) who were subject to mixing study due to isolated prolonged APTT. Additional ordered tests to identify the etiology and clinical findings were reviewed. All patients who had prolonged APTT as a result of testing with Hemosil Synthasil APTT reagent in ACL TOP analyzer were repeated with Stago Cephascreen APTT reagent in STA-R coagulation analyzer. RESULTS: APTT test was requested preoperatively in 72.2% of cases. Only 6.6% of the cases had history of bleeding. Correction with mixing study was achieved in 122 (73.5%) cases, among which 75 (45%) cases were found to have APTT test results within reference range when tested with Cephascreen reagent. In 44 (26.5%) cases, mixing study did not result in correction. Only 4 cases were confirmed to have lupus anticoagulants (LA), while 4 cases were diagnosed with hemophilia with inhibitors. CONCLUSION: Prolonged APTT results should always be retested using a different assay prior to mixing study. The clinician and the laboratory specialist should collaborate at the postanalytical phase.