Validation of a single tube 3-colour immature red blood cell screening assay for the detection and enumeration of small, medium and large paroxysmal nocturnal haemoglobinuria clones by flow cytometry.

INTRODUCTION: The reliable diagnosis of paroxysmal nocturnal haemoglobinuria (PNH) by flow cytometry is based on mandatory analysis of the erythroid, neutrophilic and monocytic lineages. In this study, we have evaluated the performance characteristics of a recently published immature red blood cell (iRBC) assay as a potential screening test for PNH by flow cytometry. METHODS: Intra- and inter-assay imprecision were determined in five replicates of small, medium and large PNH iRBC clones. Analytical and functional sensitivity was assessed by performing spiking tests for five replicates. Thirty healthy donors and 441 PNH patients were tested for evaluation of clinical specificity, sensitivity, positive and negative predictive values. RESULTS: Coefficients of variation (CV) for intra-/inter-assay imprecision analyses were 1.31/1.50, 3.19/2.61 and 3.99/1.58 for the big, medium and small clone sizes, respectively. Absolute values (100%) were found for both clinical specificity and sensitivity as well as for both positive and negative predictive values. The CV from 5 replicate results for 10 clustered events was 15.7%. The coefficient of determination (r2 ), Pearson's correlation coefficient (r) and Bland-Altman mean bias were 0.9436/0.9234/1.7 for PNH iRBC compared to PNH neutrophils and 0.9553/0.9387/2.1 for PNH iRBCs compared to PNH monocytes. CONCLUSION: Our results confirm very good performance characteristics, high analytical and functional sensitivity, absolute clinical specificity and sensitivity as well as favourable correlation between PNH iRBCs and both PNH neutrophils and monocytes, suggesting that this cost-effective 3-colour iRBC assay can be used as a reliable screening test for evaluation of small, medium and large PNH clones by flow cytometry.

WT1 Gene Expression in Peripheral Blood Before and After Allogeneic Stem Cell Transplantation is a Clinically Relevant Prognostic Marker in AML - A Single-center 14-year Experience.

BACKGROUND: This work summarizes our experience with WT1 monitoring before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT). PATIENTS AND METHODS: The expression of WT1 gene was measured by real-time polymerase chain reaction in peripheral blood according to the European Leukemia Net recommendations. Between May 2005 and August 2019, we analyzed 147 consecutive patients with acute myeloid leukemia with high WT1 expression at diagnosis, transplanted in first (CR1) or second (CR2) complete remission. RESULTS: At the time of allo-HSCT, 107 patients had WT1-normal expression (WT1 ≤ 50 copies), and 40 patients had WT1-high expression. The median follow-up was 21 months. The estimated 5-year overall survival and event-free survival was significantly better in the WT1-normal cohort (65% and 57% vs. 37% and 25%; P = .0003 and P < .0001, respectively) and 5-year cumulative incidence of relapse was significantly lower in the WT1-normal group (19% vs. 53%; P < .0001). Five-year non-relapse mortality was not significantly different (20% and 23%). Multivariate analysis revealed WT1-high expression and acute graft-versus-host disease grade 3/4 as significantly negative prognostic factors for OS. Overall, 49 patients developed WT1 molecular relapse in the post-transplant period; in 14 cases, the therapeutic intervention was done. In all but 1 relapsed patient where WT1 minimal residual disease (MRD) was monitored (38 patients), we detected WT1-high levels (sensitivity of 97%). CONCLUSION: The results of the analysis confirmed our previous experience that WT1 status before allo-HSCT is a strong prognostic factor for both OS and relapse risk. In addition, we confirmed the usefulness of this marker for MRD monitoring after allo-HSCT. The main advantage is the possibility of frequent MRD monitoring in peripheral blood and early bone marrow examination based on WT1-high expression.

CD71 improves delineation of PNH type III, PNH type II, and normal immature RBCS in patients with paroxysmal nocturnal hemoglobinuria.

BACKGROUND: The diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) relies on flow cytometric demonstration of loss of glycosyl-phosphatidyl inositol (GPI)-anchored proteins from red blood cells (RBC) and white blood cells (WBC). High-sensitivity multiparameter assays have been developed to detect loss of GPI-linked structures on PNH neutrophils and monocytes. High-sensitivity assays to detect PNH phenotypes in RBCs have also been developed that rely on the loss of GPI-linked CD59 on CD235a-gated mature RBCs. The latter is used to delineate PNH Type III (total loss of CD59) and PNH Type II RBCs (partial loss of CD59) from normal (Type I) RBCs. However, it is often very difficult to delineate these subsets, especially in patients with large PNH clones who continue to receive RBC transfusions, even while on eculizumab therapy. METHODS: We have added allophycocyanin (APC)-conjugated CD71 to the existing CD235aFITC/CD59PE RBC assay allowing simultaneous delineation and quantification of PNH Type III and Type II immature RBCs (iRBCs). RESULTS: We analyzed 24 medium to large-clone PNH samples (>10% PNH WBC clone size) for PNH Neutrophil, PNH Monocyte, Type III and Type II PNH iRBCs, and where possible, Type III and Type II PNH RBCs. The ability to delineate PNH Type III, Type II, and Type I iRBCs was more objective compared to that in mature RBCs. Additionally, total PNH iRBC clone sizes were very similar to PNH WBC clone sizes. CONCLUSIONS: Addition of CD71 significantly improves the ability to analyze PNH clone sizes in the RBC lineage, regardless of patient hemolytic and/or transfusion status.

Immunophenotyping of Paroxysmal Nocturnal Hemoglobinuria (PNH).

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare but often debilitating disease which may lead to death in up to 35% of patients within 5 years if unrecognized and untreated. Detection of PNH and assessment of PNH clone size in RBC and WBC lineages by flow cytometric analysis has increased in importance due to the availability of novel therapies. These therapies typically block the hemolysis of red blood cells and thus significantly lower the morbidities and mortality associated with this disease. This chapter describes validated, state-of-the-art, high-sensitivity flow cytometric methodologies based on latest published testing guidelines for PNH.

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.

High-sensitivity 5-, 6-, and 7-color PNH WBC assays for both Canto II and Navios platforms.

BACKGROUND: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder characterized by an inability to make Glyco-Phosphatidyl-Inositol (GPI)-linked cell surface structures. Fluorescent proaerolysin (FLAER-Alexa488) is increasingly used to detect GPI-deficient WBCs by flow cytometry. However, FLAER is not available in all countries and is expensive to obtain in others. An earlier study to compare FLAER-based and non-FLAER assays confirmed very good agreement between the two tubes suggesting a cost effective simultaneous evaluation of PNH neutrophils and monocytes is possible without FLAER. METHODS: We have used a single tube approach with a 7-color assay comprising FLAER-CD157-CD15-CD64-CD24-CD14-CD45. Conjugates were carefully selected and validated so that stained samples could be analyzed on either 10-color Navios or 8-color FACSCanto II platforms. The 6-color (minus CD14) and 5-color (minus CD24 and CD14) versions were also developed and compared with our predicate clinical lab 5-color assay comprising FLAER-CD157PE-CD64ECD-CD15PC5-CD45PC7. RESULTS/CONCLUSIONS: CD15-gated PNH neutrophil clone size was quantified using either FLAER and CD157, FLAER and CD24, or CD157 and CD24. CD64-gated PNH monocyte clone size was quantified using either FLAER and CD157, FLAER and CD14, or CD157 and CD14. Analysis of >40 PNH samples showed that the FLAER-based plots derive virtually identical data to the non-FLAER plot for neutrophils (R2 = 1) and monocytes (R2 = 0.9999) and that closely similar data can be acquired using both Canto II and Navios platforms with 7-, 6-, and 5-color versions of the assay. Assessment of non-PNH samples confirmed extremely low background rate of PNH phenotypes (neutrophils and monocytes) with all three approaches. © 2018 International Clinical Cytometry Society.

Performance Characteristics of a Non-Fluorescent Aerolysin-Based Paroxysmal Nocturnal Hemoglobinuria (PNH) Assay for Simultaneous Evaluation of PNH Neutrophils and PNH Monocytes by Flow Cytometry, Following Published PNH Guidelines.

BACKGROUND: CD157 has been recently reported as a useful glycosylphosphatidylinositol (GPI)-linked marker for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clones in patients with suspected paroxysmal nocturnal hemoglobinuria by flow cytometry as it targets both neutrophils and monocytes. The aim of this study is to test the feasibility of a non-fluorescent aerolysin (FLAER) approach and propose an alternative for laboratories, where FLAER is not available. METHODS: We validated a non-FLAER-based single-tube, 6-color assay targeting the GPI-linked structures CD157, CD24, and CD14. We determined its performance characteristics on 20 PNH patient samples containing a variety of clone sizes and compared results with a previously validated FLAER-based approach. RESULTS: Coefficient of variation (CV) for intra-/interassay precision analyses ranged from 0.1%/0.2% to 3.02%/7.58% for neutrophils and from 0.10%/0.3% to 5.39%/6.36% for monocytes. Coefficient of determination (r2 ) for linear regression analysis of PNH clones from 20 patients ranging from 0.06% to 99.7% was 0.99 in all cases, Wilcoxon ranks test showed no statistically significant differences (P > 0.05), Bland-Altman analysis demonstrated performance agreement with mean bias ranging from 0.06 to 0.2. CONCLUSION: Our results confirm very good performance characteristics for both intra- and interassay precision analyses, favorable correlation, and agreement between the FLAER and non-FLAER-based approaches, using the CD157 GPI marker. Our experience suggests that a rapid and cost-effective simultaneous evaluation of PNH neutrophils and monocytes by flow cytometry without using FLAER is possible in areas where FLAER may not be widely available. © 2016 International Clinical Cytometry Society.

ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 3 - data analysis, reporting and case studies.

Over the past several years, a diverse group of physicians and other laboratory scientists have developed various recommendations and guidelines regarding best practices for PNH testing. This manuscript is based on these previous recommendations as well as various other relevant publications of experts in the area of PNH testing. The goal is to provide flow cytometry laboratories with an updated consensus approach to analysis and reporting of PNH results and to address the most common analytical challenges for accurate reporting of this rare disease. A comprehensive case library is included in this section. © 2017 International Clinical Cytometry Society.

ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 2 - reagent selection and assay optimization for high-sensitivity testing.

Since publication in 2010 of the International Clinical Cytometry Society (ICCS) Consensus Guidelines for detection of Paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometery, a great deal of work has been performed to develop, optimize, and validate a number of high-sensitivity assays to detect PNH phenotypes in both red blood cells (RBCs) and white blood cells (WBCs, neutrophils, and monocytes). This section (Part 2) of the updated ICCS PNH Consensus Guidelines will focus on specific instrument setup for these PNH assays, the identification and proper testing of appropriate antibody conjugates and combinations therof, and basic assay design. © 2017 International Clinical Cytometry Society.

Interferon-α Revisited: Individualized Treatment Management Eased the Selective Pressure of Tyrosine Kinase Inhibitors on BCR-ABL1 Mutations Resulting in a Molecular Response in High-Risk CML Patients.

Bone marrow transplantation or ponatinib treatment are currently recommended strategies for management of patients with chronic myeloid leukemia (CML) harboring the T315I mutation and compound or polyclonal mutations. However, in some individual cases, these treatment scenarios cannot be applied. We used an alternative treatment strategy with interferon-α (IFN-α) given solo, sequentially or together with TKI in a group of 6 cases of high risk CML patients, assuming that the TKI-independent mechanism of action may lead to mutant clone repression. IFN-α based individualized therapy decreases of T315I or compound mutations to undetectable levels as assessed by next-generation deep sequencing, which was associated with a molecular response in 4/6 patients. Based on the observed results from immune profiling, we assumed that the principal mechanism leading to the success of the treatment was the immune activation induced with dasatinib pre-treatment followed by restoration of immunological surveillance after application of IFN-α therapy. Moreover, we showed that sensitive measurement of mutated BCR-ABL1 transcript levels augments the safety of this individualized treatment strategy.

Early MRD response as a prognostic factor in adult patients with acute lymphoblastic leukemia.

OBJECTIVE: To evaluate the prognostic power of minimal residual disease (MRD) monitored by polymerase chain reaction at defined time points during early treatment in adult patients with acute lymphoblastic leukemia (ALL). METHODS: Seventy-one patients were treated according to the GMALL 07/2003 protocol and evaluated for MRD in bone marrow by specific clonal rearrangements of Ig/TCR in BCR-ABL negative ALL or fusion gene transcript in BCR-ABL positive ALL. RESULTS: Three-year overall survival (OS) was 94% in patients with BCR-ABL negative ALL reaching complete molecular response (CMR) after the first course of chemotherapy (vs. 32% if MRD >10(-4) ; P = 0.001). Patients with CMR prior to the start of consolidation chemotherapy at week 11 had 3-yr OS 82% (vs. 18% if MRD >10(-4) ; P = 0.001). Patients with BCR-ABL positive ALL showed slower MRD dynamics. There was a trend to better OS in patients with ≥ 4 log reduction of BCR-ABL transcript prior to HSCT (92% vs. 50%; P = 0.065). None of the patients with detectable MRD (both BCR-ABL positive and negative) after HSCT survived 3 yr. CONCLUSION: Early MRD kinetics is an important tool for new prognostication models with direct clinical impact irrespective of standard prognostic factors in patients with BCR-ABL negative ALL.

FY*A silencing by the GATA-motif variant FY*A(-69C) in a Caucasian family.

BACKGROUND: The c.1-67C variant polymorphism in a GATA motif of the FY promoter is known to result in erythroid-specific FY silencing, that is, in Fy(a-) and Fy(b-) phenotypes. A Caucasian donor presented with the very rare Fy(a-b-) phenotype and was further investigated. STUDY DESIGN AND METHODS: Genomic DNA was analyzed by sequencing to identify the cause of the Fy(a-b-) phenotype. Samples were collected from some of his relatives to establish a correlation between the serology and genotyping results. Red blood cells were analyzed by gel column agglutination and flow cytometry. Genomic DNA was analyzed on genotyping microarrays, by DNA sequencing and by allele-specific PCR. RESULTS: In the donor, a single-nucleotide polymorphism T>C within the GATA motif was found at Position c.1-69 of the FY promoter and shown to occur in the FY*A allele. His genotype was found to be FY*A(-69C), FY*BW.01. In six FY*A/FY*B heterozygous members of the family, a perfect correlation was found between the presence vs. absence of the FY*A(-69C) variant allele and a Fy(a-) vs. Fy(a+) phenotype. CONCLUSION: The location of the c.1-69C polymorphism in a GATA motif whose disruption is known to result in a Fy null phenotype, together with the perfect correlation between the presence of the FY*A(-69C) allele and the Fy(a-) phenotype support a cause-effect relationship between the two.

Performance characteristics of consensus approaches for small and minor paroxysmal nocturnal hemoglobinuria clone determination by flow cytometry.

BACKGROUND: Evaluation of paroxysmal nocturnal hemoglobinuria (PNH) clones by flow cytometry (FCM) is not standardized and is associated with consistent inter-laboratory variability. METHODS: In order to rule out the influence of particular approach in generating final results, we analyzed the performance characteristics of individual consensus strategies for small to intermediate (1%-20%) and minor (<1%) PNH clones within the white blood cell (WBC) and red blood cell (RBC) compartments with sensitivity up to 0.1%. RESULTS: Coefficient of variation (CV) for precision/reproducibility analysis ranged from 0.67%/1.49% to 2.56%/3.09% for granulocytes, from 0.93%/3.09% to 7.76%/12.06% for monocytes and from 0.41%/4.73% to 6.53%/5.1% for RBCs. Coefficient of determination (r2) for linear regression analysis ranged from 0.95 to 0.99, Wilcoxon ranks test showed no statistically significant differences (p>0.05), Bland-Altman analysis demonstrated performance agreement with mean bias ranging from -0.18 to 1.24. CONCLUSIONS: Our results confirmed very good performance characteristics for precision and reproducibility analysis, excellent correlation and favorable agreement between strategies, suggesting that reported inter-laboratory variability is related mainly to incorrect performance and/or insufficient experience with PNH testing by flow cytometry, rather than to relevant limitations of any particular approach.

Intra- and interlaboratory variability of paroxysmal nocturnal hemoglobinuria testing by flow cytometry following the 2012 Practical Guidelines for high-sensitivity paroxysmal nocturnal hemoglobinuria testing.

BACKGROUND: Sutherland et al. recently published the Practical Guidelines for high-sensitivity detection of paroxysmal nocturnal hemoglobinuria (PNH) clones by flow cytometry (FCM), containing concise protocols for PNH testing. METHODS: Using this approach, we studied the intra- and interlaboratory variability observed in a multicenter study in which fresh blood samples containing three clinically relevant PNH clone sizes within the granulocytic, monocytic, and red blood cell (RBC) populations were shipped to each participating center. RESULTS: Coefficients of variation (CVs) for precision/reproducibility analysis ranged from 0.01%/0.02% to 0.48%/0.45% (big clone), from 0.69%/1.52% to 4.24%/5.80% (small-intermediate clone), from 1.47%/3.91% to 15.01% /17.83% (minor clone) for PNH white blood cells (WBCs) and from 0.24%/0.48% to 1.76%/1.83% (big clone), from 0.80%/1.14% to 2.39%/4.45% (small-intermediate clone), from 1.09%/3.36% to 10.54%/10.23% (minor clone) for PNH RBCs, respectively. Linear regression analysis showed excellent performance correlation between centers (r > 0.99), Wilcoxon rank test revealed no statistically significant differences for PNH granulocytes, monocytes, and RBCs (P > 0.05%), Bland-Altman analysis demonstrated good performance agreement for all target PNH clones (mean bias ranging from -1.47 to 0.71). CONCLUSION: Our results demonstrate very good intra- and interlaboratory performance characteristics for both precision and reproducibility analyses and excellent correlation and agreement between centers for all target PNH clone sizes. Our data confirm the reliability and robustness of the recently published Practical Guidelines approach for high sensitivity PNH testing by flow cytometry and suggest that such an approach represents an excellent basis for standardization of PNH testing by flow cytometry.

Novel human multiple myeloma cell line UHKT-893.

UNLABELLED: We established and characterized a new IL-6 dependent multiple myeloma (MM) cell line UHKT-893 from the bone marrow of a relapsed 57-year-old woman. RESULTS: Using nephelometry, cells with plasma cell phenotype and morphology were found to secrete IgG and free kappa (κ)-light chain of immunoglobulin. κ-Light chain was also recognized intracellularly by flow cytometry and by mass spectrometry. VH4-39 region of IgVH genes was rearranged and somatically hypermutated. Cytogenetic analysis of cells revealed new chromosome abnormalities in all breakpoints unique in both MM patients and cell lines - t(1;6), t(1;11), t(5;15), t(5;21), +der(11;15) and der(16). IL-6 independent subline UHKT-893a was established by adaptation to descending IL-6 concentration, while the original cell line keeps on maintaining its IL-6 dependency. CONCLUSION: The cell line provides a suitable material for cellular and molecular studies of tumor abnormalities, with potentially unique mutagenic features of myeloma disease. It may be utilized for human hybridoma construction and vaccine development. Both IL-6 dependent and independent cell clones represent an important model for studies of myeloma cell growth and resistance emerging during targeted therapy.

The effect of simvastatin and fenofibrate on the expression of leukocyte adhesion molecules and lipopolysaccharide receptor CD14 in type 2 diabetes mellitus.

BACKGROUND: Mixed hyperlipidemia is often associated with type 2 diabetes mellitus and contributes to atherosclerosis progression in diabetes patients. Leukocyte activation plays an important role in atherogenesis. Both statins and fibrates are used in the treatment of mixed dyslipidemia, but their specific effect on leukocyte function remains to be elucidated. We have therefore compared the effect of simvastatin and fenofibrate on several leukocyte activation markers in diabetes patients. METHODS: Twenty patients with type 2 diabetes and mixed hyperlipidemia were sequentially treated with simvastatin (20 mg/day) and fenofibrate (200 mg/day) in a randomized cross-over study (12 weeks each treatment). We measured adhesion molecules LFA-1, VLA-4 and CD18; in addition, lipopolysaccharide receptor CD14 on monocytes was analyzed as a marker of innate immunity. Leukocyte expression of these molecules was quantified using flow cytometry. Laboratory examinations were done at baseline and at the end of each treatment. Baseline values were compared to those of 29 healthy controls. RESULTS: Expression of integrin CD18 (in all leukocyte populations), lipopolysaccharide receptor and VLA-4 (on lymphocytes only) was significantly higher in patients than in controls. Both treatments resulted in significant decrease in CD18 and CD14 expression; LFA-1 and VLA-4 were not influenced. CONCLUSIONS: Both simvastatin and fenofibrate had similar favorable effect on leukocyte activation markers. This result supports the use of both statins and fibrates for the treatment of mixed hyperlipidemia in patients with type 2 diabetes mellitus.

Manifestation of atypical hemolytic uremic syndrome caused by novel mutations in MCP.

Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Mutations in genes encoding regulators of the alternative complement pathway (CFH, MCP, C3, CFI, CFB, THBD, and CFHR1-5) are connected with this disease. Polymorphisms (SNPs) in these genes might also influence the manifestation of aHUS. We have analyzed the genes of CFH, CFI, MCP, and C3 in a cohort of 10 unrelated Czech patients with clinically diagnosed familial aHUS. Surprisingly, 4 patients had mutations only in MCP, without mutations in any of the other genes that cause aHUS. Mutations, as yet unpublished, were widely distributed over the gene (SCR2 domain, signal peptide, and cytoplasmic region). The phenotype of the patients and their close relatives (14 individuals) was also investigated. Functional examination of MCP was also provided and proved lower expression on granulocytes in all mutations. Severity of disease varied, but onset was never earlier than 5 years of age. Penetrance of disease was 50% among carriers. We found that the severity and recurrence of the disease within families varied and might also be dependent on SNPs. Mutations in the MCP gene seems to be a common etiology of aHUS in Czech patients.

Unusually long survival of a 67-year-old patient with near-tetraploid acute myeloid leukemia m0 without erythroblastic and megakaryocytic dysplasia.

Patients with near-tetraploid acute myeloid leukemia (NT-AML) typically have poor survival. We present the case of a 67-year-old Caucasian male with NT-AML M0 who had an unusually long first complete remission of 51 months and an overall survival of 80 months. The only characteristic distinguishing him from other previously described patients with NT-AML was the absence of erythroblastic and/or megakaryocytic dysplasia (EMD) at diagnosis. Molecular-genetic testing for AML fusion transcripts associated with a favorable prognosis (PML/RARα,AML1/ETO, and CBFß/MYH11) were negative, as were other prognostic markers like MLL-PTD,FLT3-ITD, or mutations of FLT3-D835,NPM1, or CEBPA. Expression studies of ERG,MN1, and EVI1 revealed overexpression of ERG only. The absence of EMD may be a useful prognostic/diagnostic feature of this new rare subtype of NT-AML.

PBPC collections: Management, techniques and risks.

We evaluated the efficiency, safety and risks of three techniques which were used for autologous PBPC collections: (a) large-volume leukapheresis (LVL), (b) standard collections, and (c) a new modified technique which was named as "Mixed" collections. In spite of the fact that the standard and LVL collection techniques are used routinely, there may occur special conditions in which the procedures cannot be recommended. Some patients may suffer from serious clinical complications and they cannot tolerate either standard procedures with administration of higher doses of ACD-A, or the high extent of procedure in the course of LVL. We tried to find the safe and efficient collection technique which could help this group of patients to overcome their problems. The "Mixed" collection technique could be such a choice. The numbers of 136 autologous PBPC collections were performed in 98 patients who suffered from hemato-oncological diseases. We evaluated the results of (a) 93 LVL (more than 3 TBV, total blood volumes of the patients were processed; anticoagulation: ACD-A and Heparin), (b) 16 Standard procedures (less than 3 TBV were processed; anticoagulation: ACD-A), and (c) 27 "Mixed" collections (less than 3 TBV of patients were processed; anticoagulation: ACD-A+ Heparin). Collections were performed by the use of separator Cobe Spectra, Caridian. In patients (a) with a good effect of mobilization (precollection CD 34+ cells in blood higher than 20×10(3)/mL) we prepared almost the same median dose of CD 34+ cells from the standard and "Mixed" collections, 3.8 and 4×10(6)/kg, respectively. In LVL the median yield of CD 34+ cells was 8.2×10(6)/kg. In patients (b) who were mobilized weakly (precollection CD 34+ cells in blood lower than 20×10(3)/mL), LVL enabled to prepare 1.5×10(6) of CD 34+/kg from one collection, while the median yield of CD 34+ cells from the standard and "Mixed" collections was 0.9 and 1.2×10(6)/kg. All the standard, LVL and "Mixed" procedures were tolerated well without any serious adverse reactions. We detected 22 adverse reactions, but only three reactions were associated directly with the procedure. Mild hypocalcemia (2) and hypotensive reaction (1) were transient and treated efficiently. Procedures could continue and were finished according to the planned programme. Other reactions were related either to the insufficient function of central venous catheter or to the poor clinical condition of the patients. LVL enabled to get a higher yield of CD 34+ cells than the Standard and "Mixed" collections in well mobilized patients as well as in weakly mobilized patients. We observed the similar efficiency in standard and "Mixed" collections in well mobilized and weakly mobilized patients. We can recommend LVL in all patients who can tolerate it due to a greater chance of collecting higher yields of progenitor cells. In the weakly mobilized patients LVL offers a greater chance of collecting at least a minimum amount of CD 34+ cells needed for transplantation. "Mixed" collections may be used as an alternative technique under the circumstances in which standard or LVL cannot be recommended - like in patients who do not tolerate a high amount of citrate or a high extent of the procedure, e.g. patients with cardiac arrhytmia, impaired liver or renal function or unstable vital signs.