[The unclassifiable myeloproliferative neoplasm--morphological, cytogenetic and clinical features]. / Neklasificirana mijeloproliferativna neoplazma--morfoloske, citogenetske i klinicke karakteristike.

Myeloproliferative neoplasm, unclassifiable (MPN,U) has clinical, laboratory and morphological features of an MPN but fails to meet the criteria for any of the specific MPN entities. Because overlapping features, morphological findings in bone marrow, BCR-ABL1 fusion gene, V617F JAK2 mutation and cytogenetic abnormalities were analyzed in ten patients diagnosed with MPN,U. Bone marrow biopsy showed hypercellularity with trilineage myeloproliferation, dispersed megakaryocytes with mild pleomorphism and mature nuclei, and absence of reticulin fibrosis. All patients were BCL-ABL1 negative, while V617F JAK2 mutation was found in 6 of 8 patients. Trisomy 8 was found in two patients and t(6;12)(q12;p13) in one patient. Morphological features of MPN,U are nonspecific, however, in study cases they were most similar to diagnostic morphological features of polycythemiea vera. The high frequency of V617F JAK2 mutation in MPN,U cases analyzed revealed that its presence does not confirm a specific type of MPN.

[Association of CD34 cell surface antigen expression with cytomorphological characteristics of acute promyelocytic leukemia blasts and clinical characteristics of patients: one center experience]. / Povezanost izrazaja biljega CD34 s morfoloskim znacajkama blasta u akutnoj promijelocitnoj leukemiji i klinickim znacajkama oboljelih: iskustvo jednog centra.

The aims of the study were to investigate the association between cytomorphology and immunophenotypic expression of CD34 cell surface antigen of blasts and their relationship with clinical and laboratory characteristics of patients with acute promyelocytic leukemia (APL). Sixteen consecutive patients (male 69% and female 31%) diagnosed with APL at Department of Hematology, Merkur University Hospital between August 1998 and December 2010 were included in the study. The mean age of patients was 43.9 (range: 18-78, SD 14.9). The patients' clinical and laboratory features, cytomorphological characteristics of APL-blasts and their immunophenotype determined by flow cytometry were analyzed. Patients were divided into two groups, CD34- and CD34+, and were then compared according to clinical and laboratory characteristics. There was no difference according to age, sex or white blood cell count between two groups. The mean value of hypogranular/agranular APL-blasts was markedly higher in CD34+ group than CD34- group (34%, range 9-60, SD 24.4 vs. 11.5%, range 0-38, SD 13.7), with borderline statistical significance (P=0.055). CD34- patients had significantly better overall survival than CD34+ ones (P=0.02). Patients without Auer rods detected in APL-blasts had higher CD34 expression (69.4% +/- 33.8) compared to patients with detected Auer rods (7.3% +/- 24.8), but statistical significance was not reached (p=0.053). Our results are consistent with the results of other published studies and point to the fact that higher CD34 expression and lower cytoplasmic granularity of APL-blasts are factors that seem to define a specific subgroup of APL patients. Together with other diagnostic tools currently available, they could be of value in planning treatment of APL patients.

[Flow cytometry and acute renal rejection confirmed by histopathologic analysis]. / Protocna citometrija i akutno odbacivanje bubrega dokazano patohistoloskim nalazom.

Transplantation of solid organs, tissues or hematopoietic cells is now standard in the treatment of patients with terminal stage disease in order to cure and improve the recipients' quality of life. The study included 54 patients having undergone single or multiple organ transplantation. All patients received a combination of immunosuppressant therapy consisting of corticosteroids, calcineurin inhibitor (cyclosporine; tacrolimus), anti-CD25 (daclizumab) and mycophenolate-mofetil. In 24 patients, acute rejection was stratified by histopathologic analysis of renal biopsy. Fifteen highly sensitized patients were administered antithymocyte globulin (ATG) therapy. Absolute count and percentage of B/T lymphocyte subsets, NK cells and CD25+ or CD69+ activated T cells were measured on a flow cytometer (EPICS XL, Coulter) using single platform standardized protocol. Upon ATG therapy, rapid decline to a very low level of T and NK cell lymphocyte count was observed, as well of B lymphocytes, resulting in redistribution of lymphocyte compartment. Between consecutive measurements, kinetic changes of lymphocyte subset numbers (absolute count or percentage) did not differ in a large spectrum of immune parameters between the groups with and without rejection episode and having received quadruple immunosuppressive induction and maintenance therapy. Immunologic monitoring must be initiated prior to transplantation and continued consistently and frequently post-transplantation. Such a program is expensive and time-consuming and stressful for the patient, therefore, prospective studies should identify whether treatment decisions can be based reliably on these immune parameters. Serial measurement of immune cell counts is necessary for maintenance of ATG therapy and could be useful for monitoring patient recovery.

[Fine needle aspirate of lymph node as the analytical sample for immunophenotyping]. / Punktat limfnog cvora kao analiticki uzorak za fenotipizaciju stanica.

In modern clinical laboratory routine, cell analysis by flow cytometry means help in setting up the diagnosis by determination of B-lymphocyte clonality and thus separation of benign and malignant lymphoproliferative diseases. The aim of this study was to assess the value of cytologic diagnosis and adequacy of the material obtained by fine needle aspiration (FNA) of lymph nodes for flow cytometry analysis in cases of benign lesions and primary malignant lesions of lymph nodes. In addition, the aim was to determine B-lymphocyte clonality in different groups of benign and malignant lymph node lesions. The study was based on medical documentation, cytologic smears of FNA lymph node samples and results of flow cytometry immunophenotyping. A total of 239 patients were included over a one-year period. Patients were classified according to cytologic findings in the groups of non-Hodgkin's lymphoma of B cell origin (55%), benign lymphoproliferative disease (22%), undefined group of monomorphic population of lymphatic cells (16%), and the rest in the group of non-Hodgkin's non B cell origin. Study results showed FNA to be an appropriate method for obtaining sufficient numbers of cells for analysis by flow cytometry because there was no inadequate samples in our study group. In some cases of monomorphic lymphoid cell population, cytologic diagnosis was limited to small cell lymphomas, so determining the clonality by flow cytometry is crucial in separating malignant from benign lymphoproliferative disease. It is concluded that FNA associated with the flow cytometry method is a simple and safe method in the diagnosis of lymphoproliferative disease.

[Significance of participation in programs of external quality assessment in molecular diagnostic--our experience]. / Znacenje sudjelovanja u programima vanjske procjene kvalitete u molekularnoj dijagnostici--vlastita iskustva.

Harmonization of molecular diagnostic tests in laboratories in the Republic of Croatia has only just started. According to laboratory accreditation standard ISO 15189 participation in external quality assessment (EQA) schemes or programs is a prerequisite and support tool for clinical laboratory accreditation process. As there are no national quality assurance schemes yet, an European external quality assessment (EQA) scheme or program should be found. Because of variation in the molecular diagnostic test performance of clinical laboratories across Europe, EQA is recognized as a system whereby a set of reagents and techniques are assessed by an external provider making inter-laboratory performance comparability possible through already integrated recommendations and practice guidelines of molecular diagnostic test performance. Today, wide range of various EQA schemes and programs already in action have been available and most of them began within the last ten years. This paper is therefore intended to present and summarize the four-year EQA activities in the Institute of Clinical Chemistry, Merkur University Hospital, in three different international EQA schemes: United Kingdom National External Quality Assessment Scheme (UK NEQAS), the European Molecular Genetic Quality Network (EMQN) and Multi-National External Quality Assay program (EQUAL- qual)) and to point out their educational role in standardization of laboratory performance of any test intended for patient testing. from a laboratory point of view.

Flow cytometry immunophenotyping (FCI) of fine needle aspirates (FNAs) of lymph nodes.

Flow cytometry immunophenotyping (FCI) has an important role in the clinic work-up of fine needle aspirates (FNAs) of lymph nodes. Its standardization has been defined by proposed analytical protocols and procedures used to assure proper analytical results also in those non-routine samples. In Institute of Clinical Chemistry, "Merkur" University Hospital, FCI is accredited method according to laboratory accreditation standard ISO 15189. According to this laboratory accreditation standard, participation in external quality assessment (EQA) programs is a prerequisite for assuring integrity and quality of the entire laboratory process. A critical analysis of our institutional experience in the feasibility of FCI of the material obtained by FNA of lymph nodes with suspected lymphoma represented the purpose of the study. During an eight-year period in Institute of Clinical Chemistry, "Merkur" University Hospital, a total of 1295 FNA analysis was done, 245 of them with a possible diagnosis of B-cell Non-Hodgkin lymphomas (B-NHL) formed the basis of the study. Lymphocytes were isolated on density gradient according to Boyum et al. The average feasibility of FNAs for FCI analysis was 86% (ranged 78-93%). An acceptable total cell number in FNAs for FCI analysis (4257) was established. In total population of respondents statistical significances in expressions of cellular antigens CD3, CD5, CD22, CD23, CD19 and CD5 on B-cells (CD5+CD19+) between patient's with final diagnosis of benign, reactive lymphoid proliferations and patient's with diagnosis of B-NHL were found. EQA results analysis showed that all results were either inside target values (X +/- 1SD) or inside accepted values (X +/- 2SD). Compatibility of the restriction of immunoglobulins light chains determinated by FCI and cytomorphology diagnosis depends on the choice of criterion values of the light chains ratio which determine the monoclonality. According to the matrix of shares of all classified data of retained neural network, ranges of diagnostic sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and prevalency of 82%, 72%, 93%, 48%, and 72% were produced. As a conclusion, FCI is a reliable methodology for phenotyping FNAs of lymph nodes with suspected B-NHLs detecting their clonality easily.

External quality assessment in clinical cell analysis by flow cytometry. Why is it so important?

Participation in external quality assessment is an integral part of laboratory work and mandatory when the results have a clinical application, which is one of the requirements of standard 15189 for accreditation of medical laboratories. Institute of Clinical Chemistry, the first laboratory accredited for clinical cell analysis by flow cytometry in Croatia, participated in UKNEQAS for Leukocyte Immunophenotyping in 3 schemes: "Immune Monitoring", "CD34 Stem Cell Enumeration" and "Leukaemia Immunophenotyping". For sample processing on EPICS XL flow cytometer, lyse/no wash preparation technique with ammonium chloride (NH4Cl) or ImmunoPrep lysing reagent was employed. In "Immune monitoring" programme CD45/sideward light scatter (SSC) proposed gating strategy was adopted for lymphocyte subsets, while modified ISHAGE protocol was used for CD34+ cell enumeration. Absolute count determination was performed on flow cytometer using FlowCount beads solution. In the period from the beginning of 2006 until the middle of 2009 a total number of 100 stabilized whole blood samples were processed. The relative and absolute enumeration results for lymphocyte subsets were within tolerable limits, in 97.1 and 97.1% of cases, and 95 and 90% of CD34+ cell enumeration, respectively. In immune monitoring CD45/SSC proposed gating strategy is the most frequent analysis used (> 85% participants) and ISHAGE protocol for CD34+ cell determination with continuous rise from 76 to 83%. A number of participants who accept beads method for absolute count enumeration on flow cytometer get greater, 69 to 86%, while FlowCount was the second of bead-based techniques used (25 and 35%). Sample treatment in lyse/no wash technique using NH4Cl lysing solution was dominant procedure used by more than 1/3 participants, although its home made solution has replaced slowly by commercial reagents. The unacceptable results, 6 of 244, were obtained for 20 most frequently determined cell antigens in "Leukaemia Immunophenotyping" samples screened for leukaemia/lymphoma. Processing results of all participants showed that the deviation from laboratory guidelines and the use of older methods for cell identification, quantification of cell counting on haematology analyser, or usage an antibody conjugated with fluorochrome lesser fluorescence quantum often lead to an unacceptable result, although is noticeable trend to accept new referrals and protocols to reduce the inter-laboratory differences.