[GUIDELINES FOR DIAGNOSIS AND TREATMENT OF PATIENTS WITH MYELODYSPLASTIC SYNDROME].

Myelodysplastic syndrome Working Group of the Croatian Cooperative Group for Hematologic Diseases (CROHEM), Referral center of the Ministry of Health of the Republic of Croatia for diagnostics and treatment of MDS, as well as the Croatian Society for Haematology of the Croatian Medical Association have made Croatian guidelines for diagnosis and treatment of myelodysplastic syndrome (MDS). MDS is a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, dysplasia, cytopenia and risk of transformation to acute myeloid leukemia (AML). Diagnosis is based on morphological characteristics of hematopoietic cells supplemented with the cytogenetic analysis and bone marrow flow cytometry. Due to great differences in the natural course of the disease, i.e. time to progression to AML and the expected time of survival several scoring systems have been developed to determine the disease risk. The treatment of patients with MDS is based on the risk factors of the disease as well as the individual risk of treatment.

Validation of a laboratory and hospital information system in a medical laboratory accredited according to ISO 15189.

INTRODUCTION: The aim of the study was to present a protocol for laboratory information system (LIS) and hospital information system (HIS) validation at the Institute of Clinical Chemistry and Laboratory Medicine of the Merkur University Hospital, Zagreb, Croatia. MATERIALS AND METHODS: Validity of data traceability was checked by entering all test requests for virtual patient into HIS/LIS and printing corresponding barcoded labels that provided laboratory analyzers with the information on requested tests. The original printouts of the test results from laboratory analyzer(s) were compared with the data obtained from LIS and entered into the provided template. Transfer of data from LIS to HIS was examined by requesting all tests in HIS and creating real data in a finding generated in LIS. Data obtained from LIS and HIS were entered into a corresponding template. The main outcome measure was the accuracy of transfer obtained from laboratory analyzers and results transferred from LIS and HIS expressed as percentage (%). RESULTS: The accuracy of data transfer from laboratory analyzers to LIS was 99.5% and of that from LIS to HIS 100%. CONCLUSION: We presented our established validation protocol for laboratory information system and demonstrated that a system meets its intended purpose.

[The purpose of clinical laboratory accreditation in transplantation medicine]. / Znacenje akreditacije klinickih laboratorija u transplantacijskoj medicini.

Although transplantation of solid organs has become a more standardized method of treatment, liver transplantation represents an exceptional multidisciplinary clinical procedure requiring understanding of specific pathophysiological changes that occur in the end stage of liver disease. Liver transplantation has been performed at Merkur University Hospital since 1998, with 360 transplantations performed to date. The most common indications are alcohol liver disease, cirrhosis caused by hepatitis B and C virus, hepatocellular carcinoma and cryptogenetic liver cirrhosis. Laboratory tests required for liver transplantation are performed at Department of Clinical Chemistry, Merkur University Hospital, accredited according to ISO 15189 in 2007 for the areas of clinical chemistry, laboratory hematology and coagulation, laboratory immunology-cell immunophenotyping, and molecular diagnosis. The complexity of liver transplant patients requires constant interaction between the anesthesiologist team and clinical laboratory, which has to ensure fast and efficient intraoperative monitoring of biochemical and liver profile: electrolytes and acid-base status, complete blood count, coagulation profile and monitoring of graft function according to the individual patient's health status. Dynamics of intraoperative changes is measured in whole arterial blood samples on a Nova Biomedical Stat Profile Critical Care Xpress mobile acid-base analyzer. Frequent monitoring of ionized calcium and magnesium levels is very important because of citrated blood transfusion and for appropriate therapeutic procedure. During anhepatic stage, there is a progressive increase in lactate level concentration. After reperfusion, a rapid increase in lactate clearance is an excellent indicator of stable graft initial function and its adequate size. During the transplantation procedure, there is usually a biphasic acid-base disturbance characterized by metabolic acidosis and then by metabolic alkalosis. The loss of base equivalents starts during the dissection stage and accelerates during the anhepatic stage. Fast and efficient intraoperative monitoring of hematological tests and coagulation status is of great help in detecting the cause of possible hemorrhage and consequential complications during transplantation procedure. The possibility of organ and tissue transplantation mostly depends on well regulated international cooperation in the areas of donating, transplanting and exchange of required organs and tissues, while laboratory test results must be comparable regardless of their geographical area, methodology employed or analytical equipment used, which is mainly warranted through accreditation according to the international ISO 15189 standard.

[Diagnostic and prognostic significance of CD45 cell surface antigen expression in hematologic malignancies with main focus on acute leukemias]. / Dijagnosticko i prognosticko znacenje izrazaja biljega CD45 u hematoloskim zlocudnim bolestima s posebnim osvrtom na akutne leukemije.

CD45 cell surface antigen is a transmembrane protein with tyrosine phosphatase activity, expressed by all nucleated cells of hematopoietic origin, except erythrocytes and platelets. Monoclonal antibodies directed against CD45 represent irreplaceable tool in differential diagnosis of hematologic and other, non-hematologic low differentiated malignancies, primarily in cases of: extranodal lymphomas, non-hematologic malignancies with nodal or bone marrow localization or their metastases in mentioned sites. As cell surface immunophenotype marker, CD45 is of great value in differentiation of lymphoproliferative diseases subtypes. By flow cytometry, based on CD45 expression, the malignant cell population is being identified and that fact is used in, not only diagnosis, but also in detection of minimal residual disease, especially in cases of CD45 negative acute leukemias. Incidence of childhood CD45 negative acute lymphoblastic leukemias (ALL) is about 10%. Children diagnosed with low CD45 expression ALL generally have better prognosis than those with high CD45 expression, especially when cut-off value for CD45 expression is set on 90%. We have analyzed CD45 expression by flow cytometry in 28 consecutive patients diagnosed with ALL in our institution during a 5-year period. Among these patients 7.1% were CD45 negative. A positive correlation between CD45 and CD20 expression was found, and a negative correlation between CD45 and CD34. In our group of patients, CD45 expression did not have any influence on survival.

[Standardization in laboratory hematology by participating in external quality assurance programs]. / Standardizacija u laboratorijskoj hematologiji sudjelovanjem u programima vanjske procjene kvalitete.

Since 1985, Department of Clinical Chemistry and Laboratory Medicine, Merkur University Hospital, Zagreb, has been participating in the International External Quality Assessment Scheme for Hematology (IEQAS-H) organized by the World Health Organization (WHO). Owing to very good results, in 1987 the Department received a certificate of participation in this control scheme. Department has been cooperating in the external quality assessment program in laboratory hematology which has been continuously performed in Croatia since 1986 by the Committee for External Quality Assessment Schemes under the auspices of the Croatian Society of Medical Biochemists and School of Pharmacy and Biochemistry, University of Zagreb. Nowadays, 186 medical biochemical laboratories are included in the National External Quality Assessment program, which is performed three times per year. Our Department has participated in the international projects of the European Committee for External Quality Assurance Programs in Laboratory Medicine (EQALM).

[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.

Accreditation of medical laboratories in Croatia--experiences of the Institute of Clinical Chemistry, University Hospital "Merkur", Zagreb.

Since 2003 when the international norm for implementation of quality management in medical laboratories (EN ISO 15189, Medical laboratories--Particular requirements for quality and competence) was established and accepted, accreditation has become practical, generally accepted method of quality management and confirmation of technical competence of medical laboratories in the whole world. This norm has been translated into Croatian and accepted by the Croatian Institute for Norms as Croatian norm. Accreditation is carried out on voluntary basis by the Croatian Accreditation Agency that has up to now accredited two clinical medical biochemical laboratories in the Republic of Croatia. Advantages of accredited laboratory lie in its documented management system, constant improvement and training, reliability of test results, establishing users' trust in laboratory services, test results comparability and interlaboratory (international) test results acceptance by adopting the concept of metrological traceability in laboratory medicine.

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.

Cytological detection of lymphoma in Douglas aspirate.

Except in primary effusion lymphoma (PEL), serous effusions with lymphomatous cells in non-Hodgkin lymphoma (NHL) are not frequently seen as first manifestation of disease. In NHL lymphoplasmacytic lymphoma (LPL) the spleen, lymph nodes, and bone marrow are frequently sites of disease and this type of NHL is usually associated with a serum paraprotein of IgM type accompanied by the clinical syndrome of Waldenström macroglobulinemia. Our patient with NHL LPL type presented in this report had less frequently seen involvement of gastrointestinal tract and clinically was first manifested as effusion in Douglas space. Cytological evaluation as well immunoanalyses of effusion in such cases is essential, and various ancillary studies, because of differential diagnostic problems of lymphomas in serous effusions, first include reactive lymphocytoses and small round-cell tumors (SRCT). In our patient, cytology of effusion revealed cytomorphologically atypical lymphomatous cells with plasmocytoid differentiation. Immunocytochemical and flow cytometry analysis confirmed lymphoid cell differentiation and pathohistological diagnosis of CD20(+) LPL was set after pathohistological analysis of resected ileum.

[Single cell--definitive diagnosis! Where the profession ends and the art begins?]. / Jedna stanica--konacna dijagnoza! Gdje prestaje struka i pocinje umjetnost?

Cell is a morphologically and functionally the tiniest living organism, present from the very beginning of life on the Earth. Specialized cell types make up specific tissues and organs of the human body. Cell itself and cell elements are liable to morphological, functional, phenotypic and genotypic alterations in various physiological and pathological states. These alterations are studied by cytodiagnosis to diagnose the disease at cellular level. Cytologic examinations belong to the group of morphological, non-aggressive or minimally invasive tests that are easy to perform for both the patient and the professional. In addition, these tests are highly reliable and preferred to the related diagnostic procedures for providing immediate orientation and definitive diagnosis, thus saving both time and money. With the introduction of adjunctive technologies such as cell-surface marker analysis, computer image analysis, molecular and cytogenetic technologies performed on cytologic smears, cytology has become an ever more important factor in the diagnosis, subtyping and prognosis of malignant tumors. Thorough knowledge of cell morphology is a basis for proper performance and understanding of cytology techniques. A cytologist needs to be familiar with clinical manifestations of the disease and to be informed on all relevant data on the patient and his current and previous medical history, in order to be able to issue findings that are understandable and usable to all clinicians and patients. It requires close collaboration between the cytology laboratory and the ward, and among the cytologist, the patient and the clinician. Good collaboration with other diagnostic professions such as pathology, laboratory, molecular and cytogenetic diagnosis is by no means less important. Cytology as a profession implies knowledge of the morphological characteristics of normal cells and cells in various physiologic states, along with due knowledge of the morphology, phenotypic and genetic features of pathologic alterations. However, synchronizing and combining cytologic morphology with other sophisticated diagnostic procedures to reach an accurate diagnosis, subtyping and prognosis of tumor disease is artistry indeed.

[The disappearance of CD20 positive lymphocytes in the pleural effusion after intrapleural application of rituximab]. / Nestanak CD20 pozitivnih limfocita iz pleuralnog izljeva nakon intrapleuralne primjene rituksimaba u bolesnice s folikularnim limfomom.

A 63 year old woman with non-Hodgkin lymphoma presented with unilateral pleural effusion, which when aspirated revealed CD19 and CD20 positive malignant cells. Prior to this, the patient had received several lines of chemotherapy (CHOP, VAD, FED) with no effect on pleural effusion. Repeated percutaneous drainage procedures were unable to control the effusion either. Rituximab was therefore instilled in a dose escalating manner via repeated pleurocenteses. Fifty days after the application of rituximab, pleural effusion was still present but reduced in size. Flow cytometry and immunocytochemistry performed on the same day showed CD19 positive cells which were lacking CD20 epitope, which could be explained by either engagement or destruction of the CD20 epitope upon interaction with rituximab making the detection of the CD20 molecule impossible by routine flow cytometry. What is especially interesting is the fact that even 50 days after the application of rituximab intrapleurally no new CD20 positive cells could be found in the pleural effusion by immunochemistry or flow cytometry, opening an interesting issue concerning the length of rituximab's activity when applied locally. Although our patient had no adverse effects, further analysis of rituximab's activity and safety when applied intrapleurally is warranted.

Cholinesterase unit establishment and issuing of >>Warning Cards<< for carriers of suxamethonium sensitive serum butyrylcholinesterase variants.

Recognition of butyrylcholinesterase (EC 3.1.1.8) variants in human serum is essential to identify patients who may be susceptible to a prolonged reaction of suxamethonium and mivacurium, short-acting muscle relaxants. Thus they can be given appropriate advice along with their relatives who may be similarly affected. Therefore, Cholinesterase Unit for detection of individuals, carriers of inherited suxamethonium sensitive butyrylcholinesterase variants was established at the Institute for Clinical Chemistry of the Clinical Hospital >>Merkur<<, Zagreb, Croatia. A study was conducted on sera from patients referred to the Unit. Butyrylcholinesterase variants were determined by measuring the enzyme activity and inhibition by specific inhibitors in the sera of 384 patients and of the members of seven families. Cholinesterase Unit issued >>Warning Cards<< to the carriers of inherited serum butyrylcholinesterase variants in order to avoid prolonged apnea that suxamethonium might cause.