International Journal of Laboratory HematologyVolume 36, Issue 2 p. 222-226 Letter to the Editor Effect of EDTA-anticoagulated whole blood storage on cell morphology examination. A need for standardization Joan-Lluis Vives-Corrons, Joan-Lluis Vives-Corrons [email protected] Red Cell Pathology Unit, Hospital Clinic, University of Barcelona, Barcelona, SpainSearch for more papers by this authorCarol Briggs, Carol Briggs Department of Haematology Evaluation, University College London Hospital, London, UKSearch for more papers by this authorRamon Simon-Lopez, Ramon Simon-Lopez Beckman Coulter Eurocenter, Nyon, SwitzerlandSearch for more papers by this authorStephanie Albarede, Stephanie Albarede CTCB, 33 route de Bayonne, 31300 Toulouse, FranceSearch for more papers by this authorBarbara de la Salle, Barbara de la Salle General Haematology, UKNEQAS, Watford, UKSearch for more papers by this authorZlata Flegar-Meatrii, Zlata Flegar-Meatrii Institute of Clinical Chemistry, University Hospital Merkur, Zagreb, CroatiaSearch for more papers by this authorAida Nazor, Aida Nazor Institute of Clinical Chemistry, University Hospital Merkur, Zagreb, CroatiaSearch for more papers by this authorAnne Guyard, Anne Guyard Ansm (National agency of medicine and health product safety), Saint-Denis, FranceSearch for more papers by this authorThomas Lipsic, Thomas Lipsic St. Elisabeth Oncology Institute, Bratislava, SlovakiaSearch for more papers by this authorYukata Nagai, Yukata Nagai Faculty of Sports and Health Science, Daito Bunka University, Tokyo, JapanSearch for more papers by this authorMariana Patiu, Mariana Patiu OncologyInstitute, Cluj Napoca, RomaniaSearch for more papers by this authorJosep Piqueras, Josep Piqueras LaboratorisClínics, Hospital de la Valld'Hebron, Barcelona, SpainSearch for more papers by this authorMaria Jesus Capel, Maria Jesus Capel LaboratorisClínics, Hospital de la Valld'Hebron, Barcelona, SpainSearch for more papers by this authorMarjan Van Blerk, Marjan Van Blerk Department of Clinical Biology, Institute of Public Health, Brussels, BelgiumSearch for more papers by this authorJianbiao Wang, Jianbiao Wang Department of Medical Laboratory, Ruijin Hospital ShanghaiJiaotong, University School of Medicine, Shanghai, ChinaSearch for more papers by this authorChristophe Marzac, Christophe Marzac Hôpital Saint-Antoine, Paris, FranceSearch for more papers by this author Joan-Lluis Vives-Corrons, Joan-Lluis Vives-Corrons [email protected] Red Cell Pathology Unit, Hospital Clinic, University of Barcelona, Barcelona, SpainSearch for more papers by this authorCarol Briggs, Carol Briggs Department of Haematology Evaluation, University College London Hospital, London, UKSearch for more papers by this authorRamon Simon-Lopez, Ramon Simon-Lopez Beckman Coulter Eurocenter, Nyon, SwitzerlandSearch for more papers by this authorStephanie Albarede, Stephanie Albarede CTCB, 33 route de Bayonne, 31300 Toulouse, FranceSearch for more papers by this authorBarbara de la Salle, Barbara de la Salle General Haematology, UKNEQAS, Watford, UKSearch for more papers by this authorZlata Flegar-Meatrii, Zlata Flegar-Meatrii Institute of Clinical Chemistry, University Hospital Merkur, Zagreb, CroatiaSearch for more papers by this authorAida Nazor, Aida Nazor Institute of Clinical Chemistry, University Hospital Merkur, Zagreb, CroatiaSearch for more papers by this authorAnne Guyard, Anne Guyard Ansm (National agency of medicine and health product safety), Saint-Denis, FranceSearch for more papers by this authorThomas Lipsic, Thomas Lipsic St. Elisabeth Oncology Institute, Bratislava, SlovakiaSearch for more papers by this authorYukata Nagai, Yukata Nagai Faculty of Sports and Health Science, Daito Bunka University, Tokyo, JapanSearch for more papers by this authorMariana Patiu, Mariana Patiu OncologyInstitute, Cluj Napoca, RomaniaSearch for more papers by this authorJosep Piqueras, Josep Piqueras LaboratorisClínics, Hospital de la Valld'Hebron, Barcelona, SpainSearch for more papers by this authorMaria Jesus Capel, Maria Jesus Capel LaboratorisClínics, Hospital de la Valld'Hebron, Barcelona, SpainSearch for more papers by this authorMarjan Van Blerk, Marjan Van Blerk Department of Clinical Biology, Institute of Public Health, Brussels, BelgiumSearch for more papers by this authorJianbiao Wang, Jianbiao Wang Department of Medical Laboratory, Ruijin Hospital ShanghaiJiaotong, University School of Medicine, Shanghai, ChinaSearch for more papers by this authorChristophe Marzac, Christophe Marzac Hôpital Saint-Antoine, Paris, FranceSearch for more papers by this author First published: 12 December 2013 https://doi.org/10.1111/ijlh.12170Citations: 22Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1VivesCorrons JL, Albarede S, Flandrin G, Heller S, Horvath K, Houwen B, Nordin G, Sarkani E, Skitek M, Van Blerk M, Claude Libeer JC. Guidelines for blood smear preparation and staining procedure for setting up an external quality assessment scheme for blood smear interpretation. Part I: control material. ClinChem Lab Med 2004; 42: 922–6. 2Kennedy JB, Machara KT, Baker AM. Cell and platelet stability in disodium and tripotassium EDTA. Am J Med Technol. 1981; 47: 89. 3 NCCLS. Procedures for the Collection of Diagnostic Blood Specimens by Skin Puncture, 3rd edn. Approved Standard. NCCLS document H4-A3 (ISBN 56238-111-9) 1991. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087. 4Bates I, VivesCorrons JL. Quality Control. In Dacie and Lewis Practical Haematology, 11th edn. J Bain, I Bates, M Laffan, SM Lewis (eds). Churchill Livingstone: Elsevier; 2012: 587–602. 5Tatsumi N, Miwa S, Lewis SM; International Society of Haematology and the International Council for Standardization in Haematology. Specimen collection, storage, and transmission to the laboratory for hematological tests. Int J Hematol 2002; 75: 261–8. Citing Literature Volume36, Issue2April 2014Pages 222-226 ReferencesRelatedInformation

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.

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.

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).

Red blood cells (RBC) normally lose their nuclei before appearing in peripheral blood. After having undergone differentiation in bone marrow, blood cells must cross the blood-marrow barrier to enter the bloodstream. Erythroblasts, or nucleated red blood cells (NRBC), do not distort easily, so they cannot escape this barrier. Therefore, with the exception of the neonatal period, the presence of NRBCs in peripheral blood is always a pathologic finding. NRBCs may be found in the course of severe diseases and are associated with poor prognosis and higher mortality. The underlying pathophysiology of NRBCs in peripheral blood is not fully understood. It is hypothesized that their appearance could be provoked by either increased erythropoiesis or bone marrow micro-architectural damage mostly caused by inflammation and/or decreased tissue oxygenation. In addition, it is known that the mortality is higher in NRBC-positive patients as compared with NRBC-negative patients. Hereby we present a patient admitted to the hospital with the symptoms of cardiac failure and decompensated liver cirrhosis. The patient was already known to have liver cirrhosis of ethylic etiology, cardiac decompensation caused by hypertensive heart disease with permanent atrial fibrillation, chronic obstructive pulmonary disease, diabetes mellitus type 2, and cholelithiasis. During hospital stay, the patient developed acute pancreatitis and, soon after that, a stroke with left hemiparesis followed by cardiopulmonary arrest. Then he was transferred to the intensive care unit. Despite appropriate therapy, intensive care treatment and cardiopulmonary support, the patient's general state worsened, he developed multiple organ failure and died on day 10 of intensive care unit stay. Three days earlier, NRBCs were detected in peripheral blood and their concentration increased during the next two days before death. NRBCs are known to appear 1-3 weeks before death, but their appearance does not seem to be related to one particular cause of death. Still, detection of NRBCs is an independent risk of poor outcome, where the mortality increases with the increasing NRBC concentration. Detection of NRBCs in blood is a relatively early phenomenon prior to death, so screening for NRBCs may aid in the early identification of patients at high risk, and in making duly decision for NRBC-positive patients to obtain ongoing intensive care treatment.

Multiparameter flow-cytometrical estimation of peripheral blood progenitor cells (PBPCS) for autologous transplantation in cancer patients.

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.

In 2004, during the course of Croatia´s accession to the European Union together with implementation of accreditation standard (ISO 15189) we started with project of harmonization of laboratory results governed by the Croatian Society of Medical Biochemists (Committee for External Quality Assessment of MBLs), Croatian Chamber of Medical Biochemists and Institute of Clinical Chemistry Clinical Hospital Merkur: Reference centre of the Ministry of Health for the production of reference values. The main goals of this project were: analytical comparability of the results based on recommended analytical methods as well as clinical comparability of the results based on reference intervals produced for Croatian population. For the long-term evaluation of EQA results we calculate the performance statistics graphically using z-scores in other to demonstrate the variability of obtained results and identifies trends. In order to monitore qualitative results (in urine analysis) we use the ratio of acceptable to unacceptable results. Based on the results of these ongoing long-term evaluation, the major benefits of EQA are to (1) enhance patients care through improved analytical quality and working conditions in the medical biochemistry laboratories in Croatia ; (2) to evaluate laboratory results across different methods and different reagents ; (3) to identify medical- biochemical laboratories with poor performance and unacceptable results ; (4) to evaluate possible sources of error ; (5) to satisfy accreditation standard and regulatory requirements of Croatian Chamber of Medical Biochemists

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.

Background. The introduction of accreditation according to ISO 15189 standard in medical biochemistry laboratories in Croatia is of high priority in order to guarantee continuous quality improvement as well as the harmonization and standardization of laboratory diagnostics. The Institute of Clinical Chemistry, Merkur University Hospital, Zagreb is one of the first two laboratories accredited in Croatia. Methods. Evaluation of the impact of an electronic information system in providing safe and effective patient identification as well as in optimizing the efficiency of the entire laboratory process through the implementation of the new Laboratory Information System-LIS (BioNET application-LabNet, Josipovac, Croatia). Results. Our data showed that the application of LIS greatly contributed to the optimization of the efficiency of the whole laboratory process and significantly decrease identification errors through: application of the automatic system for patient identification and specimen labelling (bar coding) using two or more patient identifiers (name, birth date, identification number), increasing the automation of the analytical phase - work in „query-host“ with automated data entry including patient result verification, introducing computer- based medical records thus enabling critical results to reach physicians rapidly for improved clinical outcomes, applying Delta Check technology to evaluate laboratory results, improving test turnaround time, enabling health care planning and development of comprehensive electronic patient records. Conclusions. Application of electronic information system improve the overall quality and efficiency, prevent patient indentification errors and enables clinical laboratory to meet requirements according ISO 15189 standard for assessing the highest quality goals throughout the total testing process.

Primjenom odgovarajucih programa za obradu podataka vanjske procjene rada, koji osim rezultata moraju sadržavati također podatke o analitickim metodama i analitickim sustavima koje laboratoriji koriste, omogucuje se prepoznavanje uzroka međulaboratorijske varijabilnosti i dobiva uvid u uvjete rada svakog laboratorija. Iz toga proizlazi i važnost nacionalnog programa vanjske kontrole, koji se neprestano nadopunjava i osuvremenjuje u skladu s internacionalnim preporukama i tako služi kao osnova za uvođenje novih tehnologija i preporuka u svim fazama rada medicinsko-biokemijskog laboratorija te njihovu stalnu provjeru. Znacajan doprinos povecanju stupnja međulaboratorijske usporedivosti ucinjen je kroz projekt „Harmonizacija laboratorijskih rezultata“ u suradnji Hrvatske komore medicinskih biokemicara, Zavoda za klinicku kemiju KB Merkur kao Referentnog centra Ministarstva zdravstva za izradu referentnih intervala iz podrucja opce medicinske biokemije i Hrvatskog drustva medicinskih biokemicara kao organizatora nacionalnog programa vanjske procjene kvalitete. Cilj projekta je bio povecati analiticku i klinicku usporedivost rezultata između laboratorija primarne, sekundarne i tercijarne zdravstvene zastite u Hrvatskoj. Na osnovu podataka vanjske procjene kvalitete od 1998. do 2003. ustanovljeno je da se moraju revidirati neke od preporucenih metoda kako bi se omogucila klinicka usporedivost rezultata kroz primjenu jedinstvenih referentnih intervala. Realizacijom navedenih programa harmonizacije laboratorijskih metoda i pripadnih referentnih intervala stekli su se preduvjeti za prosirenje nacionalnog programa vanjske procjene kvalitete rada na izvananaliticku fazu. Prosirenjem nacionalnog programa vanjske procjene kvalitete rada na poslijeanaliticku fazu oblikovanja nalaza kao konacnog dokumenta analitickog procesa u 212 medicinsko-biokemijskih laboratorija u Hrvatskoj, a temeljem preporuka u dokumentu „ Harmonizacija laboratorijskih nalaza u podrucju opce, specijalne i visokodiferentne medicinske biokemije. Prirucnik HKMB, Medicinska naklada-Zagreb, 2007. dobit ce se podaci potrebni za standardizaciju i tog dijela rada medicinsko-biokemijskog laboratorija. Harmonizacija u dijelu izvjestavanja o rezultatima laboratorijskih analiza –poslijeanaliticka faza, kao konacnog dokumenta analitickog procesa temelji se na preporukama IFCC-a i IUPAC-a (A Proposal for an IUPAC-IFCC Recommendation. Syntax and Semantic Rules for Quantities and Units in Clinical Laboratory Sciences 1993: stage 1, draft 4.) te zahtjevima akreditacijske norme za medicinske laboratorije HRN EN ISO 15189:2006. Kako usporedivost rezultata laboratorijskih analiza ovisi o standardizaciji svih faza laboratorijskog procesa, programi vanjske procjene kvalitete rada medicinsko-biokemijskih laboratorija u dijelu izvananaliticke faze trebaju obuhvacati i predanaliticku fazu koja je izvor najveceg broja laboratorijskih pogresaka i time dati cjelovit uvid u aktualno stanje struke s ciljem stalnog poticanja poboljsanju strucnog rada i podizanju kvalitete rada medicinsko-biokemijskih laboratorija kako na nacionalnoj tako i na međunarodnoj razini.

This study aimed to assess currently used evaluation procedures for haemoglobin concentration and leukocyte concentration in European external quality assessment schemes (EQAS). Participating EQAS organizers were asked to complete a questionnaire gathering information on the statistical procedures used to assess the performance of participants, and to analyse an Excel file with 262 results for haemoglobin concentration and leukocyte concentration. Responses were received from the New York State Proficiency Testing programme in the US and from 11 European EQAS: Belgium, Croatia, Finland, France, Germany, Hungary, Russia, Slovenia, Spain (two organizers), and Switzerland. Five of the 11 European EQAS use fixed limits based upon experience, biological variation, state of the art, or professional consensus. The other EQAS use variable limits based upon statistical analysis of the performance attained. With the exception of the German, Hungarian, and Slovenian schemes, all European EQAS use consensus target values. The percentage of unsatisfactory results obtained by the European EQAS organizers for the given set of data varied between 0.4 and 15.6% for haemoglobin concentration and between 0 and 19.8% for leukocyte concentration.