Daria Pašalić
Department of Medical Chemistry, Biochemistry and Clinical Chemistry
Zagreb University School of Medicine
Šalata ul 2.
10 000 Zagreb, Croatia
Phone +385 (1) 4590 205; +385 (1) 4566 940
E-mail: dariapasalic [at] gmail [dot] com

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Verification of CD34+ stem cell analysis according to ISHAGE protocol on Beckton Dickinson FACSCanto
Sokolić I, Šiftar Z, Kardum Paro M, Flegar Meštrić Z
C.H."Merkur", Department of Clinical Chemistry and Laboratory Medicine, Zagreb, Croatia
Corresponding author: ivica [dot] sokolic [at] gmail [dot] com
Background: Implementation of new analytical equipment into routine work requires verification procedure. We have verified flow cytometer Beckton Dickinson FACSCanto II for ISHAGE protocol (Sutherland et al, 1996, J Hematotherapy, 5:213-226) stem cell (CD34+) analysis according to CLSI EP-A2-User Verification of Performance for Precision and Trueness; Approved Guideline-2nd ed.,Vol.25,No.17.,2005. in concordance with ISO 15189.
Materials and methods: Analysis was performed on peripheral blood samples, fresh leukapheresates of peripheral blood and BC StemCell Control Kit samples. Acquisition data were analyzed using BD FACSDiva 6.0 software. Verification included assessment of random error check based upon triplicate measurement of commercial samples five days in a row, assessment of measurement uncertainty, conformation of linearity, comparison of results from reference analyzer and analysis of external quality control samples.
Results: Performance characteristics: within-laboratory precision for Stem Cell Low (11.6 x 106 / L) was 9.2%, for Stem Cell High (31.5 x 106 /L) 8.0%. We confirmed linearity on peripheral blood sample 0-97.2 x 106 /L. Measurement uncertainty for Stem Cell Low was 28.4% and Stem Cell High 19.2%. Comparison of results with Beckman Coulter Cytomix FC500 was satisfactory (Passing Bablock, Y = -0.01117 + 1.0112x, P > 0.10). UK NEQAS for Immunophenotyping, Stem Cell enumeration scheme results were within target values (median ± 25 percentile) for CD34+ absolute and relative count.
Conclusion: According to our verification procedure, BD FACS Canto II is cytometer of choice for performing CD 34+ stem cell absolute count analysis.
Copper deficiencies simulating a myelodysplastic syndrome
Gallego A (1), Vera J (2), Peral E (3), Martín J (1), Bermudo C (1), Fábregas M (2)
(1) Hospital Universitario Virgen Macarena, Bioquímica Clínica, Sevilla, Spain
(2) Hospital Universitario Virgen Macarena, Hematología, Sevilla, Spain
(3) Hospital Universitario Virgen Macarena, Medicina Interna, Sevilla, Spain
Corresponding author: gallego [dot] am [at] gmail [dot] com
Introduction: Copper is an essential trace element present in a number of metalloproteins and appears to be required for the absorption and utilization of iron. Copper deficiency has been described in malnourished children, in both infants and adults receiving prolonged enteral or parenteral alimentation, and following gastric resection or bariatric gastric reduction surgery. It is already known that cupper deficiency could resemble a myelodysplastic syndrome with anemia, neutropenia and trombocytopenia.
Case report: Patient 32 years male with spastic tetraplegia after severe TBI caused by a traffic accident in 2009. It was decided to place jejunostomy in June 2010. In June 2011 a normocytic anemia was detected without signs of bleeding according both clinically and by the diagnostic tests performed and treated according to protocol. In January 2012 was readmitted for persistent leucopenia and anemia in transfusion range. It was performed bone marrow aspirate, being found a bicytopenia, dysplastic features, reactive bone and good representation of the three series. Values copper and ceruloplasmin levels were requested, being respectively: 36 mcg/dL (Normal being 65-165) and 12 mg/dL (Normal 20-40). Parenteral alimentation was maintained with supplements of copper, recovering values cupperhemic to enter the normal range and resolving the bicitopeny and symptoms secondary to it.
Conclusion: In patients with unexplained refractory cytopenias, dysplastic features and comorbidities that may trigger malnutrition such as parenteral alimentation, you should order determination of copper and ceruloplasmin levels before diagnosis of myelodysplastic syndrome.
Multiple myeloma: a case report
Cachapuz I (1), Branco A (2), Paiva A (2), Magalhaes C (1), Costa M (2)
(1) Matosinhos Local Health Unit , Clinical Pathology Service, Oporto, Portugal
(2) Matosinhos Local Health Unit, Nephrology Service, Oporto, Portugal
Corresponding author: cachapuz [dot] isabel [at] gmail [dot] com
Introduction: The multiple myeloma is a neoplasm of B cells, associated to the presence of a monoclonal protein in serum and/or urine. It is clinically manifested by different symptoms.
Case report: 58 year-old woman, with depression, obesity and hypertension, presented at the ER (Emergency Room) of Pedro Hispano Hospital on the 13th January 2012 due to nonoliguric acute kidney injury (creatinine: 7.7 mg/dL), anemia ( Hb: 7.1 mg/dL ),calcium 8.2 mg/dL, proteinuria (7 g/dia). Renal echography: normal. Blood transfusion was made. Declared having anemia for 6 months, under study, and she had already made an upper digestive endoscopic and colonoscopy both without changes. Three months before hospitalization: 10 kg loss, asthenia, anorexia, nausea, pain in the left costal margin. At the ER, abdominal CT scan revealed lytic lesions. The day after, the patient made a serum proteinogram: beta peak. The serum protein electrophoresis (SPE) revealed the presence of a monoclonal component lambda (4.54 g/L). Since there was no recovery of kidney function, haemodialysis was started. A myelogram was performed and revealed pathological plasma cells. The cytogenetic laboratory didn’t detect IGH_MMSET fusion, nor 17p13 deletion (gene TP53), at the bone marrow. She started chemotherapy with Bortezomib and Dexametazona on the 20th January 2012, and was treated with high cutoff filters without success in the recovery of kidney function.
Conclusion: This clinical case underlines the importance of the clinical-laboratory interaction for the correct and timely diagnosis of haemato-onchological pathologies. SPE is still an important auxiliary diagnostic tool.
Which value can be used as a positivity threshold for Large Unstained Cells on Advia to make slides?
László K, PatonaiZ, KellnerV, FeketeM
SynlabHungaryLtd., LaboratoryofSzékesfehérvár, Székesfehérvár, Hungary
Corresponding author: laszloki [at] yahoo [dot] com
Introduction: The Advia 2120i hematological system uses peroxidase staining for white blood cells differential testing. Peroxidase-negative cells, large lymphocytes, plasma cells, blasts are counted to large unstained cells (LUC) category. The manufacturer recommends the normal range for LUC < 4%. If LUC > 4%, it is suggested to make a slide and analyse morphology of cells by microscopy. The aim of this study was to find a real positivity threshold for LUC, where the results by microscopy give additional information to the clinician beyond the mere numerical data.
Materials and methods: ROC curve analysis was performed to obtain the appropriate results. Blood samples of 350 patients were examined by automated Advia 2120i and microscopy. Three analyses were performed and results were considered to be positive as follows, ”1”: if blasts or any number of reactive/granulated lymphocytes were found by microscopy; ”2”: if blasts or a few (4-6) or more reactive/granulated lymphocytes-, and ”3”: if blasts or several (> 7) reactive/granulated lymphocytes were counted by microscopy.
Results: In analysis ”1”, the positivity threshold for LUC > 4% was observed, the true negative rate (TNR; specificity): 74.76%; the true positive rate (TPR, sensitivity): 88.81%. High number of false positive results (FPR) were detected: 25.24%. In case of ”2” the positivity threshold for LUC > 5% was found, TNR: 83.27%, TPR: 66.25%, FPR: 16.73%. In analysis ”3” the positivity threshold for LUC > 6%, TNR: 94.08%, TPR: 56.45% and FPR: 5.92% were obtained.
Conclusion: Overall, even LUC > 6% was found to be reliable positivity threshold to request blood smears.
Role of P-selectin glycoprotein ligand-1 (PSGL-1) during G-CSF treatment in a mouse model
Miszti Blasius K (1), Felszeghy S (2), Hevessy Z (1), Kappelmayer J (1)
(1) Medical and Health Science Centre, University of Debrecen, Department of Laboratory Medicine, Debrecen, Hungary
(2) Medical and Health Science Centre, University of Debrecen, Department of Anatomy, Debrecen, Hungary
Corresponding author: cornelius [at] dote [dot] hu
Background: The effect of G-CSF (Neupogen) was investigated in wild-type (WT) and PSGL-1 knockout (KO) mice to establish the role of this mucin in myeloid cell mobilization. G-CSF activates tissue proteases that cleave several adhesion molecules, thus enhances the mobilization of myeloid cells and haemopoietic stem cells.
Materials and methods: WT and KO mice (12-16 week old, 20-25 gram body weight, N = 15) were treated with a single dose of 250 mg/kg cyclophosphamide to induce cytopenia, subsequently mice received 7.8 µg/kg G-CSF twice a day for 4 days. Retro-orbital blood samples were drawn to determine leukocyte counts by Siemens Advia 120 analyser.
Results: Neutrophil granulocyte count increased upon completion of G-CSF treatment but were significantly different in WT and KO mice (28.3 G/L vs. 47.7 G/L), while the monocyte counts were 2.0 G/L and 4.1 G/L. Four days after the last G-CSF treatment, both strains displayed considerably reduced neutrophil (1.8 G/L and 9.8 G/L) and monocyte (0.4 G/L and 1.6 G/L) counts, the values always being higher in KO animals. Contrary, eosinophil granulocyte values became elevated only at this sampling time, being 0.5 G/L (WT) and 1.2 G/L (KO). There was a similar difference in atypical cell counts always being higher in KO animals.
Conclusions: The lack of PSGL-1 results in higher mature and precursor myeloid cell release after G-CSF treatment with delayed effects on eosinophils. The differences are caused by both faster mobilization from the bone marrow and delayed extravasation in the peripheral vessels in PSGL-1-/- animals.
Usefulness of red cell indices in differentiating microcitic anemias
PavelaJ, FijačkoM, Dobrošević B, Paradinović K, Šerić V
Clinical Hospital Centre Osijek, Department of Clinical Laboratory Diagnostics, Osijek, Croatia
Corresponding author: pavela [dot] jasna [at] kbo [dot] hr
Backgound: Iron deficiency anemia (IDA) and beta-thalassemia trait (β-TT ) are the most common forms of microcytic anemia. This study was conducted to compare the validity of various discrimination indices in differentiating β-TT from IDA by calculating their sensitivity, specificity and Youden's index.
Materials and methods: A total of 146 patients (69 IDA and 77 β-TT) with microcytic anemia were involved in this study. We calculate six discrimination indices i.e. RBC count, Mentzer indeks (MI), Shine & Lal (S&L), Srivastava (SI), England & Fraser (E&F) and Red cell Distribution Width Indeks (RDWI). The number of correctly identified cases were determined and sensitivity, specifity, positive and negative predictive value and Youden's index of each discrimination index were calculated.
Results: The percentage of correctly diagnosed is highest for E&F and RDWI (86%) folowed by MI (79%) index. None of the discrimination index showed 100% sensitivity and specificity. Youden's index wich includes both, sensitivity and specificity is in descending order RDWI > E&F >MI > RBC >SL >SI.
Conclusion: RDWI, England-Fraser and Mentzer index are the most reliable formulae in discrimination between iron deficiency anemia and beta-thalassemia trait.
A method of preparing fresh blood samples for Croatian haematology external quality assessment
Nazor A (1), Flegar-Mestric Z (1), Hećimović A (2), Šiftar Z (1), Hundrić Hašpl Ž (2)
(1) University Hospital Merkur, Institute of Clinical Chemistry and Laboratory Medicine, Zagreb, Croatia
(2) Croatian Institute for Transfusion Medicine, Blood bank, Zagreb, Croatia
Corresponding author: zlata [dot] mestric [at] gmail [dot] com
Background: Croatian EQA involves participation in the National External Quality Assurance Scheme organized under the auspices of the Croatian Society of Medical Biochemists by processing of unknown samples 3 times per annum. The suitability of home-made control material for complete blood count was evaluated.
Materials and methods: The Croatian Institute for Transfusion Medicine prepared the home-made control material for complete blood count. A single portion of fresh blood is used given by voluntary donor having given consent to its intending use. A minor portion of the blood is tested for Hepatitis B and C as well as HIV and TP and was nonreactive for all these markers. After 30 minutes mixing in a way without making foam, 1.5 ml K2EDTA molarity 1 mol/L is added and the mixing was continued next 2-5 minutes. The blood is distributed into suitable 7.5 x 12 mm sterile tubes, capped and stored at +2 °C to +8 °C. The samples were analysed for homogeneity and stability according to the ISO 13528 requirements.
Results: The home made-samples are delivered within 24 hours throughout Croatia in 200 medical biochemistry laboratories and are analyzed on 37 different types of analyzers from 11 different manufacturers. Assessment against consensus values and the percent allowed difference score is introduced for evaluation of obtained results. The high degree of inter-laboratory comparability is approved.
Conclusion: The home-made control material used in Croatian EQA scheme shows applicability to all haematological analyzers and provides a long term, retrospective assessment of laboratory performance and comparability of results on national level.
Differential count in healthy newborns
Paradinović K (1), Milić M (1), Drmić S (2), Šerić V (1)
(1) University Medical Center Osijek, Department of Clinical Laboratory Diagnostics, Osijek, Croatia
(2) General Hospital of Vinkovci, Department for Biochemistry, Vinkovci, Croatia
Corresponding author: ksenija [dot] paradinovic [at] gmail [dot] com
Background: An accurate differential count is important in the diagnosis and monitoring of various clinical states. Our aim was to compare WBC differential morphology flags from haematology analyzer and differential blood count from microscopic slide examination in healthy newborns.
Materials and methods: CBC count was determined on haematology analyzer Sysmex XE2100. Differential was performed by examination of peripheral blood smear using light microscope. Slides were stained by May Grunwald Giemsa. We analysed 252 blood samples from healthy newborns aged 1-3 days.
Results: We compared flags for immature gran and left shift with nonsegmented neutrofiles, metamyelocytes, myelocytes. Average for nonsegmented neutrofiles was 6%. Compatible values (flags=pos, microscope=poz; flags=neg, microscope=neg) were in 105 cases. Noncompatibile values (flags=neg, microscope=pos) were in 11 cases and (flags=pos, microscope=neg) were in 140 cases.
Conclusions: Comparing the results, we noticed that the haematology analyser is more analytically sensitive. Microscopic slide examination should bee done in all samples flaged by haematology analayzer.
The effect of the cellularity of the bone marrow aspirate sample for cytogenetic cultures
Tegg E (1), Raik E (2)
(1) University of Tasmania, Pathology, Hobart, Australia
(2) Royal North Shore Hospital, Haematology, Sydney, Australia
Corresponding author: Elizabeth [dot] Tegg [at] utas [dot] edu [dot] au
Background: The role of cytogenetics in the diagnosis and prognosis of haematological malignancies is well recognised. The contribution made by the quality of the sample given to a cytogenetics laboratory to successfully provide diagnostic results is not.
Materials and methods: This was a retrospective control study made after a change to the bone marrow aspiration technique. The test group consisted of the first 65 patients who underwent a bone marrow biopsy requiring cytogenetic analysis after this change. The control group consisted of 65 patients who had undergone a bone marrow biopsy for cytogenetic analysis prior to this change. The parameters measured were disease type, morphological cellularity of the aspirated marrow, cell count of the cytogenetic sample, cytogenetic failure rate, number of metaphases obtained and the banding resolution achieved.
Results: Results were analysed using Microsoft Excel student t-test. The cellularity of the cytogenetic sample was significantly increased (1.23 x 109/L compared to 0.59 x 109/L; P < 0.001). The culture failure rate fell significantly from 15% to 2%. (P < 0.01). The optimal number of metaphases (20 metaphases per patient) was achieved in 75% of cases compared to 50% in the control group (P < 0.04). The banding resolution was significantly increased in the test group compared to the control group (P < 0.04).
Conclusions: Information in the literature is sparse when assessing the effect of BM cellularity on the ability to obtain a successful cytogenetic result. This study showed the positive effect of increasing the cellularity of the bone marrow aspirate sample for the successful cytogenetic analysis.
Verification of the Body Fluid Application on the Sysmex XE-5000 Hematology Analyzer
Šiftar Z (1), Kardum-Paro M (1), Nazor A (1), Sokolić I (1), Kardum Skelin I (2), Flegar Meštrić Z (1)
(1) Merkur University Hospital, Institute of Clinical Chemistry and Laboratory Medicine, Zagreb, Croatia
(2) Merkur University Hospital, Institute of Clinical Cytology and Cytogenetics, Zagreb, Croatia
Corresponding author: zoran [dot] siftar [at] gmail [dot] com
Background: Aim of this study is to investigate the performance of BF application on the Sysmex XE-5000 hematology analyzer in differential diagnosis of exudates and transudates.
Materials and methods: The XE-5000 BF application provides white blood cell (BF-WBC) and total nucleated cell (BF-TC), as well as erythrocyte (BF-ERC) count. BF-WBC is further subcategorized in mononuclear and polymorphonuclear cells, BF-MN and BF-PMN. Performance of application was evaluated according to CLSI H56-A guidelines. Comparison to reference methods: microscopic cell counting in Neubauer chamber and differential on MGG stained smears after citospin preparation were done. Additionally, measurement of cells on XS2100 analyzer was included in comparative study. Automated cell counting was performed in Institute of Clinical Chemistry and Laboratory Medicine, whilst the microscopic in Institute of Clinical Cytology and Cytogenetics. It was done on various fluids (pleural effusion and ascites, dialysis fluid within peritoneal dialysis treatment) specimens. For correlation calculation Passing and Bablock regression analysis is used.
Results: Repeatability of BF-WBC measurements was performed on 3 concentration levels. Coefficients of variation (CVs) were 21, 14 and 7.5, and met manufacturer specifications. BF-WBC linearity was proven for measurement range of 3 to 3420. Achieved coefficients of correlation with microscopic and measured counts on Sysmex XE 2100 for 35 specimens were 0.92 and 0.98, respectively. Obtained coefficient of 0.89 was found in comparison of automated and microscopic differential on 23 samples.
Conclusions: Evaluation of BF application on Sysmex XE-5000 showed significant improvement in the ability of automated hematology analyzers regarding body fluid cell count analysis.
Integration of point-of-care hematology analyzers in the hospital and laboratory information system
Baršić I (1), Brkljačić V (1), ŠrengerV (2), Majdenić K (3), Stojmilović S (3), Sertić J (1)
(1) University Hospital Center Zagreb, Clinical Institute of Laboratory Diagnostics, Zagreb, Croatia
(2) University Hospital Center Zagreb, Center for Informatics, Zagreb, Croatia
(3) IN2 Ltd, Health sector, Zagreb, Croatia
Corresponding author: ibarsic [at] kbc-zagreb [dot] hr
Background: In 2000, our Institute started with implementing point-of-care testing at sites where it was important to decrease TAT, either for critical care patients or in order to increase efficiency and patients turnaround time. University Hospital Center Zagreb has 14 blood gas analyzers (GEM Premier 3000,Rapidlab 1265), 6 hematology analyzers (Sysmex pocH-100i), 7 coagulation monitoring system analyzers (CoaguChek XS, thromboelastometry ROTEM, aggregometry Multiplate® ) and 105 glucometers (Accu Chek Inform II). Results from these devices are used to assess the current status of patients and often are not entered in the medical record, thus remaining unaccounted for in the final patient report with diagnostic and therapeutic procedures. Therefore, we initiated integration of point-of-care devices in the hospital and laboratory information system (HIS, LIS), starting with hematology analyzers.
Materials and methods: Sysmex pocH-100i is an automated hematology analyzer that provides 19-parameter complete blood counts with three-part leucocyte differential. It is connected via a local area network with LIS, further integrated into HIS. According to predefined criteria, LIS performes autovalidation and results are forwarded to HIS. In this way, results are available within a few seconds.
Conclusion: Integration of point-of-care devices in the hospital and laboratory information system allows the storage of patient results with electronic medical patient records and easier access to results when they are needed again. It also gives us the ability to have better control over reagent consumption and over results obtained from these devices.
Blood loss from laboratory tests in adult hospitalized patients
Alatas O, Kocaturk E, Bekmez M, Bayrak C, Harmansa A
Eskisehir Osmangazi University, The Medical School, Medical Biochemistry, Eskisehir, Turkey
Corresponding author: oalatas [at] ogu [dot] edu [dot] tr
Introduction: Blood loss for laboratory tests can be important especially for inpatients. The aim of this study was to determine approximate blood loss of inpatients during their stay in hospital and whether this loss contributes to changes in hemoglobin levels.
Materials and methods: All patients hospitalized for eight or nine days in twenty five clinics of University Hospital of Eskisehir Osmangazi University in 2011 were reviewed retrospectively. We estimated blood loss by multiplying the number and approximate volumes of sampling tubes that used for laboratory analysis of 1792 adult inpatients. Moreover, the first and last sample hemoglobin levels of hospitalized patients were compared.
Results: The mean blood drawn from patients was estimated 35.27 mL. The highest blood volume was observed in patients from Chest Disease Clinic. Total tube number used for 1792 patient was 22,926, and the red top tube was consisted of 36% of all tubes. Among the eight clinics which have more than fifty patients, Chest Disease Clinic was used the highest number of tubes. The first day median level of hemoglobin was reduced from 12.7 g/dL to 11.7 g/dL in eight days hospitalized patients and from 12.5 g/dL to 11.7 g/dL in nine days hospitalized patients.

Results: Blood loss from laboratory diagnostic testing is highly associated with changes in hemoglobin levels for inpatients and may contribute to anemia especially in high risk group patients. For this reason, unnecessary test requirements should be prevented and minimum test repetition time should take into consideration in order to reduce blood loss arise from laboratory diagnostic testing.