Clinica Chimica Acta 493 (2019) S379–S433

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Haematology, including haemostasis

cases of homozygous mutants T677 T, 2 cases of homozygous W034 mutants C1298C and 1 case heterozygous normal C677T with mutation in C1298C homozygosis. Prevalence of the different genotypes of the MTHFR gene performed in a South Spain health area ^ Conclusions

R. Coca Zuñigab, A. González Rayaa, G. Callejón Martína, E. Martín The study shows that 9.3% of the studied population presents Sálidoa, A. Lendinez Ramireza, M. Cantero Sancheza, M.L. Hortas the heterozygous combination of both mutations C677T + A1298C, Nietoa in these patients the activity of MTHFR will be significantly aHospital Costa del Sol, Marbella, Spain reduced. The heterozygous forms of the two alterations separately bHospital Universitario Virgen de las Nieves, Granada, Spain represent 48%, which do not present associated risk of hyperho- mocysteinemia. 6.9% and 16.2% of the cases correspond to Background-aim homozygous mutant forms for T677 T and C1298C respectively, these mutations should be considered as factors of high indepen- Methylenetetrahydrofolate reductase (MTHFR) is an enzyme dent thrombotic risk for causing high homocysteine levels. The that participates in the regulation of intracellular folate, an results of the study show that 67.3% of the studied population is essential compound for the synthesis of proteins and nucleic considered asymptomatic, because they do not present any of the acids. This enzyme catalyzes the transformation of 5,10-methy- mutation in homozygosis. It should therefore be considered to lenetetrahydrofolate to 5-tetrahydrofolate, a donor of methyl establish a right protocol to be followed in which the determination groups necessary for the conversion of homocysteine to methio- of the mutation of the MTHFR gene should be subsequent to a state nine. Deficiency in its activity can lead to hyperhomocysteinemia, of hyperhomocysteinemia. which is considered as a risk factor for both atherogenesis and thrombogenesis. A genetic polymorphism has been detected doi:10.1016/j.cca.2019.03.809 frequently in 677 nucleotide (C677T), which involves the substi- tution of alanine for valine, this variant is associated with a low enzymatic activity (up to 50%). A second polymorphism has been located at position 1298 (A1298C) which involves the substitution ^W035 of glutamine for alanine. The aim of this work is to study the prevalence of mutations in Understanding antiphospholipid antibody (APLA) syndrome in the MTHFR gene in patients from a south of Spain health area during the laboratory – Diagnostic challenges the years 2017 and 2018. S.N. Kamatham, P. Suryadeep, K. Azmathullah, S.S. Gorji, B. Jupalle, Methods B.V.L.N. Murthy, H.A. Syed CARE Hospital, Road No.1, Banjara Hills, Hyderabad, India We performed a genomic DNA study to 43 patients referred to our laboratory with clinical suspicion of hyperhomocysteinemia and Background-aim family study to the confirmed cases with genotype of high risk. To carry out the genotyping, an automated DNA extraction was ^Laboratory evidence of antibodies against phospholipids or performed on a maxwell®16 (promega) device. DNA amplification phospholipid binding protein co-factor, results in an acquired and detection of mutations with FRET probes by real-time polymer- autoimmune thrombophilia and hence essential in diagnosis of APLA. ase chain reaction (RT-PCR), in a Light 2.0 Cycler autoanalyzer (Roche Diagnostic ®). The test determines the presence or absence of Methods the mutation and distinguishes between the homozygous and heterozygous genotypes. The laboratory has algorithm approach, using both coagulation and serological studies, the former done by automation and later by Results ELISA 1. Screen for Prothrombin time and APTT with mixing studies leading to 2. identification of inhibitors by temperature incubation 3. Of the 43 cases analyzed, there were: 8 cases of normal Dilute Russel Viper Venom time 4. assays of IgG, IgM & IgA for both homozygotes C677C/A1298A, 7 cases of heterozygotes C677T, 14 anticardiolipin and antiphospholipid 5. Followed by IgG & IgM for ®2 heterozygotes A1298C, 4 cases of heterozygotes C677T/A1298C, 7 glycoprotein.

0009-8981/$ – see front matter Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

Results glomerulonephritis) was observed. In addition, the patient presented episodes of paroxysmal atrial fibrillation. She was given enoxaparin. Over the past 10 years there has been an annual increase in Since its administration, a downward trend in platelet values was referral to laboratories for investigating the APLA syndrome from appreciated. The following platelet values were obtained (range of various clinical specialities. The mechanisms and pathophysiology of normality 140.00–400.00 10^3/mL): 215.00 (11/22/2018), 154.00 the clinical symptoms are highly heterogeneous. (11/24/2018), 49.00 (11/27/2018). Upon suspicion by the laboratory, At our tertiary care hospital laboratory, the referrals have been anti heparin antibodies were determined (normal range 0.00–1.00 U/ abundant and varied, with increasing numbers every month. The mL): 12.40 (04/12/2018). incidence of numbers being as mentioned in the etiologies referred below: Results 1. Autoimmune disorders Heparin-induced thrombocytopenia was diagnosed, confirmed 2. Young strokes after positivity of the anti-heparin antibodies, so treatment with 3. Deep vein thrombosis enoxaparin was suspended until the recovery of platelet numbers for 4. Bad obstetric history subsequent initiation of oral anticoagulation. 5. Infertility 6. APLA associated with renal, neurological, cardiac presentations Conclusions 7. Catastrophic APLA (having definite criteria of clinical and laboratory) The presence of anti-heparin antibodies is an underdiagnosed Conclusions complication and should be suspected in any patient with anticoag- ulant treatment and thrombocytopenia and/or thrombotic events. After the diagnosis, treatment should be suspended and the The APLA syndrome is both a diagnostic and clinical challenge. administration of other anticoagulants such as heparinoids or direct The Global Anti Phospholipid Syndrome score (GAPPS) computation thrombin inhibitors should be evaluated. Because these incidental makes risk stratification understandable for better therapeutic findings are so rare, the laboratory must be alert to prevent possible approach. complications that put the patient's life at serious risk. Acknowledgement. All clinical specialities. doi:10.1016/j.cca.2019.03.811 doi:10.1016/j.cca.2019.03.810

^W037 ^W036 Impact of doxycycline on platelet activation during platelet Contribution from the laboratory in the diagnosis of heparin- concentrates storage – Preliminary research induced thrombocytopenia. A case study A. Rak-Pasikowskaa, A. Wrzyszczb, K. Wisniewskab, I. Bil-Lulaa J. Paco Ferreira, T. HernÁndez Lemes, M. Kassih Ibrahim, A.M. aDivision of Clinical Chemistry, Department of Medical Laboratory SÁnchez De Abajo, M. Navarro Romero, G. Garcia Aguilar, T. Dorta Diagnostics, Faculty of Pharmacy with Division of Laboratory Diagnos- Ramos, A. Cabrera Argany tics, Wroclaw Medical University, Wroclaw, Poland Servicio de Análisis Clínicos y Bioquímica Clínica, Complejo Hospitalario bDivision of Laboratory Haematology, Department of Medical Labora- Universitario Insular Materno Infantil (CHUIMI), Las Palmas de Gran tory Diagnostics, Faculty of Pharmacy with Division of Laboratory Canaria, Spain Diagnostics, Wroclaw Medical University, Wroclaw, Poland

Background-aim Background-aim

Heparin is the most commonly used anticoagulant treatment. During platelet concentrates' (PCs) storage platelets lose their Bleeding is the most common complication, however another functions and their activation increases. The reasons of these changes possible side effect is heparin-induced thrombocytopenia (HIT), a are not fully investigated. Plasma, erythrocytes and leukocytes life-threatening complication of exposure to heparin that occurs in a present in PCs may be a source of matrix metalloproteinases small percentage of patients exposed, regardless of the product, dose, (MMPs). MMPs may be one of the factors which are presumably schedule, or route of administration. HIT results from an autoanti- responsible for lesions of platelets during storage. Doxycycline is one body directed against endogenous platelet factor 4 (PF4) in complex of the MMPs inhibitor. We hypothesized that doxycycline can delay with heparin. This antibody activates platelets and can cause serious or decrease platelet activation. The aim of the study was to evaluate arterial and venous thrombosis with a mortality rate up 20%; platelet activation during storage of PCs with and without doxycy- although with improved recognition and early intervention, mortal- cline addition. ity rate have been reported as below 2%. Methods Methods Four PCs were divided into two parts and doxycycline was added Patient 79 years old woman. Generalized asthenia was reported to one of them (PC + D) (10 μM final concentration). They were from three weeks ago, analytical was performed observing acute stored simultaneously in the same conditions. Samples were renal failure with creatinine 6.60 (0.67–1.17 mg/dL). The patient was separated from main concentrate in the day of preparation (0 h), admitted as a matter of urgency at the hospital. After study, focal after 24, 48, 72 and 144 h of storage. In every sample, an expression necrotizing glomerulonephritis with crescents (pauci-immune of P-selectin and CD63 was evaluated. Statistical analysis was Abstracts / Clinica Chimica Acta 493 (2019) S379–S433 performed using Friedman's rank test and paired samples Wilcoxon at 24 °C after 6 h a decrease by 3,7% for PT and by 6,2% for APTT was test (Statistica 12 EN). noted. The changes were not statistically significant. The level of fibrinogen remained unchanged. For plasma samples stored at −20 Results °C the steady increase in PT and APTT was observed and the obtained results were on average higher by 10,2% for PT and by 19,2% for APTT Increase in expression of P-selectin and glycoprotein CD63 in PC and as compared to baseline. In contrast, fibrinogen concentration PC + D during storage has been observed: over 5-fold increase of P- decreased steadily reaching the average value lower by 10,2% after selectin expression in both PC and PC + D, over 3-fold increase of CD63 6hat−20 °C. All changes were significant (p b .01–0.05). expression in PC, and almost 3-fold in PC + D after 144 h of storage. However, platelet in PC + D show tendency to lower expression of P- Conclusions selectin compared to PC in every time point, the highest percentage difference was observed after 48 h = 45.21%. Increase of P-selectin Taking into account biological variations and total analytical expression in PC + D was observed 24 h later than in PC. Lower errors, it can be concluded that citrate plasma samples for PT, APTT expression of CD63 in PC + D compared to PC has been observed after and fibrinogen can be stored up to 6 h at room temperature but 48 h, 72 h and 144 h, and the largest percentage difference was measurement of these analytes in frozen samples is not observed after 48 h = 47.5%. However, in contrary to P-selectin, CD63 recommended. expression after 24 h storage was higher by 9.59% in PC + D than in PC. doi:10.1016/j.cca.2019.03.813 Conclusions

During PCs storage platelets activation increases regardless of W039 doxycycline addition. However, it should be considered that ^ doxycycline may lead to delay activation of platelets during storage. Immunophenotypic profile of lymphoproliferative syndromes doi:10.1016/j.cca.2019.03.812 F. Boulhen, S. Aatfaoui, H. Mimouni, S. Jaddaoui, B. Oukkache Laboratory of Haematology of CHU Ibn Rochd of Casablanca, Morocco

Background-aim ^W038

Effects of sample storage time and temperature on prothrombin Lymphoproliferative syndromes represent a heterogeneous group time (PT), activated partial thromboplastin time (APTT) and of malign hemopathies (MH) affecting mature cells of lymphoid B, T fibrinogen and other related lineages such as NK cells (1). Among these haemopathies, the most common is chronic I. Rogatko, A. Kolczyk, K. Sztefko lymphocytic leukemia (CLL), which can cause in its atypical forms Clinical Biochemistry Department IP, Jagiellonian University Collegium problems of differential diagnosis with other syndromes. Medicum, Krakow, Poland Flow cytometry (FCM) give an accurate evaluation of cell subpopulations, even when they are poorly represented. It is Background-aim therefore an essential step in the diagnosis and follow-up of the majority of hematological malignancies. The aim of our study is to Preanalytical quality control of blood samples is of critical describe the role of immunophenotyping in the diagnosis of the importance for many tests including tests for coagulation function. different types of lymphoproliferative syndromes and to evaluate Sample storage condition (time and temperature) may have strong their incidence. impact on final results. There is no agreement regarding storage condition of sample for PT, APTT and fibrinogen measurement. In Methods most guidelines blood samples may be stored unspun for 4 h without any changes of analytes of interest. No clear recommendation It is a descriptive study spread over 3 years, performed in the concerning storage time and storage temperature of citrate plasma haematology laboratory of Ibn Rochd University Hospital in for PT, APTT and fibrinogen are available for pediatric samples. Casablanca.

Methods Results

In total 102 randomly selected pediatric samples PT, APTT and 76 patients are retained, the Sex ratio (m/f) was 1.81, there was a fibrinogen were routinely measured in citrate plasma. Leftover male predominance with 49 cases (64.74%), while 27 cases were samples were split are stored for 2, 4 and 6 h at room temperature female (35.52%). Then median age was 61, with extremes of 38 years and 2,4 and 6 weeks at −20 °C, respectively. Baseline level of PT, and 63 years. The study of the immunophenotypic profile revealed APTT and fibrinogen and the results after storage at room 38 cases of chronic lymphocytic leukemia (50%), 16 cases of marginal − temperature and after storage at 20 °C after thawing were zone lymphoma (21%),^and 7 cases of mantle lymphoma (9). measurement using the same methodology (BCS XP Siemens). Statistical analysis was performed using Statistica 12. Conclusions

Results Mature lymphoid hemopathies include many entities. To precise the exact type of MH, examination of the blood smear stained with No changes for PT, APTT and fibrinogen were noted in plasma May Grüwald Giemsa is the first step in the diagnosis completed in a samples stored for 2 and 4 h at room temperature. In samples stored second time by a FCM study of blood cells. It specify the cell line of Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

the proliferation (myeloid or lymphoid) as well as its degree of time and labor and capacity build the staff exposure to the new differentiation. In the rare cases where the diagnosis remains technology. difficult, histological, cytogenetic and/or molecular studies are necessary. Conclusions

doi:10.1016/j.cca.2019.03.814 The results have shown apheresis system has reduced blood wastage and at the same time reduced the cost of labor and time used to prepare blood using manual methods. The number of times a donor can donate has improved significantly with a benefitof W040 ^ multiple products. Improvement of blood donation at Gertrude's Children's Hospital doi:10.1016/j.cca.2019.03.815 (GCH)

P. Chege Gertrude's Children's Hospital, Kenyatta University, Kenya ^W041

Background-aim Laboratory algorithm for multiple myeloma screening

GCH Laboratory is mandated by the Hospital Transfusion A. Fontana, A.C. Sopenaa, M.E. Lalanaa, M. Sancheza, A. Crespob,A. committee (HTC) to ensure Blood Safety in the Hospital. The mission Tapiaa is to provide safe, effective and adequate blood and blood products to aAnalisis Clinicos, Hospital de Barbastro, Aragon, Spain all patients in need. The Hospital Blood Transfusion committee bAnestesiología y Reanimación, Hospital San Pedro, La Rioja, Spain oversees the overall role for safety in transfusion science. The Laboratory has the main role of screening the donors and availing Background-aim safe blood and blood products. The Laboratory has used the manual methods until January 2018 for blood products collection. The Total protein (TP) is used for diagnosis and treatment of liver, automated method has been into play from January 2018. The kidney or bone marrow diseases as monoclonal gammopathies System in place is Trima Accel, this system uses a continuous flow (MG), among others. A TP level decreased is related with nutritional centrifuge to separate whole blood into individual components. deficiencies, ineffective synthesis or lost increased, etc., and an Whole blood is drawn from the donor and mixed with anticoagulant increase can take place in pathologies such as hyper- (AC). The blood and AC are pumped into a channel, then spun at high gammaglobulinemias (mono or polyclonal) or hypovolemic states speed in the centrifuge, separating the blood into its component (dehydration, sweating, vomiting or diarrhea). parts. Combinations of platelets, plasma, and red blood cells (RBCs) Multiple myeloma (MM) is part of MG, and may not cause are collected in bags and the remaining components are returned to symptoms for a long time and could not be diagnosed until it is in the donor. advanced stage. Common symptoms are unspecific pain, frequent infections, anemia (AN) and weakness. Methods Methods The aim of our laboratory is to provide save, effective and adequate blood and blood products. This has been achieved with the We show 3 cases from to the Emergency Department (ED) that use of Trima Accel; the recipient is exposed to only one donor which presented hyperproteinemia (HYP) without justified urgent disease. The reduces the infection rates. There is reduced rate of bacterial Laboratory Department (LD) has a computer algorithm, that requests a contamination by the accomplishment of leuco reduced blood. This serum sample for further studies (TP and protein electrophoresis (PE)) in system reduces labor significantly within a considerable turnaround order to screening MG in HYP from ED over 9 g/dL cases. time whereas achieving high product quality. There is insignificant wastage and the frequency within which a donor can donate is Results increased with a benefit of multiple products. 1: Male, 51 y.o., attended for dyspnea. He was diagnosed of Results respiratory infection, presenting TP: 9.4 g/dL and creatinine (CR): 1.1 mg/dL. In a second consultation, the patient presented acute We have had a total of 60 Apheresis donations each giving 2 kidney failure with preserved diuresis and CR of 5.32 mg/dL. During doses. 75% of the donations were done by male gender. Among the admission was diagnosed of MM with a M-spike (MC) IgG-Kappa of 25% females most of them had calcium supplemented due to either 3.3 g/d, with proteinuria and bad prognosis. Finally, the patient, after numbness, twitching, tingling, spasm of facial muscle, lips and heavy suffer a free light chain escape, died. tongue. Most of them responded to calcium supplementation. 2: Woman, 45 y.o., consulted for abdominal pain. AN was Although the laboratory has not fully moved from the manual observed, hemoglobin (Hb): 9.9 g/dL, HYP (10.4 mg/dL), and CR methods the uptake of the new technology is showing good levels within normality. The LD requested a study of HYP, detecting a progress. The manual trends donation were 2018(252 units), 2017 IgG-Kappa MC of 4.6 g/dL. The patient began chemotherapy without (300 units), 2016(278 units), 2015(433 units) and 2014(424umits); incidents and with good response. Currently she presents negative there is a greater improvement of transfusion practice over the years immunofixation. with some significant improvement with Apheresis system on board. 3: Male, 84 y.o., went to the ED with dyspnea; LD results: AN (Hb: We aim to in future meet the Hospital demand with increased 6.9 g/dL) and TP of 10 g/dL with normal CR. He was admitted for the platelet component quality and safety, retain a pool of consistent study of AN and finally was diagnosed of MM with bone infiltrations. donors while preventing adverse donor reactions, saving on costs, In this case the forecast was favorable. Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

Conclusions complements the derived fibrinogen obtained from PT. Furthermore, it would offer clinically relevant information for those patients to Given an HYP result in an ED blood assay, additional studies must whom only APTT is requested. Therefore, estimating fibrinogen from be run regardless of the patient's age, to find out it cause, since it can be APTT should be an option to consider in the daily routine of the a potentially serious hidden disorder such as MM. We strongly clinical laboratory. recommend performance a PE in patients from ED with TP over 9 g/dL. doi:10.1016/j.cca.2019.03.817 doi:10.1016/j.cca.2019.03.816

^W043 ^W042 Are the platelet indices useful biomarkers of arteriovenous fistula Estimation of fibrinogen concentration from activated partial thrombosis in hemodialysis patients? thromboplastine time N. Klapuh- Bukvića, E. Kiseljakovićb a S. Garcia, V. Ortiz, A. Molina, A. Merino, S. Fumanal, M. Arnau, J.L. Department of Clinical Biochemistry and Immunology, University Bedini Clinical Center Sarajevo, Sarajevo, Bosnia and Herzegovina b CORE Laboratory, Biomedical Diagnostic Center, Hospital Clinic of Department of Medical Biochemistry, Faculty of Medicine, University of Barcelona, Spain Sarajevo, Sarajevo, Bosnia and Herzegovina

Background-aim Background-aim

Fibrinogen is an essential component in the haemostasis process. Platelet indices: total platelet count (PLT), mean platelet volume It is one of the main tests in the screening of the haemostasis (MPV), platelet distribution width (PDW) and plateletcrit (PCT) are function. The technique most used is based on the Clauss method. potentially useful biomarkers for the early diagnosis of thrombosis. However, there are some laboratories that use the fibrinogen derived The native arteriovenous (AV) fistula is the gold standard of access to method, which is a low-cost technique estimated from the the blood stream for patients on hemodialysis (HD), and its frequent prothrombin time (PT) results. complication is thrombosis. This study aimed to determine the The objective of this work is to evaluate the possibility of alterations of platelet indices in hemodialysis patients with AV fistula estimating the fibrinogen concentration from the activated partial thrombosis. thromboplastin time (APTT) results. Methods Methods The clinical cross-sectional, descriptive and comparative study PT, APTT and Clauss fibrinogen were performed over a total of included 100 patients, of both sexes, average age from 20 to 65 years 100 plasma samples from normal control patients. The instrument CS of age, who are admitted to, in the period of four years, the Clinic of 5100 (Siemens) was used together with the following reagents: Hemodialysis, University Clinical Center Sarajevo (UCCS). Patients Thromborel (PT), Actin FSL (APTT) and Thrombin (fibrinogen). were divided in two groups on basis of established AV fistula In this study, we performed a linear regression model of the thrombosis. The study group included 34 patients with confirmed AV absorbance change (from both PT and APTT) against the Clauss fistula thrombosis. The control group included 66 patients without fibrinogen results. From both models we obtained an equation that AV fistula thrombosis. The venous blood sample was collected prior we used to estimate the concentration of fibrinogen. to the hemodialysis procedure.

Results Results

The results of PT, APTT and fibrinogen obtained from the samples The mean PLT (185.66 ± 60.26 × 10e9/L vs. 231.62 ± 65.56 × analyzed showed an average of 13.6 s; 33.15 s and 2.5 g/L, a 10e9/L) was significantly lower (p = .001), while the mean MPV minimum value of 12.2 s; 30.3 s and 1.61 g/L; and a maximum value (8.48 ± 0.72 vs. 6.69 ± fL 0.52 fl) was significantly higher (p = of 15 s; 36 s and 3.58 g/L, respectively. .0001) in patients undergoing chronic hemodialysis with thrombosis From the regression model performed, we obtained a coefficient of AV fistula in relation to the control group. The optimal cut-off of determination (R2) of 0.929 for the estimation based on the APTT, value for PLT in differentiating patients on chronic hemodialysis with and 0.885 for PT. The sum of the differences between the estimated established thrombosis of AV fistula amounted to 180.0 × 10e9/L value of fibrinogen and the measured one was 0.01 for the APTT with a sensitivity 81.5%, specificity 55.9%, positive predictive value model and 0.14 for PT. 77.9%, negative predictive value 61%. By using a cut-off value 7.395 fL for MPV, sensitivity was 97.1%, specificity was 95%, positive Conclusions predictive value was 97%, negative predictive value was 98% in differentiating patients on chronic hemodialysis with established fi The results of this work show that the absorbance change of the thrombosis of AV stula. APTT is a good estimator for the concentration of fibrinogen, since there is a high correlation between both variables. In addition, APTT Conclusions results show a better fit to the real fibrinogen concentration than the model based on PT. Platelet counts and mean platelet volume can be used as effective The estimation of fibrinogen from APTT may have important biomarkers of arteriovenous fistula thrombosis in patients undergo- practical applications, since it provides additional value that ing chronic hemodialysis treatment, noting that there is a greater Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

diagnostic potential for differentiation of arteriovenous fistula venipuncture. New morphological parameters such as MDW should thrombosis in mean platelet volume. be evaluated according to the EDTA tube used, since the concentra- tion of potassium salt could interfere in the results. doi:10.1016/j.cca.2019.03.818 doi:10.1016/j.cca.2019.03.819

^W044 ^W045 Influence of K2-EDTA and K3-EDTA tubes for monocyte distribu- tion width measurement Hemolytic anemia caught by every member of family caused by metallic elements M. Lopez-Molina, X. Tejedor Ganduxé, A. Martínez Iribarren, M. Espinosa, S. Torres, M. Sala, C. Fernandez, C. Abadia, M. Llopis, C. C. Malem Morales-Indiano Laboratory ¨Claudio Malem & Partners¨ Haematology-CoreLab Department, Clinical Laboratory Metropolitana Nord (LCMN), Hospital Universitari Germans Trias i Pujol, Badalona, Background-aim Barcelona, Spain Six adults patients who came to our country from the developing Background-aim country with very strong mining industry, as tourists were studied. All of them residents in an urban area, belonging to middle class Ethylenediaminetetraacetic acid (EDTA) is the anticoagulant of strata of society with good to regular nourishment. choice for haematology testing. Mainly, EDTA comes in blood tubes At the start of this study they all presented moderate to severe as dipotassium (K2) and tripotassium (K3) salts. It has been anemia, moderate leucocitosis, severe thrombocytopenia accompa- recognized that potassium's concentration salt may affect the nied with macrocitic elements with very high levels of plasmatic accuracy of cell blood counting, cell sizing and probably its stability. homocystein, hepatic profile including pancretic amilasa, CPK, LDH EDTA-salt may cause shrinkage of erythrocytes, affect mean corpus- above normal. cular volume and mean platelet volume. These effects are more Besides, an investigation about the housing conditions and family significant in K3-EDTA than in K2-EDTA. Monocyte Distribution structure (autonomy and leadership of the members) was carried Width (MDW) is a new morphology parameter available on the new out to understand the dynamic relationship of all the family DxH-900 haematology analyzer of Beckman Coulter, which could members in the house, hygiene, clothing and every day feeding potentially be useful for early sepsis detection. habits of the family group. The aim was to evaluate the differences in MDW using 2 different All these conditions contribute in a notable way to the patients EDTA-tubes (K2 and K3 salts) and to study its stability over time in welfare, but there was a fact which was not taken in to account, the both anticoagulants. presence of aquiferous effluents near the family house containing high concentrations of cadmiun (Cd) and mercury (Hg). It is caused Methods by a mining company nearby where precious metal elements are extracted. The parents even decided to have the spleen removed 25 healthy volunteers (80% women) were studied. For each one because of a mistaken diagnosis due to the high value of the we recollected two different EDTA-tubes (K2 and K3, BD enzimatic profile and the bad health condition of the parents. Vacutainer®). Samples were tested at different times (0 h (h), 1 h, It was decided to radically change the nourishing diet changing it 2 h, 4 h, 6 h, 8 h, 24 h and 48 h) and stored at room temperature for the ingestion of nutriments of protein origin, vegetables and dairy during the study. All samples were run on the DxH-900 Analyzer products and to accompany the family giving help and support. (Beckman Coulter; Miami, FL, USA). The differences between K2 and K3 at time 0 h were assessed by paired Student's t-test. Significant P Methods values were values below 0.05. To assess stability, the percentage change [(the mean of result at time X – mean of result at 0 h)/mean As a first step, it was decided to radically change the nourishing of result at 0 h]*100 was calculated. We established a significant diet for the ingestion of nutriments of protein origin, vegetables and difference for MDW when the change percentage (CP%) was N10%. dairy products. During the first 60 days ferrous fumarat and folic acid was used to Results raise the hemoglobin level whose value was between 8 and 9 g/dl for men and 7–7,5 g/dl for women, also a minimum dose of A significant 2 points difference was found between K2 and K3 for acenocumarol (adjusted to INR) was used to prevent thrombotic MDW at 0 h (K2: 16.12 ± 1.57 vs K3: 18.33 ± 1.75;p b .001). This effects in the parents. difference was maintained for most of the analyzed times. The value At the next blood control, all the hematological values were of the MDW remained stable until 8 h in both EDTA-tubes (CP%-K2: consequently changing and the presence of Cd and Hg diminished 1 h = 5.2%; 2 h = 2.6%; 4 h = 5.0%; 6 h = 6.4%; 8 h = 8.4%; 24 h = remarkably, that’s why it was decided to suspend the ferrous 19.4% and CP%-K3: 1 h = 4.2%; 2 h = 2.6%; 4 h = 5.3%; 6 h = 5.9%; fumarat, folic acid and acenocumarol, thus allowing the new diet 8 h = 8.6%; 24 h = 19.8%). perform the corresponding supply and also to control the patient's health condition in the future. Conclusions Results In our preliminary study, we found significant differences in the MDW over 2 points between K2 and K3 EDTA-tubes in most of the After 90 days, when the corresponding blood control was done, analyzed times. MDW showed CP b 10% in the first 8 h of the normal analytical values were achieved: 12–13,5 g/dl for men and Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

11,5 g/dl (in average) for women, accompanied with normocitic mutations were found (APC; 2,70 ± 0,30), while 17,8^% were elements and values of count of thrombocytes between 220 and 400 heterozygous (APC; 2,67 ± 0,26) and 1,2^% homozygotes (APC; 2,65 × 109/l, the homocystein value diminished remarkably to minor ± 0,12). Finally, no person was found with the mutation Y1702C. values as those estimated as reference ones, in the corresponding values to each patient, according to sex and age. Conclusions

Conclusions Our results confirm the previously obtained data regarding the utility of the determination of mutation C1691G N A since the All these adults, as a first step, changed their housing place, now average value of resistance to APC is b2,3. This points out a major far from the mining activity. By going on the right diet, they risk in these patients to present thrombotic episodes. Nevertheless, recovered health and activity mainly recovering bones strength and regarding mutation H1299R the average values of resistance to APC diminishing the plasmatic homocystein normal manageable values is N2,3. This questions the theory that this mutation increases such so as to prevent future thrombotic events. risk and the clinical utility of its determination. Lastly, we did not find any patient with mutations in Y1702C. doi:10.1016/j.cca.2019.03.820 doi:10.1016/j.cca.2019.03.821

^W046 ^W047 The clinical utility of the determination of the factor V Leiden mutations H1299R and Y1702C Use of flow cytometry and aggregatometry methods for the assessment of platelet haemostasis in children with essential A.B. García-Ruanoa, L. Entrenab, M.d.M. Maldonadoa, P. Antoniob,T. thrombocythemia De Haroa aHospital Universitario San Cecilio, Spain D. Polokhov, N. Ershov, A. Poletaev, N. Smetanina, M. Panteleev bHospital Universitario Virgen de las Nieves, Spain Federal Research and Clinical Centre of Pediatric Haematology, Oncology and Immunology, Moscow, Russia Background-aim Background-aim During the haemostasis, the factor V will be deactivated by the activated protein C (APC) in order to prevent the forming of a blood Childhood essential thrombocythemia (ET) is an extremely rare clot that could alter the cardiovascular stability. A mutation of the chronic myeloproliferative disorder. Patients with ET suffer from factor V Leiden leads to a synthesis of an abnormal factor V which both bleeding and ischemic complication. To get insight into the would be resistant to the deactivation forced by the APC. mechanisms of these disorders, we investigated blood samples of The mutation C1691G N A of the factor V Leiden is one of the most children with ET. common reasons of the appearance of clotting abnormalities. Approximately, 5^% of the Caucasian population has a mutated copy Methods (heterozygosity) of factor V Leiden. That is the reason why their study is requested whenever exists the suspicion that a patient could Ten children were examined. The median age of ET debut was 8 have a thrombotic risk factor. years (range 2–15 years). The gender composition was 4 boys and 6 The relationship between the mutation C1691G N A and the value girls. In 4 patients, disease progressed without clinical (group 1). In 3 of the APC was already described. But there are no data on what boys had ischemic symptoms: erythromelalgia, chest pain, head- happens with the new studied mutations H1299R and Y1702C. aches (group 2). In 3 girls hemorrhagic type: ecchymosis and/or Evaluation of the clinical utility of the determination of the factor nosebleeds of different severity (group 3). We used platelet V mutations H1299R and Y170C. aggregation (PA) with collagen, ADP and ristocetin and flow cytometry-based platelet function analysis (FC) in rest and after Methods activation with collagen+TRAP-6. Control groups (CG) for PA and FC included 14 and 47 healthy children. Retrospective study (September 2017 – February 2018) of the requests presented in the Genetics Unit of the Clinical Analysis Results Services of the Hospital Universitario San Cecilio (Granada/Spain) for the investigation of the mutations of the factor V and the resistance The median number of platelets in the debut in groups 1, 2, to the APC. and 3 were 1348 th/μl, 1594 th/μL, and 2000 th/μl, respectively. PA Detection of mutations: simultaneously with the kit Anyplex TM in response to collagen, ADP and ristocetin in the 10 examined II Thrombosis SNP Panel Assay by means of a real time PCR. patients was decreased in all patients compared with the CG. Measurement of APC resistance with the Immunochrom APC There were no differences in PA between the patients group. 9 Response assay: Immunochrom APC Response kit. patients were examined by FC. There were no significant differences in PAC1 and the number of dense granules at rest Results when compared with the CG and between the groups of patients. PAC1 and the volume of dense granule release after activation was The analysis comprised 501 patients. Relating to mutation not significantly different in the whole patient cohort compared C1691G N A, 2,76 ± 0,17 were found in the resistance value of with the CG, but both were significant decrease after activation in protein C. The protein C resistance of 7,8^% of the heterozygous were the 3 patients with hemorrhagic symptoms compared with the 6 1,74 ± 0,081. Relating to mutation H1299R, in 81^% of the patients no patients without it. Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

Conclusions ^W049

Aggregation is reduced in all patients compared to controls. In 3 Assessment of the possibility to detect samples with increased children with hemorrhagic manifestations: 1) the average number of band count on the UniCel DxH 800 Beckman Coulter in automatic platelets is higher than in children without them; 2) PAC-1 and the mode volume of dense granule release after activation are reduced in comparison with 6 patients without hemorrhages. Our data suggest V. Burovad, D. Sukhachevc, D. Kovchenkod, E. Khalyuzevad,D. possible causes of bleeding in essential thrombocythemia and the Butlitskia, V. Gerasimenkoa, E. Sukhachevab possibility of using flow cytometry for risk assessment. aBC LLC, Moscow, Russia bBeckman Coulter Eurocenter, Nyon, Switzerland doi:10.1016/j.cca.2019.03.822 cLabTech Ltd., St. Petersburg, Russia dLLC «GlobusMed», St. Petersburg, Russia

Background-aim ^W048 For high-throughput laboratories, it is difficult to identify patients Evaluation of the platelet count with the Sysmex DI-60 system with increased band count without performing blood film review, which is time-consuming and requires manual steps. UniCel DxH 800 G.L. Salvagnob, M. Puccib, D. Veneria, A. Bonalumia, D. Alzettaa,G. provides 56 @Cellular Morphometric Parameters (@CMP) for leuko- Lippib, F. Dimab cytes differentiation, which potentially may help detect the presence aHematology Section, Department of Medicine, University of Verona, of abnormal cells. The goal of the study was to investigate if @CMP, Italy together with other parameters can be used to estimate the bSection of Clinical Biochemistry, Department of Biomedicine and probability of increased band count in blood specimens. Movement Neurosciences, University of Verona, Italy Methods Background-aim

Data from 2384 patients were used as training set: 1350 women, Although automatic platelet (PLT) count is now the most ^ ^ 1034 men (1533 adults, 851 children 1–15 years old). The time from widespread approach in clinical laboratories, optical analysis of ^ ^ blood draw to sample analysis ranged from 2.5 to 8 h. Blood films were peripheral blood smears remains the methodological reference prepared for all samples and microscopic review was performed to technique for rapid and accurate diagnosis of hematological diseases. identify samples with increased band count. MedCalc statistical New semi-automatic tools have recently been introduced to improve software (ver. 17.2, Ostend, Belgium) was used for analysis. microscopic examination, thus reducing the turnaround time and inter-individual variability on optical analysis. The aim of this work is Results to compare the automated Sysmex XN (Sysmex, Kobe, Japan) and the semi-automated Sysmex DI-60 (Sysmex, Kobe, Japan) for platelet In 36 patients from the training set, increased band count was counting. detected (from 7 to 21). We developed regression model to discriminate samples with N6% of bands from samples with normal Methods band count (≤6%). This model included 2 @CMP (@MN-V-MO – Mean Monocytes Volume; @SD-V-MO – SD of Mean Monocytes Volume) We randomly selected 90 blood samples of hospitalized patients, and 2 IVD parameters NEUT# and WBC. Following results were which who did were free from platelet aggregates. Platelet counting obtained for training set: 117 true positive (TP), 4 false negative was performed with Sysmex XN system and then blood smears were (FN), 1138 false positive (FP), 1125 true negative (TN). Sensitivity assessed using DI-60 for semiautomatic platelet enumeration. ^ ^ and specificity were 96.69% and 49.71%, respectively. To verify prospectively the performance of the model, the equation was Results included in the LIS and applied to 10,192 patients. From this cohort, 327 samples with bands N6% were detected (TP-306, FP-3295, FN-21, PLT XN values (median 86.5, range 0-355 × 10^9/L) have a ^ TN-6^570). Among FP, there were samples with atypical mononuclear similar distribution to that of DI-60 (median 69.5, range 0-325 × cells, metamyelocytes and myelocytes according to morphological 10^9/L). Passing-Bablok regression analysis showed good compara- analysis; among FN –samples with Pelger cells, atypical mononuclear bility: r = 0.964, slope 0.92 (95% CI, 0.86 to 1.00), intercept 0.11 cells and myelocytes. Specificity and sensitivity in this case were − (95% CI, 4.63 to 4.55). The Bland-Altman plots revealed an average 66.6% and 93.6%, respectively. bias of −8.7 PLT x10^9/L (95% CI, −13.9 to −3.43). Conclusions Conclusions Our approach to detect samples with high band count may be In agreement with previous evidence, a high correlation and an useful in routine practice to reduce the number of unnecessary blood excellent analytical agreement emerged from our study. Less film review and improve the laboratory efficiency*. satisfactory agreement was also noted in samples with substantially *Requires validation through a controlled clinical trial. increased platelet count. @For research use only. Not for use in diagnostic procedures.

doi:10.1016/j.cca.2019.03.823 doi:10.1016/j.cca.2019.03.824 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

^W050 ^W051

The results of hemoglobin variant analysis in a routine laboratory Establishment of hematological reference intervals for healthy in a year period adults at Tribhuvan University Teaching Hospital, Kathmandu, Nepal T. Villalba Hernandezb, A. Fernandez Uriartea, J. Medina Ugarellib,M. Diaz Vallsb, J. Roige Buixadea K. Acharya, B.K. Yadav, V.K. Sharma, E.T. Tuladhar, M. Raut, A. aCatlab AIE, Terrassa, Barcelona, Spain Bhattarai bHematology, Catlab AIE, Terrassa, Barcelona, Spain Department of Biochemistry, Maharajgunj Medical Campus, TU Teach- ing Hospital, Kathmandu, Nepal Background-aim Background-aim Hemoglobin (Hb) molecule is made up of four protein subunits (globin) and four hemo groups. Aproximately 98% of erythrocyte's Reference intervals (RI) for hematological parameters are important Hb is HbA (〈2®2). Remaining 2% are HbA2 (〈2™2) and fetal for the proper diagnosis and monitoring of the patients. The right hemoglobin (HbF; 〈2©2). Globin chains are encoded in groups of interpretation of the several laboratory values depends mainly on the genes in chromosomes 11 (2 genes for ® chains) and 16 (4 genes for locally derived reference interval. Till date, there is no any single study 〈 chains). Mutations in those genes cause congenital hemoglobinop- established the reference interval for the hematological parameter in athies and the consequences may result in structural defects of the Nepal. All most all of the medical laboratory uses the reference interval protein (HbS, HbC…), decreasing of Hb synthesis (thalassemia), both provided the manufacturing company which is very different from Nepal. (thalassaemic hemoglobinopaties) or hereditary persistence of fetal Therefore, this study was designed to establish the gender and age- hemoglobin. Clinical manifestations can vary from asymptomatic specific hematological reference intervals of healthy adult population of carriers to transfusion-dependent patients with a high risk of life Nepal^ threatening complications. Also, variant hemoglobins can interfere with other measurements, as glycosilated hemoglobin (HbA1c). Methods

Methods A cross sectional study was conducted from the apparently healthy adult visiting Tribhuvan University Teaching Hospital and living nearby In our laboratory, hemoglobinopaty studies are requested in three it. About 6 ml blood samples (3 ml in EDTA-tube and 3 ml in serum settings: variant Hb peak detected in HbA1c tests, mean corpuscular separating gel tube) were collected from all the participating individuals. volume (MCV) b75 fl without iron deficiency, clinicians' requests for Hematological cell counts were analyzed by using Sysmex haematology patient diagnosis or family studies purposes and HbS quantification analyzer as per given manual. Serum iron and Ferretin were assayed in to adjust treatment. Vitrous 3600 and BT 1500 fully automated analyzer, respectively. Analyzers: Blood count (CBC): Sysmex XN (Roche), HbA1c tests and variant Hb: HPLC D-100 and D-10 (Bio-Rad), Alkaline electropho- Results 〈 〈 resis (^Hidrasys), -Thalassemia molecular study: kit globinstrip assay. In the laboratory reports, in addition to the percentages of the There was a significant difference between male and female for different hemoglobins and the description of the electrophoresis we most of the hematological parameter including serum iron and decided to scan and attach reports from the analyzers (HPLC and b ferretin (p-value ^.05). This study also demonstrated the lower level electrophoresis) in pdf format so the clinicians could have a better of Hb and MCV in Nepalese women and some were even under mild knowledge of the Hb pattern. Previously we published in our website to moderate level of anemia defined by WHO. Most of the elevated an informative document about these analyses and how to interpret hematological analytes were encountered higher in males than in the reports. females except platelets.

Results Conclusions

1042 HPLC hemoglobinopathy studies have been run in our The reference intervals derived by this study are different than laboratory during 2018. 19.6% were secondary to peak detection. 143 the currently used RI which is provided by the manufacturing patients were diagnosed as carriers of ® thalassemia and 16 of ® ™ company. Our study also demonstrated that most of the hematolog- thalassemia. 150 electrophoresis were performed: 75 het HbS, 20 het ical parameter has different RI for males and females and therefore, it HbC, a miscellanea of Hb E, D, J, Lepore… accounting for 22 samples, is advised to use locally derived RI for the proper interpretation of 12 rapid migration Hb unidentified and 5 samples without abnormal the results obtained by laboratory. bands. 〈 globin studies (n = 133) yielded 14 homo del 3.7 kb, 60 het del 3.7 kb, 8 other mutations and 50 studies without abnormalities. doi:10.1016/j.cca.2019.03.826

Conclusions

W052 In our area there is a great variety of abnormal hemoglobins, ^ about 400 new findings for a whole 395,000 CBC a year. The addition The impact of training workshops and locally adopted interna- of pdf reports offers a better traceability, ease of interpret results and tional standardized guidelines on coagulation specimen rejection improves communication between clinicians and laboratory staff. rates doi:10.1016/j.cca.2019.03.825 M. Du Toitc, A.D. Jaftaa, A.E. Zemlinb aAmpath Private Laboratories, Western Cape, South Africa Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

bDivision of Chemical Pathology, National Health Laboratory Service Department of Medical Technology, Faculty of Allied Health Sciences, (NHLS) and University of Stellenbosch, Tygerberg Hospital, Cape Town, Naresuan University, Phitsanulok, Thailand South Africa cDivision of Haematology, National Health Laboratory Service (NHLS) Background-aim and University of Stellenbosch, Tygerberg Hospital, Cape Town, South Africa Thalassemias are inherited blood disorders and hemoglobin E (Hb E) is a common type that found in Thailand, Laos, and Cambodia. Background-aim Several techniques are used to diagnose for Hb E such as dichlorophenol indophenol precipitation (DCIP), micro column The correct identification of all pre-analytical variables that can (MC) and capillary electrophoresis (CE) are useful to screen for adversely affect coagulation specimen quality is of paramount thalassemia carrier, to identify abnormal hemoglobin in prenatal importance. Heterogeneity among laboratory personnel with regards testing, and to manage thalassemia patients in hospital settings. This to the knowledge of coagulation pre-analytical variables can lead to study aims to investigate on homogeneity, stability and characteri- inconsistent identification of these variables and spurious test zation of Hb E in blood material that prepared by using donated results. The aim of this study was to determine the impact of a blood from blood bank. training workshop on coagulation specimen rejection rates and to ascertain the level of knowledge and understanding of laboratory Methods personnel concerning coagulation sample rejection criteria. Hb E was screened by DCIP and MC while Hb E typing was quantified Methods by CE. Normal blood and Hb E blood samples were used and prepared in phosphate buffered saline (PBS) with and without preserved by 0.02%

Initially, a retrospective audit was performed where coagulation NaN3. Homogeneity and stability were investigated for 8 weeks by specimen rejection data was collected over a period of three months. following the statistical analysis of ISO Guide 35 and according to Training workshops and evaluation sessions were subsequently guidelines of reference material production by ISO 17034. presented and the adherence to International Organization for Standardization (ISO) guidelines for coagulation specimen rejection Results was emphasised. A revised standard operating procedure for pre- analytical variables and coagulation specimen rejection criteria was The results shown prepared blood materials in PBS with and

then implemented and a repeat audit was performed. without 0.02% NaN3 were homogenized in those with Hb E positive and Hb E negative results by DCIP, MC and CE. HbE in blood materials Results was stable up to 8 weeks when determined by DCIP and MC while the stability of HbE positive in quantification by CE was lower than 4 The coagulation specimen rejection rate prior to the presentation days. However, the level of Hb E was in the carrier interpretation – of the training workshops was 11.34%. Following the introduction of range with Hb E 15 24^% at 8th week. Decreasing rate of Hb E in training workshops, the coagulation specimen rejection rate in- blood materials with buffer and 0.02% NaN3 was lower than those in creased to 17.42%. Evaluation sessions performed before and after buffer without 0.02% NaN3. the training workshops revealed an overall 95% improvement in knowledge among the attending laboratory personnel. Conclusions

Conclusions Blood material with and without Hb E could be prepared in PBS

with 0.02% NaN3 and used for Hb E screening by DCIP and MC up to Training workshops were performed to highlight the need for 8 weeks and lower than 4 days for CE. Prepared blood material correct identification of pre-analytical variables that impair coagula- obtained from this study may be useful for using as material in tion specimen quality. The significant increase in the coagulation proficiency testing or inter-labs comparisons for Hb E testing. specimen rejection rates following these workshops demonstrates their success in educating laboratory personnel. As many pre- doi:10.1016/j.cca.2019.03.828 analytical variables occur outside the laboratory environment, these workshops will need to be extended to phlebotomists and clinicians who are responsible for the initial blood collection. ^W054 doi:10.1016/j.cca.2019.03.827 Comparison of automatic peripheral blood smear to manual blood smear technique

b a b b b ^W053 E. Avci , B. Unver Koluman , R. Nar , H. Aybek , S. Demİr aHematology Department, Faculty of Medicine, Pamukkale University, Homogeneity and stability of hemoglobin E in prepared blood Denizli, Turkey material when characterized by DCIP, micro column and capillary bMedical Biochemistry Department, Faculty of Medicine, Pamukkale electrophoresis University, Denizli, Turkey

N. Apiratmateekula,b,J.Tokhama, K. Kongrosa,b,W.Treebuphachatsakula,b Background-aim aReference Material and Medical Laboratory Innovation Research Unit, Thailand A manual blood smear review (MBSR) is defined as the attentive bReference Material and Medical Laboratory Innovation Research Unit, and careful microscopic analysis of a well-prepared and stained Abstracts / Clinica Chimica Acta 493 (2019) S379–S433 smear of peripheral blood, with the objective of seeking morpholog- Background-aim ical changes relevant to the diagnosis and monitoring of patients. Over the last few years, the performance and abilities of automatic Patients with systemic lupus erythematosus (SLE) are subject to haematology analyzers have improved considerably. Although they significant morbidity and mortality due to atherosclerotic diseases, still cannot identify all morphological abnormalities that may occur which cannot be fully explained by traditional risk factors. Thrombin in peripheral blood, they can reliably decrease the MBSR without generation test (TGT) is a global haemostasis test providing sacrificing quality. For this purpose, we evaluated Mindray BC6800 information about the speed and amount of generated thrombin in peripheral smear performance simultaneous MBSRW by a plasma. Our aim was to find out whether results of this test might hematologist. differ in patients with SLE as compared to healthy individuals and to see whether TGT parameters are associated with thrombotic Methods episodes in SLE patients.

The examination was conducted in the haematology unit of Methods biochemistry laboratory of Pamukkale University Hospital. For each sample, a blood smear was prepared and stained using the Mindray Forty-six patients with SLE not taking anticoagulants and 70 age 6800 BCE automatic slide maker-stainer (Mindray Corporation, and sex-matched healthy controls were enrolled. Thrombin gener- China). From each sample manual blood smear was prepared ation using the calibrated automated thrombogram (CAT) assay was manually by the wedge-spread film technique, using the May- performed using platelet poor plasma and results were evaluated by Grunewald & Giemsa stains. All review was done by a single-blind the Thrombinoscope software. Lagtime, endogen thrombin potential hematologist. (ETP), peak thrombin, time-to-peak and velocity index were calculated. Clinical parameters including age, sex, BMI, smoking Results habit, traditional risk factors, thrombotic history and disease activity were registered. Thirteen slights were analyzed. Automatic peripheral smear and manual smear were good correlated. Platelet count similar to each Results other. Platelet count and images more clear than the manual smear. Erythrocyte morphology was similar in two review slights. The side In SLE patients lagtime and time-to-peak parameters were fields of the slights were not appropriate to evaluate, because of significantly prolonged, while ETP was significantly reduced as smear techniques. The central area, which the erythrocytes were compared to controls (p b .0001). more clear and selectable, was enough for the understanding the TGT parameters showed significant positive correlation with BMI erythrocyte morphology. In some areas, artifacts were present to and CRP in patients and in controls, as well. The presence of lupus inhibit the evaluating these fields. Another rare cell etc. eosinophil anticoagulant increased lagtime and time-to-peak parameters sig- was more apparent and clear in the automatic peripheral smear. nificantly, while the presence of anticardiolipin antibodies was Manual blood smear more effective to evaluate leucocytes morphol- associated with significantly lower ETP. SLE patients with history of ogy. We reached two hundred cell counts in manual blood smear, thrombotic events had significantly higher ETP values, pregnancy but in automatic smear, we sometimes could not reach this count. In morbidity was associated with elevated peak thrombin levels. some areas, neutrophil-lymphocyte discrimination was difficult to analyze. Conclusions

Conclusions In patients with SLE, the extent of TG was significantly lower as compared to controls, which might be associated with the presence The efficiency and reliability of this analyzer in performing of antiphospholipid antibodies. The history of thrombosis or peripheral smear and found acceptable with the need to review a pregnancy morbidity was associated with increased TG, indicating stained blood smear. Additional, we need further examinations to that the test might be suitable for identifying those with elevated obtain additional information on selected cases. thrombotic risk in this patient population. doi:10.1016/j.cca.2019.03.829 Funding

NKFI 128582. W055 ^ doi:10.1016/j.cca.2019.03.830 Thrombin generation in patients with systemic lupus erythema- tosus (SLE)

^W056 N.K. Tóthb, T. Tarra, P. Soltésza, L. Lóczib, L. Muszbekb, Z. Bagolyb a Department of Clinical Immunology, Faculty of Medicine, University of Bone marrow gouty tophi secondary to multiple myeloma Debrecen, Debrecen, Hungary b Division of Clinical Laboratory Sciences, Department of Laboratory H.I. Bang, T.Y. Choi, J. Kim, R. Park, W.Y. Shin, J.W. Shin Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Department of Laboratory Medicine, Soonchunhyang University Seoul Hungary Hospital, Seoul, Republic of Korea Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

Background-aim ^W057

Gout is a clinical syndrome characterized by recurrent, painful Improving D-dimer appropriateness by controlling periodicity of arthritis caused by deposition of monosodium urate crystals. A retesting: Prevention is better than cure deposit of monosodium urate crystals in patients with longstanding hyperuricemia is a tophus. The crystals that build S. Carusoa,b, D. Szökea, S. Birindellia, F.S. Falvellaa, A. Dolcia,M. up under the skin may form small white or yellow lumps known Panteghinia,b as subcutaneous tophi. We report here a rare case of gouty tophi aClinical Pathology Unit, ASST Fatebenefratelli-Sacco, Milan, Italy in the bone marrow in a patient who was diagnosed with multiple bSpecialization School in Clinical Pathology and Clinical Biochemistry, myeloma (MM). University of Milan, Milan, Italy

Methods Background-aim

A 67-yr-old woman with monoclonal gammopathy of undeter- D-dimer testing in plasma is primarily recommended for ruling out mined significance (MGUS) and end-stage renal disease (ESRD) deep vein thrombosis because of its high negative predictive value. who has been undergoing dialysis since 4 years ago, was admitted Due to the relative long half-life (~16 h), closely repeated D-dimer to the hospital for evaluation of frequent catheter occlusions by measurements are useless and retesting within 24 h must be thrombosis. After two-days from admission, she presented the discouraged. However, this recommendation is often ignored by severe pain of the distal interphalangeal joints of fingers in both clinicians. To improve the appropriateness of D-dimer request in hands. Laboratory findings were as follows: hemoglobin 9.4 g/dl, hospitals served by our laboratory (a 600-bed general hospital and a 200-bed infant-maternity setting), we issued local recommendations white blood cell count, 7^100/ul, platelet count, 146,000/ul, pro- thrombin time (PT), 9.9 s, activated partial thromboplastin time together with acting on the computerized provider order-entry (aPTT), 32.0 s, fibrinogen, 295 mg/dL, D-dimer, 9812 ng/mL, anti- (CPOE) by implementing a 24 h block on the periodicity of retesting thrombin III, 26%, protein C, 59%, free protein S, 58.8%; blood urea [the latter not exploited in the Emergency Departments (ED)]. Here we nitrogen (BUN), 47.9 mg/dL, serum creatinine, 4.88 mg/dL, uric acid, evaluated the efficacy and cost-effectiveness of these interventions. 5.0 mg/dL, IgG, 896 mg/dL, IgA, 181 mg/dL, IgM, 88 mg/dL, free light kappa chain/free light lambda chain ratio, 0.12, ®2-microglobulin, Methods 28,445.33 ng/mL. Serum protein electrophoresis showed two mono- clonal peaks in the gamma region and immunofixation electropho- We recruited data from laboratory information system about resis confirmed presence of double gammopathy, IgG-lambda. In numbers of requested D-dimer tests through the same 9-month bone marrow aspiration smear, plasma cells accounted for 45.2% of period (Mar-Nov 2017 vs. 2018), before and after the mentioned all nucleated cells. interventions. The economic impact of changes in laboratory test utilization was also estimated. Results Results Bone marrow biopsy revealed focally infiltrated plasma cells with multiple urate crystals (gouty tophi) with foreign body type giant In the two evaluated periods (2017 vs. 2018), 11,892 and 9051 D- cells. Immunophenotypic investigation revealed that plasma cells dimer tests were requested, respectively, with a − 23.9% decrease. The were CD19-, CD56dim+, and CD45-, and showed cytoplasmic percentage of tests repeated inappropriately (retesting interval b 24 h) lambda light chain restriction (tumor burden: 31.5%). No apparent was 11.4% and 3.8% before and after the CPOE modification, respectively cytogenetic abnormalities were found in a chromosome analysis and (P b .001). After the block implementation, 341 requests did not fulfil fluorescent in situ hybridization. She was diagnosed with symptom- the established minimum retesting interval. 50% of them were made by atic MM and gout additionally, and treated with bortezomib, ED, in which the CPOE modification was not implemented, and 38% in melphalan, and prednisone. However, the patient was expired due case of patients transfer in another hospital ward. Only 3.5% of tests to acute gastrointestinal bleeding with hypovolemic shock after 10 repeated after b24 h where justified because previous samples were not days of chemotherapy start. suitable or for implausible values. A saving of 30,000 €/year due to the improved prescription appropriateness was obtained, about one third of Conclusions which attributable to the control of retesting periodicity.

Gout has been reported as occurring with the hyperuricemia Conclusions found in leukemia, polycythemia vera, and MM, etc. Hyperuricemia is a classic feature of gout, but blood uric acid levels may be normal Our data show that stopping inappropriate D-dimer requests during an attack as shown in this case. Metabolic syndrome before they reach the laboratory works properly in improving test including prothrombotic and proinflammatory states with gout has cost-effectiveness without impacting on patient safety. Institutions been reported. Gout might be a contributor to increase the should fully exploit the potential of electronic requesting acting as thrombotic tendency. “enabling factor” for reinforcing educational messages and sustaining their effects over time. doi:10.1016/j.cca.2019.03.831 doi:10.1016/j.cca.2019.03.832 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

^W058 ^W059

Is red blood cell distribution width (RDW) prognostic marker in Hypercoagulable state in breast cancer patients pediatric acute leukemias? T. Denevaa, S. Stoenchevaa, D. Davchevaa, B. Deleva, E. Belevab O. Ciepielaa, J. Holkad, P. Tarnowskac, I. Kotułab aDepartment of Clinical Laboratory, Medical University, University aMedical University of Warsaw, Department of Laboratory Diagnostics, Hospital “St. George” Plovdiv, Bulgaria Poland bDepartment of Clinical Oncology, Medical University, University bMedical University of Warsaw, Department of Laboratory Medicine and Hospital “St. George” Plovdiv, Bulgaria Clinical Immunology of Developmental Age, Poland cMedical University of Warsaw, Students Scientific Group at Department Background-aim of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Poland Prothrombotic tendency is characteristic of solid tumors. In dMedical University of Warsaw, Students Scientific Group of Laboratory cancer patients there is an increased risk up to 4–6 fold for Medicine, Poland developing idiopathic thrombosis due to the tumor-associated prothrombotic status. The transmembrane protein Tissue Factor Background-aim (TF) is the physiologic trigger of coagulation in normal haemostasis. TF is present in plasma in various forms, including microparticles Red blood cell distribution width (RDW) has been already found (MPs) and alternatively spliced TF. TF also plays important roles in as a prognostic marker of many cancer and non-cancer related vasculogenesis, metastasis, and tumor-associated angiogenesis. The diseases. It was found to be a useful marker in risk stratification of aim of the study was to evaluate plasma concentration of tissue chronic myeloid and lymphocytic leukemia in adults and a marker of factor antigen (TF-Ag) and tissue factor – bearing microparticles (TF- a disease activity in hairy cell leukemia. The aim of the study was to MP) activity in patients diagnosed with breast cancer. assess diagnostic usefulness of RDW in pediatric acute lymphoblastic (ALL) and myeloblastic leukemias (AML). Methods

Methods The study included twenty-five patients with breast cancer and an age, sex-matched control group of 25 cases of benign breast The present study included 499 healthy individuals and 357 lesions. We evaluated TF-Ag, TF-MP plasma levels and coagulation children with newly recognized acute leukemia (including 303 ALL parameters including prothrombin time (PT), activated partial and 54 AML), both boys and girls, aged 0–18 years old. A thromboplastin time (APTT), fibrinogen (FIB) and D-dimer levels. retrospective analysis of red blood cell distribution width (RDW) TF-Ag and TF-MP activity were measured by ELISA assay, APTT, PT, and correlation with hemoglobin (Hgb) concentration was per- FIB and D-dimers were measured by automated coagulation system formed using Mann-Whitney test, Pearson r-coefficient and ROC Sysmex CS 2000i. analysis. An impact of RDW on survival and relapse rate was assessed by Kaplan-Meier survival analysis. A p b .05 was considered Results significant. The plasma levels of MP-TF activity and TF-Ag in the observation Results group were significantly higher than that in the control group. In the observation group, the plasma PT and APTT were shortened, and the RDW in leukemic children was 16.13 ± 2.39%, whereas healthy levels of FIB and D-dimers were increased; the differences were children had RDW of 13.3 ± 0.8%. There was a significant difference statistically significant (p b .05). After the Pearson test, the plasma in RDW between leukemic and healthy children (p b .0001). levels of MP-TF activity and TF-Ag in patients with breast cancer Interestingly, RDW from AML (16.83 ± 0.37%) and ALL patients were negatively correlated with PT and APTT, and positively (16.03 ± 0.13%) differed significantly (p = .02) and was weakly correlated with FIB and D-dimers. negatively correlated with hemoglobin concentration (r = −0.27, p b .0001). Similar correlation between RDW and Hgb was found for Conclusions healthy individuals (r = −0.28, p b .0001). RDW had a high specificity and sensitivity for diagnosis of acute leukemia. The area Aberrant tissue factor (TF) expression can lead to hypercoagula- under curve (ROC) was 0.8996. Sensitivity of the test was 82.63% and ble state in patients with breast cancer. Based on the findings it specificity 88.58% at a cut off value of 14.05%. There was no might be suggested that haemostatic parameters play crucial roles in association between mortality rate or relapses incidences and RDW invasion and metastases of malignant tumors. at a time of diagnosis, however patients with the highest values of RDW showed slight trend (p = .19) to have relapse earlier than doi:10.1016/j.cca.2019.03.834 b children with RDW 17^%.

Conclusions ^W060 Red blood cell distribution width might be helpful in detection How to deal with samples with errors on APTT, PT and fibrinogen and risk stratification in pediatric acute leukemias. due to optical clot detection? doi:10.1016/j.cca.2019.03.833 K. Eeckhout, J. Van Den Bossche, K. Deiteren, R. Malfait, M. Maes Antwerp University Hospital, Belgium Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

Background-aim Background-aim

Analysis of routine coagulation parameters PT, APTT and fibrin- The Sysmex XN-20 haematology analyzer includes a white blood ogen (FG) is performed on the automated coagulation analyzer cell differentiation channel (WDF) which identifies and counts IGs Sysmex CS5100 (Siemens Healthcare Diagnostics). A limitation of cells (metamyelocytes, myelocytes, and promyelocyte). The software optical clot detection is the inability to produce correct results in reports both the absolute and the relative IG counts. The probability turbid samples. While recollection not always solves this problem, of the presence of IGs cells is indicated by the “IG present” flag. mechanical clot detection could be an alternative method. Establish a cutoff value for the “IG present” flag reduce the blood In our laboratory, samples with no correct result on the Sysmex smear reviews. CS5100 due to non-interpretable curves, are reanalyzed with the manual benchtop analyzer STart (Stago). On both analyzers, the Methods same reagents (with equal heparin and factor sensitivity) are used. To facilitate the exchangeability and interpretation of results for 224 samples were collected in EDTA K2 tubes (BD Vacutainer®, clinicians, a correlation-equation is used to convert the results from ref.:367841). All samples were processed on a Sysmex XN-20 the STart. analyzer and microscopically reviewed using a CellaVision DM96 The aim of this study was to evaluate the stability and digital microscope. The “IG present” flag reported as a percentage applicability of the correlation-equation, used since the implemen- was studied using a ROC curve analysis to detect the presence of IGs tation of the CS 5100 (5 years ago), by correlating the converted cells. The criteria used to determine a true-positive smear finding STart results with results from the optical analyzer CS 5100 for the was a total count of metamyelocytes, myelocytes, and promyelocytes coagulation parameters APTT, PT and FG. ε3% stablish by the CellaVision DM96 review. The Sensibility(Se), Specificity (Sp), Negative Predictive Value (VPN) and Positive Methods Predictive Value (VPP) were evaluated to select the optimal “IG present” flag cutoff point. The statistical study was performed using Citrated platelet poor plasma of 30 patients, selected to cover the Analyze-it®. total measurement range, were analyzed on the Sysmex CS 5100 for APTT, PT and FG and afterwards on the Stago STart. Statistical Results analysis were calculated with MedCalc Statistical Software. From the 224 samples analyzed, 135 samples were positive with Results an IGs cells percentage ε3% and 89 samples were negative with an IGs cells percentage b 3%. The “IG present” flag Area Under the Curve For the 30 included samples, an excellent correlation between CS (AUC) reported as a percentage for the detection of IGs cellsε3% was 5100 and STart was found for APTT with a correlation coefficient of 0,97 (95% CI, 0,95-0,99). The optimal cutoff value selected was “IG b ” ε 0,97 (P-value ^0,0001). For PT (INR) there was also an excellent present 5% (Se: 0,837 (95% CI, 0,764-0,895); Sp: 0,955 (95% CI, correlation coefficient of 0,99 (P b 0,0001). A correlation coefficient 0,889-0,988); VPP: 0,966; VPN: 0,794). of 0,98 was found for FG (P b 0,0001). Conclusions Conclusions The “IG present” flag shows a high diagnostic accuracy for IGs With excellent correlation coefficients and no clinical differences cellsε3% detection with an AUC of 0,97. The implementation of the for all tested routine coagulation parameters, we can conclude that “IG present” ε5% cutoff value will reduce the false-positive rates for STart is a reliable alternative for the analysis of routine coagulation this flags with a VPP = 0,966. The “IG present” flag information will parameters for samples with errors due to optical detection on improve laboratory work flow with the implementation of “IG CS5100. The correlation-equations are stable over time (5 years) and present” ε5% cutoff value that will reduce smears reviews. different lots of reagent. With the use of the same reagents (same factor and heparin doi:10.1016/j.cca.2019.03.836 sensitivity) on both analyzers and a correlation-equation to convert the results, we can reliably report results to the clinicians even for turbid samples with reaction errors. ^W062 doi:10.1016/j.cca.2019.03.835 Interchangeability between immature granulocytes blood cell count measured by the Sysmex XN hematological analyzer and peripheral blood smear microscopic revision ^W061 B. Fernández-Cidón, I. Cachón-Suarez, A. Sancho-Cerro, C. Imperiali- “IG present” flag diagnostic accuracy of the haematology analyzer Rosario, D. Dot-Bach, L. Sánchez-Navarro Sysmex XN-20 for immature granulocytes (IGs) cells Hospital Universitary Bellvitge, Spain measurement Background-aim B. Fernández-Cidón, I. Cachón-Suarez, A. Sancho-Cerro, C. Imperiali- Rosario, D. Dot-Bach, L. Sánchez-Navarro Immature Granulocytes cells (GIs) (metamielocytes, myelocytes Hospital Universitary Bellvitge, Spain and promyelocytes) are increased in physiological conditions such as Abstracts / Clinica Chimica Acta 493 (2019) S379–S433 pregnancy, but also in pathological conditions such as bacterial current AHA (XE2100) as the reference method and 540 blood infections or myeloproliferative neoplasm. Peripheral blood smear samples were analyzed on both AHA. Outlier data were excluded microscopic revision is the reference method but he Sysmex XN prior to evaluation with the Kolmogorov-Smirnov, Passing-Bablok analyzer includes a leukocyte cell differentiation channel (WDF) that and Bland-Altman tests according to CLSI H26-A2 to compare the identifies and measures GIs cells. The objective of this study is to Systematic Error (SE) of parameters with the specifications of the assess the interchangeability between GI% measured in the Sysmex- Desirable Biological Variation (DBV) for inaccuracy. A carryover XN analyzer and the reference method. study was performed according to the CLSI H26-A2 guideline. The flagging performance of the XN-3100 and the XE-2100 were Methods compared, using two experienced laboratory technicians as the reference method. 224 blood samples were collected in EDTA K3 tubes (BD Vacutainer®). Samples were processed by Sysmex XN analyzer. Results Peripheral blood smears were stained using May Grunwald and Giemsa solutions and microscopically reviewed using a CellaVision Within-run %CV values agreed with manufacturer specifications DM96. Aberrant results were eliminated using the Bland-Altman (except eosinophils) although MCHC, eosinophils, basophils and correlation analysis. Interchangeability study was carried out using immature granulocytes showed a higher %CV than the DBV. The the Passing-Bablok non-parametric regression method. The equation between-batch %CV of MCHC and low control Hb exceeded obtained was y = ax + b, where y was the system evaluated (GI% manufacturer specification but were acceptable for the DBV. In the measured by Sysmex-XN analyzer) and x the reference method comparison study all intraclass correlation coefficients were satis- (peripheral blood smear microscopic review). Analyze-it® program factory for all parameters except for basophils. MCHC and was used for the statistical study. eosinophils had lower agreement between analysers compared to other parameters. The SE obtained from Bland-Altman was very Results low for most parameters except for MCHC, eosinophils and basophils. Eosinophils had a lower value of SE than the biological Of the 224 blood samples analyzed, 8 aberrant results (3.5%) were variability whereas MCHC and basophils exceeded the limit. eliminated. The interchangeability equation obtained with 95% Carryover was b0.4 for all parameters. The flagging performance confidence interval was: y = 0.6 [0.5–0.75] x + 1.53 [1.39–1.71] %. of the XN-3100 was satisfactory and the overall efficiency rates were high. Conclusions Conclusions Passing-Bablock regression equation for GI% measured by Sysmex-XN analyzer shows systematic and proportional errors The XN-3100 showed a strong correlation with previously used regarding to the reference method. Taking into account the results XE-2100 AHA. The XN-3100 had a comparable analytical sensitivity obtained, GI% measured by Sysmex-XN analyzer is not interchange- with the XE, with a better analytical specificity which may lead to able with peripheral blood smear microscopic revision results, so it improved turnaround time and throughput. The overall data makes can't replace peripheral blood smear microscopic review. Despite the XN-3100 suitable for integration into the routine haematology this, the implementation of a cutoff value of GI% could be very useful laboratories. criteria for smear review and thus improving laboratory workflows. doi:10.1016/j.cca.2019.03.838 doi:10.1016/j.cca.2019.03.837

^W064 ^W063 The value procalcitonin and C-reactive protein as early markers of The verification study of the new Sysmex XN-3100 automated bacteraemia among patients with hematological malignancies haematology analyzer (AHA) with Sysmex XE-2100 and micro- receiving chemotherapy: A cross-sectional study scopic examination K. Kamvumab, K. Sumbukenia, T. Kailec a S. Incirb, K.E. Palaoglua Ministry of Health, Food and Drugs, Zambia b aAmerican Hospital, Istanbul, Turkey Mulungushi University, Zambia c bKoc University Hospital, Istanbul, Turkey The University of Zambia, Zambia

Background-aim Background-aim

We performed a verification study of the new haematology The immune system of patients with hematological malignancies analyzer XN-3100 by comparing it with the XE-2100 according to is suppressed during chemotherapy. This renders them vulnerable to Clinical and Laboratory Standards Institute (CLSI) and International frequent infections especially of the bacterial type. Timely diagnosis Council for Standardization in Haematology (ICSH) guidelines. of these infections is difficult, because a severe infection may be asymptomatic or manifest only in the form of fever or malaise. There Methods is need for laboratory markers that can detect an infectious process at an early stage. This study was aimed at determining the value of Ten patient samples were tested 10 times consecutively to using Procalcitonin (PCT) and C reactive protein (CRP), for early evaluate within-run precision. Quality control materials were used diagnosis of infection in patients with hematological malignancies in duplicate over 20 days for between-batch precision. We used the receiving chemotherapy. Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

Methods and meaningful information compared to simple clotting times. Second, the test monitors total thrombin formation in a period of 1 h This was a cross sectional study consisting of sixty eight (68) that may or may not correlate with clotting time results. The third patients with hematological malignancies. Data from each participant advantage is that clotting times usually do not provide any information including sex, age, clinical and laboratory data were collected after on hypercoagulability, while TGA is useful in this respect as well. obtaining informed consent. Blood specimens were then collected for measurement of PCT, CRP and bacteriological analysis. Patients were Results divided into two groups; those with a culture positive and negative result. PCT and CRP concentrations were compared between groups Reproducibility assays carried out on plasma samples provided CV using t-test and nonparametric statistical tests respectively. The area values between 3.0 and 5.5% in different time parameters (lag time, under ROC curve, sensitivity, specificity, likelihood ratio, and Spear- timet to peak) and quantity values (Peak thrombin, ETP) that values are man's correlation coefficient were also calculated. just slightly higher than those of conventionally used clotting assays. Based on the analysis of human platelets and cultured malignant cell Results and their microparticles we drew the conclusion that the lag time and time to peak parameters are the most sensitive to detect pro- and A total of 14 (20.6%) microorganisms were isolated, of which 10 anticoagulant changes (Hudák et al., Clin Chem Lab Med, 2017, 55: were grampositive bacteria and 4 were gram-negative bacilli. The 1215–1223, Hudák et al., Biomed Res Int, 2017;2017:9795271, Tóth J et mean values of PCT which were 6.1 ng/mL in the bacteraemia group al., Thromb Res, 2017, 158:25–34). In human plasmas the parameters and 5.1 ng/mL in the non-bacteraemia group, p = .023 and median for the quantity of thrombin formation, peak thrombin and ETP values CRP values were 24.2 (6.43–48.15) in the bacteraemia and 23.5 proved more valuable in detecting hypocoagulabilty in clinical samples (6.03–75.44) in the non-bacteraemia group, p = .832. The area (Hudák et al., PLoS One. 2017;12(7):e0180477) as well as in patients under curves was 0.52 (95% CI = 0.57–0.84) for CRP and 0.70 (95% treated with anticoagulants. CI = 0.35–0.69) for PCT. PCT value of N4.7 ng/mL is diagnostic for infections (sensitivity 86%, specificity 54%) while that of CRP was 21 Conclusions mg/mL with the sensitivity and specificity of 64% and 44% respectively. Elevated levels of PCT as well as fever were significantly The analytical performance of the TGA would make it suitable as a associated with bacteremia. diagnostic test, nevertheless the lack of standardization in activating components, the variability during sample preparation in cellular Conclusions assays and the changes in stored plasma samples as well as the considerable inter-individual variability in plasma TGA values PCT was a more reliable and sensitive marker of bacteremia hampers its utilization as a diagnostic test. among patients with hematological malignancies receiving chemo- therapy than CRP. doi:10.1016/j.cca.2019.03.840

doi:10.1016/j.cca.2019.03.839

^W066

Management strategies of iron deficiency in patients undergoing ^W065 hemodialysis The thrombin generation assay: A research method or a diagnos- tic test? E. Karakoub, M. Sonikianc, A. Lekkakoua, I. Papadimitrioub,J. Skarakisd, N. Trakasb a J. Kappelmayera, I. Beke Debrecenia, G. Szabóa, R. Hudáka, J. Tótha,Z. 1st Department of Respiratory Medicine, Sismanoglio General Hospital, Bagolyb Athens, Greece b aDepartment of Laboratory Medicine, University of Debrecen, Hungary Department of Biochemistry, Sismanoglio General Hospital, Athens, bDivision of Clinical Laboratory Science, University of Debrecen, Greece c Hungary Department of Nephrology, Sismanoglio General Hospital, Athens, Greece Background-aim dResearch and Development Department, DEMO S.A. Pharmaceutical Industry, Athens, Greece The clotting time tests measure the time needed for fibrin formation and have become well standardized assays in the past years Background-aim both by mechanic or nephelometric methods. However, for fibrin to be formed a tiny fraction of the total formed thrombin is sufficient. Anemia in hemodialysis (HD) patients is common and wide- The purpose of the study was to evaluate the usefulness of the Stago spread treated with erythropoietic agents. Iron deficiency from thrombin generation assay (TGA) in human and mouse plasma, on various causes, such as erythropoietin (EPO) and blood losses, is platelets, and on cultured malignant cells and their microparticles. prevalent in HD patients leading to intravenous (IV) iron supple- mentation. The optimal strategy of IV iron therapy in terms of Methods maintenance or “load-and-hold” administration was investigated.

The TGA definitely has three advantages compared to conventional Methods clotting time assays. First the raw fluorescence data measured by a fluorimetric substrate cleavage are converted to a kinetic result by Forteen patients (M/F: 11/3, aged 60 ± 12 years) hemodialyzed using a software (Thrombinoscope) and this provides more complex since 69 ± 61 months and treated with recombinant human Abstracts / Clinica Chimica Acta 493 (2019) S379–S433 erythropoietin (EPO) alfa and iron intravenously were retrospec- reached −130 °C, −60 °C, −40 °C, and −25 °C, we evaluated CD34+ tively studied for a year. Monthly hematological parameters were cell viability by using 7-aminoactinomycin-D. recorded; trimester averages were calculated and monthly EPO and iron doses as well. Group A (GA) included 6 patients receiving iron as Results bolus “load-and-hold” therapy, when serum ferritin levels -measured every 3 months- were lower than 200 ng/ml; group B (GB) included The mean time to reach the target temperatures were as follows: 8 patients receiving consistent monthly iron therapy. -130 °C, 50.0 ± 8.2 s (range, 40–60 s); −60 °C, 355.0 ± 53.0 s (range, 260–420 s); −40 °C, 617.0 ± 59.8 s (range, 530–710 s); −25 °C, Results 958.0 ± 97.3 s (range, 800–1100 s). CD34+ cells viability was − − 85.9% ± 6.8% at 130 °C, 85.8^% ± 5.0% at 60 °C, 85.7^% ± 3.2% at There was no difference between groups in monthly EPO and iron −40 °C, 77.8% ± 5.7% at −25 °C (P = .009). doses. MCV increased in both groups (86.67 ± 13.74 to 88.48 ± 13.91 fl and 87.63 ± 2.65 to 92.35 ± 4.05 fl-p = .033 respectively), Conclusions but only GB patients showed additional increases in MCH (28.82 ± 1.38 to 30.06 ± 1.34 pg-p = .023), Ht (32.10 ± 3.51 to 35.66 ± 2.74^ When cryopreserved CB units are left at room temperature for %-p = .033) and Hb (10.60 ± 1.22 to 11.61 ± 0.99 g/dl-p = .043). N800 s, the temperature of CB rises to −25 °C. At −25 °C, CD34 + Compared with GA, GB patients had higher levels in MCHC (32.56 ± cells showed significantly lower viability. 0.92 vs 31.18 ± 1.20 g/dl-p = .031), in Ht (35.10 ± 2.60 vs 30.12 ± 4.82%-p = .03) and Hb (11.35 ± 0.88 vs 9.38 ± 1.64 g/dl-p = .013), doi:10.1016/j.cca.2019.03.842 but lower serum ferritin levels (233.38 ± 90.42 vs 600.50 ± 299.36 ng/ml-p = .006). In the total of patients, significant correlations of monthly iron doses were observed with Hb (R = 0.631-p = .016), MCHC (R = 0.713-p = .004) and ferritin (R = -0.786-p b .001). ^W068 There were significant negative correlations of Hb with monthly EPO doses (R = -0.618-p = .019) and with serum ferritin levels (R Comparison of three fold converted Hematocrit and Micro- = -0.781-p b .001). hematocrit in pregnant women

Conclusions G.T. Kiya Jimma University, Ethiopia Maintenance iron therapy was related with a major improvement of hematological parameters compared with bolus administration Background-aim but ferritin did not seem to be the most reliable index of iron status and more accurate indices are needed for monitoring iron stores in Anemia is one of the common complications during pregnancy. HD patients. Hemoglobin measurement, Hematocrit and Red cell count are used to diagnose anemia. In resource-poor setting, there is a practice of doi:10.1016/j.cca.2019.03.841 reporting Hematocrit which is threefold calculated from Hemoglo- bin. This study is aimed at assessing association and acceptability of threefold converted Hematocrit as compared to the conventional Hematocrit. ^W067 Methods Quality comparison of umbilical cord blood that experienced interruption during cryopreservation The study involved 200 pregnant women who attended ANC during the study period at Jimma University Medical Center. Three K. Kim, J. Huh, M. Kang milliliters of venous blood sample was collected with EDTA tube to Department of Laboratory Medicine, CHA Bundang Medical Center, CHA determine Hematocrit by Microhematocrit method and hemoglobin University, Republic of Korea by Hemocue method. A scatter plot, correlation coefficient, Bland and Altman plot and Area under curve were employed to assess the Background-aim agreement and acceptability of the threefold converted method as compared to the standard Microhematocrit. Umbilical cord blood (CB) banks conventionally freeze and store CB in separate devices, thus exposing frozen CB to room temperature Results during transfer from the freezer to the liquid nitrogen storage tank. Also, such exposure could occur during cryopreservation in liquid The correlation coefficient, Intraclass correlation coefficient, and nitrogen tanks by many unpredictable factors. In this study, CB units the concordance correlation coefficient were 0.91, 0.94, and 0.89 that experienced interruption during cryopreservation period were respectively. The Bland and Altman plot showed a mean difference of assessed for their temperature changes and CD34+ cell viability. 0.94 with the limit of agreement ranges from 0.6 to 1.3. The area under the receiver operating characteristics with a cut-off point of Methods Hematocrit b33% was 0.86. The sensitivity and specificity of the calculated method was 95.5% and 71.4%, respectively. The experiment used 40 CB units donated to a public CB bank between December 2014 and October 2016. The thermocouples were Conclusions then connected to a data logger thermometer, and temperatures in CB units were recorded at 10-s intervals during exposure to room Generally, there is an excellent association between the two temperature. When the temperature of the data logger thermometer methods. The two methods were identical within inherent Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

imprecision of both methods. Hence, the Hematocrit value, threefold ^W070 calculated from Hemoglobin (Hemocue method) was found to be acceptable to diagnose anemia in pregnant women. A case report: Acquired hemophilia A detected in the Laboratory of Hemostasia. Are the diagnostic algorithms necessary? doi:10.1016/j.cca.2019.03.843 E. Montero, I. Puig-Pey, M.C. Prado, L. Huete, D. Acemel, I. Ruiz, C. Banús, A. Salas, J. Laso, L. Andreoni Laboratori de Referència de Catalunya, Spain ^W069 Background-aim Detection of monoclonal free light chains (FLC) by various laboratory methods The diagnosis of hemorrhagic diathesis in the laboratory remains a challenge. The use of integrated algorithms in routine workflows P. Kušnierováa, D. Zemana, K. Revendovác, D. Stejskald,Z.Švagerad,O. would permit the detection of these rare pathologies with high Dlouhýb morbidity and mortality. aDepartment of Biomedical Sciences, Faculty of Medicine, University of Acquired hemophilia A (AHA) is a rare autoimmune coagulopathy Ostrava, Ostrava, Czech Republic caused by autoantibodies against factor VIII (FVIII). Its incidence is bDepartment of Physics, Faculty of Natural Sciences, University of estimated at 1–5 cases/million/year. Although there is no consensed Ostrava, Ostrava, Czech Republic definition about the diagnosis of AHA, but the German registry (GTH- cFaculty of Medicine, University of Ostrava, Ostrava, Czech Republic AH 01/2010) established the following basic criteria: FVIII coagulant dInstitute of Laboratory Diagnostics, University Hospital Ostrava, activity b50% (50 U/dL), inhibitor against FVIII ε0.6UB/mL detected Ostrava, Czech Republic with the Bethesda test and absence of congenital hemophilia A. Background-aim Methods

The aim of the study is determination of free light chains in In March 2018 we performed a basic coagulation study on a 62 patients with abnormal kappa free/lambda free ratio (FLC ratio) by years old outpatient. The analysis revealed an activated Partial various quantitative methods and assessment of characteristic of Thromboplastin Time (aPTT) of 44 s and normal results for the small abnormal protein bands (monoclonal bands or oligoclonal remainder remaining parameters. bands) by immunofixation electrophoresis (IMF) and isoelectric His clinical history reported: smoking history, Cacchi-Ricci focusing followed by affinity immunoblotting (IEF/AIB). syndrome, Addison's disease (1998), right testicular seminoma (orchiectomy in 2005) free of disease, thrombotic thrombocytopenic Methods purpura in 2015 due to deficit of ADAMTS 13 without inhibitor and negative study of mutations, exeresis of low grade fibrosarcoma in 20 serum samples were examined. Serum FLC levels were left pectoral muscle (2010) and idiopathic DVT (2015). The patient determined using an immunoturbidimetry and an enzyme-linked did not report a history of hemorrhagic diathesis and his previous immunosorbent assay, the monoclonal or oligoclonal bands of free analysis presented prolonged aPTT during hospitalization. light chains were examined by immunofixation electrophoresis and isoelectric focusing followed by affinity immunoblotting. Results

Results These results served to diagnose AHA. After that, an exhaustive search of underlying pathologies associated with that entity was No statistically significant correlation was found between the carried out, but none was found (N50% of the cases are idiopathic). individual FLC ratios using the Passing-Bablok regression. A statisti- The patient was referred to the reference Hospital in coagulopathies. cally significant dependence was found between FLC ratio Sebia and FLC ratio SPA using the nonparametric Spearman's correlation Conclusions coefficient (rs = 0.666, p = .001). Kappa statistic evaluated a moderate conformity between the FLC ratio Sebia and immuno- AHA is difficult to diagnose unless its diagnosis follows a clinical fixation electrophoresis (kappa = 0.468), no conformity between suspicion. Only 5% of patients debut with altered laboratory FLC ratio SPA and FLC ratio Sebia (kappa = 0.000). parameters and absence of hemorrhagic symptoms. Implementation of diagnostic algorithms in haemostasis laboratories and their Conclusions integration with the clinical data of patients would facilitate the detection of congenital or acquired coagulopathies, which can put The Binding Site diagnostic kit provides false positives of FLC patient's life at risk. ratios. It will be necessary to revise the reference limits for the respective free light chains and the FLC ratio on a larger set of data. doi:10.1016/j.cca.2019.03.845

doi:10.1016/j.cca.2019.03.844 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

^W071 ^W072

Influence of platelet-rich frozen plasma on the results of basic Analytical performances of Optilite® Freelite® Kappa and coagulation Lambda free kit on the Optilite® turbidimetry

I. Puig-Pey, M.C. Prado, E. Montero, I. Ruiz, A. Salas, D. Acemel, C. D.Y. Songb, J.H. Leeb, J. Seob, S.M. Kima, S. Juna, K. Leeb, S.H. Songb,J. Banús, L. Huete, J. Laso, L. Andreoni Songb Laboratori de Referència de Catalunya, Spain aDepartment of Laboratory Medicine, Seoul National University Bundang Hospital, Seongnam, Republic of Korea Background-aim bDepartment of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea The results of coagulation parameters can be affected by numerous preanalytical and analytical variables. Various interna- Background-aim tional guidelines (CLSI H21-A5, ISTH, BCSH, EFLM) advice working with platelet-poor plasma (PPP), with b10.000 platelets/μl (pl/uL). Kappa and Lambda free light chain (|FLC and ⌊FLC) assay has the When working with frozen plasma with a higher number of important roles of diagnosis, prognosis, and evaluation of treatment platelets, platelet-rich plasma (PRP), the basic and specialized response in monoclonal gammopathy. A new FLC analyzer, Optilite coagulation tests may be interfered due to platelets fragmentation (The Binding Site Ltd.), is a turbidimetric immunoassay analyzer and exposure to anionic phospholipids. Most of the guidelines refer which detects automatically the antigen excess by reaction kinetic to the interference results of lupus anticoagulant or the resistance to method, control addition, and sample addition. Analytical perfor- the activated protein C caused by frozen PRP. It has been mance of Optilite Freelite Kappa Free Kit and Lambda Free Kit on documented that basic coagulation tests such as prothrombin time Optilite analyzer were evaluated. (PT) and activated partial thromboplastin time (APTT) are not affected with non-frozen PRP samples, but data are limited for Methods frozen samples of PRP. Optilite's performances of precision, linearity, method compari- Methods son, sample carryover, and antigen excess detection were evaluated. In method comparison, Optilite was compared to Freelite Human Daily, our laboratory receives frozen samples for basic coagula- Kappa and Lambda Free kit on cobas 8000 c702 (Roche Diagnostics tion studies. It is not possible to control the number of platelets System, Switzerland). present in these samples, since the platelets are fragmented during the freezing process. The aim of our study was to verify the stability Results of TP and APTT results in PRP frozen samples. 75 patient samples were processed. Primary tubes were received, In precision evaluation, total coefficient of variations of |FLC and ⌊FLC at room temperature, 4 h after extraction maximum. 2 aliquots were were under 5%. Linearity was not fulfilled in the range of 3.99–112.01 done from each tube; with the first aliquot PRP was obtained mg/L in |FLC and ⌊FLC 6.66–109.03 mg/L in ⌊FLC. In method comparison, (centrifugation at 2500 rpm - 10 min) giving platelet values between it showed good correlation (r (correlation coefficient) 0.996 in |FLC; r 15.000 and 301.000 pl/uL, with the other aliquot PPP was obtained 0.993 in ⌊FLC), however both |FLC and ⌊FLCofOptiliteshowedfractional (centrifugation at 3100 rpm - 20 min) resulting b10.000 pl/uL. All and proportional deviations from cobas 8000 c702. The percentages of samples were frozen at −20 °C and after 48 h they were thawed at sample carryover were under 0.1%. Antigen excess detected in most ⌊ 37 °^C for 10 min. The TP and APTT ratios were performed with the samples, but not in a few cases especially in FLC cases. Sysmex 5100 (Siemens). Conclusions Results Optilite showed good performance in precision and carryover. The results of TP and APTT ratios in PPP and PRP are Clinicians and clinical pathologists need to aware that there are interchangeable and we can affirm that there are no significant reports that FLC changes b50% should not be taken into account as a differences between the two groups due to the impact of the prognostic value, considering non-linearity of FLC assay. Because the preanalytic phase (the confidence interval includes zero, Bland- fractional and proportional deviations with cobas 800 c702 were Altman function). observed, therefore correlation between systems should be consid- ered with care before switching to the Optilite. Conclusions doi:10.1016/j.cca.2019.03.847 These results ensure that the freezing of PRP does not affect the result of basic coagulation tests. The implementation of new models of external laboratories is W073 leading to an increase in frozen plasma samples. This work validates ^ the interference caused by the centrifugation and freezing of samples Procedure to minimize interference of hypertriglyceridemia in under uncontrolled conditions. haemogram parameters of lipemic samples in inadequate fasting doi:10.1016/j.cca.2019.03.846 X. Tejedor Ganduxé, A. Martínez Iribarren, C. Morales Indiano, À. Sala Sanjaume, M. Espinosa Nieto, A. Dueñas Márquez, M. Cánovas Bueno, M. Llopis Díaz Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

Laboratori Clínic Metropolitana Nord, Hospital Universitari Germans ^W074 Trias i Pujol, Institut Català de la Salut, Spain Comparison of automated differential blood count of Beckman Background-aim Coulter analyzers LH 780 and DxH 500 in normal and pathological samples Lipemia is characterized by turbidity of serum or plasma caused by accumulation of lipoprotein particulates. Thus, lipemia interferes M. Trticaa, Ž. Stjepanovićb,D.Bačkovića with the accurate determination of hemoglobin by spectroscopy on aDepartment for Medical Biochemistry, Faculty of Pharmacy, University most haematology analyzers and those derived calculated parame- of Belgrade, Belgrade, Serbia ters from it. bGeneral Hospital MediGroup, Belgrade, Serbia There are several case reports in literature concerning association between inadequate fasting and (hypertriglyceridemia) HTG, but few Background-aim intend to evaluate aspects of laboratory diagnosis, methodological limitations imposed by lipemia and management for resolution. CBC-Diff analysis is important in the diagnosis and follow up of This study is an approach to laboratory interference caused by various diseases. Current state-of-the-art haematology analyzers HTG in plasma from patients with inadequate fasting with the are characterized by their own technologies to provide compre- objective to present actions aimed at achieving reliable and useful hensive reports on blood cells with good level of precision and laboratory tests in clinical decisions making. accuracy. Beckman Coulter LH 780 is fully automated haematology analyzer that uses impedance method for cell count and VCS Methods technology for the leukocyte differentiation. DxH 500 is the latest 5-part diff haematology analyzer from Beckman Coulter For the study, 50 samples of patients in inadequate fasting with which is also using impedance method for the CBC and optical triglyceride values between 329 mg/dL and 11,171 mg/dL were used. impedance for the WBC differentiation. Aim of our study is Complet haemograms were determined in an automated hemocy- evaluation of DxH 500 performance compared to the high-end tometer. The lipemia correction was carried out by replacing the analyzer LH 780. plasma volume by an equal volume of saline solution (NaCl 0,9%) after centrifugation of the whole blood (with EDTA K3). Methods For each sample, it was calculated the variation between analyzes (before and after the correction) for the following parameters: leukocytes This study was conducted at the central laboratory of the General (WBC), erythrocytes (RBC), hemoglobin (Hb), hematocrit (HCT), mean hospital Medigroup in Belgrade, Serbia. 88 blood samples were corpuscular volum (MCV), mean corpuscular hemoglobin (MCH), mean obtained from the out-patient and in-patient departments. All corpuscular hemoglobin concentration (MCHC), platelets (PLT). specimens were analyzes within 4 h of blood collection. The For each parameter the reference change value (RCV) was calculated. instruments were checked with quality control samples twice daily. The concentration of triglycerides required to cause a clinically Calibration and maintenance activities were performed according to significant change in most parameters was determined by ROC curve. the manufacturer's recommendations. Correlation and regression analysis was performed between DxH 500 and LH 780 results Results according to ICSH procedure.

The ROC curve presented an Area Under Curve (AUC) of 0,9683 Results (95% CI: 0.85746–0.99491). for the concentration of TG as a predictor of interference. The The correlation coefficients and linear regression equations for optimal cutoff point was 1271 mg/dL (Se(%) = 100; Sp(%) = 88,89). CBC parameters indicate conformity between the two instruments Above this concentration the variation between analyzes (before (Table 1). The differential counts performed on DxH 500 and LH and after the correction) was N6.08% (RCV) for Hb, which directly 780 showed good agreement (Neutrophils r = 0,996, Lymphocytes impacts on RBC parameters calculated (MCH, MCHC). r =,986, Monocytes r = 0,939, Eosinophils r = 0,938) apart from By contrast the results of variation between analyses for WBC, the basophils, since most of the results were near zero. Both HCT, MCV and PLT were less than the RCV (for each one of these instruments showed no significant deviation from linearity for all parameters). parameters.

Conclusions Conclusions

Thus, since the TG levels found are greater than the interference DxH 500 system provides results equivalent to the LH 780 in our limit for Hb and RBC calculated parameters, the replacement of samples. Results obtained from both analyzers have same clinical lipemic plasma for saline solution was needed to minimize value. DxH 500 is viable solution for routine haematology parame- interference clinically significant. These data show the relevant TG ters in clinical settings. interference in blood count, and how may impact in medical decision, and the importance of correction. doi:10.1016/j.cca.2019.03.849 doi:10.1016/j.cca.2019.03.848 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

^W075 ^W076

Identification of plasma cells in peripheral blood: The role of Comparison of automated and manually method for determina- antibody-synthesizing lymphocytes AS-LYMP tion of nucleated red blood cells (NRBC) in blood samples from neonates and premature babies E. Urrechagaa, A. Vázqueza, M. Merinoa, R. Pérezb, E. De La Puertaa aHospital Galdakao Usansolo, Spain I. Vuga, F. Tomić, S. Margetić, D. Ferenec Ružić, B. GetaldićŠvarc bHospital Universitario Cruces, Spain Sestre milosrdnice University Hospital Center, Department of Clinical Chemistry, Vinogradska cesta 29, Zagreb, Croatia Background-aim Background-aim The Sysmex XN-analyzers can detect reactive lymphocytes as their cellular activity is measured by an increased in fluorescence, Nucleated red blood cells (NRBC) are precursor cells of the which can be recognized in the scattergram of leukocyte differen- erythrocytes that still contain a nucleus. In neonates and premature tials. The number of activated B lymphocytes are quantified by the babies, the presence of NRBC is a physiological condition indicating new parameter AS-LYMP (antibody-synthesizing lymphocytes). immaturity of the bone marrow so the exact number of NRBC Plasma cells (PC) are one of the end products of the B- provides key information about their health status. In our laboratory lymphocyte mediated immune response. Increased numbers in blood manual differential blood count is routinely determined for all usually indicates infection, sepsis, auto-immune diseases and newborns and the number of NRBC is also reported if present. Newer hematological malignancy, multiple myeloma (MM) plasmacytoma generation of automated haematology analyzer provides automated or PC leukemia. Therefore, the ability to detect plasma cells (PCs) on NRBC enumeration as a part of complete blood count. an automated cell analyzer might be advantageous. We evaluated The aim of this study was to compare the NRBC values counted by the value of AS-LYMP in the detection of CPs. as compared with two different methods, manual obtained by optical microscopy and lymphocyte morphology in blood smears. automated obtained by haematology analyzer.

Methods Methods

During May 2018 we recruited 100 consecutive adult patients The NRBC count in neonatal blood samples (n = 170) collected in with scattergrams suggestive of activated lymphocytes (group A), primary K3EDTA microtubes was simultaneously analyzed on the 100 patients with normal scattergrams and AS-LYMP = 0 (group B), Sysmex XN-1000 analyzer (Sysmex Corporation, Kobe, Japan) and by 160 patients with MM and 2 PC Leukemia. manual microscopy using blood smears stained by the May The blood smears were analyzed on a CellaVision 96, the number Grünwald–Giemsa method. Blood smears are examined by experi- of PC recorded. Correlation between CP counted and AS-LYMP was enced laboratory technologist and all smears with number of NRBC studied with the Spearman rank test for linear regression analysis. higher than 20 were reviewed by two tehnologists and the mean Chi squared test was applied. value is calculated as the result. Relative NRBC count (NRBC% per 100 Clinical sensitivity and specificity of were defined as its ability to WBC) obtained by haematology analyzer is compared to the relative obtain positive and negative results concordant with the results number of NRBC obtained by optical microscopic method that is used obtained by microscopy. as the reference method. Passing-Bablok regression analysis and Bland-Altman bias analysis were used for methods comparison. Results Results group A; all members of group B had AS-LYMP = 0, and no CPs were evident; none of MM patients had PCs in blood smears and AS- Passing and Bablok regression analysis showed significant both LYMP = 0; patients with PC leukemia had 38^% (30 10*9/L) and 15^% constant and proportional differences between the two methods: − fi (14.5 10*9/L) PC, AS-LYMP = 26^% and AS-LYMP = 8%, respectively. regression line equation y = 0.60 + 0.90x; 95% con dence interval Correlation R2 = 0.72. (CI) for intercept −0.83 to −0.04 (A≠0) and 95%CI for slope 0.85 to 72 samples had AS-LYMPN0 and CP detected in blood smear, 0.94 (B≠1), thus indicating statistically biased results between while in 280 AS-LYMP = 0 and no PC; 10 samples had AS-LYMP = 0 manual and automatic method for NRBC enumeration although but 1–2 PC were detected in blood smear. Cusum test for linearity indicated no significant deviation from Chi squared = 306.05, P b .0001, Contingency coefficient 0.677. linearity (P N .05). These results were also confirmed by Bland and fi Sensitivity 87.5^%, speci city 100%. Altman difference plot.

Conclusions Conclusions

Morphological examination remains the first step for rapid Comparison between manual microscopic and automated NRBC diagnosis of patients with malignant blood diseases. AS-LYMP can number has shown that these two methods of NRBC enumeration aid to select samples to review blood smears for detecting PC, with should not be used interchangeably due to significant difference high positive predictive value and PC N 3%. between them. doi:10.1016/j.cca.2019.03.850 doi:10.1016/j.cca.2019.03.851 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

^W078 ^W079

Platelet aggregation and expression of platelet activation markers Molecular characterization of B-thalassemia at the North-West in non-filtered and leukocyte-depleted platelet concentrates – region of Russia preliminary research I. Zhilenkova, V. Zimina, M. Zenina, A.V. Kozlov A. Rak-Pasikowskaa, K. Wisniewskab, A. Wrzyszczb North-Western State Medical University named after I.I. Mechnikov, aDivision of Clinical Chemistry, Department of Medical Laboratory Russia Diagnostics, Faculty of Pharmacy with Division of Laboratory Diagnos- tics, Wroclaw Medical University, Wroclaw, Poland Background-aim bDivision of Laboratory Haematology, Department of Medical Labora- tory Diagnostics, Faculty of Pharmacy with Division of Laboratory Beta-thalassemia (b-thal) is considered rare in Central and West Diagnostics, Wroclaw Medical University, Wroclaw, Poland Europe, including Russia. However, these countries also involved in thalassemia-related problems as a result of demographic changes Background-aim caused by migration of ethnic minority groups with a high frequency of thalassemic mutations. During storage the quality of platelet concentrates (PCs) decline. In This study assesses the spectrum of mutations on the b-globin order to remove leukocytes from PCs irradiation or filtration may be gene in the subjects with clinical symptoms of thalassemia of North- used during PCs preparation. Unexpected blood transfusion complica- West region of Russia. tion like transfusion-associated graft versus host disease is main reason for leukocytes removal. However, filtration may lead to increased Methods platelet activation during storage, on the other hand, leukocytes contain and release substances which may influence platelet activity. The aim of Molecular diagnostic was performed at 90 patients (from 2 to 58 the study was to evaluate filtration impact on platelets aging. years) with a diagnosis of beta-thalassemia (minor and major). The diagnosis of ®-thalassemia involves measuring of RBC parameters. Methods Samples with erythrocytosis, microcytosis (MCV b 80 fl), hypo- chromia (MCH b 27 pg) and Mentzer index (MCV/RBC) b13 selected Four PCs were divided into two parts: filtered (F) (Teruflex Imugard for hemoglobin fractions quantification. An increased level of HbA2 III) and non-filtered (NF), they were stored in the same conditions. N 3.5% and/or Hb F N 1% is considered confirmatory for beta- Samples were separated from main concentrate in the day of preparation thalassemia trait. The samples were obtained in the course of routine (0 h), after 24, 48, 72 and 144 h of storage. In every sample expression of analysis and collected in EDTA anticoagulant tubes. RBC parameters P-selectin and CD63 were evaluated and adenosine diphosphate (ADP)- were measured using a Sysmex XT-4000i haematology analyzer induced, collagen (COL)-induced platelet aggregation were measured. (Sysmex, Japan), HbA2/F quantification - by capillary electrophoresis Statistical analysis was performed using Friedman's rank test and paired (MINICAP, Sebia, France). Molecular characterization of mutations is samples Wilcoxon test (Statistica 12 EN). performed with reverse-hybridization of amplification products to a test strip containing allele-specific oligonucleotide probes Results immobilized as an array of parallel lines (®-GlobinStripAssay, ViennaLabDiagnostics, Austria). Increase in expression of CD63 was observed after 72 h storage in both F and NF PCs. However, increase expression of P-selectin in NF Results was observed after 48 h storage, and it was 24 h later than in F. P- selectin expression was higher at following time points compared to All patients living in St. Petersburg, but have a different ethnic the point 0 within each PC. Furthermore, platelets from F showed trend composition: 40% - Azerbaijanis; 33.8% - Russian, who (according to to higher expression of P-selectin and CD63 compared to NF during them) haven’t Caucasian or Mediterranean roots; 11.3% - Bulgarians, storage. The highest relative difference in P-selectin expression in F Cypriots; 8.7% - Dagestan; 6.2% - patients of mixed families (one compared to NF was observed after 24 h storage (over 2-fold increase), parent - Russian, the other - from the Caucasus or Mediterranean). 3 and in CD63 - after 48 h storage (over 5.5-fold increase). patients patient were a homozygous for b-thal mutations, 4 patients Decreased platelet COL-induced aggregation in F was observed were a double heterozygote and 78 – a heterozygote. The incidence after 48 h storage, and it was 24 h later than in NF. In turn, in ADP- of b-thal mutations: codon 8 (−AA) - 36%; IVS 1.110 (G N A) - 26%; induced aggregation continuous decrease in platelet aggregation was codon 5 (-CT) - 6%; IVS 2.1 (G N A) and IVS 1.6 (T N C) - 5%; IVS 2.745 observed. ADP-induced platelet aggregation decreased 3.9-fold in F (C N G), IVS 1.1 (G N A), IVS 1.5 (G N C) - 4%; codon 8\ 9–3%; −101 and 2.5-fold in NF, COL-induced platelet aggregation decreased 3.2- (C N T) - 1%; no mutation detected – 6%. fold in F and 2.6-fold in NF after storage. Conclusions Conclusions The most frequent b-thal mutations in this population were PCs filtration lead to increased platelet activation and decreased codon 8 (−AA) - 36% and IVS 1.110 (G N A) - 26%, that close to non- aggregation compared to non-filtered platelets. Further methods of endemic countries. leukocytes elimination and/or methods of filtration effects elimina- tion should be sought.

doi:10.1016/j.cca.2019.03.853 doi:10.1016/j.cca.2019.03.854 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

^W080 disease anemia (CDA). Hypo-He and Hyper-He are the percentage of red blood cells (RBC) with cellular hemoglobin content lower than Intrinsic anti-pathway antibodies in a hemophiliac B major: 17 pg and higher than 49 pg, respectively, and MicroR and MacroR About one case are the percentage of microcytic and macrocytic RBC, respectively. The aim of this study is to evaluate the utility of these parameters in S. Aatfaoui, S. Elhentifi, Y. Lanouari, I. Elfaiz, M. Nadi, B. Oukkache the detection of ID in patients with CDA (IDA/CDA). Laboratory of Haematology of CHU Ibn Rochd of Casablanca, Morocco Methods Background-aim We analyzed 507 anemia studies carried out between July and Anti-factor antibodies are a major iatrogenic complication that October 2018 in the Sysmex XN-1000 (Roche Diagnostics®) and 4 usually occurs in early hemophiliacs when exposed to factor groups were created based on the type of anemia. The parameters concentrates. were evaluated in each group: Group 1 (healthy adults), n = 187: hemoglobin (H) ε 12 g/dL in Methods female (f) and ε 13 g/dL in male (m). Group 2 (IDA), n = 136: H b 12 g/dL, ferritin (F) b 15 ng/mL (f) b b We report a rare case of a known major congenital hemophilia B and H 13 g/dL, F 30 ng/mL (m). b ε patient, randomly identified at 6 years of age, with recurrent Group 3 (CDA), n = 139: H 12 g/dL, F 15 ng/mL, erythrocyte ε ε epistaxis and a hematoma of the knee, without a family history of sedimentation rate (ESR) 20 mm/h, transferrin saturation (TSAT) ε b hemophilia. 20%, reticulocyte hemoglobin content (CHr) 29 pg (f) and H 13 g/ dL, F ε 30 ng/mL, ESR ε 20 mm/h, TSAT ε 20%, CHr ε 29 pg (m). b ε ε Results Group 4 (IDA/CDA), n = 45: H 12 g/dL, F 15 ng/mL, ESR 20 b b b ε mm/h, TSAT ^20%, CHr 29 pg (f) and H 13 g/dL, F 30 ng/mL, ESR ε 20 mm/h, TSAT b20%, CHr b 29 pg (m). The patient studied was victim of a pathological fracture, at the ^ Descriptive statistics of the 4 parameters in each group were age of 37 years, preoperative haemostasis assessments were re- evaluated and the Kruskal-Wallis and Mann-Whitney tests were quested objectifying elongated activated partial thromboplastin time performed. with factor IX assay collapsed to b5% (chronometric assay) with absence of circulating anti-coagulant antibodies (BETHESDA Results method). Postoperatively, the patient presented a continuous hemorrhage, the patient has developed an antibody antifactor IX: 40 units bethesda, in front of the non-improvement of the hemor- The medians of Hypo-He in groups 2 and 4 (ID) were higher (5.8% rhagic syndrome despite the administration of NOVO7, a coagulation and 2.6% respectively) with respect to groups 1 (0.1%) and 3 (0.4%). factor assay was performed with factor VIII and XI. Factor VIII: 2% We observed the same results for MicroR. factor IX: b 1% factor XI: b 1% Antibodies Anti-factor VIII, XI and XI A Kruskal-Wallis test was performed and we detected statistically fi b positive. signi cant differences among the 4 groups for each parameter (p .001). Furthermore, we carried out a Mann-Whitney test and higher Conclusions results of Hypo-He and MicroR were observed in group 4 compared with group 3 (both p b .001), but lower than those of group 2 (p = .016 and p = .028, respectively). Factor alloimmunization remains an etiological mystery to be deciphered, and which can be serious of consequence, life-threaten- Conclusions ing, in particular in our case and huge costs of curative treatments. It is underestimated because the conduct to be diagnosed is still non- standardized. Hypo-He and MicroR are useful in the detection of ID in patients with IDA/CDA, providing higher results than CDA patients. These data support doi:10.1016/j.cca.2019.03.855 the use of these parameters to identify the cause of anemia.

doi:10.1016/j.cca.2019.03.856

^W081

Detection of iron deficiency in patients with anemia of chronic ^W082 disease. Utility of the new hematological parameters Hereditary hemocromatosis: Mutations in gene HFE, TFR2 and FPN1 - Population study (2016 to 2018) M. Aliste Fernández, L. Muñoz Marín Clinical Laboratory, Haematology Department, Parc Taulí Hospital Universitari, Institut d'Investigació i Innovació Parc Taulí I3PT, M.P. Barros, F. CarriÇo, G. GaiÃo Marques Universitat Autònoma de Barcelona, Sabadell, Spain Hospital de Santa Maria - Centro Hospitalar Universitário Lisboa Norte, EPE, Lisbon, Portugal Background-aim Background-aim Iron deficiency (ID) is the most common cause of anemia (IDA). Currently, haematology autoanalysers provide parameters which can Hereditary hemochromatosis (HH) is an autosomal recessive be useful when biochemical tests are affected such as in chronic disease of iron metabolism characterized by increased iron Abstracts / Clinica Chimica Acta 493 (2019) S379–S433 absorption and its deposition in the liver, pancreas, heart, joints and ^W083 pituitary. Without treatment, death may result from cirrhosis, primary hepatocellular carcinoma, diabetes or cardiomyopathy. Stability of leucocyte research parameters over time on Sysmex Universal screening is not recommended, but should be done in XN haematology analyzer first-degree relatives of patients with HFE-related hemochromatosis in individuals with evidence of active liver disease or with changed A. Bartolinib, T. Scramoncinb, F. Sivierob, A. Padoana, G. Da Rinb results in the iron study. aDepartment of Medicine-DIMED, University Hospital of Padova, Hanson et al. in a study that analyzed 69 studies on the mutation Padova, Italy in the HFE gene C282Y and H63D concluded that in the general bLaboratory Medicine, San Bassiano Hospital, AULSS 7 Pedemontana, population the H63D/Wild mutation represents 62.4% of these Bassano del Grappa, Italy mutations, and C282Y/Wild mutation 25.1%. When considered the population with clinical diagnosis the most frequent mutation is Background-aim C282Y/C282Y (83.2%), followed by C282Y/H63D (5.7%) and H63D/ Wild mutations (5.6%). The reorganization of the hospital network according to the “hub The treatment of HH is performed through phlebotomies that, if and spoke” model led to the centralization of laboratory services and performed before the beginning of the irreversible organic lesions, the dispersion of blood collection centres in the district area. This lead to a life expectancy of these patients similar to those of the process has increased the time of blood storage before analysis with general population. possible negative consequences for the patient. For the complete blood count, it was shown that prolonged blood contact with the Objectives. Identify the mutations in the HFE gene (V53 M, V63 anticoagulant (K2-EDTA) causes morphological changes of white M, H63H, S65C, Q127H, P160delC, E168Q, E168X, W169X, C282Y, blood cells (WBC), which can be confused with pathological Q283P), TFR2 (E60X, M172K, Y250X, AVAQ594-597del) and FPN1 alterations. Therefore, it is important to assess whether the increase (n144h, v162del) more frequent in a population group studied, at of processing time of the hematological sample can alter its quality, the Haematology Laboratory of CHULN, EPE, during the period providing unreliable results to the patients. For this reason, in this between January 2016 and October 2018. study we evaluated intra-assay precision, detected over time, of conventional and research parameters of Sysmex XN. Methods Methods We studied, retrospectively, the presence of mutations in a group of 380 individuals. The mutations study was performed by Polymer- In order to evaluate intra-assay precision over time, according to ase Chain Reaction (PCR) and reverse hybridization. the International Council for Standardization in Haematology (ICSH) Statistical evaluation was performed through the IBM® SPSS® guidelines, ten random samples were collected and each sample was Statistics V24 program. analyzed ten times with the Sysmex XN (T0). This procedure was repeated analyzing other samples stored at 4 °C after 12, 24, 36, 48 h. Results Intra-assay precision, reported as coefficient of variation (CV%), was compared with quality specifications based on intra-individual Of the 380 individuals studied, 200 (52.5%) presented a mutation, biological variability (CVi), when available. 64.5% of which were male (n = 131). Of the eighteen mutations studied, four were identified, all in the Results HFE gene, namely the C282Y, H63D, S65C, H63H. The H63D/Wild fi mutation was identi ed in 51.5% of the individuals (46.4% of the The precision of Sysmex XN for the classification of WBC populations women, 54.2% of the men), the remaining mutations were distrib- falls within the limit defined for the minimum desirable precision, uted as follows: H63D/H63D - 14% (13% of women; 14% of men), except for basophils. Precision remains constant over time for all WBC C282Y/Wild - 13% (17.4% of women, 10.7% of men), C282Y/Wild- population, while there are significant variations if we consider the H63D/Wild- 10% (10.1% of women; 9.9% of men), C282Y/C282Y - positional parameters. For example for parameters NE-SFL, NE-FSC, NE- 6.5% (8.7% of women, 5.3% of men), S65C/Wild - 2% (3.1% of men), WZ and MO-Z we detect an increase of CV% at T24, compared with the C282Y/Wild-S65C H63D/H63D/S65C/S65C - 1% (1.6% of males), CV% detected at T0, of 3.4, 3.2, 2.4 and 2.6 times, respectively. H63D/Wild-S65C/Wild - 5% (1.4% of females) and H63H/Wild - 0.5% (0.8% of men). Conclusions

Conclusions Data obtained in this study show the over time stability of CVs for the classification of WBC populations by Sysmex XN. On the other In this study population, mutations in the HFE gene were hand, time dependent variations of positional parameters do not fi identi ed: C282Y, H63D, S65C, H63H. The most frequent mutation recommend to process samples stored over 12 h, especially if these was HFE H63D/Wild. parameters are used in the rules for selecting samples for microscopic review, to avoid having both false negatives and false positives results. doi:10.1016/j.cca.2019.03.857 doi:10.1016/j.cca.2019.03.858 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

^W084 ^W085

State-of-the-art for the measurement of seventeen haemostasis Method comparison for serum free light chain quantification and thrombosis variables from external quality control data between the Sebia FLC and Freelite assays

C. Bona, G. Matara, R. Meleya, C. Sottaa, M. Lopeza, J. Eynarda, B. Poggia, A. Bonnín, F. Piñol, M. Ferrera, M. Simon b Clilab Diagnòstics a PROBIOQUAL^, 7 Rue Antoine Lumière^, 69008^Lyon^, France b – Background-aim Service de Biochimie^, Hôpital de la Croix Rousse, 69317^Lyon^, France

Background-aim Serum free light chain (sFLC) analysis is well-established in routine diagnosis and management of monoclonal gammopathies, although The measurement of haemostasis variables^, performed for diagnosis poor accuracy has been reported for all the methods currently and follow-up of bleeding and thrombosis disorders^, requires reliable available. The aim of this study was to compare the recently launched tests and relevant analytical objectives. Few recent studies report state- Sebia FLC assay with our usual Freelite (Binding Site) test. of-the-art for haemostasis assays. Thence, the aim of this work is to evaluate the current performances of the main commercial methods Methods and to define analytical goals usable in the haemostasis laboratory. Data are obtained from an External Quality Assessment (EQA) program, We collected 68 random samples from patients with different organized by ProBioQual, a French proficiency testing association. pathologies referred to our laboratory for sFLC testing. Sera were aliquoted and frozen at −20 °C until analysis with both kits: Freelite Methods in the Optilite turbidimetric system and Sebia FLC in the DAS AP22 ELITE ELISA processor. Pearson correlation and linear regression Routine haemostasis variables and coagulation inhibitors were were calculated for the values of kappa (|) FLC, lambda (⌊) FLC and measured by participant laboratories (730 to 850 in each survey) for FLC-ratio using R software. Qualitative concordance was also the 2013–2017 period. Statistical evaluation was performed accord- evaluated. ing to the ISO 13528 guideline and robust algorithm A. The consensus value was calculated for all participants results (AP) and Results for each peer group (PG). The imprecision of the methods was evaluated by the inter-laboratory 90th percentile coefficient of The comparison of the results obtained using each method variation (CV90) and the accuracy quantified as 90th percentile bias showed the following correlation coefficient r: 0.94 for |FLC, 0.89 (bias90) of laboratory results from AP or PG consensus value. for ⌊FLC and 0.81 for the FLC-ratio. Slopes by linear regression were 0.43(±0.037), 0.10(±0.013) and 0.38(±0.068) for |FLC, ⌊FLC and Results FLC-ratio respectively. The qualitative concordance of the FLC-ratio was 81% with a Kappa agreement of 0.43. All the discrepancies Prothrombin Time (ProT) CV90 slighty increases from 6.2% at observed were for values close to the reference limits except in one normal level, to 6.8% at pathological level; the increase is greater case in which Freelite ratio measurement was 5 (reference range: (3.7% to 5.9%) for International Normalised Ratio (INR). Inter- 0.26–1.65) while Sebia offered a value considered as normal (range: laboratory CV90s vary between 3.1% and 4.5% for Activated Partial 0.37–1.44). This particular case was related to a follow-up point from Thromboplastin Time (aPTT), whereas they reach 8.8% for fibrinogen. a patient with myeloma multiple after the sixth treatment cycle with Antithrombin CV90s increase from 5% at normal level to 10% at bortezomib-melphalan-prednisone. Depending on the test used, the pathological level. AP biases are higher than PG biases for ProT, INR case would be classified as very good partial response or as complete and fibrinogen. APbias is 3.5 times the PG bias for aPTT. ProT response, although the therapeutic action would remain the same. differential factors biases do not change significantly with level and are lowest for factors II and X. AP bias is always higher than PG bias Conclusions for factors VIII and IX, showing a wide dispersion between methods. At a level close to 30%, AP bias90 is 18% for antithrombin, 21% for The results show a significant positive correlation between Sebia Protein C and Protein S Activity, and 13% for free Protein S. and Freelite FLC assays with a constant systematic difference. Clinical concordance was moderate as reported in previous studies. Lack of Conclusions availability of standardized reference materials hinders the clinical interpretation of the different results obtained and each case would Haemostasis assays span multiple methodologies and reagents^, require a long term follow-up evaluation. Serum FLC values must be and most tests are not standardized. In EQA programs, relevant used in combination with other biological parameters when making inaccuracy analytical objectives must be assessed by using peer medical decisions. group consensus values and are dependent on the control level. doi:10.1016/j.cca.2019.03.859 doi:10.1016/j.cca.2019.03.860 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

^W086 that this coexistence happens more frequently in males. The age of the affected is about 50 years (in both cases presented), although in Myeloproliferative syndromes and their association with lym- literature cases with this occurrence prevail in young ages. In 2015, phoid neoplasms cases of coexistence of these two syndromes in the same patients were presented in the Romanian morphological and embryological E. Çallikub, X. Ngjelab, T. Dedej-Kurtia, A. Barbullushia journals. a Clinical- Biochemical Laboratory Service, Laboratory Department^, Universitary Hospital “Mother Tereza”, Tirane, Albania doi:10.1016/j.cca.2019.03.861 bHematologji Service, Internal Departament, Universitary Hospital “Mother Tereza”, Tirane, Albania W087 Background-aim ^ Vitamin B12 screening with holo-transcobalamin is more sensi- The association of myeloproliferative syndromes with lymphoid tive than total vitamin B12 screening neoplasia is very rare. There are about 52 cases, worldwide, where these two syndromes coexist within the same patient. M. Carta, D. Giavarina This abstract will present 2 clinical cases presented at the Clinical Laboratory, St Bortolo Hospital, Vicenza, Italy Haemetology Department which were initially presented as myelo- proliferative syndrome, specifically: polycythemia vera, and subse- Background-aim quently lymphoid neoplasia, specifically: CLL, and Non-Hodgkin malignant lymphoma. Cobalamin [Vitamin B12 (VB12)], measurements in serum is frequently used as a first line assay in screening for VB12 deficiency. Methods However, diagnostic accuracy of this assay is burdened by a low sensitivity. Only the 20% of VB12 bound to the carrier protein This abstract will present 2 clinical cases presented at the transcobalamin, can be transported inside the cells and represents Haemetology Department which were initially presented as myelo- the active portion of VB12 available to cells. New assay for active B12 proliferative syndrome, specifically: polycythemia vera, and subse- measure only the cobalamin attached to transcobalamin. In this quently lymphoid neoplasia, specifically: CLL, and Non-Hodgkin study we had evaluate the effect of active B12 test in a large general malignant lymphoma. population, after a period of 18 months of use. These test are performed in Laboratory Departament, Universitary hospital” Mother Tereza”, Albania Methods

Results We compared consecutive samples arrived in the laboratory in the first six months of 2016 with the same period of 2018. Inclusion First case showing the following hematological parameters: criteria were the request of a dosage of VB12 and Complete Blood WBC 6.900/mm3 HGB 18.5 g/dl RBC 6.9 × 106/mm3, Hct 56% PLT ^ Counts (CBC), at the same time. All the measurements were performed 253000/mm3. by using chemiluminescent immuno-assays on Advia Centaur-XP WBC differential: bands 5%, segments 32%, eosinophils 4%, Analyzer (Siemens, Terrytown, USA): in the first group (2016y) VB12 bazofile 5%, lymphocytes 44%, monocytes 9%. JAK 2V617F positive. assay, in the second one, 2018y, active-B12 (holo-TC), both by A myelogram and Immunophenotyping were performed, which were Siemens. VB12 deficiency were compared with the major parameters compatible with Polycythemia Vera, and no immunophenotipically of the CBC (Hb, RBC, MCV) (Sysmex XN 9000, Kobe, Japan). pathogenic clone cells were identified. 2 months after the examina- tion the patient showed up an adenopathy. WBC differential showed Results lymphocitar proliferim (71%, 75%). The leukocyte immuno- phenotyping: CD 19 90% positive, CD 5 90% positive, CD 23 90% On 3074 samples from the first group, 4.78% were classified as positive, and have resulted compatible for probable CLL. A bone ^ deficient (cut-off 118 pmol/L). In the second group, on 4778 samples marrow biopsy showed infiltration to the Medulla Ossea of ^ measured for holo-TC concentration, 18.17% were deficient (cut-off lymphomas with small CD 20 positive cells. IHC: Glicophorina A + 35 pmol/L) (p b .001). Moreover, about 51% (2484) subjects fall in a +-,CD 61 +– ,CD 79a +–,BCl2+++Ki6710% pozitve. ^ gray zone, defined between 35 and 70 pmol/L. Second Case presented with a hematological framework compat- A reverse proportionality was noted between holo-TC concen- ible with the myeloproliferative syndrome: WBC: 20000/mm3, HGB: trations and MCV. In 95 subjects with aVB12 b 10 pmol/L, median 17.2 g/dl, RBC: 7.01 × 106/mm3, PLT: 1666000/mm3. No immuno- ^ MCV was 97.9 (95%C.I.: 95.2. to 100.7) fL; for holo-TC concentra- phenotipically pathogenic clone cells were identified. JAK 2 V617 F tions 11–20 (225), 21–30 (332) and 31–40 (457), MCV was 94.4 Pending the patient's biopsy response, the patient was given aspirin. (92.8–95.3), 92.5 (92.0–93.1) and 91.4 (90.8–92.4) fL, respectively. Based on the results of the biopsy and immunohistochemistry the Over 40 pmol/L, there was no correlation with MCV (median 91.5, patient resulted in lymphoid infiltration by non-Hodgkin's malignant 95%C.I. 90.8–91.8). Considering Hb and RBC, similar consideration lymphoma. IHC CD20 positive, CD79 positive CD 4 negative, CD 8 can be made, with a direct proportionality. negative Kappa positive, lambda positive .

Conclusions Conclusions

Screening with holo-TC identifies a greater amount of patients Myeloproliferative syndromes can precedes and associations with with vitamin B12 deficiency compared to the screening with total lymphoid neoplasms. The common denominator regarding gender, is Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

VB12 assay. A holo-TC concentration b 35 pmol/L strongly suggest a in the clinical case with ET after STEMI, balancing the risk of VB12 deficiency, while between 35-and 70 pmol/L a deficit cannot be thrombosis and hemorrhagic event. confidently excluded. doi:10.1016/j.cca.2019.03.863 doi:10.1016/j.cca.2019.03.862

^W089 ^W088 Casual finding in the laboratory of chronic myeloid leukemia Evaluation of platelet reactivity on dual antiplatelet therapy in patient with essential thrombocythemia and acute coronary K. Falcones Gracia, J.J. Ortega Huete, E. Ricart Alvarez, S. Martinez, R. syndrome Molina Gasset, J. Soler Hospital Virgen de Los Lirios, Spain D. Dinevab, I. Paskalevab, D. Todorieva-Todorovaa aClinic of Haematology, University Hospital “Geogri Stranski”, Pleven, Background-aim Bulgaria bLaboratory Department, National Heart Hospital, Sofia, Bulgaria The chronic myeloid leukemia (CML) is a chronic myeloprolifer- ative syndrome of a clonal nature, originated in the stell cell, Background-aim resulting in an excessive number of myeloid cells in all stages of maturation. Sometimes it is diagnosed incidentally. Thrombotic arterial events and bleeding complications are the most frequent life threating episodes that persist in patients with Methods essential thrombocythemia (ET). Assessment of platelet reactivity in accelerated platelet turnover may be useful in determining appro- A 73-year-old man who, after control analysis, showed extreme priate antiplatelet drug. leukocytosis (247x10E9/L) with myelia. On examination, the patient presented splenomegaly and reported asthenia of months of Methods evolution. Among his antecedents has: high blood tension, dyslipid- emia. The results of the routine laboratory report: leukocytes A 28-year-old man with bone marrow histology relevant to 247x10E9/L, platelets 399x10E9/L. Blood smear was performed, essential thrombocythemia (JAK2V6 1 7F-negative) 5 years ago, reporting Anisotrombia, leukocytosis within a severe range, neu- underwent coronary angioplasty because of STEMI (ST-elevation trophilia, lymphocytosis, monocytosis, eosinophilia, basophilia and N myocardial infarction) with drug eluting stent implantation. After immature granulocytes ^5.9%. Leukocyte formula: 4% blasts, 2% initial therapy with loading doses of clopidogrel, aspirin and GP IIb/ polymorphonuclear cells, 44% lymphomononuclear cells, 37% seg- IIIa inhibitor, dual antiplatelet therapy (DAPT) with aspirin (75 mg/ mented and segmented, 4% eosinophils, 5% basophils, 3% monocytes, day) and clopidogrel (75 mg/day) was subsequently initiated. The 1% lymphocytes. The patient is cited to expand the study; and start patient was referred to evaluation of platelet reactivity at our treatment with hydroxycarbamide + allopurinol. In the differential laboratory after one week. He was treated with interferon alpha-2b, diagnosis, other diseases that cause splenomegaly such as myelo- which he had self-medicated, because of adverse event to anagrelide proliferative disorders must be taken into account: polycythemia and refusal to any other cytoreductive therapy. Post-thraumatic vera, essential thrombocythemia and idiopathic myelofibrosis. The splenectomy had been performed at 6 years of age. The platelet diagnosis is confirmed by analyzing a blood smear combined with a function was assessed with Multiplate impedance aggregometry bone marrow (BM) analysis, cytogenetic and molecular tests. Biopsy (MEA) by ADP and ASPI tests 10 days after initiation of therapy. and aspiration results of (MO) compatible with CML (15% blasts), quantitative PCR bcr/abl (e13/e14-a2) positive (ratio 64% IS). After of Results these results, the hydroxycarbamide is suspended and treatment with imatinib is started. We found high residual ADP-induced platelet aggregation 910 aggregation units (AU), optimal inhibition at 150–450 AU, and high Results ASPI-aggregation 940 AU, optimal interval at 40–200 AU. High residual platelet activity is an independent factor for the development of early The patient presents constitutional symptoms indicating a thrombotic and ischemic events in patients with implanted coronary hypercatabolic state, with additional symptoms related to spleno- stents. Thus, we switched from P2Y12 inhibitor to more potent and megaly, this state is characteristic of the chronic phase of CML. Some effective drug - ticagrelor 2 × 90 mg/d and increased the dose of aspirin, asymptomatic patients have an incidental leukocytosis, as in our also. After 3 days we found appropriate response with ADP-aggregation case, that when performing additional molecular tests it is found that of 240 AU (73% decrease) and ASPI-aggregation 37 AU (60% decrease). it corresponds to a CML. The patient's platelet count on both visits was extremely high (1,150,000/ mm3). Further, we found impaired collagen induced aggregation, as well Conclusions as RISTO-high 21 AU (90–201 AU) or “acquired von Willebrand syn- drome” with rare episodes of epistaxis in our patient. After two months of treatment, the patient showed a clear improvement in his leukocyte count with figures of 4.08x10E9/L Conclusions (with normal formula) in the last laboratory report of December/ 2018. We evaluated platelet reactivity using MEA to determine the more aggressive and appropriate P2Y12 receptors blocker ticagrelor, doi:10.1016/j.cca.2019.03.864 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

Conclusions

W090 ^ We consider the use of this method for the detection of monoclonal components of enormous utility for the patient with Detection of monoclonal component in primary care patients by low economic cost for the laboratory. the laboratory doi:10.1016/j.cca.2019.03.865 G.D. GarcÍa Aguilar, M. Kassih Ibrahim, A. Cabrera Argany, T. Dorta Ramos, A.M. SÁnchez De Abajo, H. Cabrera Valido, T. HernÁndez Lemes, J. Paco Ferreira Servicio Análisis Clínicos y Bioquímica Clínica, Complejo Hospitalario ^W091 Universitario Insular Materno Infantil, Las Palmas de Gran Canaria, Spain Hemoglobina G Philadelphia in association with alpha thalassemia Background-aim R. Cabra Rodriguez, Á. Gragera-Martínez, M.A. Castaño Lopez, I. The monoclonal gammopathies constitute a set of diverse Vazquez Rico, A. León-Justel disorders associated to a proliferation of mature B cells. They are Juan Ramon Jimenez University Hospital, Spain characterized by the secretion of homogeneous immunoglobulin molecules, which is usually known as a monoclonal component. The Background-aim two most important clinical entities are multiple myeloma and Waldenström's macroglobulinemia. These diseases can present Structural hemoglobinopathies and thalassemias are the most numerous and important symptoms related to different organs common genetic disorders in humans. Alterations in hemoglobin problems. synthesis result in congenital hemolytic anemias, these can be of two It is known that early diagnosis increases the effectiveness of the types: Thalassemias, deficit production of globin chains, and treatment, reduces complications and improves the patients' lives structural alterations, by synthesis of abnormal globin chains. quality. Thus, the aim of this study is to evaluate the possibility of There are N30 mutations described affecting one or both of 〈- detecting monoclonal components by the laboratory as early as globin genes, there are different mutation points leading to abnormal possible. globin chains which cause abnormal hemoglobins (Hb S, Hb C, Hb E, Hb D, Hb G). Methods We present the case of a 47 years old woman, in a blood exam shows microcytosis and structural variation of 48.8% with mobilities To achieve our goal, we added a proteinogram to all those similar to Hb D but with a A2 Hb decreased 1.2%. patients form primary care with total serum proteins ε 9.5 g/dL. The study was carried out from August 2017 until December 2018. Methods

Results We carry out a molecular study of the genes involved in this type of pathology, finding two difference variations. During this time period, we detect 18 patients with total serum protein ε 9.5 g/dL. 9 of them had a proteinogram already requested Results by the clinicians. In the other 9 in which the laboratory added the proteinogram, we detect monoclonal components in 6 patients, We found a hetezygous variant c.207C N G in HBA1 gene that those patients were diagnosed as follows: cause amino acid change p.Asn69Lys with a homozygous delection of Patient 1: Multiple myeloma IgA kappa (2.65 g/dL + 2.24 g/dL) 3.7 Kb in the same gene detected by MLPA. stage IIIB of Durie and Salmon. Patient 2: Multiple myeloma IgG kappa (1.62 g/dL) minor Conclusions criterion. Patient 3: Multiple myeloma IgG lambda IgG lambda (3.64 g/dL) The presence of more than one alteration in RBCs is not an stage IIIB of Durie and Salmon. unusual event. Fortunately, HB-G has no clinical consequences, the Patient 4: Waldenstrom's macroglobulinemia IgG kappa (3.41 g/ only finding of clinical interest is microcytosis. The genetic test allow dL). us to identy mutations in hemoglobin genes, with one mutation in 〈 Patient 5: Monoclonal component IgG lambda (0.70 g/dL), gene, 25% of hemoglobin HBG and HbA a small part of HbA2 would without diagnosis at the ending date of study. be left. However, variant HBG is usually linked to an elimination of Patient 6: Diagnosed as multiple myeloma IgG kappa (6.07 g/dL) an 〈 chain and producing closer to 30% HBG in many individuals. The stage IIIA of Durie and Salmon. presence of another deletion in a gene 〈, uninvolved with the In the 3 patients remaining, no monoclonal component was structural variant could result in as high as 40% HBG. detected. The high performance liquid chromatography (HPLC) is widely In three cases (patients 1,3 and 5), we could not directly contact used for detection of hemoglobin variants. In general, the character- with the clinicians, and two of them (patients 1 and 3) were istics of peak and retentions times provide useful information on the admitted urgently one year later, with length of hospital stay longer identification of Hb variants. However, because the retention times than 2 months. The rest of patients were followed up by of many types of Hb overlap, we only can make a presumptive haematology outpatient clinic without hospitalization. diagnosis, and definitive identification requires further confirmatory Abstracts / Clinica Chimica Acta 493 (2019) S379–S433 tests, such as DNA analysis or mass spectroscopy. Our patient had Hb due to the lack of some specific antigens. CytoDiff® can be G-Philadelphia with a high percentage of variation (48.8%, normally considered a middle step between PB morphology and specific flow around 28–31%), this only can be due to the co-existence of 〈 cytometry. thalassemia and Hb D. Due to two molecular changes as a point mutation and homozygous delection. doi:10.1016/j.cca.2019.03.867 doi:10.1016/j.cca.2019.03.866

^W09^3

^W092 Early detection of primitive cells by the abnormal mono cluster and scattergram on XN Sysmex hemocytometer. A case report CytoDiff® in the diagnosis of acute leukemia: Comparison to gold standard method G. Introcasoa,T.D'erricoa, A.R. Lettieroa, C. Frassia, A. Calligarisb,L. Cadaua, M.L. Biondia J. Hernandoa, X. Nietoa, S. Gomezb, A. Marulla, G. Perezb, G. Silvab,M. aUnit of Laboratory Medicine, Centro Cardiologico “Monzino” IRCCS, Serrandoa Milan, Italy aLaboratory Dr Trueta Hospital, Spain bUnit of Telemedicine, Centro Cardiologico “Monzino” IRCCS, Milan, Italy bLaboratory Hospital 12 Octubre, Spain Background-aim Background-aim A man of 80 year old was admitted in our institute to treat a Haematology analyzers are ineffective in recognizing abnormal severe aortic stenosis with a transcatheter aortic valve implantation cells, including blasts, and may provide flag messages when such (TAVI). Patient undergoing diagnostic work up including biochemical cells are present. In those cases the gold standard method and hematological examinations shows an apparently normal (microscopic evaluation) should be done. Manual differential count hemocromocytometric test with a mild thrombocytopenia and is a laborious, time-consuming process and requires expertise. automated differential WBC count (WDF) in the normal ranges: Moreover, microscopic examination may show variable reproduc- WBC = 4.2 109/L, RBC = 5.24 1012/L, Hb = 160 g/L, PLT = 120 109/L, ibility. Blast count in a blood sample is important for the diagnosis of citrated sample PLT = 95 109/L. In a post-operative control, 17 days hematologic diseases and prognosis of patients, especially in acute later, quantitative and qualitative hematological parameters were leukemia but also in myelodisplasic syndrome. CytoDiff®, a flow not significantly different from the previous one except monocytes cytometric differential counting method introduced by Beckman count and MONO cluster. XN automatic hematological validator Coulter, may contribute to the blast detection. Our study focuses on detects a WBC abnormal scattergram and suggests a microscopic comparing manual blast count to CytoDiff® blast count and revision film. screening blast origin using CytoDiff®. Methods Methods To record clinical and analytical information we have selected 41 patients were analyzed by both Sysmex XN (^Kobbe, Japan) and from the hemocytometer XN 2000 (Sysmex, Kobe Japan) software Cytodiff®. The last one is a combination of 6 monoclonal antibodies/ the previous cell counts including abnormal scattergrams and 5 colours which performs a rapid WBC-Diff. research parameters: cell population data (CPD). A May-Grundwald 10 patients were diagnosed of acute lymphocytic leukemia (ALL) film was prepared cross-matching analytical and morphological data. and 31 were diagnosed of acute myeloid leukemia (AML). Manual An immunophenotyping by flow cytometry and a hematological WBC differential count was performed using Cellavision analyzer. evaluation were scheduled. Manual and CytoDiff® blast count were compared using Passing Bablock regression and Pearson correlation test. Results

Results We observed in the WDF analytical channel a misclassification of XN MONO cluster with an overestimation of monocytes: 30%. A Sysmex XN showed “Blast” flag in 33 samples. Passing Bablok corrected manual WBC count (%) was: neutrophil granulocytes 27, regression showed neither proportional nor systematic differences lymphocytes 27, monocytes 8, eosinophil granulocytes 1, with 95% confidence interval containing the value 1 and 0 when promyelocytes 1, primitive cells 36 (blast cells). Blood smear showed comparing manual and CytoDiff® blast count. Pearson correlation giant platelets and dysplastic WBCs. A relative reduction of CPD as test showed good correlation for blast count (r = 0.95). In 3 cases scattered signal for neutrophils and a meaningful rise for monocyte (7.3%) blast origin was identified wrongly: 1 T-ALL blast (91% CPD was found. Immunophenotyping confirmed the blast cell manual count) were found in Xn gate (86.85%), 1 AML blasts (26% phenotype as CD 34+ . manual count and 69% Xb and 2.2% Xt) were included in Xb and 1 B- ALL blast (1.3% manual count and 1,81% CytoDiff® count) were Conclusions located in Xn gate. Primitive cells were early detected even with a low WBC count. Conclusions Despite the monocyte misclassification of XN hemocytometer we highlighted that the abnormal MONO cluster may be useful to CytoDiff® blast count show good correlation with manual count schedule a peripheral blood smear analysis. Low scattered signal for and did not depends on experience. It provides more specificity than neutrophils could be probably explained by the presence of WBC_diff in blast detection although its classification is not possible dysplastic granulocytes with weak fluorescent signal. A complete Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

hematological evaluation of our case confirmed the diagnosis of acute myeloid leukemia. ^W095 doi:10.1016/j.cca.2019.03.868 Comparison of platelet quality and the effect of platelet collection in single donor platelet collected by 2 brands of apheresis machines ^W094 K. Kuahaa, P. Rattanavongjareana, S. Talabthonga, A. Jusakula,C. Measurement uncertainty of coagulation assays using the ACL Leelayuwata, C. Pomruangc, T. Warindpongb, V. Chongkolwatanab TOP 750 CTS and the Stago STA Compact haemostasis testing aFaculty of Associated Medical Sciences, Khon Kaen University, Khon systems Kaen, Thailand bFaculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, a a c b H.R. Kim , Y.K. Lim , S.R. Cho , J. Ahn Thailand a Department of Laboratory Medicine, Chung-Ang University College of cSaraburi Hospital, Saraburi, Thailand Medicine, Seoul, Republic of Korea bDepartment of Laboratory Medicine, Gachon University Gil Hospital, Background-aim Incheon, Republic of Korea c Departments of Laboratory Medicine, Ajou University School of Single Donor Platelet (SDP) prepared from platelet donors by Medicine, Suwon, Republic of Korea apheresis machines plays a role in the treatment of patients because of the large amount of platelets prepared from a single donor. Selection of Background-aim apheresis machines should be concerned in the quality of the product and the safety of blood donors. The purpose of this study was to The measurement uncertainty (MU) is a parameter associated evaluate the quality of SDP and the differences of hematological values with the dispersion of measurements. Assessment of MU is (before and after plateletpheresis) of the Amicus (version 3.21) and recommended in clinical laboratories; however, that of coagulation the Trima Accel (version 6.0) apheresis machines. tests has not been extensively studied despite the introduction of many adequate methods. Methods

Methods A total of 144 donors underwent plateletpheresis, 63 donors were processed by the Amicus and 81 donors by the Trima Accel. Platelet Activated partial thromboplastin time, prothrombin time, protein and hematological values were determined with an automated blood C and S, antithrombin III, fibrinogen, factor V, VIII and X were cell counter (Sysmex XN3000) and WBCs was counted in the selected for quantifying MUs of two haemostasis testing systems; the Nageotte chamber. ACL TOP 750 CTS (Instrumentation Laboratory, MA, USA) and the STA Compact (Diagnostica Stago, NJ, USA). To estimate the biases of Results measurements, the WHO international standards were measured according to Clinical & Laboratory Standards Institute EP29-A The platelets obtained from both apheresis machines were N 3.0 guideline, and the data of external quality assessments (EQA) were × 1011 platelets in 100% of SDP. The Amicus and the Trima Accel used when adequate standard was absent (NORDTEST method). were equivalent in PLT yields (6.31 × 1011 vs. 5.72 × 1011). Both Then, the expanded MUs were estimated with combining the machines had WBC contaminated at a lower level than that of the imprecision data from the internal quality control procedure. AABB standard. However, the platelet preparations from Amicus had Additionally, we performed the Monte Carlo Method to simulate MU. a significantly lower WBC contamination than those from Trima (0.051 vs. 0.059 × 106, P b .05). In the process of platelet preparation, Results average volume of anticoagulant used (mean 508.48 and 481.78 mL, p b .05) and duration of plasma collection (mean 104.89 and 96.81 Almost all items showed b5% of bias in a bias assessment step. min, P = .001) were significantly higher in Amicus. Hematological However, the biases of some items, such as factor V, XIII or X of the values after platelet donations were changed within the normal STA Compact, were statistically significant, and the bias corrections range. The mean differences of hematological values before and after were necessary. When applying various methods of quantifying MU, donations of Amicus (PLT, Hb, Hct and RBC were 147.78 × 103/uL, the results of MU were equivalent to each other, and the expanded 1.89 g/dL, 5.97% and 0.67 × 106/uL) were higher than those of Trima MUs varied from 6.3% to 19.3%. (131.80 × 103/uL, 1.46 g/dL, 4.44% and 0.49 × 106/uL).

Conclusions Conclusions

Using various method of calculating MU, we could quantify MU of The results indicated that both apheresis machines had an coagulation tests using automated haemostasis analyzers. There was equivalent in platelet quality and safety. However, Amicus may have no significant difference of MU between various MU estimation more blood loss in the platelet collection process, use more models, and we could conclude that all of methods was adequate for anticoagulants, and the time to separate the platelets was longer coagulation assays. However, further efforts are still needed to than Trima. achieve standardization of some coagulation tests for improving the trueness of a reported result and reducing the uncertainty. doi:10.1016/j.cca.2019.03.870

doi:10.1016/j.cca.2019.03.869 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

^W096 Center, Yeungnam University School of Medicine, Daegu, Republic of Korea Calculation of the reference range of the RET - He for a better diagnosis of the iron deficiency anemia Background-aim

S. Laura, B. Daniel, R. Manuel, P.D.A. Inmaculada, A. Carmen Cristina, Recently, many Next-generation sequencing (NGS) panels about J. Azahara, K. Imane, P. Vidal acute myeloid leukemia (AML) are accessible to screen for a Hospital Regional Universitario de Malaga, Spain pathogenic mutations of AML related genes. But the process of planning and familiarization of customized NGS panel can be difficult Background-aim for a small clinical laboratory. In this study, we evaluated the analytical performance of Oncomine Myeloid Research Assay, a Iron deficiency anemia (IDA) is one of the most common commercial NGS panel contained automated library preparation, nutritional deficiencies according to WHO. It is caused mainly by including accuracy, precision, limit of detection (LOD) and total the increase in iron losses, although other causes are decreased hands-on time for estimating labor intensity. contribution, decreased absorption or increased consumption. In his diagnosis various laboratory investigations that are routinely used, Methods we found a complete blood count where we can observe a microcytic and hypochromic anemia, with a decrease in hemoglobin, a low Total 32 reactions through 4 batches (each batch can analyze 8 mean corpuscular volumen (MCV) and a low mean corpuscular samples) were performed by Ion Torrent S5 and Ion Chef system hemoglobin (MCH); and in the biochemical analysis a low sideremia using 15 clinical specimen, 2 commercial reference materials and one and a low ferritin. specimen of external quality assessment service. The criteria of N Newer generation counters provide a new parameter, the quality assurance in sequencing were total bases ^8×108, total N N reticulocyte hemoglobin or RET - He. The RET - He is the hemoglobin reads ^4 × 106, and usable reads % x total reads ^4 × 106. content of the reticulocytes, which offers real-time information Bioinformatic pipeline was Oncomine Myeloid Research 530 w2.3.1. about the contribution of iron to erythropoiesis, allowing the The criteria of quality assurance in sequencing data were mean N N N detection of hemoglobin changes rather than through the hemoglo- depth x300, on target % 80%, and uniformity ^80%. Genetic bin content of mature red blood cells. It is not affected by the acute variations validated were single nucleotide polymorphism (SNP), phase reaction as it occurs with ferritin, so it indicates the trend of small insertion and deletion such as FLT-ITD. The specimens of LOD the current iron status. evaluation were prepared by mixing reference materials and clinical samples which were identified variant allele frequency (VAF) by Methods previous NGS analysis. Validated LOD ranged 10%, 5%, 2.5% of VAF.

We collected 122 samples from a homogeneous population of Results men and women, with hemoglobin and reticulocytes within the reference limits defined in our LIS Servolab; of an XN - 2000 device All reactions passed the criteria of quality assurance of sequenc- from the emergency laboratory of the Hospital Materno - Infantil of ing and bioinformatics process. The average of total bases, total reads Malaga, with the objective of calculating the reference ranges of RET and usable reads were 16.5 × 108, 15.9 × 106, and 9 × 106 respec- - He for our population. tively. The average of mean depth, on target rate, and uniformity were 1^697, 97.72 and 98.13. The accuracy of clinical samples and Results reference material was 100%. The precision assessment also showed consistent results in inter-run and intra-run process with similar After the calculation we obtained that the 2.5 percentile cor- VAF. A proper LOD was measured at 5% through this evaluation. But responded to the sample with order number 3, which corresponded to the results of FLT-ITD plug-in, a separated program provided by the a value of RET - He of 27.9 pg as the lower limit. And to the 97.5 manufacturer, showed higher VAF for FLT-ITD variants than that of percentile corresponded to the sample with order number 120, which main results in Ion Reporter. The average of total hands-on time was corresponding to a RET - He value of 39.1 pg as the upper limit. 83 min that accounted for 3% of the time of total process.

Conclusions Conclusions

Finally, we obtained a reference range of RET - He of 27.9–39.1 pg The Oncomine Myeloid Research Assay can provided reliable in our population, which is very useful for the diagnosis of iron results of detection of diverse variation in AML and relatively short deficiency anemia, since it provides real and early information on the hands-on time. current bioavailability of iron, even before the markers biochemical. doi:10.1016/j.cca.2019.03.872 doi:10.1016/j.cca.2019.03.871

^W098 ^W097 Understanding the possible interference of daratumumab in Performance evaluation of Oncomine Myeloid Research assay for multiple myeloma acute myeloid leukemia C. Minea, B. Garcia Corral, R. Tellez Perez, L. Peña Sanchez, A. Porres J.H. Lee, J.H. Lee, C.H. Lee Cubero Department of Laboratory Medicine, Yeungnam University Medical Fundación Jiménez Díaz, Madrid, Spain Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

Background-aim aHematology and Hemotherapy Service, University Hospital Arnau de Vilanova, Lleida, Spain b Monoclonal antibodies are nowadays important part of the Laboratory Medicine Department, University Hospital Arnau de multiple myeloma (MM) treatment. In 2008 the first patient received Vilanova, IRBLleida, Lleida, Spain daratumumab as treatment for his MM. Since then the use of daratumumab was growing till its inclusion in clinical treatment Background-aim guidelines for MM. Daratumumab is a human immunoglobulin G1 (IgG1) | that binds Monoclonal gammopathies (MG) are a group of disorders related CD38. As monoclonal antibodies, daratumumab has the potential to to plasma cell proliferative disease. With the main exception of be identified by serum. non-secretory multiple myeloma, most of the gammopathies are protein electrophoresis (SPE) and immunofixation electrophore- characterized by the secretion of electrophoretically and immuno- sis (IFE). That is way it may interfere or confuse when SPE and IFE logically homogeneous protein called monoclonal component (MC). tests are used in disease monitoring. The detection and identification of the MC is often the first clue to We investigated the detection pattern of daratumumab in serum the diagnosis since MG include a wide range of diseases such as protein electrophoresis (SPE) and immunofixation (IF). multiple myeloma (MM), Waldenström macroglobulinemia (WM) or monoclonal gammopathy of undetermined significance (MGUS). Methods The aim of this report is to describe the incidence of MC in our area during the year 2018 and its association to the clinical data. We choose a pool serum of ten patients with a normal pattern of SPE and no known hematologic diseases and we realised different Methods concentration of daratumumab: 125 μg/mL, 250 μg/mL, 500 μg/mL y 1000 μg/mL. All of that was done knowing the maximum con- The study includes a total of 9240 patients. Serum proteins certation of daratumumab in blood stream (915 μg/mL). separation was performed by capillary electrophoresis at Capillarys 2 Then we study the electrophoreric migration of daratumumab of Sebia®. Identification of monoclonal component was done by and inmunofijación. On the ¨pure ¨ daratumumab and the normal immunotyping at the same equipment. pool serum we also done SPE and IFE tests. Results Results A total number of 9240 serum electrophoresis samples were As previously described in the literature, we observed that performed in 2018. daratumumab migrate at the most catodic part of the gamma region The detection of new MC resulted in 2.54^% of the total number. in serum protein electrophoresis (SPE) and immunofixation (IF). The most frequent component was IgG kappa (|) (42.98%) followed At much more concentration of the monoclonal antibody the by IgG lambda (⌊) (25.11%) and IgM | (13.19%). Minor components spike was more easy to see and it literally fade away at lower were IgA | (7.23%), IgA ⌊ (4.68%), double components (3.83%) and μ finally IgM ⌊ (2.98%). New free | and ⌊ chain components were not concentration (1000 g/mL^). detected during this period. According to gender, 53.62% of the Conclusions components were detected in men whereas 46.38% in women. Regarding the clinical data 43.83% of the components resulted in We were able to establish a migration pattern of daratumumab. MGUS; 8.94% were MM, 1.70% quiescent MM and 1.70% WM. Clinical This may be important because it may lead at misinterpretation: data of the rest of the components (43.83%) is still not available. The average age of GMSI patients was of 69 years old and 91% of the cases 1. A monoclonal gammopathy as a new diagnostic (if we do not were over 50 years old. In case of myelomas (MM, MW and quiescent) know the clinical history of the patient), was of 74 years old and just one case occurred in a patient under 50. 2. A new monoclonal spike (^when the patient previously had another Furthermore on that classification, six cases of MGUS described in known monoclonal protein and received treatment with previous years progressed to MM: four were symptomatic MM, one daratumumab). was quiescent MM and the last was WM. 3. Persistence of the disease. Conclusions Already in use in the hospitals with IFE test is DIRA fi fi fl (daratumumab speci c immuno xation electrophoresis re ex as- Equally as described in other studies IgG kappa resulted to be the say), which is able to achieve a controlled migration of daratumumab most abundant monoclonal component, kappa light chains predom- fi using a highly speci c antibody that does not affect the endogenous inates over lambda light chains and the major incidence of monoclonal protein. monoclonal gammopathy was observed in men over fifty years old.

doi:10.1016/j.cca.2019.03.873 doi:10.1016/j.cca.2019.03.874

W099 ^ ^W100

Study of the incidence of monoclonal components in our area Diagnosis of a rare hemoglobin variant. A case study during 2018 J. Paco Ferreiraa, M. León Rodriguezb, M. Kassih Ibrahima,T. b b b b b b A. Munoz , S. Pico , A. Criado , M. Bernal , A.D. Bernat , M. Regué ,S. HernÁndez Lemesa, A.M. SÁnchez De Abajoa, H. Cabrera Validoa,M. b a b Carrasco , A. Garcia , I. Mercè RiaÑo Ruiza Abstracts / Clinica Chimica Acta 493 (2019) S379–S433 a Servicio de Análisis Clínicos y Bioquímica Clínica, Complejo ^W101 Hospitalario Universitario Insular Materno Infantil (CHUIMI), Las Palmas de Gran Canaria, Spain Measurement of hemoglobin S on CLHP automate G8 (TOSOH) bServicio de Hematología Clínica, Complejo Hospitalario Universitario and comparison with Minicap Flex Piercing (SEBIA) Insular Materno Infantil (CHUIMI), Las Palmas de Gran Canaria, Spain W. Masri, C. Petit, C. Quibon, C. Sautereau, H.A. Cung, H. Broutier, E. Background-aim Plouvier Laboratory, GHEF Meaux Site, Meaux, France, (Est – Ile de France) Hemoglobinopathies are the group of most prevalent monogenic diseases in humans. About 7% of the population is carrier and 2.7% Background-aim have the disease at birth. Within the hemoglobinopathies, we can differentiate the quantitative and the qualitative ones. In the first Hemoglobinopathies, inherited disorders, result from a quantita- ones we find the thalassemias. Within the qualitative ones we can tive deficit (〈, β thalassemias) or variants of hemoglobin (Hb). They find a wide and heterogeneous group of hemoglobin variants that make them a major public health problem and their detection has may not have clinical significance in the patient, as they may lead to important therapeutic consequences early in life. Capillary electro- mild physiological alterations (microcytosis) or serious physiological phoresis (CE) was a technique of choice to detect hemoglobin (Hb) alterations (hemolytic anemia, hydrops fetalis…). The qualitative disorders. The aim of the study was to determine the hemoglobin S hemoglobinopathies are given by variants in some of the genes that (HbS) fraction using CHLP automate G8® (TOSOH France) in encode the synthesis of a certain globin chain producing changes in comparison to Minicap Flex Piercing® (SEBIA France). the amino acid sequence of the protein. Methods Methods For each 33 patients, hemoglobin electrophoresis (EHB) and A 64-year-old male referred to the Haematology Service to HbA1c (G8) were determined. Electrophoresis was realised on measure hemoglobin A1c (glycosylated) due to his diagnosis of Minicap Flex Piercing® [capillary zone electrophoresis – SEBIA diabetes mellitus. A high resolution liquid chromatography study France: MFP] and HbA1c on the CLHP automate G8® (TOSOH France: was performed in the D-10 Testing System (Bio-Rad ®). In the G8). Hemoglobin variants such as HbS, HbC, and HbD had differents chromatogram, we observed an anomalous peak of 31.5% in the retention times then HbA and did not interfere with the HbA1c on retention time of 2.59 min, compatible with the beta chain variant of G8. The suspicion of sickle cell disease (SCD) was confirmed by Itano globin but that could not be related to the standardized patterns test. The statistical analysis used SPSS software. provided by the official supplier. To elucidate the nature of the hemoglobin variant, a molecular study was carried out. In the Results sequencing of the beta globin gene, the exons (1,2 and 3) and adjacent intron sequences were analyzed. Among the 33 patients, 23 patients were A/S, incidental finding during HbA1c analysis using the G8 TOSOH CHPL analyzer and 10 Results patients S/S before and after blood transfusion. The interval of comparison extended from 21 to 91.8%. The % hemoglobin variant As a result, a heterozygous mutation was observed by a guanine results for the two methods were similar for HbS. HbS-G8 was to adenine substitution at position 248 of the second exon of the beta significantly correlated with HbS-MFP (r = 0.991, p b .0001). Regres- globin gene. This variant produces the change of amino acid lysine by sion equation was also calculated for HbS-MFP vs HbS-G8 (y = arginine. 0.948X −2.51). Difference diagrams (Bland and Altman) showed a mean of differences in the acceptability (about 2%). Conclusions Conclusions In this case, after this workflow, the patient was determined with Taradale's hemoglobinopathy in heterozygosis by the presence of the Capillary electrophoresis (CE) and cation-exchange HPLC are variant [beta 82 (EF6) Lys - N Arg]. It affects the binding site with 2,3- technics of choice to detect hemoglobin (Hb) disorders. However, diphosphoglycerate (2,3-DPG), however, it does not seem to affect our laboratory didn't realised in urgency Hb electrophoresis. These the affinity of oxygen with hemoglobin, being the p50 normal. results showed that the HbA1C Kit of G8 has been used for the first- Even without being its main goal, the laboratory equipment that line in urgency. HbS-G8 may be suitable for the follow-up of sickle perform high resolution chromatography act as a screening method cell disease patients who sometimes required a precise HbS for the detection of unknown hemoglobin variants. Subsequently, determination before a surgery or to monitor their urgent blood the study can be extended to confirm the result. Finally, we must transfusion during a drepanocytic crisis. Each results of HbS-G8 will inform the carriers. be confirmed by an Hb electrophoresis. doi:10.1016/j.cca.2019.03.875 doi:10.1016/j.cca.2019.03.876 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

^W102 ^W103

Clonality assessment in suspected cytotoxic T cell Flow cytometry for the diagnosis of hematolymphoid neoplasms lymphoproliferations: To KIR or not to KIR in primary care systems. Role of laboratory decision rules

C. Quirós, A. Fonseca, S. Alonso-Álvarez, L. Morais, M. Moro, F.V. C. Quirós, A. Fonseca, S. Alonso-Álvarez, L. Morais, M. Moro, F.V. Álvarez, E. Colado Álvarez, E. Colado Hospital Universitario Central de Asturias, Spain Hospital Universitario Central de Asturias, Spain

Background-aim Background-aim

Clonality assessment is fundamental for the diagnosis of T cell Patients in primary care centers may be completely asymptom- neoplasms. This can be performed using either T cell rearrangement atic or paucisymptomatic. The rational use of techniques to evaluate studies, TCR-beta families expression or, Killer immunoglobulin-like and characterize potentially pathologic samples, such as multidi- receptor (CD158/KIR) isoforms analysis on cytotoxic cells. Although mensional flow cytometry (MFC) and comprehensive decision rules the WHO classification considers uniform expression of KIR family can help to identify patients with Hematolymphoid neoplasm (HLN). members or lack of expression as a surrogate marker for clonality, this has not been formally validated. Methods

Methods From January 2016 to September 2018, 338,456 complete blood counts from primary care centers were analyzed in the Laboratory We obtained samples from 39 consecutive patients with a Medicine Department. According to laboratory criteria, 5563 were suspicion of cytotoxic T cell proliferation. The origin of the samples selected for blood smear review. Persistent lymphocytosis (N5×109/ was: Lymph node biopsy, 1; Ascitic fluid, 1; Bone Marrow, 2 and L), persistent monocytosis (N1×109/L) and/or abnormal/atypical peripheral blood 35 specimens. Expanded populations were CD4- cells was found in 350 samples, which were sent for immunologic CD8+ (27 cases), CD4/CD8−/dim (11 cases) and Gamma-Delta T studies following EuroFlow Standard Operative Procedures. Statisti- cells (2 cases). Clonality was assessed using TCR-beta families cal analysis was performed using SPSS 20.0 software. expression and TCR rearrangement (3 cases, first corresponding to an hepatosplenic alpha beta T cell lymphoma and the other two to Results belonging to gamma delta T cell large granular leukemia patients). KIR phenotyping (“KIRotype”) was performed using the following All diagnoses were confirmed following the current WHO monoclonal antibodies: CD158a/h/g KIR2DL1 (REA284, Miltenyi classification. In 156 cases, reactive cell populations were found, Biotec), CD158b/j KIR2DL2 (DX27, Biolegend), CD158e KIR3DL1 ruling out HLN in a leukemic phase. In 194 cases (55.4%) HLN was (DX9, Biolegend). Homogeneous expression of one or several demonstrated, requiring Clinical Haematology or Emergency care isoforms or the complete absence of expression were considered as consultation. In brief, monoclonal B cell populations were demon- consistent with clonality for the target population. Statistical analysis strated in 141 (72.7%) cases: 110 CLL/MBLhi (including 18 biclonal was performed using Graphad Prism. cases), 25 MZL, 3 MCL, 2 FL, 1 Hairy cells Leukemia. Monoclonal T and NK cell populations were found in 25 cases (12.9%), including 7 Results CD8+ Large granular leukemia (LGL), 5 CD4+ CD8−/+d LGL, 2 CD4- CD8- LGL, 3 GammaDelta T cell LGL, 2 Sézary syndrome and 6 In 34 T cell populations, clonality was demonstrated either by TCR NK LGL. Acute leukemia was found in 10 cases (70% myeloid and 30% rearrangement or TCR-beta families expression. In 6 cases, oligo- BCP-ALL) and CMML in 12 patients. One case corresponded to clonal/reactive populations were found. KIRotype showed patterns plasma cells leukemia. Finally, 2 cases were diagnosed with compatible with clonality in 40 populations and a reactive pattern in persistent polyclonal B cell lymphocytosis. Focusing in lymphocytosis one single case. The most frequent pattern of KIR expression was cases, the combination of age, hemoglobin and platelets provided the complete absence of expression (33 cases), uniform expression of a best correlation with flow studies for the presence of an HLN (AUC single isoform (5 cases) or uniform coexpression of 2 isoforms (2 0,861, p b .001). cases). Therefore, KIRotype showed a pseudoclonal pattern in 5 cases (12.5%) with clearly reactive/oligoclonal T cell populations. Conclusions

Conclusions Clinical laboratories should develop algorithms to evaluate potentially pathological samples. MFC studies following strict Detection of populations with restricted expression of CD158/KIR standardized procedures has proven to be useful to evaluate samples isoforms is suboptimal to evaluate clonality in suspected T cell from patients in primary care centers for HLN diagnosis or reactive lymphoproliferations. conditions, providing a sensitive and rapid clinical orientation.

doi:10.1016/j.cca.2019.03.877 doi:10.1016/j.cca.2019.03.878 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

^W104 ^W105

Verification of body fluid mode on haematology analyzer Siemens Prognostic impact of serum free light chain ratio, FISH abnor- Advia 2120i malities and percentage of aberrant plasma cells in the bone marrow from patients with monoclonal gammopathy of unde- D. Vuljanić, V. Radišić Biljak, A. Šimundić termined significance (MGUS) Department of Medical Laboratory Diagnostics, University Hospital “Sveti Duh”, Zagreb, Croatia A.M. Rodrigo-Valero, D. De Miguel, D. Subirá, J. Dominguez, M. Bienvenido, J. Arbeteta, A. Martín Background-aim University Hospital of Guadalajara, Mexico

Body fluid mode on Siemens Advia 2120i (Erlangen, Germany) is Background-aim intended for counting total nucleated cells (TNC) and red blood cells (RBC) in pleural and peritoneal fluids. Our aim was to verify MGUS is an asymptomatic condition that usually precedes Multiple performance of body fluid (BF) mode on Siemens Advia 2120i Myeloma(MM). Therefore, initial risk stratification is crucial. analyzer for pleural and peritoneal fluid samples. The risk stratification model for progression of MGUS recom- mended by International Myeloma Working Group(IMWG) is based Methods on: serum M-proteinε15 g/L, non-IgG subtype, and abnormal serum free light chain(FLC) ratio. However, other possible risk factors have According to CLSI H56 (2006) and ICSH 2014 guidelines, we not been taken into account. performed verification of precision (within-run and day-to-day), The aim of the present study was to evaluate if FISH abnormal- accuracy (comparison with the routine reference analyzer, i.e. ities and high% of aberrant plasma cells in the bone marrow(BM) add second Advia 2120i), linearity, limit of quantification (LOQ) and any valuable information as compared to the risk stratification carryover using inpatient samples (pleural and peritoneal body model. fluids) and commercial complete blood count mode (CBC) control materials. Acceptance criteria were based on Siemens specifications Methods (precision for TNC δ15% and RBC δ10%; carryover b0.4%), biological variation (precision for white blood cells (WBC) 5.73%, RBC 1.6%; From 1999 to 2018, 29 MGUS patients were studied at a single bias for WBC 6.1% and RBC 1.7%) and state-of-the-art (bias for WBC institution. After excluding 13 patients with no FLC at the time of 4.4% and RBC 3.2%; proposed by Vis and Huisman, 2016). Data were diagnosis, the total population under study was 16(median age,73 analyzed using MedCalc Statistical Software version 12.5.0.0 years). Median M-protein was 9.12 g/l, 68.75% IgG subtype, 100% (MedCalc Software bvba, Ostend, Belgium). with abnormal FLC ratio(b0.26 or N 1.65), 31.25% with FISH abnor- malities(translocations, deletions and duplications) and 31.25% wih Results ε95% of aberrant BM plasma cells/total BM plasma cells. Progression to MM(median range,8 years) was observed in 6(37.5%) patients. Within-run and day-to-day precision were 16.7% and 15.6% for FLC assay was performed on The Binding Site nephelometer, TNC and 0.7% and 1.1% for RBC, respectively. Although assay principle protein electrophoresis on Sebia Capillarys2, inmunophenotypic for cell counting in BF and CBC mode on Advia 2120i is the same, analyses on FASCS Canto II flow cytometer(BDB) and FISH on declared target values for WBC count in control materials were not Termobrite stat spin. adequate for TNC count, since this control material is not suitable for We used Fisher's exact test for comparison of proportions. BF mode. Passing Bablok showed perfect agreement between two analyzers for TNC in peritoneal (N = 31; intercept −0.83 (−10.33– Results 10.74); slope 1.01 (0.98–1.04)) and in pleural fluid (N = 43; intercept −0.15 (−5.31–10.42); slope 1.00 (0.97–1.02)). Linearity Patients were classified according to the IMWG risk model as was acceptable for both TNC and RBC (r N 0.99). LOQ was 79.6 × 106/ follows: 8(50%) Low Intermediate Risk(LIR), 7(43.75%) High Inter- L for TNC and 11.1 × 109/L for RBC. Both LOQ were higher than mediate Risk(HIR) and 1(6.25%) High Risk(HR). 37.5% classified has declared by the manufacturer (27 × 106/L and 4.94 × 109/L, respec- LIR and 50% classified as HIR and HR progressed to MM(pε0.05). tively). Carryover for TNC and RBC was within declared limits. 80% of the patients that had FISH abnormalities and 18% of the patients that did not have them progressed to MM(p = .036). Conclusions 20% of the patients that had high % of aberrant BM plasma cells and 50% of the patients that did not have it, progressed to MM(pε0.05). Performance of BF mode on automated Advia 2120i analyzer meets manufacturer and state-of-the-art criteria for all tested Conclusions parameters, except for precision estimated on CBC control samples. Commercial controls for CBC mode are not suitable material for BF Risk stratification of MGUS is crucial owing to the heterogeneous mode application. progression pattern. Cytogenetic alterations in the BM identified by FISH should be doi:10.1016/j.cca.2019.03.879 considered as risk factor, together with IMWG risk model, although further studies are warranted to confirm this data.

doi:10.1016/j.cca.2019.03.880 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

^W106 Background-aim

The extending an applicability for the concept of Fibrinolysis Mucopolysaccharidoses (MPS) are hereditary diseases (mostly Shutdown autosomal recessive) caused by enzyme deficiencies resulting in accumulation of complex carbohydrates called MPS or glycosamino- E. Roitmana, A. Shabalinab, M. Tanashyanb glycans. The specific disease depends on the specific enzyme defect. aPirogov Russian National Research Medical University, Moscow, Russia Hurler syndrome is caused by mutations in the IDUAgene (4p16.3) bResearch Center of Neurology, Moscow, Russia leading to a complete deficiency in the alpha-L-iduronidase enzyme and lysosomal accumulation of dermatan sulfate and heparan sulfate. Background-aim Methods Despite the method-dependence a concept ‘Fibrinolysis Shut- down’ (SD) is of interest concept and has no single etiology (Moore, A 10 month old boy born of consanguineous marriage and was 2017). Because any concept should assume an universality we the fourth child of the couple. There was no family history of a studied the predictors pattern for Lys30 with consideration on direct similar disorder or of neonatal deaths and miscarriages. Controlled and mediated links. pregnancy, without incidents. Term newborn 41 weeks. He was derived to ophthalmology due to divergent strabismus, after the Methods exploration a corneal dystrophy was evidenced.

The study included 115 patients with myeloproliferative neo- Results plasms (MPNs), 96 patients with CCVD comorbided with Ph-negative MPNs, 174 patients with acute ischemic stroke, and 96 patients On general examination^, the boy had a rough phenotype, bulging followed up within 12 months after acute ischemic stroke. Both eyes, reductible important umbilical hernia, no inguinal hernias, standard and rapid thromboelastography were performed together Mongolian spot on the back, “Café au lait” spot on the left arm, with 98 biomarkers assays reflecting coagulation, anticoagulation, gingival hypertrophy, breathing mouth and hepatomegaly. platelets, vascular wall, angiogenesis, inflammation, blood cell count, Routine urine examination was normal, but a spot test for etc. Fibrinolysis was analyzed with plasminogen, t-PA, PAI-1, alfa-2- mucopolysaccharides excretion in urine was positive. Urine assay antiplasmin, TAFI and D-dimer. for glycosaminoglycans showed a high level (148.5 mg/mmol). Alpha L iduronidase enzyme assay revealed the absence of the enzyme Results from peripheral leukocytes compatible with Hurler syndrome. The smear shows vacuolated lymphocytes (Gasser lymphocytes) Expected correlations for Lys30 were found with PLG, Alfa-2-AP that support this diagnosis. and t-PA. With middle force Lys30 was bound with RBC, platelet count and ADP- and collagen platelet aggregations. Other indepen- Conclusions dent predictors were fVII, fVIII, fXII, TNF, IL-6. Protein C did not show a value as expected predictor. A multidisciplinary study is required for the diagnosis of MPS. In Revealed predictors having middle force indicates multistep, long addition, to the biochemical and genetic tests, the finding of Gasser nature in regulation of fibrinolysis but not due to short direct links. lymphocytes is an important element for the diagnostic orientation Seems that all long (mediated) pathways converge to thrombin and suggests the accomplishment of other tests that complement the generation. Additional evidences of this are 1) severe value of etiologic profile. inflammation shown by TNF and IL-6, and 2) fXII presence which is transmitter between coagulation and immune response. doi:10.1016/j.cca.2019.03.882

Conclusions

W108 Possibly that inflammation is in fact main regulator of fibrinolysis. ^ Additionally SD concept shows an applicability not only for ICU Utility of the hevylite assay for the paraprotein quantification patients that draws attention in a framework of the issues of thrombosis and re-thrombosis. T. Sendino Miguel, M.d.C. Mugueta Uriaque, M. Macías Conde, A. doi:10.1016/j.cca.2019.03.881 Sandúa Condado, N. Varo Cenarruzabeitia Laboratorio de Bioquímica Clínica, Clínica Universidad de Navarra, Pamplona, Spain

^W107 Background-aim

Revision of blood smear as a diagnostic tool in a case of Introduction: The heavy/light chain (HLC) immunoassay allows mucopolysaccharidoses type I quantification of the pair heavy chain/light chain of each immuno- globulin (Ig) class: IgGk, IgG⌊, IgAk, IgA⌊, IgMk and IgM⌊. It measures I. Rubio Ollo, T. Navajas Jalón, B. Fernández Da Vila, M. Unceta Suárez, both the involved (clonal) and the uninvolved (non-clonal) Ig. I. Martínez Roda, D. Armas Méndez, S. Domenech Manteca, A. López- Though promising, there is no much clinical experience about the Urrutia Fernández association with the currently used measurements in multiple Biochemistry Laboratory, Cruces Universitary Hospital, Barakaldo, Spain myeloma. Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

Objectives: 1) compare the involved immunoglobulin quantified peripheral blood, bone marrow and lymphoid tissue. Chronic by HLC with the paraprotein measured in the serum protein lymphocytic leukemia and small cell lymphoma are thought to be electrophoresis (SPE) and Ig by nephelometry, 2) quantify using the same B-malignant pathology and differ between them only by HLC the monoclonal proteins that migrate in the beta region in the the mode of blood and lymph node invasion. Within the CLL-SCL SPE and 3) investigate if HLC allows the quantitation of small spectrum about 10% of cases are presented as SCL and 90% as CLL. monoclonal bands non measurable by SPE. Chronic lymphocytic leukemia is a heterogeneous disorder, both clinically and biologically. Methods Methods 109 consecutive samples that presented a paraprotein in the SPE confirmed by immunofixation (IFE) were collected. SPE was In our study have been used data provided by the “Mother Teresa performed by agarose gel (Sebia) and densitometered employing University Hospital” Statistical Service, and data collected from the the Hydrasis software. IgG, IgA and IgM were measured by register of malignant diseases. For all patients is performed blood nephelometry in a BN ProSpec (Siemens). The involved/uninvolved smear, bone marrow examination and immunophenotype by HLC pairs were measured by turbidimetry in a SPA analyzer (Binding laboratory service. Site). Results Results A total of 627 cases diagnosed with CLL by Laboratory service and 80 patients showed a quantifiable monoclonal band in gamma by Haematology service and treated by the last one for years 2013– SPE (63 IgG, 5 IgA and 12 IgM) and 17 had a small non-measurable 2017, out of which 177 women (28.2%) and 450 males (71.7%). For band. 12 patients had a band in the ® region. The M-peak quantified the period under study are deagnosed 213 new cases with CLL. by SPE correlated with HLC for IgGk (r = 0.8, p b .001, n = 33), IgG⌊ average age of the affected was 65.5 years while the males were (r = 0.77, p b .001, n = 18) and IgMk (r = 0.95, p b .001, n = 8). No 65.47 years and women 65.58 years old. significant correlation was found for IgA or IgM⌊ probably due to the The number of new cases identified for the study period is 213 of relatively low number of samples. The composite of HLC involved which 90 females and 123 males. Regarding the stage at the time of and uninvolved pairs (Ig'k + Ig´⌊) correlated with the total amount of diagnosis for new cases, the collected data were stage 0 RAI, 41 cases Ig measured by nephelometry (IgG r = 0.44, n = 43; IgA r = 0.99, n (19.25%), stage I RAI 43 cases (20.19%), stage II RAI 57 cases = 12; IgM r = 0.61 n = 11, all p b .05). None of the bands in ® were (26.76%), Stage III RAI 39 cases (18.30%), Stage IV RAI 32 cases measurable but all of them had altered HLC ratio. HLC ratio was (15.02%). Regarding the demographic distribution, it is noticed that altered in 6 out of the 17 samples with positive IFE but small non- the largest number of cases is found in the Tirana district and measurable M-peaks. industrial areas.

Conclusions Conclusions

The HLC involved pair correlates with SPE quantitation for IgG The chronic lymphoid leukemia remains the hematologic malig- and IgM and total Ig measured by nephelometry for all Igs. Also, we nancy of the most commonly encountered. demonstrate that altered HLC ratio could help in the detection and The disease is most commonly encountered in men and there is a quantitation of monoclonal bands in the beta region. Future studies tendency for women to grow, especially after the age of 55. will be needed to validate the clinical utility of this new assay. The average age of new cases diagnosed during the study period is 65.3 for women and 64.4 for males. For the period under study we doi:10.1016/j.cca.2019.03.883 have an average of 42 new cases diagnosed per year with an incidence of 1.5 new cases per year per 100,000 inhabitants.

doi:10.1016/j.cca.2019.03.884 ^W109

Epidemiology of chronic lymphoid leukemia in Albania for the – period 2013 2017 ^W110

M. Shanib, A. Barbullushia, T. Dedej-Kurtia, E. Lamaja, P. Dajaa, A. Likaa, Comparison study between lateral flow immunochromatographic X. Ngjelab, V. Semanajc, P. Pulluqib assay and hemoglobin H staining for the screening of alpha a thalassaemia Clinical- Biochemical Laboratory Service, Laboratory Departament^, Universitary Hospital “Mother Tereza”, Tirane, Albania bHematologji Service, Internal Departament, Universitary Hospital J.Q. Tham, G.L. Wee, H.J. Yeo, P.Y. Heng, M.S. Wong “Mother Tereza”, Tirane, Albania Department of Laboratory Medicine, Khoo Teck Puat Hospital, Singapore c Immunology Laboratory Service, Laboratory Departament^, Universitary Hospital “Mother Tereza”, Tirane, Albania Background-aim

Background-aim Alpha thalassaemia is a genetic disease that is usually caused by the deletion of 1 or more 〈-globingenes.Inourscreenfor〈-thalassaemia, we Chronic lymphoid leukemia (CLL) is defined as malignant test for Hemoglobin H (HbH), by staining red blood cells supravitally with limfoproliferarive hemopathy, due to the collapse of the apoptosis brilliant cresyl blue, which precipitates HbH, allowing detection by mechanisms, which consists in the accumulation of monoclonal B microscopy. Sample preparation is simple to perform, however screening lymphocytes, with distinct immunophenotype characteristics, in for HbH inclusions is time consuming as they occur in approximately 1 in Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

10,000 RBCs in individuals with 〈-thalassaemia minor. In order to Methods increase the screening throughput and improve productivity, we evaluated the i LAB 〈THAL IC Strip Test (i + MED Laboratories, Rayong Total 194 cases were recruited. Flow cytometric analysis was Thailand) as a replacement method for HbH microscopy. performed by Beckman Coulter Cytomics FC500 cytometer. Using CD235a, CD24, CD59, CD14, CD64, and FLAER for gating and detect Methods PNH cells. PNH type is defined as three types: PNH clone(N1%), minor PNH clone(0.1–1%), rare cells with GPI-deficiency(b0.1%). One group 47 samples for routine thalassaemia studies, including 43 samples includes cytopenias in any lineage including aplastic anemia, and presenting with microcytosis, hypochromia and anemia, were another group includes HMs including AML, ALL, MDS, MDS/MPN, concurrently set up for both assay methods. The strip results were lymphoma, and multiple myeloma. compared against HbH microscopy, which was performed by experienced technologists. Five samples with discordant results were Results sent for 〈-thalassaemia genotyping. The findings were tabulated using a binary matrix and specificities and sensitivities calculated. The cytopenias were 147 cases, and HMs were 47 cases. PNH cells We also compared the turnaround times for each assay. were detected in 54 of 194 cases: 32(22%) in the cytopenias, and 22 (47%) in the HMs. Of PNH detected cytopenias, PNH cells were Results detected in 20(62.50%) of monocytes, 14(43.75%) of granulocytes, and 9(28.13%) of RBCs. Rare cells with GPI deficiency was detected in The sensitivity and specificity of the IC strip was 88% and 91% 16(50%): 11(68.8%) in monocytes, 4(25%) in granulocytes and RBCs respectively (n = 47), when compared to microscopy. 20 samples respectively. Minor PNH clones were identified in 10(31%): 5(50%) in that were negative on both methods, included patients with ®- monocytes and granulocytes respectively, and 3(30%) in RBCs. PNH thalassemia trait, Hemoglobin E trait and individuals negative for a clones were found in 6(19%): 5(83.3%) in granulocytes, 4(66.7%) in haemoglobinopathy. 22 samples were positive on both methods. monocytes, and 2(33.3%) in RBCs. Of PNH detected HMs, PNH cells Further analysis of the 5 discordant samples showed that the IC strip were detected in 15(68.18%) of granulocytes and 4(18.18%) of both test had 100% concordance with the 〈-thalassaemia genotyping monocytes and RBCs. Rare cells with GPI deficiency found in 7(32%): assay, thus implying that the sensitivity and specificity of the strip 4(57.1%) in granulocytes and RBCs respectively. Minor PNH clones assay was 100%, overall. The average turnaround time for microscopy were identified in 8(36%): 6(75.0%) in granulocytes, and 2(25.0%) in is 30 min for positive cases or between 60 and 120 min for negative monocytes. PNH clones were found in 7(32%): 5(71.43%) in cases. In contrast, the strip assay requires five minutes of hands on granulocytes, and 2(28.6%) in monocytes. time and 15 min for analysis. Conclusions Conclusions The PNH cell was detected higher in the HMs than cytopenias. Our evaluation suggests that the screening for HbH can be Among the cytopenias, PNH cells were detected with the highest expeditiously and accurately done using the IC strip assay. Due to its frequency in monocytes. However, the proportion of case where PNH simplicity, ease of use, relative low cost, short TAT and clear clones were observed was higher in granulocytes than monocytes. In interpretation, it is capable of superseding microscopy in the battery the HMs, PNH cells were detected with the most frequency in of tests for the diagnosis of 〈-thalassaemia in the routine laboratory. granulocytes. Interestingly, there was no case where PNH cells were observed 0.1% or more of RBC in the HMs. doi:10.1016/j.cca.2019.03.885 doi:10.1016/j.cca.2019.03.886 ^W111

Assessment of PNH clone by multiparameter flow cytometry in W112 cytopenia and hematologic malignancies ^

FXIII levels in patients with systemic lupus erythematosus and W.J. Kwounc, J.Y. Ahnc, J.Y. Seoc, K.H. Kimc, S.R. Choa, J.W. Huhb antiphospholipid syndrome aDepartment of Laboratory Medicine, Ajou University School of Medicine, Suwon, Republic of Korea Z. Bagolyc, N.K. Tóthc, T. Tarra, P. Soltésza, É. Katonac, Z.A. Mezeib,L. bDepartment of Laboratory Medicine, College of Medicine, Ewha Muszbekc Womans University, Seoul, Republic of Korea aDepartment of Clinical Immunology, Faculty of Medicine, University of cDepartment of Laboratory Medicine, Gachon University Gil Medical Debrecen, Debrecen, Hungary Center, Incheon, Republic of Korea bDepartment of Laboratory Medicine, Faculty of Medicine, University of Background-aim Debrecen, Debrecen, Hungary cDivision of Clinical Laboratory Sciences, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Flow cytometry is the gold standard of diagnosing paroxysmal Hungary nocturnal hemoglobinuria(PNH). Nowadays, utilizing multi- parametric flow cytometry for identifying the absence of GPI-linked Background-aim protein expression to diagnose PNH. Our study analyzed the PNH cell detection trend in cytopenia and hematologic malignancy(HM) Patients with systemic lupus erythemasosus (SLE) are subject to cases. significant morbidity and mortality due to atherosclerotic diseases, Abstracts / Clinica Chimica Acta 493 (2019) S379–S433 which cannot be fully explained by traditional risk factors. regarding manual counts and 2) analyze the automatic detection of Antiphospholipid syndrome (APS) is characterized by recurrent neoplastic cells by the UF-5000. thrombosis and pregnancy morbidity in the presence of antiphospholipid antibodies (aPL). APS can be primary or secondary, Methods often associated with SLE. It has been shown that elevated FXIII (FXIII) levels confer an increased risk of atherothrombosis in women. A total of 224 BF were analyzed from 146 patients: 125 without Our aim was to find out whether levels of FXIII might differ in neoplasia and 21 with hematological or non-hematological neopla- patients with SLE and/or APS as compared to healthy individuals and sia. The samples included 151 ascites, 57 pleural, 10 articular and 6 if levels are associated with thrombotic episodes. cerebrospinal (CBF). Manual counts of red blood cells (RBC) and nucleated cells were performed using the Neubauer chamber and Methods conventional microscope. BF in which manual cell counts were N 250/μL, or N 5/μL in CBF, the differential under the microscope was In this observational case-control study, 122 patients with SLE performed. Concurrently, percentages and absolute automatic counts and/or APS and 140 age and sex-matched healthy controls were were performed on the UF-5000 of RBC, nucleated cells, mononu- enrolled. Patients were grouped: SLE without APS (n = 60), SLE with cleated (MNC) and polymorphonucleated cells (PMNC). APS (n = 16), primary APS (n = 26), SLE with aPL without clinical Statistical analysis was performed using Pearson correlation and symptoms of APS (n = 16), aPL without clinical symptoms of APS (n Lin concordance, Mann Whitney-Wilcoxon test and ROC curve

= 4). FXIII activity, FXIII-A2B2 antigen levels, FXIII-B subunit levels (RStudio). were measured and major FXIII-A and FXIII-B polymorphisms were determined. Clinical parameters including age, sex, BMI, smoking Results habit, traditional risk factors, thrombotic history and disease activity were registered. Excellent correlation was obtained for nucleated cells (r = 0.93, p b .0001) in neoplastic samples when comparing results from the Results UF-5000 regarding to the manual method. Moreover, comparing RBC automatic and manual counts a strong correlation was found either

FXIII activity and FXIII-A2B2 antigen levels were significantly in neoplastic (r = 0.76, p b .0001) and benign samples (r = 0.74, p b elevated in APS patients (primary or secondary) as compared to .0001). controls. In SLE patients, regardless of the presence of APS, FXIII-B In neoplastic samples, the percentage of MNC was significantly levels were significantly elevated. FXIII-A2B2 and FXIII-B levels higher (mean = 69.9%) with respect to non-neoplastic (mean = significantly correlated with C4 complement levels. Among SLE 49.4%) (p b .01). Regarding to PMNC, significant high values were patients, FXIII A2B2 levels were significantly elevated in those with obtained in benign (mean = 50.6%) with respect to malignant history of atherothrombosis, while no such association was found for samples (mean = 33.1%) (p b .05). Moreover, a threshold value of FXIII-B levels. A FXIII level in the upper tertile conferred a significant 33.3% was obtained in the percentage of MNC to discriminate risk for arterial but not venous thrombotic events in SLE patients neoplastic from non-neoplastic samples, showing sensitivity and (OR:4.48; 95% CI:1.153–17.44, p = .035). Among FXIII polymor- specificity values of 92.6% and 77.2%, respectively. phisms, FXIII-B His95Arg polymorphism was found to be associated with a positive risk for venous thrombosis, while FXIII-B intron K Conclusions N c.1952 + 144^C G polymorphism conferred a protective effect against it (adjusted OR: 0.190; 95%CI: 0.04–0.79, p = .022). We found a cut-off value in the MNC percentages provided by the automatic cell counts performed in the UF-5000, which showed high Conclusions sensitivity and specificity values to discriminate malignant and benign BF samples. Our results may contribute to the optimization Elevated FXIII levels are associated with increased ath- of cellular analysis in BF demonstrating their utility in the initial erothrombotic risk in SLE patients. diagnosis of neoplasia. Funding: NKFI 128582. doi:10.1016/j.cca.2019.03.888 doi:10.1016/j.cca.2019.03.887 ^W114

A retrospective study to assess the relative value of peripheral W113 ^ blood and CBC results that indicate flow cytometric analysis for the diagnosis lymphoproliferative disorders (LPD) Detection of neoplastic cells in body fluid samples on the Sysmex UF-5000 analyzer A. Cortés Bosch De Basea, R. Güell Miró, E.A. Pérez Hernández, C. Romero Gutiérrez, C. Romero García L. Boldú, J.L. Bedini, A. Merino Laboratori Clínic de L'Hospitalet, Institut Català de la Salut, Spain Core Laboratory, Hospital Clinic of Barcelona, CDB, Spain Background-aim Background-aim

Advances in automated haematology analyzers have decreased Cellular analysis of body fluids (BF) has clinical relevance in the the samples that require microscopic smear review or manual diagnosis of infections, hemorrhages and neoplasia. differential count. However, peripheral blood smear review The objectives of this work are: 1) assess the correlation between remains an important diagnostic tool, and consensus guidelines automatic counts provided by the Sysmex UF-5000 analyzer have been developed for when such review should be triggered. Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

fi Peripheral blood smear review is useful for con rming instrument ^W115 flags. Flow cytometric immunophenotyping has a well-established role Uncommon hemoglobin variants description and their interfer- in the evaluation of peripheral blood, evaluating white blood cells ence in glycated hemoglobin determination (WBC) as part of the multiparameter evaluation for LPD. The medical indications for peripheral blood flow cytometric M. Ortíz Espejob, S. Díez Espigab, N. Fañanás Rodríguezb, A. Moyano immunophenotyping include some abnormal CBC findings and Martínezb, R. García Sardinab, C. Esparza Del Valleb, G. Pérez clinical symptoms and signs that suggest the possibility of a Vázqueza, M.J. Muruzabal Sitgesb, C. Fernández Cuestab hematolymphoid neoplasm. aHospital Sierrallana, Torrelavega, Spain Laboratory professionals are in an ideal situation to identify bHospital Universitario Marqués de Valdecilla, Santander, Spain CBC and peripheral blood smear findings that raise the possibility of an LPD, and based on this information make recommen- Background-aim dations for additional studies, such as flow cytometric immunophenotyping. Glycated hemoglobin (HbA1c) determination is used in the This study intends to show how many LPDs have been diagnosed routine control of diabetic patients and reflects blood glucose levels fl adding a ow citometry immunophenotype when the CBC results or for the past 2–3 months. High performance liquid chromatography the morfology of the peripheral blood smear suggested so. (HPLC) allows the quantification of HbA1c and reveals the presence of hemoglobin (Hb) variants. Hemoglobinopathies are the result of Methods alterations in the genes that encode the globin chains. The presence of any of these variants in the chromatogram may interfere with the We have reviewed all the immunophenotype studies during 2017 measurement of HbA1c. and 2018. Description of rare variants of Hb that interfere in the routine All the samples were processed in Sysmex XN analyzers for the control of HbA1c. Complete Blood Count (CBC). May-Grundwald Giemsa stain was performed in those that Methods required blood smear revision and were reviewed under optic fi microscopy to nd any possible hematologic disorder. The measurement was made by HPLC cation exchange in the Immunophenotype testings were added when needed and the automated analyzers Variant II Turbo and D-100 Hemoglobin Testing samples were sent to a central laboratory in the Universitary System, Bio-Rad Laboratories ®. Possible Hb variants were reprocessed in Hospital of Bellvitge. the D-10 analyzer and sent to the Haematology Service where they were studied by capillary electrophoresis (Minicap de Sebia®). The cases with Results clinical repercussion were sent to an external center for genetic study.

In total we studied 676 patient samples, 332 women and 344 Results men with an average age of 66.8 years. 309 (45.7%) of the samples studied resulted in a Chronic B- The following Hb variants are uncommon in our environment and Lymphoproliferative Disorder. 60% of which were diagnosed as their presence interferes with the determination of HbA1c. Chronic Lymphocytic Leukemia (CLL). Hb Le Lamentin: The chromatogram shows an anomalous peak 58 (8.6%) were Chronic T Lymphoproliferative Disorders, 48% of that causes underestimation of the HbA1c value. The genetic study which resulted in LGL Leukemia. shows the CCA N CAA mutation in codon 20 of the 1st exon of the When looking into the results of overall lymphocitosis we can see Alfa2 gene. that most of the samples sent to immmunophenotype had overall Hb Stanleyville - II: A possible variant of Hb overestimates the lymphocitosis over 5 × 109/L which is the target established in our HbA1c determination. Capillary electrophoresis reveals a peak in Z1. laboratory to make a revision of the peripheral blood smear. The AAC N AAA mutation is found in codon 78 of the 2nd exon of the In addition, we also review those samples that had an instrument alpha2 gene. fl ag of atypical lymphocytes even though the absolute number may Hb Niigata: An abnormal peak is detected by HPLC that produces be within the normality range. We have studied 70 samples with a negative interference in the measurement of HbA1c. It presents the overall lymphocytes below 5 × 109/L and 42 (60%) were diagnosed GTG N CTG mutation in codon 1 of the exon of the beta gene. with LPD. Hb Korle-Bu: An atypical peak is detected by HPLC, which underestimates the HbA1c value. Capillary electrophoresis presents Conclusions a variant that migrates in Z6. The GATN AAT mutation is found in codon 73 of the 2nd exon of the beta gene. In summary, the combination of manual peripheral blood smear review, CBC results and flow cytometric studies provides very useful Conclusions information for the evaluation for LPD. Even though, it is important to integrate peripheral blood smear Chromatograms review is crucial to avoid reporting erroneous fi fl review ndings with the clinical information before adding ow results. Hemoglobin variants can interfere in the measurement and cytometry testing. can lead to inadequate treatments administration. Laboratory has the chance to discover unknown hemoglobinopathies, contributing to doi:10.1016/j.cca.2019.03.889 improve patient care.

doi:10.1016/j.cca.2019.03.890 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

b ^W116 Department of Internal Medicine Merkur University Hospital and School of Medicine, University of Zagreb, Croatia Clinical evaluation of a new SFLC assay for monoclonal gammopathies patients Background-aim

M. Haddad, B. Pegourié, B. Lardy Flow cytometric immunophenotyping has been widely used to University Hospital of Grenoble, France identify neoplastic plasma cell populations (PCs) in patients with multiple myeloma (MM). Markers that have been associated with Background-aim informative aberrant antigen expression profiles for MM according to European Myeloma Network (EMN) guidelines are: CD19, CD56, Since now N15 years, the importance of free light chains (FLC) CD45, CD38, CD27, CD20, CD28, CD33, CD117 and to a less extent quantification has increased steadily. Combined with serum protein also CD81 and CD200. In combination with cytoplasmic immuno- electrophoresis (SPE) and immunofixation, the FLC assay contributes globulin (cytIg) KAPPA and LAMBDA light chain staining, they to the diagnosis, prognosis and follow-up of patients with monoclo- contribute to establish the clonal nature of a population of suspicious nal gammopathies. For multiple myeloma diagnosis, FLC concentra- PCs. The aim of this study was to evaluate the diagnostic utility of tions, and particularly the ratio between the involved and flow cytometric imunophenotyping (FCI) for MM analysis. uninvolved (i/u) became a diagnostic criterion in combination with signs of organic damage (CRAB) and medullar clonal plasma cells Methods assessment. In 2018 FCI was done on bone marrow specimen from 40 Methods patients, 18 females (49–83 years) and 22 males (52–85 years), characterized as newly diagnosed (13), stationary MM (11) or Currently, two groups of reagents are available on the market relapsed MM (16), respectively. Multiparametric flow cytometric based on turbidimetric and/or nephelometric technologies: (1) immunophenotyping was performed using monoclonal antibodies Freelite marketed by The Binding Site, using polyclonal antibodies against CD138, CD38, CD45, CD56, CD19, CD20, CD33, CD117, CD200, and (2) N-Latex FLC marketed by Siemens, using monoclonal CD27 and CD28, cytKAPPA, cytLAMBDA following the procedure for antibodies. The results obtained by these two reagents were shown intracytoplasmatic determination. Backbone markers CD38, CD45 to be globally correlated but non-transposable. Recently, a new and CD138 were included in all tests. All measurements were method of analysis of light free chains has been proposed by Sebia. performed on a flow cytometer Navios, Beckman-Coulter, in 10 color This technique (Sebia FLC) relies on ELISA detection using polyclonal analysis. Gating procedure was based on CD38 vs. CD138 antigen antibodies. We wanted to assess the sebia FLC in the context of expression. follow up of patients with monoclonal gammopathies. Results Results PCs population accounted 0.4–64% of the total nucleated cell We analyzed 360 samples from 80 patients' one year follow up count by flowcytometry. The abnormal antigen expression pattern as with both Sebia FLC and Freelite techniques. Our results showed a defined by EMN in 40 MM cases, was found for: CD45 (75%), CD56 good correlation between the two techniques despite significantly (82.5), CD19 (90%), CD20 (8.1%), CD33 (45%), CD117 (60%), CD200 higher absolute values for the Freelite assay. Additionally, we show (82.1%), CD27 (57.5%) and CD28 (37.5%), respectively. Light chain that Sebia FLC units are strictly identical than the concentration of restriction was demonstrated in all of them, with either KAPPA (30) the FLC peak quantified with the tangent mode on SPE for samples or LAMBDA (10) clonal expression. with quantifiable peak on SPE. The study of patients monitoring show that the iFLC trend is strictly comparable for the 80 tested Conclusions patients. For the diagnosis of myeloma, the i/u FLC ratio of 100 obtained by Freelite cannot be used for the Sebia FLC technique. Although CD19, CD56 and CD200 represented as the most valuable antigens for identifying neoplastic population in patients Conclusions with MM, they can't stand alone to distinguish normal/reactive vs. tumor cells. The immunophenotyping panel proposed by EMN is SEBIA's technique for assaying light free chains is a promising powerful for distinguishing neoplastic from reactive plasma cells in technique that can be integrated with the two techniques currently clinical practice. available on the market. Standardization in processing, analysis and reporting increases objectivity of FCI findings in cases when MM is suspected. doi:10.1016/j.cca.2019.03.891 doi:10.1016/j.cca.2019.03.892

^W117 ^W118 Diagnostic utility of multiparameter flow cytometric immuno- phenotyping in multiple myeloma The Abbott Alinity hq accurately identifies the presence of immature neutrophils M.M. Kardum Paroa, I. Taradia, D. Radić Krištob, I. Mandac Roguljb,S. a c b d Ostojić Kolonićb, M. Kursarb,Z.Šiftara Z. Mukhtar , C.L. Slim , B.A. Wevers , M.W.H.J. Demmers , H.J. d b c a a aDepartment of Clinical Chemistry and Laboratory Medicine, Merkur Adriaansen , J.A. Kooren , H. Storm , H.J. Hoffmann , G. Lakos a University Hospital, Zagreb, Croatia Abbott Diagnostics, Santa Clara, CA, United States Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

bDepartment of Clinical Chemistry, Atalmedial Medical Diagnostic aDepartment of Clinical Biochemistry, Hospital Universitario 12 de Centers, Hoofddorp, The Netherlands Octubre, Madrid, Spain cDepartment of Clinical Chemistry, Certe, Groningen, The Netherlands bTBS Iberia, Spain dDepartment of Clinical Chemistry, Gelre Hospital, Apeldoorn, The Netherlands Background-aim

Background-aim The diagnosis, follow-up and treatment monitoring of asymp- tomatic monoclonal gammapathies (MG) and multiple myeloma The Alinity hq high throughput haematology analyzer (Abbott (MM) is based on the quantification and identification of the Diagnostics, Santa Clara, CA) reports the absolute concentration and monoclonal paraprotein by electrophoresis and immunofixation percentage of Immature Granulocytes (IG), defined as techniques in serum and urine. Monoclonal free light chains in MM promyelocytes, myelocytes and metamyelocytes, as part of a 6-part patients have traditionally been monitored in 24 h urine by WBC differential. In addition, Alinity hq displays a Left Shift (LS) flag electrophoretic measurements of Bence Jones protein (BJP); how- if an increased proportion of band neutrophils (BN) is detected. ever, the impact of renal metabolism, limited analytical sensitivity and low urine samples compliances limit the usefulness of Methods urinalysis. We aim to assess the usefulness of 24-h urine measurements versus the use of serum free light chains (sFLC) The ability of Alinity hq to detect neutrophilic left shift was determination in the follow-up of patients with monoclonal assessed in comparison to CELL-DYN Sapphire (Abbott) and manual gammapathies. WBC differential (reference method). The study cohort consisted of 1461 clinical samples from the routine workload of the Departments Methods of Clinical Chemistry at Atalmedial Diagnostic Center, Gelre Hospital and Certe (The Netherlands), and comprised of patients with various We compared on a retrospective analysis the results obtained by conditions, including hematological malignancies. the determination of sFLC (SpaPlus, The Binding Site) against urine electrophoresis (UPE) and immunofixation (UIFE) in 24-h urines Results (SAS 1&2, Helena) of 977 samples of patients with MG. We have calculated the negative predictive value (NPV) between the deter- Manual and Alinity hq %IG comparison resulted in a correlation minations in both matrixes. coefficient of 0.68 and a Passing-Bablok slope of 1.3. Total agreement between the Alinity hq LS flag and the combination of the CELL-DYN Results Sapphire Band Alert (bndAlrt) and IG Alert (igAlrt) was 89.8%. Considering ε 5% BN alone, the sensitivity of the Alinity hq LS flag Between 2016 and 2018, we made an average of 1865 UIFE per and the CELL-DYN Sapphire bndAlrt were 52.6% and 34.8%, with a year, of which only 26.06% were positive for BJP. We have calculated specificity of 91.2% and 95.8%. the probability of having normal urinalysis when sFLC were also Alinity hq detected the presence of 1% and 2% IG, respectively, normal by evaluating NPV of the following comparisons:i)UIFE vs with a sensitivity of 72.3% and 67.0% and a specificity of 90.7% and altered sFLC ratio = 0.9481(CI:0.9258–0.9652);2)UPE vs altered K/L 95.1%, compared to a sensitivity of 59.9% and 66.7% and a specificity ratio = 0.9926 (CI:0.9811–0.9980);iii)UIFE vs iFLCN100 mg/L = of 90.5% and 88.9% of the CELL-DYN Sapphire igAlrt. 0.9538 (CI:0.9351–0.9684);iv)UPE N200 mg/24 h vs iFLCN100 mg/L When left shift was defined as the combination of ε 5% BN and/or = 0.9657 (CI:0.9490 to 0.9781). ε 1% IG, it was detected with 64.3% sensitivity and 91.1% specificity by the LS flag and/or ε 1% IG in the automated differential. Conclusions Samples with LS flag had significantly higher WBC count (17.8 vs 7.36 × 109/L) and neutrophil count (12.23 vs 4.73 × 109/L). The The results obtained by UIFE and UPE can be highly subjective and – fl median (inter-quartile range) of %BN and %IG was 2.76^% (1.00 6.96) in uenced by the type of manipulation of the sample (concentration, vs 0.25% (0.00–1.00) and 0.97% (0.00–3.35) vs 0.00% (0.00–0.25) for precipitation, etc.). With a NPV close to 95% in all comparisons, we can samples with and without LS flag, respectively (p b .0001 for both). assume that patients who present negative results in urine studies can be followed by determination of serum free light chains with the Conclusions greater comfort and objectivity that this supposes. Our results suggest that sFLC might be an assay that could guide to more selectively Alinity hq effectively identifies the presence of clinically signifi- requests of urinalysis. However, it is necessary to consider as cant concentration of immature forms of neutrophil granulocytes. inflammatory processes, intercurrent infections or alterations of the Quantitation and reporting of IG concentration represents an renal function could alter chain ratios in these patients. advancement compared to qualitative flagging. doi:10.1016/j.cca.2019.03.894 doi:10.1016/j.cca.2019.03.893

^W120 ^W119 Agreement study between the response criteria of the interna- Can the sFLC assay help on the optimization of the use of 24h tional Myeloma Working Group vs the Intergroupe Francophone urines in patients with monoclonal gammapathies? du Myelome

I. Liria Gonzáleza, D. Melero Lópeza, N.M. Barbosa De Carvalhob,C. D. Melero Lópeza, I. Liria Gonzáleza, N.M. Barbosa De Carvalhob,C. Tejedor Díaza, P. Puerta Fonolláa Tejedor Díaza, P. Puerta Fonolláa Abstracts / Clinica Chimica Acta 493 (2019) S379–S433 a Department of Clinical Biochemistry, Hospital Universitario 12 de ^W121 Octubre, Madrid, Spain bTBS Iberia, Spain Unusual presence of green neutrophil inclusions in peripheral blood as a marker of impending patient death Background-aim A. Merinoa, A. Molinaa, P. Castrob The treatment response criteria in patients with multiple aCore Laboratory, Hospital Clinic of Barcelona, CDB, Spain myeloma (MM) have varied with the introduction of new more bMedical Intensive Care Unit, Hospital Clinic of Barcelona, Spain sensitive techniques and a greater knowledge of this pathology. The International Myeloma Working Group (IMWG) introduced serum Background-aim free light chains in 2009 as a biomarker to define response criteria along with electrophoresis and immunofixation both in serum and Green inclusions (GI) in the cytoplasm of neutrophils has been urine. In 2018, the Intergroupe Francophone du Myelome (IFM) reported in critically-ill patients as an indicator that death is published a proposal to modify the response criteria based only on imminent. We report the so called “green crystals of death” within serum studies and replacing the 24-h urine proteinuria studies by neutrophils in a 58-year-old woman with a sepsis, alongside with a serum free light chains (sFLC). review of the literature, with the goal of increasing the meaning and The objective of this study is to compare the agreement of the awareness of their detection. treatment response categories according to the criteria of IMWG and IFM. Methods

Methods A 58-year-old woman was admitted into the Hospital because an obstructive pyelonephritis and sepsis. Automatic cell counts were We randomly selected 33 patients with multiple myeloma (MM): done in the analyzer Advia 2120 (Siemens). A film review was 20 intact immunoglobulin, 11 light chains and 2 oligosecretory. We indicated because of the increased number of leukocytes. Blood analyzed the results in serum by electrophoresis (V8, Helena) and smears were reviewed using May Grünwald-Giemsa staining and the immunofixation (Interlab G26, Biometa), the same in urine (SAS 1 CellaVision DM96. and 2, Helena) and serum free light chains (SpaPlus, The Binding Site) at diagnosis/pretreatment in relapse and after treatment. We Results classified the response of each patient according to the criteria of both groups and calculated their agreement using the weighting Patient arrived in bad general conditions and hemodynamically kappa. unstable, being referred to the medical intensive care unit. At admission, blood tests showed an impaired renal function. An Results automatic blood cell count showed high white blood cell count (31.8 x10e9/L) with neutrophilia (17 x 10e9/L, normal 2.5–7), low Quadratic weighting kappa is 0.8061 (CI 95% 0.4061–1). hemoglobin values (115 g/L) and low platelet count (101x10e9/L). Coagulation parameters were abnormal (PT: 48.1% and APTT: 41.9 s). Conclusions In the blood film examination on day 2, some green inclusions were seen within neutrophils (5%). We considered them as a “critical We found a substantial agreement between the response criteria finding”, being reported to the clinicians. Elevation of liver transam- of both groups. In the analysis, 6 of the 7 patients' discordant results inases AST/ALT was found (671/341 UI/L, normal 5–40), alongside (85.7%) have an altered glomerular filtration, b60 ml/min/1.73 m2 by with lactic acidosis (51 mg/dL, normal 5–20). The diagnosis was the CKD-EPI eq. (VR:N 90 ml/min/1.73 m2). One IgA Kappa MM obstructive pyelonephritis and acute liver failure in the context of patient (ISS 2) presented at diagnosis a iFLC b50 mg/L; according to sepsis. She recovered after antibiotics and support treatments. the IMWG criteria, the response to treatment was a complete A total of 41 cases have been reported in the literature, so that the response, however by the IFM criteria it was classified as stable finding of these GI is unusual. Clinically, GI have been associated with disease; this discrepancy can be explained by the use of the IFM elevated transaminases, hepatic failure, and a high early mortality group of the decrease in dFLC as a response criterion (ε50% and ε90% rate. The mortality in 18 patients with GI was 13/18 (72%) within 72 for partial response and very good partial response, respectively), h of their detection in 12 of them. which in this case was not fulfilled. Further studies are necessary to evaluate the replacement of 24 h Conclusions urine studies by the determination of serum free light chains in the response criteria of MM patients, mainly those with renal The detection of green inclusions within neutrophils is a marker involvement. associated with acute liver damage, lactic acidosis and high mortality. They could represent a lipofuscin-like substance taken up doi:10.1016/j.cca.2019.03.895 by phagocytosis following ischaemic injury to the liver. Their report may alert to the clinicians to an ischaemic insult or lactic acidosis.

doi:10.1016/j.cca.2019.03.896 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

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Rationalization of Bence Jones protein analysis based on a serum False automatic basophil counts associated with lymphoprolifer- algorithm including free light chain testing ative disorders with expression in peripheral blood

E. Moga Naranjoc, L. Alserawan De Lamoc, T. Franco Leyvac, M.V. A. Molina, A. Merino, J. Alcaraz, M. Arnau, S. Fumanal, V. Ortiz, S. Rubialesc, A. Baucellsc, E. Zapicoa, M. Granell Gorrochateguib Garcia, J.L. Bedini aBiochemistry Department, Hospital de la Santa Creu i Sant Pau, CORE Laboratory, Biomedical Diagnostic Center, Hospital Clinic of Barcelona, Spain Barcelona, Spain bHematology Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Background-aim cImmunology Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Haematology analyzers count thousands of leukocytes in every aspiration and, therefore, they offer high reliable estimates of the Background-aim different leukocyte subsets. However, many studies show a poor performance of the automatic basophil count when comparing with Bence Jones protein identification (BJP) has been a hall mark for the other leukocyte subtypes. diagnosis and monitoring Multiple Myeloma patients. However, The goal of this work is to perform a retrospective study of assay limitations associated both with poor sample compliance, and samples in which an increased value of automatic basophil count renal interference, result in a high percentage of negative results was observed, but where the manual differential count revealed while disease is still active. Finally, it is also rather time-consuming normal values. for the lab. The objective of this work was to evaluate the possibility of Methods rationalizing the use of BJP determinations using a serum algorithm that includes the highly sensitive Free light Chain assay to identify A retrospective search (January to December 2018) was per- the best moment to perform urine anaylis. N formed for samples with an automated basophil count ^5^% (criteria for peripheral blood smear review in our laboratory) being the total Methods N leukocyte count ^1×109/L. All blood samples were collected in tubes with EDTA as Retrospective evaluation of all patient's samples arriving at Sant anticoagulant and processed by the automated haematology instru- Pau Hospital (Barcelona) from January 2017 to November 2018, ment ADVIA 2120 (Siemens). The diagnosis of the different patients, where results for urine immunofixation (IFU) (Sebia), urine electro- who the samples belonged to, was reviewed. phoresis (UPE) (Sebia) or serum free light chains (FLC) (The Binding Site, UK) were available. Data for Serum protein electrophoresis Results (SPE) (Sebia), and immunofixation (IFS) (Sebia) were also retrieved. Statistical analysis was done with Excel and Graph Pad v.5. A total of 148 samples from 69 different patients showed the inclusion criteria for the study. It is important to mention that 62/69 Results (90^%) of the patients were diagnosed of different types of hematological diseases. Data from 2103 samples corresponding to 934 patients were Surprisingly, half of the patients (34/69) in which the automatic retrieved. From the 1784 IFU retrieved, 68% were negative, and from basophil count was high, alongside with normal counts in the the 282 UPE, 73% were b 200 mg/24 h. Considering a serum algo- manual differential, were diagnosed of some of the following rithm based on FLC, IFS, and EPS, only 2% (N = 9) of 555 results, lymphoproliferative disorders: chronic lymphocytic leukemia (24/ where IFU positive and negative by the three serum techniques, 34), multiple myeloma (3/34), splenic marginal zone lymphoma (1/ simultaneously. On the other hand, 49% (N = 273) of the samples 34), mantle cell lymphoma (1/34), Sezary syndrome (2/34) and were negative by IFU and positive by the serum algorithm. other T-cell lymphomas (3/34). This group of patients was charac- Comparing the 281 results where both UPE and serum FLC were terized by the following findings: 1) high leukocyte counts (average available, it was found that 55 UPE and 168 FLC met the criteria of of 128 × 109/L); 2) Abnormal lymphocytes with expression in measurable disease of each technique. Only 7 samples were positive peripheral blood; 3) high automated basophil count (in many cases N by UPE and negative by FLC while the opposite was observed in 120 ^20^%); and 4) normal basophil count in the manual differential samples. counts.

Conclusions Conclusions

Our analysis suggests that this serum algorithm could be helpful In this study an unusual phenomenon of pseudobasophilia to make a more selective use of IFU and UPE determinations. More associated to lymphoproliferative disorders is described. One possi- studies are necessary, but these data suggest that an IFU could be ble explanation to this finding may be related to the increased reserved for confirming the negative result of the serum algorithm. resistance of the abnormal lymphocytes membrane to lysis in the basophil/lobularity channel measurements. doi:10.1016/j.cca.2019.03.897 This significant finding should be considered when reviewing cell blood count results.

doi:10.1016/j.cca.2019.03.898 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

W124 ^W125

Performance evaluation of the new Beckman Coulter DxH-900 Alinity hq reference ranges for reticulocytes and related haematology analyzer parameters

A. Martínez Iribarren, X. Tejedor Ganduxé, M. Sala, M. Lopez-Molina, M. Patelb, H. Hoshinob, R. Chandrasa,K.Qua, Z. Mukhtarb, G. Lakosb L. Galiano, C. Juan, I. Sierra, A. Castillo, K. Qyyum, M. Llopis, C. aAbbott Diagnostics, Lake Forest, IL, United States Morales-Indiano bAbbott Diagnostics, Santa Clara, CA, United States Haematology-Core Lab Department, Clinical Laboratory Metropolitana Nord (LCMN), Hospital Universitari Germans Trias i Pujol, Badalona, Background-aim Barcelona, Spain Alinity hq (Abbott Diagnostics, Santa Clara, CA) is a high Background-aim throughput haematology system that utilizes advanced MAPSS™ technology and fluorescence flow cytometry. When used in the CBC DxH900 is a new fully automated analyzer that provides cell + RETIC mode, the analyzer reports the concentration and percent- blood count, white blood cell (WBC) differential, reticulocyte count age of reticulocytes (R and %R), the immature reticulocyte fraction (RET) and NRBC enumeration. The aim of this study was to validate (IRF), percent reticulated platelets (%rP) and the hemoglobin content DxH900 and compare it with its predecessor DxH800. of reticulocytes (MCHr). IRF is an indicator of erythropoietic activity, and is used to Methods monitor bone marrow regeneration or response to therapy. MCHr is a marker of bone marrow iron status and availability and is a 321 samples (EDTA-K3 blood specimen) were analyzed over a 1- predictor of response to anemia therapy. Reticulated platelets reflect month period. Following the guidelines of the Clinical and Laboratory megakaryopoietic activity in the bone marrow and have been shown Standards Institute (CLSI, H26-A2) we evaluated background (BG), to be a non-invasive predictive marker in patients with thrombocy- carryover (CO), reproducibility (R), intrinsic interference (I), linear- topenia. Previous publications have noted that reference intervals for ity (L), limit of quantification (LQ), abnormality messages (flags), reticulocytes and related parameters are technology-dependent. differential count (DC) and correlation with DxH800. Methods Results Reference ranges for the above-mentioned parameters were The results for BG, CO, R and I met manufacturer's specifications. established on Alinity hq using samples from up to 213 apparently Excellent linearity was obtained for red blood cells (RBC) (y = healthy adult subjects (46.6 ± 12.0 years, 19 to 91 years). Gender − − 0.03 + 1.01×;R = 1.00), WBC ( 0.04 + 1.01×;R = 0.99), hemo- and age-dependency were also assessed.^ globin (Hg) (0.11 + 1.01×;R = 1.00) and platelet count (PLT) (−4.87 + 0.98×;R = 0.99). The samples used to assess LQ presented Results WBC values between 0.236 and 0.311 × 109/L with coefficient of variation (CV) b15%, while the samples used to quantify PLT (with Data distributions were Gaussian for IRF and MCHr, while %R and values between 4.1 and 8.35 × 109/L) yielded a CV b 20%. Passing %rP results followed a non-normal distribution. The central 95th Bablok method showed no differences between both analyzers for percentile ranges were calculated according to CLSI EP28-A3c WBC (y = 0.05 + 1.00×;R = 0.99), Hg (y = −0.25 + 1.02×;R = guideline using a parametric method for normally distributed 0.99), RBC (y = 0.10 + 0.98×;R = 0.99) and RET (y = 0.06 + measurands, and a non-parametric method for non-normally 1.04×;R = 0.99). PLT showed a good correlation (y = 1.29 + 0.92×; distributed data. A small difference was observed between genders R = 0.99). PLT measured on the DxH-900 was higher in comparison for %rP values only (p = .012). Age dependency was noted for IRF to the DxH-800 with a 7% bias (3.1 to 16.5%). The comparison of 50 and %rP in males, and for %R values only in females. A statistically leukocyte DC results analyzed in parallel with blood smear significant positive correlation was found between IRF and %R (r = observation (reference method), revealed a good correlation for 0.208, p = .0023) and a negative correlation between IRF and MCHr neutrophils (R = 0.95), lymphocyte (R = 0.97), monocyte (R = (r = −0.381 (p b .0001). 0.90) and eosinophils (R = 0.81), while basophils showed a poor The cumulative reference ranges for %R, IRF and MCHr, respec- correlation (R = 0.52). Sensitivity and specificity for the detection of tively, were 0.86–2.82%, 0.17–0.44, and 25.3–35.5 pg. The reference flags were 0.75 and 0.92 for blasts, 0.91 and 0.91 for immature range was 1.14–6.87% for %rP. granulocyte, 0.95 and 0.77 for left shift and 0.95 and 0.87 for variant lymphocytes, respectively. The overall performance of the flags Conclusions showed a negative predictive value (NPV) of 0.99. Reference ranges for %R, IRF, MCHr and %rP were established on Conclusions anility hq. These ranges can serve as guide for Alinity hq users and as basis for comparison to other analyzers^. DxH900 provides reliable results and fully comparable to DxH800. One of the highlights is sensitivity and NPV of flags, which doi:10.1016/j.cca.2019.03.900 has been improved. DxH900 is an accurate, highly precise analyzer with good analytical performances to be used effectively in high- volume laboratories. doi:10.1016/j.cca.2019.03.899 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

^W126 ^W127

A patient with unexplained eosinophilia Monocytic subpopulation analysis by multidimensional flow cytometry outperforms classical variables for CMML diagnosis R. Palmab, A. Escobara, P. Fuentesb, C. Morenob, P. Picazob,D. Melguizob, M.Á. Ruizb, M.C. Lorenzob C. Quirós, A. Fonseca, S. Alonso-Álvarez, L. Morais, M. Moro, E. Colado aHospital San Pedro de Alcántara, Cáceres, Spain Hospital Universitario Central de Asturias, Spain bHospital Virgen de la Salud, Toledo, Spain Background-aim Background-aim Chronic Myelomonocytic leukemia (CMML) is characterized by Eosinophilia refers to an absolute eosinophil count in the peripheral persistent peripheral blood (PB) monocytosis, accounting for ε10% blood of ≥500 eosinophils/μL and hypereosinophilia is defined as leukocytes. Conditions associated with reactive monocytosis must be moderate (1500 to 5000 eosinophils/μL) to severe eosinophilia (N5000 ruled out and this diagnosis may not be straightforward when eosinophils/μL). Peripheral blood eosinophilia can be caused by myelodysplasia or genetic alterations are not found. Moreover, the numerous allergic, infectious and neoplastic disorders, which require a temporal criteria for “persistent” monocytosis is not clear. Based on variety of different treatments. It is important to define the main cause current limitations, we aimed to develop a diagnostic algorithm. of this disorder in order to begin treatment as soon as possible. Methods Methods

EDTA-K3 anticoagulated PB from 57 consecutive patients (21 A 23 years old male patient presented to the emergency CMML and 36 reactive monocytosis) with persistent monocytosis department with odynophagia, fever, difficulty breathing and left (ε1x109/L) maintained for at least 3 months were stained with the cervical swelling. following monoclonal antibodies CD36-FITC (CLB-IVC7, Sanquin), CD64-PE (10.1, Cytognos), CD34-PerCP-Cy5.5 (8G12, BDB); CD16- Results PE-Cy7 (3G8, BDB); CD300e- APC (UP-H2, Immunostep); CD14- APC-H7 (MOP9, BDB); HLADR-PacB (L243, BDB) and CD45-HV500 6 The clinical analysis revealed a mild hypertransaminasemia, an (2D1, BDB). At least 10 viable cells were acquired in a FACSCanto elevated CRP and a complete blood count with mild anemia and a II (BDB, San Jose, CA). Instrument setup and sample preparation differential of 68,9% eosinophils (10 × 10^3/μL) without any blast was performed following EuroFlow Standard Operative Procedures. cells on the peripheral smear. Laboratory advised to dismiss other Boolean gating selecting cells co-expressing CD64 and HLADR and/ reactive eosinophilia causes. A bilateral pleural effusion was or expressing CD300e, HLADR and CD16 was used to select PB observed on the admission chest X-ray and the patient was admitted monocytes. These cells were further divided on classical (M1) to the internal medicine service. Additional testing revealed normal monocytes (CD14+ CD16-), intermediate (M2) monocytes (CD14+ serum IgE levels, negative Epstein Barr Virus and Strongyloides CD16 + d) and non-classical (M3) monocytes (CD14- CD16+) stercoralis antibodies. An anterior mediastinal mass concerning for using Infinicyt v2.0 (Santa Marta, SA). Statistical analysis was lymphoma disease was seen on the thorax- abdominal computed performed using GraphPad Prism software (ROC curve and Mann- axial tomography (CAT) scan. Fine-needle aspiration (FNA) with Whitney test). cytology showed increased eosinophilia, small lymphocytes and atypical Reed-Sternberg giant cells that were PAX5+, CD20+ (rare Results cells), CD30+, CD15+, p53+, bcl2+, ALK- and EBER- phenotype. Bone narrow aspiration revealed high levels of eosinophilia without CMML patients showed increased M1 population with decreasing lymphoproliferative infiltration. Molecular genetic analysis showed numbers of M2 and/or M3 monocytes (p b .001). The percentage of polyclonal rearrangement of IgH and TCRG genes and negative M1 in the monocytic compartment was the most informative expression of the FIP1L1/PDGFRA transcript. He was thus diagnosed parameter to classify CMML patients vs reactive monocytosis (AUC with classical Hodgkin lymphoma. = 0.99) with a cutoff of ε94% M1 monocytes (Specificity 97%, Sensitivity 95%). All other variables, including hemoglobin (AUC = Conclusions 0.75), platelets (AUC = 0.81), absolute monocytes (AUC = 0.84) or absolute M1 number (AUC = 0.83) had lower discriminative Hodgkin lymphoma and B cell non-Hodgkin lymphoma can be potential. associated with eosinophilia. The clinician should consider many possible causes of eosinophilia. The degree of eosinophilia is rarely Conclusions helpful for identifying the cause except at extremes of eosinophil counts (e.g., very mild eosinophilia is more likely to be seen with CMML is a rather heterogeneous condition. A flow cytometry- asthma or allergic rhinitis; severe eosinophilia [e.g., ε20,000 based analysis can be used to perform differential diagnosis of CMML eosinophils/μL] is more likely to be caused by a myeloid neoplasm). and reactive conditions.

doi:10.1016/j.cca.2019.03.901 doi:10.1016/j.cca.2019.03.902 Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

b ^W128 Section of Clinical Biochemistry, Department of Biomedicine and Movement Neurosciences, University of Verona, Italy Evaluation of health outcomes after the implementation of rotational thromboelastometry in patients undergoing cardiac Background-aim surgery The infusion of stem cells collected from peripheral blood by I. Rodríguez Martín, C. Sánchez Mora, V. Sánchez Margalet apheresis has considerably increased during the last decade, often Clinical Biochemistry Department, Virgen Macarena University Hospital, replacing the use of bone marrow as a cellular source for Seville, Spain reestablishment of hematopoietic activity in patients undergoing autologous and allogeneic transplantation. This study describes the Background-aim results of comparing HPC (Hematopoietic Progenitor Cell) Sysmex XN technique (Sysmex, Kobe, Japan) for stem cell count in peripheral Viscoelastic tests (rotational thromboelastometry, ROTEM®), blood transplantation (PBSCT) samples with the reference method together with the implementation of a specific algorithm for based on CD34+ monoclonal antibodies cytofluorimetry. coagulation management in cardiac surgery, enable perioperative coagulopathy to be better controlled. Methods

Methods HPC count is based on cytofluorimetric analysis of fluorochrome labeled cells after selective cell lysis according to their relative Retrospective cohort study including 675 patients who under- maturity. HPC has been compared with CD34+ count (BD Stem Cell went cardiac surgery with cardiopulmonary bypass. The incidence of kit Enumeration on flow cytometer BD FACSCanto, ISHAGE protocol) allogeneic blood transfusions and clinical postoperative complica- in 125 apheresis samples collected from patients undergoing tions were analyzed before and after ROTEM® implementation. autologous or allogeneic bone marrow transplantation. The compar- ison was carried out with Passing and Bablok regression and Bland- Results Altman plots, using Analyze-it software (Ltd, Leeds, UK).

Following viscoelastic testing and the implementation of a specific Results algorithm for coagulation management, the incidence of any allogeneic blood transfusion decreased (41.4% vs 31.9%, p = .026) during the HPC values (median 2110 × 10^9/L, range 830-7362 × 10^9/L) perioperative period. In the group monitored with ROTEM®, decreased have a similar distribution as assessed with CD34+ cytofluorimetry incidence of transfusion was observed for packed red blood cells (31.3% (median 2156 × 10^9/L, range 932-8666 × 10^9/L). The Passing and vs 19.8%, p = .002), fresh frozen plasma (9.8% vs 3.8%, p = .008), Bablok regression analysis revealed good comparability, with r = prothrombin complex concentrate administration (0.9% vs 0.3%, p = 0.882, slope 0.99 (CI 95%, 0.89–1.09), intercept −93.5 (95% CI, .599) and activated recombinant factor VII (0.3% vs 0.0%, p = .603). −332.2 to 120.3) and p = .656. The Bland-Altman plots demon- Increased incidence was observed for platelet transfusion (4.8% vs 6.8%, p strated a mean bias of −48.7 × 10^9/L (95% CI, −159.3 to 61.7 × = 0,530) and fibrinogen concentrate (0.9% vs 3.5%, p = .066), tran- 10^9/L) for HPC compared to CD34+ cytofluorimetry. examic acid (0.0% vs 0.6%, p = .370) and protamine administration (0.6% vs 0.9%, p = .908). Similar results were observed in the postoperative Conclusions period, but with a decreased incidence of platelet transfusion (4.8% vs 3.8%, p = .813). In addition, statistically significant reductions were These results suggest that Sysmex XN HPC may be considered a detected in the incidence of postoperative bleeding (9.5% vs 5.3%, p = reliable system for stem cell enumeration during apheresis. Owing to .037), surgical reexploration (6.0% vs 2.9%, p = .035), and length of its rapidity, ease of use and possibility to simultaneously performing Intensive Care Unit (ICU) stay (6.0 days vs 5.3 days, p = .026). complete blood counts, this method can be a reliable surrogate of cytofluorimetry for both CD34+ cell collection and for investigating Conclusions their kinetics, also enabling a much greater degree of standardization. The monitoring of haemostasis by ROTEM® in cardiac surgery, was associated with decreased incidence of allogeneic blood transfusion, doi:10.1016/j.cca.2019.03.904 clinical hematologic postoperative complications and lengths of ICU stay. doi:10.1016/j.cca.2019.03.903 ^W130

Cell blood counts jointly with cell population data are suitable to point out patients with sepsis and septic shock ^W129

Comparison of automated Hematopoietic Progenitor Cell (HPC) F. Spolaore count between Sysmex XN and the CD34+ ISHAGE in apheresis Department of Laboratory Medicine, University-Hospital of Padova, Italy samples Background-aim G.L. Salvagnob, G. Lippib, E. Barisona, M. Midoloa, F. Benedettia,F. Dimab Cell size and complexity, fluorescence intensity and width of aHematology Section, Department of Medicine, University of Verona, dispersion of those events are provided by the leukocyte population Italy data (CPD) that represent novel parameters determined by some Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

hematological analyzers, obtained with the cell blood and leukocyte titer performed by the same technique, can detect an increased differential counts (CBC). production of maternal antibody during pregnancy. This study evaluates a new automated alloantibody titration Methods system, IH-500 System (BioRad) by comparison with the manual standard technique in column gel card, assessing the accuracy and WeevaluatedforCBCandCPDan overall of 331 patients, (158 M, concordance with this established manual reference method. 173F, 18-85ys) 110 presenting different sickness without infections (A), 53 with infections (B), 85 with sepsis (C), 32 with septic shock (D) and Methods 51 treated with granulokines, due to hematological diseases (E). CBC and CPD were assessed by the Sysmex XN1000 (Kobe, Japan software 0015). Prospective study in which a total of 43 samples of patients, from the Blood and Tissue Bank, were processed, which contained some Results irregular antibody for which the prior identification and manual titration were carried out. They were then processed by a fully Logistic regression and post-hoc analysis showed that, among all automated random access system for ABO blood grouping, reverse patients, in those with sepsis and septic shock the following parameters testing, phenotype, Rh-subgroups, single antigen testing, direct AHG were statically different: leukocytes count (P = .0197), hemoglobin testing (DAT), antibody screening, antibody identification and concentration (P = .0001), platelets count (P = .002), immature plate- antibody titration. let fraction (IPF) (P ≤.0001), neutrophils count (P = .094), monocytes Each sample was tested a total of 3 times, on different days, by count (P = .0031), monocytes percentage (P = .0186), immature each method for precision as an indicator of accuracy. Samples were granulocytes (IG) (P = .0186) and neutrophils fluorescence intensity stored frozen between test days. (NE-SFL channel) (P = .0406). For the statistical analysis, the assessment of reproducibility for The median values obtained for each patient's group were: variables with more than two categories was used with the weighted Leukocytes (109/L) A:8.45, B:10.06, C:10.5, D:12.96, E:8.96; Hemoglo- kappa index. bin (g/L) A:110, B:105, C:95, D:105, E: 108; Platelets (109/L): A:255; B:272, C:180, D:164, E:147; IPF (%): A:3.9, B:3.65, C:5.9, D:8.5, E:4.3; IG Results (%): A:1.5, B:1.5, C:2, D:3, E:2.75; Neutrophils (109/L): A:5.52, B:6.44, C:7.76, D:9.62, E:4.41; Monocytes (109/L): A:0.84, B:0.9, C:0.79, The precision of repeat titer results performed in the automated D:0.82, E:1.04; Monocytes (%): A:9.2, B:9.2, C:8.25, D:7.15, E:11.65; system was slightly more consistent (p = .0095) when compared to NE-SFL: A:50.95, B:52, C:53.80, D:51.65, E:52.5. By stratifying patients manual standard titration. There was no difference in titer in 26 of 43 by age, in those N75 years there's predictability only for platelets (P = samples (60,5%) when testing was performed by automated and .0081), IG (P = .0124) and neutrophils complexity (NE-SSC) (P = manual method, while 17 of 43 samples (39,5%) showed a difference .0116). In comparison to patients with infections, those with sepsis in titer of 1 dilution when repeat testing was performed using showed higher values for the width of the neutrophils size (NE-WY), manual standard titration. The assessment of agreement for variables 730 vs 679 (P ≤.001), the width of the monocyte complexity (MO- with more than two categories that uses the weighted kappa index, WX):291 vs 256 (P = .018) and lymphocytes fluorescence (LY-Y) 74.8 showed values of 0.9304 (0.8979 to 0.9629) (p = .0000). vs 72.9 (P = .01). In comparison to patients with sepsis, those with septic shock showed also a significant different values of the MO-WX: Conclusions 282 vs 291 (P = .013). Although the titers in the automated system could be slightly Conclusions higher than manual standard method, performing antibody titration using this automated technology presents acceptable values in terms CPD parameters provide information about cell functions and of precision and agreement with the manual reference method. jointly with hematological parameters can be suitable to improve clinical outcomes in patients with sepsis and septic shock, on the doi:10.1016/j.cca.2019.03.906 perspective of personalized medicine.

doi:10.1016/j.cca.2019.03.905 ^W132

Markers of hypochromia in the study of erythropoiesis and iron fl ^W131 availability in patients with In ammatory Bowell Disease

Evaluation of an automated alloantibody titration system E. Urrechagaa, M. Merinoa, P. De La Herab, A. Francisco Javierb aCore Laboratory, Hospital Galdakao Usansolo, Galdakao, Vizcaya, Spain X. Tejedor Ganduxéb, A. Martínez Iribarrenb, C. Morales Indianob,À. bCore Laboratory, Hospital Universitario Basurto, Bilbao, Vizcaya, Spain Sala Sanjaumeb, A. Dueñas Márquezb, T. Lema Puñalb, E. Alonso Noguésa, J.R. Grífols Rondaa, M. Llopis Díazb Background-aim aBanc de Sang i Teixits, Spain bLaboratori Clínic Metropolitana Nord-Hospital Universitari Germans Anemia due to lack of iron, absolute or functional, is a common Trias i Pujol, Institut Català de la Salut, Spain complication of Inflammatory Bowell Disease (IBD). The assessment of iron supply and erythropoiesis in patients with inflammatory Background-aim diseases, such as IBD using common biochemical assays is insuffi- cient as these are affected by the inflammatory response. The titration of an alloantibody to a red cell antigen is a useful We investigated the potential utility of reticulocyte hemoglobin semi-quantitative screening tool that, when compared to a previous content (RetHe) and percentage of hypochromic cells (HypoHe) in Abstracts / Clinica Chimica Acta 493 (2019) S379–S433 the assessment of the erythropoiesis status in terms of iron Methods availability in patients with inflammatory bowel disease (IBD). Three hundred and thirty samples collected in K2EDTA anti- Methods coagulant were run sequentially on both Sysmex XN-20 and Mindray BC 8600+ counters. The scope of the pathology included a variety of We recruited 100 consecutive outpatients with IBD non receiving diseases representative of the daily workload: 80 healthy subjects, 84 therapy. C reactive protein (CRP), Serum iron, transferrin saturation, iron deficiency anemia, 87 anemia of chronic disease, 79 thalassemia ferritin and soluble transferrin receptor (sTfR) were tested. Complete carriers. blood counts were performed on a XN9000 system (Sysmex C reactive protein, Serum iron, transferrin saturation, ferritin and Diagnostics). CRP, S-Iron, Transferrin, ferritin and sTfR were assayed soluble transferrin receptor (sTfR) were assayed in a chemical in a chemical analyzer Cobas c 502 (Roche Diagnostics). analyzer Cobas c 502 (Roche Diagnostics). Differences among groups were studied using analysis of Kolmogorov-Smirnoff was used to verify normal. Spearman's rank variance, considering P b .05 to be significant. For post hoc compar- correlation coefficient was used. Receiver operating characteristic isons of outcomes between each pair of groups Scheffé correction analysis was used to assess the diagnostic performance of CHr, and % was applied. Receiver operating characteristic analysis was used to Hypo for detecting iron deficient erythropoiesis. Gold standard for assess the diagnostic performance of RetHe and HypoHe for low iron availability was sTfR N52 nmol/L. detecting iron deficient erythropoiesis. Gold standard for low iron availability was sTfR N52 nmol/L. Results

Results Sweked distribution was proven for the four parameters. – – – Whole range RetHe 20.6 42.5 pg, CHr 25.0 46.1 pg^; HypoHe 0.1 fi – Seventy one patients were anemic; 37 had iron de ciency anemia 14^% %Hypo 0.1 30^%. (IDA), 17 anemia of chronic disease (ACD), 17 had mixed ACD + IDA. Median and 25-75th quartiles in healthy subjects CHr 33.3 pg, – – RetHe and HypoHe had lower values in the anemic patient (all 32.0 34.5 pg; %Hypo 0.1^% 0.1 0.3%. b – – groups) when compared to the non-anemic group (P .0001), RetHe 30.0 pg 29.3 32.3 pg HypoHe 0.2^% 0.1 0.6%. whereas no differences were found in RetHe among the anemic Linear correlation Ret-He and CHr y = 1.054×-1.86. (95%CI -5.2- groups, irrespectively to inflammation status. 1.7 slope; 0.95–1.1 intercept). RetHe IDA/Mixed P = .032 Mixed/ACD P = .038 IDA/ACD P = Correlation between HypoHe and %HYPO can be described by a .040 2nd degree polynomial equation y = 0.0082 × 2 + 0.765× + 0.446. HypoHe IDA/Mixed P = .048 Mixed/ACD P = .068 IDA/ACD P = CHr Area under curve (AUC) 0.934 (95% CI 0.892–0.963 cut off .041 b29.0 pg, sensitivity 82.9%, specificity 98.6%. %Hypo AUC 0.918 (95% – – N fi RetHe Area under curve was (AUC) 0.858 (95% CI 0.816 0.952), CI 0.872 0.951 cut off 0.6^%, sensitivity was 76.4%, speci city 97.3%. considering a cut off 30.0 pg, sensitivity 76.8%, specificity 99.8%. – HypoHe AUC 0.737 (95% CI 0.634 0.824), considering a cut off 4.4^ Conclusions %, sensitivity 72.0%, specificity 72.5%. RetHe and CHr are directly comparable. Hypo-He and %HYPO Conclusions fi are not exchangeable, due the different de nition of hypochromia^, b in terms of Hemoglobin (Hb) content ^17 pg (Sysmex) or Hb RetHe and HypoHe are reliable tests to assess iron supply for concentration b 280 g/L (Mindray). CHr and %Hypo are reliable erythropoiesis in patients with IBD, useful to evaluate long term markers to identify iron deficient erythropoiesis. (HypoHe) and short term (RetHe) periods. doi:10.1016/j.cca.2019.03.908 doi:10.1016/j.cca.2019.03.907

^W134 ^W133 Method comparison of the new Sebia serum free light chain Clinical utility of reticulocyte hemoglobin and hypochromic assay, against the Binding Site Freelite assay erythrocytes reported by Mindray BC8600^+^counter in the study of erythropoiesis M.A. Blaylock North Cumbria University Hospitals NHS Trust, United Kingdom E. Urrechaga, S. Bilbao, M. Merino Hospital Galdakao Usansolo, Spain Background-aim

Background-aim The clinical utility of serum free light chain (sFLC) measurement is well recognized for both the diagnosis and monitoring of Erythrocyte and reticulocyte indices, reticulocyte hemoglobin monoclonal gammopathies. The limitations of current nephelometric content, and percentage of hypochromic red cells have been and turbidimetric assays are well reported: This has led to the extensively used in diagnosing different erythropoiesis conditions. development of a novel enzyme linked immunosorbent assay Mindray BC 8600+ counter reports both parameters CHr and %Hypo; (ELISA) for sFLC measurement. The new quantitative assay designed we study the utility of these indices for the diagnosis of iron deficient by Sebia was compared to the The Binding Site's Freelite assay, using erythropoiesis and compare with similar parameters, RetHe and routine patient serum samples from a busy UK clinical immunology HypoHe, reported by Sysmex analyzers. laboratory. Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

Methods respect to their particular impact on diagnostic and therapeutic decisions and associated with biological properties of plasma cells. Method comparison was performed on 458 patient serum samples; 107 ‘normal’ patients (23%) and 351 ‘abnormal’ 77%. Serum Results free kappa and lambda measurements were compared, as well as the kappa/lambda ratio. Statistical analysis was used to determine The median proportion of BMPC was median 30% (quartiles: 15– overall percentage agreement (OPA) and statistical significance. 70%) in biopsy specimens, 7% (quartiles: 2–16%) on aspirate smears, Concordance with serum protein electrophoresis (SPE) was deter- and 3% (quartiles: 1–10%) by FACS analysis. At the limit of 10% BMPC, mined by individual review. The proportion of samples which which is critical to discriminate plasma cell myeloma from MGUS, 74% required further testing by either method was compared. of cases would have been missed for diagnosis if classified by smear morphology only. The difference of plasma cell numbers between core Results biopsy and aspirate smear counts was significantly (all p b .001) associated with a clonal BMPC phenotype, aberrant expression of Good OPA between the two assays was seen; Kappa 82.75%, Lambda CD56 and bone marrow fibrosis. The expression of CD56 was also 72.93% and Ratio 86.24%. The SebiaELISAshowedanegativebias associated with a significantly lower yield of BMPC by FACS analysis. compared to the Freelite assays, which was most profound at higher sFLC concentrations. Analysis showed statistically significant differences Conclusions between absolute values for both Kappa and Lambda serum concentra- tions, when comparing the two methods (p ≤.0001). Good agreement Histopathologic enumeration of BMPC is mandatory to correctly between the Sebia ELISA results and SPE was seen on individual sample classify plasma cell dyscrasias. The expression of the adhesion review. Two specific examples showed reduced overestimation of the molecule CD56 is associated with aspiration failure in first pull as sFLC concentration by the Sebia ELISA, compared to the Freelite assay. well as second pull aspirates. 23.31% of samples required additional testing (re-test %) by the Freelite assay, compared to 6.54% by the Sebia ELISA method. doi:10.1016/j.cca.2019.03.910

Conclusions

W136 This study provides evidence that the overall aims for an improved ^ sFLC assay outlined by Sebia were met. A good overall concordance Two-site evaluation of high-fluorescent cells for the detection of between the two sFLC assays was seen, however a negative bias in sFLC malignant cells: The importance of clinical information concentration was observed when comparing the Sebia assay to the FreeLite assay. The phenomenon of sFLC overestimation was reduced J. Favresseb, L. Bolanda, M. Schellena, B. Chatelainb, J. Defoura,F. using the Sebia assay compared to the Freelite assay. Mullierb, H. Jacqminb a doi:10.1016/j.cca.2019.03.909 St-Luc University Hospital and Catholic University of Louvain, Depart- ment of Laboratory Medicine, Haematology Laboratory, Brussels, Belgium bUniversité catholique de Louvain, CHU UCL Namur, Namur Thrombosis ^W135 and Haemostasis Center, Haematology Laboratory, Yvoir, Belgium

Choice of proper approach for the assessment of plasma cells in Background-aim the bone marrow of patients with monoclonal gammapathies The presence of high fluorescent cells (HF-BF) on the XN-1000 S. Demyanets, A. Kaider, R. Thalhammer, G. Bayer, M. Krauth, H. Agis, analyzer (Sysmex Corporation) has gained interest in literature to I. Schwarzinger predict the presence of malignant/atypical cells in body fluids (BF) Medical University of Vienna, Austria but is suffering from a lack of sensitivity. In this study, we aimed to evaluate the performances of the Background-aim optimal HF-BF cut-off for the detection of malignant cells along with the integration of clinical information. Monoclonal gammapathies are categorized into monoclonal gammopathy of undetermined significance (MGUS), smoldering Methods multiple myeloma (SMM) or multiple myeloma (MM) based on the number of monoclonal plasma cells in the bone marrow (BM). One thousand six-hundred and eighty-six serous fluids were Quantification and characterization of BM plasma cells (BMPC) guides analyzed on the Sysmex XN body fluid mode in two haematology therapeutic decisions at diagnosis and has gained increasing impor- laboratories. All samples were microscopically screened on cytospin tance for assessment of minimal residual disease (MRD). Methods to slides for the presence of malignant cells. First, optimal HF-BF cut- enumerate BMPC basically include core biopsy examination, BM offs to predict the presence of malignant cells in serous fluids were aspirate smear examination as well as flow cytometry (FACS). determined based on ROC curve analysis. Thereafter, the added value of clinical information (history and/or high suspicion of a neoplastic Methods disorder) on the performance characteristics was evaluated.

We retrospectively compared the results of plasma cell enumer- Results ation performed in parallel by core biopsy count, BM aspirate smear count and FACS analysis in 238 patients with MM, SMM or MGUS. The optimal HF-BF cut-off in the first haematology center was 108 Discrepancies of results between methods were evaluated with cells/μL and yielded a sensitivity/specificity of 61.1%/93.4%. The Abstracts / Clinica Chimica Acta 493 (2019) S379–S433 optimal HF-BF cut off in the second haematology center was 45 cells/μL We analyzed a total of eleven unrelated patients belonging to six and yielded a sensitivity/specificity of 86.8%/66.6%. The sensitivity/ different families characterized by bleeding history and laboratory specificity for malignant cells detection evolved to 100.0%/68.9% in the coagulation findings suspecting vWD (aPTT, RIPA, PFA, RiCoF, vWF: first center when adding clinical information to the local HF-BF cut-off Ag, FVIII:C). criteria. In the second center, adding clinical information also improved the sensitivity to 100%. The specificity was however not Results determined. These data highlights that smear review for detecting malignant cells is only useful when HF-BF N cut-off and/or when there After multimer analysis, we identified eight type1 vWD patients is clinical suspicion for malignancy. If neither of these two criteria is (multimers equally reduced) and two patients with significant HMW fulfilled, manual review for malignant cell detection seems not useful. reduction (one type 2A and one type 2B). vWF gene contains 52 exons, For our data, this would have led to 55.0% reduction in microscopic BF and 2A and 2B mutations invariably cluster in the large exon 28. Then, review. More importantly, this would also have improved turnaround we firstly sequenced the full-length exon 28 of those type 2 vWD time for the BF's that wouldn't need manual review. patients and next that of their first degree relatives. Sequence analyses revealed two different missense mutations: V553 M (V1316 M; nt3946 Conclusions G N A) and L817P (L1580P; nt4739 T N C) respectively in A1 and A2 domain causing type 2B and 2A phenotype respectively. Finally, We propose an algorithm for BF review based on HF-BF results repeated External Quality Assessment Surveys (UK-NEQAS codes: 18/ and clinical information. This algorithm could avoid useless slide 53; 18/26; 17/53) perfectly matched with the reference results. review and would allow to efficiently report automated parameters from the XN-1000 BF mode. Prospective evaluation of this algorithm Conclusions is needed to confirm our results. We validated this method by genotype-phenotype correlation doi:10.1016/j.cca.2019.03.911 and UK-NEQAS evaluations, concluding that it is a robust diagnostic procedure improving vWD typing in the laboratory and clinical practise, respectively helping technicians and clinicians in the complex vWD approach. ^W137 doi:10.1016/j.cca.2019.03.912 Genotype-phenotype correlation in von Willebrand disease by automated von Willebrand multimer analyzer [Hydrasys 2] and UK-NEQAS validation ^W138 D. Gemmatia, G. Longoa, T. Lupianoa, V. Tisatob, M.L. Serinoa,B. Bonaric, S. Longoc, L. Ghisoc, C. Balbonic, M. Etroc, P. Pellegattic,E. Extending the storage time of Dithiothreitol-treated red blood Montanaric cells used to eliminate daratumumab interference in alloantibody aCentre Haemostasis & Thrombosis, Section of Medical Biochemistry, testing Molecular Biology & Genetics, University of Ferrara, Italy b Department of Morphology, Surgery and Experimental Medicine and E. Jaime-Lara, M.J. García Bueno, R. Barquero Jimenez, N. González LTTA Centre, University of Ferrara, Ferrara, Italy López, A. Redruello Alonso, M.L. Casas Losada c Laboratories of Clinical Chemistry & Microbiology, Sant'Anna Univer- Hospital Universitario Fundación de Alcorcón, Madrid, Spain sity Hospital, Ferrara, Italy Background-aim Background-aim Daratumumab (DARA) interferes with pretransfusion tests like von Willebrand disease (vWD) is the most common inherited alloantibody screening by binding to CD38 on red blood cells (RBCs). bleeding disorder caused by mutations in the vW-Factor (vWF) gene Treat RBC with Dithiothreitol (DTT) is a practical way to solve this (locus, 12p13.31). vWF is a large multimeric glycoprotein produced problem but it is a time-consuming procedure. An important by megakaryocytes and endothelial cells. It binds activated platelets limitation is the hemolysis observed during storage. Instead, DTT to the damaged endothelium and is the circulating carrier of treated RBC must be prepared and immediately used, increasing our coagulation FVIII. Multimers vary in size from the less active low- turnaround times. The objective of this study is to extend the molecular-weight (LMW) to the more active high-molecular-weight stability of DTT-treated RBCs to 15 days using a RBC preservative (HMW). vWD classification is based on reduction, dysfunction, or solution (Alsever's solution (AS)). RBCs could be treated at once, absence of vWF (type 1, 2 and 3 respectively). Types 1 and 3 are stored, and used later with reliable results, decreasing our turn- quantitative defects while type 2 (2A, 2B, 2 M, 2 N) qualitatively around times and improving laboratory efficiency. affects vWF. Multimer analysis, essential for vWD classification, is currently long and laborious. Methods

Methods Antibody-screening RBCs were treated with DTT at a ratio of 4:1 and incubated at 37 °C for 30 min. Then, RBCs were washed 4 times A new IVD-method (Hydragel von Willebrand multimers; SEBIA, (3 times with normal saline and a final wash with AS). Finally, RBCs France), performed by electrophoresis Hydrasys_2 technology, includes were resuspended to 5% in AS. ready to use buffers, agarose gels, and antibodies. It allows the evaluation On day 1, we tested the efficacy of the treatment by testing and quantification of vWF multimeric pattern after immune-fixation against anti-K. Also, the treated- RBCs were tested against plasma of directly on the gel including integrated densitometry and scan. DARA-treated patients to ensure the interference was eliminated. Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

On day 5, 10 and 16 we tested the ability of the treated RBC to detect clinical samples selected from the routine workload of the Depart- alloantibodies using Blood Bank Controls (containing anti -c, -Fya, ments of Clinical Chemistry at Atalmedial Diagnostic Center, Gelre −D). A control of untreated RBCs was performed for parallel testing. Hospital and Certe (The Netherlands), and included patients with a Also, we evaluated the degree of hemolysis of treated and untreated wide variety of diseases and conditions. RBCs measuring Hemolysis Index (HI) with Advia Chemistry XPT. A RBC phenotype was performed on day 1 and 16, to assess the Results surface antigen integrity during the study. RBC, HGB, MCV and % reticulocyte results from the two analyzers Results showed a high level of correlation and agreement (r = 0.97 to 1.00, slope = 0.98 to 1.09). The efficacy of DTT treatment was evidenced by denaturation of Good correlation (r = 0.71) and a small negative bias was the K antigen. Also, the panreactivity previously detected disap- observed between Alinity hq MCHr and CELL-DYN Sapphire MCHr, peared after DTT treatment, proving the interference was cleared. with a Passing-Bablok slope of 0.91 and intercept of 1.8. The No decrease in the reaction strength was observed during the predicted bias at the 29 pg medical decision level was −0.8 pg study (3+). Also, the strength was equal compared to untreated (−2.9%) and at 26 and 32 pg, respectively, the predicted bias was RBCs. The phenotype remains unaltered. −0.6 and − 1.1 pg (−2.2% and − 3.5%). A significant negative The hemolysis was higher in treated cells (mean HI was 65 correlation of MCHr was demonstrated with %hypochromic RBC (treated RBC) vs 20 (untreated RBCs) on day 16). However, did not and %microcytic RBC (p b .0001 for both), especially on samples with b b reach a critical cut off value ( 235). MCHr ^29 pg (r = 0.71 and 0.69).

Conclusions Conclusions

AS preserves the integrity of the DTT-treated RBCs for at least 15 MCHr values, as well as RBC and reticulocyte parameter results days. By having DTT-treated RBCs prepared in advance we will that were obtained by Alinity hq showed close correlation and reduce alloantibody detection time by 45 min. agreement with those from CELL-DYN Sapphire. In this study, the MCHr value of 28.2 pg on the Alinity hq analyzer was equivalent to doi:10.1016/j.cca.2019.03.913 the MCHr value of 29 pg measured by CELL-DYN Sapphire.

doi:10.1016/j.cca.2019.03.914

^W139

Abbott Alinity hq reticulocyte hemoglobin cutoff for diagnosing ^W140 functional iron deficiency in chronic kidney disease Three novel fibrinogen mutations (Fibrinogen Innsbruck II, III Z. Mukhtara, B.A. Weversb, C.L. Slimc, M.W.H.J. Demmersd, H.J. and IV) causing hypodysfibrinogenemia Adriaansend, J.A. Koorenb, H. Stormc, H.J. Hoffmanna, G. Lakosa aAbbott Diagnostics, Santa Clara, CA, United States L. Loacker, S. Schmidt, A. Griesmacher bDepartment of Clinical Chemistry, Atalmedial Medical Diagnostic University Hospital Innsbruck, Austria Centers, Hoofddorp, The Netherlands cDepartment of Clinical Chemistry, Certe, Groningen, The Netherlands Background-aim dDepartment of Clinical Chemistry, Gelre Hospital, Apeldoorn, The Netherlands Congenital dysfibrinogenemia is an infrequent hereditary disor- der affecting either the quantity (afibrinogenemia, hypo- Background-aim fibrinogenemia) or the quality (dysfibrinogenemia) of circulating fibrinogen. On a genetic basis this disorder is caused by heterozy- Assessment of iron status is key for management of anemia in gous, homozygous or compound heterozygous mutations in the chronic kidney disease (CKD). Adequate iron status in CKD has been fibrinogen A alpha (FGA), B beta (FGB) or G gamma (FGG) genes. We defined as either b6% hypochromic red blood cells or a reticulocyte describe here three patients carrying novel fibrinogen mutations hemoglobin content of b29 pg (or equivalent). In addition to CKD, recently diagnosed at the University Hospital Innsbruck, Austria. this parameter has been shown to be clinically relevant in the diagnosis of iron deficiency and functional iron deficiency in other Methods chronic diseases, such as inflammatory bowel disease. Depending on the technology and platform, the hemoglobin content of reticulo- Three patients (a 27 year old female patient, 76 and 31 year old cytes has been referred to by different names. Examples include CHr male patients) were examined because of bleeding tendency. (Siemens), Ret-He (Sysmex) and MCHr (Abbott). Due to the different Routinely performed coagulation tests revealed in all patients methods that are used to determine this measurand, studies show a significantly decreased fibrinogen plasma levels (108, 104, and 113 lack of interchangeability between values. mg/dl respectively) as well as prolonged thrombin time results (24, 23 and 25 s respectively). Consequently we performed DNA Methods sequencing of the complete FGA, FGB and FGG genes. In each patient one presently unknown mutation was discovered: a heterozygous MCHr results obtained by the Alinity hq haematology analyzer missense mutation p.Met257Arg (c.770 T N G) in FGA Exon 5 (patient (Abbott Diagnostics, Santa Clara, CA), together with RBC and 1), a heterozygous nonsense mutation p.Arg445X (c.1333A N T) in reticulocyte parameters, were compared to those generated with FGB Exon 8 (patient 2) and a heterozygous splice site mutation the CELL-DYN Sapphire (Abbott). The study cohort consisted of 1461 (c.851 + 3A N G) in FGG (patient 3). Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

Results laboratory for genotyping. The analysis was performed by the direct sequencing of the DNA extracted from peripheral blood leukocytes. All three detected variants were analyzed by multiple bioinformatical The specific regions analyzed were the nucleotide sequence −110 of conformation analysis methods (MutationTaster, Polyphen, SIFT/ the 5’ UTR to IVS II-90 of the ® gene, the sequence −100 to +20 3’ PROVEAN, Ensembl Variant Effect Predictor, HumanSplicingFinder or UTR of the 〈2 gene and the sequence −140 to +20 3’ UTR of the 〈1 NNSPLICE) and predicted to be diseasecausing.Previously published gene. The diagnostic molecular investigation confirmed the presence fi N N brinogen variants carrying mutations in similar DNA regions (e.g. FGA of the c.20 A T mutation of the ® gene (HbS) and of the c.344^C T Trp248X, FGB Arg445Thr or FGG c.851 +1GN A) are also associated with mutation of the 〈2 gene (Hb Nouakchott). No defects were found in haemorraghes. the 〈1 gene.

Conclusions Conclusions

We describe here three novel fibrinogen mutations causing The high-resolution of the CE and the clarity of the electropho- hypodysfibrinogenemia and bleeding tendency. Performing gene retic profile enabled the identifi cation of the double heterozygous sequencing after several screening coagulation tests is a suitable HbS/Hb Nouakchott defect, visible only in the CE, which was algorithm to establish the definite diagnosis of dysfibrinogenemia. fundamental for the correct diagnosis of the patient. doi:10.1016/j.cca.2019.03.915 doi:10.1016/j.cca.2019.03.916

W142 ^W141 ^

HbS/Hb Nouakchott: Double heterozygosity, observed for the first Higher percentage of dimeric form of FLC kappa type is connected time in Italy, visible only in capillary electrophoresis with worse renal function in patients with multiple myeloma

G. Antonellob, M.T. Belgerib, P. Ferrarab, R. Fontanab, D. Gasparetb,C. J. Tisonczyk, P. Dumnicka, R. Drozdz Lo Monacob, C. Molinob, P. Napolib, M. Nigraa, C. Panerob, L. Pezzatib, Department of Medical Diagnostics, Faculty of Pharmacy, Jagiellonian S. Rossettib, M. Scavuzzob, A. Sfrisob, D. Solimandob University Medical College, Krakow, Poland aASL Citta’ di Torino Presidio Ospedaliero Martini, Direttore SSD Laboratorio Analisi, Spain Background-aim bASL Citta’ di Torino Presidio Ospedaliero Martini, Laboratorio Analisi, Spain Renal failure complicates a significant number of monoclonal gammapathies (MG) cases. Many data indicates that the free light Background-aim chains themselves are nephrotoxic with significant differences between individual clones. Prediction of nephrotoxicity of individual A woman, age 64, of Moroccan origin came under our observation clone of FLC may be of great benefit in selecting the most appropriate for a glycated hemoglobin test. The sample showed an “Atypical” treatment regimen for each patient. The confirmation of nephrotox- profile due to the presence of anomalous fractions corresponding to icity of the FLC clone to date can only be done by a kidney biopsy. a variant, so additional tests were performed to identify the The procedure of renal biopsy is invasive and connected with the hemoglobin variant. possibility of side effects. There is still need for more simple parameters that could help to predict the FLCs impact on renal Methods function. The study on FLC dimer/monomer (D/M) pattern has revealed the significance of dimerization process of FLC. But the The test was performed using the Tosoh HLC-723G8 HPLC question whether the D/M pattern of FLC may determine the severity analyzer in the “HbA1c Variant analysis mode” as well as using the of MG disorder still remains open. In our previous study we Capillarys 2 Flex Piercing (Sebia) capillary electrophoresis (CE) demonstrated that the D/M pattern of FLC is an intrinsic character- instrument with the Capillarys HbA1c Kit. The HPLC measured 68 istic of individual clone of FLC. The aim of this study was to mmol/mol of HbA1c, with the detection of a 33% hemoglobin variant, determine if renal impairment may be correlated with D/M pattern identified as the H-V1 fraction with a retention time of 1.17 min. of individual FLC clone. Additional tests were performed including a full blood count, a blood^ −iron assessment and a hemoglobin analysis with dedicated Methods programmes (“®-thalassaemia Analysis mode” for the G8 and Capillarys Hemoglobin(e) using the Capillarys 2 Flex Piercing (CAP The SDS electrophoresis of urine of 178 patients with diagnosed 2FP) instrument). multiple myeloma and the presence of Bence-Jones proteinuria was performed. The dimer/monomer pattern of FLC and the type of Results proteinuria (glomerular proteinuria: selective/non-selective or/and tubular proteinuria) were defined. The relationship between per- The patient's haematochemical parameters showed no significant centage of FLC dimeric form and the condition of kidney was alterations. Yet, the chromatogram confirmed the presence of an determined. additional 33.1% fraction identified as S+, which was presumably HbS in heterozygosis. Instead, the electropherogram showed the Results presence of 3 additional fractions, which confirmed the initial hypothesis of the presence of an variant (7.3%), a variant (32.4%) Among all patients taken together, the presence of glomerular and an 2* 2* hybrid (4.7%). The sample was sent to a genetics proteinuria was significantly associated with higher percentages of Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

FLC's dimer [median 66 (39–100)% versus 52 (9–94)%; p = .023]. But 0.77–0.82, p = .0001) and HF-BF# 0.83 (95%CI, 0.81–0.85, p = when we divided the group according to the FLCs form (kappa or .0001). The cut-off points chosen based on the best sensitivity were lambda) it turned out that the difference was highly significant TC-BF# ε245/ L and HF-BF# ε17/ L. The combination of TC-BF# ε245/ among patients with FLC kappa [median 47 (27–61)% versus 24 (0– L and HF-BF# ε17/ L presented a sensitivity, specificity, PPV, NPV 47)%; p = .002], and was not significant among those with FLC and LR + of 86, 75.16%, 99% and 3.4 respectively. These results were lambda [median 100 (78–100) versus 100 (77–100); p = .5]. improved by excluding cirrhotic patients and ambulatory peritoneal dialysis. The same happened when adding the study of cellular atypia Conclusions through optical microscopy performed in the Biochemistry labora- tory, finally achieving sensitivity, specificity, VPP, NPV and LR+ of The results suggest that the higher percentage of dimeric form of 84, 99, 97%, 99% and 570 respectively. kappa light chain is connected with worse renal function. There is need for more detailed study taking into consideration other markers Conclusions of renal function and median survival for patients. The results of this study show that the study of ascitic fluids using doi:10.1016/j.cca.2019.03.917 high fluorescence cells is useful for the screening of carcinomatous ascites. Although the cut-off point for HF-BF# ε17/⎧L has been described above, our study provides an extensive number of samples and clinical and radiological variables that give this hypothesis W143 ^ greater strength. fl fl High uorescence cell count in ascitic body uids for carcinoma- doi:10.1016/j.cca.2019.03.918 tosis screening

J. Wong-Artetaa, E. Gila, R. Cabezón-Vicentea, J.M. Banalesb,L. b Bujanda ^W144 aDepartment of Biochemistry, Donostia University Hospital, San Sebastián, Spain Comprehensive diagnosis of neoplastic effusions from the clinical bDepartment of Gastroenterology, Donostia University Hospital-Bio- laboratory donostia Health Research Institute, University of the Basque Country (UPV/EHU), CIBERehd, San Sebastián, Spain J. Wong-Artetaa, M. Reyc, L. Aragónc, E. Gila, L. Bujandab aDepartment of Biochemistry, Donostia University Hospital, San Background-aim Sebastián, Spain bDepartment of Gastroenterology, Donostia University Hospital - Malignant ascites is a serious condition, with overall survival Biodonostia Health Research Institute, University of the Basque Country between 1 and 4 months after diagnosis, and accounts for 7 to 10% of (UPV/EHU), CIBERehd, San Sebastián, Spain all cases of ascites. The etiology is diverse, but approximately two cDepartment of Immunology, Donostia University Hospital, San thirds of cases are secondary to carcinomatous ascites. Therefore, it is Sebastián, Spain important to recognize the presence of malignant cells. The Sysmex analyzers provide in the cell count the high fluorescence cells (HF- Background-aim BF), where macrophages, mesothelial and neoplastic cells are grouped. The main objective of this work was to evaluate the 10% of ascites and up to 35% of pleural effusions have a neoplastic usefulness of HF-BF in the screening of peritoneal carcinomatosis. origin. Flow cytometry (FC) through the use of new antibodies such as EpCAM is able to accurately identify carcinomatous cells. Using an Methods algorithm that we have designed in a previous study, we are able to correctly screen the ascites samples that arrive to the Biochemistry We analyzed all consecutive ascitic fluid samples obtained as of laboratory, obtaining sensitivity, specificity, PPV, NPV and LR+ of 84, November 2015. The total number of high fluorescence cells per 99, 97%, 99% and 570 respectively for the screening of carcinoma- microliter was designated HF-BF# and the total nucleated cell count tosis. The main objective of this study is to evaluate the usefulness of per microliter TCBF#. A “gold standard” was designed based on FC for the diagnosis of neoplastic effusions. clinical, radiological and imaging criteria, thus determining the presence of carcinomatosis when at least one of the following Methods criteria was found: (1) tomography (CT) with report of peritoneal carcinomatosis and compatible clinical course, (2) cytology by All ascitic and pleural fluids that arrive the Biochemistry laboratory pathological anatomy (PA) positive for malignant cells. were studied consecutively and prospectively. After the cell count, a screening for neoplastic infiltration was performed through the Results algorithm we have previously developed, which is based on micro- scopic and auto-analyzer criteria. To the positive samples for this A total of 3128 samples of serous fluids were collected, 46% of screening are performed FC determining the presence of carcinoma- these (1432/3128) corresponded to ascitic fluids. Carcinomatosis tous cells. The gold standard (GS) for the diagnosis of neoplastic was identified in 5.4% (78/1432) of the cases. All of them underwent effusions was designed combining the following criteria: (1) clinical CT, which was positive in 96% (75/78); while 73% (57/78) picture compatible with neoplastic disease, (2) tomography with underwent PA, being positive in 65% (37/57). The areas under the report of carcinomatosis and free abdominal fluid, and or (3) cytology curve for the detection of carcinomatosis were: TC-BF# 0.79 (95%CI, positive for malignant cells from the Pathology department (PA). Abstracts / Clinica Chimica Acta 493 (2019) S379–S433

Results Methods

A total of 465 fluids were analyzed (ascites 283, pleural 182). Polyclonal antibodies (anti-IgG, -IgA, -IgM, −| and -⌊) covalently Using the screening algorithm, 34 samples were included (ascites 11, attached to paramagnetic microparticles were incubated with serum, pleural 23). Based on the criteria described for GS, neoplastic effusion washed and treated to simultaneously elute and reduce patient has been identified in 76% (26/34). Although there were discrepan- immunoglobulins. Mass spectra were generated on a MALDI-TOF-MS cies in the results of 4 samples, the areas under the curve of FC and system, and specificity was assessed using normal human serum PA for the identification of malignant cells were similar: 0.904 (95% (NHS) and patient sera containing monoclonal immunoglobulins. CI, 0.75–0.98, p = .0001) and 0.923 (95%CI, 0.78–0.98, p = .0001), Accelerated stability was assessed at 22 °C over 12 weeks using NHS. respectively; without finding a difference among them (p = .77). LoB and LoD were determined by serial titrations of NHS diluted in The sensitivity and specificity were also similar: 81% and 100% (FC), sheep serum. T-mAbs daratumumab or elotuzumab were spiked at 84% and 100% (PA). The median response time for FC was 1 day (1– 0.2 g/L into NHS or myeloma patient sera. Sensitivity was compared 4) and for PA was 5 days (2–26). to CZE and IFE.

Conclusions Results

The preliminary results of this work show that a comprehensive Polyclonal molecular mass distributions for the light chains from study of neoplastic effusions is possible from the Clinical laboratory, NHS: IgG (median IgG|/IgG⌊ ratio 2.3:1 (CV = 4.4%)), IgA (IgA|/IgA⌊ starting with the screening of samples through basic cytometry and = 1:1 (CV = 3.5%)) and IgM (IgM|/IgM⌊ = 1.5:1 (CV3.7%)), total |, optical microscopy until the diagnosis by advanced cytometry. and total ⌊ were observed. Stability assessed at 22 °C for 12 weeks Applying the FC in the diagnosis of neoplastic effusions achieves a gave reproducible signal intensities for the polyclonal molecular lower delay in the response and traceable results compared to the mass distributions from NHS, without any loss of activity. The LoD for cytology from PA. monoclonal proteins diluted into sheep serum were: 0.7 mg/L for IgG, 1.4 mg/L for IgA, IgM and total |, and 0.17 mg/L for total ⌊.Ina doi:10.1016/j.cca.2019.03.919 blind study, QIP-MS had a greater sensitivity for the detection of monoclonal immunoglobulins than either SPE (100×) or IFE (10×). QIP-MS was able to distinguish the monoclonal light chains originating from patient's M protein from those of the t-mAb at W145 ^ therapeutically relevant concentrations (0.2 g/L) in all samples analyzed. QIP-MS: A specific, sensitive, accurate, and quantitative alterna- tive to electrophoresis that can identify endogenous m-proteins Conclusions and distinguish them from therapeutic monoclonal antibodies in patients being treated for multiple myeloma QIP-MS provides a highly reproducible and sensitive alternative to conventional electrophoresis. The ability to determine a unique S. Northa, D. Barnidgeb, S. Brusseaua, R. Patela, M. Haseltonb,B.Du molecular mass for any myeloma paraprotein offers an innovative Chateaub, G. Wallisa, S. Hardinga, D. Sakrikarb, J. Ashbya addition for the identification and quantification of monoclonal aThe Binding Site Group Ltd., Birmingham, United Kingdom immunoglobulins. T-mAbs daratumumab and elotuzumab were bThe Binding Site Inc., Rochester, MN, United States easily distinguishable from the patient M-protein, even in the presence of high polyclonal background and at levels below the Background-aim detection limit of IFE.

Here we present performance data for quantitative Immunopre- doi:10.1016/j.cca.2019.03.920 cipitation mass spectrometry (QIP-MS), a polyclonal antibody-based assay to identify intact immunoglobulins and to distinguish them from therapeutic monoclonal antibodies (t-mAb).