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Aim To assess the correlations of B regulatory cells (Bregs) and monocyte subsets in peripheral blood with the National Institutes of Health (NIH)-consensus-defined clinical manifestations of chronic graft-vs-host disease (cGvHD), in an attempt to establish their role as cellular biomarkers. Methods This multidisciplinary prospective study enrolled adult cGVHD patients treated in the University Hospital Center Zagreb and University of Zagreb School of Medicine. Immunophenotypic subpopulations of CD24highCD38high Bregs (CD27-, CD27+, and total) and monocyte (classical, intermediate, and non-classical) counts were correlated with demographic, transplant, and cGVHD-related data. Bivariate correlation analysis was performed to evaluate the correlations between Bregs and monocytes subsets and cGVHD organ involvement, as well as cGVHD severity and immunosuppression intensity. Results Twenty-two adult patients (54.5% female) with cGVHD were enrolled. The median (range) age was 44.5 years (24-65). All patients were transplanted for hematologic malignancies and 40.9% had severe NIH cGVHD global score. The median time from cGVHD diagnosis to the analysis was 16.6 months (0-176). The organ most frequently affected with cGVHD were the eyes (68.2%), skin (45.5%), lungs (45.5%), and liver (40.9%). Lower total and CD27-Bregs counts were correlated with worse cGVHD severity, higher immunosuppression intensity, and lung cGVHD, in terms of cell count, but also with skin cGVHD, in terms of percentages. Patients with liver and joint/fascia cGVHD had a lower percentage of non-classical monocytes and patients with more severe global NIH score had a higher classical monocytes count. Conclusion Different organs affected by cGVHD are differently associated with different subpopulations of Bregs and monocytes.

Objective Foundation project for the first human milk banks (HMB) in Croatia was launched in 2017 as the result of a collaboration between the Ministry of Health, University Hospital Centre (UHC) Zagreb, UNICEF and the Croatian Association of Breastfeeding Support Groups. The HMB was opened in UHC Zagreb on November 15, 2019as a part of the Croatian Tissue and Cell Bank, a division of the Department of Transfusion Medicine and Transplantation Biology. The specific technical side is based on the PATH standards and Guide to the quality and safety of tissues and cells for human application, Council of Europe. On January 10, 2020, HMB passed a Competent Authority (CA) inspection, conducted in accordance with the requirements of national tissue legislation that has been adapted to EU Directive 2004/23/EC on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells. The specific technical part is based on the PATH standards and Guide to the quality and safety of tissues and cells for human application, Council of Europe. The aim of this study was to review the very first experience in human milk banking in Croatia. Methods We retrospectively analysed the data of the first 28 human milk donors. Results From the opening to mid-February, 135 women showed interest in donating human milk, of which 28 became donors. In February 2020, we had 24 still active donors. Four women stopped donating, with a median donation period of 2 months. All donors were tested for blood borne viruses by serology and NAT and were negative. In total, we received 79 L of donated human milk.We started pasteurizing the milk after obtaining a license. Of the 30 controlled pre-pasteurization milk pools, 10% was over the allowed microbial contamination. Microbiological controls were performed for each pasteurization cycle. They were all sterile. The nutritional values of milk were all within the expected range. Conclusions Human milk is recognised as the optimal feeding for all newborn infants. When mothers’ own milk is not available, donor human milk provided by HMB is the second-best choice, especially for premature or sick infants. The opening of the HMB in Croatia is highly important in helping to provide the best possible medical care for prematurely born babies and infants with a serious medical condition when they cannot receive their mother’s milk.

Neželjene reakcije prilikom transplantacije alogeničnih krvotvornih matičnih stanica

Validacija i provedba periodičke provjere metode za određivanje CD34+ stanica na protočnom citometru

Indikacije i učinkovitost liječenja hospitaliziranih hematoloških bolesnika koncentratima trombocita

Testiranje antiheparinskih protutijela u bolesnika na izvantjelesnoj membranskoj oksigenaciji.

Kretanje učestalosti odgođenih seroloških i odgođenih hemolitičkih transfuzijskih reakcija u prošlom desetljeću

Prikupljanje i krioprezervacija autolognih mononuklearnih stanica iz periferne krvi za potrebe proizvodnje car-t stanica

Pandemija COVID-19 dovela je do izazova u liječenju bolesnika kojima je potrebna transplantacija krvotvornih matičnih stanica (KMS). Porastom broja oboljelih širom svijeta smanjena je dostupnost KMS zbog infekcije darivatelja, ograničenja u međunarodnom prometu koja su onemogućila redovitu dostavu pripravaka iz stranih registara, uz rizik od nedostupnosti novih darivatelja. Sukladno tome, početkom pandemije uveden je niz mjera u cilju zaštite darivatelja i bolesnika tijekom postupaka prikupljanja i transplantacije KMS kako bi se osigurala dostupnost transplantata na dan transplantacije i izbjeglo kondicioniranje bolesnika u slučaju njegove nedostupnosti. Sukladno preporukama European Society for Blood and Marrow Transplantation (EBMT) (1) i World Marrow Donor Association (WMDA) (2) i u našem centru su uvedene ove mjere.

Ekstrakorporalna fotofereza nakon transplantacije pluća u KBC Zagreb (2014–2021.)

Program terapijske eritrocitafereze u Kliničkom bolničkom centru Zagreb

Učestalost antitrombocitnih aloprotutijela i genotipizacija 12 specifičnih trombocitnih antigena (HPA) u trudnica

To gain insight into the peculiarities of clinical presentation and time of diagnosis in the population of patients with combined immunodeficiency treated in our center. We retrospectively analyzed 22 patients with combined immunodeficiency treated in our center in the past 25 years. Clinical findings were collected, including age of presentation and age of diagnosis, genetic defects, consanguinity, family history, number and site of infections, presence of diarrhea, failure to thrive, skin rash, vaccine complications and type of treatment. Immunological findings included WBC with absolute counts, lymphocyte subsets and immunoglobulin levels. Patients (n=22) with severe combined immunodeficiency (n=17) and other combined immunodeficiencies (n=5) were included in the study. Sixteen patients (77%) were male. The most common clinical manifestations were opportunistic infections. Most of the patients were diagnosed after 3 months of age. Prenatal diagnosis was performed in one patient (4.5%), two patients (9%) did not undergo genetic analysis. The spectrum of genetic defect in our cohort show wide heterogeneity. Seventeen patients (77%) underwent HSCT, one patient (4.5%) is on enzyme replacement therapy. None of our patients have undergone gene therapy. Twelve patients (54.5%) died, of which four (18%) died before HSCT. Combined immunodeficiencies are fatal without definitive therapy (HSCT or gene therapy). The outcomes are better if infants are diagnosed and treated in pre-symptomatic stage, preferably before 3.5 months of age. More and more countries are implementing newborn screening for primary immunodeficiency diseases in their health care systems to diagnose patients as early as possible and to prevent infections. For the same reason, we advocate for expanding our national neonatal screening program

Both procedures used in our laboratory, RBC treatment with 0.2 M dithiothreitol-treated RBCs and neutralization test using DaraEx reagent, have not been shown to be sufficiently reliable in resolving the interference with the routine blood compatibility testing caused by the anti-CD38 monoclonal antibody. Therefore, for blood transfusion of these patients it is necessary to provide antigenmatched RBC units. Good collaboration between the ward and the transfusion service, with protocols in place for blood compatibility testing and blood transfusion, remains a precondition for timely and safe blood transfusion for this group of patients.

Background: Chronic Graft-versus-Host Disease (cGvHD) is a serious late complication after allogeneic hematopoietic stem cell transplantation (alloHSCT) with heterogeneous presentation and still poorly understood pathophysiology including inflammation and endothelial dysfunction. Factor VIII (FVIII) and von Willebrand Factor (VWF) are coagulation factors but also known indicators of endothelial dysfunction and inflammation in different settings, and therefore could serve as interesting candidate biomarkers of cGvHD. Methods: Since 2013 patients after alloHSCT were assessed by the Multidisciplinary cGvHD team at the University Hospital Center Zagreb, Croatia, using established NIH cGvHD-related measurements. An extensive history, physical and laboratory evaluations were performed, including FVIII, VWF:Ag and VWF:Ac analysis. Descriptive statistic and non-parametric analyses were performed. Variables that showed significant univariate correlations were used in multivariate logistic regression (MLR) to identify the most predictive for FVIII, VWF:Ag and VWF:Ac in cGvHD patients. Results: 70 cGvHD patients and 41 controls (subjects after alloHSCT without cGvHD) were analysed. Median age of cGvHD patients was 42 (9-65) years, 50% females, 91.5% underwent alloHSCT for hematologic malignancies, 55.7% had myeloablative conditioning and 52.9% matched related donor. Median time from HSCT to study was 450.5 days and from cGvHD diagnosis to study 82 days. There were no demographic neither transplant related significant differences between cGvHD patients and controls beside stem cell source (peripheral blood 71.4% vs 51.2%, p=0.041) and history of acute GvHD (70.0% vs 22.0%, p< 0.001). Majority of patients had moderate (52.9%) or severe (42.6%) NIH global cGvHD score, 57.2% active cGvHD by clinician´s impression. Median number of organs involved by cGvHD was 3 (1-6), and the most frequently involved organs were mouth, skin and eyes (52.0% each). cGvHD patients compared to controls had higher FVIII levels (median 206 (52-453)% vs 182 (51-406)%, p=0.044, reference range 50-149%) and higher VWF:Ag (median 261.6 (76.6-601)% vs 203.2 (51.9-600)%, p=0.030, reference range 50-160%), while VWF:Ac showed a trend toward higher levels among patients (median 253.4 (54-601)% vs 178 (48.6-601)%, p=0.084, reference range 50-150%). Patients had higher GGT (p=0.002), lower anticardiolipin IgG (p=0.001) and IgM (p=0.003), and lower albumin (p=0.018) than controls, without differences between other laboratory parameters. Univariate analysis showed that among cGvHD patients higher FVIII was associated with worse Karnofsky score (KS) (p=0.031) and performance score (PS) (p=0.030), higher leukocytes (p=0.031), cholesterol (p=0.003), triglycerides, AST, ALT, GGT, LDH, and lower albumin. Higher VWF:Ag and VWF:Ac in cGvHD patients were associated with worse KS and PS (p< 0.001), with more active cGvHD (p< 0.001), worse NIH cGvHD liver (p=0.042 ; p=0.039) and NIH cGvHD mouth (p=0.012 ; p=0.009), higher total NIH score (p=0.044 ; p=0.005), higher number organs involved (p=0.013 ; p=0.003), higher ESR, monocytes, D-dimers, AST, ALT, GGT, LDH, triglycerides, β-2-microglobulin, ferritin, total proteins, IgA and lower albumin. MLR analysis showed leukocytes (p=0.018) and cholesterol (p=0.010) as the strongest predictor of FVIII (r2=49.8% ; p< 0.001), while strongest predictor of VWF:Ac was number of organs involved by cGvHD (r2=71.7% ; p=0.031). Conclusions: Results of this study detected high FVIII and VWF levels in cGvHD patients with possible reflections to cGvHD manifestations, what needs to be further confirmed in larger longitudinal studies. Disclosure: This work was supported, in part, by the Unity Through Knowledge Fund project entitled “Clinical and biological factors determining severity and activity of chronic Graft-versus-Host disease after allogeneic hematopoietic stem cell transplantation”, and also, in part, by the Croatian Science Foundation project entitled “New biomarkers for chronic Graft-versus-Host disease”. Antonela Samardzic - Work financed by the Croatian Science Fondations` “Young researchers` career development project - training of doctoral students”

Sažetak. Unatrag nekoliko godina u hematologiji i onkologiji globalno sve češći problem postaje prikladna opskrba „starijim i manje zanimljivim“ kemoterapeuticima. Zbog povremene nestašice karmustina, jednog od osnovnih kemoterapeutika pri kondicioniranju prije autologne transplantacije krvotvornih matičnih stanica (ATKS) u oboljelih od limfoma, u našem se centru od 2016. godine on zamjenjuje bendamustinom. U ovom radu retrospektivno analiziramo tijek ATKS-a u 41 bolesnika koji su primili bendamustin u sklopu protokola BeEAM te ga uspoređujemo s tijekom ATKS-a u 40 bolesnika koji su primili karmustin u sklopu protokola BEAM. Medijan oporavka vrijednosti neutrofila (> 0,5 × 109/l) u skupini koja je primila bendamustin iznosio je 11 dana, dok je u skupini kondicioniranoj karmustinom iznosio 10 dana. Medijan oporavka vrijednosti trombocita (> 20 × 109/l) bio je duži kod skupine koja je primala bendamustin (16 prema 13 dana) te su ti bolesnici bili duže ovisni o transfuzijama eritrocita (7 prema 5 dana). Infektivne komplikacije nisu bile češće nakon primjene bendamustina, ali smo nakon primjene karmustina imali veću pojavu mukozitisa II. – III. stupnja (35% prema 12%). Nakon primjene bendamustina zabilježen je jedan slučaj nefrotoksičnosti i kardiotoksičnosti terapije, dok kod primjene karmustina te komplikacije nisu zabilježene. Pri upotrebi bendamustina kod kondicioniranja u naših bolesnika u ovom trenutku nije utvrđena znatnija hematološka toksičnost u odnosu prema karmustinu, ali su prisutni dulji period oporavka vrijednosti trombocita te niža incidencija mukozitisa., Summary. Inadequate supply of „old and less interesting“ chemotherapeutic agents is becoming a global issue in hemato-oncology today. In 2016 we were faced with occasional carmustin shortage, one of the most commonly used in autologous transplant conditioning regimens for lymphoma in our centre, so we decided to use bendamustin instead. We performed a retrospective analysis of 41 patients treated at our centre who had received bendamustin within BeEAM protocol and compared them with 40 patients who had received carmustin within BEAM protocol. Both protocols were used as conditioning protocols before autologous stem cell transplantation. Neutrophil recovery median following transplantation (ANC>0,5x109/l) was 11 days in the bendamustin group in comparison to 10 days in the carmustin group.Platelets recovery median following transplantation (PLT>20x109/l) was longer in the bendamustin group (16 vs.13 days) as was blood transfusion dependency (7 vs. 5 days). Infectious complications were not more frequent after bendamustin, but grade II–III mucositis was more frequent in patients who received carmustin (35% vs.12%). Following bendamustin we had one reported case of nephrotoxicity and cardiac toxicity, not reported with carmustin. Bendamustin has shown similar hematologic toxicity compared to carmustin but a longer platelet recovery period and a lower mucositis incidence.

In patients treated with allogeneic HSCT, there is a risk ofdeveloping new alloimmunization, due to exposure of donor lymphocytes to RBC ofthe recipient, or transfusion therapy with antigen incompatible blood components.After allogeneic HSCT, our patients developed alloantibodies of the Rhesus, Kell, MNS and Lutheran blood group systems that can cause significant hemolysis andcomplicate the course of recovery. Since some patients develop alloantibodiesdespite intensive immunosuppressive treatment, continuous immunohematologicalmonitoring is required for patients following HSCT.

Inadequate supply of „old and less interesting“ chemotherapeutic agents is becoming a global issue in hemato- oncology today. In 2016 we were faced with occasional carmustin shortage, one of the most commonly used in autologous transplant conditioning regimens for lymphoma in our centre, so we decided to use bendamustin instead. We performed a retrospective analysis of 41 patients treated at our centre who had received bendamustin within BeEA M protocol and compared them with 40 patients who had received carmustin within BEA M protocol. Both protocols were used as conditioning protocols before autologous stem cell transplantation. Neutrophil recovery median following transplantation (AN C>0,5x109/l) was 11 days in the bendamustin group in comparison to 10 days in the carmustin group.Platelets recovery median following transplantation (PLT >20x109/l) was longer in the bendamustin group (16 vs.13 days) as was blood transfusion dependency (7 vs. 5 days). Infectious complications were not more frequent after bendamustin, but grade II–III mucositis was more frequent in patients who received carmustin (35% vs.12%). Following bendamustin we had one reported case of nephrotoxicity and cardiac toxicity, not reported with carmustin. Bendamustin has shown similar hematologic toxicity compared to carmustin but a longer platelet recovery period and a lower mucositis incidence.

Danas se u svijetu sve veći broj bolesnika s malignim i nemalignim bolestima liječi transplantacijom alogeničnih krvotvornih matičnih stanica (aloTKMS). Kronična bolest presatka protiv primatelja (eng. Chronic graft versus host disease - cGVHD) vodeći je uzrok morbiditeta i mortaliteta koji nije povezan s relapsom osnovne bolesti u osoba nakon aloTKMS‑a. Na KBC Zagreb u 2013. godini formiran je multidisciplinarni tim za liječenje cGVHD-a i dugotrajnih komplikacija nakon aloTKMS-a u suradnji s National Cancer Institute (NCI), NIH, SAD. Donosimo izvješće o daljnjem radu tima i nedavno održanom međunarodnom simpoziju.

Introduction: Predictive, diagnostic, and risk stratification biomarkers of chronic Graft- versus-Host disease (cGVHD) are critically needed to improve treatment of this serious late complication of allogeneic hematopoietic stem cell transplantation (HSCT). Since elevated von Willebrand’s factor (VWF) and factor VIII (FVIII) levels have been described as indicators of endothelial dysfunction and inflammation in different settings, the goal of this study was to assess possible role of VWF and FVIII as potential biomarkers of cGVHD. Materials (or patients) and methods: Multidisciplinary cGVHD clinical infrastructure was organized in 2013 at the Division of Hematology, University Hospital Center Zagreb, Croatia, using established cGVHD-related scales and measurements in collaboration with the National Cancer Institute, National Institutes of Health (NIH), USA. An extensive demography, history, physical and laboratory evaluations were obtained in this cross-sectional prospective study using standardized methods, including measurement of VWF:Ag, VWF:RCo, and FVIII. Descriptive statistic and non-parametric analyses were performed, results with p- valueso0.05 were considered as statistically significant, and variables with Spearman rank correlation coefficient 0.5o|r|o0.7 were considered as moderately correlated. Results: Twenty cGVHD patients were assessed (median age 38.5 years, 55% females). Median time from HSCT to study enrollment was 16.4 months. Median time from HSCT to cGVHD diagnosis was 9.5 months, and median time from cGVHD diagnosis to study enrollment was 5.5 months. Majority of patients (90%) were transplanted for hematologic malignancies. Myeloablative conditioning was used in 50%, 11 (55%) received peripheral blood stem cells, and 12 (60%) transplants were from matched related donors. Eight patients had de novo cGVHD, 5 quiescent, 7 progressive, and 16 (80%) classic cGVHD. Ten (50%) patients had moderate and 10 (50%) severe NIH global cGVHD score. Most involved organs were skin, lung, liver, and eyes. At the time of evaluation, clinician’s impression was in 50% that cGVHD was active. Elevated level of FVIII (median 191%, range 52-297%, reference range 50-149%) and VWF:Ag (median 208%, range 108-600%, reference range 50-160%) was found. Median level of VWF:Rco was 138% (range 75- 547%, reference range 50-150%). Patients after unrelated HSCT had higher FVIII, VWF:Ag and VWF:RCo. Shorter time from cGVHD diagnosis to study enrollment was associated with higher VWF:Ag (P¼0.015) and VWF:Rco (P¼0.018). NIH cGVHD liver score (P¼0.013), average NIH score (r¼0.535), AST (r¼0.668), ALT (r¼0.603), beta-2 microglobulin (r¼0.510), triglyceride (r¼0.596) and cGVHD activity by therapeutic intent (P¼0.019) were associated with higher FVIII. NIH cGVHD mouth score (P¼0.016), clinician impression of cGVHD activity (P¼0.023) and AST (r¼0.563) were associated with higher VWF:Ag. Ferritin (r¼0.506) was associated with higher VWF:RCo, while cGVHD activity by therapeutic intent, beta-2 microglobulin, D-dimers, and triglyceride were associated with both higher VWF:Ag and VWFR:Co. Other markers of inflammation (C-reactive protein, fibrinogen, erythrocyte sedimentation rate) did not correlate neither with VWF:Ag, VWF:RCo nor with FVIII. Conclusion: Results of this pilot study suggest that VWF and FVIII could represent interesting candidate biomarkers of cGVHD. However, these need to be further investigated in larger studies. Disclosure of Interest: None declared.

Svrha ovog rada je procjeniti rizik javljanja akutnih transfuzijskih reakcija (ATR) prema vrsti krvnih pripravaka i skupinama bolesnika nakon uvođenja transfuzijskog liječenja samo s krvnim pripravcima sa smanjenim brojem leukocita (FKP). Uvođenje transfuzijskog liječenja samo s krvnim pripravcima sa smanjenim brojem leukocita značajno je smanjilo rizik akutnih transfuzijskih reakcija u našoj ustanovi. Uvođenje univerzalne leukofiltracije trombocita na nacionalnoj razini povećalo bi sigurnost I kvalitetu transfuzijskog liječenja u Hrvatskoj.

Implementation and dissemination of chronic Graft- versus-Host Disease NIH criteria and recommendations - formation of a multidisciplinary team and program at the University Hospital Center Zagreb, Croatia.