MESENCHYMAL STEM CELLS: IMMUNOMODULATORY PROPERTIES AND CLINICAL APPLICATION

Mezenhimske matične stanice (MMS) multipotentne su nekrvotvorne stanice prvobitno otkrivene u koštanoj srži. MMS su imunoprivilegirane stanice koje u in vitro sustavu izražavaju imunomodulatorne značajke, što ih čini vrlo pogodnima za kliničku primjenu. Ex vivo umnožene MMS novi su oblik stanične terapije koji ima vrlo širok raspon potencijalne kliničke primjene. Do sada se u kliničkim studijama potvrdila sigurnost primjene MMS i njihova učinkovitost u tri kliničke situacije: u suzbijanju reakcije presatka protiv primatelja, poticanju prihvata transplantiranih alogenih krvotvornih matičnih stanica i poticanju rasta bolesnika s osteogenesis imperfecta. Iako mehanizmi imunomodulatornog djelovanja MMS nisu potpuno razjašnjeni, potencijalna korist terapije s MMS opravdava njezinu kliničku primjenu u nekoliko bolesti. U ovom članku dan je povijesni pregled razvoja kliničke primjene MMS i trenutačna znanstvena i klinička dostignuća u tom području. Bolje poznavanje bioloških značajki i mehanizama djelovanja MMS nesumnjivo će omogućiti njihovu širu kliničku primjenu.Mesenchymal stem cells (MSCs) are multipotent nonhematopoietic cells that were first identified in bone marrow. Clinical interest for MSCs was initiated by the observation that MSCs are immunoprivileged cells that display immunomodulatory properties in vitro. Ex vivo expanded MSCs have therefore become a new type of cellular therapy in development with a wide range of potential clinical applications. So far many clinical studies confirmed safety of their use and showed that infused MSCs suppress graft versus host disease, support engraftment of transplanted allogeneic hematopoietic stem cells and stimulate growth in patients with osteogenesis imperfecta. Although underlying immunomodulatory mechanisms of action are not completely understood, potential benefit of MSC therapy justifies its clinical use in a broad range of disorders. In this report we give historical overview of MSC discovery and current scientific and clinical achievements in this field. Better insight into their biological properties and mechanisms of action are needed

CHARACTERISTICS, AUTOANTIBODIES AND TREATMENT OF PATIENTS WITH AUTOIMMUNE HAEMOLYTIC ANAEMIA

Uvod: Autoimunosna hemolitička anemija (AIHA) je vrlo rijetka autoimunosna bolest uzrokovana autoprotutijelima usmjerenima na bolesnikove eritrocite koja se dokazuje kliničkom slikom, biokemijskim pokazateljima hemolize uz isključenje ostalih uzroka hemolize i pozitivnim direktnim antiglobulinskim testom (DAT), osim kod DAT negativnih AIHA. Pozitivan DAT mogu imati i zdrave osobe i bolesnici bez AIHA-e. Liječenje ovisi o vrsti AIHA-e i osnovnoj bolesti, a sastoji se od više linija terapije. Transfuzijsko liječenje je rizik zbog moguće prisutnih aloprotutijela prikrivenih autoprotutijelima koja mogu uzrokovati hemolitičku transfuzijsku reakciju (HTR). Cilj rada bio je istražiti obilježja bolesnika, serološka obilježja autoprotutijela, hematološke pokazatelje anemije, biokemijske pokazatelje hemolize i liječenje bolesnika s AIHA-om. Postupci: U ovo retrospektivno istraživanje uključeno je 27 bolesnika kojima je od 1.siječnja 2018. do 31.prosinca 2020. godine dijagnosticirana AIHA u Kliničkom bolničkom centru Zagreb. Za dijagnostiku su provedena imunohematološka ispitivanja i laboratorijski pokazatelji hemolize. Učinkovitost transfuzijskog liječena defi nirana je kao porast vrijednosti hemoglobina (Hb) ≥5 g/L po dozi koncentrata eritrocita. Rezultati: Najviše je bolesnika dijagnosticirano s toplom AIHA-om (70,4 %), zatim s bolesti hladnih aglutinina (14,8 %) i miješanom AIHA-om (7,4 %), a najmanje s paroksizmalnom hladnom hemoglobinurijom (PCH, engl. Paroxysmal cold hemoglobinuria) i DAT-negativnom AIHA-om (po 3,7 % svaka). Prva linija terapije sastojala se od primjene kortikosteroida, a u drugoj i trećoj liniji 37 % bolesnika je primilo rituksimab. U 7,4 % bolesnika otkrivena su aloprotutijela. Transfuzijsko liječenje primilo je 81,5 % bolesnika, bez prijavljene transfuzijske reakcije. Učinkovitost transfuzijskog liječenja zabilježena je u 76,8 % slučajeva, s medijanom porasta Hb od 7,5 g/L po dozi krvi. Zaključak: Najviše je bilo dijagnosticirano toplih AIHA, a najmanje PCH i DAT-negativnih AIHA. U prvoj liniji terapije primjenjivani su kortikosteroidi, dok je u drugoj i trećoj liniji terapije najčešće primijenjen rituksimab uz imunosupresivnu terapiju. Transfuzijsko liječenje bilo je uspješno u većine bolesnika, bez prijavljenih transfuzijskih reakcija.Introduction: Autoimmune haemolytic anaemia (AIHA) is a very rare autoimmune disease caused by autoantibodies directed at patient\u27s red blood cells as evidenced by a clinical picture, biochemical indicators of hemolysis, excluding other causes of hemolysis, and a positive direct antiglobulin test (DAT), except for DAT-negative AIHA. Both healthy people and patients without AIHA can have a positive DAT. Treatment depends on the type of AIHA and the underlying disease, and consists of multiple lines of therapy. Blood transfusion poses a risk due to the possible presence of alloantibodies masked by autoantibodies, that may cause a haemolytic transfusion reaction (HTR). The aim: To analyse patient’s characteristics, serological characteristics of autoantibodies, laboratory parameters for anaemia, biochemical parameters for haemolysis and treatment of patients with AIHA. Methods: This retrospective study included 27 patients who were diagnosed with AIHA from1 January 2018 to 31 December 2020 in Clinical Hospital Centre Zagreb. For diagnosis, immunohematological tests and laboratory parameters of haemolysis were performed. The effi cacy of transfusion was defi ned as haemoglobin (Hb) value increase of ≥5g/L per unit of blood. Results: Most patients were diagnosed with warm AIHA (70.4%), then with cold agglutinin disease (14.8%) and mixed AIHA (7.4%), and the least number of patients with paroxysmal cold haemoglobinuria (PCH) and DAT-negative AIHA (3.7% each). The fi rst line of therapy consisted of corticosteroids, and in the second and third lines 37% of patients received rituximab. In 7.4% of patients alloantibodies were detected. Transfusion was administrated in 81.5% of patients, with no reactions reported. Effi cacy of transfusion was noted in 76.8% of the cases, with median increase of Hb of 7.5 g/L per unit of blood. Conclusion: Warm AIHA was diagnosed the most and PCH and DAT-negative AIHA the least frequently. Corticosteroids were used in the fi rst line of therapy, while rituximab was the most commonly used in combination with immunosuppressive therapy in the second and third lines of therapy. Transfusion was successful in most patients, with no transfusion reactions reporte

CHARACTERISTICS, AUTOANTIBODIES AND TREATMENT OF PATIENTS WITH AUTOIMMUNE HAEMOLYTIC ANAEMIA

Uvod: Autoimunosna hemolitička anemija (AIHA) je vrlo rijetka autoimunosna bolest uzrokovana autoprotutijelima usmjerenima na bolesnikove eritrocite koja se dokazuje kliničkom slikom, biokemijskim pokazateljima hemolize uz isključenje ostalih uzroka hemolize i pozitivnim direktnim antiglobulinskim testom (DAT), osim kod DAT negativnih AIHA. Pozitivan DAT mogu imati i zdrave osobe i bolesnici bez AIHA-e. Liječenje ovisi o vrsti AIHA-e i osnovnoj bolesti, a sastoji se od više linija terapije. Transfuzijsko liječenje je rizik zbog moguće prisutnih aloprotutijela prikrivenih autoprotutijelima koja mogu uzrokovati hemolitičku transfuzijsku reakciju (HTR). Cilj rada bio je istražiti obilježja bolesnika, serološka obilježja autoprotutijela, hematološke pokazatelje anemije, biokemijske pokazatelje hemolize i liječenje bolesnika s AIHA-om. Postupci: U ovo retrospektivno istraživanje uključeno je 27 bolesnika kojima je od 1.siječnja 2018. do 31.prosinca 2020. godine dijagnosticirana AIHA u Kliničkom bolničkom centru Zagreb. Za dijagnostiku su provedena imunohematološka ispitivanja i laboratorijski pokazatelji hemolize. Učinkovitost transfuzijskog liječena defi nirana je kao porast vrijednosti hemoglobina (Hb) ≥5 g/L po dozi koncentrata eritrocita. Rezultati: Najviše je bolesnika dijagnosticirano s toplom AIHA-om (70,4 %), zatim s bolesti hladnih aglutinina (14,8 %) i miješanom AIHA-om (7,4 %), a najmanje s paroksizmalnom hladnom hemoglobinurijom (PCH, engl. Paroxysmal cold hemoglobinuria) i DAT-negativnom AIHA-om (po 3,7 % svaka). Prva linija terapije sastojala se od primjene kortikosteroida, a u drugoj i trećoj liniji 37 % bolesnika je primilo rituksimab. U 7,4 % bolesnika otkrivena su aloprotutijela. Transfuzijsko liječenje primilo je 81,5 % bolesnika, bez prijavljene transfuzijske reakcije. Učinkovitost transfuzijskog liječenja zabilježena je u 76,8 % slučajeva, s medijanom porasta Hb od 7,5 g/L po dozi krvi. Zaključak: Najviše je bilo dijagnosticirano toplih AIHA, a najmanje PCH i DAT-negativnih AIHA. U prvoj liniji terapije primjenjivani su kortikosteroidi, dok je u drugoj i trećoj liniji terapije najčešće primijenjen rituksimab uz imunosupresivnu terapiju. Transfuzijsko liječenje bilo je uspješno u većine bolesnika, bez prijavljenih transfuzijskih reakcija.Introduction: Autoimmune haemolytic anaemia (AIHA) is a very rare autoimmune disease caused by autoantibodies directed at patient\u27s red blood cells as evidenced by a clinical picture, biochemical indicators of hemolysis, excluding other causes of hemolysis, and a positive direct antiglobulin test (DAT), except for DAT-negative AIHA. Both healthy people and patients without AIHA can have a positive DAT. Treatment depends on the type of AIHA and the underlying disease, and consists of multiple lines of therapy. Blood transfusion poses a risk due to the possible presence of alloantibodies masked by autoantibodies, that may cause a haemolytic transfusion reaction (HTR). The aim: To analyse patient’s characteristics, serological characteristics of autoantibodies, laboratory parameters for anaemia, biochemical parameters for haemolysis and treatment of patients with AIHA. Methods: This retrospective study included 27 patients who were diagnosed with AIHA from1 January 2018 to 31 December 2020 in Clinical Hospital Centre Zagreb. For diagnosis, immunohematological tests and laboratory parameters of haemolysis were performed. The effi cacy of transfusion was defi ned as haemoglobin (Hb) value increase of ≥5g/L per unit of blood. Results: Most patients were diagnosed with warm AIHA (70.4%), then with cold agglutinin disease (14.8%) and mixed AIHA (7.4%), and the least number of patients with paroxysmal cold haemoglobinuria (PCH) and DAT-negative AIHA (3.7% each). The fi rst line of therapy consisted of corticosteroids, and in the second and third lines 37% of patients received rituximab. In 7.4% of patients alloantibodies were detected. Transfusion was administrated in 81.5% of patients, with no reactions reported. Effi cacy of transfusion was noted in 76.8% of the cases, with median increase of Hb of 7.5 g/L per unit of blood. Conclusion: Warm AIHA was diagnosed the most and PCH and DAT-negative AIHA the least frequently. Corticosteroids were used in the fi rst line of therapy, while rituximab was the most commonly used in combination with immunosuppressive therapy in the second and third lines of therapy. Transfusion was successful in most patients, with no transfusion reactions reporte

Prijetransfuzijsko ispitivanje i transfuzijsko liječenje pri primjeni monoklonskog protutijela anti-CD38

Daratumumab je prvo monoklonsko protutijelo anti-CD38 koje se primjenjuje u liječenju multiplog mijeloma. Njegova primjena uzrokuje panreaktivnost u testovima prijetransfuzijskog ispitivanja. Panreaktivnost je posljedica vezanja monoklonskog protutijela anti-CD38 na protein CD38 na površini eritrocita, što u standardnom testiranju onemogućuje otkrivanje antieritrocitnih aloprotutijela i osiguranje podudarne krvi za transfuzijsko liječenje. Cilj rada bila je retrospektivna analiza vlastitih iskustava u rješavanju smetnja prijetransfuzijskog ispitivanja uzrokovanih monoklonskim protutijelom anti-CD38 i u transfuzijskom liječenju tih bolesnika. Prikazani su postupci za prijetransfuzijsko ispitivanje i transfuzijsko liječenje bolesnika liječenih monoklonskim protutijelom anti-CD38 koji su provedeni u Kliničkome bolničkom centru Zagreb. U istraživanju je analizirano 10-ero bolesnika liječenih daratumumabom. Prije i poslije primjene daratumumaba pretražena su antieritrocitna protutijela i određen je direktan antiglobulinski test. Pri transfuzijskom liječenju napravljeni su test pretraživanja antieritrocitnih protutijela i križne reakcije standardnim testiranjem i specifičnim postupcima imunohematoloških ispitivanja za uklanjanje smetnja monoklonskog protutijela anti-CD38. Postupci su uključivali obradu eritrocita ditiotreitolom koncentracije 0,2 M i neutralizacijski test uz primjenu reagensa DaraEx. Kod svih bolesnika testovi pretraživanja antieritrocitnih protutijela i križne reakcije bili su nakon primjene daratumumaba pozitivni, dok je direktan antiglobulinski test zbog primjene daratumumaba bio pozitivan u gotovo polovine bolesnika. Nakon obrade eritrocita ditiotreitolom 0,2 M učestalost lažno pozitivnih rezultata testova pretraživanja antieritrocitnih protutijela i križnih reakcija iznosila je oko 40%, a poslije primjene reagensa DaraEx oko 20%. Oba specifična postupka, obrada eritrocita ditiotreitolom 0,2 M i neutralizacijski test primjenom reagensa DaraEx, nisu se pokazala dovoljno pouzdanima u rješavanju smetnja uzrokovanih monoklonskim protutijelom anti-CD38. Zato je za transfuzijsko liječenje tih bolesnika nužno osigurati eritrocitne pripravke podudarne prema klinički najvažnijim antigenima u sustavima krvnih grupa Rh, Kell, Kidd, Duffy i MNS. Dobra suradnja između odjela i transfuzijske službe te postojanje protokola za prijetransfuzijsko ispitivanje i transfuzijsko liječenje ostaju preduvjet za pravodobno i sigurno transfuzijsko liječenje te skupine bolesnika

Validacija testova prijetransfuzijskog ispitivanja pretraživanja i identifikacije antieritrocitnih protutijela primjenom DG Gel kartica

Background Pre-transfusion tests vary in sensitivity and specificity and should be evaluated before their implementation into routine use. The aim of this study was to evaluate the diagnostic accuracy of the Grifols DG Gel column agglutination system and to compare the data with two other column agglutination systems used in our laboratory: Ortho BioVue and Bio-Rad. Special attention was focused on using more vials of reagent red blood cells by Grifols DG Gel system when compared to other systems in order to investigate whether it increases the sensitivity for clinically significant antibodies. Methods All samples were tested in parallel with Grifols DG Gel, Ortho BioVue and Bio-Rad cards according to manufactures’ instructions. Samples were processed through manual instrumentation. Tests were performed by trained and experienced staff. A total of 419 tests were performed on 302 samples. Results Concordant results between Grifols DG Gel system and the other two systems were obtained in 93.8% of the tests. For antibody screening by Grifols DG Gel system, sensitivity was 97.53%, specificity was 100%, predictive positive value was 100% and predictive negative value was 97.73%. For antibody identification, the accuracy was 96.03% for Grifols DG Gel system, 97.22% for Ortho BioVue and 94.44% for Bio-Rad. Conclusions The Grifols DG gel system shows high diagnostic accuracy and is safe for pre-transfusion compatibility procedures in blood transfusion laboratories. Using more vials of reagent red blood cells by Grifols DG Gel system when compared to other systems increases the sensitivity for some antibody specificities, particularly anti-Jka. This could have major impact on the prevention of delayed transfusion reactions.Uvod Testovi za predtransfuzijsko ispitivanje razlikuju se prema osjetljivosti i specifičnosti i trebalo bi ih validirati prije primjene za rutinski rad. Cilj ove studije bio je procijeniti dijagnostičku točnost sustava aglutinacije u mikrostupcu Grifols DG Gel i usporediti podatke s još dva sustava aglutinacije u mikrostupcu koji se primjenjuju u našem laboratoriju: Ortho BioVue i Bio-Rad. Posebna pozornost posvećena je upotrebi više bočica test eritrocita kod sustava Grifols DG Gel u usporedbi s drugim sustavima kako bi se istražilo povećava li se osjetljivost za klinički značajna protutijela. Materijal i metode Svi uzorci testirani su paralelno karticama Grifols DG Gel, Ortho BioVue i Bio-Rad prema uputama proizvođača. Uzorci su obrađeni ručno. Ispitivanja su obavili obučeni i iskusni djelatnici. Ukupno je provedeno 419 ispitivanja na 302 uzorka. Rezultati Podudarni rezultati između sustava Grifols DG Gel i ostala dva sustava dobiveni su u 93,8% ispitivanja. Za pretraživanje antieritrocitnih protutijela sustavom Grifols DG Gel osjetljivost je bila 97,53%, specifičnost 100%, prediktivna pozitivna vrijednost 100% i prediktivna negativna vrijednost 97,73 %. Za identifikaciju specifičnosti protutijela točnost za sustav Grifols DG Gel bila je 96,03%, 97,22% za Ortho BioVue i 94,44% za Bio-Rad. Zaključci Sustav Grifols DG gel pokazuje visoku dijagnostičku točnost i siguran je za predtransfuzijsko ispitivanje. Upotrebom više bočica test eritrocita sustavom Grifols DG Gel u usporedbi s drugim sustavima povećava se osjetljivost za neke specifičnosti protutijela, posebno anti-Jka. To bi moglo imati velik utjecaj na prevenciju odgođenih transfuzijskih reakcija

Human Milk Bank in Croatia: initial experiences

Darovano humano mlijeko je najbolja zamjena za majčino mlijeko u okolnostima kad ona ne može hraniti svoje dijete. Sigurno i kvalitetno darovano humano mlijeko osiguravaju banke humanog mlijeka. S tim ciljem u Hrvatskoj banci tkiva i stanica Kliničkog bolničkog centra Zagreb osnovana je Banka humanog mlijeka u siječnju 2020. Rad Banke u skladu je sa Zakonom o primjeni ljudskih tkiva i stanica. U ovom radu prikazujemo rezultate njenog rada od otvaranja do lipnja 2020. godine. Iz logističkih razloga uzrokovanih epidemijom COVID-19 i potresom u Zagrebu Banka humanog mlijeka nije prikupljala mlijeko 43 dana. Mlijeko je darovala 31 majka. Medijan dobi bio je 31 godina a 81% ih je bilo visokoobrazovanih. U 52% slučajeva majke su počele darivati mlijeko tri mjeseca nakon porođaja. Najviše darivateljica darovalo je mlijeko samo jedan put (45%). Medijan razdoblja darivanja bio je 46 dana. Većina (52%) darivateljica rodilo je prvi put, u očekivanom terminu porođaja (94%), djecu porođajne mase >2 500 gr. Samo troje od njihove djece (9%) bilo je na intenzivnom liječenju. Ukupno je prikupljeno 175,5 L mlijeka (prosječno 5,7 L/darivateljici), od kojih je 151,5 L zadovoljilo zahtjeve ulazne kontrole kakvoće, a 141 L je bila i pasterizirana. Kritičan broj vijabilnih, aerobnih i fakultativnih bakterija imalo je 32,6% mješavina mlijeka pripremljenih za pasterizaciju, a nakon nje 8,9%. Za kliničku primjenu izdano je 78,7 L u tri jedinice intenzivnog liječenja novorođenčadi. Već u prvim mjesecima rada Banka humanog mlijeka pokazala je važnost svog djelovanja. Kako bi mogli zadovoljiti potrebe za darovanim humanim mlijekom na nacionalnoj razini, potrebno je trajno podučavati/educirati majke o važnosti humanog mlijeka i promicati njegovo darivanje.Donated human milk is the best substitute for breast milk in the case when the mother cannot feed her baby. Human milk banks provide safe and high quality donated human milk. That was the reason why the Human Milk Bank was established in the Croatian Tissue and Cell Bank at the Zagreb University Hospital Centre in January 2020. The Bank works in accordance with the Law on the Application of Human Tissues and Cells. In this paper, we present the results of the Bank work since from its opening until June 2020. Due to logistic reasons caused by the COVID-19 epidemic and the earthquake in Zagreb, the Human Milk Bank did not collect milk for 43 days. Milk was donated by 31 mothers. Their median age was 31 years and 81% of them had high education level. In 52% of cases, mothers started donating milk three months after giving birth. Most donors donated milk only once (45%). The median period of donation was 46 days. The majority (52%) of donors gave birth for the fi rst time, in the expected term of childbirth (94%), birth weight was >2500 g. Only three of donors’ children (9%) were in intensive care. A total of 175.5 L of milk were collected (mean 5.7 L per donor), of which 151.5 L met the requirements of input quality control, and 141 L were pasteurized. A critical number of viable aerobic and facultative bacteria were identifi ed in 32.6% of milk pools prepared for pasteurization, and 8.9% after pasteurization. For clinical use, 78.7 L were dispensed in three neonatal intensive care units. The Human Milk Bank has already shown the importance of its activities during the fi rst months of operation. In order to be able to meet the needs for donated human milk at the national level, it is necessary to constantly inform mothers about the importance of human milk and to promote its donation

Collection and composition of autologous peripheral blood stem cells graft in patients with acute myeloid leukemia: influence on hematopoietic recovery and outcome [Skupljanje i sastav transplantata autolognih krvotvornih matičnih stanica periferne krvi u bolesnika s akutnom mijeloičnom leukemijom: utjecaj na hematološki oporavak i ishod]

Hematopoietic stem cell (HSC) transplantation is a standard approach in the treatment of hematological malignant diseases. For the last 15 years the main source of cells for trasplantation have been peripheral blood stem cells (PBSC). With the availability of hematopoietic growth factors and understanding the advantages of treatment with PBSC, the application of bone marrow (BM) was supplanted. The aim of this survey was to explore the success of PBSC collection, the factors which influence the success of PBSC collection, the composition and the quality of graft and their infuence on hematopoietic recovery and outcome after transplantation in patients with acute myeloid leukemia (AML). PBSC were collected by the method of leukapheresis after applying a combination of chemotherapy and growth factors or only growth factors. The quality of graft was determined with the clonogenic progenitor cell assay and with the flow citometry analysis. Of the total 134 patients with AML, who were submitted to HSC mobilization, the collection was successful in 78 (58.2%) patients. The collection was more successful after the first than after the second attempt of HSC mobilization (49% vs. 11%). The criteria for effective mobilization were the number of leukocytes >3´109/L and the concentration of CD34+ cells >20´103/mL in the peripheral blood on the first day of leukapheresis. The number of CD34+ cells infused had the strongest impact on hematopoietic recovery. We noted significantly faster hematological recovery of neutrophils and platelets, fewer number of transfused units of red blood cells and platelets, shorter duration of the tranfusion support, shorter treatment with intravenous antibiotic therapy and shorter hospitalization after PBSC compared to BM transplantation. These advantages could provide their standard application in the treatment of patients with AML

EXTRACORPOREAL PHOTOPHERESIS IN TREATMENT OF CHRONIC GRAFT VERSUS HOST DISEASE

Ekstrakorporalna fotofereza (EF) jest imunomodulatorna terapija koja se rabi u liječenju kronične reakcije transplantata protiv primatelja (engl. chronic graft versus host disease, cGVHD). Tijekom EF-a leukaferezom se iz krvi izdvajaju mononuklearne stanice, ex vivo im se dodaje 8-metoksipsoralen, stanice se ozrače UVA-zrakama i potom reinfundiraju bolesniku. Cilj istraživanja bio je procijeniti klinički i imunomodulatorni učinak postupka EF-a u bolesnika s cGVHD-om. Analiziran je 341 postupak EF-a u 7 bolesnika s cGVHD-om s medijanom EF-a po bolesniku 37 (raspon 13–131). U svih bolesnika cGVHD se manifestirao kožnim promjenama u kombinaciji sa simptomima drugih organskih sustava. EF su provođene dva dana za redom: prvih mjesec dana svaki tjedan, sljedeća 2 mjeseca svaki drugi tjedan, a potom jednom na mjesec. Medijan trajanja liječenja postupkom EF-a iznosio je 10 mjeseci (raspon 2 do 58). EF je većinom povoljno utjecao na simptome cGVHD-a pa je u 6 bolesnika došlo do poboljšanja i/ili stabilizacije promjena kože te bolje pokretljivosti zglobova, a u 2 bolesnika s ulceracijama sluznice usne šupljine promjene su se u cijelosti povukle. Do kliničkog poboljšanja došlo je 2 do 3 mjeseca nakon početka EF-a, što je omogućilo značajno smanjenje ili prestanak primjene glukokortikoida. Neželjene reakcije javile su se tijekom 4,9% postupaka. U bolesnika u kojih je došlo do poboljšanja kliničkog stanja normalizirale su se vrijednosti omjera CD4+/CD8+ stanica, kao i broj NK-stanica. Rezultati našeg istraživanja pokazuju da primjena EF-a povoljno utječe na simptome cGVHD-a i omogućuje sniženje doze kortikosteroida uz poboljšanje kvalitete života bolesnika pa se stoga može preporučiti za bolesnike koji ne odgovaraju na standardno liječenje.Extracorporeal photopheresis (ECP) is an immunomodulatory therapy which has been used in the treatment of chronic GVHD (cGVHD). ECP involves separation of the mononuclear cells with leukapheresis, followed by ex vivo administration of 8-methoxypsoralen and UV-A radiation and reinfusion to the patient. Aim of the study was to evaluate clinical and immunomodulatory effect of ECP procedures in patients with cGVHD. We analyzed 341 ECP procedures performed in 7 patients with cGVHD; median ECP per patient was 37 (range 13–131). All patients suffered from skin changes in combination with impaired joint mobility and symptoms of oral disease. ECP procedures were performed for two consecutive days: in initial phase weekly, followed by every two weeks and than monthly according to clinical response. Median of ECP treatment duration was 10 months (range 2–58). The effect of ECP in patients with cGVHD with skin and joint involvement was mostly beneficial: 6 patients experienced either improvement or stabilization in skin changes and joint mobility. In 2 patients who suffered from oral disease, the total recovery was observed. Clinical response was typically delayed until 2 to 3 months, and reduction in glucocorticoid dose was observed. Adverse reactions were observed in 4.9% procedures. In patients who responded to ECP treatment, CD4+/CD8+ ratio and number of NK cells were normalized. ECP proved to be an efficient and safe procedure that may be recommended for patients with cGVHD who do not respond to conventional therap

Leukocitafereza u liječenju teškog, o steroidima ovisnog ulceroznog kolitisa

Ulcerative colitis (UC) is a multifactorial disease of unknown precise etiology and immunopathogenesis. Peripheral blood granulocytes and monocytes/macrophages are the major sources of cytokines, which regulate inflammation. Leukocytapheresis (LCAP) is a method where blood is processed by apheresis system that removes lymphocytes and plasma before being returned to the body. We report the first case in Croatia where we used LCAP in the treatment of a patient with severe steroid-dependent UC. After 12 LCAP procedures, good clinical response was obtained and there were no significant adverse side effects noticed. The patient remained in clinical remission over two years in which he underwent regular follow ups at outpatient clinic. Over a 10-year follow-up period after LCAP, the patient had only occasional clini-cal symptoms of disease activity. The clinical course was complicated with the development of metastatic colorectal carcinoma, which points to the importance of regular disease monitoring rather than the in-creased risk of malignant disease after LCAP. Patients with UC are a demanding group of patients that warrant the search for novel treatment strategies other than conventional pharmacological therapies. Alt-hough LCAP is still not a common treatment modality in our daily practice, data from recent studies sug-gest it to be an effective and safe procedure in the management of active UC patients.Ulcerozni kolitis (UC) je kronična bolest multifaktorske etiologije čiji detaljan mehanizam imunološkog procesa još nije sasvim razjašnjen, ali ključnu ulogu svakako imaju granulociti i monociti/makrofazi koji reguliraju i pojačavaju upalni proces lučenjem proupalnih citokina. Leukocitofereza (LCAP) je terapijski postupak kojim se prolaskom krvi kroz sustav za aferezu odstranjuju limfociti i plazma prije nego što se krv ponovno vrati u krvotok. U ovom radu je prikazan o steroidima ovisan bolesnik s teškim relapsom UC-a koji je, prvi put u Hrvatskoj, liječen protokolom LCAP. Nakon 12 terapijskih protokola LCAP kod bolesnika je došlo do značajnog kliničkog poboljšanja bez razvo-ja nuspojava. Bolesnik je ostao u kliničkoj remisiji tijekom dvije godine ambulantnog praćenja, a unutar 10 godina praćenja nakon LCAP bolesnik je imao tek povremene simptome aktivnosti bolesti. Klinički tijek bio je kompliciran razvojem metastatskog karcinoma debelog crijeva, što prvenstveno upućuje na važnost redovitog praćenja bolesti, a ne na povećan rizik maligne bolesti nakon LCAP. Bolesnici s UC-om su zahtjevna skupina pacijenata koja zahti-jeva potragu za novim terapijskim strategijama osim onih konvencionalnih farmakoloških. Iako LCAP nije čest modalitet liječenja u svakodnevnoj kliničkoj praksi, novije studije upućuju na to da je postupak učinkovit i siguran u liječenju bolesnika s aktivnim UC-om

Collection and Composition of Autologous Peripheral Blood Stem Cells Graft in Patients with Acute Myeloid Leukemia: Influence on Hematopoietic Recovery and Outcome

Hematopoietic stem cell (HSC) transplantation is a standard approach in the treatment of hematological malignant diseases. For the last 15 years the main source of cells for trasplantation have been peripheral blood stem cells (PBSC). With the availability of hematopoietic growth factors and understanding the advantages of treatment with PBSC, the application of bone marrow (BM) was supplanted. The aim of this survey was to explore the success of PBSC collection, the factors which influence the success of PBSC collection, the composition and the quality of graft and their infuence on hematopoietic recovery and outcome after transplantation in patients with acute myeloid leukemia (AML). PBSC were collected by the method of leukapheresis after applying a combination of chemotherapy and growth factors or only growth factors. The quality of graft was determined with the clonogenic progenitor cell assay and with the flow citometry analysis. Of the total 134 patients with AML, who were submitted to HSC mobilization, the collection was successful in 78 (58.2%) patients. The collection was more successful after the first than after the second attempt of HSC mobilization (49% vs. 11%). The criteria for effective mobilization were the number of leukocytes >3´109/L and the concentration of CD34+ cells >20´103/mL in the peripheral blood on the first day of leukapheresis. The number of CD34+ cells infused had the strongest impact on hematopoietic recovery. We noted significantly faster hematological recovery of neutrophils and platelets, fewer number of transfused units of red blood cells and platelets, shorter duration of the tranfusion support, shorter treatment with intravenous antibiotic therapy and shorter hospitalization after PBSC compared to BM transplantation. These advantages could provide their standard application in the treatment of patients with AML