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

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Golubić-Čepulić B.S07-1: Cord blood as a source of stem cells. Biochemia Medica 2009;19(Suppl 1):S55.
Department of Clinical Transfusiology, Zagreb University Hospital Center, Zagreb, Croatia
The author did not provide an abstract.
Kelečić J. S07-2: Haematopoetic stem cell transplantation in primary immunodeficiencies. Biochemia Medica 2009;19(Suppl 1):S56-S57.
Department of Pediatrics, Zagreb University Hospital Center, Zagreb, Croatia
Corresponding author:jadranka [dot] kelecic [at] zg [dot] t-com [dot] hr
Primary immunodeficiencies (PIDs) are complex congenital disorders characterised by impairment of innate or adaptive immunity, and usually a poor prognosis. Hematopoietic stem cell transplantation (HSCT) represents a curative approach in many of these disorders, including severe combined immunodeficiencies (SCIDs), Wiskott-Aldrich syndrome, phagocyte disorders such as chronic granulomatous disease and leucocyte adhesion deficiency, Chediak-Higashi syndrome, hemophagocytic syndromes, X-linked hyper IgM syndrome, X-linked lymphoproliferative syndrome. The first hematopoetic stem cell transplantation with durable success in humans were reported in 1968 in three patients with primary immunodeficiencies who received grafts from HLA-matched siblings (two with severe combined immunodeficiency, and one with Wiskott-Aldrich syndrome).Since then significant progress has been made with HSCT for PIDs. There are several factors that influence the outcome of HSCT in PIDs patients: 1) high resolution tissue typing, 2) ability to phenotype and quantitate hematopoetic stem cells, 3) availability of closely matched unrelated donor bone marrow, peripheral blood stem cells and cord blood, 4) the aplication of reduced intensity conditioning regimens pre-transplant associated with significantly reduced early post-transplant mortality. The most important factors are the age at transplant and general health of the patient. Young age is associated with fewer comorbidities and less frequent pre-transplant exposure to herpes family and enteric viruses, thus lowering the risks of related post-transplant complications. The optimal results of HSCT in primary immunodeficiencies have long been obtained with related HLA-identical donors. HSCT using HLA-identical healthy sibling donors can provide correction of PIDs such as SCID, Wiskott-Aldrich syndrome (WAS) and other X-linked immunodeficiencies in approximately 90% of the cases. This option is limited to a minority of patients. Transplantation from mismatched related donors has been used with good results mainly in infants with severe combined immunodeficiency, but has been associated with significantly delayed or incomplete immune reconstitution. Recent data indicate that transplantation from matched unrelated donors and cord blood tranplantation represent valid alternatives, which can be used in all forms of severe primary immunodeficiencies. Optimal donor choise, monitoring of infection and preemptive treatment has resulted in a significant improvement for severe form of primary immunodeficiencies.
Čolić-Cvrlje V.S07-3: Hepatitis C i orthotopic liver transplantation. Biochemia Medica 2009;19(Suppl 1):S57-S58.
Department of Internal Medicine, Merkur University Hospital, Zagreb, Croatia
Corresponding author:vesnacoliccvrlje [at] yahoo [dot] com
HCV infection in Europe and USA is the most common indication for liver transplantation 40–60%. Outcome of HCV related OLT is universal viral infection and recurrent hepatitis C with progression to cirrhosis. The frequency of disease progression and the risk for severe HCV hepatitis postOLT are increasing in recent years.
Indication for OLT are liver decompensation CTP > 7, refractory ascites, encephalopathy, history of spontaneous bacterial peritonitis, varicel bleeding unresponsive to treatment, hepatocellular carcinoma. HCV + HCC coexistence are listed in the absence of hepatic decompensation according the Milan criteria: 1 nodule less than 5 cm, 3 nodules less than 3 cm, abscence of vascular invasion, absence of extrahepatic metastases. Pts are given added priority with minimum MELD score 22 that increases every three months.
Posttransplant HCV recurrent infection is evident by reappearence of HCV in serum, decrease of HCV RNA in anhepatic phase. HCV RNA level increases rapidly 2 weeks postOLT and is 10-20 fold higher 1 year after OLT. 75% patients develop acute hepatitis in 6 months post OLT, 80% chronic hepatitis in 1-2 years, 30% cirrhosis in 5 years and < 10% accelerated FCH. Factors influencing disease severity, progression and survival postOLT are related to host, virus, enviroment, donor. PreLT and early postLT viral loads are predictors of severe liver disease. Higher preLT viral load – lower is postLT survival.
Donor factors are: age > 50, steatosis - worse outcome, prolonged ischemic time, CMV coinfection. Corticosteroids increase levels of hepatitis C viremia in nontranspant HCV infected pts and postOLT boluses are harmful to HCV recipients. They increase HCV levels. Corticosteroide treatment for rejection have increased risk of death. Greater immunosupression leads to more sever postOLT HCV hepatitis. There is no difference among patients receiving tacrolimus or cyclosporin. MMF treatment has no impact on HCV levels. With higher RNA levels preOLT there is 30% greater mortality. In cirrhosis Child B and C antiviral therapy is poor tolerated with serious AE. SVR is only 13% in genotype 1. Antiviral therapy is limited to Child A cirrhosis. Posttransplant antiviral therapy is preemptive therapy for prophylaxis is tolerable but not efficient. Therapy for established HCV recurrence is PEG IFN monotherapy or PEG IFN+RBV combination. Dose reduction of PEG IFN is 80% patients. Low initial doses of ribavirin are required with gradual dose escalation. Retransplantation for HCV is routine. Poor outcome predictors are: age > 50, high bilirubin, renal failure, FCH. Indications for ReTx is therefore without consensus. In hospital “Merkur” 300 OLT were performed for different indications. 41 pts for HCV cirrhosis. 9 pts died post OLT, 32 pts lives 3 months to 6 years. Some pts are treated with PEG IFN + RBV. 2 pts were retransplanted for HCV cirrhosis recidive.
In conclusion the worse outcome of HCV related patients in recent years is associated with the all pretransplant and posttransplant variables: host, donor, viral, enviroment, immunosupression, corticosteroid therapy, success of pre and postOLT antiviral therapy, retransplantation.
Šiftar Z.S07-4:Immune profile in solid organ transplantation. Biochemia Medica 2009;19(Suppl 1):S58-S60.
Institute of Clinical Chemistry, Merkur University Hospital, Zagreb, Croatia
Corresponding author:zoran [dot] siftar [at] hdmb [dot] hr
Introduction: Transplantation is now widely accepted method of choice in the treatment of patients with terminal stage kidney, liver, pancreas disease etc who can resist transplantation procedure and long-term intake of immunosuppressive therapy, necessary for graft survival and the patient.
Assessment of the optimal dosage of combined therapy, the initial and maintenance therapy, is still one of the greatest problems in kidney transplantation. Acute rejection may lead to graft lost and it is an important risk factor for the emergence of chronic rejection, in which T cells play an important role in the pathogenesis of rejection. At least certain groups of lymphocyte in circulation should reflect the image of active cells that are found locally within graft. So far, there is no single marker that would safely predict rejection except PHD analysis of renal biopsy, and continuous monitoring of lymphocyte population distribution may have clinical significance. To directly measure the effective impact of immunosuppressive therapy at the cellular level previously were used T cell number and the number of CD4+T lymphocytes, CD4/CD8 ratio, double positive (CD3+CD4+CD8+) and double negative (CD3+CD4-CD8-) populations of T lymphocytes, as the number of active T lymphocytes (CD25+).
Material and methods: During 2007 and 2008 there were 72 transplanted patients in “Merkur” University Hospital (54 kidney, 13 kidney/pancreas, /SPKT/ and 5 kidney/liver/LKT/), 42 male and 30 female, average age 44 years (range 16-68), who have received a combined, initial, prior transplantation: corticosteroids, calcineurin inhibitor, either anti-CD25 or polyclonal antythymocyte globulin (ATG) and maintenance therapy, after transplantation: corticosteroids, calcineurin inhibitor, either anti-CD25 or polyclonal antythymocyte globulin (ATG), mycophenolate mofetil. Histological findings of indicated renal biopsy during the first year has confirmed acute rejection in 27 patients (21 kidney, 4 SPKT, 2 LKT). The multiple determination of the number of lymphocyte populations in venous blood: T lymphocytes, B lymphocytes, NK cells, CD4+ and CD8+ T lymphocytes, active T lymphocytes: CD25+, CD69+, CD127+, activated (CD25+) CD4 and CD8 T lymphocytes, and specific population of T lymphocytes, CD3+CD4+CD25high+ was done using flow cytometry. Quantification of lymphocyte population is made on a flow cytometer Epics XL, Coulter, according to “lyse/no wash” standard procedure with FlowCount calibration particles, with the analyses conditions according to CD45/side scatter protocol (SSC). The staining has been done with antibodies for flow cytometry, directly conjugated, by Beckman-Coulter, and Dako. Measurement results were in accordance and within the target value of internal and external quality assurance programme UKNEQAS Immune Monitoring.
After the induction phase, as expected, reducing the numbers occurs in all groups in lymphocyte compartment, with drastic decrease when ATG was applied instead of anti-CD25. During maintenance therapy, gradually recover of the same occurs, until normalization within 3 months, where diversity in behaviour is not observed between the 2 groups of patients. Number of activated CD25+ T lymphocytes grows during the 1 month and mainly is located in population of CD4+ T lymphocytes. Population of CD3+CD4+CD25high+ is a part of CD25+ activated CD4+ T lymphocytes, and according to literature has a positive effect on the graft stability unlike CD69+ activated T lymphocytes. Positivity of CD25 on CD8+ occurs in parallel with the expression on CD4+ T cells, and then speaks in favour of treating inflammation complications, like infection. The results of measurements on the Epics XL are repeatable and proven in practice to another cytometer, FC500, Beckman-Coulter.
Conclusion: Longitudinal monitoring the number of immune cells in circulation is helpful in assessing the efficiency of immunosuppressive therapy at the cellular level and the recovery of patients after surgical procedures in kidney transplantation, although insufficiently sensitive for prediction of graft rejection.
Ožvald I, Šurina B.S07-5: Monitoring of immunosuppressed therapy in transplated patients. Biochemia Medica 2009;19(Suppl 1):S60-S61.
Institute of Clinical Chemistry, Merkur University Hospital, Zagreb, Croatia
Corresponding author:iozvald [at] gmail [dot] com
Monitoring of successful transplantation is an interdisciplinary task of clinicians and medical biochemists. In Merkur University Hospital 300 orthotopic liver transplantations (OLT) and 100 kidney transplantations have been performed since 1998. Therefore, we strongly believe that the development of transplantation medicine in our hospital has contributed to the Croatian membership in Eurotransplant since May 2006.
Laboratory medicine monitors the success and/or rejection of transplant by established “transplantation follow-up protocol“ which includes CBC, PT, PT-INR, fibrinogen, AST, ALT, GGT, ALP, ChE, total and direct bilirubin, total cholesterol, total proteins, albumins, CRP, serum electrolytes, (K, Na, total and ionized Ca, total and ionized Mg), urea, creatinin, glucose, pH and blood gasses, directed towards follow-up of general patient’s state, especially towards liver and kidney function.
Beside these tests monitoring of immunosuppressed therapy is very important, which means lifelong therapy for these patients. Each patient needs titration and introduction of the optimal and efficient therapy dosage of appropriate immunosuppressive with purpose to achieve the therapeutic goal without exposing patient to immunodeficiency complications like infections, malignancy or organ rejection.
Therefore, in our Department of Clinical Chemistry we determine following immunosuppressives: calcineurin inhibitors (cyclosporin and tacrolimus), selective immunosuppressives – sirolimus (the second generation of immunosuppressive drugs which affect immunofilins and enable the alternative to the standard immunosuppressive therapy); as well as estimation of nucleotide synthesis inhibitors: micofenolate, applied as combination of mono- or polyvalent therapy.
By its selective and specific impact on cells of immune system, especially on T-lymphocytes immunosuppressives provide tolerance to these cells to the transplant. Due to strong variability in bioavailability and distribution it is necessary continuously to measure drug concentrations in circulation, because every immunosuppressive has its therapeutic range within narrow limits and the dosage is titrated by the measured levels in circulation.
Mentioned tests cannot differ between changes caused by acute rejection and transplant dysfunction of other etiology, nor can they determine the severity of rejection. Therefore, the final answer is provided by histological test method as golden standard which is not always possible. That is what makes the follow-up of a transplantation outcome an interdisciplinary task.
In our Department we determine levels of: cyclosporin since 1998 (FPIA), tacrolimus since 2002 (MEIA), everolimus (FPIA) 2006-2008, sirolimus since 2008 (MEIA) and micofenolat since 2006 (EMIT).
From 300 OLT the overall one year survival rate was 85% and survival rate for several years was 80% what concurs with results from other European transplantation centers. Liver transplantations were performed completely in concordance with Eurotransplant criteria (patient selection) on patients with different liver diseases: alcohol-induced liver disease, liver diseases caused by Hepatitis C virus infection, primary biliary cirrhosis, criptogen liver disease, hepatocellular carcinoma, metabolic alpha 1 antitripsin deficiency, Budd-Chiari syndrome, mesenchymal hemangioendothelioma, nonalcoholic steatohepatitis, autoimmune hepatitis, Wilson’s disease.
Our final goal is supported by routine follow-up of immunosuppressive therapy which is already in usage in Merkur University Hospital to offer the “optimal algorithm“ of immunosuppressive therapy for a patient in correlation with clinical efficiency in prediction of acute or chronic transplant rejection.
Based on diagnostic efficiency in determination of “critical drugs“, i.e. immunosuppressives, we would like to contribute to the final goal setting for clinicians as well as for medical biochemists and this goal is a successful outcome of a transplantation.