Current Practice in FFP Preparation and Use in Greece: A National Survey

Objective: Fresh frozen plasma (FFP) transfusion is widely used in modern clinical settings. Practices regarding its use vary due to lack of guidelines from randomized trials. The aim of this study was to assess both the current practices regarding FFP production, use, and wastage and the implementation of quality control (QC), female donor plasma production policies, and use of pharmaceutical hemostatic agents in Greece. Materials and Methods: The study was conducted during February-April 2018. For the first part of the study, data including FFP transfusion indication, hospital department, diagnosis, FFP units/transfusion episode, ABO compatibility, blood donor's sex, and reasons for discarding were collected. For the second part, questionnaire data were analyzed. Results: According to data from 20 Greek hospitals, 12655 FFP units were transfused to 2700 patients during 5069 transfusion episodes in the studied period of time. Most patients were hospitalized in internal medicine, general surgery, and intensive care unit departments. Each patient received on average 4.69 units (2.5 units/episode). Transfusion requests were in accordance with international guidelines in 63.44% of cases and 99.04% of the units were given to ABO-identical patients. Main reasons for discarding included failure to meet quality requirements (30.06%), female donors (22.17%), and other causes (27.26%). Among 96.9% of all transfusion services across the country, 28.26% perform QC according to the directions of the European Directorate for the Quality of Medicines & Health Care and 68.83% discard plasma from female donors. Pharmaceutic hemostatic agents are used in 37.23% of the hospitals. Conclusion: This is the first national survey regarding FFP production and transfusion in Greece. Staff of internal medicine, general surgery, and ICU departments, where most FFP-transfused patients are hospitalized, should be regularly involved in training on contemporary transfusion guidelines. Upcoming centralization of FFP production and inventory management could help in homogenizing practices regarding FFP use and improve product quality. Strengthening the use of pharmaceutic hemostatic agents could improve patients' management.

A modified flow cytometry method for objective estimation of human CD4+ regulatory T cells (CD4+ Tregs) in peripheral blood, via CD4/CD25/CD45RO/FoxP3 labeling.

BACKGROUND: Several methods exist for flow-cytometric estimation of human peripheral blood CD4+ T regulatory cells (CD4+ Tregs). METHODS: We report our experience with the estimation of human CD4+ Tregs via three different characterizations using flow cytometry (CD25high FoxP3+ , CD25high CD127low/- FoxP3+ , and CD4+ CD25high/int CD45ROFoxP3+ ) in normal subjects. We have used these methods on the control populations from two studies (32 and 36 subjects, respectively), the latter two methods retrospectively on the subjects of the first study. The six CD4+ T cell fractions obtained by the third method were differentially colored to ascertain the distribution of these cell fractions in the CD25/FoxP3, CD45RO/FoxP3, and CD25/CD127 dot plots from CD4/CD25/CD45RO/FoxP3 and CD4/CD25/CD45RO/CD127 panels. RESULTS: Each approach gives significantly different estimates of Tregs (expressed as percentage of CD4+ T cells), with the second almost invariably yielding higher percentages than the other two. Only the third approach can distinguish among effector and naïve Tregs and FoxP3+ non-Tregs. Analysis of CD25/CD127 dot plots reveals that Treg delineation via the widely used definition of CD4+ CD25high CD127low/- cells unavoidably yields a mixture of nearly all effector and most of naïve Tregs, as well as FoxP3+ non-Tregs plus other cells. Delineation of effector/naïve Tregs and FoxP3+ non-Tregs is possible via CD45RO/CD25 dot plots but not by CD45RO/FoxP3 counterparts (as done previously) because of overlapping FoxP3 intensities among Tregs and non-Tregs. CONCLUSION: Our comparison shows that CD4/CD25/CD45RO/FoxP3 panels are an objective means of estimating effector and naïve Tregs via colored dot plots, aiding thus in Treg delineation in health and detecting aberrations in disease.

Platelets transfusion in Greece: Where, when, why? A national survey.

BACKGROUND: Platelet transfusion is among the most useful therapeutic tools in modern clinical settings which mean that ensuring an adequate supply is of paramount importance. AIM: The aim of our study was to record the use and wastage of platelet concentrates (PCs) in Greece, so as to come up with evidence-based interventions. METHODS: The study was conducted during May and June 2015. We evaluated the use of random-donor platelets (RDPs) and single-donor apheresis platelets (SDPs). We analyzed such parameters as hospital department and diagnosis, indication for transfusion, PCs' age at the time of transfusion, and wastage rate. RESULTS: We used data from 21 hospitals across the country. A total of 12,061 RDPs and 1189 SDPs were transfused, with an average of 4.84 (±2.72) and 1.12 (±2.73) units per episode, respectively. Most patients had been admitted to the internal medicine and hematology departments. The transfusions were mostly given prophylactically, usually in cases of acute leukemia, and mostly on the day before expiration. Wastage rate was 16.75% for RPDs and 2.70% for SDPs, primarily because of the expiration of the use-by date. CONCLUSIONS: This is the first national survey regarding platelet transfusion in Greece. Since most patients were admitted in internal medicine and hematology departments, we recommend that the staff of the abovementioned departments should undergo training on contemporary transfusion guidelines. Platelet discard rate could further be lowered through the centralization of inventory management along with the extension of the lifetime of PCs by means of emerging technologies.

Molecular characterization of occult hepatitis B cases in Greek blood donors.

The use of sensitive nucleic acid testing for hepatitis B virus in blood donors revealed a number of HBV DNA(+) cases among HBsAg(-) donors, a status known as occult HBV infection. The purpose of this study was the serological and molecular characterization of occult HBV infection in Greek blood donors. A prospective study was undertaken in order to identify occult HBV infection cases in blood donors. As part of the routine screening of blood donations in Greece, blood units were screened individually by a multiplex HIV-1/HCV/HBV nucleic acid assay. Initially reactive samples were retested with discriminatory assays. HBV DNA(+)/HBsAg(-) samples were tested further for HBV serological markers and HBV DNA was quantified by real-time PCR. Molecular characterization was performed by sequencing the envelope and polymerase genes of HBV. Preliminary screening revealed 21 occult cases with the following patterns: anti-HBc only: 7 donors, anti-HBc/anti-HBs: 7 donors, anti-HBc/anti-HBe: 5 donors, anti-HBc/anti-HBs/anti-HBe: 2 donors. In all cases, the HBV DNA load was <351 IU/ml. Sequencing was successful in 10 donors (classified within genotype D) revealing several amino acid substitutions related to diagnostic escape and antiviral resistance. HBsAg diagnostic failure and low viral replication in occult HBV infection carriers could possibly be attributed to multiple changes in envelope and polymerase regions, respectively.

High prevalence of elevated liver enzymes in blood donors: associations with male gender and central adiposity.

OBJECTIVE: Nonalcoholic fatty liver disease is an increasingly recognized condition, but its exact prevalence is unknown. In this prospective, multicenter study, we evaluated the prevalence of elevated alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyl-transpeptidase levels as indirect markers of nonalcoholic fatty liver disease in volunteer blood donors as well as their associations with epidemiological and anthropometrical characteristics. METHODS: Alanine aminotransferase, aspartate aminotransferase and gamma-glutamyl-transpeptidase levels were determined in blood donors from four transfusion centers during the morning sessions of a 3-month period. Cases with positive hepatitis B surface antigen, anti-hepatitis C virus, anti-HIV or elevated liver enzymes and alcohol abuse were excluded. RESULTS: Abnormal liver enzymes were found in 17.6% of 3063 participants (alanine aminotransferase: 14.5%, aspartate aminotransferase: 4.6%, gamma-glutamyl-transpeptidase: 4.7%). Individuals with abnormal compared with those with normal liver enzymes or alanine aminotransferase values were more frequently men and had higher weight, body mass index, waist, hip and neck circumference (P<0.001 for all comparisons). The prevalence of abnormal liver enzymes was also associated with the transfusion center ranging between 8.8 and 22.1% (P<0.001) and alcohol consumption (P=0.001). In multivariate analysis, presence of elevated enzymes was independently associated with male sex, higher weight or body mass index, higher waist circumference and transfusion center. CONCLUSIONS: More than 15% of Greek blood donors exhibit elevated liver enzymes, most likely as a result of unrecognized nonalcoholic fatty liver disease. The prevalence of nonalcoholic fatty liver disease is mainly associated with male sex, obesity and waist circumference, but it may range significantly among different population groups.

Antiplatelet antibodies in patients with chronic viral hepatitis receiving interferon-alpha.

BACKGROUND/AIMS: The aim of this study was to investigate the possible role of interferon-alpha in the development of antiplatelet IgG antibodies in patients with chronic viral hepatitis B or C. METHODOLOGY: Ninety-one consecutive patients with chronic viral hepatitis (51 with chronic hepatitis B and 40 with chronic hepatitis C) were investigated for the presence of antiplatelet IgG antibodies in their serum immediately prior to IFN-alpha therapy and after six months of therapy. The method used was the solid phase red cell adherence test (Immucor, Norcross, USA), which is a sensitive tracer of antiplatelet antibodies. Some of the results were confirmed using an indirect immunofluorescence test for the detection of antiplatelet antibodies RESULTS: Overall, we found that antiplatelet antibodies were present in 37.54% (19/51) of patients with chronic hepatitis B before IFN-alpha therapy and in 35.29% (18/51) after therapy. Moreover, antiplatelet antibodies were found in 20% (8/40) of patients with chronic hepatitis C before and after IFN-alpha therapy. CONCLUSIONS: Therapy with IFN-alpha did not induce antiplatelet antibodies in patients with chronic viral hepatitis B or C. Thrombocytopenia observed during IFN-alpha therapy in our study was not due to the development of antiplatelet antibodies.

HCV-RNA qualitative assay based on transcription mediated amplification improves the detection of hepatitis C virus infection in patients on hemodialysis: results from five hemodialysis units in central Greece.

BACKGROUND: End-stage renal disease patients (ESRD) on maintenance hemodialysis (HD) are at increased risk of acquiring hepatitis C virus (HCV) infection. An early and accurate diagnosis of HCV infection is important for the prevention of viral transmission and the management of ESRD patients on HD but conventional ELISA and PCR have often failed to reveal active HCV infection. OBJECTIVES: This study evaluated the prevalence of HCV infection in ESRD patients from all HD units in central Greece using a sensitive HCV-RNA transcription mediated amplification (TMA) assay and compared its sensitivity with that of anti-HCV ELISA. STUDY DESIGN: Anti-HCV antibody (third generation ELISA), HCV-RNA (TMA) and HCV genotypes (HCV TMA-LiPA) were determined in 366 ESRD Greek patients. RESULTS: In total, 132 (36%) ESRD patients were HCV positive by ELISA or TMA; 44 by TMA alone, 16 by ELISA alone and 72 positive by both assays. More than half of the viraemic patients had genotype 3a. CONCLUSIONS: HCV-RNA (TMA) assay appears to increase the accuracy in the diagnosis of HCV infection in HD patients compared to the anti-HCV ELISA and could serve as an additional screening tool in these patients.

Intrafamilial spread of hepatitis B virus infection in Greece.

OBJECTIVE: No study has investigated the intrafamilial spread of hepatitis B virus (HBV) in Greece. We conducted a 9-year prospective study to determine the rate of HBV spread in family members when a member is identified as an HBV carrier, the possible routes and risk factors for transmission of HBV and the family members with the highest risk of infection according to kinship degrees. METHODS: A total of 387 family members of 166 hepatitis B surface antigen (HBsAg) carriers were investigated for the detection of HBV infection markers using standard enzyme immunoassays; 6.696 blood donors from the same area were used as controls. RESULTS: Serological markers of past or current HBV infection were detected significantly more frequently among family members of HBsAg carriers (23.2 and 15.8%, respectively) compared with blood donors (14.1 and 0.85%, respectively). The prevalence of the above markers was higher among siblings, husbands and parents of the carriers. Offspring of the female index cases had higher rates of current or past infection. HBV infection markers were significantly increased in family members who reported common use of syringes (P<0.001), birth in rural areas (P<0.001) and a low level of education (P<0.001). CONCLUSIONS: We demonstrated a high risk of HBV transmission among family members of HBsAg carriers, which was associated with special risk factors for contracting HBV. Our findings indicate the need for strict adherence to the universal guidelines of vaccination against HBV and also the need for an immediate investigation of other potentially infected relatives among family members of HBsAg carriers.

Changes of serum adhesion molecules and cytokines in post-ERCP pancreatitis: adhesion molecules and cytokines in acute pancreatitis.

OBJECTIVES: To assess the early changes of soluble IFN-γ, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, TNF-α, TNF-ß, IL-17A, IL-22, soluble (s) P-Selectin, sE-Selectin and sICAM-1 in post-ERCP pancreatitis (PEP). METHODS: Single center, prospective study of 318 ERCP procedures. Serum samples were acquired from all patients prior to ERCP, 6 hours and 24 hours after the procedure. For every PEP case, another patient was chosen as a control, matched for gender, age and time period in which ERCP took place. RESULTS: Totally, 28 cases and 28 controls were studied. Except for significantly higher IL-1b levels in cases at baseline, no significant differences were observed between cases and controls after Bonferroni corrections. An increase in IL-6 was noted between baseline and 6 h in cases alone (p=0.016). There was a significant fall in sP-selectin levels at 6 and 24 hours compared to baseline in all patients (corrected p=0.008 and 0.016 for cases and 0.016 and 0.048 for controls respectively). An increase of sE-selectin in cases was observed between 6 and 24 hours post-ERCP (corrected p=0.03). CONCLUSIONS: Soluble forms of cytokines and adhesion molecules studied seem not to play a major role in PEP.

Platelet IgG antibodies are significantly increased in chronic liver disease.

BACKGROUND: The aim of this study was to investigate the presence of IgG antiplatelet (anti-P) IgG antibodies in patients with chronic liver disease (CLD) of diverse but well defined etiology. METHODS: One-hundred fifty-six consecutive patients with CLD (65 with chronic hepatitis B, 57 with chronic hepatitis C, 23 with alcoholic liver disease and 11 with primary biliary cirrhosis), and 240 healthy blood donors were investigated for the presence of anti-P antibodies. RESULTS: Anti-P antibodies were present in 36.5% (57/156) of patients with CLD, and 2.9% (7/240) of controls (P=0.0001). In detail, anti-P antibodies were detected in 35.4% (23/65) of patients with chronic hepatitis B, 26.3% (15/57) of patients with chronic hepatitis C, 47.8% (11/23) of patients with alcoholic liver disease and 72.7% (8/11) of those with primary biliary cirrhosis. The study also demonstrated the significantly higher prevalence of anti-P antibodies in patients with cirrhosis (53.0%) than in non cirrhotic patients (26.4%, P=0.0018). The association of anti-P antibodies with thrombocytopenia was inconsistent. CONCLUSIONS: This study showed a high prevalence of anti-P IgG antibodies in patients with CLD compared to healthy controls.

Inflammatory bowel disease and hepatitis B and C in Western Balkans: a referral centre study and review of the literature.

BACKGROUND AND AIMS: There is limited data on IBD patients diagnosed with viral hepatitis B and C. The aim of the study was to assess the prevalence of chronic HBV or HCV infection in IBD patients followed by our centre and to describe and review the course of bowel and liver disease during therapy. METHODS: Single centre retrospective study on 482 consecutive IBD patients. Laboratory investigation for HBV and HCV was performed with routine methods. Treatment protocols for HBV included IFNa and nucleot(s)ide administration and for HCV combined IFNa and ribavirin. RESULTS: We diagnosed 15 patients (15/482, 3.1%) with HBV or HCV. Of these, 11 were HBV (11/482, 2.3%) and 4 were HCV (4/482, 0.8%). Nine of eleven HBV patients received antiviral therapy (8 lamivudine, 1 IFNa). Five lamivudine patients were switched to tenofovir and in another one adefovir dipivoxil were added. Bowel disease was in remission in ten of the eleven HBV patients. One patient was diagnosed with carcinoid tumor. Two HCV patients received IFNa that was well tolerated. One HCV patient denied therapy and one died from hepatocellular cancer. Of the seven patients on azathioprine only one achieved sustained response. Four patients on Infliximab achieved bowel disease remission but experienced biochemical or virological flare. CONCLUSIONS: This study demonstrates that prevalence of HBV and HCV infection in a large IBD cohort from Western Balkans is compared to that of the background population. IBD patients under immunosuppressants may apparently be treated with safety if preventive antiviral treatment is administered.

Hepatitis B remains a major health priority in Western Balkans: results of a 4-year prospective Greek-Albanian collaborative study.

OBJECTIVE OF THE STUDY: To report on the results of two projects on chronic hepatitis B in Western Balkans lead by Ioannina, Northwest Greece and Tirana, Albania. METHODS: In two prospective projects, HEPAGA I and HEPAGA II which lasted 4 years. In HEPAGA I, serum samples from 410 Albanians were tested for HBV. In HEPAGA II, health care consumption was recorded in hospitalized patients with chronic hepatitis B. RESULTS: HEPAGA I showed that 11.89% of the Albanians was HBsAg(+) and only 21.19% had HBV immunoprotection. HEPAGA II study included 101 patients. There was a significant difference in hospitalization costs per patient between centers. The Greek patients were significantly older (p=0.027) and there was a significant correlation between age >50 years and hospitalization costs (p=0.035). In Greece, hospitalization costs, number of patients admitted and number of hospitalization days per patient were in a remarkable position compared to other causes of hospitalization. CONCLUSIONS: The HEPAGA I study showed a decrease in the prevalence of chronic HBV infection in Albania compared to that of the previous decade. The HEPAGA II study demonstrated that health care consumption due to HBV infection is still an important determinant of the overall health consumption in Western Balkans.