The Use of Blood Products in Adult Patients with Burns.

INTRODUCTION: Burn anemia represents a common complication following a burn injury. Burn anemia etiology carries distinct features occurring at each stage of the post-injury and treatment periods resulting from different causes. We aimed to analyze the use of blood components in Finnish burn victims and to identify patient- and injury-related factors influencing their use. METHODS: To study the use of blood products in burn patients, we used data collected from the Optimal Use of Blood registry, developed through co-operation between 10 major hospital districts and the Finnish Red Cross Blood Service. Burn patients ⩾18 years treated at the Helsinki University Hospital between 2005 and 2011 with an in-hospital stay ⩾1 day who received at least one transfusion during their hospital stay were included in this study. RESULTS: Among all 558 burn patients, 192 (34%) received blood products during their hospital stay. The transfused cohort comprised 192 burn patients. The study cohort received a total of 6087 units of blood components, 2422 units of leukoreduced red blood cells, 1728 units of leukoreduced platelets, and 420 units of single-donor fresh frozen plasma or, after 2007, 1517 units of Octaplas(®) frozen plasma. All three types of blood components were administered to 29% of patients, whereas 45% received only red blood cells and 6% received only Octaplas. Transfused patients were significantly older (p < 0.001), experienced fire-/flame-related accidents and burns to multiple locations (p < 0.001), and their in-hospital mortality exceeded that for non-transfused burn patients fivefold (p < 0.05). DISCUSSION: We show that Finnish adult burn patients received ample transfusions. The number of blood components transfused varied according to the anatomical location of the injury and patient survival. Whether the additional mortality is related directly to transfusions or is merely a manifestation of the more severe burn injury remains unknown.

CARD15/NOD2 gene variants are associated with familially occurring and complicated forms of Crohn's disease.

BACKGROUND: Variants of the caspase activating recruitment domain 15/nucleotide oligomerisation domain 2 (CARD15/NOD2) gene have been associated with susceptibility to Crohn's disease (CD). AIM: Our aim was to evaluate the allele frequencies of the CARD15 variants R702W, G908R, and 1007fs in Finnish inflammatory bowel disease (IBD) patients and to search for possible associations between CARD15 variants and occurrence of familial forms of IBD or complicated forms of CD. PATIENTS AND METHODS: We investigated 198 sporadic CD patients, 46 probands with familial CD, 27 CD probands from mixed IBD families, 99 unrelated patients with ulcerative colitis (UC), and 300 control individuals for the occurrence of the CARD15 gene variants R702W, G908R, and 1007fs. RESULTS: In CD patients, the allele frequencies for the rare variants of these polymorphisms were 3.3%, 0.6%, and 4.8% (total 8.7%), and the corresponding frequencies in healthy controls were 1.8%, 0%, and 1.7% (total 3.5%) (8.7% v 3.5%; p<0.01). In UC patients allele frequencies were comparable with those in controls. The frequency of the 1007fs polymorphism variant allele was significantly higher among all CD patients than in controls (4.8% v 1.7%; p<0.01) but there was no significant difference in allele frequencies between the CD and UC groups. The 1007fs allele frequency was higher in familial CD than in non-familial cases with CD (10.9% v 3.5%; p<0.01). There were no significant differences in the allele frequencies of the R702W and G908R polymorphisms between CD patients, UC patients, and controls. We found that 15.5% of CD patients, 9.1% of UC patients, and 6.7% of controls carried at least one of the CARD15 variants. In CD patients carrying at least one of the three NOD2 variants, the ileum was affected more often than in non-carrier CD patients (90% v 73%; p<0.05), they had stricturing or penetrating disease more often than non-carriers (88% v 56%; p<0.01), and they had an increased need for bowel surgery. CONCLUSIONS: The frequency of NOD2 gene variants was lower in genetically homogenous Finns than in other populations. The 1007fs variant was associated with CD. The occurrence of CARD15 variants predicted ileal location as well as stricturing and penetrating forms of CD.

Initial P-glycoprotein expression in childhood acute lymphoblastic leukemia: no evidence of prognostic impact in follow-up.

Treatment results in childhood acute lymphoblastic leukemia (ALL) have improved remarkably during the past 20 years, but still 25% of children cannot be permanently cured. Drug resistance is a major cause of poor outcome. One of the most investigated resistance mechanisms is the P-glycoprotein (P-gp)-mediated multiple-drug resistance (MDR). The authors prospectively analyzed P-gp using flow cytometry with monoclonal antibody JSB1 in a population-based series of 103 children with ALL treated according to intensive Nordic ALL protocols. Increased P-gp expression was detected in 55 patients (53%). With a cutoff value of 1% P-gp-positive blasts in bone marrow, no difference was found in event-free survival (EFS) or overall survival between children with low vs. increased P-gp expression. The 4-year EFS in the whole series was 77%. Patients with T-ALL had higher P-gp levels than the others, 3.6% vs. 1.0% (p = .002). P-gp expression did not correlate with the white blood cell count, age, sex, or cytogenetics. The authors conclude that the level of P-gp expression cannot be used as a tool for treatment stratification in childhood ALL.

Viral markers and use of factor products among Finnish patients with bleeding disorders.

In the seventh national voluntary cross-sectional survey (in 1999) of Finnish patients with haemophilia A or B, type 3 von Willebrand disease or factor XIII deficiency, a plasma sample was received from 193 patients (67%). The samples were tested for hepatitis B and C, human immunodeficiency virus (HIV) and human T-cell leukaemia virus (HTLV) antibodies. Fifty-one percent of the patients were hepatitis C antibody positive and 34% hepatitis B core antibody positive. None of the patients had antibodies against HIV or HTLV. Eighteen percent of the patients had an elevated alanine aminotransferase activity. Abnormal alanine aminotransferase was significantly associated with hepatitis C seropositivity. No new seroconversions were detected among the haemophiliacs or patients with type 3 von Willebrand disease when compared with the last two surveys in 1993 and 1996, and there was no seroconversion in sole users of solvent/detergent-treated factor products. Currently, 32% of the patients use prophylactic factor treatment as their principal mode of therapy, particularly the younger patients with severe forms of the bleeding diseases.

Multiple drug resistance mediated by P-glycoprotein is not a major factor in a slow response to therapy in childhood ALL.

In childhood acute lymphoblastic leukemia (ALL), early response to treatment is an important prognostic factor and drug resistance is a major cause of poor outcome. One of the most investigated resistance mechanisms is P-glycoprotein (P-gp)-mediated multiple drug resistance (MDR). We analyzed P-gp using flow cytometry with monoclonal antibody JSB1 in a series of 118 children with ALL, 103 at diagnosis and 15 at relapse. Increased P-gp expression was found in 55 (53%) patients at diagnosis and in 11 (73%) at relapse. We also analyzed the bone marrow aspirate slides for early response to treatment in a central review. No correlation was found between P-gp and early response. Patients with T-ALL had higher P-gp levels than the others, 5.3% versus 1.0% (P = .002). We conclude that P-gp-mediated multiple drug resistance is not a factor in a slow response to ALL induction therapy.

Retesting of bone donors 2 months after donation guarantees sufficient safety of bone allografts.

Both allogeneic bone grafting and blood transfusion may transmit infections from the donor to the recipient. The most effective means to reduce the risk of infection is careful donor selection and screening of donors for markers of infection. The risk of blood transfusion-transmitted HIV infection in Finland, calculated with the incidence/window period model, is approximately 1:3,300,000. The calculated risk for hepatitis B (HBV) and C (HCV) is 1:217,000 and 1:147,000 donations, respectively. In bone banking we can further reduce the risks by retesting the living donors. Retesting 2 months after donation seems to be sufficient, at least in countries with a low incidence of transplantation-transmitted infections.

Clinical significance of hepatitis C antibodies in blood donors.

The clinical significance of hepatitis C antibodies (anti-HCV) in a healthy population was studied by liver function tests and liver biopsies. The patient population consisted of 195 (96.1%) of the 203 blood donors found to be either anti-HCV positive or indeterminate by a recombinant immunoblot assay (RIBA) during the first year of anti-HCV screening of 307,606 donors in Finland using a first generation enzyme-linked immunosorbent assay. Alanine aminotransferase (ALT) levels in 67 donors reacting positively and in 128 reacting indeterminately by a second generation RIBA (RIBA-4) were monitored to evaluate the prevalence of liver damage. Serum N-terminal type III procollagen (PIIINP) concentrations were measured in all donors who fulfilled our criterion for possible hepatitis C (ALT values over two times the normal upper limit on two occasions or over five times the normal upper limit on one occasion) and in 23 randomly selected RIBA-4 positive donors without ALT abnormalities (control group). Two (1.6%) of the RIBA-4 indeterminate donors had ALT values compatible with possible hepatitis C (negative by polymerase chain reaction) whereas there were 25 (37.3%) such individuals among the RIBA-4 positive donors (P < 0.0005). Twenty (80%) of the latter 25 RIBA-4 positive donors with possible hepatitis C consented to liver biopsy. Of these 20 donors, 11 (55.0%) were found to have chronic persistent hepatitis, four (20.0%) mild, three (15.0%) moderate, and two (10.0%) severe chronic active hepatitis.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric analysis of glutathione-S-transferase-pi in acute leukemia.

Increased expression of glutathione-S-transferase isoenzyme pi (GST-pi) may account for drug resistance and treatment failure in hematologic malignancies when alkylating agents like cyclophosphamide, chlorambucil, busulfan and melphalan, or doxorubicin are used. We have studied the expression of GST-pi in peripheral blood lymphocytes of healthy blood donors. In peripheral and bone marrow lymphocytes/blasts of patients with other diseases than hematologic malignancies, and of patients with acute leukemia by using flow cytometry. We studied bone marrow cells of 35 patients diagnosed as having acute leukemia at initial presentation, 16 patients in the refractory stage, 20 in morphological remission and 15 controls. None of the samples obtained in remission contained more GST-pi-positive cells than the controls, whereas 51% of the samples obtained at diagnosis and 56% of those obtained in the refractory stage were GST-pi-positive. The mean proportion of GST-pi-positive cells in the lymphocyte/blast cell gate of bone marrow cells of controls was 2.6% and of patients with acute leukemia studied at diagnosis 16.6%, respectively. We analyzed the samples also for P-glycoprotein expression. There was a significant positive association between GST-pi and P-glycoprotein expression in acute leukemia.

Hepatitis C antibodies in dialysis patients and patients with leukaemia.

The prevalence of hepatitis C antibodies (anti-HCV) among multitransfused patients was studied and compared with predicted values obtained from a post-transfusion hepatitis study and from data on the prevalence of anti-HCV among blood donors. The prevalence of hepatitis B core antibodies (anti-HBc) was also studied to determine the routes of transmission of hepatitis C virus. The patients consisted of 65 dialysis patients (57 on haemodialysis and 8 on continuous ambulatory peritoneal dialysis) and 71 leukaemia patients in long-term remission [49 with acute myeloid leukaemia (AML) and 22 with acute lymphatic leukaemia (ALL)]. The presence of anti-HCV was investigated using a second generation enzyme-linked immunosorbent assay. Reactive samples were confirmed by a second generation recombinant immunoblot assay. Anti-HBc was studied in the 65 dialysis patients and in 40 of the leukaemia patients. Three (4.6%) of the 65 dialysis patients and 12 (24.5%) of the 49 AML patients were anti-HCV positive whereas all of the ALL patients were seronegative. The total number of blood units transfused to 134 patients (data on two dialysis patients were not available) was 18,148, out of which 17,575 units had been transfused prior to the initiation of anti-HCV screening of blood donors. On the basis of the anti-HCV prevalence among blood donors and the incidence of post-transfusion hepatitis, the predicted number of seropositive patients was 11 and 18, respectively. Five of the 65 dialysis patients were anti-HBc positive, compared with only one of the 40 leukaemia patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical significance of P-glycoprotein expression in acute leukaemia as analysed by immunocytochemistry.

Multidrug resistance, mediated by the overexpression of an energy-dependent transport protein, P-glycoprotein, has been one of the major targets of interest in solving the mechanisms of clinical drug resistance of malignant cells. To evaluate the correlation between P-glycoprotein overexpression and the response to chemotherapy, we analysed cytospin preparations of gradient-separated blood or bone marrow mononuclear cells from 79 patients with acute leukaemia by means of the P-glycoprotein-directed monoclonal antibody JSB-1 and immunocytochemistry using the alkaline phosphatase-antialkaline phosphatase technique. P-glycoprotein expression was detected in all disease phases of acute leukaemia. Thirteen out of 51 patients at diagnosis, 10/29 patients in relapse or during residual disease and 8/27 patients in remission overexpressed P-glycoprotein. Seven out of the 8 positive remission samples were collected between the cycles of consolidation treatment. Our results suggest that increased P-glycoprotein expression in samples collected between the cycles of consolidation treatment during remission may be induced in normal leukocytes by cytotoxic drug treatment, infections, or by some physiological mechanisms related to the disease. Patients older than 45 years of age were significantly more often P-glycoprotein-positive (11/25) at diagnosis than younger patients (2/26). P-glycoprotein expression at diagnosis was significantly correlated with a low remission rate after the first cycle of induction therapy. Of 34 P-glycoprotein-negative patients, 25 achieved remission after the first cycle as compared to 4/12 of the P-glycoprotein-positive patients. Our results indicate that the method used is specific and sensitive enough for the analysis of P-glycoprotein expression and that the expression at initial presentation is inversely correlated with the outcome of induction therapy.

Flow cytometric analysis of terminal deoxynucleotidyl transferase. A simplified method.

The authors evaluated a new cell membrane permeabilization method for the flow cytometric detection of terminal deoxynucleotidyl transferase (TdT). In this method, gradient-separated leukocytes or unseparated blood or bone marrow cells were incubated in a commercially available diethylene glycol-based red blood cell lysing solution, which not only lyses red blood cells, but also permeabilizes leukocyte cell membranes; the light scattering properties of the cells are retained. The validity of the current method was demonstrated by the good concordance of the findings with previously published data as follows: (1) practically identical results were obtained when an established method for cell permeabilization was used in parallel on the same samples; (2) the proportion of TdT-positive cells in normal peripheral blood was negligible; (3) the proportion of TdT-positive cells in normal bone marrow averaged 1%, and a significant portion of TdT-positive cells in normal bone marrow expressed CD10 and CD34; and (4) TdT-positive cell populations were seen with the expected frequencies in various types of leukemia. This method for TdT flow cytometry provides significant advantages over previously used methods and is especially suitable for TdT detection in routine laboratories.

Flow cytometric analysis of P-glycoprotein in normal and leukemic cells.

Classical multidrug resistance is characterized by overexpression of a membrane protein, P-glycoprotein, which acts like a drug-extruding pump, reducing accumulation of cytotoxic drugs inside malignant cells. We have developed a simple method for detecting an intracellular epitope of P-glycoprotein in normal and leukemic cells by the monoclonal antibody JSB-1 and fluorescence-activated flow cytometry. Permeabilization of blood and bone marrow cells in unprocessed samples is achieved by a commercially available red blood cell lysing solution which excellently preserves the light scatter properties of leukocytes. The method is suitable for analyzing samples in clinical routine. Lower than 1% reactivity was seen in the lymphoid gate of normal peripheral blood and bone marrow samples as compared with over 60% of reacting cells in some leukemic samples. Twelve patients with acute de novo leukemia were studied at presentation, 13 patients at a refractory stage, and 28 in remission. There was a positive correlation between the P-glycoprotein and the CD34 expression in acute myelogenous leukemia and an association between the P-glycoprotein expression and the blast count in both acute myelogenous and lymphatic leukemias.

Expression of decorin in human tissues and cell lines and defined chromosomal assignment of the gene locus (DCN).

Earlier studies had shown that the expression of the gene coding for one eminent connective tissue proteoglycan, decorin (DCN), is deficient in the fibroblasts of 3 out of 15 Marfan patients (Pulkkinen et al., 1990). To obtain more information on the expression of this gene, various human tissues and cell lines were studied. High mRNA levels of decorin were detected in aorta, lung, skin, kidney, smooth muscle, and placenta, whereas significantly lower mRNA levels were found in the rest of the tissues analyzed. Two sizes of transcripts were observed in all tissues. The two transcripts of decorin most probably do not represent two different genes, since in situ hybridization gave only one strong signal, placing the gene in 12q21----q22. No tissue-specific differences in the two mRNA species of decorin were detected. This is in contrast to the gene of versican, another connective tissue proteoglycan gene, that was analyzed as a control; high expression of a longer transcript of the versican gene was found in brain and smooth muscle, whereas the shorter transcript was predominant in all other tissues studied. DCN was actively transcribed in cultured mesenchymal cells, whereas in cells of endothelial or epithelial origin, the transcription level was undetectable. These tissue- and cell type-specific variations in the expression of DCN may help to explain the complex phenotypic variation typical of individuals with Marfan syndrome.

Risk factors for hepatitis C virus antibody positivity in blood donors in a low-risk country.

Demographic variables, sexual risk behavior and prevalence of parenteral risk factors were studied in 305 randomly selected donors seronegative for hepatitis C virus, in 170 randomly selected donors reactive on solely enzyme-linked immunosorbent assay (ELISA C-100), in 71 consecutive donors reacting indeterminately according to the second-generation recombinant immunoblot assay (RIBA II) and in 46 consecutive donors found to be positive using the RIBA II. Donors who were positive by RIBA II had significantly more often a risk factor, for example use of intravenous drugs or previous blood transfusion, than donors reacting indeterminately (34 out of 46) (73.9%) versus 14 out of 71 (19.7%, p = 0.0000). Donors reacting indeterminately by RIBA II had one of those risk factors significantly more often than seronegative donors (14 out of 71) (19.7%) versus 23 out of 280 (7.8%, p < 0.005). When donors either positive or indeterminate by RIBA II were compared with donors negative for hepatitis C antibodies, the odds ratio for a possible parenteral source of infection was 7.6 (p = 0.0000). Subjects who had received a poor education (odds ratio 0.3, p < 0.001) or who lived in southern Finland (odds ratio 2.3, p < 0.05) were also at higher risk for being positive or indeterminate in RIBA II. First-time donors were also prone to having antibodies according to RIBA II (odds ratio 2.2, p = 0.1), whereas sexual risk behavior, gender, age, occupational class and type of residential area were not risk factors for hepatitis C antibodies in RIBA.(ABSTRACT TRUNCATED AT 250 WORDS)

Multidrug resistance.

Resistance of malignant cells to cytotoxic agents is often a limiting factor to successful chemotherapy. The classical multidrug resistance is characterised by overexpression of a membrane protein, P-glycoprotein, which acts like a drug extruding pump reducing accumulation of cytotoxic agents inside malignant cells, thereby preventing their function. Resistance is expressed simultaneously towards several structurally unrelated drugs. P-glycoprotein is also expressed in many normal human tissues, e.g., in the gastrointestinal tract, and this may be the reason for intrinsic resistance observed clinically in cancers derived from certain tissues. More often multidrug resistance is acquired during chemotherapy. The physiological function of P-glycoprotein is still unknown but it may have a role in cellular detoxification and secreting mechanisms. Interest in the phenomenon of multidrug resistance centres on the correlation of P-glycoprotein expression to clinical drug resistance. Another goal is to find mechanisms by which the function of P-glycoprotein as a multidrug transporter is prevented and drug resistance reversed.