Colorectal carcinoma: influence of systemic cytokine concentrations and pathohistological characteristics on anemia and thrombocytosis

U studiji su ispitivane sistemske koncentracije imunomodulacijskih proteina/interleukina i kliničko-patohistološke karakteristike bolesti i povezane su sa anemijom/trombocitozom kod obolelih od kolorektalnog karcinoma (CRC). Ispitanici sa CRC-om, su razvrstani u grupu sa anemijom, odnosno sa trombocitozom (eksperimentalne grupe) i grupu bez anemije, odnosno bez trombocitoze (kontrolne grupe). U ovako determinisanim skupovima ispitanika analizirane su kliničke (kompletna krvna slika sa brojem krvnih ćelija, vrednost hemoglobina, preocena aktivnosti bolesti, kompletan radiološki pregled, prisustvo komplikacija i pojava bolesti van gastrointestinalnog trakta) i patohistološke karakteristike bolesti (tip tumora, nuklearni i histološki gradus, TNM klasifikacija), kao i koncentracija citokina i imunomodulacijskih proteina (IL-1, IL-4, IL-10, IL-17, IL-33, galektina-1, galektina-3, TNF-α i INF-γ) i biohemijskih belega maligne bolesti (AFP,.CEA,.CA.19-9) u serumu bolesnika. Bolesnici sa CRCom i anemijom, odnosno trombocitozom imaju teži kliničko-patohistološki oblik bolesti (viši nuklearni i histološki gradus tumora, veći stepen invazije limfnih i krvnih sudova, viši TNM stadijum bolesti, više otkrivenih metastaza u plućima, jetri ili peritoneumu), u poređenju sa bolesnicima sa CRC-om bez anemije, odnosno bez trombocitoze. Takođe, utvrđena je pozitivna korelacija anemije, odnosno trombocitoze i sistemskih vrednosti interleukina, imunomodulacijskih proteina i biohemijskih belega maligne bolesti (AFP,.CEA,.CA.19-9) uhserumu bolesnika. Postoji pozitivna korelacija između sistemskih vrednosti imunomodulacijskih proteina i težeg kliničko-patohistološkog oblika bolesti, u odnosu na prisustvo anemije, odnosno trombocitoze kod bolesnika sa kolorektalnim karcinomom.We investigated systemic concentrations of immunomodulatory proteins/interleukins, as well as clinical and histopathological characteristics of the disease and correlated them with anemia/thrombocytosis in patients with colorectal cancer (CRC). The study included patients with colorectal cancer, divided into a group with anemia and a group with thrombocytosis (experimental groups) and a group without anemia, or without thrombocytosis (control groups). In such determined groups, clinical (complete blood count with blood cell count, hemoglobin value, disease activity, complete radiological examination, complications and occurrence of diseases outside the gastrointestinal tract) and pathohistological characteristics of the disease (tumor type, nuclear and histologic grade, TNM classification), as well as concentrations of cytokines and immunomodulatory proteins (IL-1, IL-4, IL-10, IL-17, IL-33, galectin-1, galectin-3, TNF-α and INF-γ) and biochemical indicators of malignant disease (AFP,.CEA,.CA.19-9) in the patients’ serum were analyzed. Patients with CRC and anemia/thrombocytosis, have a more severe clinical-pathohistological form of the disease (higher nuclear and histological grade of tumor, higher lymph and blood vessels invasion, higher TNM stage of the disease, more detectable lung, liver or peritoneum metastasis) compared to CRC patients without anemia or thrombocytosis. Moreover, a positive correlation between anemia/thrombocytosis, and cytokines, AFP,.CEA and.CA.19-9 values in the patients’ serum was determined. There is a positive correlation between the systemic values of immunomodulatory proteins and severe clinical-pathohistological form of the disease, based on the presence of anemia or thrombocytosis in CRC patients

Mirasol PRT system inactivation efficacy evaluated in platelet concentrates by bacteria-contamination model

Background/Aim. Bacterial contamination of blood components, primarily platelet concentrates (PCs), has been identified as one of the most frequent infectious complications in transfusion practice. PC units have a high risk for bacterial growth/multiplication due to their storage at ambient temperature (20 ± 2°C). Consequences of blood contamination could be effectively prevented or reduced by pathogen inactivation systems. The aim of this study was to determine the Mirasol pathogen reduction technology (PRT) system efficacy in PCs using an artificial bacteria-contamination model. Methods. According to the ABO blood groups, PC units (n = 216) were pooled into 54 pools (PC-Ps). PC-Ps were divided into three equal groups, with 18 units in each, designed for an artificial bacteria-contamination. Briefly, PC-Ps were contaminated by Staphylococcus epidermidis, Staphylococcus aureus or Escherichia coli in concentrations 102 to 107 colony forming units (CFU) per unit. Afterward, PC-Ps were underwent to inactivation by Mirasol PRT system, using UV (l = 265-370 nm) activated riboflavin (RB). All PC-Ps were assayed by BacT/Alert Microbial Detection System for CFU quantification before and after the Mirasol treatment. Samples from non-inactivated PC-P units were tested after preparation and immediately following bacterial contamination. Samples from Mirasol treated units were quantified for CFUs one hour, 3 days and 5 days after inactivation. Results. A complete inactivation of all bacteria species was obtained at CFU concentrations of 102 and 103 per PC-P unit through storage/ investigation period. The most effective inactivation (105 CFU per PC-P unit) was obtained in Escherichia coli setting. Contrary, inactivation of all the three tested bacteria species was unworkable in concentrations of ≥ 106 CFU per PC-P unit. Conclusion. Efficient inactivation of investigated bacteria types with a significant CFU depletion in PC-P units was obtained - 3 Log for all three tested species, and 5 Log for Escherichia coli. The safety of blood component therapy, primarily the clinical use of PCs can be improved using the Mirasol PRT system

Principles of NAT technology in view of the results obtained at the Institute of Transfusiology of MMA from 2007 till 2011

Nucleic-acid Amplification Testing (NAT) consist of extraction, amplification, hybridization and detection. Polymerase Chain Reaction (PCR) is method for amplification of viral genetic material (DNA/RNA) in enough number of copies, so we can detect them. Since year 1943, when transmission of hepatitis via transfusion was reported till beginning of 21st century, when NAT was implemented, large number of tests for detection of hepatitis B and C virus (HBV, HCV) and human immunodeficiency virus (HIV) were developed. In this article, we describe basic princples of detection of these viruses by NAT. We also show results from testing samples of blood donors in Institute of Transfusiology from April 2007 till June 2011. From 50 369 donors testing in Mini Pools (MP) of 24, we found two HCV RNA positive samples, one HBV DNA positive sample and all pools were HIV RNA negative. From 2 689 samples which were testing by Individual Donation (ID) NAT, 135 were HBV DNA positive, 108 were HCV RNA positive and 7 were HIV RNA positive. All samples also were tested by Enzyme Link Immunosorbent Assay (ELISA) and Confirmatory tests. Comparison of our results show validity of NAT testing, especially when ELISA tests of less specificity and sensitivity were used.Da bi testiranje nukleinskih kiselina bilo moguće, neophodno je izdvojiti ih iz virusa (ekstrakcija), zatim umnožiti (amplifikacija), obeležiti specifičnim probama (hibridizacija) i detektovati. To se obavlja korišćenjem metode 'Polymerase Chain Reaction' (PCR) ili lančane reakcije polimeraze koja nam omogućava da umnožimo deo virusnog genoma do količine koju je moguće detektovati. Od 1943. godine, kada je transmisija hepatitisa preko transfuzije prvi put prijavljena, do početka 21. veka, kada je masovno uvedena NAT tehnologija (eng. Nucleic-acid Amplification Testing - testiranje amplifikovanih nukleinskih kiselina), došlo je do značajnog poboljšanja u otkrivanju virusa uzročnika hematogenih transmisivnih bolesti. U ovom radu dati su osnovni principi NAT-a, odnosno detekcije genetskog materijala virusa uzročnika hepatitisa tipa B, C i virusa humane imunodeficijencije (HIV-a). Takođe, opisani su NAT rezultati dobijeni kod uzoraka krvi davalaca u Institutu za transfuziologiju VMA u periodu od aprila 2007. do juna 2011. godine. Od 50.369 testiranih davalaca u pulu, detektovana su dva HCV RNK pozitivna uzorka, jedan HBV DNK pozitivan uzorak, a za HIV RNK svi pulovi su bili negativni. Od 2.689 pojedinačno obavljenih NAT testiranja, najviše pozitivnih bilo je za HBV DNK (135 uzoraka), zatim za HCV RNK (108 pozitivnih) i 7 pozitivnih za HIV RNK. Za sve navedene uzorke paralelno su rađeni određeni enzimoimunski i potvrdni testovi. Komparacija rezultata primenjenih testova na našim ispitanim uzorcima potvrdila je opravdanost primene NAT-a, posebno prilikom korišćenja manje specifičnih i osetljivih enzimoimunskih testova.nul

Morphometric Analysis of Nonsclerosed Glomeruli Size and Connective Tissue Content during the Aging Process

Number of sclerotic glomeruli increases during the aging process. Consequently, majority of remained nonsclerosed glomeruli become hypertrophic and some of them sclerotic, too. The aim of this study was to quantify the size and connective tissue content of nonsclerosed glomeruli and to evaluate the percentage of hypertrophic ones in examined human cases during the aging. Material was right kidney's tissue of 30 cadavers obtained during routine autopsies. Cadavers were without previously diagnosed kidney disease, diabetes, hypertension, or any other systemic disease. Tissue specimens were routinely prepared for histological and morphometric analysis. Images of the histological slices were analyzed and captured under 400x magnification with digital camera. Further they were morphometrically and statistically analyzed with ImageJ and NCSS-PASS software. Multiple and linear regression of obtained morphometric parameters showed significant increase of glomerular connective tissue area and percentage. Cluster analysis showed the presence of two types of glomeruli. Second type was characterized with significantly larger size, connective tissue content, and significantly lower cellularity, in relation to the first type. Such glomeruli might be considered as hypertrophic. First type of glomeruli was predominant in younger cases, while second type of glomeruli was predominant in cases older than 55 years

Colorectal carcinoma: influence of systemic cytokine concentrations and pathohistological characteristics on anemia and thrombocytosis

U studiji su ispitivane sistemske koncentracije imunomodulacijskih proteina/interleukina i kliničko-patohistološke karakteristike bolesti i povezane su sa anemijom/trombocitozom kod obolelih od kolorektalnog karcinoma (CRC). Ispitanici sa CRC-om, su razvrstani u grupu sa anemijom, odnosno sa trombocitozom (eksperimentalne grupe) i grupu bez anemije, odnosno bez trombocitoze (kontrolne grupe). U ovako determinisanim skupovima ispitanika analizirane su kliničke (kompletna krvna slika sa brojem krvnih ćelija, vrednost hemoglobina, preocena aktivnosti bolesti, kompletan radiološki pregled, prisustvo komplikacija i pojava bolesti van gastrointestinalnog trakta) i patohistološke karakteristike bolesti (tip tumora, nuklearni i histološki gradus, TNM klasifikacija), kao i koncentracija citokina i imunomodulacijskih proteina (IL-1, IL-4, IL-10, IL-17, IL-33, galektina-1, galektina-3, TNF-α i INF-γ) i biohemijskih belega maligne bolesti (AFP,.CEA,.CA.19-9) u serumu bolesnika. Bolesnici sa CRCom i anemijom, odnosno trombocitozom imaju teži kliničko-patohistološki oblik bolesti (viši nuklearni i histološki gradus tumora, veći stepen invazije limfnih i krvnih sudova, viši TNM stadijum bolesti, više otkrivenih metastaza u plućima, jetri ili peritoneumu), u poređenju sa bolesnicima sa CRC-om bez anemije, odnosno bez trombocitoze. Takođe, utvrđena je pozitivna korelacija anemije, odnosno trombocitoze i sistemskih vrednosti interleukina, imunomodulacijskih proteina i biohemijskih belega maligne bolesti (AFP,.CEA,.CA.19-9) uhserumu bolesnika. Postoji pozitivna korelacija između sistemskih vrednosti imunomodulacijskih proteina i težeg kliničko-patohistološkog oblika bolesti, u odnosu na prisustvo anemije, odnosno trombocitoze kod bolesnika sa kolorektalnim karcinomom.We investigated systemic concentrations of immunomodulatory proteins/interleukins, as well as clinical and histopathological characteristics of the disease and correlated them with anemia/thrombocytosis in patients with colorectal cancer (CRC). The study included patients with colorectal cancer, divided into a group with anemia and a group with thrombocytosis (experimental groups) and a group without anemia, or without thrombocytosis (control groups). In such determined groups, clinical (complete blood count with blood cell count, hemoglobin value, disease activity, complete radiological examination, complications and occurrence of diseases outside the gastrointestinal tract) and pathohistological characteristics of the disease (tumor type, nuclear and histologic grade, TNM classification), as well as concentrations of cytokines and immunomodulatory proteins (IL-1, IL-4, IL-10, IL-17, IL-33, galectin-1, galectin-3, TNF-α and INF-γ) and biochemical indicators of malignant disease (AFP,.CEA,.CA.19-9) in the patients’ serum were analyzed. Patients with CRC and anemia/thrombocytosis, have a more severe clinical-pathohistological form of the disease (higher nuclear and histological grade of tumor, higher lymph and blood vessels invasion, higher TNM stage of the disease, more detectable lung, liver or peritoneum metastasis) compared to CRC patients without anemia or thrombocytosis. Moreover, a positive correlation between anemia/thrombocytosis, and cytokines, AFP,.CEA and.CA.19-9 values in the patients’ serum was determined. There is a positive correlation between the systemic values of immunomodulatory proteins and severe clinical-pathohistological form of the disease, based on the presence of anemia or thrombocytosis in CRC patients

Expression of collagen type IV in human kidney during prenatal development

Background/Aim. Type IV collagen belongs to the group of nonfibrillar collagens and is an important component of the basement membranes, where it accounts for approximately 50% of its structural elements. The aim of the study was to describe the expression and distribution of collagen type IV in the embryonic and fetal metanephric kidney and to determine the volume density of collagen type IV in kidney tissue in each trimester of development. Methods. The material consisted of 19 human embryos/fetuses, in the gestational age from 8th to 37th week. Kidney tissue specimens were routinely processed to paraffin molds, stained immunohistochemically using polyclonal anti-collagen IV antibody and counterstained with Mayer hematoxylin and eosin. Stained slides were examined using a light microscope, and images of the selected areas under different lens magnification were captured with a digital camera. Volume density of collagen type IV was determined using ImageJ 1.48v and a plugin of the software, which inserted a grid system with 336 points. For the data comparison, the One-Way Analysis of Variance (ANOVA) was used. Results. Strong collagen IV immunopositivity was seen in all specimens, with a distribution in the basement membranes of urinary bud, parietal leaf of Bowman’s capsule, glomerular basement membrane, basement membrane of interstitial blood vessels, and basement membranes of nephron tubules and collecting ducts. No statistically significant difference in the volume density of type IV collagen was found among the different trimesters of the embryonic and fetal development. Conclusion. The synthesis and secretion of collagen type IV simultaneously follow the development of nephron structures, collecting system and blood vessels. The volume density of collagen type IV remains constant throughout all the trimesters of metanephric kidney development, indicating that it plays a crucial role in the normal development of nephron and collecting system structures, as well as in maintaining the normal kidney function

Stem cell harvesting protocol research in autologous transplantation setting: Large volume vs. conventional cytapheresis

Background/Aim. The use of peripheral blood as a source of hematopoietic stem cells (SCs) is progressively increasing and has nearly supplanted bone marrow transplantation. Interpatient variability in the degree and kinetics of SC mobilization into peripheral blood is an expected event after conventional chemotherapy-based treatment, followed by sequential administration of recombinant granulocyte-colony- stimulating factor (rHu-CSF). In this study, specific factors associated with the application of two different SC-harvesting approaches, including the use of large volume leukapheresis (LVL) vs. repetitive conventional apheresis (RCA), were analyzed. The basic goal of the study was to evaluate the influence of apheresis protocol (collection timing, processed blood volume and cell yield) upon the clinical outcome of transplantation. Methods. Results obtained by LVL (76 pts) and RCA (20 pts - control group) were compared. The SC mobilizing regimen used was cyclophosphamide (4-7 g/m2) or polychemotherapy and rHuG-CSF 10-16 μg/kg of body mess (bm) per day. Cell harvesting was performed using COBE-Spectra (Caridian-BCT, USA). The volume of processed blood in LVL setting was ≥ 3.5 - fold of the patient's circulating blood quantity (ranged from 12.7 to 37.8 l). All patients tolerated well the use of intensive treatment, without any side or adverse effects. Our original controlled-rate cryopreservation was carried out with 10% dimethyl sulfoxide (DMSO) using Planer R203/200R or Planer 560-16 equipments (Planer Products Ltd, UK). Total nucleated cell (NC) and mononuclear cell (MNC) counts were examined by flow cytometry (Advia-2120 Bayer, Germany; Technicon H-3 System, USA). The CD34+ cell surface antigen was investigated by the EPICS XL-MCL device (Coulter, Germany). Results. Performing LVL-apheresis, high-level MNC and CD34+ cell yields (7.6±4.6 × 108/kg bm and 11.8±6.5 × 106/kg bm, respectively) were obtained. As a result, rapid hematopoietic reconstitution ("graft-healing") - on the 9.4th and 12.4th day for granulocytes and platelets, respectively was achieved. Using repetitive conventional apheresis (2-3 procedures), the total MNC count was high (8.2±7.0 × 108/kg bm), but the total CD34+ yield was lower 10.8±9.9 due to inferior CD34+ vs. MNC ratio. Conclusion. The results obtained suggest that well-timed LVL-apheresis increased SC-yield in cell harvest, resulting in faster bone marrow repopulation and hematological reconstitution, as well as better overall clinical outcome of transplantation. These results necessitate additional examinations of CD34+ subsets ratio in cell harvest