Dynamic light scattering analysis of immune complexes in sera of rheumatoid arthritis patients.

The size of circulating immune complexes (CICs) in rheumatoid arthritis (RA) could be an emerging criterion in disease diagnosis. This study analyzed size and electrokinetic potential of CICs from RA patients, healthy young adults, and RA patients age-matched controls aiming to establish their unique CIC features. Pooled CIC of 30 RA patients, 30 young adults, and 30 RA group's age-matched controls (middle-aged and oldеr healthy adults), and in vitro IgG aggregates from pooled sera of 300 healthy volunteers were tested using dynamic light scattering (DLS). Size distribution of CIC in healthy young adults exhibited high polydispersity. RA CIC patients and their age-matched control showed distinctly narrower size distributions compared with young adults. In these groups, particles clustered around two well-defined peaks. Particles of peak 1 were 36.1 ± 6.8 nm in RA age-matched control, and 30.8 ± 4.2 nm in RA patients. Particles of peak 2 of the RA age-matched control's CIC was 251.7 ± 41.2 nm, while RA CIC contained larger particles (359.9 ± 50.5 nm). The lower zeta potential of RA CIC, compared to control, indicated a disease-related decrease in colloidal stability. DLS identified RA-specific, but also age-specific distribution of CIC size and opened possibility of becoming a method for CIC size analysis in IC-mediated diseases.

The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method.

Flow cytometry (FC) analysis of erythrocyte shape and related biomechanical properties, such as osmotic fragility, have not moved from a research tool to regular clinical testing. The main reason is existing evidence that various pre-analytical factors influence the mathematical interpretation of the data obtained. With an aim to contribute to the standardization and broaden the use of FC for human erythrocyte shape assessment, freshly prepared peripheral blood erythrocytes isolated from healthy donors were incubated in iso and hypo-osmotic solutions (pure saline, saline with potassium and calcium, and phosphate buffered saline) and examined by FC using values of forward scatter (FSC) and side scatter (SSC). Kurtosis, skewness, Pearson's second skewness coefficient of dissymmetry (PCD), and spherical index, calculated from FSC distributions, were used for the erythrocyte shape evaluation. In all isotonic media FSC distribution and FSC-based morphology parameters showed huge inter-individual and inter-medium variation. With decreasing osmolality, in all media and samples, the size of the erythrocytes increased, and swelling index and kurtosis decreased. However, changes in skewness and PCD were influenced by the medium used and the sample tested. Compared to FSC, SSC signal in isotonic and its change in hypotonic media showed lower inter-individual variation and was not influenced by the type of medium. We propose a spherical index and kurtosis as FSC-based indicators of erythrocyte shape. As more resistant to the influence of the preanalytical treatment, SSC data appeared to be unfairly neglected for the assessment of erythrocyte shape, in comparison to the usually employed FSC data.

Platelet gel in wound treatment in patient with pemphigus vulgaris and Type 2 diabetes mellitus: Case report.

Treatment of wounds, especially in patients with systemic diseases, is very difficult and time consuming, and it represents great challenge. Complications like infections and impaired healing are regularly seen in these patients, sometimes leaving almost no other treating option, but amputation surgery. In wound healing process, platelets play one of key roles, both in hemostasis, and, by releasing many growth factors, in reepithelization and tissue remodeling. Platelet concentrates are defined as autologous or homologous platelet derivatives with a platelet concentration higher than it is usually in human blood. Concentration of platelets needed to affect wound healing should be 1,000,000/µl. This is a report about patient with pemphigus vulgaris and Type 2 diabetes mellitus, who got injury of the left lower leg. Traditional surgical methods of wound closure and vacuum-assisted closure therapy were ineffective. Homologous platelet gel was used topically, resulting in wound closure, without any adverse effects.

Transfusion-transmissible infections among Serbian blood donors: declining trends over the period 2005-2017.

BACKGROUND: The mass migrations experienced by the Western Balkans in the past decades have significantly changed the demographic structures and have probably altered the prevalence of transfusion-transmitted infections (TTIs) among blood donors. However, data on the prevalence of TTIs in the Western Balkans countries remain incomplete. This study reports the prevalence of TTIs among blood donors in Serbia in the period 2005-2017. MATERIALS AND METHODS: Between January 2005 and December 2017, in the four largest Serbian transfusion centres, mandatory serology tests for screening HBV, HCV, HIV and syphilis infection were used for all blood donations. RESULTS: Of the total of 1,660,019 blood donations made, 3,377 (0.203%) were positive for one of the TTIs: 1,440 (0.087%), 1,055 (0.064%), 215 (0.013%), and 667 (0.040%) were positive for HBV, HCV, HIV and syphilis, respectively. Serbia showed a declining trend of prevalence of HBV and HCV infection, while prevalence of HIV and syphilis remained unchanged. Prevalence of TTIs varied between different transfusion centres and showed a north-to-south upward trend. DISCUSSION: The reported prevalence of TTIs among blood donors in Serbia was low and continued to follow a declining trend over the period of study.

Flow cytometry analysis of platelet populations: usefulness for monitoringthe storage lesion in pooled buffy-coat platelet concentrates.

BACKGROUND: Early detection of the platelet storage lesion is still a challenge in transfusion practice. Using flow cytometry, we evaluated the appearance of the storage lesion, based on the expression of platelet activation markers, in total platelets and platelet populations. MATERIALS AND METHODS: Buffy-coat-derived platelet concentrates were stored under standard conditions for 5 days. The expression of activation antigens CD42b, CD36, CD62p and phosphatidylserine on total platelets and populations of small, medium-sized and large platelets was analysed by flow cytometry on storage days 1, 3 and 5. RESULTS: The activation/lesion on total platelets and each platelet population was detected on storage day 3, by the increased expression of CD36. On the same day, increased expression of CD42b and CD62p was detected, but only on large platelets. Small and medium-sized platelets had increased CD62p expression only on day 5. Externalisation of phosphatidylserine was not detected. DISCUSSION: Evaluation of the level of expression of various activation markers on different platelet populations could be an additional valid analysis in cell quality control of platelet concentrates, and in the assessment of novel approaches to platelet concentrate manipulation.

Comparative studies on osmosis based encapsulation of sodium diclofenac in porcine and outdated human erythrocyte ghosts.

The objective of our study was to develop controlled drug delivery system based on erythrocyte ghosts for amphiphilic compound sodium diclofenac considering the differences between erythrocytes derived from two readily available materials - porcine slaughterhouse and outdated transfusion human blood. Starting erythrocytes, empty erythrocyte ghosts and diclofenac loaded ghosts were compared in terms of the encapsulation efficiency, drug releasing profiles, size distribution, surface charge, conductivity, surface roughness and morphology. The encapsulation of sodium diclofenac was performed by an osmosis based process - gradual hemolysis. During this process sodium diclofenac exerted mild and delayed antihemolytic effect and increased potassium efflux in porcine but not in outdated human erythrocytes. FTIR spectra revealed lack of any membrane lipid disorder and chemical reaction with sodium diclofenac in encapsulated ghosts. Outdated human erythrocyte ghosts with detected nanoscale damages and reduced ability to shrink had encapsulation efficiency of only 8%. On the other hand, porcine erythrocyte ghosts had encapsulation efficiency of 37% and relatively slow drug release rate. More preserved structure and functional properties of porcine erythrocytes related to their superior encapsulation and release performances, define them as more appropriate for the usage in sodium diclofenac encapsulation process.

Blood donors' positivity for transfusion-transmissible infections: the Serbian Military Medical Academy experience.

BACKGROUND: Members of armed forces worldwide are considered to be very susceptible to sexually transmitted infections, thus falling into a high-risk group of blood donors regarding transfusion-transmissible infections. In the Serbian Military Medical Academy a significant number (44% for the period 2005-2013) of blood donations were from members of the Serbian Army. The aim of this study was to determine the significance of military blood donors for the safety of blood transfusion. MATERIAL AND METHODS: Between January 2005 and December 2013, a total of 155,479 blood donations were tested for hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV) and syphilis using serological assays (enzyme immunoassays, chemiluminescent microparticle immunoassay and western blot) and molecular testing (polymerase chain reaction analysis). RESULTS: The percentage of blood donations positive for transfusion-transmissible infections in the estimated period was 0.38%, and the percentage of HBV, HCV, HIV and syphilis positive blood donations was 0.20%, 0.12%, 0.005% and 0.06%, respectively. During that period, the percentage of all transfusion-transmissible infections, and in particular of HBV and HCV, declined significantly. In contrast, the percentage of HIV and syphilis positive blood donations remained unchanged. Higher rates of positivity for transfusion-transmissible infections in blood donations from members of the Serbian Army were not found, especially after mandatory military service was abolished in 2009. DISCUSSION: The reported rate of positivity for transfusion-transmissible infections in blood donations from the Military Medical Academy was considered low. This information is of great significance for further implementation of public health measures.

[HIV/AIDS and other sexually transmitted infections among military members of the Armed Forces of Serbia].

BACKGROUND/AIM: Military personnel is a population group at special risk of exposure to sexually transmitted diseases (STD). In peacetime, STD infection rates among service members are generally 2 to 5 times higher than among civilian population. In time of conflict, the differences can be 50 or more times greater. This study describes sexual behavior as a risk factor for STD in the Armed Forces of Serbia. METHODS: The sample of 5 617 voluntary blood donors from the Armed Forces of Serbia gave blood and filled World Health Organization Questionnaire about sexual behavior within January 2007 - December 2008 period. The mandatory testing of voluntary blood donors was performed in the Institute of Transfusiology Military Medical Academy in Belgrade, by the specific immunoenzyme tests and polymerasa chain reaction tests for HIV, hepatitis B, C and syphilis. Statistical analysis of data was done using State for Windows 93, USA, 1996. RESULTS: We identified 36 soldiers with some form of STDs. This study showed that 1 668 (29.7%) tested soldiers reported always using condoms, 1 725 (30.72%) almost always, 1 238 (20.04%) sometimes, 495 (8.81%) almost never and 490 (8.73%) never. Among the sample, 449 (7.99%) soldiers reported sexual contacts with partners with high risk of sexual behavior, whilst 22 (0.37%) of them reported homosexual and bisexual contacts. CONCLUSION: This study reported STDs found in voluntary blood donors among the service members of the Armed Forces of Serbia, but none of them was identified to be HIV positive. Soldiers with the most frequent risk behavior were reported to be those with inconsistent condom use. In the future, the STD Control and Prevention Program should be more intensively conducted among the members of the Armed Forces of Serbia.

Human cartilage glycoprotein 39--biomarker of joint damage in knee osteoarthritis.

Human glycoprotein of cartilage (YKL-40) synthesizes chondrocytes and synovial cells in inflammatory conditions or remodels the outer cell matrix in osteoarthritis. The aim of this study was to conduct a parallel analysis between thickness of cartilage and length of osteophytes, ultrasound indicators of joint destruction, with levels of YKL-40 in serum in patients with primary osteoarthritis. Ultrasound findings and concentration of YKL-40(ng/ml) were examined in 88 patients. The average value cartilage thickness measured on medial condyles of the femur was 1.30 +/- 0.23 mm and on lateral was 1.39 +/- 0.27 mm. Median YKL-40 in patients with shorter osteophytes was 62.0 (44.5-90) ng/ml, and with longer osteophytes was 119 (range 80-171) ng/ml (p = 0.000). YKL-40 can be a marker for the appearance of longer osteophytes (sensitivity = 79.1%; specificity = 61.9%; cut off = 75.0 ng/ml). The duration of illness is very much connected to values of YKL-40 (r = 0.651, p = 0.000). After an illness duration of five years, the concentration of YKL-40 was 83.68 +/- 33.65 ng/ml, after ten years it was 138.22 +/- 48.88 ng/ml, and after 15 and 20 years it was 209.30 +/- 79.36 ng/ml and 218.50 +/- 106.51 ng/ml, respectively. Higher concentrations of YKL-40 indicate the level of cartilage destruction and can be used for assessment of destruction.

Microprocessor-controlled vs. "dump-freezing"platelet and lymphocyte cryopreservation: a quantitative and qualitative comparative study.

BACKGROUND/AIM: Thermodynamical and cryobiological parameters responsible for cell damages during cryopreservation (cryoinjuries) have not yet been completely explained. Thus, freezing procedures should be revised, exactly optimized to obtain an enhanced structural and functional recovery of frozen-thawed cells. The aim of this study was to compare microprocessor-controlled (controlled-rate) with the compensation of the released fusion heat and "dump-freezing" (uncontrolled-rate) of the platelet and lymphocyte cryopreservation efficacy. METHODS: Platelet quantitative recovery (post-thaw vs. unfrozen cell count), viability (using hypotonic shock response--HSR), morphological score (PMS), ultrastructural (electron microscopy) properties and expression of different surface antigens were investigated. In lymphocyte setting, cell recovery and viability (using trypan blue exclusion test) as well as functionality (by plant mitogens) were determined. Controlled-rate freezing and uncontrolled-rate cryopreservation were combined with 6% (platelets) and 10% (lymphocytes) dimethyl sulfoxide (DMSO). RESULTS: Platelet recovery and functionality were superior in the controlled-rate system. The majority of surface antigen expression was reduced in both freezing groups vs. unfrozen cells, but GP140/CD62p was significantly higher in controlled-rate vs. uncontrolled-rate setting. Controlled-rate freezing resulted with better lymphocyte recovery and viability (trypan blue-negative cell percentage). In mitogen-induced lymphocyte proliferative response no significant intergroup difference (controlled-rate vs. uncontrolled-rate) were found. CONCLUSION: The data obtained in this study showned the dependence of cell response on the cryopreservation type. Controlled-rate freezing provided a superior platelet quantitative and functional recovery. Lymphocyte recovery and viability were better in the controlled-rate group, although only a minor intergroup difference for cell proliferative response was obtained.

Controlled-rate versus uncontrolled-rate freezing as predictors for platelet cryopreservation efficacy.

BACKGROUND: Cryobiologic variables responsible for cell injuries and freezing techniques applicable in medical cryopractice should be revised and/or reengineered for minimizing cryoinjuries and maximizing cell recovery. In this study, the efficacy of different cryopreservation protocols based on platelet (PLT) recovery was evaluated. STUDY DESIGN AND METHODS: PLTs (n = 33) were prepared from whole-blood units. Cell count and viability, PLT morphologic score (PMS), and hypotonic shock response were determined. PLT surface antigens were measured by flow cytometry. Controlled-rate (with compensated fusion heat) and uncontrolled-rate freezing methods combined with 6 percent dimethyl sulfoxide were used. RESULTS: PLT recovery was superior in the controlled-rate setting (91.0 +/- 5.5 vs. 86.0 +/- 6.5; p < 0.05). PMS was significantly better in controlled-rate freezing (p < 0.01). GPIb/CD42b expression was reduced in both freezing groups versus control. GP140/CD62p expression was significantly (p < 0.05) lower in the controlled-rate group and in both frozen groups was significantly higher than in the control groups. CONCLUSION: The use of strictly equalized (1 degrees C/min) controlled-rate freezing, combined with an intensified cooling rate (2 degrees C/min) during the liquid-to-solid-phase transition period, allows advanced quantitative and qualitative PLT recovery, even though the minor intergroup differences for some variables were observed.

Quantitative, functional, morphological and ultrastructural recovery of platelets as predictor for cryopreservation.

Cryopreservation of platelets is of great interest, since it could extend the shelf life of therapeutic platelet concentrates and facilitate stockpiling and inventory control in blood banking. Despite the use of many cryopreservation procedures the optimal cryopreservation procedure is not defined yet. We have compared the cryopreservation of human platelets by various protocols employing controlled-rate and non-controlled-rate freezing procedures in combination with different concentrations of DMSO (6% and 10%) or 5% DMSO + 6% HES combination. After storage for 1 to 3 months, samples were thawed and analyzed. Measurements included cell recovery, platelet viability according to hypotonic shock response (HSR), platelet aggregation with ADP, morphological and ultrastructural properties of defrozen platelets. Our findings show that the application of our original procedure for controlled-rate freezing consisting of six cooling steps (cooling rate 1 degree C/min) with compensation of released heat of fusion (cooling rate 2 degrees C/min) has significantly influenced the quality of thawed platelets. At the same time, a concentration of 6% DMSO proved to be the most effective. In summary, cryopreservation of human platelets using controlled-rate freezing procedure in combination with lower (6%) DMSO concentration resulted in less damage from freezing and higher recovered function of platelets.