The cryopreservation protocol optimal for progenitor recovery is not optimal for preservation of marrow repopulating ability.

The efficiency of five different cryopreservation protocols (our original controlled-rate and noncontrolled-rate protocols) was evaluated on the basis of the recovery after thawing of very primitive pluripotent hemopoietic stem cells (MRA(CFU-GM), pluripotent progenitors (CFU-Sd12) and committed granulocyte-monocyte progenitors (CFU-GM) in mouse bone marrow. Although the nucleated cell recovery and viability determined immediately after the thawing and washing of the cells were found to be similar, whether controlled-rate or noncontrolled-rate cryopreservation protocols were used, the recovery of MRA(CFU-GM), CFU-Sd12 and CFU-GM varied depending on the type of protocol and the cryoprotector (DMSO) concentrations used. It was shown that the controlled-rate protocol was more efficient, enabling better MRA(CFU-GM), CFU-Sd12 and CFU-GM recovery from frozen samples. The most efficient was the controlled-rate protocol of cryopreservation designed to compensate for the release of fusion heat, which enabled a better survival of CFU-Sd12 and CFU-GM when combined with a lower (5%) DMSO concentration. On the contrary, a satisfactory survival rate of very primitive stem cells (MRA(CFU-GM)) was achieved only when 10% DMSO was included with a five-step protocol of cryopreservation. These results point to adequately used controlled-rate freezing as essential for a highly efficient cryopreservation of some of the categories of hematopoietic stem and progenitor cells. At the same time, it was obvious that a higher DMSO concentration was necessary for the cryopreservation of very primitive stem cells, but not, however, for more mature progenitor cells (CFU-S, CFU-GM). These results imply the existence of a mechanism that decreases the intracellular concentration of DMSO in primitive MRA cells, which is not the case for less primitive progenitors.

[Transfusion-infusion therapy in modern wars]. / Transfuziono-infuziono lecenje u savremenim ratovima.

All previous experiences have shown that the application of blood, blood components and intravenous solutions presents an unreplaceable therapeutical measure in modern surgical-resuscitative management of war injuries. Together with the broad application of the whole blood, there have been also used other blood components (even such as cryoprecipitate and platelet rich plasma). Among intravenous solutions the most frequently mentioned were isotonic saline, Ringer's lactate solution, glucosaline, 5% dextrose solution, dextran solutions and, recently, human albumin solutions. Due to a high risk of transmission of hepatitis virus, the dried pooled human plasma is less frequently used. There is the generally accepted agreement that availability of the sufficient quantity of blood, blood components and intravenous solutions resulted in the decreased mortality of the wounded. The role of intravenous solutions is of particular importance in the initial phase of management of the wounded and in the situations when it is necessary to wait for blood.

[Detection of anti-HCV antibodies in patients on long-term hemodialysis]. / Detekcija anti-HCV antitela kod bolesnika na dugotranoj hemodializi.

Hepatitis C virus (HCV) infection is highly prevalent among chronic dialysis patients and is the most common cause of chronic liver disease. Ninety six patients on chronic hemodialysis in our institute of transfusiology at the Military Medical Academy were evaluated for the presence of HCV infection. There was a significant relationship between presence of anti-HCV antibodies and number of blood transfusion received by examined patients. We concluded that hepatitis C is a common problem among patients on chronic hemodialysis in our institution: HCV infection is proved in about 48.95% of all patients on hemodialysis.

[Transfusion-infusion methods in the treatment of acute poisoning]. / Transfuziono-infuzione metode u tretmanu akutnih trovanja.

For modern acute poisoning management there are several therapeutic transfusion-induced methods (forced diuresis, haemodialysis, peritoneal dialysis, haemoperfusion, plasmapheresis, exsanguino-transfusion, blood and blood component transfusion, intravenous solution infusion etc), each of them having their advantages, disadvantages and limitations. No one of these methods is so efficacious enough that its application could be justified in all poisonings. However, some of them are considered to be method of choice in the treatment of certain acute poisonings.

[Blood transfusion therapy at the Military Medicine Academy--present possibilities]. / Usmerena hemoterapija u Vojnomedicinskoj akademiji--sadasnje mogucnosti.

Blood component therapy refers to the transfusion of the specific part of blood that a patient needs, as opposed to the routine transfusion of whole blood (WB) in the past. This not only maintains blood resources, but also provides the optimal method of transfusing patients who require large amounts of a specific blood component. Since this concept have been accepted, the Institute of Transfusiology of Military Medical Academy (MMA) possess appropriate equipment for blood collection and processing of WB in components. Mainly, all kind (except frozen) of packed red cells (RBCs), platelet concentrates (random-donor buffy coat or apheresis donation), single-donor (apheresis) or random-donor (buffy coats) granulocytes, fresh frozen plasma (FFP), single-donor cryoprecipitate are prepared. Recently, fibrin glue (obtained by recycled cryoprecipitation from single-donor or autologous plasma), and some of new generation of blood components: hematopoietic stem and progenitor cells (fresh or cryopreserved), collected from bone marrow or harvested from peripheral blood after mobilization and donor-specific mononuclear cells for cell therapy, i.e. immunomodulation during relapse of leukemia after bone marrow transplantation, have become the routine. Analysis of blood component therapy done at the MMA during the past 11 years (1989-1999) showed that: a) participation of WB transfusion in the group of surgical clinics was permanently decreased (from 59.60% in 1989 to 0.37% in 1999); b) WB transfusion (in the last few years) practically was not used in the group of internal medicine clinics (0.82% in 1993 and 0.45% in 1999); c) overall WB transfusion in MMA is extremely rare (0.37%).

[Advantages of polyethylene glycol in comparison to bovine serum albumin in the indirect antiglobulin test in pregnant women]. / Prednost polietilen glikola u odnosu na govedji serumski albumin u indirektnom antiglobulinskom testu kod trudnica.

Results of indirect antiglobulin test (IAT) adding polyethylene glycol (PEG) were compared with conventional IAT performed using bovine serume albumin (BSA) with the aim of prenatal protection of Rh(D) negative pregnant women. Investigation enrolled 986 samples of pregnant women sera and confirmed that the use of PEG-IAT increased the degree of detection of clinically significant antierythrocyte antibodies. Above all, form the Rhesus blood groups system (using exclusively PEG-IAT) was detected by one antibody of anti-D, anti-C, anti-e and two anti-E), while by using BSA, anti-e was not detected at all. Besides, the need for additional serologic techniques has been reduced (treating of erythrocytes by enzymes) and the work of laboratories for prenatal protection has been alleviated, and at the same time the quality of analyses was not diminished, which gave the preference to PEG-IAT compared to BSA-IAT in prenatal testing.

[Albumin and its therapeutic use - part I]. / Albumin i njegova terapijska primena -- I deo.

Albumin is most abundant and most studied protein of the circulation. Its biosynthesis is closely dependent on the nutrition, amino acid supply, hormonal millieu, environment, osmotic equilibrium, diseases and some other factors. Albumin is synthetized in the liver on a polyscmes and delivered into the blood streem. Degradation of albumin is practically still unknown and is one of many biological puzzles. Albumin prepared for therapeutic use almost contains dimers, oligomers and polymers which are very important, because they are one of the parameters for evaluation of albumin products quality.

[Albumin and its therapeutic use - Part II]. / Albumin i njegova terapijska primena -- II deo.

Albumin is the most effective oncotic agent and has wide clinical application. In the resuscitation of injured patients with hypovolemic shock (during war and peace) the use of supplemental albumin is purported to be more effective in restoring plasma volume, increasing cardiac output, preventing pulmonary oedema and maintaining organ perfusion than resuscitation without supplemental albumin. Most important in a study of albumin are criterias for appropriate use because it was sometimes unjustified. Therapeutic use of albumin should not be dictated by arbitrary or pragmatic restriction but rather by rational prescribing from physicians well educated in its use.

Early plasma amino acid pool alterations in patients with military gunshot/missile wounds.

Plasma amino acid profiles in patients during the early period (first 18 hours) following military gunshot/missile wounds were investigated. Patients (n = 29) were casualties from the war in the former Yugoslavia with injury severity scores ranging from 4 to 18. They were divided into three groups: soft tissue (muscle) damage, wounds with fractures, and vital structures injured. Controls were normal blood donors (n = 17). Free amino acids were analyzed in venous plasma. Increased concentrations of phenylalanine and glutamine associated with increased molar phenylalanine/tyrosine ratio in plasma indicated increased net protein catabolism in the peripheral tissues, regardless of the type of injured tissues. Decreased plasma arginine, ornithine and citrulline levels, accompanied with increased molar glutamine/valine ratio, suggested disturbance in urea cycle activity, although urea level was not altered. We concluded that early changes in plasma amino acid pool characteristics after wounds were of systemic origin, not related to the type of injured tissues.

Application of therapeutic plasma exchange in hematology.

Therapeutic plasma exchange (TPE) was used in 146 patients with hematologic disorders: hyperviscosity syndrome, 74; cryoglobulinemia, 53; porphyria, 9; immune complex disease, 3; cold agglutinin disease, 1; hemolytic uremic syndrome, 1; autoimmune hemolytic anemia, 1; autoimmune thrombocytopenia, 1; autoimmune neutropenia, 1; Clq deficiency, 1; and secondary immunodeficiency, 1. It was shown that TPE applied in patients with hyperviscosity syndrome resulted in rapid reduction of paraprotein concentrations, and normalization or significant decrease of serum viscosity associated with marked clinical improvement (regression of neurologic, renal, hematologic, visual and other disturbances). Application of TPE in patients with cryoglobulinemia resulted in plasma cryoglobulin reduction and clear clinical effects (blood flow improvement, skin ulcer healing, reversal of impaired renal function and disappearance of purpura and other abnormalities). Very good results were obtained in patients with porphyria (decreased sensitivity to sunlight) and also in patients with Clq deficiency. Satisfactory clinical improvement and better laboratory findings were also seen in patients with immune complex disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia and hemolytic uremic syndrome.

[Biochemical changes in thrombocyte concentrates stored for 5 days]. / Biohemijske promene u jedinicama koncentrovanih trombocita skladistenih pet dana.

The purpose was to evaluate biochemical and functional changes in platelet concentrates prepared from buffy-coat (PC-BC), stored up to five days at temperature of 20 +/- 2 degrees C. Forty-two PC-BC units from random blood donors were examined. In order to determine the biochemical changes we studied the release of lactate dehidrogenase (LDH), changes in pH value, pCO2, pO2, glucose and lactate concentrations. In addition, the aggregation of platelets with adenosine diphosphate (ADP), and hypotonic shock response were examined. The concentration of LDH markedly increased from 138.8 IU/l (day 1st) up to 234 IU/l (day 5th). The lactate concentration increased significantly (p < 0.001) from 2.1 to 8.6 mmol/l, and consequently pH decrease was observed (from 7.31 to 7.13). The level of glucose decreased gradually from 22.8 to 19.4 mmol/l. The pCO2 decreased progressively from 36.8 mmHg to 24 mmHg during first two days of storage and after that gradually to 20 mmHg. In contrast, pO2 fluctuated between 108 and 133 mmHg during five day of storage. Recovery of hypotonic shock response was 70% on day 1st and 35.7% on day 5th, respectively (p < 0.001). Platelet aggregation using ADP showed significant increase from 6.6 sec (day 1st), to 11.2 sec (day 4th) and 14.6 sec (day 5th), too. Significant differences in biochemical parameters and platelet functions, with confirmed relationship between changes obtained during five days stored PC-BC did not affect the efficacy of applied platelets.

Plasma exchange in myasthenia gravis and multiple sclerosis.

During the last twenty-year period therapeutic plasma exchange (TPE) was used in the treatment of 68 patients with myasthenia gravis and 61 patients with multiple sclerosis. The therapeutic effects were evaluated on the basis of neurologic deficit changes, electrophysiological findings, necessary laboratory analyses and patient's general conditions. It was shown that the therapeutic effects mosty depended on the nature and stage of the basic disease, adequate selection of the patients and timely applied therapeutic procedure. Significant positive effects of the TPE treatment applied with the anti-inflammatory and immunosuppressive therapy were observed in patients with myasthenia gravis and multiple sclerosis upon clinical findings and some paraclinical tests.

[Significance of blood transfusion in the development of cytotoxic antibodies in patients on hemodialysis]. / Znacaj transfuzija krvi za razvoj citotoksicnih antitela kod bolesnika na hemodijalizi.

The aim was to evaluate the influence of red blood cells (RBC) transfusion on the development of cytotoxic antibodies (C-Ab) in patients subjected to hemodialyses (HD) and planned for the kidney transplantation. The group of 71 HD patients, of mean age 42 years (19-65), 48 males and 23 females, planned for the kidney information was examined. Out of 71 HD patients, only 42 (59.19%) HD patients (group I) received subcutaneously recombinant human erythropoietin--rhuEPO (Eprex--epoetin-alpha or Recormon SE--epoetin-beta in dosage of 4,000 IU during every HD; i.e. one to three times a week) and they were not treated by RBC transfusion. The other 29 (40.85%) HD patients (group II) received RBC transfusion: 18 (62.07%) HD patients received < 10 units 18 of RBC, 8 (27.59%) HD patients received 10-20 units of RBC; 3 (10.35%) HD patients received > 20 units of RBC. Testing of C-Ab was done in all patients every three months by standard lymphocytotoxicity test on the panel from 20 different lymphocyte donors with definite class I phenotype of antigen HLA. C-Ab was not found in HD patients who were not treated by RBC transfusion. Out of 18 HD patients who received < 10 units of RBC only 3 (16.67%) HD patients developed C-Ab; out of 8 HD patients who received 10-20 units of RBC, in 4 (50%) patients was proved C-Ab; and C-Ab was proved in all 3 HD patients who received > 20 units of RBC. RhuEPO administration is very important for the transfusiologic treatment of HD patients; especially those who are planned for the kidney transplantation. Development of C-Ab is in direct correlation with the number of transfunded units of RBC. HD patients who received 10 or more units of RBC were at great risk to develop C-Ab.

Locally produced specific cytomegalovirus immunoglobulin for intramuscular use as a therapy in renal allograft recipients.

Immunomodulator, i.e. specific hyperimmune anticytomegalovirus immunoglobulin for intramuscular administration, produced in 1999 with the aim of prevention of CMVI, and the development of the disease, was for the first time applied in kidney transplant recipients in January 2000, in the Center for kidney transplantation at the Military Medical Academy. Therapy was administered in four cytomegalovirus (CMV)--seronegative kidney recipients from CMV-seropositive donors--the combination that in the majority of cases lead to the development of CMVI/disease resulting in transplant rejection. Patients received 0.2-0.3 ml/kg of cytomegalovirus immunoglobulin (CMVIG) 6 hours before the transplantation, and subsequently the same dose during the following 5 weeks. Simultaneously, they received ganciclovir in therapeutic doses adjusted according to creatine clearance during the first three post-transplantation months (2 weeks parenterally, the rest orally). Kidney transplant recipients tolerated well i.m. applied CMVIG without any adverse effects. Test result obtained from the Paul-Erlich Institute, Germany in 1999 spoke in favor of the quality of the first national CMVIG preparation.

[Use of fibrin glue prepared from recycled cryoprecipitate in experimental liver surgery]. / Primena fibrinskog lepka pripremljenog reciklaznom krioprecipitacijom u eksperimentnoj hirurgiji jetre.

Fibrin glue (FG) is a two-component biologic system with adhesive, sealant and topical hemostatic properties, containing fibrinogen (Fg), factor XIII (FXIII), fibronectin (Fn), thrombin, some antifibrinolytic agent if needed and ionized calcium. In this study, FG component 1 was prepared by recycling cryoprecipitation from single-donor plasma. The mean concentrations of Fg, FXIII and Fn were: 54.2 +/- 19.9 g/l, 8.03 +/- 2.3 IU/ml and 3103.1 +/- 148.91 mg/l, respectively. Horizontal tensile strength of FG was 1.076 +/- 0.18 N/cm2 in the average. Using a rat model, the efficacy of the FG-treatment in liver surgery was evaluated on the basis of the 24 hour survival ratio and hematological parameters of the experimental animals and control group. Survival of rats subjected to partial and total lobectomy and FG-treated was significantly (p < 0.001) higher than in FG-non-treated animals. Survival of animals subjected to liver incision was not significantly different, although the differences in hematological parameters were significant (p < 0.001 to p < 0.09) in favor of FG-treated animals. Our findings confirmed that high quality FG can be prepared by recycling cryoprecipitation from single-donor plasma--with sufficient yield of fibrinogen, FXIII and fibronectin and with the risk of disease transmission not greater than with the use of single unit of blood or plasma--which have efficient hemostatic and therapeutic properties.

Donor leukocyte infusion--the effect of mutual reactivity of donor's and recipient's peripheral blood mononuclear cells on hematopoietic progenitor cells growth.

Donor leukocyte infusions are an effective therapy for patients who relapse with leukemia after bone marrow transplantation. We report the case of 14-year-old boy who relapsed 34 months after sibling donor bone marrow transplant for Philadelphia-positive chronic myeloid leukemia. Subsequently, he received three infusions of donor mononuclear cells (DMNC) harvested in steady state hematopoiesis and one G-CSF mobilized-peripheral blood mononuclear cells (PBMC) infusion. Simultaneously, test named as--"Test of Mixed Progenitors" (TMP) was performed for the assessment whether the outcome of donor leukocyte infusion treatment could be predicted. Prior to DMNC infusions, the CFU-GM and BFU-E colony assays were performed for donor's and recipient's PBMC individually, as well as for the mixture of these cells at 1:1 ratio. The cells were plated either directly in the semisolid medium or after 24 h preincubation treatment. Significantly lower values for CFU-GM derived colonies were determined in TMP in comparison to the CFU-GM values obtained for the recipient's cells. The reduced number of CFU-GM was determined both in TMP performed without preincubation treatment, app. 80% and after the 24 h preincubation, app. 55%. The reduced number of BFU-E derived colonies (app. 44%) was observed only related to recipient's cells and after the preincubation treatment of the cells. The patient did not develop GVHD and currently (40 months after the first infusion). He remained well in complete hematological, cytogenetic, molecular and clinical remission, which was the most direct evidence of the GVL effect. The novel in vitro TMP test in which the specific contribution of donor's leukocytes to the growth of recipient's hematopoietic precursor cell growth was determined, correlated with the clinical outcome.

[The quality of preserved blood]. / Kvalitet konzervisane krvi.

The examinations of 30 blood samples each preserved with three Yugoslav different ACD-solutions were performed. The blood samples were stored at 2-6 degrees C and examinations were performed at the day of blood donation and after on the 7th, 14th and 21st day during the storage. Differences in hematocrit (well known dilution effect of the ACD-solutions used) and intensive morphological and chemical changes were found in all blood samples regardless the type of ACD-solution used. It was shown that the permanently increasing number morphologically altered erythrocytes (echinocytes and spherocytes) and the excessive release of hemoglobin and potassium from erythrocytes were occurred during the storage of blood samles. Too, there were noticed significant decrease of pH values enormous accumulation of ammoniac and other metabolic producta.

Evaluation of cryopreserved murine and human hematopoietic stem and progenitor cells designated for transplantation.

The influence of five different cryopreservation protocols on the quality and/or quantity of frozen cells was investigated on mouse bone marrow cells and human peripheral blood mononuclear cells (MNC). The efficiency of the protocols was evaluated on the basis of the recovery of very primitive pluripotent hematopoietic stem cells (MRA), pluripotent progenitors (CFU-Sd12), committed granulocyte-monocyte progenitors (CFU-GM) of mouse cells after thawing. The recovery of MRA, CFU-Sd12 and CFU-GM varied depending on the type of freezing procedure and cryoprotector (DMSO) concentrations used. It was shown that the controlled-rate protocol was more efficient, enabling better recovery of all categories of progenitor cells in frozen samples. The most efficient was the controlled-rate protocol of the cryopreservation designed to compensate for the release of fusion heat, which enabled the best recovery of CFU-GM (73.0 +/- 8.8%) and CFU-Sd12 (90.0 +/- 15.9%) when combined with 5% DMSO concentration (protocol 4). On the contrary, a better recovery (79.8 +/- 13.5%) of very primitive stem cells (MRA) was achieved only when the higher concentration (10%) DMSO was used in combination with a five-step protocol of cryopreservation (protocol 1). These results pointed out the adequately used controlled-rate freezing to be essential for a highly efficient cryopreservation of some of the categories of hematopoietic stem and progenitor cells. At the same time, it was obvious that a higher DMSO concentration was necessary for the cryopreservation of MRA, but not for more mature progenitor cells (CFU-S, CFU-GM). These results imply the existence of a mechanism that decreases the intracellular concentration of DMSO in MRA cells, which is not the case in less primitive progenitors. For human MNC, the recovery and viability of the cells, as well as the engraftment potential of cryopreserved cells after thawing were investigated. Cryopreservation protocol 1 resulted in better MNC recovery (82.7 +/- 10.4%) than protocol 3 (49.9 +/- 15.1%). The mean recovery of MNCs (collected from patients for autologous transplantation) was 78.5 +/- 7.3% (protocol 1) and 53.1 +/- 26.2% (protocol 3). The obtained favorable recovery of thawed cells and rapid reconstitution of hematopoiesis (on the day 11th following the transplantation) in patients confirmed that the controlled-rate freezing in combination with optimal DMSO concentration was able to obtain sufficient progenitor cryoprotection.