[Crypreservation of biological samples unit].
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Biological Specimen Banks↗
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[Electron microscopy study of HES cryopreserved erythrocytes].
Using electron microscopy (EM), it was examined whether cryopreservation with HES causes shape changes of erythrocytes. Each of 11 erythrocyte suspensions (Hct = 40; HES 200,00/0.5/12.5%; 60 mM NaCl) was separated into 40-ml samples, cooled down to -196 degrees C and finally stored. In addition, 11 samples were stored at -80 degrees C for 3 months. The preparation for EM was done immediately after thawing or in case of native cells shortly after donation. On EM micrographs, there was no visible difference between native and cryopreserved erythrocytes. In every case the preparations showed normocytes, either as single cells or having attracted other ones, forming rouleau. Packed cells were attached tightly to each other without any gap in between. The tangent count method neither revealed an excess of convexity nor of concavity. The erythrocyte membrane looked normal, and the cytoplasmatic space was filled with electron-dense material (hemoglobin) homogeneously; Heinz bodies were not seen. Scanning microscopy portrayed native as well as cryopreserved cells as discocytes with the characteristic bioconcave resting shape of human erythrocytes. It is concluded that cryopreservation of erythrocytes with HES does not cause shape changes. Therefore, a sequestration into the reticulo-endothelial system (RES) by means of identification of morphological abnormalities may not be expected.
Blood Preservation↗
Human motile sperm recovery after cryopreservation: freezing in nitrogen vapor vs the direct plunge technique.
Objectives: The following will be tested: 1) Freezing in nitrogen vapor prior to submerging will have a higher motile sperm recovery rate compared to directly submerging in liquid nitrogen, 2) The length of time a sample spends in nitrogen vapor will not affect the motile sperm recovery, and 3) The volume of sample to be frozen will not affect the recovery of motile sperm.Design: This is a prospective study performed through a university infertility clinic. The semen samples were obtained from 20 male partners of couples being screened for in vitro fertilization.Materials and Methods: Semen was collected and washed with 5 volumes of modified Ham's F10 medium and centrifuged at 120xg for 10 minutes. The pellet was resuspended in 5 mL of medium and centrifuged in a similar manner. The swim-up sperm was prepared with the second pellet overlaid with 1 mL of medium, containing 1% human serum albumin, and incubated for 1 hour at 37 C. Equal volumes of sperm suspension and cryoprotectant were mixed by gentle stirring. Sperm density and motility were recorded before freezing. Each vial contained 1 mL of the sperm/cryoprotectant mixture. All vials were eventually submerged in liquid nitrogen for 40 minutes and thawed at room temperature for 1 hour. The percent of motile sperm recovered was assessed. Hypothesis 1: Comparing freezing in nitrogen vapor versus direct plunge, one vial was frozen for 6 minutes in the nitrogen vapor 2 cm above the liquid surface prior to submerging. The paired vial was kept at room temperature for 6 minutes before submerging. Hypothesis 2: Two vials were frozen in vapor for 6 and 12 minutes prior to submerging them. Hypothesis 3: To assess the effect of sample volume on motile sperm recovery, three separate vials were prepared, each containing either 1.0, 0.5, or 0.25 mL of the sperm/cryoprotectant mixture. Each vial was suspended in vapor for 6 minutes prior to submerging into the liquid. Paired t test analysis was used for objectives 1 and 2, with repeated measures analysis of variance used to determine the significance of objective 3.Results: Pure motile sperm density and motility were assessed for 20 samples after the addition of cryoprotectant. The sperm density and motility ranged from 0.9 to 120 million sperm/mL (33.2, SD +/- 35.0) and 78-100% (86.5, SD +/- 12.4), respectively. The mean motile sperm recovered for vapor freezing prior to plunge was 54.9% (SD +/- 15.4), compared to submerging directly, which was 21.5% (SD +/- 10.0); this was significant with a P <.05. The initial sperm density, <20 or >/=20 million/mL, did not influence the percent of motile sperm recovered (P >.05). Motile sperm recovered from samples in vapor for 6 and 12 minutes was 49.7% (SD +/- 8.71) and 51.8% (SD +/- 10.4), respectively; this was not significant. The volume of purified motile sperm frozen did not significantly alter the percent of motile sperm recovered.Conclusion: Directly submerging a sample from room temperature into liquid nitrogen has shown a reduction of motile sperm recovery by 65.0%, compared to exposing the sample to liquid nitrogen vapor prior to submerging, which only showed a reduction of 31.7%. Motility appears to be influenced by the rate at which the sample is frozen. There was no significant impact on motile sperm recovery compared to the amount of sperm preserved, length of time the sample was in liquid nitrogen vapor, or the initial sperm density. This is the first study to isolate the motile faction from semen and examine the recovery rate between the two cryopreservation techniques. The clinical application implies that increasing the recovery of motile sperm could improve pregnancy rates and this simplified technique is possible without obtaining special equipment that could impose a significant financial burden with similar results.
Journal Article↗
Preliminary evaluation of a unique freezing technology for bovine spermatozoa cryopreservation.
Artificial insemination (AI) and semen cryopreservation has significantly improved the breeding potential of male animals. However, current freezing techniques commonly result in reduced semen quality. Ten years ago, a unique freezing technology (UFT) was developed for the freezing of foodstuffs and other materials. Previous work from this laboratory has demonstrated the UFT to be a superior method of freezing for a number of cell types. In a preliminary study, the UFT was compared with the conventional freezing methodology of bovine semen. Semen samples were collected from an angus (Bull A) and a gelbivich bull (Bull B), prepared using a conventional bovine cryoprotectant, and frozen in the UFT or in liquid nitrogen (LN) mist. The samples were stored in LN before being thawed and assessed for the semen parameters of motility and forward progression. Preliminary results suggest the UFT is equivalent to current techniques in the cryopreservation and recovery of bovine semen, and with modification, possibly a superior technique for semen freezing. Further studies using larger sample populations, and using a CASA system to evaluate motility, forward progression and linearity are merited.
Animals↗
Controlled preparation of the endometrium with exogenous oestradiol and progesterone: a novel regimen not using a gonadotrophin-releasing hormone agonist.
In women having inactive ovaries, controlled preparation of the endometrium has been achieved with exogenous oestradiol and progesterone. We report on the feasibility and practicality of using a similar regimen for timing transfers of cryopreserved embryos in women whose ovaries have not been suppressed. A total of 91 women having cryopreserved embryos from previous in-vitro fertilization (IVF) attempts received 4 mg/day of oestradiol valerate, starting on cycle day 1 of spontaneous (n = 85) or induced (n = 6) menstruation. A single blood sample was obtained on cycle day 14 for the measurement of plasma progesterone, oestradiol and luteinizing hormone (LH). Vaginal administration of micronized progesterone (300 mg/day) was started on day 15. Cryopreserved embryos were transferred on day 17 or 18 provided that day 14 plasma progesterone remained < or = 0.5 ng/ml, thereby confirming the absence of spontaneous ovulation prior to the administration of exogenous progesterone. Out of 91 cycles studied, plasma progesterone was found to be elevated (> 1 ng/ml) in only three (3.2%). Of the 88 scheduled transfers, 31 did not take place because no embryo survived thawing. In the remaining 57 cycles, 116 embryos were transferred resulting in 10 pregnancies, giving pregnancy and embryo implantation rates of 17.5 and 8.6% respectively. When a positive beta human chorionic gonadotrophin (HCG) titre was obtained, supplementation with oral oestradiol and vaginal progesterone was continued until placental autonomy was achieved. Of the 10 pregnancies, five (50%) were lost during the first trimester (biochemical, n = 1; miscarriage, n = 3; ectopic, n = 1).(ABSTRACT TRUNCATED AT 250 WORDS)
Abortion, Spontaneous↗
Spermatogenesis and steroidogenesis in mouse, hamster and monkey testicular tissue after cryopreservation and heterotopic grafting to castrated hosts.
Retrieval, extracorporal storage and autotransplantation of testicular tissue could become an important strategy for preserving male gonadal function. The present study used syngeneic and immunodeficient nude mice as hosts, and immature and adult mice, neonatal and adult photoregressed Djungarian hamsters and neonatal marmosets to identify the potential of testicular tissue grafting to maintain the morphological and functional integrity of the testis. Testicular tissue was grafted s.c. either as fresh tissue or after cryopreservation into adult, orchidectomized hosts. The mice that received rodent testis tissue were autopsied 50 days later, and blood samples were collected. Sixty-five per cent of mouse isografts contained morphologically normal testicular tissue and seminiferous tubules with some degree of spermatogenic recovery. Mature spermatozoa were recovered after enzymatic disaggregation. Although the recovery of spermatogenesis was limited in adult mouse and hamster tissue, complete spermatogenesis was observed in grafts from immature rodents. Testicular tissue from neonatal marmosets developed up to the stage of spermatocytes at day 135 after xenografting. Androgen concentrations were comparable in intact control mice and in mice receiving fresh mouse and hamster grafts, slightly lower in mice receiving cryopreserved grafts and adult photoregressed hamster tissue, and low in castrated control mice and in mice receiving marmoset tissue. These results show that isografts and xenografts of immature and adult testicular tissue become functionally active as a s.c. graft in the mouse and that this approach might be useful in combination with cryopreservation as a tool for storage and activation of the male germ line and androgen replacement therapy in patients.
Animals↗
Biomedical testing of cryopreserved tissues.
The Laboratory of Biomechanics of the Testing of Materials Institute of the Uruguayan Engineering School has ongoing biomechanical research for thr last 25 years. First about fixators employed in osteosynthesis and now also on the characterisation of biological tissues. A multidisciplinary group with physicians, chemists and statistical and mechanical engineers was integrated for that purpose. Research of biological tissues is carried out together with the National Organs and Tissues Bank. All materials are provided from cadaver donors. The objective is the biomechanical evaluation of tissues to be used as all allografts and the improvement of preservation methods. Elastic properties are determined for example in compression, tensile and bending tests. Sample extraction and preparation, equipments, testing procedures and some results for tendons and bones are detailed. Evaluation of fresh and cryopreserved vascular tissues is described and conclusions about their biomechanical difference between them are drawn.
Biomechanical Phenomena↗
[Quantitative assessment of fibroblast viability in cryopreserved aortic valve allografts].
The purpose of this study is to clarify whether serum (fetal bovine serum or human serum) is necessary or not for cryopreservation of aortic valve allografts. The protective effects of fetal bovine serum compared with human serum were evaluated by means of quantitative assessment of fibroblast viability. Porcine aortic valves were excised and rinsed immediately after death, followed by treatment with low concentration antibiotics. Valves were gradually frozen at a control-rate of -1 degree C/min and then stored in liquid nitrogen vapor-phase. The samples were classified into three groups by nutrient medium as follows. Group A (n = 5); Dulbecco's Modified Eagle Medium (DMEM), Group B (n = 5); DMEM containing 10% fetal bovine serum, Group C (n = 5); DMEM containing 20% human serum. At 1 week, 1 month, and 3 months after initiation of storage, the valves were thawed rapidly and examined for fibroblast viability, which was assessed quantitatively by means of autoradiography with tritiated proline. After 1 week storage, the fibroblast viability rate reduced from 98% to 80.7% in group A, 79.4% in group B and 79.2% in group C respectively, but there were no significant differences among three groups. The viability rate decreased with the passage of time, and remained around 72% in all groups after 3 months storage. The histological examination did not show any changes in the structure of the valves in all samples up to 3 months of storage. The results demonstrate that the fibroblasts are still viable after 3 months storage by cryopreservation. The addition of fetal bovine serum or human serum does not improve fibroblast viability.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Decreased non-MHC-restricted (CD56+) killer cell cytotoxicity after spaceflight.
Cytotoxic activity of non-major histocompatibility complex-restricted (CD56+) (NMHC) killer cells and cell surface marker expression of peripheral blood mononuclear cells were determined before and after spaceflight. Ten astronauts (9 men, 1 woman) from two space shuttle missions (9- and 10-day duration) participated in the study. Blood samples were collected 10 days before launch, within 3 h after landing, and 3 days after landing. All peripheral blood mononuclear cell preparations were cryopreserved and analyzed simultaneously in a 4-h cytotoxicity (51)Cr release assay using K562 target cells. NMHC killer cell lytic activity was normalized per 1,000 CD56+ cells. When all 10 subjects were considered as one study group, NMHC killer cell numbers did not change significantly during the three sampling periods, but at landing lytic activity had decreased by approximately 40% (P < 0.05) from preflight values. Nine of ten astronauts had decreased lytic activity immediately after flight. NMHC killer cell cytotoxicity of only three astronauts returned toward preflight values by 3 days after landing. Consistent with decreased NMHC killer cell cytotoxicity, urinary cortisol significantly increased after landing compared with preflight levels. Plasma cortisol and ACTH levels at landing were not significantly different from preflight values. No correlation of changes in NMHC killer cell function or hormone levels with factors such as age, gender, mission, or spaceflight experience was found. After landing, expression of the major lymphocyte surface markers (CD3, CD4, CD8, CD14, CD16, CD56), as determined by flow cytometric analysis, did not show any consistent changes from measurements made before flight.
Adrenocorticotropic Hormone↗
Post-thaw evaluation of dog spermatozoa using new triple fluorescent staining and flow cytometry.
A new triple fluorescent staining method was developed to evaluate frozen-thawed dog spermatozoa. This method was used to compare functional parameters of canine spermatozoa cryopreserved using 2 different freezing-thawing protocols. One ejaculate from each of 10 dogs was split into 2 aliquots and processed using the Andersen method or the CLONE method. Semen samples were evaluated immediately after thawing and after 3 h of incubation at 37 degrees C. Plasma membrane integrity and acrosomal status of the spermatozoa were evaluated simultaneously by flow cytometry using a combination of 3 fluorescent dyes: Carboxy-SNARF-1 (SNARF), to identify the live spermatozoa; propidium iodide (PI), which only stains dead cells or cells with damaged membranes; and fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA), which binds to the acrosomal content of spermatozoa with damaged plasma and outer acrosomal membranes. The accuracy of this new staining method in quantifying the proportions of live and dead spermatozoa by flow cytometry was evaluated by comparing it with the staining technique using carboxyfluorescein diacetate and propidium iodide (CFDA-PI), which yielded high correlation coefficients. The triple-stained sperm samples were also analyzed by epifluorescence microscopy, and both methods proved to be highly correlated. Post-thaw progressive motility and plasma membrane integrity were similar for the 2 freezing procedures, but the proportion of damaged acrosomes after thawing was lower using the Andersen method and the spermatozoa had a higher thermoresistance. This new triple staining method for assessing canine sperm viability and acrosomal integrity provides an efficient procedure for evaluating frozen-thawed dog semen samples either by flow cytometry or fluorescence microscopy.
Acrosome↗
Osteoarthritis chondrocytes die by apoptosis. A possible pathway for osteoarthritis pathology.
OBJECTIVE: To determine which kind of cell death occurs in cartilage from patients with osteoarthritis (OA). METHODS: Seven normal and 16 OA cartilage samples were collected at autopsy or during joint replacement surgery, respectively. A piece of cartilage was cryopreserved until histologic studies were done. The rest of the cartilage was used to isolate chondrocytes. Apoptotic chondrocytes were analyzed by light and fluorescence microscopy using nuclear 4',6-diamidino-2-phenylindole dihydrochloride stain. Apoptotic chondrocytes were quantified by fluorescence-activated cell sorter (FACS) analysis. The TUNEL technique was used to study histologic apoptosis in situ. Superficial cartilage was processed for ultrastructural study by electron microscopy. RESULTS: OA chondrocytes displayed nuclear and cytoplasmic changes consistent with apoptotic cell death. FACS analysis showed that the OA cartilage had a higher proportion of apoptotic chondrocytes than did normal tissue (51% versus 11%; P < 0.01). In situ study of DNA fragmentation in the cartilage showed that apoptotic cells were located in the superficial and middle zones. Ultrastructural analysis of the superficial OA cartilage revealed some empty lacunae, lysosomal-like structures, matrix vesicle-like structures, fragmented chondrocytes, and nuclear condensation. CONCLUSION: Chondrocytes in OA cartilage demonstrated morphologic changes that are characteristic features of apoptosis. This mechanism of cell death plays an important role in the pathogenesis of OA and could be targeted for new treatment strategies.
Aged↗
A simplified [3H] serotonin release assay for the detection of platelet antibodies.
The [3H] platelet serotonin release assay is a sensitive means for detecting antiplatelet antibodies. We have found that by using prelabeled frozen platelets which have been treated with the cryoprotective agent dimethyl sulfoxide (DMSO) the performance of this assay can be facilitated without interfering with its sensitivity. There was 100% correlation between the standard [3H] platelet serotonin release assay using fresh platelets and the modified assay using prelabeled, frozen, cryopreserved platelets when sera from 12 patients with known antiplatelet antibodies were tested. When normal serum samples (n = 111) were tested against ten platelet donors, a false-positive rate of 3.9% was observed. This modification provides a simple means for quickly screening large numbers of potential platelet donors at one time.
Antibodies↗
Evidence for genetic restriction in the suppression of erythropoiesis by a unique subset of T lymphocytes in man.
The suppression of erythropoiesis by lymphocytes from patients with a T cell lymphoproliferative syndrome and pure erythrocyte aplasia has been previously demonstrated. To study the nature of the suppressor cell and possible genetic restriction of this suppression, we investigated a patient with severe anemia, splenomegaly, lymphocytosis, and erythroid aplasia. A 3-mo course of low-dose daily oral cyclophosphamide achieved a complete remission for over 12 mo. The surface phenotype of his lymphocytes was analyzed by means of antibodies to lineage, differentiation, and activation-specific surface antigens. The cells expressed mature T cell antigens T3, T8, and T11, while lacking T1. Immature T cell, B cell, and the monocyte-specific antigen Mo2 were absent, while Mo1, a monocyte-associated antigen not normally expressed on T cells, was present. T10 and Ia expressed as activation antigens were also present. The cells, cryopreserved at diagnosis, were thawed and co-cultured in plasma clot with patient remission marrow samples at T cell/bone marrow ratios of 1:1 and 2:1. There was nearly 90% suppression of erythroid colony-forming unit expression and 60% suppression of erythroid burst-forming unit expression at 2:1 T cell to bone marrow ratios and somewhat less suppression at 1:1. Granulocyte/macrophage progenitor expression was unaffected. Erythroid progenitor differentiation in the marrows of two HLA identical siblings was similarly suppressed. The cells were co-cultured with the marrows of nine nonrelated donors to investigate the potential genetic restriction of this suppression. Colony suppression equal to that observed in the marrow of the patient and his siblings was found in studies of two partially HLA identical individuals. No suppression was detected in marrow co-cultures of two entirely HLA dissimilar individuals. These results show that suppression of erythropoiesis by a unique subset of T8, Mo1, Ia-positive lymphocytes isolated from a patient with lymphocytosis and erythrocyte aplasia is genetically restricted.
Adult↗
Antitumor immune response and interleukin 2 production induced in colorectal cancer patients by immunization with human monoclonal anti-idiotypic antibody.
The immunogenicity of human anti-idiotypic antibody has been investigated using a human monoclonal anti-idiotypic antibody (105AD7) which interacts with the binding site of 791T/36, a mouse monoclonal antibody against gp72 antigen. This antigen is frequently expressed in gastrointestinal cancer, therefore, six patients with advanced colorectal cancer have been immunized with 105AD7 as an aluminum hydroxide gel precipitate in a phase I clinical study. Cryopreserved blood mononuclear cells were tested for in vitro proliferative responses by [3H]thymidine incorporation; plasma samples were tested by enzyme-linked immunosorbent assay for anti-anti-idiotypic and antitumor antibodies, and for interleukin 2. Proliferative responses to gp72 positive tumor cells were seen in four of five patients tested; parallel in vitro responses to 105AD7 anti-idiotypic antibody were seen in most of these patients. Interleukin 2 was detected in the plasma of four of six patients after 105AD7 immunization, with peak levels up to 7 units/ml. No toxicity related to anti-idiotype immunization and no antitumor or anti-anti-idiotype antibodies were seen. This study shows that human monoclonal anti-idiotype 105AD7 is immunogenic in cancer patients, inducing cellular antitumor responses and interleukin 2 production. This suggests that human monoclonal anti-idiotype antibodies may have considerable potential for immunotherapy of human cancer.
Adult↗
Cytotoxicity of lymphocytes and antibodies against autologous tumor cells in patients with myeloid leukaemias and preleukaemic disorders. III. Stage-dependence of oncovirus-specific immune response.
Lymphocyte and plasma samples from the quiescent and blastic phase of chronic granulocytic leukaemia (CGL) and from the blastosis and remission of acute myeloid leukaemia (AML), were compared for cytotoxic activity. Target cells were collected from the blastic phases of diseases. 51Cr-release tests showed that the lymphocytes and plasma samples from blastic crisis of CGL had no cytotoxic activity for autologous blast cells. In contrast, cryopreserved lymphocytes and plasmas from the quiescent phase of CGL proved to be cytotoxic for the autologous tumor cells, and their effect could be blocked by native gp70 antigens of gibbon ape leukaemia virus (GaLV) and baboon endogenous virus (BaEV). A blocking effect was less frequently exerted by carbohydrate-free gp70 and p15(E) antigens. A similar relationship was found between the blastosis and remission stage of AML, however, out of the antigens of BaEV only the native gp70 showed a marked blocking effect.
Antibodies, Neoplasm↗
The number of circulating CD34+ blood cells predicts the colony-forming capacity of leukapheresis products in children.
In children, only a few guidelines are available for optimizing peripheral blood progenitor cell (PBPC) harvesting. We analyzed by means of flow cytometry and clonogenic assays 60 harvest products obtained from 20 children by standardized leukapheresis after treatment with chemotherapy and CSF. In addition, 27 fresh blood samples obtained prospectively during the mobilization phase were studied. CFU-GM/kg significantly correlated with MNC/kg, CD34+ cells/kg and CD34+33- cells/kg in apheresis products (P < 0.001). In fresh blood samples, CFU-GM/ml significantly correlated with MNC/ml, CD34+ cells/ml and CD34+33- cells/ml (P < 0.001). The numbers of CD34+ cells/ml, CD34+33- cells/ml and MNC/ml in 19 blood samples taken prior to leukapheresis were compared with CFU-GM/kg harvested and thawed after cryopreservation applying multiple regression analysis with stepwise variable selection. The number of circulating CD34+ cells/ml prior to leukapheresis highly correlated with and was predictive for the number of collected CFU-GM/kg (P < 0.001). In addition, a significant correlation (P < 0.05) between the number of progenitor cells/kg reinfused and the time to myeloid and platelet recovery was found in children undergoing high-dose therapy. Our data indicate that a single leukapheresis will be sufficient to obtain a minimum number of 5 x 10(4) CFU-GM/kg if the pre-harvest number of circulating CD34+ cells is > or = 10(5)/ml. Thus, our results will help to optimize PBPC transplantation in children.
Adolescent↗
Quick-freezing of cultured cardiac cells in situ with special attention to the mitochondrial ultrastructure.
A new method has been developed which allows quick-freezing in situ of primary, cardiac cell cultures grown to confluence on gas-permeable membranes (Petriperm dishes). Small pieces of the growth substratum, with rhythmically beating myocardial cells, were slam-frozen, without cryoprotectants, against the surface of a helium-cooled copper block at approximately 16 K. The quality of the cellular cryopreservation, as judged by ultrastructural criteria, was studied in freeze-substituted specimens processed for transmission electron microscopy. The ultrastructure of cryofixed cardiac cells was compared with that of unfrozen, chemically fixed samples. The severity of cryodistortions increased progressively with increasing distance from the point of first impact. Of particular interest were the dramatic alterations of the mitochondrial ultrastructure. The concept that the reticular and the outer mitochondrial membranes are intimately and strongly associated was clearly demonstrated. Optimally frozen material revealed cryopreserved ultrastructure of high quality. The method described appears to offer an ideal model system for correlating the information gained by phase-contrast microscopy of living cell cultures with the ultrastructure of the same samples fixed in situ by chemical or physical techniques. Cryofixation would be particularly useful for studying dynamic cellular processes associated with physiological and pathophysiological conditions, e.g. metabolic inhibition, anoxia and substrate deprivation.
Animals↗
Enhanced circulatory parameters of human platelets cryopreserved with second-messenger effectors: an in vivo study of 16 volunteer platelet donors.
Platelet transfusion represents an important component of the therapy for thrombocytopenic patients. Prolonged storage capabilities for platelets would alleviate many problems associated with blood banking. Unfortunately, current cryopreservation methods are complex to implement and result in loss of cell number and functional activity. Previous in vitro studies have shown that the use of ThromboSolTM, a platelet-stabilizing formulation, in the cryopreservation of platelets results in significant retention of cell number and in vitro functional activities in addition to reducing the DMSO requirement to only 2%. We evaluated the in vivo circulatory parameters of platelets cryopreserved with ThromboSol. Single donor platelet units were obtained from healthy volunteers (n = 16); the units were then split and cryopreserved with either ThromboSol and 2% DMSO or 6% DMSO alone. Following storage at -80 degrees C for 7-10 d the samples were thawed, washed and radiolabelled with either 51Cr or 111In. The paired samples were then mixed and reinfused into the autologous volunteer. At various time intervals following transfusion a blood sample was drawn and the quantity of circulating labelled platelets was determined. The percent recovery and survival time was determined by multiple-hit analysis. The ThromboSol-treated platelets, as compared to the 6% DMSO-treated platelets, displayed statistically higher percent recovery (40.2% v 28.8%) and survival time (166.3 h v 152.1 h). These results demonstrated that platelets cryopreserved with ThromboSol displayed superior in vitro and in vivo characteristics as compared to the standard 6% DMSO method. The use of ThromboSol allowed for a 3-fold reduction in the DMSO concentration in conjunction with a 40% increase in circulating cell number and normal survival times.
Blood Platelets↗