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PubMed1997

Effect of artificial stimulants on cryopreserved spermatozoa from cancer patients.

PURPOSE: We determined whether cryopreserved sperm samples obtained from cancer patients before treatment respond to artificial motility stimulants and if this response is related to the extent of disease. MATERIALS AND METHODS: Pre-freeze distribution of disease stage in the different types of cancer and the percentage of the population with or without oligospermia before cryopreservation were examined. Cryopreserved semen samples from 17 cancer patients (10 with testicular cancer, 5 with Hodgkin's disease and 2 with other metastatic disease) were examined for a relationship between post-thaw sperm motion characteristics and patient age or status (survived versus died) and type of disease. Motion characteristics (curvilinear velocity, straight line velocity, average path velocity, linearity and amplitude of lateral head displacement) were analyzed on a computer assisted semen analyzer before (time 0), and 30 and 60 minutes after addition of a 2.5 mM. concentration of pentoxifylline and 2-deoxyadenosine. RESULTS: Post-thaw sperm motion characteristics were not correlated with patient age or status, whether they did or did not have oligospermia, or type of cancer. Compared to baseline values, sperm motion characteristics increased significantly after stimulation at time 0 (p < 0.02) and at 60 minutes (p < 0.05). Oligospermic or nonoligospermic specimens responded to the same extent with pentoxifylline and 2-deoxyadenosine. A negative correlation was noted between overall stage, and type of disease and motion characteristics. CONCLUSIONS: Sperm banking should be encouraged at cancer diagnosis regardless of semen quality. Artificial stimulation of sperm motility results in significant improvement in sperm motion characteristics.

Adult↗
PubMed2003

Immune function of cryopreserved avian peripheral white blood cells: potential biomarkers of contaminant effects in wild birds.

Contaminants can cause detrimental effects in wild birds. However, these effects are difficult to measure in all but the most severe cases. Immune function is a sensitive and meaningful biological marker of contaminant-induced effects in captive birds but has more limitations in wild birds due in part to the lack of a proven blood preservation method. We developed methods to assess ex vivo immune function in wild birds using cryopreserved peripheral white blood cells (WBCs). We assessed the effects of cryopreservation on WBC viability and functionality in two immunoassays (concavalin A-induced T lymphocyte proliferation and macrophage phagocytosis) in domestic chickens (Gallus spp.: white Wyandottes and Dominiques) and validated this approach on cryopreserved WBC samples from wild American coots (Fulicia americana). Cryopreservation of chicken WBCs caused a slight but significant decrease in cell viability (99% +/- 0.2 SE for fresh cells versus 84% +/- 2 SE for cryopreserved cells, p = 0.001, Mann-Whitney U, n = 8). No difference was detected in viability between cells that were cryopreserved for less than 10 days (88% +/- 3.7 SE) and more than 50 days (89% +/- 1.3 SE) (n = 6). Overall, there was no statistical difference in the performance of cryopreserved cells compared to fresh cells. Across multiple experiments, cryopreserved T lymphocytes exhibited 200-900% stimulated proliferation above nonstimulated cells, and 40-80% of cryopreserved macrophages ingested yeast. 9,10,Dimethyl-1,2-benz-anthracene (DMBA) reduced proliferation and phagocytosis in cryopreserved cells over an ex vivo exposure range of 0-170 microM DMBA. Tests of immune function on American coot WBCs cryopreserved for up to 10 months (viability of 72% +/- 2.5 SE, n = 24) were similar to the cryopreserved chicken WBCs. This study will facilitate greater use of ex vivo immune function assays as tools to study effects of contaminant exposure in wildlife by demonstrating the viability and functionality of cryopreserved avian cells.

Animals↗
PubMed1993

The isolation and genetic heterogeneity of Trypanosoma brucei gambiense from north-west Uganda.

Fifty-two samples of blood were taken from sleeping sickness patients in north-west Uganda. All samples failed to infect immunosuppressed mice. Ten cryopreserved blood samples were fed to laboratory bred Glossina morsitans morsitans; eight flies developed midgut infections from which procyclic cultures were established in vitro. Isoenzyme electrophoretic analysis of 9 enzymes revealed that 7 of the 8 trypanosome isolates had a combination of enzyme patterns already described for Trypanosoma brucei gambiense. The eighth isolate had a different aspartate aminotransferase polymorphism which placed it in a new zymodeme. Analysis of polymorphisms in genes for 3 variant surface glycoproteins (VSGs) confirmed that the 8 Ugandan trypanosome isolates were T.b.gambiense and revealed further heterogeneity. The VSG 117 gene was present in all the isolates in a pattern of fragments (equivalent to AnTat 1.8) characteristic for T.b.gambiense. For two other VSG genes characteristic of T.b.gambiense, the LiTat 1.3 gene was present in all the isolates, while the AnTat 11.17 gene was present in only 2 of the 8 isolates.

Animals↗
PubMed1997

Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa.

Mitochondrial function and sperm viability were quantified in samples of cryopreserved bovine spermatozoa from 12 bulls using fluorometric techniques. The active mitochondria of the spermatozoa were fluorescently stained using three different fluorophores: rhodamine 123 (R123), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyan ine iodide (JC-1) or MitoTracker Green FM (MITO). The stained spermatozoa, and companion aliquots that had been stained with SYBR-14 (a living-cell nucleic acid stain) and propidium iodide to assess viability, were quantified using flow cytometry. The resulting fluorescent measurements of mitochondrial function were compared with microscopic assessments of progressive sperm motility immediately after thawing, with motility after 3-h incubation at 37 degrees C, and with the fluorescent assessment of sperm viability. Staining with either R123 or MITO resulted in a single green population. In contrast, the JC-1 staining of mitochondria produced both green and red-orange populations of spermatozoa and sometimes a progressive gradient between the two populations. The ability of JC-1 to discriminate between mitochondria exhibiting high membrane potential from those having low to medium membrane potential provided a more rigorous estimate of metabolic function than the other two fluorescent stains. Overall, the three fluorometric measurements of mitochondrial function were highly correlated with each other, with the SYBR-14 assessment of viability, and with the microscopic estimates of motility.

Animals↗
PubMedDate not supplied

Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a mode of sublethal cryodamage to human sperm during cryopreservation.

Cryopreservation of human sperm, now generally required in donor insemination programs, adversely affects the sperm in terms of standard sperm evaluation parameters and fertilizing ability. The freeze-thaw process appears to produce sublethal damage that appears only after a delay. The authors hypothesized that cryopreservation enhanced peroxidation of sperm membrane lipids, based on previous studies of sperm lipid peroxidation, which showed that the effects of peroxidative damage became evident only after a delay, depending on the peroxidation rate. The effect of cryopreservation on the phospholipid content, the composition of the acyl moieties of the phospholipids, and the activities of the peroxidation protective enzymes, superoxide dismutase (SOD) and glutathione peroxidase plus reductase, in human sperm were examined to test the hypothesis. Parallel determinations were made of the percent motility, the average path velocity of the motile cells, and the time to loss of motility under specified aerobic incubation conditions, which gives a good estimate of the lipid peroxidation rate. The phospholipid content decreases after cryopreservation, with loss of phosphatidylcholine and phosphatidylethanolamine being the more pronounced. Polyunsaturated acyl moieties were also preferentially lost. This loss pattern is observed also from lipid peroxidation. The activities of glutathione peroxidase plus reductase remained unchanged. The sperm SOD activities varied widely between samples before cryopreservation. In all samples there was a decline in SOD activity after freeze-thaw, but the extent of the decline was also widely variable. The time to loss of motility declined in parallel with SOD activity, and a strong correlation (R2 greater than 0.9) between SOD activity and time to loss of motility was found for all samples, before and after freeze-thaw. The authors conclude that cryopreservation does enhance lipid peroxidation in human sperm, as hypothesized, and that this enhancement is mediated at least in part by the loss of SOD activity occurring during the process.

Adult↗
PubMed1999

Stability of transbilayer phospholipid asymmetry in viable ram sperm cells after cryotreatment.

The transbilayer dynamics of lipids in the plasma membrane of mammalian sperm cells is crucial for the fertilization process. Here, the transbilayer movement and distribution of phospholipids in the plasma membrane of fresh, ejaculated and cryopreserved ram spermatozoa was studied by labeling cells with fluorescent analogues of phosphatidylserine and phosphatidylcholine. By co-labeling cells with the DNA-binding dye propidiumiodide as well as by employing fluorescence microscopy and flow cytometry we were able to determine the transbilayer redistribution of fluorescent phospholipid analogues in intact (propidiumiodide-negative) and in impaired (propidiumiodide-positive) spermatozoa. The transbilayer distribution of the fluorescent phosphatidylserine and phosphatidylcholine analogues was not perturbed in intact sperm cells after cryopreservation. In those cells, the phosphatidylserine analogue became rapidly enriched on the cytoplasmic leaflet by the activity of a putative aminophospholipid translocase similar to intact cells of fresh, ejaculated samples. However, upon cryopreservation the activity of the putative aminophospholipid translocase was significantly reduced in intact cells. Employing annexin V-FITC, we found that even after cryopreservation the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet is maintained in intact cells, but not in impaired cells. The phosphatidylcholine analogue redistributed very slowly remaining essentially confined to the exoplasmic leaflet of the plasma membrane of intact cells from both fresh, ejaculated and cryopreserved samples. The physiological consequences of a perturbed transbilayer asymmetry in sperm plasma membranes is discussed.

4-Chloro-7-nitrobenzofurazan↗
PubMed2002

Semen production in adolescent cancer patients.

BACKGROUND: The influence of an accompanying person (parent, guardian or nurse) on the ability of an adolescent (post-pubescent, <20 years of age) to produce a semen sample for cryopreservation, is undetermined, as is the potential for use of urine samples to retrieve sperm in those adolescents that are unable to produce a semen sample. METHODS: The records from 1991-2000 inclusive were reviewed to derive those adolescent patients who were unable to produce semen for cryopreservation prior to undergoing treatment for a malignant condition. RESULTS: During the study period 238 adolescents attended our unit of whom 205 (86.1%) banked semen ('producers'). The remaining 33 adolescents (13.9%) were initially unable to produce a sample ('non-producers'), four of these provided a urine specimen for analysis (12.1%) and of these one had sufficient sperm for cryopreservation. Of the 'accompanied' patients 29.7% (19/64) were non-producers while in the 'unaccompanied' patients only 8.0% (14/174) were non-producers (chi(2) = 16.58, P < 0.001). The relative risk (RR) of not producing a semen sample for the accompanied group of patients was greater than that for the unaccompanied group (RR = 3.689, 95% confidence interval: 2.0-6.9). One patient returning alone successfully provided a semen sample for storage. CONCLUSION: Units should consider the effect of the presence of an accompanying person when an adolescent is unable to produce a semen sample and should consider requesting urine to retrieve sperm.

Adolescent↗
PubMed2005

Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT.

BACKGROUND: Cryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC), and ELISPOT. In this study, we compared the performance of these assays on leukapheresed PBMC shipped overnight in medium versus cryopreserved PBMC from matched donors. RESULTS: Using CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p <or= 0.001). The differences in response magnitude between cryopreserved and shipped PBMC specimens were not significant for most antigens and assays. There was significant correlation between CFC and ELISPOT assay using pp65 peptide pool stimulation, in both shipped and cryopreserved samples (p <or= 0.001). Strong correlation was observed between CFC (using HLA-A2-restricted pp65 peptide stimulation) and tetramer staining (p < 0.001). Roughly similar sensitivity and specificity were observed between the three assays and between shipped and cryopreserved samples for each assay. CONCLUSION: We conclude that all three assays show concordant results on shipped versus cryopreserved specimens, when using a peptide-based readout. The assays are also concordant with each other in pair wise comparisons using equivalent antigen systems.

Antigen Presentation↗
PubMed2006

Substantial improvements in performance indicators achieved in a peripheral blood mononuclear cell cryopreservation quality assurance program using single donor samples.

Storage of high-quality cryopreserved peripheral blood mononuclear cells (PBMC) is often a requirement for multicenter clinical trials and requires a reproducibly high standard of practice. A quality assurance program (QAP) was established to assess an Australia-wide network of laboratories in the provision of high-quality PBMC (determined by yield, viability, and function), using blood taken from single donors (human immunodeficiency virus [HIV] positive and HIV negative) and shipped to each site for preparation and cryopreservation of PBMC. The aim of the QAP was to provide laboratory accreditation for participation in clinical trials and cohort studies which require preparation and cryopreservation of PBMC and to assist all laboratories to prepare PBMC with a viability of >80% and yield of >50% following thawing. Many laboratories failed to reach this standard on the initial QAP round. Interventions to improve performance included telephone interviews with the staff at each laboratory, two annual wet workshops, and direct access to a senior scientist to discuss performance following each QAP round. Performance improved substantially in the majority of sites that initially failed the QAP (P = 0.002 and P = 0.001 for viability and yield, respectively). In a minority of laboratories, there was no improvement (n = 2), while a high standard was retained at the laboratories that commenced with adequate performance (n = 3). These findings demonstrate that simple interventions and monitoring of PBMC preparation and cryopreservation from multiple laboratories can significantly improve performance and contribute to maintenance of a network of laboratories accredited for quality PBMC fractionation and cryopreservation.

Blood Preservation↗
PubMed1992

High-dose combination alkylating agents with autologous bone-marrow support in patients with breast cancer: preliminary assessment of DNA damage in individual peripheral blood lymphocytes using the single cell gel electrophoresis assay.

The single cell gel (SCG) assay is a sensitive electrophoretic technique for detecting the presence of DNA single strand breaks and alkali-labile damage in individual cells. This technique was used to evaluate the levels of DNA damage in cryopreserved peripheral blood lymphocytes (PBLs) from 11 breast cancer patients treated with high doses of cyclophosphamide and cisplatin and provided autologous bone marrow transplantation after treatment. PBL specimens for the SCG study were obtained just prior to treatment, following the administration of cyclophosphamide and cisplatin for 2 days, and upon lymphocytic recovery. Based on a concurrent analysis of DNA damage in cryopreserved and non-cryopreserved PBL samples from three patients, the mean level of DNA migration or the dispersion of damage among cells was not affected by the process of cryopreservation. The pre-treatment samples of several patients contained PBL with increased levels of DNA damage, presumably reflecting persistent DNA damage induced by previous treatment regimens. Chemotherapy resulted in a significant but variable increase in DNA damage in PBL samples from all patients. In this limited study, the level of damage did not correlate with serum levels of cyclophosphamide or with lymphocyte toxicity. Among the post-treatment samples, increased levels of DNA damage were absent in most but not all patients. The presence of damaged cells in the post-treatment samples may be indicative of an inadequate therapy regimen or of DNA damage resulting from non-therapy related processes. Because of its simplicity and short processing time, the SCG assay can be used to evaluate levels of DNA damage during the course of therapy, allowing the dose schedule to be altered to achieve a desired effect level.

Adult↗
PubMed1995

Can the immunobead assay for detecting sperm antibodies in fresh samples be reproduced in cryopreserved/thawed human spermatozoa?

PROBLEM: To evaluate the reproducibility of the Immunobead Assay (IBA) on sperm samples before and after cryopreservation. METHOD: Sperm samples (fresh and post-thaw) from known antibody negative donors (N = 20) were evaluated for percent immunobead binding by IBA following incubation with known antibody-positive serum. RESULTS: In both fresh and thawed negative samples, the mean sperm head binding was 0.5% +/- 0.5, the mean sperm tail binding was 2.0% +/- 2.0 and the mean sperm head-tail binding was 3.0% +/- 2.0 for IgG, IgA and IgM type antibodies, respectively. The same samples exposed to positive sera showed 40.0% +/- 10.0 mean head binding, 7.0% +/- 8.0 mean tail binding and 47.0% +/- 11.0 mean head-tail binding. CONCLUSIONS: IBA is highly reproducible for detecting sperm antibodies in both fresh and cryopreserved/thawed samples of human spermatozoa.

Autoantibodies↗
PubMedDate not supplied

Effects of cryopreservation on bull sperm head morphometry.

Artificial insemination using cryopreserved semen is a common management tool of the contemporary livestock producer. However, cryopreservation is detrimental to sperm function and fertility, killing some 50% of the spermatozoa during the process. Prediction of cryopreservation damage from prefreeze samples remains elusive. Computer-automated sperm head morphometry was used in this study to determine the effects of cryopreservation on bovine sperm head morphometry. Semen was collected from 18 bulls and was divided. One portion was extended to 200 x 10(6) sperm/ml and a microscope slide was prepared, while the remaining portion was cryopreserved in a Triscitrate-yolk extender. After thawing, the cryopreserved samples were prepared on microscope slides. All slides were air dried and were stained with hematoxylin and rose bengal. The morphometric dimensions for length, width, width/length, area, and perimeter for a minimum of 200 sperm heads were analyzed from each slide by computer-aided sperm head morphometry analysis, and the mean measurements were recorded. Bull sperm heads were significantly (P < 0.01) smaller in cryopreserved spermatozoa than in the companion extended samples for length (8.56+/-0.07 vs. 8.63+/-0.08 microm), width (4.39+/-0.05 vs. 4.48+/-0.05 microm), area (28.42+/-0.07 vs. 29.14+/-0.08 microm), and perimeter (23.33+/-0.21 vs. 23.70+/-0.23 microm) for all bulls. Width/length was also different (0.513 vs. 0.519). In addition, differences (P < 0.05) were found within 14 of 18 bulls for at least four of the morphometric parameters. The percent change in measures after cryopreservation were correlated (P < or = 0.05) to the variability of the extended sample. Variations in sperm head measurements were lower (P < or = 0.05) in extended samples of the four bulls in which no changes occurred than in extended samples of the remaining 14 bulls. These data suggest that the variability in sperm head measurements of individual bulls, or ejaculates, may be an indicator of sperm cryosurvivability.

Animals↗
PubMed2001

Improved human sperm recovery using superoxide dismutase and catalase supplementation in semen cryopreservation procedure.

The aim of this work was to evaluate the effects of ROS scavenger supplementation in human semen samples undergoing cryopreservation procedures.After screening out andrological pathologies, we selected 25 male partners of infertile couples with the following semen profile: volume >/= 2.0 ml, normal viscosity, sperm count >/=20 x 10(6)/ml, straight progressive motility (classes 1 and 2) >/= 40% (Mazzilli, Rossi, Delfino and Nofroni (1999) Andrologia 31: 187-194), atypical forms </= 70%, WBCs < 1.0 times 10(6)/ml.After liquefaction, standard semen analysis and the Hypoosmotic Viability Test (HVT) were carried out; the samples were then divided into 4 aliquots. The first was untreated as a control; 200 U/ml of superoxide dismutase (SOD) was added to the second, 200 U/ml of catalase to the third and both SOD (100 U/ml) and catalase (100 U/ml) were added to the fourth aliquot. Each aliquot was mixed (v/v) with TEST yolk buffer freezing medium (Irvine Scientific) and then frozen at -196 degrees C. The percent recovery of progressive motile and swollen spermatozoa was evaluated after thawing.No significant variation in the recovery of progressive motility was seen in the aliquots with added SOD or catalase alone, compared to the control group. On the other hand, a significant improvement in sperm parameter recovery was seen in the aliquot with both SOD and catalase supplementation; perhaps because of their combined and simultaneous action on superoxide anion and hydrogen peroxide. These results suggest that, in some selected cases, SOD and catalase supplementation can contribute greatly to the prevention of sperm membrane lipid peroxidation by ROS and thus allow good sperm parameter recovery after freezing-thawing procedures.

Journal Article↗
PubMed1985

Variation in peripheral blood T cell subsets in serial assays.

No significant differences were found in the T cell subsets of fresh and frozen peripheral blood mononuclear cells (PBM) from six healthy donors analysed with the Ortho series of monoclonal antibodies and a fluorescence-activated cell sorter. Analysis of replicates of cryopreserved PBM showed that considerably higher variation in T cell subsets occurred when samples were assayed in serial assays than when the samples were analysed together under the same conditions. These results indicate that errors introduced into a longitudinal study by serial analysis of samples may be reduced if samples are cryopreserved and subsequently thawed and analysed together at the end of the study.

Antibodies, Monoclonal↗
PubMedDate not supplied

Do conventional CASA-parameters reflect recovery of kinematics after freezing? CASA paradox in the analysis of recovery of spermatozoa after cryopreservation.

Conventional kinematic parameters (KPs) are averages of values obtained from analysing the entire motile fraction of cells in a sample. Occasionally, in spite of overall deterioration of semen samples after cryopreservation and other 'damaging' manipulations, the average relative (derived) KPs such as linearity (LIN), dance (DNC), dance mean (DNM) etc., may show apparently elevated values. Similarly, the absolute (actual) KPs such as straight line velocity (VSL), curvilinear velocity (VCL), average velocity path (VAP), average lateral head displacement (ALH), beat-cross frequency (BCF), etc., may also be higher after damaging treatments depending on the tolerance of various sub-populations in a single sample. The conditions for this CASA-paradox are discussed. Simple modifications of actual CASA-parameters are proposed to correct the pseudo-enhancement of kinematics characteristics in processed semen samples

Journal Article↗
PubMed2003

Evaluation of donor skin viability: fresh and cryopreserved skin using tetrazolioum salt assay.

Cell viability assessment in allograft skin is an essential step to ensure a supply of good quality allograft skin for clinical repair of wounds. It is widely recognised that 'take' of allografts is strongly influenced grafted by tissue viability. The aim of this study was to set-up storage protocols that maintain high viability of the allograft after harvest, treatment and storage. In this study, the viability of post-mortem allografts (n=350) harvested from 35 different donors, was investigated using the MTT salt assay. The conditions of preparation and storage of the allograft included: 1. Fresh skin samples (about 12, 30, and 60h after harvesting). 2. The same specimens (stored at 4 and 37 degrees C) tested for at least 1 month. 3. Samples after cryopreservation and thawing. 4. Thawed specimens tested daily for at least 6 days. Parallel histomorphological analysis performed, under each of these conditions, showed a correlation between changes in structure and changes in viability as measured by the MTT quantitative assay. The viability index (VI) of skin is expressed as the ratio between the optical density (O.D.) produced in the MTT assay by the skin sample and its weight in grams. The percentage viability index is the ratio of the VI of the fresh sample (considered as 100% viability) and the value of specimens from the same harvest batch after storage or cryopreservation. The results indicated that samples tested within 12-30h from harvesting have an average viability index of about 75 with little variation. Samples tested within 60h have an average viability index of 40, showing a viability decrease of about 50%. A protocol to treat skin within a maximum of 30h was, therefore, set-up. The data suggested that skin stored at 37 degrees C, undergoes a viability increase during the first 2 days after harvesting. However, the viability under these conditions then decreased very quickly. After 6 days of preservation at this temperature the samples were no longer viable (PVI = 0). The tissue structure started to become damaged after 3 days. On the other hand, skin stored at 4 degrees C, showed a very slow viability decrease. After 15 days, viability was still almost 25% of the fresh sample. The tissue architecture showed no signs of damage under these conditions until day 7 from harvesting. MTT analysis was performed on the specimens cryopreserved with DMSO at 10%. These measurements were compared to viability assessment of the same fresh skin samples (considered as 100%) that were analysed within 30h from harvesting. The average PVI of thawed skin was 54% of the fresh sample. This result demonstrates that the viability of cryopreserved skin is comparable to the viability of fresh skin stored at 4 degrees C for 4 days. The PVI of thawed skin samples decreased dramatically within 24h, and had reached 0% within 6 days.

Adolescent↗
PubMed2004

Effect of dendritic cells on flow cytometric quantification of cytomegalovirus-specific, interferon-gamma-producing T cells.

Flow cytometric measurement of intracellular cytokines in T cells exposed to antigen is a widely used method for quantification of an antigen-specific T-cell response. As the frequency of antigen-specific T cells is often very low, any improvement in signal to noise ratio is of great importance. Thus, in this study, the ability of antigen-pulsed dendritic cells (DCs) to increase the number of antigen-specific, interferon-gamma (IFN-gamma)-producing CD4+ T cells measurable both in fresh peripheral blood and in reconstituted frozen blood mononuclear cell (MNC) samples was evaluated. Cytomegalovirus (CMV) was used as antigen in a 10 h assay, using cells from both CMV-seropositive and -seronegative donors. When reconstituted frozen samples were analysed, the general response towards CMV lysate in CMV-seropositive donors was 23-86% lower compared to the corresponding fresh blood samples. Antigen-pulsed DCs could not improve the sensitivity of the intracellular cytokine-detection assay when fresh peripheral blood samples were used. Interestingly, however, the addition of CMV lysate-pulsed DCs to cryopreserved MNC samples substantially increased the frequency of specifically induced IFN-gamma-producing cells to a level comparable to the frequency found in the corresponding fresh blood samples.

Adult↗