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PubMed2002

Microsatellite analysis of cryopreserved stallion semen stored on FTA paper.

The aim of this study was to establish and validate a method to permit microsatellite analysis of DNA profiles obtained from frozen-thawed stallion sperm cells. This would provide reliable and accurate verification of the identification of a semen donor. Ejaculates from 5 pony stallions were collected, processed and frozen in 0.5 ml plastic straws. Aliquots of 100 microl of the frozen-thawed semen thus obtained were either placed directly, or diluted (1:10; 1:100; and 1:1000) and placed on slides of FTA paper. Similarly, blood samples obtained from each of the stallions were placed onto slides of FTA paper. A punch was removed from each sample after drying Each sample was mixed with FTA purification reagent, Dithiothreitol and Proteinase K before incubation and processing. All samples were processed with a set of 13 microsatellite markers. Further analysis permitted a comparison of the DNA profiles of the frozen-thawed semen and the blood samples. A full profile of markers was obtained from the 1:10 and 1:100 dilutions of the frozen-thawed semen samples as well as from the blood samples. The DNA profiles from the frozen-thawed semen and blood samples obtained from the stallions matched in all cases.

Animals↗
PubMed2006

Seminal plasma improves cryopreservation of Iberian red deer epididymal sperm.

We tested the protective action of seminal plasma on epididymal spermatozoa from Iberian red deer, especially considering cryopreservation, as a means for germplasm banking improvement. We obtained seminal plasma by centrifuging electroejaculated semen, and part of it was thermically inactivated (denatured plasma; 55 degrees C 30 min). Epididymal samples (always at 5 degrees C) were obtained from genitalia harvested after regulated hunting, and pooled for each assay (five in total). We tested three seminal plasma treatments (mixing seminal plasma with samples 2:1): no plasma, untreated plasma and denatured plasma; and four incubation treatments: 32 degrees C 15 min, 5 degrees C 15 min, 5 degrees C 2h and 5 degrees C 6h. After each incubation, samples were diluted 1:1 with extender: Tes-Tris-Fructose, 10% egg yolk, 4% glycerol; equilibrated for 2h at 5 degrees C, extended down to 10(8) spz./mL and frozen. Sperm quality was evaluated before 1:1 dilution, before freezing and after thawing the samples, assessing motility (CASA) and viability (percentage of viable and acrosome-intact spermatozoa; PI/PNA-FITC and fluorescent microscopy). Plasma treatment, both untreated and denatured, rendered higher viability before freezing and higher results for most parameters after thawing. The improvement was irrespective of incubation treatment, except for viability, which rendered slightly different results for untreated and denatured plasma. This may be due to the presence of thermolabile components. We still have to determine the underlying mechanisms involved in this protection. These results might help to improve the design of cryopreservation extenders for red deer epididymal sperm.

Acrosome Reaction↗
PubMed1992

Laboratory determination of chemotherapeutic drug resistance in tumor cells from patients with leukemia, using a fluorometric microculture cytotoxicity assay (FMCA).

An automated fluorometric microculture cytotoxicity assay (FMCA) based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) to fluorescein was employed for chemotherapeutic-drug-sensitivity testing of tumor-cell suspensions from patients with leukemia. Fluorescence was linearly related to cell number, and reproducible measurements of drug sensitivity could be performed using fresh or cryopreserved leukemia cells. A marked heterogeneity with respect to chemotherapeutic drug sensitivity was observed for a panel of cytotoxic drugs tested in 43 samples from 35 patients with treated or untreated acute and chronic leukemia. For samples obtained from patients with chronic lymphocytic and acute myelocytic leukemia, sensitivity profiles for standard drugs corresponded to known clinical activity and the assay detected primary and acquired drug resistance. Individual in vitro/in vivo correlations indicated high specificity with respect to the identification of drug resistance. The results suggest that the FMCA may be a simple and rapid method for in vivo-representative determinations of chemotherapeutic drug resistance in tumor cells obtained from patients with leukemia.

Antineoplastic Agents↗
PubMed1995

Characterization of a new human glioblastoma cell line that expresses mutant p53 and lacks activation of the PDGF pathway.

We have established and characterized a new glioblastoma cell line, termed GT9, from a biopsy sample of a female adult patient with glioblastoma multiforme. The line has now undergone over 60 passages and has been successfully cultured after cryopreservation. Immunofluorescence analyses with a panel of monoclonal antibodies were positive for glial fibrillary acidic protein and vimentin, and negative for neurofilament, galactocerebroside, and fibronectin, a pattern typical of glial cells. Based on a tetraploid, the composite karyotype of GT9 cells included the loss of chromosome 10, gain of chromosome 7, and the presence of double minute chromosomes, three of the most common karyotypic abnormalities in glioblastoma. Sequence analysis of p53 cDNA revealed a homozygous double mutation at codon 249 (commonly mutated in aflatoxin-associated hepatocellular carcinoma) and codon 250. Moreover, there was a complete absence of wild-type p53. However, unlike the majority of human glioblastomas previously described, the expression of platelet-derived growth factor-B (PDGF-B), a potent mitogenic autocrine factor, was low in GT9 cells. The expression and phosphorylation of c-Jun and Jun-B, downstream mediators of the PDGF pathway, were also low. Thus, deregulation of the PDGF pathway does not appear to be involved in the pathogenesis of the GT9 glioblastoma. Conversely, Jun-D, a negative regulator of cell growth, was also low. In addition, Phosphorylated Egr-1, a recently reported suppressor of PDGF-B/v-sis-transformed cells, was also low, suggesting that the lack of activation of the PDGF pathway was not due to these suppressive mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1999

Is there a risk of cytomegalovirus transmission during in vitro fertilization with donated oocytes?

OBJECTIVE: To define the risk of human cytomegalovirus (HCMV) transmission from donated oocytes. DESIGN: Prospective study. SETTING: University IVF program. PATIENT(S): Sixty-seven couples undergoing 72 cycles of IVF-ET. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Serum from both partners (women: n = 71; men: n = 60) was obtained for detection of antibodies to HCMV. Semen before preparation (n = 53), sperm after preparation (Percoll gradient; n = 47), cervical mucus aspirated at the time of oocyte aspiration (n = 70), and uninseminated oocytes and embryos not suitable for cryopreservation (n = 568) were frozen in liquid nitrogen. Polymerase chain reaction was used for detection of HCMV (immediate early 1 gene) in all samples collected. RESULT(S): Serum antibodies to HCMV were found in 62% of the women and 37% of the men tested. Human cytomegalovirus DNA was detected in 25% of the ejaculates and in 19% of the cervical mucus samples. There was no amplification of HCMV DNA from oocytes or embryos. CONCLUSION(S): Because we were unable to amplify HCMV DNA from any of the oocytes or embryos, it seems unlikely that HCMV is transmissible through oocyte or embryo donation.

Antibodies, Viral↗
PubMed1993

Rapid diagnosis of enterovirus infection by magnetic bead extraction and polymerase chain reaction detection of enterovirus RNA in clinical specimens.

We describe a rapid method for extraction and detection of enterovirus RNA in clinical samples. By using magnetic bead technology, enterovirus RNA was efficiently and rapidly extracted from cerebrospinal fluid, stool, saliva, blood, pericardial fluid, urine, and cryopreserved or formalin-fixed solid tissue. Enterovirus RNA was then detected by reverse transcription followed by polymerase chain reaction amplification with primers designed to allow detection of most enterovirus serotypes. For detection of enteroviruses in specimens from patients with acute enteroviral disease, the overall sensitivity of enzymatic RNA amplification was greater than that of cell culture isolation, especially in blood specimens and in stool specimens from patients with acute cardiac disease. Enterovirus RNA was also detected in cryopreserved and archival formalin-fixed myocardial tissue from patients with acute myocarditis and chronic dilated cardiomyopathy. The ability to study archival specimens is of particular value in conducting retrospective investigation. The RNA extraction procedure used was considerably faster than extraction methods using organic reagents, used less hazardous reagents, and was of similar sensitivity. This detection protocol may therefore be useful both for the diagnosis of enterovirus infection and in studying the pathogenesis of acute and chronic enterovirus-induced disease.

Adult↗
PubMed2004

Cryopreservation and sexing of in vivo- and in vitro-produced bovine embryos for their practical use.

My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GL-Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex pre-determination.

Animals↗
PubMed2000

Freezing of human ovarian tissue--not the oocytes but the granulosa is the problem.

The freezing of human ovarian tissue may be the key for restoring fertility after systemic therapy of cancer. In contrast to others we investigated the survival rate of whole follicles, and had a special look at the granulosa cells. Ovarian tissue was collected laparoscopically (n = 10) and divided into equal parts for freezing (n = 1570) or as control (n = 1660). The cryopreservation was done slowly, or as a ultrarapid freezing. After thawing the number of follicles, oocytes and granulosa cells surviving was counted and corrected for equal volumina of the samples. While 84.5% of the oocytes survived freezing, only 40.4% of the follicles were intact after thawing. The data show that the procedure damaged follicles, which mainly affected the granulosa cells. As the intactness of follicles may play a critical role for the maturation of the oocytes after thawing the protocols should be optimised to meet the needs of oocytes and granulosa cells.

Adult↗
PubMed2004

Cryopreservation of human spermatozoa: comparison of two cryopreservation methods and three cryoprotectants.

OBJECTIVE: To evaluate the ability of two cryopreservation methods and three cryoprotectants to preserve sperm quality. DESIGN: A prospective clinical study. SETTING: Male infertility clinic at a tertiary healthcare center. PATIENT(S): Twenty infertile men and 10 healthy donors. INTERVENTION(S): In the first experiment, semen was cryopreserved by either the Irvine Scientific method (IS) or the Cleveland Clinic Foundation (CCF) method. In the second experiment, semen was cryopreserved by the IS method and one of three cryoprotectants: TES and Tris yolk buffer, Sperm Freezing Medium, or Enhance Sperm Freeze. MAIN OUTCOME MEASURE(S): Postthaw sperm motility, cryosurvival, and kinematics. RESULT(S): Percentages of postthaw sperm motility and cryosurvival were higher in the IS cryopreservation method compared with in the CCF method (15.94 +/- 9.19 vs. 12.07 +/- 7.31 and 47.42 +/- 17.44 vs. 35.76 +/- 17.56). However, the CCF method resulted in significantly better sperm kinematics. Postthaw motility in the donors and patients was highest in the samples frozen in TES and Tris yolk buffer medium. CONCLUSION(S): The IS method was associated with more flash freezing compared with the CCF method and resulted in better preservation of sperm motility and a higher cryosurvival rate. TES and Tris yolk buffer was most effective at protecting sperm from the negative effects of the cryopreservation process. This may be due to the presence of egg yolk along with glycerol.

Cryopreservation↗
PubMedDate not supplied

[Functional status of cryopreserved rat liver mitochondria].

Mitochondria from the rat liver have been frozen down to -196 degrees C under protection of dimethylsulfoxide, after which they were subjected to periodical increase and decrease in temperature (cycling) in the ranges of -105 degrees C to -196 degrees C or -135 degrees to -196 degrees C. There have been observed some non-lethal damages of mitochondria which were revealed in the decrease in the respiratory control and in the ADP/O ratio after the cycling. The damage rate increased with the increase of the number of temperature decrease-increase cycles. The damages were also greater in the case when the upper limit of cycling temperature was higher than the glass transition temperature of the freezing medium (Tg = -126 degrees C). The non-lethal damages of mitochondria are explained by the influence of electric fields, appearing in the frozen sample during the temperature increase or decrease.

Animals↗
PubMed1997

Studies on the pathogenicity of Babesia bovis in water buffaloes after cryopreservation and resuscitation.

Packed erythrocytes infected with Babesia bovis were mixed with an equal volume of 16% dimethyl sulphoxide (DMSO) in Alsever's solution and dispensed into 1.5 or 5 ml cryotubes. The vials were kept in liquid nitrogen (-196 degrees C) for 26, 78, 142 or 149 days. The samples were removed from the liquid nitrogen container and rapidly thawed in a 40 degrees C water bath. The thawed blood successfully infected splenectomised buffalo calves by injection via subcutaneous or intravenous or via intravenous and subcutaneous routes. The parasites, typical B. bovis, were discovered in red blood cells 5, 8 or 9 days after inoculation. The highest percentage of parasitised erythrocytes (PPE) was 15%. The babesiosis resulting from cryopreserved parasites was the same as that resulting from fresh parasites inoculated by ticks. Typical clinical signs were found, such as continuous fever (the highest temperature was 41.3 degrees C), anaemia, icterus and haemoglobinuria. Infected calves, which were not treated, died. Cryopreservation is a simple and reliable method for longterm preservation of B. bovis of water buffaloes.

Animals↗
PubMed1996

Myelination by transplanted human and mouse central nervous system tissue after long-term cryopreservation.

Human and mouse oligodendrocytes were transplanted, after a long period of cryostorage, into newborn mouse brain. Tissue fragments were obtained from brain and spinal cord of 10-week-old human fetuses and from the periventricular zone of embryonic and newborn mouse brains. Samples were stored at -180 degrees C for periods of 3 days to over 5 years. Frozen or fresh fragments were transplanted into the brains of newborn shiverer mutant mice, which are deficient in myelin basic protein (MBP). Normal myelin, produced by grafted oligodendrocytes, was detected by immunohistochemistry with an anti-MBP antiserum. The best results were obtained with isospecific grafts. The timing of myelin appearance did not depend significantly on the species or age of the donor. Myelination obtained with mouse grafts was more profuse when the donor was younger (embryonic versus newborn). Cryopreservation over 5 years did not impede the graft's ability to produce myelin and can be considered for long-term storage of oligodendrocytes in view of cell therapy.

Animals↗
PubMed2006

Influence of temperature during glycerol addition and post-thaw dilution on the quality of canine frozen semen.

Contents The aim of the present study was to compare the influence of room temperature (27 degrees C) and 4 degrees C during glycerol addition on canine semen cryopreservation and verify the effect of different post-thawing dilutions on canine semen. Ten ejaculates from five stud dogs were collected by digital manipulation. Semen samples were evaluated and further divided into two aliquots. The first aliquot was extended in Tris-egg yolk-glycerol at 27 degrees C and the second one received glycerol at 4 degrees C. Samples were frozen and stored in liquid nitrogen. After 1 week, samples were thawed and submitted to evaluations of progressive sperm motility, morphology, acrosomal integrity, hypo-osmotic swelling (HOST) and thermoresistance tests. For thermoresistance test, aliquots were divided in two portions: one portion was kept undiluted (1 : 0) and the other one was diluted in a 1 : 4 ratio (one part semen to four parts extender). No differences were observed between temperatures for glycerol addition regarding seminal parameters evaluated. Furthermore, post-thawing dilutions demonstrated similar effect on canine semen longevity. Correlations among post-thaw sperm motility and HOST and results from thermoresistance test were observed for both temperatures for glycerol addition. In conclusion, glycerol could be added to canine semen at room temperature (27 degrees C) or at 4 degrees C. Moreover, there is no need to extend canine semen after thawing for the thermoresistance test, but if we need to increase the inseminating volume for artificial inseminations, the addition of extender will not damage the semen.

Acrosome↗
PubMed2006

Rapid-cooling and storage of plant nematodes at -140 degrees C.

Low temperatures can assure the long-term or even indefinite preservation of important biological specimens. Nematode cryopreservation allows for the availability of large numbers of living nematodes at any one time, especially for experimental purposes. New isolates of Bursaphelenchus have recently been collected, including Bursaphelenchus eremus (Rühm) Goodey. This species was identified in north-central Italy on dying oak trees and from the bark beetle Scolytus intricatus Ratzeburg as dauer larvae. We therefore, sought to develop a cryopreservation technique for the long-term storage of all available Bursaphelenchus spp. The technique consists of a rapid-cooling protocol involving immersion in a liquid nitrogen bath before storage of the frozen samples in a mechanical freezer at -140 degrees C. The survival of nematodes subjected to this rapid-cooling protocol was higher than previously reported using slow-cooling methods and is suitable for several species of Bursaphelenchus and other phytoparasitic nematodes.

Animals↗
PubMed2004

Activation pattern of caspases in human spermatozoa.

OBJECTIVE: We investigated the activation of caspases 8, 9, 1, and 3 in human ejaculated spermatozoa to study main pathways of apoptosis. Potential functional impact of this phenomenon and possible activation mechanisms were examined by [1] subjecting cells to freezing and thawing, and [2] testing the dependence of caspase activity on membrane integrity. DESIGN: Experimental study. SETTING: Andrology Center Leipzig (European Academy of Andrology). PATIENT(S): Ten healthy volunteers, 40 semen samples. INTERVENTION(S): Sperm populations were separated by annexin V magnetic activated cell sorting (MACS) according to externalized phosphatidylserine. MAIN OUTCOME MEASURE(S): Active caspases 8, 9, 1, and 3 were examined in human ejaculated spermatozoa before and after cryopreservation. RESULT(S): The active caspases were localized in the postacrosomal region (caspases 8, 1, and 3) or in the midpiece (caspase 9). Cryopreservation led to a significant increase in the number of cells showing activation of all caspases. The MACS separation resulted in a significant depletion of sperm positive for activated caspases within the externalized phosphatidylserine-negative fraction. CONCLUSION(S): Caspases 8, 9, 1, and 3, representing the main pathways of apoptosis, are present in human spermatozoa and can become activated, particularly after freezing and thawing, and are associated with changes in the outer cell membrane. Spermatozoa showing these changes and caspase activation can be efficiently removed from semen samples by the annexin V MACS technique.

Annexin A5↗
PubMed1989

Correlation of hematologic recovery with CFU-GM content of autologous bone marrow grafts treated with 4-hydroperoxycyclophosphamide. Culture after cryopreservation.

We previously described an exponential correlation of the CFU-GM content of 4-hydroperoxycyclophosphamide-treated autologous bone marrow grafts and the rate of hematologic recovery of the recipients after transplantation. Those grafts were assayed before cryopreservation. We now describe a similar analysis based upon cultures of the thawed bone marrow. In a proportional hazards model, the CFU-GM content of the grafts sampled after thawing predicted time to achieving a peripheral blood count of 1 X 10(9)/l leukocytes, 0.5 X 10(9)/l granulocytes, 2% reticulocytes, and last platelet transfusion (p less than 0.001). Using linear regression analysis, the logarithm of the CFU-GM content/kg of recipient weight also was correlated with the time to achieving 1 X 10(9)/l leukocytes (r = -0.62), and 0.5 X 10(9)/l granulocytes (r = -0.66). The hazard ratios (Cox model) and regression coefficients (linear regression model) for the equations calculated from the post-thaw cultures were usually smaller than the corresponding coefficients based upon the pre-freeze cultures, suggesting that pre-freeze cultures may be more accurate predictors of aplasia duration. However, patients with poor post-freeze survival of CFU-GM were more likely to experience delayed engraftment. Therefore, the correlation of post-thaw cultures with engraftment may permit the analysis of cryopreservation techniques.

Adolescent↗
PubMed1997

Selection and transplantation of autologous hematopoietic CD34+ cells for patients with multiple myeloma.

Here we review our recent experience addressing the issue of positive selection and transplantation of hematopoietic CD34+ cells to reduce neoplastic contamination in peripheral blood (PB) autografts from patients with multiple myeloma (MM). We evaluated PB samples from 30 pretreated MM patients following the administration of high dose cyclophosphamide (Cy; 7g/m2 or 4g/m2) and granulocyte-colony stimulating factor (G-CSF), for collection of circulating stem cells (PBSC) to support hematopoietic reconstitution following myeloablative radio-chemotherapy. Twenty six patients showed adequate mobilization of CD34+ progenitor cells and were submitted to PBSC collection. Circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, cryopreserved, and used to reconstitute BM function after myeloablative therapy in 13 patients. The median purity of the enriched CD34+ cell population was 89.5% (range 51-94%) with a 75-fold increase compared to the pretreatment samples. The median overall recovery of CD34+ cells and CFU-GM was 58% (range 33-95%) and 45% (range 7-100%), respectively. Positive selection of CD34+ cells resulted in 2.5-3 log of plasma cells and CD19+ B-lineage cells depletion as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene demonstrated the persistence of minimal residual disease (MRD) in 5 out of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (TBI, 1000 cGy) and high dose Melphalan (140 mg/m2) or Melphalan (200 mg/m2) alone. They received a median of 5 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis: the median time to 0.5 x 10(9) neutrophils, 20 and 50 x 10(9) platelets/L of PB was 10, 11 and 12 days, respectively. When we analyzed the immunological reconstitution of this group of patients, we observed a rapid and full recovery of total lymphocyte and NK cell count, although the absolute CD4+ cell count was lower than pretreatment level. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (=13) receiving unmanipulated PBSC following the same pretransplant conditioning regimen. The results of this trial demonstrate that positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3 log and provides a cell suspension capable of restoring a normal hematopoiesis after a TBI-containing conditioning regimen. Based on this pilot trial, we have recently started a clinical study involving a double autotransplant, conditioned with melphalan (200 mg/m2) followed by melphalan (140 mg/m2) and busulphan (14 mg/kg), supported by the reinfusion of highly purified CD34+ cells.

Antigens, CD34↗
PubMed1991

Cryopreservation and long-term liquid nitrogen storage of peripheral blood mononuclear cells for flow cytometry analysis: effects on cell subset proportions and fluorescence intensity.

The effect of cryopreservation and long-term liquid nitrogen storage on peripheral blood mononuclear cell (PBMC) subsets was prospectively analyzed using monoclonal antibodies and flow cytometry. Brief cryopreservation did not significantly alter the proportion of positively stained cells for CD3+, CD4+, CD8+, CD14+, CD16+, and CD19+ cells. A small but statistically significant increase in the proportion of positive cells was observed for HLA-DR+ and HLe-1+ cells. Brief cryopreservation was associated with a decrease in the mean fluorescence intensity (MFI) values for CD3+, CD4+, and CD8+ cells; an increase in MFI values for CD14+ and HLA-DR+ cells; and no change for CD16+, CD19+, and HLe-1+ cells. There was no significant change in the proportion of CD3+, CD4+, or CD16+ cells during 20 months of storage in liquid nitrogen. Small but statistically significant decreases in the proportion of CD8+ and CD19+ cells were observed over the same interval, and the proportion of CD14+ cells (monocytes) was highly variable. Chronologic changes in fluorescence intensity during long-term storage were observed for all cell subsets except CD16+ and CD19+ cells. Cryopreservation is a valuable technique for long-term storage of viable cells. For many laboratory applications, the small changes noted in the present study will have no practical importance. However, for clinical and epidemiological investigations encompassing large numbers of samples, statistical techniques to adjust for small changes during storage should be considered.

Antigens, CD↗