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PubMed1997

Assessment of the acrosomal status of membrane-intact ram spermatozoa after freezing and thawing, by simultaneous lectin/Hoechst 33258 staining.

We have evaluated the effect of freezing and thawing on the acrosomal status of ram spermatozoa, especially those that withstood cryopreservation as assessed by membrane integrity. To this end, we performed simultaneous lectin/Hoechst 33258 staining, and compared the ability of three fluoresceinated lectins. Ram spermatozoa were treated with fluorescein isothiocyanate-labelled Pisum sativum lectin (PSA), fluorescein isothiocyanate-labelled Arachis hypogea lectin (PNA) and fluorescein isothiocyanate-labelled Triticum vulgaris lectin (WGA) and simultaneously with Hoechst 33258 for determination of membrane integrity and acrosomal status. In all cases, three forms were readily distinguished by their distribution pattern. For both PSA and PNA, the most abundant form found in fresh semen consisted of fluorescence on the acrosomal area. This form corresponds to acrosome-intact spermatozoa, as assessed by Differential Interference Contrast (DIC) microscopy. Two minor forms showed weak fluorescence on the equatorial segment or no fluorescence on the head. DIC microscopy revealed that both forms were associated with acrosome-lost spermatozoa. WGA labelling showed two forms, one of which consisted of fluorescence on the entire head, albeit more intensely on its anterior segment. Spermatozoa in this form were acrosome-intact by DIC. The other form lacked fluorescence on the acrosomal region, but still showed faint fluorescence in the posterior region. This form was acrosome-lost by DIC. Incubation of fresh spermatozoa with calcium ionophore A23187 for up to 1 h significantly increased the percentage of those forms identified as acrosome-reacted as described above. This was confirmed by the time-dependent accumulation of these forms, as well as by DIC microscopy. At all times, differences among values obtained using these three lectins were not significant. Freezing and thawing led to a decrease of both membrane integrity and acrosomal integrity, irrespective of the lectin used. However, almost all spermatozoa that withstood cryopreservation, as evaluated by Hoechst exclusion, showed intact acrosomes. In this case, no differences between fresh and frozen/thawed samples were observed. These results suggest that the structural integrity of ram spermatozoa is mostly unaffected after cryopreservation, suggesting that it is damage to the plasma membrane that is primarily responsible for the low fertility of cryopreserved samples.

Acrosome↗
PubMed2005

Molecular epidemiology biomarkers--sample collection and processing considerations.

Biomarker studies require processing and storage of numerous biological samples with the goals of obtaining a large amount of information and minimizing future research costs. An efficient study design includes provisions for processing of the original samples, such as cryopreservation, DNA isolation, and preparation of specimens for exposure assessment. Use of standard, two-dimensional and nanobarcodes and customized electronic databases assure efficient management of large sample collections and tracking results of data analyses. Standard operating procedures and quality control plans help to protect sample quality and to assure validity of the biomarker data. Specific state, federal and international regulations are in place regarding research with human samples, governing areas including custody, safety of handling, and transport of human samples. Appropriate informed consent must be obtained from the study subjects prior to sample collection and confidentiality of results maintained. Finally, examples of three biorepositories of different scale (European Cancer Study, National Cancer Institute and School of Public Health Biorepository, University of California, Berkeley) are used to illustrate challenges faced by investigators and the ways to overcome them. New software and biorepository technologies are being developed by many companies that will help to bring biological banking to a new level required by molecular epidemiology of the 21st century.

Automation↗
PubMed1991

Feasibility of semen collection and cryopreservation during chemotherapy.

Collection and cryopreservation of semen was undertaken in a 40-year-old man undergoing combination cyclophosphamide-doxorubicin-vincristine-prednisone chemotherapy for diffuse large cell lymphoma. Semen samples worthy of cryopreservation could be obtained through 39 days of therapy, with azoospermia observed after 70 days. Although pregnancy has not been attempted in the current case, reports by others suggest that normal outcomes can be expected with thawed semen collected during chemotherapy.

Adult↗
PubMedDate not supplied

Bovine mucus penetration test and routine semen analysis of fresh and cryopreserved human spermatozoa.

Semen samples from 18 fertile men and 18 infertile men were evaluated both fresh and after freezing and thawing using traditional semen parameter, the hypoosmotic swelling test (HOS-test) and the bovine cervical mucus penetration test (BMP-test). Major differences in the semen analysis between the fertile and infertile men were observed for motility, morphology (head and tail defects), the bovine mucus penetration test and the hypoosmotic swelling test. Cryopreservation resulted in a decrease regarding motility, the BMP-test and the HOS-test. Percentagewise the losses due to cryopreservation were higher in the infertile group than in the fertile group. The results of this study show that the bovine mucus penetration test as well as the HOS-test are valuable additions to the routine semen analysis of fresh and cryopreserved spermatozoa.

Adult↗
PubMed2003

Risk of cytomegalovirus transmission by cryopreserved semen: a study of 635 semen samples from 231 donors.

BACKGROUND: The hypothetical responsibility of sperm donation in cytomegalovirus (CMV) transmission to recipients and precautions to prevent this transmission are widely discussed. The aim of this French CECOS Federation study was to evaluate both the reality and the importance of the CMV risk due to donor sperm and the relevance of measures used to screen it. METHODS: We conducted a prospective multicentric study. CMV was detected by rapid and conventional cultures and by PCR in the frozen sperm of donors who met the normal criteria required of semen donors, irrespective of their CMV serological status. RESULTS: 635 samples from 231 donors (39.4% IgG(+)) were obtained and tested by culture; 551 samples from 197 donors were also tested by PCR. From those samples, 0.78% were culture(+), 1.57% culture(+) and/or PCR(+); 3.3% of seropositive donors and 0.72% of initially seronegative donors were culture(+), but in the latter seroconversion occurred during the quarantine period; of the 197 PCR-tested donors, 3.5% (6.2/1.7) were PCR(+), 3.3% (5.3/1.45) culture(+) and/or PCR(+). PCR(+) samples can be culture(-) and vice versa. The most strongly positive sample corresponded to an initially seronegative donor. CONCLUSION: The best strategy to prevent potential CMV risk is to test donors for CMV IgG and IgM antibody at the outset and after a 6 month period of quarantine and to reject initially IgM seropositive donors or donors who seroconvert during the quarantine period.

Antibodies, Viral↗
PubMed1984

Cryopreservation of viable Giardia intestinalis trophozoites.

A technique is described for the cryopreservation of Giardia intestinalis trophozoites. The most satisfactory results were obtained when organisms were preserved with either 7.5 or 10% dimethyl sulphoxide (Me2SO) and cooled using a liquid nitrogen controlled freezer. Under these conditions more than 70% of organisms were motile after thawing. Lower recovery rates were obtained using glycerol as the cryopreservant or when samples were placed directly into a -70 degrees C refrigerator to cool. Cultures were successfully re-established from material cooled under controlled conditions using either 7.5% Me2SO or glycerol as the cryopreservant. However, the former had an initial generation time of 12.0 hours compared to 24.5 hours for the latter.

Animals↗
PubMed2002

CML leukapheresis products can be enriched for CD34+ cells and simultaneously depleted of CD15+ cells using a simple Ab cocktail.

BACKGROUND: CML progenitor-cell studies would be greatly facilitated if samples could be repeatedly accessed from a source of well-characterized cells. The present study was designed to develop a simple, inexpensive Ab cocktail that would provide subpopulations of cells enriched for CD34+ cells and simultaneously depleted of CD15+ mature myeloid cells. METHODS: Cells from leukapheresis products from CML patients at diagnosis were incubated with each of two Ab cocktails. The standard cocktail (debulking, DB), containing 11 Abs, is recommended for obtaining a highly enriched population of CD34+ cells. The efficacy of an alternative, simpler cocktail (CML custom, CC), containing only four Abs was tested. The recoveries of CD34+ cells, CD15+ cells, colony-forming unit granulocyte-macrophage, and LTCIC were monitored. The samples were then cryopreserved, thawed, and the recoveries remeasured. RESULTS: The purity of CD34+ cells was significantly superior using the DB cocktail than with the CC cocktail. Conversely, using the CC cocktail, the yield of CD34+ cells was significantly higher compared to the DB cocktail. These results were maintained even when the amount of Ab was reduced 10-fold. Both Ab cocktails consistently removed > 99% of the CD15+ cells. Consistent with the CD34+ cell-enrichment data, higher colony-forming cell (CFC) frequencies were obtained with the DB cocktail, although superior yields of CFC were obtained with the CC cocktail. After cryopreservation and thawing the yield of CD34+ cells remained high, and a further reduction in the number of CD15+ cells was obtained. DISCUSSION: A method is described that allows the rapid and efficient debulking of large CML samples. This strategy will provide a source of well-characterized CML stem/progenitor cells that can be repeatedly accessed.

Antigens, CD34↗
PubMed2003

Karyotype of cryopreserved bone marrow cells.

The analysis of chromosomal abnormalities is important for the study of hematological neoplastic disorders since it facilitates classification of the disease. The ability to perform chromosome analysis of cryopreserved malignant marrow or peripheral blast cells is important for retrospective studies. In the present study, we compared the karyotype of fresh bone marrow cells (20 metaphases) to that of cells stored with a simplified cryopreservation method, evaluated the effect of the use of granulocyte-macrophage colony-stimulating factor (GM-CSF) as an in vitro mitotic index stimulator, and compared the cell viability and chromosome morphology of fresh and cryopreserved cells whenever possible (sufficient metaphases for analysis). Twenty-five bone marrow samples from 24 patients with hematological disorders such as acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, megaloblastic anemia and lymphoma (8, 3, 3, 8, 1, and 1 patients, respectively) were selected at diagnosis, at relapse or during routine follow-up and one sample was obtained from a bone marrow donor after informed consent. Average cell viability before and after freezing was 98.8 and 78.5%, respectively (P < 0.05). Cytogenetic analysis was successful in 76% of fresh cell cultures, as opposed to 52% of cryopreserved samples (P < 0.05). GM-CSF had no proliferative effect before or after freezing. The morphological aspects of the chromosomes in fresh and cryopreserved cells were subjectively the same. The present study shows that cytogenetic analysis of cryopreserved bone marrow cells can be a reliable alternative when fresh cell analysis cannot be done, notwithstanding the reduced viability and lower percent of successful analysis that are associated with freezing.

Bone Marrow Cells↗
PubMed1991

Biotransformation activity in vitrified human liver slices.

In vitro testing of human liver for biotransformation or xenobiotic metabolism studies has been limited by unpredictable acquisition of samples. Consequently, it has become necessary to consider methods to cryopreserve and store these samples whenever they do become available for culture of the revived tissue at a more convenient time. Human liver slices were cryopreserved by vitrification, which allows for the transfer of aqueous media to low temperatures (-196 degrees C) without the formation of ice crystals. Human liver slices were exposed to increasing concentrations of 1,2-propanediol up to a final concentration of 4.76 M in fetal calf serum. Slices were then vitrified by direct immersion into liquid nitrogen and warmed by submersion in 37 degrees C fetal calf serum. Warming was done either immediately or after 4 and 8 weeks of storage under liquid nitrogen. The effects of vitrification, storage time, and warming on biotransformation were determined by assessing the integrated metabolism of 7-ethoxycoumarin (7-EC). Vitrified or fresh human liver slices were exposed to 50 microM 7-EC and its primary metabolite 7-hydroxycoumarin (7-HC) in organ culture for up to 6 hr. Metabolite production of both fresh and vitrified liver slices was compared. Retention of the inherent biotransformation rate was usually high and seemed independent of storage time. Integration of both cytochrome P450-mediated and secondary conjugation processes was retained in cryopreserved tissue. Vitrification offers a way to cryopreserve human liver slices for the study of xenobiotic metabolism in humans.

Biotransformation↗
PubMed2003

[Effects of different cooling rates on cryopreservation of hematopoietic stem cells from cord blood].

Clinical evidence of hematopoietic restoration with umbilical cord blood (UCB) grafts indicates the UCB can be a useful source of hematopoietic stem cells for routine bone marrow reconstitution. Considering (10 +/- 5) x 10(8) nucleared cells per cord blood unit, there is a potential limitation for the use of cord blood in adults, which, however, can be overcome by ex vivo expansion of cells. A prerequisite for expansion is the significantly higher recovery of MNC, CD34+ cells and colony-forming cells (CFC) by thawing cryopreserved MNC. Cooling rate always acts as a critical factor that can affect the recovery of cells. Although the rate of - 1 degrees C/min is adopted in most of the cryopreservations, no data has been reported about the detailed effects of different cooling rates. The aim of the study was to reveal the different effects of cooling rates on cryopreservation of hematopoietic stem cells from cord blood. UCB samples were collected, and cryopreserved as mononuclear cells (MNC) with different cooling rates of - 0.5 degrees C/min, - 1 degrees C/min, - 5 degrees C/min, and the recovery and viability of MNC and CD34+ cells, the clonogenic capacity and the ex vivo expansion potential of UCB progenitor cells were evaluated after thawing. With - 1 degrees C/min cooling rate, the recovery of MNC reached 93.3% +/- 1.8% , viability 95.0% +/- 3.9% , recovery of CD34+ cells 80.0% +/- 17.9% , and clonogenic recovery were 87.1% +/- 5.5%, 88.5% +/- 8.9%, 86.2% +/- 7.4% for BFU-E CFU-GM CFU-MK, respectively. After 14 days of liquid culture, no significant difference was detected in CFC expansion between fresh and cryopreserved MNC cells with - 1 degrees C/min cooling rate, but this was not the case with - 0.5 degreesC/min and - 5 degrees C/min. In conclusion, it was demonstrated that controlling the rate at - 1 degrees C/min is more suitable for cryopreservation of hematopoietic stem cells than - 0.5 degrees C/min and - 5 degrees C/min.

Cell Survival↗
PubMed2006

Smooth muscle cell injury after cryopreservation of human thoracic aortas.

The cryopreservation protocol we use for arterial reconstructive surgery has been studied to evaluate smooth muscle cell (SMC) structural integrity and viability before implantation. Samples of human thoracic aortas (HTA) were harvested from five multi-organ donors. Sampling included unfrozen and cryopreserved specimens. Cryopreservation was performed using RPMI with human albumin and 10% Me(2)SO in a controlled-rate freezing apparatus. Thawing was accomplished by submerging bags in a water bath (39 degrees C) followed by washings in cooled saline. In situ cell preservation as investigated by light and transmission electron microscopy showed that SMCs from cryopreserved HTA had nuclear and cytoplasmic changes. A TUNEL assay, performed to detect DNA fragmentation in situ, showed increased SMC nuclear positivity in cryopreserved HTA when compared to unfrozen samples. 7-AAD flow cytometry assay of cells derived from cryopreserved HTA showed that an average of 49+/-16% cells were unlabeled after cryopreservation. Organ cultures aimed to study cell ability to recover cryopreservation damage showed a decreasing number of SMCs from day 4 to day 15 in cryopreserved HTA. In conclusion, the cryopreservation protocol applied in this study induces irreversible damage of a significant fraction of arterial SMCs.

Aorta, Thoracic↗
PubMedDate not supplied

Preservation of vein allograft viability during long-term storage.

BACKGROUND: The aim of this study was to compare the vein allograft viability following cryopreservation with that remaining after prolonged refrigerated storage. MATERIALS AND METHODS: Great saphenous vein biopsies had been cryopreserved, and the samples were divided into two matched groups and stored in tissue culture medium for 42 days at +4 degrees C, either with or without regular medium replacement. Each vein allograft was biopsied and assayed for viability every third day by the methyltetrazolium reduction assay. Viability indexes of vein allografts harvested from brain-dead multi-organ donors and from cadavers whose warm ischemic periods were maximally 24 h were also compared. RESULTS: Vein allografts stored for 42 days at +4 degrees C showed a similar viability (58.9 +/- 1.2%) to that of cryopreserved veins (59.7 +/- 2.3%). This was true even when cryopreserved and thawed allografts were subjected to 3 days of post-thaw incubation under presumably favorable conditions (58.7 +/- 1.6%). There was no viability index difference between the samples with medium replaced and not replaced and the specimens harvested from two different donor groups. CONCLUSIONS: Long-term storage of vein allografts at +4 degrees C is a valuable option for regular banking practice. Sufficient amounts can be procured from cadavers similar to tissue donors.

Adult↗
PubMed2006

Lack of evidence of disease contamination in ovarian tissue harvested for cryopreservation from patients with Hodgkin lymphoma and analysis of factors predictive of oocyte yield.

Ovarian cryopreservation is a promising technique to preserve fertility in women with Hodgkin lymphoma (HL) treated with chemotherapy. Thus, the aim of this study was to examine harvested ovarian tissue for subclinical involvement by HL by morphology/immunohistochemistry, and to define patient and treatment factors predictive of oocyte yield. This was a retrospective analysis of 26 ovarian tissue samples harvested for cryopreservation from women with HL. Histology, immunohistochemistry and follicle density (number mm(-3)) was examined. Disease status and preharvest chemotherapy details were obtained on 24 patients. The median age was 22 years (range 13-29). Seven of 24 patients had infradiaphragmatic disease at time of harvest. Nine of 20 patients had received chemotherapy preharvest (ABVD (Adriamycin), Bleomycin, Vinblastine and Dacarbazine) = 7, other regimens = 2). The seven receiving ABVD showed no difference in follicle density compared to patients not receiving treatment (n = 14); (median = 1555 vs 1620 mm3 P = 0.97). Follicle density measurement showed no correlation with patient age (R2 = 0.0001, P = 0.99). There was no evidence of HL involvement in the 26 samples examined (95% CI = 0-11%). In conclusion, subclinical involvement of HL has not been identified in ovarian tissue, even when patients have infradiaphragmatic disease. Furthermore, the quality of tissue harvested does not appear to be adversely affected by patient's age or prior ABVD chemotherapy.

Adolescent↗
PubMed1993

Effect of natural antioxidants on frozen bovine semen preservation.

The influence exerted by natural antioxidants (Vitamin E or sodium ascorbate) was studied in various thermal treatments of semen and their effect on respiratory activity and membrane integrity during cryopreservation. Frozen bovine semen samples of diverse quality were employed in the presence and absence of antioxidants. Both in good-quality samples subjected to cold shock and in those of poor-quality standard-cooled, low superoxide dismutase activity was observed concomitantly with high malondialdehyde production; as regards oxygen uptake there was no evidence of mitochondrial coupling. In good-quality samples standard-cooled in the presence of antioxidants, greater superoxide dismutase activity, intact acrosome percentage and mitochondrial coupling were recorded as well as lower malondialdehyde production than in the controls. Natural antioxidants would seem to exert a protective effect on the membrane of the cryopreserved spermatozoon in samples from good-quality semen.

Journal Article↗
PubMed2004

Fundamentals of cryobiology in reproductive medicine.

The aim of this review will be to provide a basic understanding of the biophysical processes that accompany the application of cryopreservation in reproductive medicine. The ability to store cells in 'suspended animation' outside the body has become a keystone practice in the development of many modern clinical therapies, and, in fact, the sciences of cryobiology and IVF have developed in parallel over the past 50 years. During this time, some of the underlying principles of the quantitative biophysical aspects of cryobiology have been clarified. Water is the universal biocompatible solvent, but also possesses unique properties for stability of living cells. Whilst low temperatures themselves have defined effects on cell structure and function, it is the phase transition of water to ice that is the most profound challenge for survival. The thermodynamics of dilute aqueous solutions dictate how cells and tissues respond to the freezing process. Current concepts of nucleation, ice crystal growth and solute exclusion from the ice lattice will be discussed to illustrate what cells must negotiate to avoid lethal damage, and the role of cryoprotectants in enhancing recovery. Quantitative formalisms now exist to model and predict how water and solutes move across cell membranes before and during freezing, or how nucleation events will proceed, and these will be outlined. Cryoprotectants have both positive and negative effects on cell function depending on the kinetics of exposure. The concept of tolerable osmotic excursion of cell volume will be discussed, along with the evidence for a 'pseudo-glassy' state for cells during traditional cryopreservation. This will be compared with the recent interest in promoting glassy states in the whole sample using vitrification protocols, outlining the advantages and drawbacks of each approach. Additional methods for controlling ice nucleation have a role to play here, and a brief outline of current technologies will be given. Finally, issues of safety and stability of cryopreserved samples will be set out.

Cell Physiological Phenomena↗
PubMed2000

Hidden effects of cryopreservation on quality of human spermatozoa.

The effects of cryopreservation on two characteristics of human spermatozoa were investigated: the early phases of disturbed plasma membrane function and the activity of enzymes in intact spermatozoa. The membrane function was detected by means of the calcium-dependent binding of fluorescein isothiocyanate (FITC)-conjugated Annexin V to sperm plasma membranes. Annexin V monitors the translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane, which is one of the earliest features of membrane disintegration. For the second aim synthetic fluorogenic substrates for peptidases, proteinases, esterases, elastases and collagenases were applied. These substrates, CellProbe trade mark reagents consist of different peptide sequences, specific for the enzymes, and a fluorescein- or rhodamine 110-dye moiety. They enter the cells without previous membrane permeabilisation and exhibit fluorescence after cleavage depending on enzyme activity. The number of positive cells and the intensity of the fluorescence were determined by flow cytometric analysis comparing fresh spermatozoa with cryopreserved ones. Thirty-five semen samples collected from 35 donors were cryopreserved using the freezing medium TEST yolk buffer. All specimens showed normal spermiogram parameters. Twenty-five of the samples were used for detection of Annexin V-FITC binding and 10 semen samples for investigations of the intracellular enzymes. The Annexin V-assay applied two fluorescent dyes (Annexin V, AN and propidium iodide, PI) which led to three groups of spermatozoa (a) viable spermatozoa (AN V-negative and PI-negative), (b) dead spermatozoa (AN V-positive and PI-positive) and (c) cells with impaired but integer plasma membrane (AN V-positive and PI-negative). The percentage of vital Annexin V-negative spermatozoa (x +/- SEM) decreased significantly (p < 0.001) from fresh spermatozoa (51.6 +/- 3.1) to cryopreserved spermatozoa (26.6 +/- 2.2%) and was associated with their motility (57.9 +/- 1.9% motile fresh spermatozoa vs. 22.6 +/- 3.9% motile sperm after cryopreservation). Of the spermatozoa 28.2% were Annexin V-positive before and 44.4% after cryostorage even though they did not bind to PI. Thus, vital spermatozoa showed a disturbed membrane function indicating viability before as well as after cryostorage. Moreover, after cryopreservation the spermatozoal fluorescence increased applying substrates for butyryl esterase (p < 0.05), prolyl-aminopeptidase (p < 0.001) and val-lys-(VK)-cathepsin (p < 0.001). In contrast, the activities of fluorescein diacetate (FDA)- and FDA/sodium fluoride (NAF)-esterase (p < 0.05), ala-ala-pro-val-(AAPV)-elastase (p < 0.001), gly-pro-leu-gly-pro-(GPLGP)-collagenase (p < 0.05) and gly-gly-leu-(GGL)-subtilisin (p < 0.001) decreased after cryopreservation. The substrates for arg-gly-glut-ser-(RGES)-elastase, gly-phenyl-gly-ala-(GFGA)-collagenase and threo-pro-(TP)-cathepsin were not cleaved before as well as after cryostorage. In addition to the known effects of sperm cryopreservation our results showed two further alterations of human ejaculated spermatozoa: (a) disturbed plasma membrane function, which is not detectable by supravital staining and (b) a changed pattern of intracellular enzyme activities.

Journal Article↗
PubMed1998

Changes in L-selectin expression on CD34-positive cells upon cryopreservation of peripheral blood stem cell transplants.

Several studies have pointed out that L-selectin on CD34-positive cells plays a role in haematopoietic reconstitution after peripheral blood stem cell (PBSC) transplantation. Since it is known that a decrease in L-selectin expression in lymphocytes and granulocytes can be induced by a variety of stress situations, we have investigated in this study whether the freeze-thawing procedure, used in PBSC transplantation, would affect L-selectin expression on CD34+ stem cells. Flow cytometry was performed by labelling the cells with anti-CD34 (HPCA2 PE) and anti-CD62L (FMC46 FITC). The leucapheresis procedure itself caused a slight decrease of L-selectin expression on CD34 cells in 11 out of 12 cases (mean decrease of the percentage of positive cells 11.9; range 6-23). A much larger decrease was found upon freeze-thawing: a mean of 39% (range 4-78% in 27 cases) compared to fresh material. To determine if L-selectin expression might be up-regulated after cryopreservation, thawed transplant samples (n = 11) were incubated at 37 degrees C in RPMI with 10% FCS at 5% CO2. Already early in the course of incubation two CD34-positive populations appeared in the blast region, characterized by either a low or high forward scatter. Simultaneous viability staining with the DNA dye 7-Amino Actinomycin D and the DNA/RNA dye Syto16 revealed that the population with low forward scatter was apoptotic while the population with the high forward scatter was non-apoptotic. The latter population is considered to be most relevant for transplantation. In this population an increase of L-selectin expression after overnight incubation was observed in 8/11 samples up to values of 46-120% of the values of the fresh nonfrozen samples. In addition, the mean fluorescence intensity was significantly increased in 10/11 cases. Kinetic experiments with shorter incubation times revealed that only part of the leucapheresis samples (two out of 8) showed an increase of L-selectin expression within 4 h. In addition, a decrease of L-selectin expression was found upon CD34 purification from fresh leucapheresis material by magnetic isolation (decrease ranging from 59 to 92%, n = 5). In contrast to frozen samples, L-selectin reappearance was seen already within 4 h of incubation in all samples. Both the loss of L-selectin expression on CD34 cells occurring upon freeze-thawing, the emergence of apoptosis, as well as the recovery of L-selectin expression on non-apoptotic cells varies largely between individual leucapheresis samples, and therefore it is concluded that such processes should be considered when correlations with clinical outcome after transplantation are made.

Antigens, CD34↗
PubMed1995

Flow cytometry quantification of CD34+ cells and other leukocyte subpopulations in frozen-thawed blood cell suspensions: investigation of a new teflon container for cryopreservation of hematopoietic progenitor cells.

BACKGROUND: Cryopreservation is the only available method for the long-time maintenance of blood cells. The present study was designed to prove: (i) the reliability of multiparameter flow cytometry (MFC) for estimation of CD34+ cells in frozen-thawed cell suspensions and (ii) the acceptability of a new teflon container for the cryopreservation of hematopoietic progenitor cells. MATERIALS AND METHODS: Each of 15 ABO-compatible buffy coats (BC) were pooled, and mononuclear cells (MNC) were then separated with the Fresenius AS 104 device (n = 10). MNC harvested by apheresis were then divided into 2 portions and transferred pairwise into either the new Fresenius or into Gambro cryopreservation containers. Paired samples were frozen at controlled rates (9% DMSO final concentration) and stored at -196 degrees C in liquid nitrogen for 2 weeks. Leukocyte, MNC and differential blood counts and proportions of CD3+, CD4+, CD8+, CD14+ and CD34+ cells were assessed from the pooled BC, the apheresis products, and the frozen-thawed samples. Methyl cellulose culture assays as well as trypan blue viability staining were also carried out. RESULTS: The mean content of the divided apheresis products was 4.9 x 10(9) leukocytes with 86% MNC, 6.89 x 10(6) CD34+ cells, 2.1 x 10(5) granulocyte-macrophage colony-forming units (CFU-GM) and 7.1 x 10(5) erythroid burst-forming units (BFU-E). As expected, there were virtually no granulocytes after freezing in both types of container. The corresponding mean cell content was as follows: 6.3 x 10(6) CD34+ cells, 2.5 x 10(5) CFU-GM, and 8.1 x 10(5) BFU-E in Fresenius containers, and 6.1 x 10(6) CD34+ cells, 1.3 x 10(5) CFU-GM, and 7.7 x 10(5) BFU-E in Gambro containers. The mean MNC viability of the samples frozen in Fresenius was 81.5% and 82.7% in the Gambro containers. MFC was found to compare with stained smear differentials. CD34+ cell counts correlated with CFU-GM (0.69, p = 0.03) and BFU-E (0.63, p = 0.02) colony formation. CONCLUSIONS: The study reported here revealed no significant differences between the 2 types of storage containers. The new Fresenius teflon container could thus be recommended for cryopreservation of hematopoietic progenitor cells. MFC provided reliable data on CD34+ cell content and leukocyte subset composition of the frozen-thawed cell suspension.

Antigens, CD↗