Subsequent Cryopreservation Research Articles

Retrieval of immature oocytes followed by in vitro maturation and vitrification: A case report on a new strategy of fertility preservation in women with borderline ovarian malignancy

Background. We report a novel fertility preservation strategy in a woman with borderline ovarian tumors involving retrieval of immature oocytes, in vitro maturation (IVM) and subsequent cryopreservation. Case. A 43-year-old woman underwent laparotomy for cystic ovarian masses on day 18 of her menstrual cycle. A diagnosis of bilateral borderline ovarian tumors was made following histological frozen section analysis. Left salpingo-oophorectomy, right ovarian cystectomy, omentectomy and lymph node sampling were performed. All visible follicles on the surface of the removed ovary were aspirated. Four immature oocytes were retrieved and underwent IVM. Three oocytes matured after 48 h and were cryopreserved. Conclusion. Immature oocytes can be successfully isolated from the oophorectomy specimen regardless of the day of menstrual cycle, matured in vitro and cryopreserved, providing a possible strategy for fertility preservation in this group of women.

Live birth following transfer of a cryopreserved embryo generated from a cryopreserved oocyte and a cryopreserved sperm: Case report

As is the case with non-frozen oocytes, the efficient and successful use of cryopreserved oocytes in human assisted reproduction is, in part, dependent on the ability to apply selection criteria when choosing the 'best' embryos for transfer from a cohort. In many cases this, in turn, will necessitate the cryopreservation of non-transferred embryos to minimise the risk of multiple pregnancy. It is therefore important to establish that an embryo, generated by fertilization of a frozen-thawed oocyte, can be capable of surviving subsequent cryopreservation while retaining the potential for normal development. In this case report, we document the delivery of a normal male infant following transfer of a frozen-thawed embryo, generated by the fertilization of a frozen-thawed oocyte by a frozen-thawed sperm.

PROLIFERATION AND COLD HARDENING OF IN VITRO GROWN APPLE SHOOT TIPS

Shoot tips of apple cultivar 'Rubin' were successfully established in vitro. Five proliferation MS (Murashige and Skoog) media containing different concentrations of BAP (0.25, 0.5, 1, 2 mg L -1 ) or a combination of 1 mg L -1 BAP and 0.1 mg L -1 IBA were tested. The highest proliferation rate was obtained on MS medium with 1 mg L -1 BAP, which produced 4.2 ±0.3 shoots/explant after one month of cultivation. In vitro plants of cv. 'Rubin' were cultivated for 4 weeks at low temperature (4°C) and short photoperiod (8/16-h - light/dark). The assessment of frost resistance of cold hardened and non-hardened in vitro apple plants was done by freezing tests. In vitro plants were exposed to the controlled cooling rate 4°C/h. The lethal temperature (LT) was -13.0°C for cold hardened and -6.5°C for non-hardened in vitro plants. Cold hardened in vitro cultures were used for subsequent cryopreservation. Cryopreservation was made by encapsulation-dehydration vitrification method. The survival and regeneration of cv. 'Rubin' was 78% after thawing in 40°C water.

Cryosurvival of bovine blastocysts is enhanced by culture with trans-10cis-12 conjugated linoleic acid ( 10t, 12c CLA)

An excessive lipid content in embryo cells is a consequence of embryo culture in the presence of serum which is suggested to be responsible for their high susceptibility to cryopreservation. The objective of the present study was to examine the effects of supplementing serum-containing culture media with trans-10 cis-12 conjugated linoleic acid ( 10t, 12c CLA) on embryo lipid accumulation and its subsequent cryopreservation. Abattoir-derived oocytes were matured and fertilized in vitro (IVF = day 0). On day 1, presumptive zygotes ( n = 3390) were randomly placed in: (I) (MS), granulosa cell monolayer cultured with M199 and 10% serum; (II) (SCLA), granulosa cell monolayer cultured with M199, 10% serum and 100 μM 10t, 12c CLA and (III) (SOF), modified synthetic oviduct fluid, where embryo culture proceeded for 8 days. Cleavage rates or D7/D8 embryo quality did not vary among treatments. D7/D8 embryo production rate was significantly ( P < 0.001) lower in SOF (17.9 ± 1.6%) than in groups MS (29.8 ± 2.5%) and SCLA (27.8 ± 2.0%). After cytoplasmic lipid droplets observation under Nomarski microscopy, classified embryos were the leanest when cultured in SOF, intermediate in SCLA and the fattest in MS ( P < 0.02). Post-thawing intact blastocyst rates where significantly higher in the SCLA group (84.7 ± 4.1%) than in SOCS (50.3 ± 4.8%, P = 0.0007) or SOF (65.3 ± 6.9%, P = 0.03) groups. Post-thawing re-expanding rates were significantly lower when embryos were cultured in MS (34.7 ± 3.7%) than in SCLA (63.7 ± 5.3%, P = 0.0006) or SOF (49.0 ± 4.6%, P = 0.04). Moreover, re-expanding rates were lower ( P = 0.05) in SOF than in SCLA cultured embryos. These results clearly show that addition of CLA to serum-containing media reduced lipid accumulation during in vitro culture and significantly improved cryopreservation survival.

Cryosurvival of Testicular Spermatozoa From Obstructive Azoospermic Patients: The Cleveland Clinic Experience

CryoTESE is the modality of choice in the management of non-reconstructable obstruction of the excurrent duct system in cases where epididymal sperm aspiration is unsuccessful. It also serves as a back-up source of sperm cells for future ICSI attempts in obstructive azoospermic patients undergoing corrective surgery. However, there is very little information in the literature documenting the cryosurvival of spermatozoa obtained from surgically retrieved testicular tissue. The purpose of our study was to evaluate the cryosurvival of testicular spermatozoa after the freeze-thaw cycle and to scrutinize the effectiveness of freezing protocol used in our institution. Retrospective study at a tertiary care institution. This study involved 21 cases of obstructive azoospermia (OA) who underwent testicular biopsy and subsequent cryopreservation of retrieved tissue at our institution between April 2002 and May 2004. The patients presented with primary or secondary infertility following obstruction due to previous surgery, infection or inflammation. Before surgery, all patients underwent a physical examination and work-up by a male infertility specialist. Testicular tissue samples (n= 2 -14) were retrieved upon confirmation of the presence of sperm cells by frozen section histopathology and microscopic examination of wet preparation during surgery. These samples were then labeled and sent for cryopreservation to the Andrology laboratory. Tissue samples were individually mixed with fresh HTF and manually mashed to produce a suspension for cryopreservation. Before freezing, a droplet (5μL) of this suspension was assessed at 400 X magnification on an inverted microscope (Nikon Diaphot, Japan) for presence of motile spermatozoa. Similarly, the presence of motile spermatozoa was determined immediately after the thawing process. All 21 patients who underwent testicular biopsy followed by cyropreservation at our center demonstrated motile spermatozoa after the freeze-thaw process. Only one out of 21 patients had rare motile sperm in his post thaw sample. The etiology of obstructive azoospermia or the previous fertility status had no influence on motility outcomes in these patients. The presence of motile sperm in all of our patients after cryopreservation and thawing as seen in this series is in agreement with few similar studies reported in the literature. These results validate that testicular spermatozoa from OA patients could be frozen successfully for use in future assisted reproduction.

The Use of Ischaemic Vessels as Prostheses or Tissue Engineering Scaffolds After Cryopreservation

Objective: to evaluate the condition of organ donor arteries subjected to prolonged cold-ischaemia followed by cryopreservation, for their possible use as vascular grafts.Materials and methods: fresh specimens of human iliac artery from organ donors were used as controls. These arteries were divided into two portions, one of which was cryopreserved in an automated freezer. A further group of arteries was immersed in Wisconsin solution and kept for 4 days at 4°C (cold-ischaemia). After this period, the arteries were also cut into two, and one of these portions was cryopreserved. All the cryopreserved arterial segments were stored for a month and then subjected to automated gradual thawing. The thawed specimens were evaluated by light microscopy, scanning and transmission electron microscopy, immunohistochemical analysis (MMPs, elastin, CD31, von Willebrand factor) and the in situ detection of fragmented DNA (TUNEL method).Results:the most marked changes induced by cryopreservation were partial vessel deendothelialisation and morphological changes in cells of the intima that were in the process of detachment. No significant changes were observed in the medial layer, other than discrete elastic fibre fragmentation. Following cold-ischaemia, the endothelium was the most affected layer, with large denuded areas and exposure of the fibroelastic layer. Increased MMP-2 expression was also noted after cold-ischaemia. When subjected to both cold-ischaemia and cryopreservation, a large proportion of endothelial cells showed positivity for the TUNEL technique, however, no significant difference was observed between the ischaemic and the ischaemic/cryopreserved specimens.Conclusions:prolonged cold-ischaemia causes some additional damage to the arterial wall compared to cryopreservation alone. However, the structural component of the ischaemic vessel remains in a condition that is suitable for subsequent cryopreservation and use as a vessel substitute or a scaffold for tissue engineering.

Storage of cane toad (Bufo marinus) sperm for 6 days at 0 degrees C with subsequent cryopreservation.

We investigated the recovery of motility of cane toad (Bufo marinus) sperm after storage for 6 days at 0 degree C (on ice) and after subsequent cryopreservation. Sperm suspensions were prepared from testes macerated in either simplified amphibian Ringer (SAR) or 10% (w/v) sucrose diluents, with 15% or 20% (v/v) glycerol or Me2SO as cryoprotectants, and were stored for 6 days. Alternatively, suspensions were prepared in either SAR or 10% (w/v) sucrose diluent and stored for 6 days, after which some of these suspensions were kept in diluents alone or, alternatively, had 15% or 20% (v/v) glycerol or Me2SO added. All treatments (suspensions) were then cryopreserved. Sperm motility was measured at Day 1 and Day 6 (before and after cryopreservation). A substantial and variable proportion (range 0%-40%) of sperm was immotile in suspensions immediately after preparation from testes macerates. Sperm stored in either SAR or 10% (w/v) sucrose diluent maintained approximately 75% motility for 6 days, but few sperm survived cryopreservation. After storage and cryopreservation, recovery of motility was substantially higher with Me2SO than in glycerol. However, both cryoprotectants exhibited toxicity at high concentrations. Glycerol was more toxic than Me2SO in 10% (w/v) sucrose than in SAR diluent, both before and after cryopreservation. The addition of some cryoprotectants to suspensions before storage gave greater recovery both before and after cryopreservation. After cryopreservation, the highest rate of sperm recovery was in suspensions with 10% (w/v) sucrose and 15% (v/v) Me2SO added prior to storage (mean (+/-SEM) 46 +/- 7% relative to initial; 39 +/- 6% absolute). Sperm were also stored for 6 days at 0 degrees C in suspensions or testes (with suspensions then prepared from testes) and cryopreserved. Sperm maintained higher recovery and membrane integrity both before and after cryopreservation when stored in suspensions rather than in testes.

Permeabilization ofCochliomyia hominivorax(Diptera: Calliphoridae) Embryos

Embryos of the primary screwworm, Cochliomyia hominivorax (Coquerel), were successfully permeabilized for use in subsequent cryopreservation studies. Mortality was greater for eggs incubated for < 5 h before treatment. The mean survival of embryos to first instars was 55.7, 61.1, and 62.6% when the embryos were incubated for 5, 5.5, and 6 h before treatment, respectively. The survival to the pupal and adult stages was low. An improved media for culturing the embryos during and immediately after treatment needs to be devised and the procedure for rearing the larval stages also needs to be altered to improve survival for emerging adults.

Cryopreservation of entrapped monoxenically produced spores of an arbuscular mycorrhizal fungus.

A study was conducted to quantify the ability of entrapped, monoxenically produced spores of an arbuscular mycorrhizal fungus to germinate and reproduce the fungal life cycle after cryopreservation. No germination was obtained after incubation of entrapped spores in glycerol and mannitol and subsequent cryopreservation at -70 °C, regardless of the concentration of cryoprotectants and duration of incubation. Incubation for 1 d in 0.5 M sucrose, and for 1 and 2 d in 0.5 M trehalose, led to spore germination after cryopreservation at -70 °C. Lower cryopreservation temperatures were tested with entrapped spores incubated for 1 d in 0.5 M trehalose. The highest germination rate, estimated by the percentage of potentially infective beads (%PIB), was obtained at -100 °C. A %PIB of 95% (water agar medium) to 100% (Strullu-Romand medium) was obtained at this temperature. Thereafter, %PIB rapidly decreased at -140 and -180 °C. Heavy sporulation and high internal root colonization were obtained after re-association of the entrapped spores, incubated for 1 d in 0.5 M trehalose and subsequently cryopreserved at -100 °C, with transformed carrot roots. This demonstrates the ability of entrapped spores to reproduce the fungal life cycle following cold treatment.

Isolation of monocytes from whole blood by density gradient centrifugation and counter-current elutriation followed by cryopreservation: six years' experience

Background: Monocyte purification by means of counter-current elutriation and subsequent cryopreservation for future use was initiated in 1986 and has been established as a routine since 1993. Aim: To sum up and evaluate our method for the isolation and preservation of monocytes. Materials and methods: Peripheral blood mononuclear cells (PBMC) were isolated from healthy donor blood by density gradient centrifugation, and monocytes were isolated from the PBMC by counter-current elutriation centrifugation using the Beckman J-6M/E centrifuge. The monocytes were then cryopreserved at -135°C and thawed when required for experimental use. Results: Results are given for the last 6 years, including 59 elutriations and the fractions containing monocytes. The mean purity of monocytes was 93% (range 64-98%); mean recovery was 51% (range 22-55%). Studies of CD14 expression and Annexin V indicate that there are no differences between elutriated fractions immediately upon purification or after freezing and thawing. The studies also indicate that interdonor variations are much larger than intradonor variations. Discussion: Although it differs from other reports in certain respects, our procedure has nevertheless produced results in line with other findings. After extensive testing and use in different contexts we feel confident that we have established a method for producing a large number of purified and well-preserved monocytes. Conclusion: The goal of being able to perform a large number of experiments with monocytes of high purity and good functionality has been reached.

Reduced survival after human embryo biopsy and subsequent cryopreservation.

Preimplantation genetic diagnosis (PGD) is performed in couples at risk of genetic disease, so as to avoid transfer of embryos which are affected by a monogenic disease or which carry chromosomal aberrations. As in all in-vitro fertilization (IVF) cycles, supernumerary non-affected good-quality embryos may be available after PGD. These embryos can be cryopreserved. So far, limited data on survival after cryopreservation of biopsied human embryos are available. In this study, human embryos of good morphological quality derived from abnormal fertilization were used to evaluate the influence of the embryo biopsy procedure on survival after cryopreservation. Embryos were allocated to three different groups: control (n = 20), drilling-only (n = 16), and biopsy (n = 29). After freezing and thawing, a significantly lower number of blastomeres was intact in the drilling-only group (46/118, i.e. 39.0%, P < 0.01) and in the embryo biopsy group (46/156, i.e. 29.5%, P < 0.0001) than in the control group (85/151, i.e. 56.3%). This difference was reflected in survival rates of embryos. Fifty-five per cent of the control embryos, 37.5% of the drilling-only group, and 33.3% of the biopsy group had at least 50% of their blastomeres intact. After further in-vitro culture, four blastocysts, three from the drilling-only group and one from the biopsy group, developed from the surviving embryos. From this study it can be concluded that current cryopreservation procedures are less successful when biopsied human embryos are cryopreserved, but that surviving embryos can develop to the blastocyst stage and thus may have the potential to develop to term.

Controlled Comparison of Percutaneous and Microsurgical Sperm Retrieval in Men With Obstructive Azoospermia

A controlled comparison of the efficacy and reliability of sperm retrieval by testicular fine needle aspiration (TFNA), percutaneous testicular needle biopsy (PercBiopsy) and microsurgical epididymal sperm aspiration (MESA) was performed in nine patients with obstructive azoospermia. During a planned MESA procedure, sperm retrieval was attempted on the same testis with TFNA and PercBiopsy. Spermatozoa were obtained from all patients using MESA and PercBiopsy. Spermatozoa were retrieved using TFNA from 6/9 (67%) men. The mean number of epididymal spermatozoa retrieved with MESA (15 x 106) was significantly higher (P = 0.003) than that retrieved percutaneously from the testis. The mean number of spermatozoa obtained by PercBiopsy was 0.116 x 10(6) while TFNA recovered 0.014 x 106 spermatozoa (P = 0.025). MESA is the optimal choice to retrieve the greatest number of spermatozoa with highest motility for assisted reproduction and subsequent cryopreservation. However, percutaneous testicular retrieval does not require microsurgical expertise and is less invasive. Our results suggest that the optimal percutaneous procedure for sperm retrieval from the testis involves percutaneous testicular needle biopsy with an automatic biopsy gun.

Effect of fatty acid-free bsa, replacement of serum supplementation in culture medium on the biopsyand subsequent cryopreservation of in vitro-produced bovine embryos

Effect of fatty acid-free bsa, replacement of serum supplementation in culture medium on the biopsyand subsequent cryopreservation of in vitro-produced bovine embryos

Determination of acrosin amidase activity in equine spermatozoa

Acrosin amidase activity of spermatozoa has been been associated with in vitro fertilization success in humans and has been proposed as an additional method for assessing sperm function in vitro. In this study, acrosin amidase activity was determined in equine spermatozoa by the hydrolysis of an arginine amide substrate. This assay includes a detergent to release acrosomal enzymes into a medium of basic pH to activate proacrosin to acrosin, which subsequently hydrolyses N-α-benzoyl-DL-arginine para-nitroanilide-HCl (BAPNA) to a chromogenic product. Spermatozoa (n = 3 ejaculates from each of 4 stallions) were washed free from seminal plasma by centrifugation through Ficoll and incubated with a detergent-substrate mixture (BAPNA in triton X-100; pH = 8.0) at room temperature for 3 h in the dark. At the end of the 3-h incubation, benzamidine was added to test samples to stop the reaction, and samples were centrifuged to remove spermatozoa. Absorbance at 410 nm was measured to determine acrosin amidase activity (μIU acrosin/10 6 sperm). Acrosin amidase activity increased with sperm concentration (P < 0.001; r 2 = 0.75), and there were significant effects (P < 0.001) of stallion and ejaculate within stallion on acrosin activity. Acrosin activity detectable in equine seminal plasma was 312 ± 49 μU/ml (n = 3 ejaculates). Addition of a cryopreservation medium containing egg yolk, skim-milk, glycerol and sucrose to equine spermatozoa and subsequent cryopreservation significantly (P < 0.05) increased acrosin amidase activity compared with spermatozoa from raw semen. This result is in contrast to that previously reported for frozen-thawed human spermatozoa. Determination of acrosin amidase activity in equine spermatozoa may provide an alternative method for assessing sperm function in vitro; however, further studies are needed to determine the relationship between acrosin activity and fertility in the horse.

The Effect of Moisture Content on the Low Temperature Responses of Araucaria hunsteinii Seed and Embryos

The effect of moisture content on the low temperature responses of 'recalcitrant' Araucaria hunsteinii seed and excised embryos was investigated in relation to germination and storage. At 6°C seed longevity was decreased as mean moisture content was reduced from 45 to 30% (fresh weight basis); the time predicted for the loss of one probit of germination, σ, fell from about 2000 to 20 d. The estimated base temperature for germination rate, Tb, was near 6°C. Germination occurred during storage at 6°C unless the initial seed moisture content was below about 45%. Embryo freezing point decreased from -6 to -19°C as moisture content was decreased from 44 to 27%. The unfrozen water content of the embryo was estimated by thermocouple and differential scanning calorimetry methods to be around 25%. Embryos exhibited reduced growth in vitro following drying below the moisture content range 34-41%, equivalent to water potentials below about -4 MPa. Moisture was unevenly distributed among component tissues of the embryo; in undried material, the radicle had a higher moisture content than the hypocotyl and the hypocotyl was in turn moister than the cotyledons. Individual embryo tissues completely lost the ability for organized growth when dried below 25% moisture content, although some cells survived, as shown by callus production from the radicle. This type of survival on drying was also seen in embryos after their subsequent cryopreservation in liquid nitrogen and after 4 years storage at -20°C within 'non-germinable' seeds. Practical storage conditions for seeds and embryos are considered in relation to these findings.

Fertilization and early embryology: The quarantine of fertilized donated oocytes

The fertilization and subsequent cryopreservation of donated oocytes have enabled the resulting embryos to be quarantined for a minimum of 6 months, and to be thawed and replaced only after the donor has had a second negative human immunodeficiency virus (HIV) test. In the study described here, a total of 39 women had 56 embryo transfers, and 12 pregnancies (21% per transfer) were achieved. The logic of the protocol for minimizing the risk of infection of the recipient, which is in line with that of semen donation, is presented, together with an argument for the feasibility of such an approach.

Arterial Smooth Muscle Function after Prolonged Exposure to a Medium Containing Dimethyl Sulfoxide (Me2SO) and Storage at -196°C

Clumges in smooth muscle responsiveness were investigated in vitro after storage of canine femoral arteries at room temperature (21°C) and in liquid nitrogen (-196°C) in Krebs-Henseleit solution containing 50% fetal calf serum, 2.0 M dimethyl sulfoxide, and 0.1 M sucrose as cryoprotectants. Both contractile responses to noradrenaline and relaxant effects of bimakalim were unchanged after exposure of arterial smooth muscle preparations for 1 h to the cryomedium without freezing. However, after exposure of the tissues for increasing time periods to the cryomedium with subsequent cryopreservation the post-thaw functional recovery was progressively diminished. Optimal post-thaw functional recovery was obtained with tissues that had been frozen within 10 min of being placed in the cryomedium. The results suggest that exposure to the cryomedium without freezing is well tolerated by the arterial smooth muscle, whereas a progressive reduction of the contractile activity occurs with prolonged exposure of the preparations to room temperature cryomedium before starting the cooling process.

Cryopreservation of semen, oocytes, and embryos

Human embryo cryopreservation is widely applied by programs of assisted reproductive technology. However, recent surveys have shown that relatively few in vitro fertilization programs in the United States substantially increase their per retrieval delivery rates through cryopreservation. While embryos may be successfully frozen and thawed at a range of developmental stages using several different cryoprotectants, the majority of in vitro fertilization programs use 1,2 propanediol to freeze early (pronuclear four-cell) embryos. Ultrarapid freezing techniques are under continued study; they have not yet been shown to offer any clinical advantage over slow freezing protocols. Although interest in oocyte cryopreservation remains high, experimental evidence shows that cryoinjury to human oocytes results in chromosome sorting defects, reduced fertilization, and compromised embryonic development. Micromanipulation of mammalian oocytes appears to have no deleterious effect on subsequent cryopreservation.

In vitro and in vivo activity of murine lymphokine‐activated killer cells after cryopreservation

The in vitro and in vivo effects of cryopreservation on the cytotoxic activity of murine lymphokine-activated killer (LAK) cells were studied. LAK cells were generated by incubation of spleen lymphocytes of BALB/c mice for 3 days with recombinant interleukin-2 (rIL-2) and subsequent cryopreservation. Cytotoxicity was determined in a 51Cr release assay. After thawing, cytotoxic activity was reduced (40.4% 51Cr release at an effector:target cell ratio of 40:1 as compared to 68.5% 51Cr release before freezing) and could be restored to precryopreserved levels by reincubation with rIL-2 for 2 days after thawing (78.8% 51Cr release). These cells were then tested in BALB/c mice injected with RAW 112 cells, a pre-B-cell lymphoma line. The results demonstrate that the survival rate of mice injected with cryopreserved and restimulated LAK cells (50% survival greater than 180 days after injection) did not differ significantly from that of mice injected with fresh unfrozen LAK cells (60% survival greater than 120 days, 50% survival greater than 180 days). Cryopreserved LAK cells have potential use in adoptive immunotherapy.

Collection and cryopreservation of human stem and progenitor cells for bone marrow transplantation

Bone marrow collection was undertaken from human organ donors (Group 1: n = 7) to develop a closed-system single-step technique for stem and progenitor cell enrichment, using the Cobe 2997 continuous-flow blood-cell separator. The effects of programmed freezing, storage in liquid nitrogen, and thawing were then defined using these grafts. Once standardised, this method was extended to autografting following cryopreservation of a comparable fraction (Group 2: n = 8) and then to allogeneic transplantation after ex vivo exposure to the lytic monoclonal antibody. Campath-1 IgM and human complement, but without cryopreservation (Group 3; n = 9). The median number of mononuclear cells harvested was 5.0 x 10(6)/mL (n = 24), and this was not significantly different in the three groups. The ex vivo graft, composing marrow rich anticoagulated whole blood, was recirculated in the separator at a flow rate of 60 mL/minute, with a centrifuge speed of 1,100 r.p.m., and the mononuclear cell fractions were collected at the rate of 1.5 mL/minute. The average procedure time from formation of the interface in the single disposable channel to achievement of the final volume was 90 minutes. The mean recovery of the mononuclear cells was 101.4% (SD 38.0) and the GM:CFUc was 91% (SD 43.86). These figures were not significantly influenced by subsequent cryopreservation (Group 1; n = 7 and Group 2; n = 8) or following exposure to the monoclonal antibody. Campath-1 IgM (Group 3; n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)