Cord blood banking: volume reduction of cord blood units using a semi-automated closed system.
(9/2910)
Clinical evidence indicates that placental/umbilical cord blood (CB) is an alternative source of haematopoietic stem cells for bone marrow reconstitution. To establish a CB bank large panels of frozen, HLA-typed CB units need to be stored. Cryopreserved, unprocessed CB units require vast storage space. This study describes a method, using the Optipress II Automated Blood Component Extractor (Opti II) from Baxter Healthcare Corporation, to reduce the volume of the CB collection, preserving the quantity and quality of the progenitor cells, in a closed system. The CB collection was transferred to a triple bag system, centrifuged to produce a buffy coat layer and processed using a standard Opti II protocol to separate the whole blood into three components: plasma, buffy coat and buffy coat-depleted red cell concentrate. The buffy coat volume was standardised to 25 ml; mean reduced volume of 24.5 ml (s.d. 1.5 ml) with 53% red cell depletion. Good recovery of cells was observed: 92%, 98%, 96% and 106% recovery of nucleated, mononuclear, CD34+ and total colony-forming cells, respectively. Using this method for processing CB units reduces storage requirement by two-thirds but preserves the quantity and quality of the progenitor cells. (+info)
Effects of long-term cryopreservation on hematopoietic progenitor cells in umbilical cord blood.
(10/2910)
There is considerable interest in developing banks of frozen umbilical cord blood cells for transplants but it is uncertain how long frozen cells survive. Our objective was to determine the recovery of frozen umbilical cord blood cells. We quantitated recovery of hematopoietic progenitor cells (CFU-GM, BFU-E, and CFU-GEMM) from frozen umbilical cord blood cells stored for up to 12 years. Decay rates of CFU-GM, BFU-E and CFU-GEMM (d, expressed as percent of viable cells recovered (95% confidence interval) were 0.9930 (0.9889-0.9970), 0.9840 (0.9769-0.9911) and 0.9817 (0.9707-0.9927). Time-dependent recoveries, calculated by the formula d(k), (k = frozen storage interval in years) were >90% at 10 years. We conclude that frozen cord blood cells can be stored safely for prolonged intervals without substantial loss in hematopoietic progenitor cells. (+info)
Optimal timing for processing and cryopreservation of umbilical cord haematopoietic stem cells for clinical transplantation.
(11/2910)
Some of the factors that may influence the number and quality of cord blood haematopoietic progenitor cells available for transplantation have been investigated including site of collection, delayed processing after collection and cryopreservation protocol. We used the granulocyte-macrophage progenitor (CFU-GM) and erythroid burst-forming unit (BFU-E) assays to quantify progenitors. The capacity of CFU-GM to produce secondary colonies was used as a measure of progenitor cell quality. We found that: (1) there were no significant differences in total nucleated cells (TNC), mononuclear cells (MNC), CFU-GM or BFU-E numbers in paired specimens from the umbilical vein or veins at the base of the placenta. The potential of the CFU-GM to produce secondary colonies from the two sites was similar; (2) storing cord blood at room temperature or at 4 degrees C resulted in a significant reduction in progenitor cell numbers beyond 9 h; and (3) cryopreservation following either controlled rate freezing or passive cooling reduced MNC numbers, viability and CFU-GM survival insignificantly but the potential of CFU-GM to produce secondary colonies was significantly reduced post cryopreservation (P = 0.04). We conclude that the yield of CB progenitor cells is not affected by the site of collection, but is adversely affected by delays between collection and cryopreservation. Furthermore, cryopreservation reduced the CFU-GM potential to produce secondary colonies. Measures of progenitor cell quality as well as quantity may be relevant to assessing CB blood collections. (+info)
Adverse events occurring during bone marrow or peripheral blood progenitor cell infusion: analysis of 126 cases.
(12/2910)
Bone marrow (BM) and/or peripheral blood progenitor cells (PBPC) given after high-dose chemo-radiotherapy are commonly cryopreserved. Re-infusion of the thawed product can cause cardiovascular and other complications. We compared two groups of adult patients receiving autologous BM or PBPC transplant to assess the incidence of adverse events occurring during infusion. Fifty-one patients received BM, and 75 PBPC. The two groups were comparable in respect of age, total volume infused, quantity of dimethylsulfoxide (DMSO) and number of polymorphonuclear neutrophils. Patients receiving PBPC had a higher number of nucleated cells per kg of body weight; those in the BM group received a significantly greater quantity of red cells. Non-cardiovascular complications occurred in 19% and 8% of patients rescued by BM and PBPC respectively. The incidence of hypertension was 21% in the BM and 36% in the PBPC group. Asymptomatic hypotension was more frequent in PBPC patients (P<0.001). Bradyarrhythmia was noticed in two of 75 PBPC patients and in 14 of 51 BM patients (P<0.001). In the former group one patient had heart block; he died of renal failure 10 days later. Bradycardia and hemoglobinuria were more common in patients receiving BM where a higher concentration of red cells was present (P<0.001). Since bradyarrhythmias may be a life-threatening complication we advise continuous careful monitoring during infusion of thawed BM. The strong correlation between bradycardia and red blood cell contamination suggests the use of purified products with a very low red cell content. (+info)
The cryopreservation protocol optimal for progenitor recovery is not optimal for preservation of marrow repopulating ability.
(13/2910)
The efficiency of five different cryopreservation protocols (our original controlled-rate and noncontrolled-rate protocols) was evaluated on the basis of the recovery after thawing of very primitive pluripotent hemopoietic stem cells (MRA(CFU-GM), pluripotent progenitors (CFU-Sd12) and committed granulocyte-monocyte progenitors (CFU-GM) in mouse bone marrow. Although the nucleated cell recovery and viability determined immediately after the thawing and washing of the cells were found to be similar, whether controlled-rate or noncontrolled-rate cryopreservation protocols were used, the recovery of MRA(CFU-GM), CFU-Sd12 and CFU-GM varied depending on the type of protocol and the cryoprotector (DMSO) concentrations used. It was shown that the controlled-rate protocol was more efficient, enabling better MRA(CFU-GM), CFU-Sd12 and CFU-GM recovery from frozen samples. The most efficient was the controlled-rate protocol of cryopreservation designed to compensate for the release of fusion heat, which enabled a better survival of CFU-Sd12 and CFU-GM when combined with a lower (5%) DMSO concentration. On the contrary, a satisfactory survival rate of very primitive stem cells (MRA(CFU-GM)) was achieved only when 10% DMSO was included with a five-step protocol of cryopreservation. These results point to adequately used controlled-rate freezing as essential for a highly efficient cryopreservation of some of the categories of hematopoietic stem and progenitor cells. At the same time, it was obvious that a higher DMSO concentration was necessary for the cryopreservation of very primitive stem cells, but not, however, for more mature progenitor cells (CFU-S, CFU-GM). These results imply the existence of a mechanism that decreases the intracellular concentration of DMSO in primitive MRA cells, which is not the case for less primitive progenitors. (+info)
A novel approach to sperm cryopreservation.
(14/2910)
Human spermatozoa have unusual cryobiological behaviour and improvements in their survival have not been achieved by the standard approaches of cryobiology. Conventional approaches to cryopreservation impose a linear change of temperature with time; however, the stresses that cells encounter during cryopreservation are all non-linear with time. In this paper it is shown that improved methods of cryopreservation may be developed by specifically manipulating the manner in which cells experience physical changes instead of imposing a linear temperature reduction. Several treatments were compared: control of solidification to achieve constant ice formation with time was more damaging than the standard linear reduction in temperature. However, treatments which followed a chosen non-linear concentration profile, referred to as 'controlled concentration' allowed recovery of almost all the cells which were motile before freezing. The biophysical basis of these different responses was examined using the cryostage of a scanning electron microscope and freeze substitution and it was found that, surprisingly, all samples of spermatozoa in the frozen state were neither osmotically dehydrated nor had any visible intracellular ice. Viability on thawing did not appear to correlate with conventional theories of cellular freezing injury, which suggests that for human spermatozoa other factors determine viability following freezing and thawing. (+info)
Diagnostic testicular biopsy and cryopreservation of testicular tissue as an alternative to repeated surgical openings in the treatment of azoospermic men.
(15/2910)
Between May 1996 and May 1998, 64 azoospermic patients underwent an investigative testicular biopsy combined with the cryopreservation of spermatozoa which were retrieved from a simultaneously examined fresh sample. Testicular tissue cryopreservation was carried out in 43 cases (67%) for late intracytoplasmic sperm injection (ICSI) attempts. In all, 23 couples underwent 26 assisted conception cycles; the fertilization rate was 64% with spermatozoa (139/218, 24 cycles), 40% with round spermatids (2/5, one cycle), and 69% with elongated spermatids (9/13, one cycle). The embryo cleavage rate was 84%. A mean number of 2.7 +/- 0.7 embryos were replaced in 24 patients. In two cases, embryo quality was very poor and they were not transferred. Eight clinical pregnancies resulted (35% per patient and 33% per transferred cycle) with an implantation rate of 14.1%: two patients have already delivered and six are ongoing. In conclusion, the cryopreservation of testicular tissue during the first diagnostic biopsy is an alternative to repeated surgical openings and permits patients to initiate an ovarian stimulation cycle with the certitude of having spermatozoa available. Moreover, since only one straw is routinely used for each ICSI cycle, the frozen tissue remains as a sperm source for multiple attempts. (+info)
Transfer technique and catheter choice influence the incidence of transcervical embryo expulsion and the outcome of IVF.
(16/2910)
We examined the influence of the procedures used in embryo transfer on pregnancy rates. Over a period of 15 months (Nov. 1996-Jan. 1998), 320 patients were recruited. They were randomized on an alternate basis either to a Wallace catheter or an Erlangen metal catheter. Randomization was also applied within the same groups for embryo transfer at 48 h and 72 h post-insemination. Fifty patients randomly selected from each group were subjected to speculum examination 15 min following embryo transfer, during which any fluid leaking from the cervix was examined for the presence of embryos. In five patients a transvaginal-transmyometrial transfer was performed. The pregnancy rate appeared to be slightly higher in patients who had their embryos transferred at 72 h than in those patients who had their transfers at 48 h, but this difference was not significant in either group. (The ease with which the Erlangen catheter was used compared with that of the Wallace catheter was reflected in a significantly lower incidence of uterine sounding of cervical dilatation and bleeding.) Also there was a significant increase (P = 0.0001) in the mucus attached to the tip of the Wallace catheter and the embryos trapped compared with those of the Erlangen group (P = 0.0007). The pregnancy rate per embryo transfer was apparently higher in the Erlangen group than in the Wallace group but this difference was not significant. In eight (16%) patients of the Wallace group, 1-3 embryos were found in the fluid sucked from the external os, compared with three (6%) patients in the Erlangen group, but again this difference was not significant. In 92% of patients who became pregnant, the transfer procedure was smooth and easy. Successful embryo transfer was not influenced by the time of transfer post-insemination. The choice of catheter did not affect pregnancy rate. In cases in which transcervical transfer is very difficult or impossible, transvaginal-transmyometrial transfer is a viable option. The significance of early or late expulsion of transferred embryos into the vagina needs to be addressed in larger controlled studies. (+info)