Nucleated Cell Content Research Articles

Very late ANTIGEN-5 and leukocyto functional-associated ANTIGEN-1 are critical for early stage hematopoietic progenitor cell homing

Abstract Adhesion molecule interactions of hematopoietic stem cells (HSC) and stromal cells including bone marrow microvascular endothelial cells (BMMEC) are critical for hematopoiesis. However, most of the identified HSC receptors mediate interactions between HSC and stromal cells in the extravascular space. We studied the interaction between HSC and BMMEC in the early period of homing with blocking monoclonal antibodies. Splenectomized C57BL mice were given 100 cGy of total body irradiation (TBI) followed by infusion into the tail vein of the bone marrow mononuclear cells (BMMNC). For the study of seeding kinetics early after transplantation, 2 × 10 6 BMMNC were infused 2 hours after TBI. At 0.5, 1, 2, 12, 18 and 24 hours after infusion, the content of total nucleated cells and colony-forming units granulocyte/macrophage (CFU-GM) in each of two femurs were determined. For the study of posttransplant circulating progenitor cells early after transplantation, 0.5 to 1.0 ml peripheral blood (PB) was aspirated in a heparinized syringe from the inferior vena cava. Experiments were repeated eight to ten times to ensure the reproducibility of results. Homing assay was performed after pretreatment with an antibody specific for VLA-4, VLA-5, LFA-1, or L-selectin. Briefly, BMMNC were incubated with an antibody (5 μg/ml) for 30 minutes at room temperature in IMBM and used immediately for transplantation studies. Posttransplant kinetics of CFU-GM from BM were evaluated at 0.5, 2, 12, 18 and 24 hours after infusion of BM cells. CFU-GM was the highest at 30 minutes. The number of CFU-GM decreased at first, reached a nadir at 12 hours, and then increased rapidly. As for posttransplant kinetics of CFU-GM from PB, 50 colonies/ml of CFU-GM was found 30 minutes at a frequency of 50 colonies/ml after infusion. These declined rapidly in 2 hours, and were not detectable after 24 hours in PB. Effects of pretreatment of BMMNC with antibodies against adhesion molecules on seeding efficiency of CFU-GM after transplantation are shown in the following table. Seeding efficiency is a ratio of the number of CFU-GM in two femurs to the number of transplanted CFU-GM. Blockage by anti-VLA5 and anti-LFA-1 antibodies caused a significant decrease in CFU-GM seeding efficiency at the early period of homing. Therefore, VLA-5 and LFA-1 may be involved in the recognition of BMMEC and/or adhesion to BMMEC.

Umbilical cord blood processing with the Optipress II blood extractor

We have previously shown that UC blood (UCB) units can be volume-reduced manually, in a closed system, without major losses of nucleated and CD34(+) cells and without the addition of exogenous material. Our aim was to use an automated method for the separation of the UCB components using the Optipress II, extractor, with the 'buffy-coat' collection in a standardized volume. After centrifugation, the 51 UCB units were separated into the three blood components, plasma, buffy coat (BC) and red cells, using the Optipress II. The final volume of the BC fraction, rich in nucleated and progenitor CD34(+) cells, was set at 30 mL. The nucleated and CD34(+) cell content of the UCB collections and the resulting BC were evaluated. The UCB units were grouped according to the volume collected: Group I < 80 mL and Group II > or = 80 mL. Standardization of the BC at 30 mL resulted in significant volume reduction for both groups, with median values of 51% in Group I and 70% in Group II. The nucleated and CD34(+) cell recoveries in the BC from Group I were 88% and 99% respectively; for Group II they were 80% and 97%. This semi-automated method of volume reduction efficiently reduces low, as well as high volume UCB units, with good nucleated- and progenitor-cell yields. Being a closed system and free of external material, the risk of contamination is minimized. The resulting fractions are then available for validation studies of the unit, effectively fulfilling the main requisites for UCB banking.

The effect of granulocyte colony-stimulating factor administration in healthy donors before bone marrow harvesting.

To investigate whether granulocyte colony-stimulating factor (G-CSF) administration to donors before harvest may lighten the burden imposed on them and accelerate the bone marrow (BM) recovery, we administered 2 microgram/kg/d of G-CSF for five consecutive days before the marrow harvest. All of the donors tolerated the G-CSF administration well without severe adverse events. After 5 d of G-CSF treatment, CD34+ cells and granulocyte-macrophage colony-forming units (GM-CFU) in the donors' BM exceeded baseline values by 4.2-fold (range 0.71-316) and 1.6-fold (0.28-118) respectively. The concentration of total nucleated cells (x 107/ml) in the graft increased from 1.61 (0.95-3.23) to 2.44 (1.27-4.01). Although we collected 1020 ml of BM and obtained 1.50 x 1010 nucleated cells from unprimed donors, 940 ml of BM were sufficient to obtain 2.14 x 1010 nucleated cells from primed donors. However, G-CSF-primed BM did not shorten the time to tri-lineage engraftment and the duration of hospitalization compared with unprimed BM, although primed BM contained more CD34+ cells than baseline values. We consider that the advantages of BM priming are not the acceleration of BM recovery but rather the reduction of blood loss during BM harvesting.

The number of nucleated cells reflects the hematopoietic content of umbilical cord blood for transplantation.

A single umbilical cord blood (UCB) collection may contain sufficient hematopoietic stem cells to achieve engraftment and repopulation of the hematopoietic system of children and adults after myeloablative therapy. The hematopoietic potential of a UCB unit is often defined by the number of CD34+ cells or the number of colony-forming units as measured in semisolid hematopoietic progenitor cell (HPC) cultures. However, these assays are relatively difficult to standardize between UCB banks. The number of nucleated cells infused per kilogram body weight of the recipient is also reported to be a significant factor in the speed of recovery of neutrophils and platelets after transplantation. To analyze which parameters could be used to evaluate the hematopoietic potential of a UCB graft, we evaluated almost 300 UCB units that were collected for banking for unrelated transplantation. A strong correlation was found between the frequencies of CD34+ cells and the HPC as measured in semi-solid medium cultures. From the various leukocyte subpopulations, the concentration and total numbers of nucleated cells correlated best with both the HPC content and the number of CD34+ cells. Differentiation of these nucleated cells into subsets of leukocytes offered no advantage for better prediction of HPC or CD34+ cells. These results indicate that the nucleated cell count probably reflects the hematopoietic potential of a UCB graft, and may for that reason correlate with the speed of engraftment after transplantation.

Nuclei-size distributions as predictive tools of hematopoietic cell proliferation

Current protocols for transplantation of hematopoietic stem and progenitor cells may be limited by donor-cell availability and the long time needed to restore neutrophil and platelet counts to normal levels. Ex vivo expansion of hematopoietic cells has the potential to decrease the required harvest size, and to enhance the transplant outcome, by providing greater numbers of progenitor and post-progenitor cells. However, widespread application of ex vivo expansion in the clinical setting is complicated by sample-to-sample variability in the extent and kinetics of cell expansion. For example, the lag time before active cell expansion may vary by several days and some samples may never expand under the culture conditions employed. An early determination regarding the fate of a culture would save time and resources, and would allow corrective action to be taken if desired. Furthermore, anticipation of the onset of cell cycling should prove useful in the development of culture-feeding strategies, as well as for maximizing transduction efficiency in gene-therapy protocols that employ retroviral vectors. We demonstrate that the nuclei-size distribution, which is obtained at the same time as the total nucleated cell concentration, can be used to predict the onset of cell proliferation. The formation of a second peak (with diameter > 4 microm) in the nuclei-size distribution, in addition to the smaller diameter peak (< 4 microm) present for quiescent cells, precedes total cell expansion. In particular 94% of all MNC and CD34+ cell cultures that we have observed to exhibit a second peak in the nuclei-size distribution have realized total cell expansion. Furthermore, only one of 67 observed cultures that did not exhibit the formation of a second peak realized total cell expansion. The formation of a second peak in the nuclei-size distribution is evident, either before or on the same day as the presence of a significant fraction of cells in the S-phase of the cell cycle.

Characterization of human bone marrow stromal cells with respect to osteoblastic differentiation.

Human bone marrow was harvested by means of iliac crest aspiration and cultured under conditions that promote an osteoblastic phenotype. Human bone marrow aspirates from 30 normal subjects, ages 8-80 years, with no systemic illness, yielded a mean of 92 +/- 65 x 10(6) nucleated cells per 2 ml of aspirate. The prevalence of potential osteoblastic progenitors was estimated by counting the number of alkaline phosphatase-positive colonies. This assay demonstrated a mean of 43 +/- 28 alkaline phosphatase-positive colonies per 10(6) nucleated cells, which was about one per 23,000 nucleated cells. The prevalence of these colonies was positively correlated with the concentration of nucleated cells in the original aspirate (p = 0.014) and was negatively correlated with donor age (p = 0.020). The population of alkaline phosphatase-positive colonies in this model sequentially exhibited markers of the osteoblastic phenotype; essentially all colonies (more than 99%) stained positively for alkaline phosphatase on day 9. Matrix mineralization, which was associated with the synthesis of bone sialoprotein, was demonstrated on day 17 with alizarin red S staining. On day 45, cells that were stimulated with 1,25-dihydroxyvitamin D3 synthesized and secreted osteocalcin at concentrations consistent with known osteoblastic cell lines. This model provides a useful method for the assay of progenitors of connective tissue from human subjects, examination of the effects of aging and selected disease states on this progenitor population, and investigation into the regulation of human osteoblastic differentiation.

Analysis of apheresis content of progenitor cell collections from normal donors to whom granulocyte-colony-stimulating factor is administered.

Allogeneic blood cell transplantation is a new alternative to bone marrow transplantation. Preliminary data suggest that granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells from normal donors can provide rapid hematopoietic engraftment without significant increases in transplant-related morbidity. Potential advantages to donors include the elimination of an operation under general anesthesia. Twenty-one normal donors underwent high-dose (16 pg/kg/day for 5 days) G-CSF mobilization. Apheresis was performed on the fifth and sixth days of G-CSF administration, and apheresis components were analyzed by flow cytometry. Donor characteristics in relationship to apheresis yields were also analyzed. Apheresis components were analyzed according to donor weight. The median total numbers of white cells per kg, CD34+ cells per kg, and CD3+ cells per kg were 10.8 x 10(6), 7.2 x 10(8), and 295 x 10(8), respectively. Day 5 collections had significantly higher nucleated cell content and median CD34+ percentages than did Day 6 collections (0.71% on Day 5 vs. 0.58% on Day 6, p < 0.01). CD34+ content and total white cells were not related to age or sex. No donors experienced toxic effects that required their removal from the study. Day 5 is the optimal day for the harvest of normal-donor peripheral blood progenitor cells after mobilization with high-dose G-CSF. Older individuals are acceptable donors of allogeneic progenitor cells.

Stimulation of the DNA replication and murine bone marrow cell proliferation with exogenous zinc-metallothionein

It is shown that zinc-metallothionein from rat liver increases 1.5-fold thein vitro incorporation of3H-thymidine in murine bone marrow cells. The same concentrations of zinc chloride and a mixture imitating zinc-metallothionein (zinc, cysteine, and albumin) inhibit DNA synthesis. In mice receiving an intraperitoneal injection of zinc-metallothionein 10–15 min before γ-irradiation, the incorporation of3H-thymidine and the content of nucleated cells in the bone marrow is 1.5- to 2-fold higher than those in unprotected animals, the number of endogenous splenic colonies in pretreated mice being 2.7-fold higher.

The purification of CD34 cells from human cord blood: comparison of separation techniques and cytokine requirements for optimal growth of clonogenic progenitors.

This study was performed to assess the methods which gave maximal recovery of purified CD34/45+ cells from a cord blood specimen and optimal growth of progenitors cultured from the purified cells. Cord blood samples were separated using Percoll gradients (either one (1.080) or two successive (1.080 and 1.068) gradient(s)) and commercially available devices for CD34+ cell isolation (affinity columns as manufactured by CellPro Inc. or immunomagnetic separation procedure as devised by Baxter Inc.). "CellPro' or "Baxter' techniques gave similar results in terms of nucleated, CD34/45+ and progenitor cell concentration; however, the yield of CD34/45+ cells in the CD34+ enriched fraction was significantly higher when using the "CellPro' technique. We also found significantly higher numbers of CD34/45+ cells in the CD34+ enriched final fraction when using only one, 1.080, Percoll density gradient in the first separation step. Using one density separation step followed by the "CellPro' technique, we obtained an average of 3 x 10(6) purified CD34/45+ cells from samples containing 8.5 x 10(8) nucleated cells. Granulomonocytic progenitors (CFU-GM) and mixed progenitors (CFU-GEMM) cells from light-density and purified CD34/45+ cell fractions were evaluated. We found that 20-30% of the light-density cells and the purified CD34/45+ cells, yielded a granulomonocytic colony in serum free medium in the presence of interleukins 3 and 6, erythropoietin, granulomonocytic and granulocytic colony-stimulating factors and stem cell factor. The addition of tumour-necrosis factor alpha to the cocktail significantly improved the growth of CFU-GEMM allowing 10% of the purified CD34/45+ cells to yield a mixed colony, which confirms the role of this cytokine on CD34+ cells from cord blood. This study provides an improved method for recovery of CD34/45+ purified cells and their colony formation. These methods may serve as a basis for studies on CD34/45+ cell amplification and gene transfer.

Immunotoxicity study of repeated small doses of dimethoate and methylparathion administered to rats over three generations.

Immunotoxic effects of chronic, equitoxic doses (1/50, 1/75, and 1/100 LD50) of two organophosphorus pesticides dimethoate (DM) and methylparathion (MPT) (14.1, 9.39, and 7.04 mg kg-1 DM; and 0.436, 0.291, and 0.218 mg kg-1 MPT) were investigated in a three generation study in outbred Wistar rats. Treatment of the first generation (G1) with these doses began in animals 4 weeks of age; the parental males were dosed until separation of females, and after mating the females were treated until separation of their G2 offspring (at the age of 4 weeks), and the G3 generation was produced in the same way from treated parental G2 animals. Selected 4 week old males from each generation were also treated with DM and MPT for 4 weeks (experimental groups) before determination of certain conventional toxicological (body weight gain, birth weight and number, organ weights), haematological (absolute and differential WBC, RBC, Ht, MCV, nucleated cell content of femoral bone marrow), and immune function parameters (IgM-PFC number of spleen, DTH reaction). Effects of both substances on immunological variables were detectable at the 1/75 LD50 dose level, but different parameters were affected in the three consecutive generations.

Comparison of bronchoalveolar lavage findings and measurements of gas exchange during exercise in horses with poor racing performance

Twenty-four Thoroughbred and twelve Standardbred racehorses aged between 2 and 6 years, presented for reported poor racing performance, underwent clinical exercise testing. During the last 10 s of exercise at each speed throughout an incremental speed exercise test on a treadmill inclined at a 10% slope, samples of arterial blood and expired gases were collected. Maximum oxygen uptake and the partial pressures of oxygen and carbon dioxide in arterial blood were determined. These values were compared between the two breeds of horses and also with reference to cytological findings of bronchoalveolar lavage samples, including neutrophil, erythrocyte and haemosiderophage percentage and the total nucleated cell concentration. The results revealed an inverse relationship (Spearman R = -0.45, p < 0.05) between the total nucleated cell count in bronchoalveolar lavage samples and arterial oxygen partial pressure during exercise at 11 m.s(-1). This result suggests that subclinical pulmonary disease may be a more important cause of poor racing performance than previously thought. Also of note was a positive correlation (Spearman R = 0.50, p < 0.05) between maximum oxygen uptake and the percentage of erythrocytes.

Effect of Cell Concentration on Bone Marrow and Peripheral Blood Stem Cell Cryopreservation

AbstractThe effects of cell concentration during cryopreservation on bone marrow (BM) or peripheral blood (PB)-derived hematopoietic progenitor cells have not been described. The much greater numbers of cells harvested for autologous PB stem cell (PBSC) transplantation requires that the cells be frozen at higher cell concentrations, or in much greater volumes, compared with BM. We cryopreserved 108 PBSC collections from 30 patients at an average (±SD) cell concentration of 3.7 ± 1.9 × 108 nucleated cells per mL in 127 ± 45 mL. The proportion of mononuclear cells was 52.9% ± 27.2%. The products also contained 2.9 ±2.1 × 109 platelets/mL and an average red cell proportion of 12.9% ± 7.2%. The nucleated cell recovery after thawing was 75.4% ± 13.0%. The nucleated cell concentration during freezing was not predictive for the postthaw recoveries of nucleated cells (P = .38), granulocyte-macrophage colony-forming unit (P = .06) or CD34+ cells (P = .54), or for the viability of mononuclear cells (P = .81). The platelet and red cell concentrations similarly were not predictive for these endpoints. Samples (3 BM, 7 PBSC) from 10 patients were simultaneously cryopreserved at twofold, and from 5 additional patients (PBSC) at 6- to 24-fold differing cell concentrations. A lower recovery of erythroid burst forming unit was found for samples frozen at higher cell concentrations (P = .04), but no significant differences were found in the other endpoints listed above. The average cell concentration during freezing for each patient’s PBSC collections (n = 34 patients) did not predict time to achieve a PB count of >500 granulocytes/μL (P = .51) or platelet transfusion independence (P = .39). Patients achieved these endpoints of engraftment at medians of 12 and 13 days, respectively. The infusion of these products was generally well tolerated. Similarly, the cell concentration at which BM cells were frozen did not predict for the duration of granulocyte (P = .63) or platelet (P = .36) aplasias for 54 patients undergoing autologous BM transplantation. These data suggest that PBSC or BM cells collected for transplantation may be cryopreserved at very high cell concentrations without loss of engraftment potential or undue infusion-related toxicity.

Effect of cell concentration on bone marrow and peripheral blood stem cell cryopreservation

The effects of cell concentration during cryopreservation on bone marrow (BM) or peripheral blood (PB)-derived hematopoietic progenitor cells have not been described. The much greater numbers of cells harvested for autologous PB stem cell (PBSC) transplantation requires that the cells be frozen at higher cell concentrations, or in much greater volumes, compared with BM. We cryopreserved 108 PBSC collections from 30 patients at an average (+/- SD) cell concentration of 3.7 +/- 1.9 x 10(8) nucleated cells per mL in 127 +/- 45 mL. The proportion of mononuclear cells was 52.9% +/- 27.2%. The products also contained 2.9 +/- 2.1 x 10(9) platelets/mL and an average red cell proportion of 12.9% +/- 7.2%. The nucleated cell recovery after thawing was 75.4% +/- 13.0%. The nucleated cell concentration during freezing was not predictive for the postthaw recoveries of nucleated cells (P = .38), granulocyte-macrophage colony-forming unit (P = .06) or CD34+ cells (P = .54), or for the viability of mononuclear cells (P = .81). The platelet and red cell concentrations similarly were not predictive for these endpoints. Samples (3 BM, 7 PBSC) from 10 patients were simultaneously cryopreserved at two-fold, and from 5 additional patients (PBSC) at 6- to 24-fold differing cell concentrations. A lower recovery of erythroid burst forming unit was found for samples frozen at higher cell concentrations (P = .04), but no significant differences were found in the other endpoints listed above. The average cell concentration during freezing for each patient's PBSC collections (n = 34 patients) did not predict time to achieve a PB count of &gt; 500 granulocytes/microL (P = .51) or platelet transfusion independence (P = .39). Patients achieved these endpoints of engraftment at medians of 12 and 13 days, respectively. The infusion of these products was generally well tolerated. Similarly, the cell concentration at which BM cells were frozen did not predict for the duration of granulocyte (P = .63) or platelet (P = .36) aplasias for 54 patients undergoing autologous BM transplantation. These data suggest that PBSC or BM cells collected for transplantation may be cryopreserved at very high cell concentrations without loss of engraftment potential or undue infusion-related toxicity.

Lmmunotoxicological Investigation of SCMF, a New Pyrethroid Pesticide in Mice

The toxicity of a new pyrethroid pesticide Supercypermethrin Forte (SCMF) was studied in male CFLP mice using classic toxicological (body weight, organ weights) and haematological (white blood cell count, haematocrit, nucleated cell content of femoral bone marrow) methods and immune function tests (PFC assay, DTH reaction). Four weeks of oral treatment in a 5 days per week system at doses of 1/10, 1/20, or 1/40 x LD50 did not cause evaluable changes in the measured parameters. When single calculated LD20, LD10, or LD5 doses of SCMF were administered on different days before termination to different groups of mice the two higher doses caused a time- and dose-dependent decrease in the splenic PCF number. Apart from some temporary toxic signs and an increase of haematocrit at the top dose the other examined parameters did not show evaluable changes. Under these experimental conditions toxic changes appeared only at the high dose range and, of those applied, the PFC assay proved to be the most sensitive method for detecting the toxicity of SCMF.

Effects of perioperative granulocyte colony-stimulating factor on horses with ascending colonic ischemia.

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that regulates the proliferation and maturation of hematopoietic progenitor cells and modulates the function of mature neutrophils. The responses to administration of G-CSF alone, and in combination with antimicrobials, were studied in an equine model of ascending colon ischemia. Complete segmental colonic ischemia (3.75 hours) with pelvic flexure enterotomy was created in four treatment groups. Group 1 horses received recombinant canine G-CSF (10 micrograms/kg, every 24 hours, intramuscularly), gentamicin sulfate (2.2 mg/kg, every 8 hours, intravenously), and potassium penicillin G (40,000 IU/kg, every 6 hours, intravenously). Group 2 horses were treated with the G-CSF vehicle and antimicrobials as for group 1. Group 3 horses received G-CSF and the antimicrobial drug vehicles, and group 4 horses served as the untreated control receiving G-CSF vehicle and antimicrobial vehicles. The results for 20 horses, five horses in each group, were compared. Treatment with G-CSF was associated with an increased concentration of white blood cells, band neutrophils, neutrophils, lymphocytes, and monocytes in the peripheral blood after surgery. Antimicrobial administration had no detectable effect on cell concentrations after surgery. Administration of G-CSF was associated with an increased concentration of nucleated cells in the peritoneal fluid including neutrophils, small mononuclear cells and large mononuclear cells. Horses that developed incisional infections had lower neutrophil concentrations in the peripheral blood on postoperative day 2 than horses without infected incisions. These results suggested that the prophylactic administration of G-CSF may be useful in the treatment of patients at risk for developing neutropenia after surgery.

Enumeration of cells in bone marrow and peripheral blood stem cell collections: technical issues and prospects for standardization.

Enumeration of total nucleated cells and mononuclear cells is a fundamental part of the laboratory evaluation and quality control program for bone marrow and peripheral blood stem cell collections intended for transplantation. Measurement of the total nucleated cell content is especially useful for providing rapid feedback about the bone marrow product during or immediately after the harvest. However, the mononuclear cell content may be more informative because the nucleated cell population contains a variable number of mature granulocytes and nucleated red cells, which do not contribute to hematopoietic engraftment. The lack of comparative data among various laboratories and among different types of cell counting methods has hindered standardization of these assays among bone marrow processing laboratories. Specific issues needing attention in assay standardization include sample preparation and handling, identification and elimination of artifacts in automated counting, relative advantages and disadvantages of manual and automated counting methods, and criteria for differential counting of nucleated cells. The establishment of standards for bone marrow and other stem cell counting methods, as well as other evaluation procedures, should be preceded by collection and analysis of comparative data, and followed by a proficiency testing program for bone marrow processing laboratories.

Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells.

The purpose of this study was to evaluate human umbilical cord blood as an alternative to bone marrow in the provision of transplantable stem/progenitor cells for hematopoietic reconstitution. Although no direct quantitative assay for human hematopoietic repopulating cells is at present available, the granulocyte-macrophage progenitor cell (CFU-GM) assay has been used with success as a valid indicator of engrafting capability. We examined greater than 100 collections of human umbilical cord blood for their content of nucleated cells and granulocyte-macrophage, erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells, in many cases both before and after cryopreservation. First it was determined that granulocyte-macrophage, erythroid, and multipotential progenitor cells remained functionally viable in cord blood untreated except for addition of anticoagulant for at least 3 days at 4 degrees C or 25 degrees C (room temperature), though not at 37 degrees C, implying that these cells could be satisfactorily studied and used or cryopreserved for therapy after transport of cord blood by overnight air freight carriage from a remote obstetrical service. Granulocyte-macrophage progenitor cells from cord blood so received responded normally to stimulation by purified recombinant preparations of granulocyte-macrophage, granulocyte, and macrophage colony-stimulating factors and interleukin 3. The salient finding, based on analysis of 101 cord blood collections, is that the numbers of progenitor cells present in the low-density (less than 1.077 gm/ml) fraction after Ficoll/Hypaque separation typically fell within the range that has been reported for successful engraftment by bone marrow cells. Another observation of practical importance is that procedures to remove erythrocytes or granulocytes prior to freezing, and washing of thawed cells before plating, entailed large losses of progenitor cells, the yield of unwashed progenitor cells from unfractionated cord blood being many times greater. The provisional inference is that human umbilical cord blood from a single individual is typically a sufficient source of cells for autologous (syngeneic) and for major histocompatibility complex-matched allogeneic hematopoietic reconstitution.

Variability in 4-Hydroperoxycyclophosphamide Activity During Clinical Purging for Autologous Bone Marrow Transplantation

We examined the effects of varying incubation conditions on the in vitro activity of 4-hydroperoxycyclophosphamide (4HC). 4HC activity against CFU-GM and against the K562 tumor cell line decreased with increasing the RBC concentration of the incubation mixture. Increasing the concentration of nucleated bone marrow cells in the incubation mixture also decreased the 4HC activity. Evaluation of 53 consecutive patients undergoing autologous bone marrow transplantation (BMT) revealed that the incubation RBC concentration during clinical purging showed a similar effect on CFU-GM recovery. Aldehyde dehydrogenase content of RBCs and nucleated marrow cells appears to be the cause of the inhibition of 4HC activity. Although there was no difference in individual CFU-GM sensitivity to 4HC among normals, previously treated patients undergoing autologous BMT showed significant variability in CFU-GM sensitivity to 4HC. The combined effects of incubation RBC concentration and individual patient 4HC sensitivity appear to account for most of the variability in CFU-GM recovery and speed of hematologic recovery after clinical purging with 4HC.

Variability in 4-hydroperoxycyclophosphamide activity during clinical purging for autologous bone marrow transplantation

We examined the effects of varying incubation conditions on the in vitro activity of 4-hydroperoxycyclophosphamide (4HC). 4HC activity against CFU-GM and against the K562 tumor cell line decreased with increasing the RBC concentration of the incubation mixture. Increasing the concentration of nucleated bone marrow cells in the incubation mixture also decreased the 4HC activity. Evaluation of 53 consecutive patients undergoing autologous bone marrow transplantation (BMT) revealed that the incubation RBC concentration during clinical purging showed a similar effect on CFU-GM recovery. Aldehyde dehydrogenase content of RBCs and nucleated marrow cells appears to be the cause of the inhibition of 4HC activity. Although there was no difference in individual CFU-GM sensitivity to 4HC among normals, previously treated patients undergoing autologous BMT showed significant variability in CFU-GM sensitivity to 4HC. The combined effects of incubation RBC concentration and individual patient 4HC sensitivity appear to account for most of the variability in CFU-GM recovery and speed of hematologic recovery after clinical purging with 4HC.

Prenatal Asphyxia in Growth Retarded Fetuses

Members of the Department of Obstetrics at King’s College Hospital, London SE5 have measured the umbilical venous oxygen and carbon dioxide tensions, pH, lactate and glucose concentrations, nucleated red cell (erythroblast) content, and haemoglobin concentration in 38 fetuses with intra-uterine growth retardation in which blood sampling was performed by cordocentesis.