Nucleated Cell Content Research Articles

High Chance of Finding “Suitable” Unrelated Cord Blood Donations (UCBD) in the Australian Network of Cord Blood Banks (AUSCORD).

Aims: To determine the value of a CB inventory in the country of citizenship of the patient, we investigated the potential to find suitable UCBD in the Auscord inventory for Australian patients who could not identify a matched unrelated bone marrow donor (MUBMD) amongst donors registered either in the ABMDR or international registries.Methods: Between 7/2003 and 6/2005, search was commenced for 776 Australian patients; 32% of searches resulted in a BM or CB transplant, 22% were cancelled, 46% remained open. Fifty-two patients were unable to find an 8/8 MUBMD (intermediate level class 1 (A, B) and allelic level class 2 (DRB1, DQB1)). The Auscord inventory contained 17,761 UCBD with an ethnic mix of 65% Caucasian, and the remainder with ethnic backgrounds reflective of those in the Australian community. Four, 5 and 6 of 6 matched UCBD with intermediate or higher resolution typing were identified and their nucleated cell (NC) content recorded.Results: Six patients were 16 years or younger and 46 were older, 19 were female and 33 were male and mean weight was 72.2kg (range 16–85.9kg). Ethnic mix was 68% Caucasian, 8% Asian/Indian, 6% multiple ethnicity, 4% Pacific Islander, 4% Middle Eastern and was not recorded for 14%. One patient (1.9%), of New Guinean background, had no 4, 5 or 6/6 matched UCBD. Two patients (3.8%) had a single 6/6 HLA matched UCBD identified; one had >20 5/6 matched and the other had no 5/6 and >20 4/6 matched UCBD. Twenty-nine patients (55.8%) had between 1 and 15 5/6 and between 1 and >20 4/6 matched and 20 patients (38.5%) had between 2 and >20 4/6 matched UCBD. To determine the potential for these patients to proceed to transplant only those UCBD where allelic DRB1 typing (15,279) was available were evaluated. The NC content of the best matched UCB donation was a mean of 159.2x107 (range 59–281) and provided a NC dose of 2.5x107/kg (range 0.8–13.5) and for the second UCBD was 168.8x107 (range 43–274) with a NC dose of 5.2x107/kg (range 1.3–27.0). Five patients (9.6%; 2 adults and 3 children) had a single best matched UCBD which provided a NC dose of ≥3.5x107/kg, and a further 6 patients (11.5%; 2 children and 4 adults) had a single best matched which provided a NC dose of between 3.0 and 3.49x107/kg. For the 46 patients who had a single best matched which provided a NC dose of <3.5x107/kg, an analysis was then performed combining the NC content of the best match single with a second UCBD which had the highest NC content, whether this UCBD was the same degree of matching or 1 HLA mismatch lower. Thirty-nine of 46 patients (84.4%) had 2 UCBD which provided a combined NC dose of ≥3.5x107/kg, 4 (8.7%) had 2 UCBD which provided a combined NC dose of between 3.0 and 3.49x107/kg, and 3 (6.5%) did not have 2 UCBD which provided a combined NC dose of ≥3.0x107/kg.Conclusions: These data indicate that for Australian patients who could not identify a MUBMD 92.3% found suitable UCBD in an Australian inventory of 17,716 UCBD that provided an acceptable NC dose. This reality highlights that the ethnic mix of a CB inventory in the country of nationality of the patient is reflected in the community from which the donations are sourced, and that suitable donations could be identified for 89% of adult patients. However, 39% of patients had only 4/6 matched UCBD available and thus a larger inventory may result in identifying more closely matched UCBD.

Efficacy of the “MarrowMiner” a Novel, Minimally Invasive Bone Marrow Harvesting Device in a Large Animal Model.

INTRODUCTION: Bone marrow (BM) contains a rich supply of adult stem & progenitor cells. Traditional percutaneous large bore needle aspiration methods for harvesting marrow for bone marrow transplantation (BMT) are crude, tedious and expensive usually requiring general anesthesia. A novel device, the MarrowMiner (MM), was developed for the minimally invasive harvest of BM which may enable the rapid, convenient outpatient harvest of large quantities of BM derived cells under local anesthesia for use in BMT and an increasing array of developing marrow based regenerative therapies. The MarrowMiner utilizes a single marrow entry site into the iliac bone, through which the flexible, powered, MM catheter is able to access the majority of the accessible marrow space and aspirate rich marrow. The device has been tested extensively in human cadavers and in large animal trials.METHODS: In 5 juvenile pigs (70–87kg), standard 1- and 6-port needles were used to aspirate marrow at three locations along one iliac crest. On the opposite iliac, the MM device was used to collect BM either along the crest or longitudinally down the plane of the iliac wing. Harvests were collected in: small (2–5ml), medium (5–20ml), & large (>20ml) volumes. BM cell viability, counts & CFU-F assays were performed.RESULTS: The MM was observed to be safe, and despite long aspiration paths (up to 20cm), the catheter remained within the marrow cavity without penetrating the cortical walls. Marrow volumes >60 ml could be aspirated in a single pass using either a syringe or vacuum suction. No significant differences were observed in viability, or nucleated cell concentration among BM harvested by 1-port, 6-port needles, and MM in a longitudinal manner, with the exception that BM aspirated along the iliac crest by the MM contained a higher concentration of nucleated cells (p<.05). As expected, the CFU-F concentration decreased with increasing BM volume harvested when standard harvest needles were used. In contrast, no significant decrease in CFU concentration was observed with increasing marrow volume aspirates with the MM (Figure 1). The MM yielded significantly higher CFU concentration than the standard 1-port and 6-port aspiration needles at the medium harvest volume. When a large volume of marrow was aspirated, MM harvested significantly higher [9 fold increase] in CFU-F concentration than the 6-port needle. As aspiration volume increased from medium to large, MM aspirate maintained CFU concentration while the 6-port needle resulted in an approximately 7.5 fold reduction. Marrow aspirated along the crest yielded significantly higher concentrations of CFU-F.CONCLUSIONS: The study demonstrated that the MM can successfully & safely harvest large volumes of BM, with significantly higher stem cell content than traditional needle aspiration. The flexible shaft allowed for aspirating bone marrow of the highest cellularity along the iliac crest, a path unfeasible for rigid needles. This study suggests that this novel device may enable significantly improved clinical BM harvest in a more rapid, reproducible and less invasive manner, particularly if directed to harvest along the iliac crest. [Display omitted]

Concentration of Bone Marrow Total Nucleated Cells by a Point-of-Care Device Provides a High Yield and Preserves Their Functional Activity.

Stem and progenitor cell therapy is a novel strategy to enhance cardiovascular regeneration. Cell isolation procedures are crucial for the functional activity of the administered cellular product. Therefore, new isolation techniques have to be evaluated in comparison to the Ficoll isolation procedure as the current gold standard. Here we prospectively evaluated a novel point-of-care device (Harvest BMAC System) for the concentration of bone marrow total nucleated cells (TNC) in comparison to the Ficoll isolation procedure for bone marrow mononucleated cells (MNC). The yield in total numbers of TNC was 2.4-fold higher for Harvest compared to Ficoll. Despite significant differences in their cellular compositions, the colony-forming capacity was similar for both products. Intriguingly, the migratory capacity was significantly higher for the Harvest TNC (164 ± 66%; p = 0.007). In a mouse model of hind limb ischemia, the increase in blood flow recovery was similar between Harvest BM-TNC and Ficoll BM-MNC (0.53 ± 0.20 vs. 0.46 ± 0.15; p = 0.88). However, adjustment of the injected cell number based on the higher yield of Harvest TNC resulted in a significant better recovery (0.64 ± 0.16 vs. 0.46 ± 0.15; p = 0.003). Cells concentrated by the Harvest point-of-care device show similar or greater functional activity compared to Ficoll isolation. However, the greater yield of cells and the wider range of cell types for the Harvest device may translate into an even greater therapeutic effect.

Recovery, viability and clinical toxicity of thawed and washed haematopoietic progenitor cells: analysis of 952 autologous peripheral blood stem cell transplantations

Cryopreservation and thawing of haematopoietic stem cells are associated with cell loss and infusion-related toxicities. We analysed viability, total nucleated cell (TNC) and CD34+ cell recovery, and infusion-related toxicities of 952 thawed and washed products. Mean TNC and CD34+ viable cells recoveries were 55.9+/-18.6 and 98.0+/-36.5%, respectively. Mean cell viability was 68.25+/-18.9%. TNC recovery was correlated with viability but independent of the initial nucleated cell concentration. No difference in TNC recovery or viability was observed according to underlying diseases, except for myeloma, for which these variables were significantly lower (P<0.05). CD34+ cell recovery was not correlated with viability or CD34+ initial count and was similar for all diseases. Cryostorage duration was not associated with cell loss. Immediate adverse events occurred in 169 patients (19%) and were moderate (grade I or II) for the majority of patients. Clinical toxicity was associated with a higher infused cell number and the presence of clumps in infused bags. The washing procedure of cell products lead to a low rate of adverse events, but patients transplanted with high cell numbers or bags in which clumps were identified are predisposed to such complications.

Clinical side effects during peripheral blood progenitor cell infusion

There are several side effects which have been reported during the infusion of peripheral blood progenitor cells (PBPCs) either due to the infusion or the content of the infusate. We have evaluated the side effects detected during PBPCs infusion in 194 autologous and 25 allogeneic transplantations. In autologous cryopreserved PBPCs infusion, we detected a total of forty-nine (25.25%) side effect events during and after the infusion period. Forty-six (23.71%) of these side effects were detected during the infusion period including fifteen (7.73%) cardiac side effects, which required stopping the infusion, and thirty-one (15.97%) non-cardiac side effects, which did not require cessation of the infusion. Sinus bradycardia after a minimum of 45 min after completing the infusion was seen in three (1.54%) patients. The median volume, dimethyl sulfoxide (DMSO) and total nucleated cell (TNC) content of the product were found to be significantly higher in patients with side effects compared to the group without any side effects ( P < 0.05). The median volume and DMSO content were found to be significantly higher in patients with cardiac side effects compared to non-cardiac side effects ( P < 0.05). There was no cardiac side effects in patients treated with an infusate containing 100 × 10 9 L −1 leukocytes. We did not observe any infusion-related side effects in patients given allogeneic non-cryopreserved PBPCs. We have concluded that the volume, DMSO and TNC content of autologous cryopreserved PBPCs product are directly related to clinical side effects.

Dosing of Post-Thaw Colony Forming Units (CFU) and CD34 Cells Predict Engraftment and Survival in Recipients of Banked Unrelated Donor Umbilical Cord Blood (UCB) for Allogeneic Transplantation.

A single umbilical cord blood unit (CBU) often doses patients at the threshhold for successful hematopoietic cell transplantation. While both cryopreserved and infused total nucleated cell (TNC) content is somewhat predictive of outcomes, there has been no reliable "potency" assay developed for UCB. Frequently, pre-cryopreserved and post-thaw recoveries are inconsistent and unpredictable. The lack of standardization from cord blood bank to bank and transplant center laboratory to laboratory further complicates interpretation of the data that is currently available. Our program is in the unique position to operate both a public cord blood bank, "The Carolinas Cord Blood Bank" (CCBB) and adult and pediatric blood and marrow transplant programs performing >100 unrelated donor cord blood transplants (UCBT) per year, placing us in the unique position of evaluating cord blood pre-cryopreservation and post-thaw TNC, viability, CD34 and colony forming assays (CFU-GM, CFU-GEMM, BFU-E) in the same laboratory, eliminating the potential for differences in laboratory practices. We have evaluated the results of 218 transplants performed from CBUs obtained from the CCBB and transplanted at our center. These units did not require transfer from the bank to the transplant center (TC) eliminating the possibility that warming of CBUs during shipping could compromise cell potency. In this dataset, the cummulative incidence (CINC) of neutrophil engraftment (ANC 500/uL by day +42) was 79.4% (95% CI 74–84.8) and platelet engraftment (PLT 50K by day 180) was 61.3% (95% CI 54.8–67.8). The CINC of event-free survival (EFS) at day 180 and 1 year was 68.7% (95% CI 62.5–75) and 62.5% (95% CI 56–69.1), respectively. The CI of acute grades II–IV graft-versus-host disease at days 100 and 150 was 10.6% (95% CI 6.5–14.7%). The CI of relapse at 1 year was 8.1% (95% CI 4.4–11.8%). Thus, most deaths occurred from graft failure, infection or regimen related toxicity all of which could be influenced by graft potency. We also expanded our dataset to include an additional 405 transplants where the CBU was shipped from other cord blood banks and transplanted at Duke. In this group, the CINC of neutrophil engraftment was 78.2% (95% CI 75–81.2) and the CI of EFS at day 180 and 1 year was 60–2 (95% CI 56.3–64) and 51% (95% CI 47–55), respectively. We hypothesized that post-thaw CFU and or CD34 would be better predictors of engraftment and survival than TNC. We examined the impact of post-thaw total CFU and CD34 recoveries and dosing on neutrophil engraftment and overall survival in these patients. Dosing of post-thaw CFU/kg infused (median 3.5x10e4/kg) correlated with neutrophil engraftment (p=<0.01). Dosing of post-thaw CD34/kg (median 1.8x10e5/kg) correlated with both neutrophil engraftment (p=<0.01) and event-free survival (p=<0.01). These data suggest that it would be possible to create a potency assay that could be tested prior to final CBU selection allowing for transplantation of the highest quality unit to the patient. This would require correlation of post-thaw recovery from the CBU with either a segment or cryovial from the same unit. These studies will allow for comparison of results from shipped versus local CBU transplants to ask whether shipping of CBUs affects potency at transplant. These studies should be planned for the National Cord Blood Inventory program.

Evaluation of Biological Features of Cord Blood Units Collected With Different Methods After Cesarean Section

Introduction Cord blood banks are established worldwide as a result of the increased use of umbilical cord blood (UCB) transplantation. The outcomes of this procedure relate to the cell dose of the UCB unit and the UCB collection. The aim of this study was to evaluate whether the mode of collection influenced the biological features of the UCB units. Materials and methods We studied 151 UCB units consecutively collected in the cesarean setting with two different methods: in utero after infant delivery and before delivery of the placenta, and ex utero after delivery of placenta. Results Sixty-nine UCB units were collected in utero and 82 ex utero. The two groups were comparable for maternal and obstetric factors. The proportion of banked UCB units was similar in the two groups (38% vs 40%, respectively). No statistically significant differences were observed between the methods of collection in term of volume, white blood cell count, total nucleated cell content, CD34 + cells, and CFU-GM. Conclusion This preliminary study showed that the two methods of collection in the cesarean setting were overlapping and valid if performed according to standard operating procedures.

Evaluation of ex vivo expansion and engraftment in NOD-SCID mice of umbilical cord blood CD34+ cells using the DIDECO ‘Pluricell System’

The Dideco "Pluricell System" is a commercially available closed device composed of an expansion chamber and a kit of certified reagents that allow haematopoietic stem cell expansion. We have expanded seven umbilical cord blood (UCB) samples following the manufacturer's instructions; two groups of irradiated NOD-SCID mice have been transplanted with expanded and nonexpanded cells from the same UCB, and bone marrow was analysed for the presence of human cells. Average UCB volume was 61.6+/-8.8 ml; mean nucleated cell content was 1090.5+/-189.9 x 10(6). Percentage and number of CD34+ cells were 0.37+/-0.13% and 3.9+/-1.2 x 10(6). After separation, CD34+ cell purity was 82+/-11%. Mean number of inoculated cells was 760 000; mean NC and CD34+ fold expansion at 12 days was 230.4+/-91.5 and 21.0+/-11.9. Both groups of mice showed successful engraftment: the percentage of human cells was higher in the group receiving expanded cells (3.4+/-2.01%) compared to the group receiving nonexpanded cells (1.5+/-0.66%) (P<0.00018, Mann-Whitney test). The cell population obtained after 12 days expansion consisted mainly of myeloid and megakaryocytic progenitors. The CD34+ antigen reached the maximum expression level at day 12 (7.5+/-2.0%). Analysis of lineage-markers for human myelomonocytic, megakaryocytic, B, T, CD34 and erythroid cells, gave evidence that all the lineages were represented in the marrow of transplanted mice.

Cord blood hematopoietic progenitor cell concentration and infant sex

Characterization of cord blood facilitates understanding of the factors affecting cord blood transplant quality and improvement of transplantation results. Cord blood obtained from male and female infants has not been thoroughly characterized. A study was performed to test the hypothesis that the cord blood hematopoietic progenitor cell content-of which the CD34+ cell and colony-forming unit (CFU) concentrations were taken as markers-would not only associate with birth weight but also with sex. The hematopoietic progenitor cell concentrations of 1999 healthy infants (47% female) were analyzed in a cord blood bank setting. Male infants had significantly higher median CD34+ cell concentrations than female infants (31.8/microL vs. 30.2/microL, respectively; p = 0.03). Although the disparity in absolute concentrations was small, it was 5.3 percent. In CFU subgroup analysis, the median CFU-mixed concentration of male infants (11.1/microL) was higher than in female infants (9.9/microL; p = 0.03). The difference was more pronounced when cumulative frequencies of the CFU-mixed concentrations from cesarean section deliveries were compared. In multivariate linear regression analysis, the positive influence of male sex on the CD34+ cell concentration was significant (p < 0.05). The expected higher median nucleated cell concentration of female compared to male infants (13.9 x 10(9)/L vs. 13.3 x 10(9)/L, respectively; p = 0.0001) was mainly due to the higher neutrophil concentration of female infants (7.1 x 10(9)/L vs. 6.5 x 10(9)/L, respectively, p < 0.0001). Cord blood hematopoietic progenitor cell concentration was higher in male infants, even after correcting for birth weight. Sex may affect the hematopoietic potential of cord blood transplants.

A Modified Cord Blood Collection Method Achieves Sufficient Cell Levels for Transplantation in Most Adult Patients

Umbilical cord blood transplantation (UCBT) has been used increasingly in both pediatric and adult patients. The total nucleated cell (NC) dose infused is the most critical factor in determining speed of engraftment and survival. Using standard collection techniques, the mean NC content of UCB units is about 10 x 10(8) and only 25% of these units reach the target cell dose of 2 x 10(7)/kg in UCBT patients weighing 50-70 kg. We have designed a modified placental/umbilical two-step collection method in which a standard blood fraction obtained by umbilical venipuncture is combined with a second fraction harvested after placental perfusion with 50 ml heparinized 0.9% saline. This second fraction contributed 32% volume and 15% NCs to the whole UCB unit (123.7 +/- 50.1 ml and 1.26 +/- 0.52 x 10(9) NC). The proportion of progenitor cells in both fractions was not significantly different, indicating that the hematopoietic potential of these larger units is 20% (range, 2%-100%) higher than UCB units collected by standard methods. In addition, the bacterial contamination rate associated with this novel collection method (2.78%) compares favorably. Since 1998 we have further enriched our units by processing only UCB units over 0.8 x 10(9) NCs, resulting in a 36% cell increment (1.46 +/- 0.52 x 10(9) NCs). Thus, 84% and 54% of the Madrid UCB Bank inventory would fulfill the target cell dose of 2 x 10(7)/kg in patients weighing 50 and 65 kg, respectively. This significant UCB banking improvement gives larger pediatric and adult patients a greater chance of finding adequate grafts in order to achieve better clinical outcomes after UCBT.

The viability of cryopreserved PBPC depends on the DMSO concentration and the concentration of nucleated cells in the graft

DMSO is widely used as a cryoprotectant for PBPC. It is desirable to reduce the amount of DMSO without jeopardizing the quality of the stem cell product. The present study was undertaken to investigate whether recovery and survival of CD34+ cells would be significantly altered when PBPC used for autologous transplantations were cryopreserved with four different DMSO concentrations. Apheresis samples of PBPC from 20 consecutive patients were mixed in parallel with 2%, 4%, 5% and 10% DMSO, frozen with identical cell concentrations at a controlled rate, and stored in liquid nitrogen for 6-8 weeks. PBPC samples from 11 consecutive patients were also cryopreserved with two different cell concentrations (150 and 300 x 10(6) nucleated cells/mL) to investigate the effect of increasing the cell concentrations while decreasing the DMSO concentration. The flow cytometric absolute count method, based on ISHAGE guidelines, was used to measure the absolute count of total and viable CD34+ cells in the post-thaw samples. PBPC cryopreserved at 150 x 10(6) cells/mL with 2% DMSO yielded significantly inferior CD34+ cell recovery (P < 0.001) and survival (P < 0.001) compared with cryopreservation with 4% and 5% DMSO. This was also observed when comparing higher cell concentrations. However, a reduced cell survival (P = 0.02) was observed when the nucleated cell concentration was increased from 150 to 300 x 10(6) cells/mL in samples cryopreserved with 5% DMSO. We conclude that 5% DMSO may be the optimal dose for cryopreserving PBPC as long as the cells have not been concentrated at much more than 200 x 10(6) nucleated cells/mL.

The Impact of Variability among Banks on the Quality of Umbilical Cord Blood for Transplantation.

INTRODUCTIONAs the frequency of umbilical cord blood (UCB) stem cell transplantation has increased, the quality of UCB available in banks is an important part of the success of UCB stem cell transplants.METHODSUCB units from outside banks were received in the Clinical Cell Therapy Laboratory where accompanying documentation, transfusion-transmitted disease test results, histocompatibility and ABO typing results, medical history, sterility testing, and processing records including cell counts were reviewed. Deviations were categorized based on cord blood regulations and the potential impact of deviations using a system developed to categorize according to the medical history, quality control testing, or labeling and documentation and for their potential risk to the patient.RESULTSBetween January 1, 2001 and December 31, 2003, 268 UCB units were received for transplantation from 14 UCB banks. Of these 268 units, 151 (56%) from 12 banks (86%) had a total of 246 quality issues (Table). Most (54%) of the issues were in quality control. Examples included: 21 units with transmissible diseases tests that were incomplete, pending, or with positive results; 4 with a positive microbiological screening culture at the shipping UCB bank from a sample obtained prior to cryopreservation; 20 from mothers thought to be colonized with group B streptococcus; 31 from mothers who received antibiotics during labor; and 7 units arrived with inadequate temperature monitoring so it could not be documented that the units were maintained at an acceptable temperature. 11 units had questionable quality control test results for cell viability < 50% (2), poor CFU growth (2), nucleated red cell content > 20% (5), leaking segment (1) and confirmatory HLA typing not performed from attached segment (1). Of the 132 units with quality control issues, 25 (19%) were likely to pose a risk to patients. Documentation was inadequate for five units because related records did not have a complete numbering system. Reports of transmissible disease testing, cell content, donor medical history, or other details were sent on sheets of paper that did not contain the unique number of the UCB unit or other identifying information to assure that the data on that page were from the unit in question. Very few (4%) medical history issues posed a risk to patients. Examples of the medical history issues included: maternal exposure to malaria, recent tattoo, body piercing, maternal HIV risk factors, and maternal history of herpes, chlamydia, human papilloma virus (HPV) or use of donor sperm for the pregnancy.CONCLUSIONBecause standards have evolved over time, UCB banks contain units that have different levels of quality. Some units have been placed in the usable inventory with incomplete test results and/or documentation or that may not meet the banks own current criteria. Since many are only recognized after a transplant decision has been made, information about any quality or operating procedure deviation should be provided in sufficient detail and at the initiation of the search process so that transplant physicians can consider these quality issues when making a transplant decision.TotalPotential Impact#%LikelyPotentialUnlikelyMedical history994043758Quality control13254253077Labeling/documentation1564110TOTAL2461003378135

Birthweight of full-term infants is associated with cord blood CD34+ cell concentration

CD34+ cell counts are used to define the haematopoietic stem cell potential of a given cord blood transplant. The aim was to test the hypothesis that high concentration of cord blood haematopoietic progenitor and stem cells could be a reflection of intrauterine growth, of which birthweight is an indicator. Simple and multiple regression analyses were applied to test cord blood bank data on 1368 infants for associations of selected obstetric factors and cellular contents of cord blood. When groups were formed based on the extreme values (5th versus 95th percentiles) of a given variable, e.g. birthweight, the term infants having the highest birthweights were found to have statistically significantly higher median cord blood CD34+ cell concentrations. Also, infants in the top 50th percentile of relative birthweight had higher median CD34+ cell concentration than infants in the low 50th percentile. In multiple regression analysis, the correlation between birthweight and CD34+ cell concentration was statistically clearly significant. Notably, while an expected correlation between gestational age and nucleated cell concentration was found, there was no association between infant gestational age and CD34+ cell concentration. Haematopoietic progenitor and stem cells may reflect intrauterine growth and have a more central role in foetal development than has been reported earlier.

Birthweight of full‐term infants is associated with cord blood CD34+ cell concentration

Aim: CD34+ cell counts are used to define the haematopoietic stem cell potential of a given cord blood transplant. The aim was to test the hypothesis that high concentration of cord blood haematopoietic progenitor and stem cells could be a reflection of intrauterine growth, of which birthweight is an indicator. Methods: Simple and multiple regression analyses were applied to test cord blood bank data on 1368 infants for associations of selected obstetric factors and cellular contents of cord blood. Results: When groups were formed based on the extreme values (5th versus 95th percentiles) of a given variable, e.g. birthweight, the term infants having the highest birthweights were found to have statistically significantly higher median cord blood CD34+ cell concentrations. Also, infants in the top 50th percentile of relative birthweight had higher median CD34+ cell concentration than infants in the low 50th percentile. In multiple regression analysis, the correlation between birthweight and CD34+ cell concentration was statistically clearly significant. Notably, while an expected correlation between gestational age and nucleated cell concentration was found, there was no association between infant gestational age and CD34+ cell concentration. Conclusion: Haematopoietic progenitor and stem cells may reflect intrauterine growth and have a more central role in foetal development than has been reported earlier.

Comparison of total nucleated cell measurements of UC blood samples using two hematology analyzers

The total nucleated cell (TNC) content of umbilical cord blood (UCB) units currently serves as the most important measure for determining suitability for transplantation. Hence it is important that TNC measurements are performed in an accurate manner. TNC content is evaluated routinely by hematology analyzers (HA) as WBC counts. The objective of the study was to compare TNC content utilizing two different HA, one utilizing an impedance channel and optical channel, and the other using only an optical channel. The HA utilized in this study used two different modes of operation for lysis, regular mode (RM) and extended lysis mode (ELM). Cell-Dyn 3200 (CD3.2) utilizes optical technology for WBC measurements, involving WBC optical count (WOC) and nuclear optical count (NOC), whereas the Cell-Dyn 3700 (CD3.7) utilizes both the impedance (WIC) and optical technology (WOC) for WBC measurements. TNC content was determined with 17 identical samples using CD3.2 in one laboratory and CD3.7 in the other laboratory. Cord blood samples processed to concentrate nucleated cells by either of the laboratories were sent by overnight courier and assays were performed on the same day by both laboratories. For CD3.7, the WOC values were consistently lower than the WIC using the regular mode, but showed no significant differences (P>0.05). The WIC and WOC values were comparable on using the ELM and RM. For CD3.2, WOC values using RM and NOC values using ELM showed no significant differences (P>0.05), even though the WOC measurement was lower than the NOC values for most samples. The best comparison of TNC measurement between the two HA could be achieved by comparing CD3.7-WIC with CD3.2-NOC values. The results were equivalent (P>0.05) and 12 of 17 samples had equal to or less than 10% difference (mean 9.5%). TNC measurements of UCB samples were essentially identical using the WIC channel of the Cell-Dyn 3700 and the NOC channel of the Cell-Dyn 3200.

Method of separation and concentration of fetal nucleated red blood cells in maternal blood and its application to fetal diagnosis.

A fetal diagnostic method that is without risk to the embryo has been long awaited in the field of gene diagnosis. Establishment of non-invasive fetal diagnosis using maternal peripheral blood will greatly contribute to perinatal medical care. The lectin method that we have studied and developed selectively recovers nucleated red blood cells (NRBC) among fetal cells mixed in maternal peripheral blood. Maternal blood, 7 mL, was collected with ethylene diamine tetraacetic acid (EDTA) treatment in each week of gestation and subjected to preliminary concentration by density centrifugation (Histopaque [Histopaque-1077; Sigma Diagnostics, MO, USA], specific gravity: 1.095), and NRBC were separated and collected on slide glasses by the lectin method (soybean agglutinin [SBA]: 50 microg/mL). To investigate selective adhesion of the erythrocyte fraction, the SBA concentration was set to 50 microg/mL, and the cells were labeled with CD11a and CD33 (anti-white blood cell antibodies) and investigated by flowcytometry. Erythrocytes adhered at a high rate (87.0 +/- 9.7%) while the adhesion rates of granulocytes, monocytes, and lymphocytes were low, confirming the usefulness of the method for separation and recovery of the erythrocyte fraction. When recovery of NRBC was investigated using this method, a mean of 6.57 +/- 7.12 cells were recovered from 1 mL of maternal blood (May-Gluüwald-Giemsa stain). The number of recovered NRBC increased slowly with pregnancy, but differences were not significant. To confirm that recovered NRBC were derived from the fetus, NRBC were recovered by the lectin method in four patients suspected of 18 trisomy by echography and analyzed by fluorescence in situ hybridization. Three hybridization signals were detected in NRBC at a high frequency, showing that most cells were derived from the fetus and thus, fetal diagnosis may be possible. Since the procedure of the lectin method we have developed is simple, and high concentration efficiency can be obtained at a low cost, it may be clinically applicable.

Associations among nucleated cell, CD34+ cell and colony-forming cell contents in cord blood units obtained through a standardized banking process.

Nucleated cell content is one of the main components used when evaluating cord blood (CB) units for clinical use. However, other indicators of the haematopoietic potential of a CB unit, such as CD34+ cell and colony-forming cell (CFU-TOT) content, have also been investigated. The aim of this study was to determine whether the CD34+ cell content could be used in selecting CB collections for banking. The collection data, as well as cellular contents of 588 CB collections obtained using a standardized CB banking process, were analysed. Altogether, 526 CB units from the 588 collections accepted for processing were included in international search registries. The volume collected was, as expected, 69 ml (range 28-116 ml). The correlation between total CD34+ cell and CFU-TOT (n = 88) content in the CB collection was higher (r = 0.87) than the correlation between the total nucleated cell and CFU-TOT content (r = 0.69, both P < 0.0001). The correlations of pre- and postvolume reduction values of the total nucleated cell and CD34+ cell numbers were highly significant (r = 0.96, P < 0.0001, both). The total CFU-TOT content of the CB collection correlated significantly with the total CD34+ cell content of the CB unit before cryopreservation (but after volume reduction) (r = 0.89, P < 0.0001). CD34+ cell content predicts the haematopoietic potential of a CB unit better than nucleated cell content. Accordingly, the CD34+ cell content of CB could be used to select CB for banking purposes and for transplantation.

Engraftment capacity of umbilical cord blood cells processed by either whole blood preparation or filtration.

Umbilical cord blood (UCB) preparation needs to be optimized in order to develop more simplified procedures for volume reduction, as well as to reduce the amount of contaminating cells within the final stem cell transplant. We evaluated a novel filter device (StemQuick((TM))E) and compared it with our routine buffy coat (BC) preparation procedure for the enrichment of hematopoietic progenitor cells (HPCs). Two groups of single or pooled UCB units were filtered (each n = 6), or equally divided in two halves and processed by filtration and BC preparation in parallel (n = 10). The engraftment capacity of UCB samples processed by whole blood (WB) preparation was compared with paired samples processed by filtration in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse animal model. Filtration of UCB units in the two groups with a mean volume of 87.8 and 120.7 ml, respectively, and nucleated cell (NC) content of 9.7 and 23.8 x 10(8) resulted in a sufficient mean cell recovery for mononucleated cells ([MNCs] 74.2%-77.5%), CD34(+) cells (76.3%-79.0%), and colony-forming cells (64.1%-86.3%). Moreover, we detected a relevant depletion of the transplants for RBCs (89.2%-90.0%) and platelets ([PLTs] 77.5%-86.1%). In contrast, the mean depletion rate using BC processing proved to be significantly different for PLTs (10%, p = 0.03) and RBCs (39.6%, p < 0.01). The NC composition showed a highly significant increase in MNCs and a decrease in granulocytes after filtration (p < 0.01), compared with a less significant MNC increase in the BC group (p < 0.05). For mice transplanted with WB-derived progenitors, we observed a mean of 15.3% +/- 15.5% of human CD45(+) cells within the BM compared with 19.9% +/- 16.8% for mice transplanted with filter samples (p = 0.03). The mean percentage of human CD34(+) cells was 4.2% +/- 3.1% for WB samples and 4.5% +/- 3.2% for filter samples (p = 0.68). As the data of NOD/SCID mice transplantation demonstrated a significant engraftment capacity of HPCs processed by filtration, no negative effect on the engraftment potential of filtered UCB cells versus non-volume-reduced cells from WB transplants was found. The StemQuick((TM))E filter devices proved to be a useful tool for Good Manufacturing Practices conform enrichment of HPCs and MNCs out of UCB. Filtration enables a quick and standardized preparation of a volume-reduced UCB transplant, including a partial depletion of granulocytes, RBCs, and PLTs without the need for centrifugation. Therefore, it seems very probable that filter-processed UCB transplants will also result in sufficient hematopoietic reconstitution in humans.

Content of stromal precursor cells in heterotopic transplants of bone marrow in CBA mice of various ages.

Efficiency of colony formation of stromal precursor cells in cultured bone marrow transplants from old (24 month) CBA mice implanted to young (2-month-old) mice almost 3-fold surpassed that in cultured transplants implanted to old recipients. The content of nucleated cells in bone marrow transplants from senescence accelerated mice SAMP increased more than 2-fold, if SAMR mice with normal aging rate were used as the recipients instead of SAMP mice. Bone marrow taken from old and young CBA mice endured the same number of transplantations if the recipient mice were of the same age (5 month). It was concluded that stromal tissue considerably changes with age and is under strict control of the body.

Effect of preeclampsia on umbilical cord blood hematopoietic progenitor-stem cells

Objective: The aim of the present study was to determine the influence of preeclampsia on cord blood hematopoietic progenitor-stem cells obtained at delivery because cord blood is increasingly used clinically for stem cell retrieval as an alternative to bone marrow. Study Design: Umbilical cord blood was collected from patients fulfilling the criteria for preeclampsia and from gestational age– and birth weight–matched control subjects at delivery (patient/control subjects ratio, 1:2). Cord blood volume and nucleated cell content were measured, and the number of hematopoietic progenitor-stem cells was determined by means of fluorescence-activated cell sorting with the CD34+ epitope and by means of colony assays with different hematopoietic growth factors. In addition, the expression of adhesion molecules by CD34+ progenitor-stem cells was examined. Results: In pregnancies affected by preeclampsia, volume and nucleated cell and total CD34+ cell contents in the collected cord blood were significantly smaller compared with those of control subjects. Furthermore, there was a trend toward a smaller relative number of CD34+ cells and colony-forming units per nucleated cell in cord blood samples from preeclamptic patients. No difference in the expression of the cell-adhesion molecules leukocyte function–associated antigen 1, very late activation antigen 4, and L-selectin by CD34+ cells could be found. Conclusion: This study shows that preeclampsia affects umbilical cord blood volume and nucleated cell and progenitor-stem cell numbers obtained at birth. (Am J Obstet Gynecol 2001;185:725-9.)