Ayuko Narita

Quality assessment of umbilical cord blood units at the time of transplantation

Total nucleated cell (TNC) count, CD34+ cell count, colony-forming unit-granulocyte–macrophage (CFU-GM) content, and cell viability impact the outcome of umbilical cord blood (UCB) transplantation. Assessments of unit quality have usually been provided by cord blood banks (CBBs), but it is unclear whether pre-freezing tests or pre-transplant release tests performed by CBBs are reproducible. The aim of this study was to compare the UCB characteristics analyzed at the site of infusion of the UCB with those provided by CBBs. Samples were taken from 54 UCB units for assessment of post-thaw characteristics. TNC counts and CD34+ cell contents measured at our hospital before infusion showed good correlations with values assessed in pre-freezing tests (r = 0.900 and 0.943, respectively) and pre-transplant release tests (r = 0.829 and 0.930, respectively). Our data reveal that the TNC counts and CD34+ cell contents determined by pre-freezing and pre-transplant release tests, which are the most important UCB unit selection criteria, accurately reflected the quality of infused UCB units. However, CFU-GM content was poorly correlated (r = 0.560 and 0.606). Correlation of post-thaw cell viabilities measured before infusion and during the pre-transplant release tests was also poor (r = 0.308). We suggest that the TNC count and CD34+ cell content estimated before cryopreservation and in pre-transplant release tests provided by CBBs are reproducible and can assist the transplant physicians in selection of appropriate UCB units.

A simple controlled-rate freezing method without a rate-controlled programmed freezer provides optimal conditions for both large-scale and small-scale cryopreservation of umbilical cord blood cells.

BACKGROUND:  Umbilical cord blood (CB) is being used as a source of alternative HPCs for transplantation with increasing frequency. The goal of CB banks for unrelated transplantation is to provide good quality‐controlled CB units that can be transplanted for HPCs into the largest possible number of patients.

Quality evaluation of umbilical cord blood progenitor cells cryopreserved with a small-scale automated liquid nitrogen system☆

The performance of a small-scale automated cryopreservation and storage system (Mini-BioArchive system) used in the banking of umbilical cord blood (UCB) units was evaluated. After thawing the units, the viability and recovery of cells, as well as the recovery rate of hematopoietic progenitor cells (HPCs) such as CD34+ cells, colony-forming unit-granulocyte-macrophage (CFU-GM), and total CFU were analyzed. Twenty UCB units cryopreserved using the automated system and stored for a median of 34 days were analyzed. Mean CD34+ cell viabilities before freezing were 99.8+/-0.5% and after thawing were 99.8+/-0.4% in the large bag compartments and 99.7+/-0.5% in the small compartments. The mean recovery values for total nucleated cells, CD34+ cells, CFU-GM, and total CFU were 94.8+/-16.0%, 99.3+/-18.6%, 103.9+/-20.6%, and 94.3+/-12.5%, respectively in the large compartments, and 95.8+/-25.9%, 106.8+/-23.9%, 101.3+/-23.3%, and 93.8+/-19.2%, respectively in the small compartments. A small-scale automated cryopreservation and storage system did not impair the clonogenic capacity of UCB HPCs. This cryopreservation system could provide cellular products adequate for UCB banking and HPC transplantation.

Quality of umbilical cord blood cd34+ cells in a double-compartment freezing bag cryopreserved without a rate-controlled programmed freezer

The aim of this study was to evaluate how a simple method of cryopreservation influences the quality of CD34+ cells in umbilical cord blood (UCB). The cells were dispensed into a double-compartment freezing bag, cryopreserved at -85°C without a rate-controlled programmed freezer, and stored in the liquid phase of nitrogen. The viability of the CD34+ cells before freezing and after thawing was assessed by flow cytometry with 7-aminoactinomycin D and by colony-forming assays. Twenty UCB units cryopreserved for a median of 92 days were analyzed. Mean CD34+ cell viabilities before freezing were 99.8% ± 0.4% and after thawing were 99.5% ± 0.8% in large chambers, 99.6% ± 0.5% in small chambers, and 99.4% ± 0.6% in sample tubes. The mean values from colony-forming assays of the viable CD34+ cells before freezing were 30.7 ± 6.8 (colony-forming units-granulocyte-macrophage [CFU-GM] per 100 viable CD34+ cells) and 68.5 ± 14.8 (total CFUs per 100 viable CD34+ cells). The CFU-GM and total CFU values after thawing were, respectively, 32.7 ± 9.0 and 66.0 ± 13.4 in large chambers, 32.4 ± 8.1 and 64.5 ± 16.1 in small chambers, and 30.9 ± 5.4 and 64.7 ± 12.4 in sample tubes. The results of the colony-forming assays before freezing and after thawing were not significantly different. Our findings overall indicated that our simple method for the cryopreservation of UCB cells without a rate-controlled programmed freezer does not impair the clonogenic capacity of UCB progenitor cells. This cryopreservation method could provide cellular products adequate for hematopoietic stem cell transplantation.

Elevation of Serum Acid Sphingomyelinase Activity in Acute Kawasaki Disease.

Kawasaki disease (KD) is an acute systemic vasculitis that affects both small and medium-sized vessels including the coronary arteries in infants and children. Acid sphingomyelinase (ASM) is a lysosomal glycoprotein that hydrolyzes sphingomyelin to ceramide, a lipid, that functions as a second messenger in the regulation of cell functions. ASM activation has been implicated in numerous cellular stress responses and is associated with cellular ASM secretion, either through alternative trafficking of the ASM precursor protein or by means of an unidentified mechanism. Elevation of serum ASM activity has been described in several human diseases, suggesting that patients with diseases involving vascular endothelial cells may exhibit a preferential elevation of serum ASM activity. As acute KD is characterized by systemic vasculitis that could affect vascular endothelial cells, the elevation of serum ASM activity should be considered in these patients. In the present study, serum ASM activity in the sera of 15 patients with acute KD was determined both before and after treatment with infusion of high-dose intravenous immunoglobulin (IVIG), a first-line treatment for acute KD. Serum ASM activity before IVIG was significantly elevated in KD patients when compared to the control group (3.85 ± 1.46 nmol/0.1 ml/6 h vs. 1.15 ± 0.10 nmol/0.1 ml/6 h, p < 0.001), suggesting that ASM activation may be involved in the pathophysiology of this condition. Serum ASM activity before IVIG was significantly correlated with levels of C-reactive protein (p < 0.05). These results suggest the involvement of sphingolipid metabolism in the pathophysiology of KD.