Post-thaw viable CD34(+) cell count is a valuable predictor of haematopoietic stem cell engraftment in autologous peripheral blood stem cell transplantation.

BACKGROUND AND OBJECTIVES: In peripheral blood stem cell transplantation, the number of CD34(+) cells infused is considered a predictor of haematopoietic engraftment. However, the currently accepted minimal threshold of CD34(+) cells/kg was determined by counting CD34(+) cells before freezing, and the loss of viable CD34(+) cells during freezing, cryopreservation or thawing prior to reinfusion has not been assessed. MATERIALS AND METHODS: Total and viable CD34(+) cells were quantified using single platform flow cytometry and viability dye, 7-amino actinomycin D (7-ADD), at the time of collection and prior to reinfusion in 46 peripheral haematopoietic stem cell grafts from 36 patients. The time to engraftment of neutrophil and platelet was assessed by routine peripheral blood cell counts performed daily. RESULTS: The median number of viable CD34(+) cells harvested was 3.6 x 10(6)/kg (range 0.05-21.2), and the median viability was 98% (range 70-100%) before freezing. After thawing, the median number of viable CD34(+) cells was reduced to 2.2 x 10(6)/kg (range 0.04-14.8) and the median viability was reduced to 71% (range 31-89%). The number of viable CD34(+) cells/kg before freezing and after thawing significantly correlated with engraftment of neutrophils (P < 0.0001 both) and platelets (P = 0.007 and 0.006, respectively). Although the minimum dose for engraftment (2.0 x 10(6) CD34(+) cells/kg) was harvested in 37 of 46 cases (85%), only 25 cases (54%) met this threshold at the time of reinfusion. For platelet engraftment, determination of viable CD34(+) cells prior to reinfusion was more important than enumeration at the time of collection. CONCLUSION: Quantification of post-thaw viable CD34(+) cells better represents the actual composition of the graft and may be a more accurate predictor of haematopoietic engraftment than post-thaw total CD34(+) cell counts, or prefreeze determinations, especially for platelet engraftment. It is necessary to develop good quality controls for freezing and thawing procedures to minimize variance in cell viability.

Influence of air exposure and storage condition on serum ionized magnesium level.

The aim of this study was to evaluate whether reporting serum level of ionized magnesium (iMg) is appropriate when affected by various conditions such as exposure to air and delayed measurement. Serum levels of pH, iMg and normalized magnesium (nMg, normalized or adjusted concentration of iMg to pH 7.40) from 28 inpatients were measured at intervals of 3 min after exposing the samples to air at room temperature. Serum from 30 inpatients was stored in closed tubes at 4 degrees C and -20 degrees C and iMg and nMg levels were measured after 2 days. It was found that serum iMg and nMg concentrations exposed to air were decreased by 0.0023 mmol/l and 0.0001 mmol/l per minute, respectively. nMg did not display any significant changes compared with iMg at 0 min, whereas iMg showed significant changes, which exceeded between-day precision. For the stored serum, only iMg of serum at -20 degrees C showed no statistically significant changes (p = 0.169). It is concluded that to report the result as iMg, the sample should be kept anaerobically, and if exposed to air, the result should be reported as nMg. For storage, iMg of serum kept anaerobically at -20 degrees C is reliable.

Changes in enzyme activity and expression of DHFR of Toxoplasma gondii by antifolates.

The responses to antifolates of Toxoplasma gondii were investigated by measuring the dihydrofolate reductase (DHFR) activity, quantity of DHFR mRNA, and single-strand conformational polymorphism (SSCP) pattern. Pyrimethamine (PYM) and methotrexate (MTX) were tested as antifolates. When T. gondii was treated with PYM, the viability was decreased by the increasing concentration of PYM, DHFR activity tended to increase as the passage proceeded, and the quantity of mRNA expressed was also increased according to passages. The viability of T. gondii was decreased by the increasing concentration of MTX, but it was maintained over 40% up to 100 microM MTX. DHFR activity was 77.4% in the 1st passage (1 microM). 82.2% in the 4th passage (10 microM), and 141.3% in the 7th passage (100 microM). But no changes were detected in SSCP pattern of T. gondii exposed to PYM and MTX, both. These results suggested that the response of T. gondii to PYM was regulated by transcriptional level and that, in MTX, the viability of T. gondii was derived from increasing DHFR activity.

Analysis of antigenic domain of GST fused major surface protein (p30) fragments of Toxoplasma gondii.

Antigenic domain of major surface protein (p30) of Toxoplasma gondii was analyzed after polymerase chain reaction (PCR) of its gene fragments. Hydrophilic or hydrophobic moiety of amino acid sequences were expressed as glutathione S-transferase (GST) fusion proteins. Fragments of p30 gene were as follows: T37, total p30 open reading frame (ORF); S28, total ORF excluding N-terminal signal sequence and C-terminal hydrophobic sequence: A19, N-terminal 2/3 parts of S28; P19, C-terminal 2/3 of S28; X9, N-terminal 1/3 part of S28; Y10, middle 1/3 of S28; and Z9, C-terminal 1/3 of S28, respectively. Primer of each fragment was synthesized to include clamp sequence of EcoR I restriction site. PCR amplified DNA was inserted into GST (26 kDa) expression vector, pGEX-4T-1 to transform into Escherichia coli (JM105 strain). GST fusion proteins were expressed with IPTG induction as 63, 54, 45, 45, 35, 36, and 35 kDa proteins measured by SDS-PAGE. Each fusion protein was confirmed with GST detection kit. Western blot analysis with the serum of a toxoplasmosis patient revealed antigenicity in proteins expressed by T37, S28, and A19 but not those by P18, X9, Y10, and Z9. Antigenicity of p30 seems to be located either in N-terminal 1/3 part in the presence of middle 1/3 part or in the oligopeptides between margins of the first and second 1/3 parts.

Lymphadenitis in experimental murine toxoplasmosis induced by intramuscular injection of tachyzoites.

When tachyzoites (RH strain) of Toxoplasma gondii are injected intramuscularly, experimental mice survive up to 7 days, 1-2 days longer than those infected intraperitoneally. We observed sequential histopathological changes in inguinal lymph nodes after intramuscular injection of tachyzoites to thighs of specific pathogen free (SPF) mice. Initial findings on 1 or 3 days after the injection were reactive germinal centers, distended sinuses and epithelioid cell clusters in cortical and paracortical regions. Later on 5 days after the injection, however, effacement of nodal structure with depletion of cells and focal necrosis were observed. Necrotizing lymphadenitis in the experimental murine toxoplasmosis suggests the causal relation between T. gondii infection and the human disease.