A flow cytometric technique using thiazole orange to detect platelet engraftment following pediatric stem-cell transplants.

BACKGROUND: Thiazole orange (TO) is a nucleic-acid-specific dye that enters cells without pretreatment. When it binds to either RNA or DNA, there is an increase in fluorescence emission. This property has been utilized to measure the amount of newly released platelets using flow cytometry. These newly released platelets differ from more mature platelets because they still contain residual amounts of RNA, and have become known as reticulated platelets. METHODS: Peripheral blood samples were collected at least 48 h following platelet infusion. For validation, manual reticulocyte counts obtained in the laboratory were compared with results obtained using TO and flow cytometry. Following validation, experiments using platelet-rich plasma were performed to evaluate the presence of reticulated platelets in the sample. RESULTS: Validation experiments comparing the manual and flow cytometric reticulocyte counts gave a strong relationship between the two values (r(2) = 0.92). Reticulated platelet studies performed on platelet-rich plasma samples yielded the following results. Patients who did not engraft within 4 days were significantly different from patients who did engraft within 4 days, idiopathic thrombocytoperic purpura (ITP) patients, and donor platelet segments (all P < 0.0008). Patients who engrafted within 4 days, ITP patients, and donor platelet segments were all statistically similar (all P > 0.08). DISCUSSION: The statistical difference between patients who did engraft within 4 days and those who did not suggests that this method could have an important clinical impact in determining those patients who are still in need of platelet support. However, great care must be taken when performing and analyzing the results.

Analysis of parameters affecting engraftment in children undergoing autologous peripheral blood stem cell transplants.

Eighty-three pediatric patients underwent autologous peripheral blood stem cell transplants at a single institution and were included in a study evaluating the correlations between five engraftment parameters and the time to both neutrophil and platelet recovery. The parameters included: the number of nucleated cells per kg (TNC/kg), the absolute CD34+ cell content per kg (CD34+/kg), the number of mononuclear cells per kg (MNC/kg), the number of BFU-E/kg, and the number of CFU-GM/kg. A two-tailed Mann-Whitney test (alpha = 0.05) was used to determine if there were significant differences between patients with neuroblastoma (n = 45) and patients with other diagnoses (n = 38). No statistically significant differences existed between neuroblastoma patients and patients with other diagnoses. Therefore, the two groups of patients were pooled together. Data were analyzed using both a univariate and multivariate correlation method and Student's t-test (alpha = 0.05). Two statistically significant logarithmic relationships were found. The first relationship was between MNC/kg and time to ANC reconstitution (P = 0.05). The second relationship was between CFU-GM/kg and time to platelet recovery (P = 0.01). Based on the statistical data, we conclude that there is no correlation between nucleated cell dose, CD34+ cell dose, and BFU-E content with either neutrophil or platelet recovery. Accordingly, in this study MNC cell dose per kilogram was the most important parameter predicting the length of time between graft infusion and neutrophil recovery while CFU-GM content per kilogram was the most important parameter predicting the length of time until platelet recovery.

Analysis of parameters affecting engraftment in children undergoing allogeneic BM transplants.

BACKGROUND: Animal studies performed on mice have shown that the number of cells infused following ablative regimens affected the speed and quality of engraftment. Similar studies on humans have resulted in contradicting results. We present our experience assessing multiple parameters. METHODS: Fifty-eight pediatric patients underwent allogeneic BM transplants at a single institution and were included in a study evaluating the correlation between five engraftment parameters and the time to either neutrophil, or platelet recovery. The parameters included: the number of total nucleated cells per kg (TNC/kg), the absolute CD34(+) cell content per kg (CD34(+)/kg), the number of mononucleated cells per kilogram (MNC/kg), the number of NFU-E/kg and the number of colony-forming units-granulocyte-macrophage (CFU-GM)/kg. Data were analyzed using both multivariate and univariate correlation method and a Student's t-test. RESULTS: Three satistically-significant logarithmic relationships were found. The first relationship was between TNC/kg and time to ANC reconstitution (p=0.01). The second and third relationship correlated the CD34(+) cell dose with both ANC and platelet recovery. DISCUSSION: Based on the statistical data, we conclude that there is no correlation between MNC dose, CFU-GM/kg and BFU-E/kg content, with either neutrophil or platelet recovery. However, TNC and CD34(+) cell dose per kilogram are the most important parameters predicting the length of time between graft infusion and neutrophil recovery, while CD34(+)/kg is the most important parameter predicting the length of time until platelet recovery. We recommend the use of CD34(+) cell dose as the most reliable parameter to determine the size of the graft in pediatric hematopoietic stem cell recipients.

The frequency of human papillomavirus detection in postmenopausal women on hormone replacement therapy.

Postmenopausal women enrolled in the Iowa portion of the postmenopausal estrogen/progestin interventions randomized clinical trial (n = 105) during 1989-1991 were studied for (i) the prevalence of human papillomavirus (HPV) in this older age population (ages 45-64), and (ii) the association between hormone replacement therapies (HRTs) and changes in detection of HPV over a 2-year time period. HPV is causative in most cervical and some other genital cancers and in the presence of steroid hormones has been shown to increase neoplastic transformation by HPV in vitro. Using PCR to detect HPV DNA, the overall frequency of the virus regardless of time period was 50.3% (n = 53) with a baseline (BL) frequency of 38.1% and the second year follow-up (FU) of 22.9%. The oncogenic types HPV-16 (75.5%) and HPV-31 (20.8%) were the most commonly reported. All those with persistently detected infection (10.5%), defined as HPV+ at both BL and FU, were identified with HPV-16 or -18. Between these two time periods there were no significant differences in HPV frequency between the placebo and combined HRT groups (BL-/FU+, 21% vs 18%; BL+/FU-, 71% vs 80%). While the study is based on a small sample, the findings suggest that short-term use of HRTs is not associated with an increased risk of HPV detection, but assessment of effects from long-term use is needed. The data also indicate that the frequency of HPV found in older women is higher than previously suspected but that short-term changes in HPV detected in this age group are unrelated to the development of precancerous cervical lesions.