Attitude to extended use and long‐term storage of newborn screening blood spots in Japan

Background: Residual dried blood spots (DBS) remaining after routine newborn screening (NBS) tests are candidate specimens for extended uses such as quality assurance and the development of new technology. A trial of NBS using tandem mass‐spectrometry was launched in 2004 in Japan. The aim of the present study was to analyze the attitudes of the public, patient families, and medical professionals toward the extended use and long‐term storage of residual DBS, and to construct a standardized informational brochure. Methods: A questionnaire was sent to randomly selected members of the public, members of the Japanese Phenylketonuria (PKU) Association, medical staff of a general hospital, staff of a children's hospital, obstetricians and gynecologists, pediatricians and NBS personnel. Associated responses, which were given in a free comment format, were analyzed by text mining. Results: The awareness ratio of NBS was low in the public (26.6%), but despite this, when a brief explanatory note on NBS was provided, 71.7% of them recognized the necessity of NBS. They were less positive than medical professionals and PKU patient families regarding the extended use of DBS for forensic investigation, for the study of health problems, or long‐term storage of residual DBS, regardless of whether these factors affected them personally or not. Among the medical professionals, obstetricians and pediatricians exhibited a higher ratio of negative responses toward the extended use and long‐term storage of DBS than others. Conclusion: The general public is more conservative than PKU patients and their families or medical professionals about the extended use or long‐term storage of residual DBS. Presentation to the public, particularly to couples of childbearing age, of appropriate explanatory information on NBS itself, or the extended use or long‐term storage of residual DBS, is recommended.     

Extraction of DNA from human embryos after long‐term preservation in formalin and Bouin's solutions

The “Kyoto Collection of Human Embryos” at Kyoto University was begun in 1961. Although morphological analyses of samples in the Kyoto Collection have been performed, these embryos have been considered difficult to genetically analyze because they have been preserved in formalin or Bouin's solution for 20–50 years. Owing to the recent advances in molecular biology, it has become possible to extract DNA from long‐term fixed tissues. The purpose of this study was to extract DNA from wet preparations of human embryo samples after long‐term preservation in fixing solution. We optimized the DNA extraction protocol to be suitable for tissues that have been damaged by long‐term fixation, including DNA‐protein crosslinking damage. Diluting Li₂CO₃ with 70% ethanol effectively removed picric acid from samples fixed in Bouin's solution. Additionally, 20.0 mg/mL proteinase was valuable to lyse the long‐term fixed samples. The extracted DNA was checked with PCR amplification using several sets of primers and sequence analysis. The PCR products included at least 295‐ and 838‐bp amplicons. These results show that the extracted DNA is applicable for genetic analyses, and indicate that old embryos in the Kyoto Collection should be made available for future studies. The protocol described in this study can successfully extract DNA from old specimens and, with improvements, should be applicable in research aiming to understand the molecular mechanisms of human congenital anomalies.     

Diagnosis and incidence of Fetal Parvovirus infection in an Autopsy Series: II. DNA Amplification

This study evaluates the practical utility of the polymerase chain reaction (PCR) as a diagnostic method for intrauterine fetal parvovirus infection in cases of hydrops fetalis. Paraffin-embedded, formalin-fixed fetal tissues from cases of hydrops fetalis were assessed for parvovirus B19 by histology and PCR in conjunction with ³²P hybridization. Of 673 fetal and neonatal autopsies performed at Women and Infants' Hospital for the years 1985 through 1990, 32 cases were determined to have hydrops fetalis, of which five were positive for parvovirus infection by both histology and the PCR. PCR was not used in seven (22%) of the 32 hydrops cases because 1 µg of DNA was not available for study. Histology was as sensitive as PCR in detecting parvovirus B19 in fetal autopsy tissues from cases of hydrops fetalis, and could be used reliably in each case to diagnose parvovirus infection. In our hands, histology is as sensitive as PCR and less labor-intensive. We would reserve PCR for cases without inclusions and with a strong suspicion of parvovirus infection, or for fluids in which histological analysis is not available.     

婴儿血中四种疱疹类病毒DNA及特异性IgM抗体检测

目的 建立1种能快速区分4种疱疹类病毒DNA的特异性酶切图谱。方法 在疱疹类病毒高度同源序列DNA多聚酶基因中设计1对通用引物,该引物能同时扩增单纯疱疹病毒Ⅰ型与Ⅱ型(HSVⅠ／Ⅱ)、爱泼斯坦-巴尔病毒(EBV)、人巨细胞病毒(CMV)4种病毒。选择BamHI和BstUI2种内切酶对同一PCR产物进行酶切,建立了能区分4种疱疹类病毒的PCR限制性内切酶片段长度多态性分析(RFLP)法。采用PCR-RFLP和ELISA法,对75例怀疑病毒感染的患儿和38名健康儿童的血标本进行上述4种病毒DNA及IgM抗体检测。结果 4种标准病毒株PCR扩增后产物为510～592bp,经酶切后能区分病毒种类,其最小检出量为0．1fg,与乙型肝炎病毒、新型隐球菌、金黄色葡萄球菌、大肠埃希氏菌和人类基因组：DNA无交叉反应。用该方法检测临床75份血标本,阳性23份,其中CMV、EBV和HSVⅡ感染分别为13、4和5例,HSVⅠ为1例,同时用ELISA法检测该标本,10例阳性,分别为CMV5例、EBV3例和HSVⅡ2例。在38例健康体检儿童的血标本中上述4种病毒DNA及IgM抗体均为阴性。结论 本研究建立的4种疱疹类病毒特异性酶切图谱具有特异敏感,快速准确的特点,能检测到ELISA法不能检测到的疱疹病毒感染。     

Long‐term monitoring of Epstein–Barr virus DNA load and humoral parameter abnormalities in pediatric liver transplant recipients before development of malignancy

Gregorek H, Jankowska I, Dzier?anowska‐Fangrat K, Teisseyre J, Sawicka A, Kasztelewicz B, Paw?owska J. Long‐term monitoring of Epstein–Barr virus DNA load and humoral parameter abnormalities in pediatric liver transplant recipients before development of malignancy.
Pediatr Transplantation 2010: 14:629–635. © 2010 John Wiley & Sons A/S. Abstract: EBV loads and abnormalities of humoral responses were monitored in 51 pediatric liver transplant recipients as a proposed non‐invasive laboratory tool for early detection of changes preceding severe clinical complications. EBV DNA load, concentrations of IgM, IgG, IgA, and monoclonal proteins were determined in each blood sample. EBV DNA was detected in 70.6% of the children, dysgammaglobulinemia of one or more Ig isotype was present in 41.2% of them. MG detected in 43.1% of patients correlated with the presence of EBV DNA (p = 0.003) and was usually preceded by hypergammaglobulinemia. The median maximum EBV load was significantly higher in EBV DNA+/MG+ patients than in EBV DNA+/MG‐ patients (p = 0.04), although there was no correlation between current viral load and appearance of MG. Four of 15 EBV DNA‐negative patients developed MG, preceded by hypergammaglobulinemia in two. Minimization or cessation of immunosuppression in 42 patients, in whom abnormal biomarkers and/or clinical symptoms raised suspicion of disease progression, permitted complete resolution of abnormalities in all but one patient who developed B‐NHL and died. Simultaneous monitoring of protein profiles and EBV DNA load together with thorough physical evaluation of children after LTx is important for early implementation of suitable preemptive therapy.     

Effects and long‐term follow‐up of using umbilical cord blood–derived mesenchymal stromal cells in pediatric patients with severe BK virus‐associated late‐onset hemorrhagic cystitis after unrelated cord blood transplantation

This is a retrospective study to evaluate the efficacy and safety of umbilical cord blood–derived mesenchymal stromal cells (MSCs) for the treatment of pediatric patients with severe BK virus–associated late‐onset hemorrhagic cystitis (BKV‐HC) after unrelated cord blood transplantation (UCBT). Thirteen pediatric patients with severe BKV‐HC from December 2013 to December 2015 were treated with MSCs. The number of MSCs transfused in each session was 1 × 10⁶/kg once a week until the symptoms improved. The median follow‐up time was 1432 (89‐2080) days. The median frequency of MSC infusion was 2 (1‐3), with eight cured cases and five effective cases; the total efficacy rate was 100%. The copy number of urine BKV DNA was 4.43 (0.36‐56.9) ×10⁸/mL before MSC infusion and 2.67 (0‐56.3) ×10⁸/mL after MSC infusion; the difference was not significant (P = .219). There were no significant differences in the overall survival, disease‐free survival, and the incidence of relapse and acute and chronic graft‐versus‐host disease between the MSC infusion group and non‐MSC infusion group. There was also no significant difference in the cytomegalovirus, Epstein‐Barr virus (EBV), and fungal and bacterial infection rates between the two groups. Although umbilical cord blood–derived MSCs do not reduce the number of BKV DNA copies in the urine, the cells have a high efficacy rate and minimal side effects in treating severe BKV‐HC after UCBT among pediatric patients. MSCs do not affect the rates of relapse, long‐term infection, or survival of patients with leukemia.