We enrolled 150 patients in a prospective multicenter study of children with acute myeloid leukemia undergoing hematopoietic stem cell transplantation (HSCT) to compare the detection of measurable residual disease (MRD) by a "difference from normal" flow cytometry (ΔN) approach with assessment of Wilms tumor 1 (WT1) gene expression without access to the diagnostic specimen. Prospective analysis of the specimens using this approach showed that 23% of patients screened for HSCT had detectable residual disease by ΔN (.04% to 53%). Of those patients who proceeded to transplant as being in morphologic remission, 10 had detectable disease (.04% to 14%) by ΔN. The disease-free survival of this group was 10% (0 to 35%) compared with 55% (46% to 64%, P < .001) for those without disease. The ΔN assay was validated using the post-HSCT specimen by sorting abnormal or suspicious cells to confirm recipient or donor origin by chimerism studies. All 15 patients who had confirmation of tumor detection relapsed, whereas the 2 patients with suspicious phenotype cells lacking this confirmation did not. The phenotype of the relapse specimen was then used retrospectively to assess the pre-HSCT specimen, allowing identification of additional samples with low levels of MRD involvement that were previously undetected. Quantitative assessment of WT1 gene expression was not predictive of relapse or other outcomes in either pre- or post-transplant specimens. MRD detected by ΔN was highly specific, but did not identify most relapsing patients. The application of the assay was limited by poor quality among one-third of the specimens and lack of a diagnostic phenotype for comparison.

Background: In addition to ⁵¹chromium release assay, flow cytometric methods have been described to assess in vitro cell‐mediated cytotoxicity. In this report, we describe a new flow cytometric approach for determination of in vitro cell‐mediated cytotoxicity utilizing three‐color flow cytometric assay.Methods: This method is based on monoclonal antibody staining of either effector or target cells to evaluate cytotoxicity with increased accuracy by utilizing fluorospheres for calibration. The basic strategy involves labeling effector or target cells with a specific fluorescent‐conjugated monoclonal antibody, in addition to staining with annexinV‐FITC and propidium iodide to identify apoptotic/dead cells. The effector and target cell populations as well as conjugates were clearly and easily identified by this approach.Results: We obtained significant correlation between cytotoxicity calculated by this technique and ⁵¹chromium release assay results. The integration of fluorospheres allowed us to determine the absolute number of events reflective of the cumulative cell death rather than a cross‐sectional, percentage‐based cytotoxicity assessment in the target cell population at the time of analysis.Conclusions: This method provides additional advantages to other methods and enables the study of target cell fate in more detail, as well as providing a potential contribution to understanding the mechanisms of cell elimination. Cytometry Part A 56A:53–60, 2003.

A 13 year old girl with haemoglobin Sβ⁺thalassaemia developed simultaneous aplastic crisis and encephalopathy associated with parvovirus B19 (PB19) infection. Brain magnetic resonance imaging findings were consistent with central nervous system (CNS) vasculitis and her symptoms resolved with steroid therapy. Thus, PB19 induced CNS hypersensitivity vasculitis must be considered in the differential diagnosis of encephalopathy.

Clostridium difficile is a well-known cause of hospital-acquired infection such as antibiotic associated diarrhoea or pseudomembranous colitis. Extraintestinal infections caused by this pathogen are described rarely. A case of post-traumatic wound infection caused by C. difficile in an immunocompetent, young and otherwise healthy trauma patient is reported. A 31-year-old female, a car accident victim, was admitted to hospital because of polytrauma. After open reduction and internal fixation of a supracondylar femoral fracture by means of the dynamic condylar screw (DCS) system, a purulent fistula occurred. Microbiological examination of the pus revealed C. difficile as the single aetiological factor of this infection. Empirical antibiotic treatment with cefazoline and metronidazole had been administered right after the surgery, but was found to be ineffective. The strain isolated from the patient was sensitive to most antimicrobials except for clindamycin, and amoxicillin/clavulanic acid was chosen for the guided therapy. Such treatment combined with the removal of the DCS system produced a desirable effect.