Real-time monitoring of non-viable airborne particles correlates with airborne colonies and represents an acceptable surrogate for daily assessment of cell-processing cleanroom performance.

BACKGROUND AIMS: Airborne particulate monitoring is mandated as a component of good manufacturing practice. We present a procedure developed to monitor and interpret airborne particulates in an International Organization for Standardization (ISO) class 7 cleanroom used for the cell processing of Section 351 and Section 361 products. METHODS: We collected paired viable and non-viable airborne particle data over a period of 1 year in locations chosen to provide a range of air quality. We used receiver operator characteristic (ROC) analysis to determine empirically the relationship between non-viable and viable airborne particle counts. RESULTS: Viable and non-viable particles were well-correlated (r(2) = 0.78), with outlier observations at the low end of the scale (non-viable particles without detectable airborne colonies). ROC analysis predicted viable counts ≥ 0.5/feet(3) (a limit set by the United States Pharmacopeia) at an action limit of ≥ 32 000 particles (≥ 0.5 µ)/feet(3), with 95.6% sensitivity and 50% specificity. This limit was exceeded 2.6 times during 18 months of retrospective daily cleanroom data (an expected false alarm rate of 1.3 times/year). After implementing this action limit, we were alerted in real time to an air-handling failure undetected by our hospital facilities management. CONCLUSIONS: A rational action limit for non-viable particles was determined based on the correlation with airborne colonies. Reaching or exceeding the action limit of 32 000 non-viable particles/feet(3) triggers suspension of cleanroom cell-processing activities, deep cleaning, investigation of air handling, and a deviation management process. Our full procedure for particle monitoring is available as an online supplement.

Clinical implementation of a procedure to prepare bone marrow cells from cadaveric vertebral bodies.

AIMS: Vertebral bone marrow is a rich and easily accessible source of hematopoietic and mesenchymal stem cells that has been used to promote chimerism and transplantation tolerance in connection with cadaveric organ transplantation. The purpose of this study is to provide a detailed account of the procedure used to prepare the first five vertebral bone marrow products for infusion in conjunction with the first hand/hand-forelimb transplants performed at the University of Pittsburgh (PA, USA). MATERIALS & METHODS: The cell separation and release testing were performed at the University of Pittsburgh Cancer Institute's Hematopoietic Stem Cell Laboratory, a Good Manufacturing Practice-compliant facility accredited for clinical cell processing by the Foundation for Accreditation of Cellular Therapy (FACT) and for clinical flow cytometry by the College of American Pathologists (CAP).

Alteration of the fragile histidine triad gene in intrahepatic cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma is the second most common intrahepatic neoplasm, accounting for 10-30% of primary liver cancers. Since little is known about the development of this cancer, we searched for alterations to the fragile histidine triad (FHIT) gene, a putative tumour suppressor gene on chromosome 3p14.2. In addition, we investigated oncogenic mutations in beta-catenin, which lead to an activated Wnt signalling pathway and microsatellite instability as a consequence of mismatch repair deficiency. METHODS AND RESULTS: Loss of heterozygosity at the FHIT/FRA3B locus was detected in two out of ten informative cases (20%) using the marker D3S1300 and in one out of seven informative cases (14%) by marker D3S1234. Furthermore, immunohistochemistry revealed loss of Fhit expression in most of the 19 intrahepatic cholangiocarcinomas analysed, although to varying degrees. Oncogenic mutations were excluded in exon 3 of the beta-catenin gene by restriction enzyme digest and DNA sequence analysis. Microsatellite instability could not be detected in the tumour specimens tested using a validated marker panel. In two out of nine informative cases (22%), loss of heterozygosity was displayed close to the adenomatous polyposis coli (APC) gene. CONCLUSIONS: Alterations to FHIT/FRA3B were initially detected by allelic losses of genomic DNA in intervening sequences of this tumour suppressor gene. Immunohistochemistry extended this initial observation by demonstrating that seven of the 19 intrahepatic cholangiocarcinomas had entirely lost expression of FHIT. Loss of Fhit protein in only a subpopulation of tumour cells due to oligoclonality may explain the varying portions of negative staining observed in the other tumour samples. Microsatellite instability did not appear to contribute to the development of intrahepatic cholangiocarcinoma in the cohort of tumours we analysed. Furthermore, the Wnt pathway may be constitutively turned on by inactivation of the APC gene due to deletion of genomic DNA but not by oncogenic mutations within exon 3 of the beta-catenin gene.