RESUMO
BACKGROUND: Although widely used, commercially available automated culture methods are not US Food and Drug Administration-approved for sterility testing of cell-therapy products. For cell-therapy products regulated under Section 351 of the Public Health Service Act, sterility testing must be performed by the methods described in 21 CFR 610.12 and USP <71> (CFR/USP method), or by methods demonstrated to be equivalent. METHODS: Two automated methods, BacT/Alert (BTA; bioMerieux) and Bactec (Becton Dickinson), were compared with the CFR/USP method. Representative mononuclear cell (MNC) products were formulated using six different product media. MNC product aliquots containing 10-50 x 10(6) cells in a 0.5 mL volume were seeded with organisms, and cultured for 14 days in aerobic and anaerobic bottles of each system. Ten different organisms at target concentrations of 10 and 50 colony-forming units (CFU) per bottle were tested. RESULTS: Positives were detected in a mean (range) of 72% (7-100%) of cultures for CFR/USP, 82% (0-100%) for BTA, and 93% (57-100%) for Bactec. For nine of the 10 organisms tested, overall detection rates for BTA and Bactec were equivalent to or higher than CFR/USP. Of the six product media tested, detection of organisms was impaired only by the medium containing multiple antibiotics: this occurred in all three systems. Both BTA and Bactec had shorter times to detection than the CFR/USP method, with overall means (ranges) of 87 (24-264) h for CFR/USP, 24 (12-54) h for BTA, and 33 (12-80) h for Bactec. Detection occurred consistently within 7 days for both BTA and Bactec, but not for CFR/USP. DISCUSSION: Both BTA and Bactec are superior to the CFR/USP method for overall detection and time to detection of organisms in MNC products suspended in commonly used media. These data support general use of either BTA or Bactec for sterility testing of a variety of cell-therapy products, and suggest that a 7-day culture period is sufficient to detect clinically relevant organisms. These results confirm the need for bacteriostasis and fungistasis testing of antibiotic-containing products, even when antibiotic-binding substances are used.
Assuntos
Automação/métodos , Técnicas de Cultura de Células/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Contaminação de Medicamentos/prevenção & controle , Esterilização/métodos , Antibacterianos , Automação/instrumentação , Automação/normas , Bactérias Aeróbias/efeitos dos fármacos , Bactérias Aeróbias/crescimento & desenvolvimento , Bactérias Anaeróbias/efeitos dos fármacos , Bactérias Anaeróbias/crescimento & desenvolvimento , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/normas , Terapia Baseada em Transplante de Células e Tecidos/instrumentação , Terapia Baseada em Transplante de Células e Tecidos/normas , Células Cultivadas , Contagem de Colônia Microbiana , Meios de Cultura/farmacologia , Fidelidade a Diretrizes , Humanos , Laboratórios/normas , Leucócitos Mononucleares/fisiologia , Esterilização/instrumentação , Esterilização/normas , Fatores de TempoRESUMO
BACKGROUND: Container integrity is critical for maintaining sterility of cryopreserved cellular therapy products. We investigated a series of catastrophic bag failures, first noticed in early 2001. METHODS: Process records were reviewed for all PBPC and lymphocyte products cryopreserved in bags from January 2000 through April 2002. Patient charts were also reviewed. RESULTS: One thousand two hundred and four bags were removed from storage for infusion to 261 patients. All products had been cryopreserved in Cryocyte poly(ethylene co-vinyl acetate) (EVA) bags in either 10% DMSO or 5% DMSO and 6% pentastarch. Product volumes were 25-75 mL, and bags were stored with overwrap bags in a liquid nitrogen tank. From January 2000 to April 2001, failure occurred in 10 of 599 (1.7%) bags. From May 2001 to April 2002, 58 of 605 (9.6%) bags failed, typically with extensive fractures that were visible before thaw. Of the 58 that failed, 24 were salvaged by aseptic methods and infused to patients under antibiotic coverage; 10 of those 24 (42%) had positive bacterial cultures. Bag failures were not related to product type, cryoprotectant solution, liquid versus vapor storage, or freezer location. Failures were linked to use of four Cryocyte bag lots manufactured in 2000 and 2001. After replacing these lots with a 1999 Cryocyte lot and with KryoSafe polyfluoroethylene polyfluoropropylene (FEP) bags, no more failures occurred in 75 and 102 bags, respectively, thawed through April 2002. DISCUSSION: High rates of bag failure were associated with four Cryocyte bag lots. No serious adverse patient effects occurred, but bag failures led to microbial contamination, increased product preparation time, increased antibiotic use, and increased resource expenditure to replace products.
Assuntos
Criopreservação/instrumentação , Criopreservação/métodos , Células-Tronco/metabolismo , Adolescente , Adulto , Idoso , Antígenos CD34/metabolismo , Assepsia/instrumentação , Assepsia/métodos , Bacillus/isolamento & purificação , Criança , Corynebacterium/isolamento & purificação , Criopreservação/estatística & dados numéricos , Contaminação de Equipamentos/economia , Contaminação de Equipamentos/estatística & dados numéricos , Falha de Equipamento/estatística & dados numéricos , Humanos , Infusões Intravenosas , Pessoa de Meia-Idade , Transplante de Células-Tronco de Sangue Periférico/métodos , Transplante de Células-Tronco de Sangue Periférico/estatística & dados numéricos , Plásticos/metabolismo , Plásticos/uso terapêutico , Staphylococcus/isolamento & purificação , Células-Tronco/microbiologia , Preservação de Tecido/instrumentação , Preservação de Tecido/métodos , Preservação de Tecido/estatística & dados numéricos , Coleta de Tecidos e Órgãos/efeitos adversos , Coleta de Tecidos e Órgãos/métodos , Coleta de Tecidos e Órgãos/estatística & dados numéricosRESUMO
Choriocarcinoma has been reported in association with endometrial carcinoma and as a metaplastic change in multiple carcinomas, including liver, urinary bladder, lung, and the gastrointestinal tract. We report choriocarcinoma in conjunction with a carcinosarcoma (also called malignant müllerian mixed tumor) in a 71-year-old woman whose hysterectomy specimen revealed two polypoid lesions of the endometrium, one arising from the anterior endometrium and one arising from the posterior endometrium. Histologic examination revealed three histologic patterns. The anterior endometrial lesion showed a FIGO grade 2 endometrioid endometrial adenocarcinoma. The posterior endometrial lesion showed a carcinosarcoma composed of a high-grade adenocarcinoma and scant homologous stromal sarcoma. In addition, a choriocarcinoma was identified intermixed with the adenocarcinoma. The syncytiocytotrophoblasts and cytotrophoblasts stained strongly with 0 human chorionic gonadotropin (beta-hCG) and human placental lactogen (hPL). The patient's beta-hCG levels on postoperative days 14, 27, and 42 were 283, 32, and 7 mIU/mL, respectively. This unusual case suggests the importance of identifying the choriocarcinomatous component, since the serum beta-hCG can serve as a marker of tumor recurrence postoperatively.
Assuntos
Carcinoma Endometrioide/patologia , Carcinossarcoma/patologia , Coriocarcinoma/complicações , Coriocarcinoma/patologia , Neoplasias do Endométrio/patologia , Tumor Mulleriano Misto/patologia , Neoplasias Ovarianas/patologia , Neoplasias Uterinas/complicações , Neoplasias Uterinas/patologia , Idoso , Biomarcadores Tumorais/isolamento & purificação , Carcinoma Endometrioide/complicações , Carcinossarcoma/complicações , Coriocarcinoma/cirurgia , Gonadotropina Coriônica Humana Subunidade beta/sangue , Neoplasias do Endométrio/complicações , Feminino , Humanos , Histerectomia , Tumor Mulleriano Misto/complicações , Neoplasias Ovarianas/complicações , Resultado do Tratamento , Neoplasias Uterinas/cirurgiaRESUMO
INTRODUCTION: Measurement of hemoglobin A1c (HbA1c) is used as an objective measure of long-term blood glucose control in diabetic patients. Recent improvements in automation combined with new recommendations for precision and accuracy have caused us to reevaluate our methods for measuring HbA1c. OBJECTIVE: We evaluated a newly automated high-performance liquid chromatography (HPLC) instrument for measurement of HbA1c (Tosoh A1c 2.2 Plus Glycohemoglobin Analyzer, Tosoh Medics, Foster City, Calif) and compared the results obtained by HPLC to those obtained with an immunoassay (Hitachi 911, Boehringer Mannheim Corporation, Indianapolis, Ind). RESULTS: The Tosoh analyzer was found to be linear in a range of 5.3% to 17% and had a throughput of 20 samples per hour. HbA1c results for 102 patient samples by the 2 techniques showed good correlation, with a slope of 0.87 and an intercept at 1.27% +/- 0.15%. Both the total and within-run coefficients of variation were consistently lower for the HPLC method compared with the immunoassay method. The HPLC method produces a chromatogram that shows the different hemoglobin fractions, allowing identification of abnormal hemoglobin variants. In heterozygous individuals, HbA1c measurements are made with no interference from the hemoglobin variant. In the case of homozygous or doubly heterozygous hemoglobin variants, the Tosoh HPLC identifies the hemoglobin variants as such and correctly does not report a HbA1c value in the presence of a markedly decreased amount of hemoglobin A. CONCLUSIONS: The Tosoh HPLC provides adequate throughput and improved precision, and the method is traceable to the Diabetes Control and Complications Trial.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Hemoglobinas Glicadas/análise , Diabetes Mellitus/sangue , Estudos de Avaliação como Assunto , Hemoglobinas Anormais/análise , Humanos , Imunoensaio , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Although approximately 15.7 million Americans have diabetes mellitus, with the vast majority having type 2 diabetes, it is estimated that as many as 5.4 million are undiagnosed. The present case illustrates that undiagnosed diabetes can be a factor in otherwise unexplained deaths. A 39-year-old white male with no significant past medical history other than alcohol abuse was found deceased at his residence. The manner of death appeared to be natural, but no anatomic cause was found. Toxicological analysis revealed a blood ethanol level of 0.02 g/dL and was negative for drugs of abuse. Analysis of the vitreous fluid revealed a glucose level of 502 mg/dL. The blood glucose level was 499 mg/dL, and the hemoglobin A1c (HbA1c) level was 10.6%. Only trace urine ketones were detected, suggesting that the death was the result of hyperglycemic hyperosmolar non-ketosis (HHNK) from unsuspected diabetes. The postmortem HbA1c value serves as a definitive indicator of prolonged hyperglycemia. In order to aid the interpretation of the clinical data, this case is discussed in conjunction with a similar case of a known diabetic patient.