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Background Standardization of the manufacturing and processing of cellular immunotherapy products is necessary to ensure patient safety, establish efficacy, and demonstrate potency. Recognition of the autologous donor as a likely source of microbial contamination of cellular immunotherapy products may improve patient care and reduce expense to the laboratory. Methods Data on 243 immunotherapy products manufactured in the authors' institution between December 1997 and June 2001 were retrospectively reviewed. Also reviewed were the case reports of four patients whose autologous immunotherapy products were contaminated. Results Twenty-five (10%) of the 243 immunotherapy products processed were positive on one or more tests for microbial contamination. In six (24%) of these products, the source of microbial contamination was the autologous donor. In 17 of the remaining 19 products, test results were judged to be false-positive. Discussion The unique processing techniques and stringent controls involved in the manufacture of cellular immunotherapy products may result in changes in the sources of microbial contamination routinely encountered. The identification of the autologous donor as a potential source of microbial contamination of the product may assist the clinician and the laboratory in troubleshooting products with positive results on microbial sterility testing. Also, the number of false-positive results in this study indicates that further research is needed to maximize the specificity of testing while maintaining the present high sensitivity. [ABSTRACT FROM AUTHOR]

Background: There are no effective treatments that slow the progression of neurodegenerative diseases. A major challenge of treatment in neurodegenerative diseases is appropriate delivery of pharmaceuticals into the cerebrospinal fluid (CSF) of affected individuals. Mesenchymal stromal cells (MSCs-either naïve or modified) are a promising therapy in neurodegenerative diseases and may be delivered directly into the CSF where they can reside for months. In this preclinical study, we evaluated the safety of intrathecal autologous MSCs in a rabbit model., Study Design and Methods: Autologous adipose-derived MSCs (or artificial CSF) were delivered intrathecally, either with single or with repeated injections into the foramen magnum of healthy rabbits and monitored for 4 and 12 weeks, respectively., Results: Rabbits tolerated injections well and no definitive MSC-related side effects were observed apart from three rabbits that had delayed death secondary to traumatic foramen magnum puncture. Functional assessments and body weights were equivalent between groups. Gross pathology and histology did not reveal any abnormalities or tumor growth. Complete blood count data were normal and there were no differences in CSF interleukin-6 levels in all groups tested., Conclusion: Our data suggest that intrathecal delivery of autologous MSCs is safe in a rabbit model. Data from this study have supported two successful investigational new drug applications to the Food and Drug Administration, resulting in the initiation of two clinical trials using autologous MSCs in amyotrophic lateral sclerosis and multiple system atrophy., (© 2014 AABB.)

Case Report: A 50-year-old male with T-cell lymphoma presented for autologous peripheral blood stem cell transplantation. After granulocyte-colony-stimulating factor (G-CSF) mobilization, his peripheral blood CD34+ cell count was 166 × 10(6) /L on the day before collection, which predicted a high yield of CD34+ cells in the apheresis product. The first two collections had yields much lower than expected, triggering an investigation and changes to the apheresis collection methods since mobilization appeared adequate from the peripheral CD34+ values., Results: Changes to the apheresis collection variables and instrumentation did not improve the yields in the next three collections. The laboratory investigation demonstrated that there was an interfering substance in the patient's plasma that was causing falsely high peripheral blood CD34+ cell values and that the low CD34+ cell yields in the collections were consistent with the actual peripheral blood CD34+ cell value. Based on this finding and after discussion with the clinical service, the patient then received plerixafor to increase the number of circulating CD34+ cells before Collections 6 and 7. The patient went on to achieve the target CD34+ cell dose and subsequently underwent a successful autologous transplant with full hematopoietic engraftment., Conclusion: This case demonstrates the importance of timely and critical review of laboratory results in the context of the specific patient. This case exemplifies how diligent review of laboratory results and open communication among the various teams can positively affect patient outcomes., (© 2013 American Association of Blood Banks.)

Background and Objectives: The aim of the collaborative study was to evaluate a panel of plasma samples containing different genotypes of parvovirus B19 (B19V) for use in nucleic acid amplification technology (NAT)-based assays., Materials and Methods: The panel of samples [Center for Biologics Evaluation and Research Parvovirus B19 Genotype Panel 1; National Institute for Biological Standards and Control (NIBSC) code number 09/110] comprises four different members, i.e. Member 1, Member 2, Member 3, and Member 4 (M1-M4); these represent genotypes 1, 2, 3a B19V, and a negative plasma control, respectively. Thirty-five laboratories from 13 different countries participated in the study. Participants assayed the panel members concurrently with the 2nd World Health Organization (WHO) International Standard for B19V DNA (NIBSC code 99/802) on four separate occasions., Results: A total of 44 sets of data were returned, 34 from quantitative assays and 10 from qualitative assays. The majority of assays used were in-house and based on real-time PCR. The results showed that all three genotypes were detected consistently by the majority of participants, although a small number of assays detected genotypes 2 and 3 less efficiently, or not at all. Real-time stability studies have indicated that the panel of B19V samples is stable under normal conditions of storage, i.e. ≤-70°C., Conclusions: The four-member panel is intended for use in evaluating the ability of NAT assays to detect different B19V genotypes (M1-M3). Based on the results of the collaborative study, the panel was established as the 1st WHO International Reference Panel for parvovirus B19 genotypes., (© 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.)

With favorable regenerative and immunotolerant profiles, patient-derived human mesenchymal stem cells (hMSCs) are increasingly considered in cell therapy. Derived from bone marrow (BM) and standardized with culture in fetal bovine serum (FBS), translation of hMSC-based approaches is impeded by protracted expansion times, risk of xenogenic response, and exposure to zoonoses. Here, human platelet lysate adherent to good manufacturing practices (GMP-hPL) provided a nonzoonotic adjuvant that enhanced the capacity of BM-hMSC to proliferate. The nurturing benefit of GMP-hPL was generalized to hMSC from adipose tissue evaluated as an alternative to bone marrow. Long-term culture in GMP-hPL maintained the multipotency of hMSC, while protecting against clonal chromosomal instability detected in the FBS milieu. Proteomic dissection identified TGF-β, VEGF, PDGF, FGF, and EGF as highly ranked effectors of hPL activity, revealing a paradigm of healing that underlies platelet lysate adjuvancy. Thus, GMP-adherent human platelet lysate accelerates hMSC proliferation with no chromosomal aberrancy, through an innate repair paradigm.

In order to protect tissue recipients, the Food and Drug Administration drafted Title 21, Section 1271 of the Code of Federal Regulations 1271 (21 CFR 1271) to address infectious disease risk. These regulations apply to tissues but not vascularized organs. Pancreatic islet cells are regulated under 21 CFR 1271. These regulations require qualification of suppliers of critical materials and services with regard to 21 CFR 1271 compliance. As part of supplier qualification, all organ procurement organizations (OPOs) in the United States were sent a questionnaire covering the key components of these regulations. Of the 57 OPOs, 29 (51%) were in compliance based upon survey results. Twelve (21%) were not compliant in one or more areas. All indicated plans to become compliant. The remaining 15 (27%) either failed or refused to complete the survey, some indicating 21 CFR 1271 did not apply to OPOs. Using 2006 data, OPOs compliant with 21 CFR 1271 recovered 50% of the organs procured in the United States. These findings represent a challenge for allogeneic islet cell transplant programs whose raw material must comply with 21 CFR 1271. OPOs should work toward understanding and complying with 21 CFR 1271. Regulatory agencies should work toward enhancing safety of the pancreas supply by facilitating compliance through harmonization of requirements.

Background: In order to harmonize results for the detection and quantification of Plasmodium falciparum DNA by nucleic acid amplification technique (NAT)-based assays, a World Health Organization (WHO) collaborative study was performed, evaluating a series of candidate standard preparations., Methods: Fourteen laboratories from 10 different countries participated in the collaborative study. Four candidate preparations based upon blood samples parasitaemic for P. falciparum were evaluated in the study. Sample AA was lyophilized, whilst samples BB, CC and DD were liquid/frozen preparations. The candidate standards were tested by each laboratory at a range of dilutions in four independent assays, using both qualitative and quantitative NAT-based assays. The results were collated and analysed statistically., Results: Twenty sets of data were returned from the participating laboratories and used to determine the mean P. falciparum DNA content for each sample. The mean log10 "equivalents"/ml were 8.51 for sample AA, 8.45 for sample BB, 8.35 for sample CC, and 5.51 for sample DD. The freeze-dried preparation AA, was examined by accelerated thermal degradation studies and found to be highly stable., Conclusion: On the basis of the collaborative study, the freeze-dried material, AA (NIBSC code No. 04/176) was established as the 1st WHO International Standard for P. falciparum DNA NAT-based assays and has been assigned a potency of 10(9) International Units (IU) per ml. Each vial contains 5 x 10(8) IU, equivalent to 0.5 ml of material after reconstitution.

The common conception of a drug is that of a chemical with defined medicinal effect. However, cells used as drugs remain critical to patient care. Cell therapy's origins began with the realization that complex tissues such as blood can retain function when transplanted to the patient. More complex transplantation followed, culminating with the understanding that transplantation of some tissues such as bone marrow may act medicinally. Administration of cells with an intended therapeutic effect is a hallmark of cellular therapy. While cells have been used as drugs for decades, testing a specific therapeutic effect of cells has begun clinical testing relatively recently. Lessons learned during the establishment of blood banking (including the importance of quality control, process control, sterility, and product tracking) are key components in the assurance of the safety and potency of cell therapy preparations. As more academic medical centers and private companies move toward exploiting the full potential of cells as drugs, needs arise for the development of the infrastructure necessary to support these investigations. Careful consideration of the design of the structure used to manufacture is important in terms of the significant capital outlay involved and the facility's role in achieving regulatory compliance. This development perspective describes the regulatory environment surrounding the infrastructure support for cell therapy and practical aspects for design consideration with particular focus on those activities associated with early clinical trials.

Background: Administration of culture-positive hematopoietic progenitor cells (HPCs) causing adverse events has been a hypothesized yet largely unmeasured risk of the clinical practice of HPC transplantation. To enhance patient safety, the FDA has issued regulations prohibiting the use of culture-positive HPCs. Numerous studies have reported the infusion of culture-positive HPCs; however, the low frequency of adverse events prevents accurate determination of this risk., Study Design and Methods: Product culture results and clinical outcomes from January 1998 through March 2006 representing 7233 HPC collections for 2118 transplants at a single institution were reviewed., Results: A total of 119 units of HPCs (1.6%) intended for 95 patients were culture-positive. Of the 69 patients transplanted with culture-positive HPCs, 5 received products with cultures pending, and 64 received products with the positive culture results known. One of 69 patients had a new positive blood culture 5 days after infusion with the same species as the product. There was not a clinically relevant difference in the rate of infusion-related symptoms reported for patients who received culture-positive products compared to all infusions. The survival of patients who received culture-positive products (n = 69) was not different from all HPC recipients (n = 2046; p = 0.419)., Conclusion: No infusion-related risks of culture-positive HPCs to patient safety were identified. Our data suggest that the decision to use culture-positive HPCs must be made in the context of the global risks associated with transplants such as remobilization, replacement product availability, and the nature of the organism.

Purpose: Autograft absolute lymphocyte count (A-ALC) affects survival after autologous stem cell transplantation (ASCT) in non- Hodgkin lymphoma (NHL). AMD3100, a CXCR4 antagonist, mobilizes CD34+ stem cells in patients with NHL undergoing ASCT. We sought to study the impact of AMD3100 on A-ALC collection in patients with NHL undergoing ASCT., Patients and Methods: The primary endpoint of the study was to assess the association between AMD3100 and A-ALC collection. We compared 7 consecutive patients with NHL mobilized with AMD3100 and granulocyte colony-stimulating factor with 29 control patients with NHL mobilized with granulocyte colony-stimulating factor alone., Results: Higher median A-ALCs were observed in the AMD3100 group compared with the control group (4.16 x 10(9) lymphocytes/kg vs. 0.288 x 10(9) lymphocytes/kg; P < 0.0001). With a median follow-up of 20 months (range, 4-24 months), no relapses were reported in the AMD3100 group compared with 15 of 29 in the control group (P < 0.02)., Conclusion: Our data suggest that AMD3100 affects A-ALC and clinical outcome in patients with NHL undergoing ASCT.

Background: Dendritic cells (DCs) have been used as vaccines in clinical trials of immunotherapy of cancer and other diseases. Nonetheless, progress towards the use of DCs in the clinic has been slow due in part to the absence of standard methods for DC preparation and exposure to disease-associated antigens. Because different ex vivo exposure methods can affect DC phenotype and function differently, we studied whether electroporation-mediated transfection (electrotransfection) of myeloid DCs with in vitro expanded RNA isolated from tumor tissue might be feasible as a standard physical method in the preparation of clinical-grade DC vaccines., Methods: We prepared immature DCs (IDCs) from CD14+ cells isolated from leukapheresis products and extracted total RNA from freshly resected melanoma tissue. We reversely transcribed the RNA while attaching a T7 promoter to the products that we subsequently amplified by PCR. We transcribed the amplified cDNA in vitro and introduced the expanded RNA into IDCs by electroporation followed by DC maturation and cryopreservation. Isolated and expanded mRNA was analyzed for the presence of melanoma-associated tumor antigens gp100, tyrosinase or MART1. To test product safety, we injected five million DCs subcutaneously at three-week intervals for up to four injections into six patients suffering from stage IV malignant melanoma., Results: Three preparations contained all three transcripts, one isolate contained tyrosinase and gp100 and one contained none. Electrotransfection of DCs did not affect viability and phenotype of fresh mature DCs. However, post-thaw viability was lower (69 +/- 12 percent) in comparison to non-electroporated cells (82 +/- 12 percent; p = 0.001). No patient exhibited grade 3 or 4 toxicity upon DC injections., Conclusion: Standardized preparation of viable clinical-grade DCs transfected with tumor-derived and in vitro amplified mRNA is feasible and their administration is safe.

Autograft absolute lymphocyte count (A-ALC) is an independent prognostic factor for survival after autologous peripheral blood hematopoietic stem cell transplantation (APHSCT) for non-Hodgkin's lymphoma (NHL). Factors enhancing A-ALC collections are unknown. We hypothesize that apheresis instrument settings could affect A-ALC. Data from 127 NHL patients collected from 15 January 1999 to 30 July 2004 using a single apheresis instrument (COBE Spectra (SP), Baxter Amicus (AM), and CS3000 Plus (CS)) were analyzed. The primary end point of the study was to assess the correlation between apheresis instrument settings and A-ALC. The secondary end point was to determine the effect of apheresis instrument on survival post-APHSCT. Patients collected using SP achieved higher A-ALC compared to AM (with modified settings) or CS (P<0.05) and demonstrated superior overall (OS) and progression-free survival (PFS) (P<0.03). Multivariate analysis demonstrated A-ALC and not the apheresis instrument as an independent prognostic factor for OS and PFS, cancelling the prognostic effect of the apheresis instruments observed in the univariate analysis. The survival advantage observed by SP was from the higher A-ALC collected compared to AM and CS. These data suggest that apheresis instrument settings should be optimized to collect CD34(+) cells as well as an A-ALC target, with direct impact on survival post-APHSCT., (Bone Marrow Transplantation (2006) 37, 811-817. doi:10.1038/sj.bmt.1705338; published online 13 March 2006.)

Background: We conducted a phase I clinical immunotherapy trial of CML to evaluate the safety of a clinical-grade leukemic DC product standardized for purity and mature phenotype., Methods: We injected autologous DC into patients in late chronic or accelerated phases of CML. The patients received mature CD83+ and bcr-abl+ DC prepared from CD14+ cells. Two cohorts of three patients received four injections each of 3 x 10(6) DC and 15 x 10(6) DC/injection, respectively. The first patient was studied before imatinib mesylate (IM) was available, four patients were treated concurrently with IM therapy and one did not tolerate the IM and was off the drug at the time of DC therapy. IM effects on WBC counts precluded DC preparation in numbers sufficient for further dose escalation. The first patient received DC s.c. and all subsequent patients received DC into a cervical lymph node under ultrasound guidance., Results: DC injections were well tolerated. We observed no clinical responses. T cells drawn later in the course of therapy were more sensitive to stimulation by CML DC in vitro., Discussion: The increase in T-cell sensitivity to CML-specific stimulation that accompanied active immunization by CML DC justifies further clinical studies, possibly with modifications such as an increased frequency and number of DC injections.

Absolute lymphocyte count at day 15 (ALC-15) after autologous peripheral blood hematopoietic stem cell transplantation (APHSCT) is an independent prognostic factor for survival in multiple myeloma (MM); however, factors affecting ALC-15 in MM remain unknown. We hypothesized that the dose of infused peripheral blood autograft lymphocytes (autograft absolute lymphocyte count: A-ALC) impacts ALC-15 recovery. Between 1989 and 2001, 267 consecutive MM patients underwent APHSCT. We set out to determine the correlation between A-ALC and ALC-15 and the utility of A-ALC as a marker for ALC-15 recovery. A-ALC was found to be both a strong predictor for area under curve (AUC=0.93; P=0.0001) and strongly correlated with (r(s)=0.83; P=0.0001) ALC-15 recovery. Higher infused A-ALC was significantly correlated with an ALC-15>/=500/microl. In addition, median post-transplant overall survival (OS) and time to progression (TTP) were longer in patients who received an A-ALC>/=0.5 x 10(9) lymphocytes/kg versus A-ALC <0.5 x 10(9) lymphocytes/kg (58 vs 30 months, P=0.00022; 22 vs 15 months, P<0.00012, respectively). Multivariate analysis demonstrated A-ALC as an independent prognostic indicator for OS and TTP. These results indicate that an infused dose of autograft lymphocytes significantly impacts clinical outcome post-APHSCT in MM.

Absolute lymphocyte count at day 15 (ALC-15) after autologous peripheral blood hematopoietic stem cell transplantation (APHSCT) is an independent prognostic factor for survival in non-Hodgkin's lymphoma (NHL). Factors affecting ALC-15 remain unknown. We hypothesized that dose of infused autograft lymphocytes (A-ALC) directly impacts upon ALC-15. A total of 190 consecutive NHL patients received A-ALC between 1993 and 2001. The primary end point was correlation between A-ALC and ALC-15. A strong correlation was identified (r=0.71). A higher A-ALC was infused into patients achieving an ALC-15 > or =500/microl vs ALC-15 <500/microl (median of 0.68 x 10(9)/kg (0.04-2.21 x 10(9)/kg), vs 0.34 x 10(9)/kg (0.04-1.42 x 10(9)/kg), P<0.0001). The median follow-up for all patients was 36 months (maximum of 109 months). The A-ALC threshold was determined at 0.5 x 10(9)/kg. The median overall survival (OS) and progression-free survival (PFS) times were longer in patients who received an A-ALC >/=0.5 x 10(9)/kg vs A-ALC <0.5 x 10(9)/kg (76 vs 17 months, P<0.0001; 49 vs 10 months, P<0.0001, respectively). Multivariate analysis demonstrated A-ALC to be an independent prognostic indicator for OS and PFS. These data support our hypothesis that ALC-15 and survival are dependent upon the dose of infused A-ALC in NHL.

Background: We evaluated a clinical-grade protocol for the manufacture of mature DC from CD14 + precursors derived from normal donors and patients suffering from CML and stage IV malignant melanoma. We manufactured six products for CML patients and five for melanoma patients and administered them as vaccines in phase I clinical trials., Methods: We isolated CD 14+ cells from apheresis products by immunomagnetic separation and incubated them in X-VIVO 15' medium supplemented with human AB serum, GM-CSF and IL-4 for 7 days, and with additional tumor necrosis factor (TNF)-a, IL-lIf, IL-6 and prostaglandin E2 for 3 days. Some cells were electroporated and transfected with mRNA isolated from melanoma tissue. DC were characterized by flow cytometry for the expression of CD83, CD86 andCD14., Results: CD14+ cells constituted 14.4+/-6.2% (mean + SD) of nucleated cells in apheresis products and 98.3+/- 3.6% of isolated cells. Normal DC and CML DC were 77.4+/-7.3% CD83+ and 93.5+/- 7.0% CD86+. Corresponding values for electroporated DC from melanoma patients were 66.1 + 7.2% and 94.1 + 7.8%. The yield of CD83+ DC from isolated CD14+ cells was 18.1 + 7.2% for normal and CML patients and 9.8 + 3.7% for melanoma patients. DC viability was 92.7 + 5.8%; after cryopreservation and thawing it was 77+/-13.5%., Discussion: Our method yielded viable and mature DC free of bacteria and mycoplasma. This robust and reproducible method provides cells of consistent phenotype and viability. Cryopreservation in single-dose aliquots allows multiple DC vaccine doses to be manufactured from a single apheresis product.

Detection of viral nucleic acids in blood samples from cadavers is often difficult because of inhibition of the reverse transcriptase (RT) or polymerase chain reaction (PCR) steps by substances present in the samples. A robust method for the extraction and detection of hepatitis C virus (HCV) RNA from cadaver blood samples by polymerase chain reaction RT-PCR has been developed on the basis of the Qiagen QIAamp DNA mini kit extraction system (Basel, Switzerland). Twenty of 36 samples tested were positive for HCV RNA. Six of the 16 HCV-antibody- and RNA-negative samples contained inhibitors that were successfully removed by pretreatment of samples with the Qiagen AX matrix before extraction.

We attempted to induce therapeutic immunity against prostate-derived tissues in patients suffering from progressive hormone-refractory metastatic prostate carcinoma. Thirteen patients were treated with two infusions, 1 month apart, of autologous dendritic cells (APC8015) preexposed ex vivo to PA2024, a fusion protein consisting of human granulocyte/macrophage-colony stimulating factor (GM-CSF) and human prostatic acid phosphatase (PAP). The infusions were followed by three s.c. monthly doses of PA2024 without cells. Three groups of patients each received PA2024 at 0.3, 0.6, or 1.0 mg/injection. All Ps were two-sided. Treatment was well tolerated. After infusions of APC8015, patients experienced only mild (grade 1-2) short-lived fever and/or chills, myalgia, pain, and fatigue. One patient developed grade 3 fatigue. Four patients developed mild local reactions to s.c. PA2024. Twelve patients were evaluable for response to treatment. Circulating prostate-specific antigen levels dropped in three patients. T cells, drawn from patients after infusions of APC8015, but not before, could be stimulated in vitro by GM-CSF (P = 0.0004) and PAP (P = 0.0001), demonstrating broken immune tolerance against these two normal proteins. Injections of PA2024 did not influence the reactivity of T cells against PAP and GM-CSF. However, antibodies to GM-CSF and, to a much lesser extent, to PAP reached maximum titers only after two or even three injections of PA2024, showing that directly injected PA2024 was involved in stimulation of humoral immunity. Dendritic cells exposed to antigen ex vivo can induce antigen-specific cellular immunity in prostate cancer patients, warranting further studies of this mode of immunotherapy.