IRX-2, a novel immunotherapeutic, protects human T cells from tumor-induced cell death.

IRX-2 is a cytokine-based biologic agent that has the potential to enhance antitumor immune responses. We investigated whether IRX-2 can protect T cells from tumor-induced apoptosis. Tumor-derived microvesicles (MV) expressing FasL were purified from supernatants of tumor cells and incubated with activated CD8(+) T cells. MV induced significant CD8(+) T-cell apoptosis, as evidenced by Annexin binding (64.4+/-6.4%), caspase activation (58.1+/-7.6%), a loss of mitochondrial membrane potential (82.9+/-3.9%) and DNA fragmentation. T-cell pretreatment with IRX-2 prevented apoptosis. IRX-2-mediated cytoprotection was dose and time dependent and was comparable to effects of IL-2, IL-7 or IL-15. IRX-2 prevented MV-induced downregulation of JAK3 and TCRzeta chain and induced STAT5 activation in T cells. IRX-2 prevented MV-induced Bax and Bim upregulation (P<0.005-0.05), prevented cytochrome c release and Bid cleavage, and concurrently restored the expression of Bcl-2, Bcl-xL, FLIP and Mcl-1 (P<0.005-0.01) in T cells. In addition, IRX-2 reversed MV-induced inhibition of the PI3K/Akt pathway. An Akt inhibitor (Akti-1/2) abrogated protective effects of IRX-2, suggesting that Akt is a downstream target of IRX-2 signaling. Thus, ex vivo pretreatment of CD8(+) T cells with IRX-2 provided potent protection from tumor-induced apoptosis. IRX-2 application to future cancer biotherapies could improve their effectiveness by bolstering T-cell resistance to tumor-induced immunosuppression.

Retention of water-borne bacteria by membrane filters. Part III: Bacterial challenge tests on 0.1 micron rated filters.

Clear performance differences were observed between different 0.1 micron rated filters in terms of their microbial removal efficiency when challenged with naturally occurring waterborne bacteria from a water source. Penetration occurred with three 0.1 micron rated "sterilizing grade" filter types tested, from three different filter manufacturers, that did not have a specific high titer reduction claim for Acholeplasma laidlawii. Bacteria shown to penetrate these 0.1 micron rated filters were quite similar to those recovered downstream of 0.2.0.22 micron rated filters (described in Part II). All of the isolates identified via FAME analyses were common environmental or ubiquitous organisms, and some, such as Acidovorax sp. and Hydrogenophaga pseudoflava, have also been isolated from pharmaceutical water systems. In contrast, four different 0.1 micron rated "sterilizing grade" filter types from two different manufacturers, which had been qualified with both B. diminuta and A. laidlawii, consistently produced sterile effluents under similar test conditions. This study thus highlights the need for an industry or regulatory standard method of defining the microbial removal performance of 0.1 micron rated filters, and supports the use of functionally qualified 0.1 micron rated filters as sterilizing grade filters in pharmaceutical operations for enhanced sterility assurance.

Retention of water-borne bacteria by membrane filters. Part II: Scanning electron microscopy (SEM) and fatty acid methyl ester (FAME) characterization of bacterial species recovered downstream of 0.2/0.22 micron rated filters.

The results of scanning electron microscopic (SEM) and fatty acid methyl ester (FAME) characterization of the bacterial species shown to penetrate conventional 0.2/0.22 micron rated "sterilizing grade" filters are presented. SEM data suggest that retention of bacteria by these filters appears to be strongly influenced by the morphology, and especially the width of bacteria and less so by length. When the bacterial cell width is small, less than 0.3 micron or so, the cell length does not appear to limit the ability to penetrate 0.2/0.22 micron rated filters. As the bacterial width increases, there is also a strong, almost exponential, decrease in the allowable length for penetration, with most penetrative cells tending to be coccoid beyond a width of 0.5 micron. Significant percentages of the bacteria (40-50%) that were observed downstream of these filters were larger than B. diminuta, the standard organism used to qualify 0.2/0.22 micron rated filters. The average sizes of natural waterborne bacteria that penetrated the filters tested were 20-40% larger in width, and 40-70% larger in length, compared to B. diminuta. These results indicate that size exclusion is not the sole mechanism governing bacterial retention. All isolates identified via FAME analyses were common environmental or ubiquitous organisms, and some, such as Acidovorax sp. and Hydrogenophaga pseudoflava, have also been isolated from pharmaceutical water systems. Most of the bacteria recovered downstream of 0.2/0.22 micron rated filters were gram negative, oxidase positive, motile, nonfermentors.

Retention of water-borne bacteria by membrane filters. Part I: Bacterial challenge tests on 0.2 and 0.22 micron rated filters.

The results of bacterial challenge tests conducted on several 0.2 and 0.22 micron rated "sterilizing grade" filter cartridge types with bacteria from a natural water source are presented. Eight different 0.2/0.22 micron rated "sterilizing grade" filter types from four different filter manufacturers, claimed to be capable of retaining Brevundimonas diminuta at a challenge level of 10(7) CFU/cm2, were tested. The filters tested included nylon 6.6 and polyamide filters from two manufacturers, modified or hydrophilic PVDF filters from two manufacturers, modified or asymmetric PES filters from three manufacturers, and cellulose acetate filters from a single manufacturer. Consistent bacterial penetration was observed, over the 18-24 h challenge period, for all twenty-five integral 0.2 and 0.22 micron rated filter cartridges tested, at challenge levels of about 10(1)-10(4) CFU/cm2, indicating that natural waterborne bacteria were more penetrative than B. diminuta. The observed penetration was thus qualitatively independent of filter media type or manufacturer. These results add to the growing body of evidence that shows 0.2 and 0.22 micron rated filters may not remove all microorganisms under all conditions. These results further establish that bacterial penetration of 0.2/0.22 micron rated filters is not limited just to (1) specific membrane types, or (2) extended duration challenges (>> 24 h), or (3) extremely high challenge levels, or (4) bacteria that can only exist in a penetrative state in an artificial laboratory setting.

Method for qualifying microbial removal performance of 0.1 micron rated filters. Part I: characterization of water isolates for potential use as standard challenge organisms to qualify 0.1 micron rated filters.

Although 0.1 microm rated filters intended for pharmaceutical sterilization applications have been commercially available for at least 15 years, there is no industry-wide standard for qualifying the microbial removal performance of these filters. In this article, we report on the bacterial challenge methodology used to screen four bacterial species for potential utility as a standard challenge organism to qualify 0.1 microm rated filters. These isolates were, in their natural state, demonstrated to penetrate 0.2/0.22 microm rated filters in prior studies. In the screening challenges described in this study, three out of these four candidates tested demonstrated consistent penetration of one 0.22 microm rated filter type tested (when cultured in a low nutrient medium under standard laboratory conditions). These included 6204-22 (FAME ID Acidovorax avenae citrulli), 6266-15 (FAME ID Comamonas acidovorans), and 6266-34 (FAME ID Hydrogenophaga pseudoflava). Of these, H. pseudoflava (6266-34) was chosen for additional experiments with other 0.2 microm rated filter membranes. In total, seventeen 0.2 and 0.22 microm rated filter discs, spanning five different "sterilizing grade" filter types from three different filter manufacturers were tested. H. pseudoflava penetration was observed for every filter tested. Under the same challenge conditions, H. pseudoflava was consistently retained by a 0.1 microm rated hydrophilic PVDF (polyvinylidenefluoride) filter with a specified high titer reduction claim for Acholeplasma laidlawii. In order to ensure selection of the most stable penetrative phenotype (i.e., select for nonrevertants), H. pseudoflava was subjected to three rounds of "filter cloning," and these results are described herein. The advantages of using H. pseudoflava for qualifying the microbial removal performance of 0.1 microm rated filters are also discussed.

Method for qualifying microbial removal performance of 0.1 micron rated filters. Part II: preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for use as a standard challenge organism to qualify 0.1 micron rated filters.

In this article, we report on the preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for potential use as a standard challenge organism to qualify 0.1 microm rated filters. Filter-cloned H. pseudoflava (ATCC 700892) was easily cultured in a low nutrient broth (R2A broth) under standard laboratory conditions, reaching high titers of 10(8)-10(9) cfu/mL within 48-65 hours of incubation at 25+/-5 degrees C. Under these conditions, H. pseudoflava is a rod-shaped bacterium, averaging 0.25+/-0.03 microm by 1.65+/-0.35 microm, and appears to be smaller than Brevundimonas diminuta in width (0.31+/-0.03 microm), but somewhat longer in length (0.88+/-0.19 microm), which may partly explain the observed penetration. In total, thirty-five 0.2/0.22 microm rated filter discs, spanning five different "sterilizing grade" filter types from two different filter manufacturers were challenged with H. pseudoflava. In all cases, H. pseudoflava was shown to consistently penetrate every 0.2/0.22 microm rated filter disc tested. These tests also spanned three different challenge durations, including short-term challenges (30-40 minutes), and two different challenge fluids. The use of serial (double) 0.22 mm rated filters, which is a common industry practice to reduce the prefiltration bioburden to the final "sterilizing" filter, was also shown to be inadequate to fully retain H. pseudoflava under the challenge condition used. In contrast, two different 0.1 microm rated filter types functionally qualified with a specified high titer reduction claim for Acholeplasma laidlawii, were shown to consistently and fully retain H. pseudoflava, and retention by these two filter types was shown to be robust and independent of the challenge duration.

Method for qualifying microbial removal performance of 0.1 micron rated filters. Part III: bacterial challenge tests on 0.2/0.22 and 0.1 micron rated filter cartridges with Hydrogenophaga (formerly Pseudomonas) pseudoflava.

We have previously reported on the preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for potential use as a standard challenge organism to qualify 0.1 microm rated filters. This article reports on the retention efficiencies of a large panel of 0.2/0.22 microm and 0.1 microm rated filter cartridges for H. pseudoflava (ATCC 700892) versus the retention capabilities of the same filters for Brevundimonas diminuta (ATCC 19146). A total of thirty-two 0.2/0.22 microm rated filter cartridges, spanning nine different "sterilizing grade" filter types from four different filter manufacturers, were challenged with H. pseudoflava at challenge levels exceeding 10(7) cfu/cm2. H. pseudoflava was shown to penetrate every 0.2/0.22 microm rated filter tested, with log titer reduction (LTR) values ranging from 3.5 to 7.7 logs. H. pseudoflava was shown to be more penetrative than B. diminuta under the same challenge conditions. B. diminuta was fully retained by nineteen of the twenty 0.2/0.22 microm rated filters that were challenged with both organisms. In the case of 0.1 microm rated filters, eighteen filter cartridges, spanning five different filter types from three manufacturers were tested. H. pseudoflava was consistently retained by four out of the five filter types tested, with LTR values in excess of 11.5 to 12.2 logs. The 0.1 microm rated filter type that was penetrated by H. pseudoflava has been previously demonstrated to be not fully retentive for naturally occurring bacteria. The data show that H. pseudoflava penetrates 0.2/0.22 microm rated filters just as readily as B. diminuta penetrates 0.45 microm rated filters. In addition, titer reductions provided by 0.2/0.22 microm rated filters for H. pseudoflava are comparable to those reported for A. laidlawii mycoplasma, albeit under different conditions. This study demonstrates that H. pseudoflava meets all criteria for use as a standard organism for qualifying the microbial removal performance of 0.1 microm rated filters for enhanced sterility assurance.

Membrane filtration for virus removal.

Incorporation of adequate clearance methodologies for viral safety assurance constitutes an integral part of the manufacturing process for plasma-derived biologicals. Filtration, being non-invasive, non-destructive and a <> mechanism for virus removal is often the method of choice. This paper discusses the virus filter performance characteristics and other process-related issues specifically applying to the Pall Ultipor VF filters (Grade DV50 and Grade DV20), which are operated in the direct flow filtration (DFF) mode and the Pall Filtron Omega VR filters (the Omega 300 K and Omega 100 K), which are operated in the tangential flow filtration (TFF) mode.

Process control procedures to augment quality control of leukocyte-reduced red cell blood products.

Quality control of leukocyte-reduced packed red cell units (LRprc) produced in blood facilities must conform to regulatory criteria, which state that units may not contain more than 1 x 10(6) to 5 x 10(6) white blood cells (WBC) per unit. The post-filtration WBC content of a total of n = 386 LRprc units was counted with a Nageotte chamber to model the probability that a unit would not meet the regulatory criteria. The distribution of the residual leukocyte counts is close to a negative binomial distribution (NBD) and is independent of the packed red cell volume filtered. The observed probability that a unit of blood has a residual WBC greater than 5 x 10(6) is 2.6 +/- 2.6 x 10(-3). A power analysis of the two-sample Kolmogorov-Smirnov (KS) test in this application shows that a sample size of 20 is sufficient for determining that the process is in control when an out of control process has a k NBD parameter greater than or equal to that of the in control process. The three out of control processes observed to date appear to have this property. A sample of size 80 may be necessary for confirming that process validation data sets conform to the larger 'reference' database (n = 386) for processes that are out of control in such a way that their k NBD parameter is less than the k parameter of the in control process.

Application of bacteriophages as surrogates for mammalian viruses: a case for use in filter validation based on precedents and current practices in medical and environmental virology.

Infectivity-based assays are the assays of choice for the detection of pathogenic mammalian viruses. While it is intuitively appropriate to conduct testing and validation studies with the known viral burden or a closely related mammalian species, logistic considerations often dictate otherwise. Consequently, bacteriophages have served as suitable surrogates for mammalian viruses in both medical and environmental virology applications. The wide range of bacteriophages available offers a powerful analytical tool amenable to several different applications: filter validation studies (where removal is based on size exclusion), investigations into virus contamination control issues, evaluation of barrier materials, etc. There is a considerable body of evidence to suggest and support the use of bacteriophages as surrogates for mammalian viruses. Use of appropriately sized bacteriophages provides an innocuous, efficacious and expeditious method for economical testing and validation of viral clearance capabilities of virus removal filters, thus facilitating performance of filter validation studies in biopharmaceuticals under product- and process-specific conditions in an overall effort towards ensuring the virological safety of biologicals. This paper discusses the limitations associated with mammalian virus assays and provides a rationale for the use of bacteriophages as surrogates for mammalian viruses. Data from published literature documenting applicability of bacteriophages in filter validation studies, especially when removal is based on size exclusion, is reviewed along with examples of studies from the fields of medical and environmental virology.

Application of membrane filtration for removal of diminutive bioburden organisms in pharmaceutical products and processes.

In this report, we present results of a recent investigation in our laboratories demonstrated the effect of process conditions and/or drug product composition on the ability of 0.2 micron and 0.22 micron sterilizing grade filters to fully retain Ralstonia (formerly Burkholderia, formerly Pseudomonas) pickettii. R. pickettii is a opportunistic pathogen widely distributed in nature as well as clinical specimens and there have been several reports of nosocomial infections due to intrinsic manufacture-related R. pickettii contamination in filter-sterilized parenteral fluids. This study documents the penetration of 0.2 micron nylon 66 and 0.22 micron modified PVDF sterilizing grade filters by R. pickettii (grown and challenged) in a drug solution under conditions that simulated a pharmaceutical filling operation. Penetration was not observed for every filter disc tested, and this may be explained, in part, by the stochastic nature (i.e., governed by the rules of probability) of the retention mechanisms involved. Scanning electron microscopy revealed significant changes in the microorganism's size and morphology as a result of exposure to the drug solution; these changes are consistent with those reported for bacteria subjected to nutrient deprivation. The SEM analyses of R. pickettii challenge suspensions in the drug solution showed that the average cell length decreased from 1.25 +/- 0.27 microns to 0.84 +/- 0.17 micron between zero and 24 hours. In addition, significant changes were observed in the size (length) distributions, with approximately 35% of the cells at 24 hours being smaller than any cell observed at the start of the challenge. These data suggest that the significant reduction in bioburden size and morphology that occurred as a result of exposure to the drug solution may play a role in the reduced ability of the 0.2 micron and 0.22 micron filters tested in this study to retain these organisms. Under the same test conditions where penetration of 0.2/0.22 micron filters was observed, 0.1 micron rated membrane filters qualified with both B. diminuta and Acholeplasma laidlawii mycoplasma consistently provided sterile effluent. Bacterial penetration of 0.2 (or 0.22) micron sterilizing grade filters was not observed under identical test conditions with either R. pickettii in a standardized solution (saline lactose broth) routinely used in challenge testing filters, or with the standard test organism, B. diminuta, in the drug solution. This study thus supports the renewed emphasis on both product- and process specific validation as well as routine bioburden monitoring expressed by regulatory agencies, and the use of enhanced bacterial removal efficiency 0.1 micron rated filters to provide enhanced sterility assurance in pharmaceutical processes.

White cell subsets in apheresis and filtered platelet concentrates.

BACKGROUND: White cell (WBC)-reduced platelet concentrates (PCs) are defined by their absolute WBC count, a criterion which provides no information regarding the various WBC subsets contained in the PC. These heterogeneous cells are known to mediate different physiologic and pathophysiologic functions and account for distinct adverse transfusion responses. This study describes a method which allows the detection and quantification of these subsets and characterizes their presence in a variety of platelet components. STUDY DESIGN AND METHODS: Random-donor pooled PCs (RD PCs) and single-donor apheresis PCs (SD PCs) were studied. RD PCs consisting of 6 units of 2- to 3-day old PCs were randomly assigned to be filtered with one of four WBC-reduction filters from three different manufacturers (n=34). The residual WBCs were pelleted by centrifugation and isolated on a density gradient. The various WBC subsets were quantified by flow cytometry in unfiltered and filtered PCs using fluorescence and two-angle light scatter. SD PCs obtained with two manufacturer's systems and three processing protocols (n=30) were studied in like manner. RESULTS: WBC counts for non-WBC-reduced PCs averaged 3 x 10(8) in RD PCs and ranged from 8.6 to 9.6 x 10(6) per SD PC. Residual WBC counts in filtered PCs ranged from 2.3 x 10(4) to 2.2 x 10(5) and those in WBC-reduced SD PCs averaged 2.2 x 10(5) per unit. The data demonstrate significant phenotypic differences among PCs produced with various procedures. All SD PCs and two of four filtered RD PCs contained five WBC populations including granulocytes and monocytes, while RD PCs filtered with the remaining manufacturer's devices contained only lymphocytes. CONCLUSION: The data confirm that distinct phenotypic differences exist among PCs prepared with different devices and/or procedures. It is suggested that as for non-generic pharmaceuticals, the clinical benefits of these various PCs should be individually proved.

Lack of clinically significant contact system activation during platelet concentrate filtration by leukocyte removal filters.

When blood (plasma) contacts certain foreign surfaces, factor XII can activate and trigger a series of reactions leading to cleavage of kininogens with subsequent release of bradykinin. In this study, we investigated two different widely used leukocyte removal filters, Pall PXL8K (A) and Asahi PLS-5A (B), to test whether clinically significant contact activation occurred during leukodepletion of platelet-rich plasma (PRP). Kininogens were measured by particle concentration fluorescence immunoassay (PCFIA), which can detect cleavage of high and low molecular weight kininogens (HK and LK), the parent molecules of bradykinin, to determine if contact activation had occurred. A slight, nonsignificant decrease in HK and LK was observed with filter A after the first 5 mL was filtered that returned to prefiltration levels by the end of the filtration. Specific TotK (the combined measurement of HK and LK heavy chains divided by plasma protein concentration) showed a small, significant decrease with filter A after the first 5 mL of platelet concentrates was filtered that returned to prefiltration levels by the end of the filtration. There were no significant increases or decreases in the cleaved kininogen index (CKI), an index of HK proteolytic activation or HK and LK destruction (with release of bradykinin). These data suggest that small amounts of both HK and LK initially adsorb to filter A and then desorb, primarily intact. These data also indicate that no significant contact activation, as measured by PCFIA, occurs during leukodepletion of platelet concentrates with either filter A or B.

Virus retention by a hydrophilic triple-layer PVDF microporous membrane filter.

Retention of bacteriophages (phi 6, PR772, T1, and PP7) and mammalian viruses (poliovirus and influenza A virus) by a hydrophilic triple layer PVDF microporous membrane, the Ultipor VF grade DV50 membrane, was evaluated. Challenges of membrane discs or pleated filter cartridges were performed at concentrations of 10(6)-10(8) PFU/mL in one or more of the following carrier fluids: water, saline, gelatin (0.1%) in phosphate buffer, Dulbecco's Modified Eagle Medium (MEM), and MEM supplemented with 10% fetal bovine serum (MEM + 10). The data demonstrate a minimum log titer reduction (LTR) of 6 for viruses larger than 50 nm irrespective of the carrier fluid. Protein transmission levels of greater than 95% for IgG and albumin were achieved. For integral pleated filter cartridges, correlation between a nondestructive integrity test (using the forward flow integrity test method) and virus retention was demonstrated. The Ultipor VF grade DV50 filter can be applicable in the manufacture of biologicals and biopharmaceuticals, where high protein transmission and consistent viral titer reduction are desired.

Removal of Mycobacterium species by breathing circuit filters.

Breathing circuit filters (BCFs) are used in respiratory and critical-care settings for humidification of air and to limit transmission of respiratory pathogens. Three types of BCFs (Pall BB 25A, BB 100, and HME 15-22) were evaluated (in triplicate) for removal of Mycobacterium species. Filters were challenged with aerosolized Mycobacterium bovis (a surrogate for Mycobacterium tuberculosis), at minimum total concentrations of 10(4) colony-forming units. No M bovis was recovered in the effluent, providing removal efficiencies of > 99.99% to > 99.999% for the filters tested.

Practical guidelines for process validation and process control of white cell-reduced blood components: report of the Biomedical Excellence for Safer Transfusion (BEST) Working Party of the International Society of Blood Transfusion (ISBT).

BACKGROUND: The increased use of white (WBC)-reduced blood components has prompted many institutions to develop quality assurance programs directed to such component preparation processes. For consistent preparation of WBC-reduced blood components that meet clinical needs as well as national standards, a program of process validation and control should be instituted. This involves controlling key factors that affect WBC reduction as well as periodic monitoring of the residual cellular content of components. Practical guidelines for the implementation of such a program are provided. STUDY DESIGN AND METHODS: A program involving three phases of monitoring was developed by individuals belonging to an international working party of the International Society of Blood Transfusion. RESULTS: The first phase, process validation, evaluates a minimum of 20 consecutive units (a minimum of 60 units when nonparametric measurements are used) to document the successful local implementation of a new or substantially modified process. Ongoing process control employing Levey-Jennings type control charts is used to demonstrate that the process remains stable over time. Process capability assessment and conformance with standards are evaluated once residual WBCs are counted in a sufficient number of units. This enables a facility to claim with a specified degree of confidence that a stated proportion of WBC-reduced units will meet national standards. Two approaches to determine the number of units that should be selected for counting are presented. The first approach considers units as either acceptable or not acceptable and assumes that the distribution of failed (or nonconforming) units approximates the Poisson distribution. The second approach takes into consideration the observed WBC content of the tested units, with the assumption that the residual WBC content in WBC-reduced components follows a lognormal distribution. A method to assess the lognormal distribution of residual WBCs is presented. Specific tables based on each of these approaches are provided to guide the reader in the design of a program that will verify conformance with any national standard at specific confidence levels. The approach can be generalized to other process control applications. CONCLUSION: Guidelines are presented for process validation, process control, and assessment of conformance in the production of WBC-reduced blood components. Policy makers retain the responsibility to establish, on the basis of the expected use of WBC-reduced components, requirements for the frequency of testing and for the proportion of prepared units that are expected with a stated degree of confidence to meet the standards. Facilities preparing WBC-reduced components can monitor key factors that influence the preparation of WBC-reduced blood, can periodically assess their conformance with the standards, and can intervene to correct adverse changes in the process. This approach can be used to ensure the consistent quality of WBC-reduced blood components.

Multicenter evaluation of the 3% paraformaldehyde method for white cell counting in leukocyte-reduced red blood cells. BEST (Biomedical Excellence for Safer Transfusion) Working Party of the International Society of Blood Transfusion.

BACKGROUND/AIM: The 3% paraformaldehyde (PFA) method is a simple technique for counting residual white blood cells (WBC) in leukocyte-depleted red blood cells (RBC). Preliminary data suggested that its sensitivity is at least equal to PCR and flow cytometry. We report the results of a multicenter study conducted by the BEST Working Party to determine precision and accuracy of the 3% PFA method. STUDY DESIGN: In the 7 participating laboratories, 5 sets of samples containing nominal concentrations of 200, 100, 50, and 10 WBC/ml were prepared by diluting whole blood into 'WBC-free' RBC. Ten milliliters of each sample were processed using the 3% PFA method, which is based on erythrocyte lysis and WBC concentration into 5% of the original sample volume; a Nageotte chamber is used to count concentrated WBC. RESULTS: The precision of the technique varied according to the nominal concentration, ranging from a CV of 12% at 200 WBC/ml to 57% at 10 WBC/ml. The technique measured fewer than the nominal WBC concentrations (mean of all laboratories, -12.4%); underestimation was probably due to cell loss during sample manipulation. Overall accuracy was however acceptable, because statistical considerations establish that the actual WBC concentration would unlikely exceed 2 times the estimated count. CONCLUSIONS: The 3% PFA method is suitable for the enumeration of residual WBC at concentrations > or = 50/ml. It represents a useful tool for evaluation of high performance filters by reference laboratories.

Chemiluminescent detection of DNA on nylon membranes.

Two types of nonradioactive DNA detection systems were optimized for use with nylon membranes in Southern transfers. A luminol substrate system (consisting of an enhanced chemiluminescent reaction utilizing luminol enzyme substrate) was used with peroxidase-labeled probe DNA, and a dioxetane-based substrate was used with alkaline phosphatase/antibody and digoxigenin-labeled probe DNA. Chemiluminescence was detected by autoradiography. Methods for reprobing the membranes were also optimized for both systems; blots could be reprobed at least ten times. Results showed that excellent sensitivity and low background can be achieved on both amphoteric and positively charged nylon membranes, using either detection system.

Blinded, two-laboratory comparative analysis of Escherichia coli heat-stable enterotoxin production by using monoclonal antibody enzyme-linked immunosorbent assay, radioimmunoassay, suckling mouse assay, and gene probes.

Heat-stable enterotoxin (ST)-producing enterotoxigenic Escherichia coli (ETEC) can be identified by a variety of assays, including the suckling mouse assay (SMA), radioimmunoassay (RIA), polyclonal or monoclonal antibody enzyme-linked immunosorbent assay (ELISA), and DNA hybridization with STh and STp gene probes. To compare the sensitivity and reliability of these assays, 100 coded ETEC and non-ETEC isolates were blindly tested in two independent laboratories. SMA, RIA, and monoclonal ELISA were performed in Cincinnati, Ohio, while gene probe analysis was performed in Baltimore, Md. The method of storage of organisms had a profound effect on the stability of plasmids in certain strains. Hybridization experiments to determine the presence or absence of the enterotoxin gene showed that strains stored on Dorset egg medium at room temperature better retained their plasmids than strains stored frozen in skim milk. Forty-four of the 100 organisms obtained from the skim milk stock were found to produce STa in liquid culture by the RIA, SMA, and monoclonal ELISA (100% agreement). However, 50 of 54 of the strains stored on Dorset egg medium which were originally classified as STa+ or ST+ LT+ (positive for both heat-stable and heat-labile [LT] enterotoxins) were found to produce STa and retain the plasmid by each of these assays. Three additional strains were found which harbored the plasmid but did not elaborate STa by any of the assays (3% discrepancy). The monoclonal antibody ELISA appears to be highly reliable for determination of STa production by ETEC and can be easily scored visually even by untrained personnel. Furthermore, when this STa assay is coupled with a polyclonal antibody assay, it is possible to predict the genotype of STh- and STp-producing organisms.