Multimodal Biomarkers That Predict the Presence of Gleason Pattern 4: Potential Impact for Active Surveillance.

PURPOSE: Latent grade group ≥2 prostate cancer can impact the performance of active surveillance protocols. To date, molecular biomarkers for active surveillance have relied solely on RNA or protein. We trained and independently validated multimodal (mRNA abundance, DNA methylation, and/or DNA copy number) biomarkers that more accurately separate grade group 1 from grade group ≥2 cancers. MATERIALS AND METHODS: Low- and intermediate-risk prostate cancer patients were assigned to training (n=333) and validation (n=202) cohorts. We profiled the abundance of 342 mRNAs, 100 DNA copy number alteration loci, and 14 hypermethylation sites at 2 locations per tumor. Using the training cohort with cross-validation, we evaluated methods for training classifiers of pathological grade group ≥2 in centrally reviewed radical prostatectomies. We trained 2 distinct classifiers, PRONTO-e and PRONTO-m, and validated them in an independent radical prostatectomy cohort. RESULTS: PRONTO-e comprises 353 mRNA and copy number alteration features. PRONTO-m includes 94 clinical, mRNAs, copy number alterations, and methylation features at 14 and 12 loci, respectively. In independent validation, PRONTO-e and PRONTO-m predicted grade group ≥2 with respective true-positive rates of 0.81 and 0.76, and false-positive rates of 0.43 and 0.26. Both classifiers were resistant to sampling error and identified more upgrading cases than a well-validated presurgical risk calculator, CAPRA (Cancer of the Prostate Risk Assessment; P < .001). CONCLUSIONS: Two grade group classifiers with superior accuracy were developed by incorporating RNA and DNA features and validated in an independent cohort. Upon further validation in biopsy samples, classifiers with these performance characteristics could refine selection of men for active surveillance, extending their treatment-free survival and intervals between surveillance.

Microencapsulated islet allografts in diabetic NOD mice and nonhuman primates.

OBJECTIVE: Our goal was to assess the efficacy of encapsulated allogeneic islets transplanted in diabetic NOD mice and streptozotocin (STZ)-diabetic nonhuman primates (NHPs). MATERIALS AND METHODS: Murine or NHP islets were microencapsulated and transplanted in non-immunosuppressed mice or NHPs given clinically-acceptable immunosuppressive regimens, respectively. Two NHPs were treated with autologous mesenchymal stem cells (MSCs) and peri-transplant oxygen therapy. Different transplant sites (intraperitoneal [i.p.], omental pouch, omental surface, and bursa omentalis) were tested in separate NHPs. Graft function was monitored by exogenous insulin requirements, fasting blood glucose levels, glucose tolerance tests, percent hemoglobin A1c (% HbA1c), and C-peptide levels. In vitro assessment of grafts included histology, immunohistochemistry, and viability staining; host immune responses were characterized by flow cytometry and cytokine/chemokine multiplex ELISAS. RESULTS: Microencapsulated islet allografts functioned long-term i.p. in diabetic NOD mice without immunosuppression, but for a relatively short time in immunosuppressed NHPs. In the NHPs, encapsulated allo-islets initially reduced hyperglycemia, decreased exogenous insulin requirements, elevated C-peptide levels, and lowered % HbA1c in plasma, but graft function diminished with time, regardless of transplant site. At necropsy, microcapsules were intact and non-fibrotic, but many islets exhibited volume loss, central necrosis and endogenous markers of hypoxia. Animals receiving supplemental oxygen and autologous MSCs showed improved graft function for a longer post-transplant period. In diabetic NHPs and mice, cell-free microcapsules did not elicit a fibrotic response. CONCLUSIONS: The evidence suggested that hypoxia was a major factor for damage to encapsulated islets in vivo. To achieve long-term function, new approaches must be developed to increase the oxygen supply to microencapsulated islets and/or identify donor insulin-secreting cells which can tolerate hypoxia.

PTEN loss in Gleason grade 7 prostate tumors exhibits intratumoral heterogeneity and is associated with unfavorable pathological features

Background: PTEN loss is observed in 20­30% of prostate cancers and is associated with a poor outcome, but clinical details of the impact of this biomarker are unclear for intermediate grade tumors. Methods: We investigated 43 radical prostatectomy-derived grade 7 prostate tumors from the Clinics Hospital of Ribeirão Preto. Tissue microarray (TMA) blocks were constructed and PTEN copy number status was determined for all patients through fluorescence in situ hybridization (FISH). To determine the presence of PTEN protein loss in our study cohort, we performed immunohistochemistry (IHC) in TMA sections. We then developed an automated algorithm in HALO™ to identify regions of PTEN protein loss in whole prostate scanned sections from ten patients with known PTEN deletion status by FISH. Clinical analyses were conducted to determine the associations between PTEN loss and patient outcome. All statistical analyses were conducted in R v3.4.3 with P-values below 0.05 being considered statistically significant. Results: In this study of 43 grade 7 tumors, we found PTEN deletions by FISH in 18.9% of tumors, and PTEN protein loss by IHC in 16.3% of tumors. Both techniques were highly concordant and complementary. Clinical analysis demonstrated that PTEN deletion by FISH was significantly associated with positive margin invasion (P = 0.04) and Gleason score upgrade (P = 0.001). Digital image analysis of ten representative tumors demonstrated distinct intratumoral heterogeneity for PTEN protein loss in four tumors. Conclusions: This study shows that PTEN loss in Gleason grade 7 tumors can be heterogeneous and that a systematic analysis of this biomarker using a combination of FISH, IHC, and digital imaging may identify patients with a greater risk of poor outcome (AU)

Postpartum hemoptysis as presenting sign of longstanding vasculitis.

We present the case of a 26-year-old postpartum patient who presented with an episode of desaturation and hemoptysis on postpartum day three after an uncomplicated spontaneous vaginal delivery. The patient came to our attention in the postpartum area after she experienced massive hemoptysis and we were called by the obstetric team. The patient was subsequently intubated, mechanically ventilated, and underwent bronchoscopy, demonstrating diffuse alveolar hemorrhage. She was brought to the intensive care unit, placed on high-dose steroids and plasmapheresis was initiated. Her intensive care unit course was complicated by acute respiratory distress syndrome, acute kidney injury and a pulmonary embolism, but she recovered well and was discharged on postpartum day 23. This report describes a rare case of medium vessel vasculitis diagnosed in the peripartum period, and describes the diagnostic dilemmas underlying making a rare diagnosis, and the difficulties initiating appropriate therapy in a postpartum patient.

Severe congenital anomalies of the kidney and urinary tract: epidemiology can inform ethical decision-making.

OBJECTIVE: Decision-making for pregnancies complicated by severe congenital anomalies of the kidneys and urinary tract (CAKUT) are ethically challenging, partly because the outcomes are not well studied. STUDY DESIGN: Retrospective cohort study of severe cases of CAKUT over 14 years. RESULTS: Seventy-one of the 108 cases could be completely analyzed. Forty-six percent (n=33) infants were live-born; one-third (n=11) survived to 12 months. Twice as many non-surviving infants received a trial of therapy vs comfort care only. Two-thirds of non-survivors who received a trial of therapy died within the first 9 h of life. Live-born infants faced morbidities such as pneumothorax and neonatal dialysis. CONCLUSIONS: Over half of pregnancies complicated by severe CAKUT ended in termination or stillbirth, but one-third of live-born infants survived to 12 months and the majority of non-survivors died within hours. This may allay concerns about prolonged and futile intensive care for parents considering a trial of therapy.

Team-based model for non-operating room airway management: validation using a simulation-based study.

BACKGROUND: Non-operating room (non-OR) airway management has previously been identified as an area of concern because it carries a significant risk for complications. One reason for this could be attributed to the independent practice of residents in these situations. The aim of the present study was to ascertain whether differences in performance exist between residents working alone vs with a resident partner when encountering simulated non-OR airway management scenarios. METHODS: Thirty-six anaesthesia residents were randomized into two groups. Each group experienced three separate scenarios (two scenarios initially and then a third 6 weeks later). The scenarios consisted of one control scenario and two critical event scenarios [i.e. asystole during laryngoscopy and pulseless electrical activity (PEA) upon post-intubation institution of positive pressure ventilation]. One group experienced the simulated non-OR scenarios alone (Solo group). The other group consisted of resident pairs, participating in the same three scenarios (Team group). RESULTS: Although the time to intubation did not differ between the Solo and Team groups, there were several differences in performance. The Team group received better overall performance ratings for the asystole (8.5 vs 5.5 out of 10; P<0.001) and PEA (8.5 vs 5.8 out of 10; P<0.001) scenarios. The Team group was also able to recognize asystole and PEA conditions faster than the Solo group [10.1 vs 23.5 s (P<0.001) and 13.3 vs 36.0 s (P<0.001), respectively]. CONCLUSIONS: Residents who performed a simulated intubation with a second trained provider had better overall performance than those who practised independently. The residents who practised in a group were also faster to diagnose serious complications, including peri-intubation asystole and PEA. Given these data, it is reasonable that training programmes consider performing all non-OR airway management with a team-based method.

Exposure-response estimate for lung cancer and asbestosis in a predominantly chrysotile-exposed Chinese factory cohort.

BACKGROUND: Cumulative fiber exposures, predominantly chrysotile, were estimated in a Chinese asbestos worker cohort and exposure-response relationships with lung cancer mortality and cumulative incidence of asbestosis were determined. METHODS: Individual time-dependent cumulative exposures were estimated for 577 asbestos workers, followed prospectively for 37 years. Occupational history and smoking data were obtained from company records and personal interviews; vital status and causes of death were ascertained from death registries and hospital records. Hazard ratios were generated for disease outcomes, with adjustments for smoking and age. RESULTS: Median cumulative fiber exposure for the cohort was 132.6 fiber-years/ml (IQR 89.3-548.4). Exposure-response relationships were demonstrated for both disease outcomes, with nearly sixfold and threefold increased risks seen at the highest exposure level for lung cancer deaths and asbestosis, respectively. CONCLUSION: Evidence using quantitative exposure estimates was provided for increased risks of lung cancer mortality and development of asbestosis in a predominantly chrysotile-exposed cohort.

In prostate cancer needle biopsies, detections of PTEN loss by fluorescence in situ hybridization (FISH) and by immunohistochemistry (IHC) are concordant and show consistent association with upgrading.

The prognostic value of phosphatase and tensin homolog (PTEN) loss in prostate cancer has primarily been evaluated by either fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC). Previously, we found that PTEN loss by IHC was associated with increased risk of upgrading from biopsy (Gleason 3 + 3) to prostatectomy (Gleason 7+). Now, using an evaluable subset of 111 patients with adjacent biopsy sections, we analyzed the association between PTEN deletion in cancer and the odds of upgrading by a highly sensitive and specific four-color FISH assay. We also compared the concordance of PTEN loss by IHC and PTEN deletion by FISH. PTEN deletion was found in 27 % (12/45) of upgraded cases compared with 11 % (7/66) of controls (P = 0.03). Cancers with PTEN deletions were more likely to be upgraded than those without deletions (adjusting for age odds ratio = 3.40, 95 % confidence interval 1.14-10.11). With respect to concordance, of 93 biopsies with PTEN protein detected by IHC, 89 (96 %) had no PTEN deletion by FISH, and of 18 biopsies without PTEN protein by IHC, 15 had homozygous or hemizygous PTEN deletion by FISH. Only 4 biopsies of the 93 (4 %) with PTEN protein intact had PTEN deletion by FISH. When the regions of uncertainty in these biopsies were systematically studied by FISH, intra-tumoral variation of PTEN deletion was found, which could account for variation in immunoreactivity. Thus, FISH provides a different approach to determining PTEN loss when IHC is uncertain. Both FISH and IHC are concordant, showing consistent positive associations between PTEN loss and upgrading.

The safety and feasibility of probiotics in children and adolescents undergoing hematopoietic cell transplantation.

Hematopoietic cell transplantation (HCT) has become a standard treatment for many adult and pediatric conditions. Emerging evidence suggests that perturbations in the microbiota diversity increase recipients' susceptibilities to gut-mediated conditions such as diarrhea, infection and acute GvHD. Probiotics preserve the microbiota and may minimize the risk of developing a gut-mediated condition; however, their safety has not been evaluated in the setting of HCT. We evaluated the safety and feasibility of the probiotic, Lactobacillus plantarum (LBP), in children and adolescents undergoing allogeneic HCT. Participants received once-daily supplementation with LBP beginning on day -8 or -7 and continued until day +14. Outcomes were compliance with daily administration and incidence of LBP bacteremia. Administration of LBP was feasible with 97% (30/31, 95% confidence interval (CI) 83-100%) of children receiving at least 50% of the probiotic dose (median 97%; range 50-100%). We did not observe any case of LBP bacteremia (0% (0/30) with 95% CI 0-12%). There were not any unexpected adverse events related to LBP. Our study provides preliminary evidence that administration of LBP is safe and feasible in children and adolescents undergoing HCT. Future steps include the conduct of an approved randomized, controlled trial through Children's Oncology Group.

Size- and type-specific exposure assessment of an asbestos products factory in China.

This study describes fibre size and type-specific airborne asbestos exposures in an asbestos product factory. Forty-four membrane filter samples were analysed by scanning electron microscopy to determine the size distribution of asbestos fibres, by workshop. Fibre frequencies of bivariate (length by width) categories were calculated and differences between workshops were tested by analysis of variance. Data were recorded for 13,435 chrysotile and 1075 tremolite fibres. The proportions between size metrics traditionally measured and potentially biologically important size metrics were found to vary in this study from proportions reported in other cohort studies. One, common size distribution was generated for each asbestos type over the entire factory because statistically significant differences in frequency between workshops were not detected. This study provides new information on asbestos fibre size and type distributions in an asbestos factory. The extent to which biologically relevant fibre size indices were captured or overlooked between studies can potentially reconcile currently unexplained differences in asbestos-related disease (ARD) risk between cohorts. The fibre distributions presented here, when combined with similar data from other sites, will contribute to the development of quantitative models for predicting risk and our understanding of the effects of fibre characteristics in the development of ARD.

Identification of proteomic biomarkers predicting prostate cancer aggressiveness and lethality despite biopsy-sampling error.

BACKGROUND: Key challenges of biopsy-based determination of prostate cancer aggressiveness include tumour heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. The resulting uncertainty in risk assessment leads to significant overtreatment, with associated costs and morbidity. We developed a performance-based strategy to identify protein biomarkers predictive of prostate cancer aggressiveness and lethality regardless of biopsy-sampling variation. METHODS: Prostatectomy samples from a large patient cohort with long follow-up were blindly assessed by expert pathologists who identified the tissue regions with the highest and lowest Gleason grade from each patient. To simulate biopsy-sampling error, a core from a high- and a low-Gleason area from each patient sample was used to generate a 'high' and a 'low' tumour microarray, respectively. RESULTS: Using a quantitative proteomics approach, we identified from 160 candidates 12 biomarkers that predicted prostate cancer aggressiveness (surgical Gleason and TNM stage) and lethal outcome robustly in both high- and low-Gleason areas. Conversely, a previously reported lethal outcome-predictive marker signature for prostatectomy tissue was unable to perform under circumstances of maximal sampling error. CONCLUSIONS: Our results have important implications for cancer biomarker discovery in general and development of a sampling error-resistant clinical biopsy test for prediction of prostate cancer aggressiveness.

Curative therapy for bladder cancer in routine clinical practice: a population-based outcomes study.

AIMS: Definitive therapy of bladder cancer involves cystectomy or radiotherapy; controversy exists regarding optimal management. Here we describe the management and outcomes of patients treated in routine practice. MATERIALS AND METHODS: Treatment records were linked to the Ontario Cancer Registry to identify all cases of bladder cancer in Ontario treated with cystectomy or radiotherapy in 1994-2008. Practice patterns are described in three study periods: 1994-1998, 1999-2003, 2004-2008. Logistic regression, Cox model and propensity score analyses were used to evaluate factors associated with treatment choice and survival. RESULTS: In total, 3879 cases (74%) underwent cystectomy and 1380 (26%) were treated with primary radiotherapy. Cystectomy use increased over time (66, 75, 78%), whereas radiotherapy decreased (34, 25, 22%), P < 0.001. There was substantial regional variation in the proportion of cases undergoing radiotherapy (range 16-51%). Five year cancer-specific survival (CSS) and overall survival were 40 and 36% for surgical cases and 35 and 26% for radiotherapy cases (P < 0.001). In multivariate Cox model and propensity score analyses, there was no significant difference in CSS between surgery and radiotherapy (hazard ratio 0.99, 95% confidence interval 0.91-1.08); radiotherapy was associated with slightly inferior overall survival (hazard ratio 1.08, 95% confidence interval 1.00-1.16). CONCLUSION: Utilisation of cystectomy for bladder cancer in routine practice has increased over time with no evidence of a significant difference in CSS between radiotherapy and cystectomy.

Causal versus spurious spatial exposure-response associations in health risk analysis.

Many recent health risk assessments have noted that adverse health outcomes are significantly statistically associated with proximity to suspected sources of health hazard, such as manufacturing plants or point sources of air pollution. Using geographic proximity to sources as surrogates for exposure to (possibly unknown) releases, spatial ecological studies have identified potential adverse health effects based on significant regression coefficients between risk rates and distances from sources in multivariate statistical risk models. Although this procedure has been fruitful in identifying exposure-response associations, it is not always clear whether the resulting regression coefficients have valid causal interpretations. Spurious spatial regression and other threats to valid causal inference may undermine practical efforts to causally link health effects to geographic sources, even when there are clear statistical associations between them. This paper demonstrates the methodological problems by examining statistical associations and regression coefficients between spatially distributed exposure and response variables in a realistic data set for California. We find that distance from "nonsense" sources (such as arbitrary points or lines) are highly statistically significant predictors of cause-specific risks, such as traffic fatalities and incidence of Kaposi's sarcoma. However, the signs of such associations typically depend on the distance scale chosen. This is consistent with theoretical analyses showing that random spatial trends (which tend to fluctuate in sign), rather than true causal relations, can create statistically significant regression coefficients: spatial location itself becomes a confounder for spatially distributed exposure and response variables. Hence, extreme caution and careful application of spatial statistical methods are warranted before interpreting proximity-based exposure-response relations as evidence of a possible or probable causal relation.

A cautionary tale: the characteristics of two-dimensional distributions and their effects on epidemiological studies employing an ecological design.

In recent years, many spatial epidemiological studies that use proximity of subjects to putative sources as a surrogate for exposure have been published and are increasingly cited as evidence of environmental problems requiring public health interventions. In these studies, the simple finding of a significant, positive association between proximity and disease incidence has been interpreted as evidence of causality. However, numerous authors have pointed out limitations to such interpretations. This, the first of two companion studies, examines the effects of analyzing (real and simulated) spatial data using logistic regression. Simulation is also employed to explore the statistical power of such analyses to detect true effects, quantify the probabilities of Type I and Type II errors, and to evaluate a proposed mechanism that explains the observed effects. Results indicate that, even when the odds ratios of cases and controls are regressed against random or nonsense sources, significant, positive associations are observed at frequencies substantially greater than chance. These frequencies increase when targets are highly non-uniformly distributed such that, for example, false-positive associations are more likely than not when odds ratios are regressed against the actual distribution of ultramafic rocks in California. The coefficients of true, causal associations are substantially attenuated under realistic conditions so that, absent corroborating analyses, there is no non-arbitrary means of distinguishing causal from spurious or real but non-causal associations. Factors affecting where people choose to live act as powerful confounders, creating spurious or real but non-causal associations between exposure and response variables (as well as between other pairs of variables). Consequently, future epidemiological studies that use proximity as a surrogate for exposure should be required to include adequate negative control analyses and/or other kinds of corroborating analyses before they are accepted for publication.

Overreliance on a single study: there is no real evidence that applying quality criteria to exposure in asbestos epidemiology affects the estimated risk.

A critical need exists for reliable risk management policies and practices that can effectively mitigate asbestos-related health threats, and such policies and practices need to be based on sound science that adequately distinguishes hazardous situations from those that are not. Toward that end, the disparate means by which study quality has been addressed in recent meta-analyses used to establish potency factors (K ( L ) and K ( M ) values) for asbestos cancer risks were compared by conducting additional sensitivity analyses. Results suggest that, other than placing undue emphasis on the influence of the K ( L ) and K ( M ) values reported from a single study, there appears to be little to no evidence of a systematic effect of study quality on K ( L ) or K ( M ) values; none of the findings warrant excluding studies from current or future meta-analyses. Thus, we argue that it is better to include as much of the available data as possible in these analyses while formally addressing uncertainty as part of the analysis itself, rather than sequentially excluding studies based on one type of limitation or another. Throwing out data without clearly proving some type of bias is never a good idea because it will limit both the power to test various hypotheses and the confidence that can be placed in any findings that are derived from the resulting, truncated data set. We also believe that it is better to identify the factors that contribute to variation between studies included in a meta-analysis and, by adjusting for such factors as part of a model, showing that the disparate values from individual studies can be reconciled. If such factors are biologically reasonable (based on other evidence) and, if such a model can be shown to fit the data from all studies in the meta-analysis, the model is likely to be predictive of the parameters being evaluated and can then be applied to new (unstudied) environments.

Counting rules for estimating concentrations of long asbestos fibers.

Mounting evidence that long asbestos fibers (e.g. >20 or even 40 µm) pose the greatest cancer risk underscores the need for accurate measurement of concentrations of such fibers. These fiber lengths are of the same order of magnitude as the size of openings in the grids (typically ≈90 µm per side) used to analyze asbestos samples by transmission electron microscopy. This means that a substantial proportion of long fibers will cross the edge of a grid opening (GO) and therefore not be completely visible. Counting rules generally deal with such fibers by assigning a length equal to twice the visible length. Using both theoretical and simulation methods, we show that this doubling rule introduces bias into estimates of fiber concentrations and the amount of bias increases with fiber length. We investigate an alternative counting rule that counts only fibers that lie completely within a GO and weights those fibers by the reciprocal of the probability that a fiber of that length lies totally within a GO. This approach does not have the bias inherent in the doubling rule and is essentially unbiased if the stopping rule specifies a fixed number of GOs to be scanned. However, a stopping rule based on successively scanning GOs until a fixed number of fibers have been counted will introduce bias into any counting method, although this bias may typically not be large enough to be of practical concern. We recommend use of the weighted approach as a supplement to use of the doubling rule when estimating concentrations of long fibers, irrespective of the stopping rule employed.

Chamber for testing asbestos-containing products: validation and testing of a re-created chrysotile-containing joint compound.

Joint compound products containing chrysotile asbestos were commonly used for building construction from the late 1940s through the mid-1970s. Few relevant data exist to support reconstructing historical worker exposures to fibers generated by working with this material. Therefore, we re-created 1960s-era chrysotile-containing joint compound (JCC) and compared its characteristics to a current-day asbestos-free joint compound (JCN). Validation studies showed that a bench-scale chamber with controlled flow dynamics, designed to quantify particulate emissions from joint compound products, provided precise and reliable measurements of generated airborne dust mass, chrysotile fiber concentrations, and corresponding activity-specific emission rates. Subsequent chamber studies characterized fibers counted by phase contrast microscopy (PCM) per mass of respirable dusts and total suspended particulate dusts (total dusts), generated during JCC sanding or sweeping, as well as corresponding dust emission rates for JCC and JCN, and the ratio of total to respirable dust mass for JCN. From these data we estimated factors, F(CH-rd) and F(CH-td) (in units of f cm(-3) per mg m(-3)), by which respirable JCN dust mass concentrations collected during construction use can be converted to corresponding airborne PCM fiber concentrations generated by sanding or sweeping JCC. For sanding, median values (95% confidence limits) of F(CH-rd) and F(CH-td) were estimated to be 0.044 (0.039-0.050) and 0.212 (0.115-0.390) f cm(-3) per mg m(-3), respectively. The F(CH-td) to F(CH-rd) ratio indicates that approximately five times as many airborne PCM fibers are anticipated per unit air volume sampled when JCC dust is collected on cassettes (as done historically), than when respirable JCC dust is collected on cyclones. As the sizes of individual fibers collected appear to be primarily respirable, this difference may be a sampling artifact and suggests caution in interpreting historical fiber concentration measures made using cassettes during work with JCC-like materials. F(CH-rd) can be used with published and newly generated field measurements of respirable dust mass concentrations associated with the use of JCN or equivalent JCN materials to better characterize historical worker exposures to PCM fibers from use of JCC or equivalent JCCs. The experimental process described also can be used to develop conversion factors for other combinations of modern-day asbestos-free and historical chrysotile-containing products.

Potential artifacts associated with historical preparation of joint compound samples and reported airborne asbestos concentrations.

Airborne samples collected in the 1970s for drywall workers using asbestos-containing joint compounds were likely prepared and analyzed according to National Institute of Occupational Safety and Health Method P&CAM 239, the historical precursor to current Method 7400. Experimentation with a re-created, chrysotile-containing, carbonate-based joint compound suggested that analysis following sample preparation by the historical vs. current method produces different fiber counts, likely because of an interaction between the different clearing and mounting chemicals used and the carbonate-based joint compound matrix. Differences were also observed during analysis using Method 7402, depending on whether acetic acid/dimethylformamide or acetone was used during preparation to collapse the filter. Specifically, air samples of sanded chrysotile-containing joint compound prepared by the historical method yielded fiber counts significantly greater (average of 1.7-fold, 95% confidence interval: 1.5- to 2.0-fold) than those obtained by the current method. In addition, air samples prepared by Method 7402 using acetic acid/dimethylformamide yielded fiber counts that were greater (2.8-fold, 95% confidence interval: 2.5- to 3.2-fold) than those prepared by this method using acetone. These results indicated (1) there is an interaction between Method P&CAM 239 preparation chemicals and the carbonate-based joint compound matrix that reveals fibers that were previously bound in the matrix, and (2) the same appeared to be true for Method 7402 preparation chemicals acetic acid/dimethylformamide. This difference in fiber counts is the opposite of what has been reported historically for samples of relatively pure chrysotile dusts prepared using the same chemicals. This preparation artifact should be considered when interpreting historical air samples for drywall workers prepared by Method P&CAM 239.