Dual proline labeling protocol for individual "baseline" and "response" biosynthesis measurements in human articular cartilage.

OBJECTIVE: The heterogeneity of biosynthesis in human-derived cartilage explants poses a challenge to its use in experiments. The aim of this study was to determine the consistency with which two consecutive measures of biosynthesis could be made in individual human articular cartilage explants using a dual proline radiolabeling protocol. METHODS: Full-thickness cartilage explants were harvested from young bovine or human (total knee replacement) tibial plateaus. Two consecutive measurements of biosynthesis were obtained by measuring (3)H-proline and (14)C-proline incorporation. Each sample's ratio of (14)C-/(3)H-proline incorporation was computed. For comparison to traditional experimental designs, the (14)C-proline incorporation ratio was computed for adjacent cartilage samples. The number of samples needed to observe a change in the proline incorporation ratio of 10, 20, and 50% was determined for both methods. RESULTS: The dual-label ratio was consistent across samples from the same plateau [95% confidence interval (CI): +/-20% (human) and +/-30% (bovine) of median]. Adjacent human sample pairs had much greater variability in their (14)C-proline incorporation (95% CI: +/-50% of median). Adjacent bovine sample pairs had CIs that were similar in magnitude to those for the dual-label approach. In the human plateaus, ratio changes of 10, 20 and 50% could be detected using dramatically fewer samples than the adjacent pair method. For bovine samples, the two methods required a similar number of samples per group. CONCLUSION: The consistency of the dual-label approach may overcome the difficulties in studying the effects of interventions on biosynthesis in human cartilage in vitro.

Sputum cytology within and across laboratories. A reliability study.

A total of 11 cytotechnologists at sites in Texas (TX1, TX2), California (CA) and Arkansas (AR) were assessed for agreement of six-category diagnoses of sputum cytology slides prepared by the method of Saccomanno. For three observers at TX1 there was more agreement within observers (27-60%) than across observers (13-50%). Within-1 category intraobserver agreement underwent a twofold to threefold increase, to 77-93%; within-2 category agreement was 90-100%. Interobserver within-1 category agreement was 47-92%; within-2 category agreement was 83-100%. Agreement was significantly greater than chance (using kappa) in 69% of all intraobserver and interobserver pairings. Intralaboratory agreement was 40% for CA and 40-57% for TX2. Among pairings of the four sites, the range of interlaboratory agreement was 13-60% over several occasions. The overall range of agreement with the TX1 standard was 17-50% over observers/occasions. Within certain categories, outside agreement with the TX1 standard was 53-90% for normal, 39-80% for squamous metaplasia, 68-84% for mild atypia, 80-100% for moderate atypia and 93-100% for severe atypia or carcinoma. We conclude that agreement is acceptable for extreme atypia, but more training or refinement of the guidelines may be needed, if justified, to better differentiate the lowest categories. Good agreement appears to be as likely for observers with many years of overall experience as for those with high exposure to the Saccomanno method. For potential statistical analyses, the scale should probably be condensed into three to four categories to reduce extraneous variability.

Presentation of Sporothrix schenkii in pulmonary cytology specimens.

Macrophages containing small ovoid intracytoplasmic yeast cells, surrounded by a slight "halo," were observed in Papanicolaou-stained smears of sputum submitted for cytologic examination from two patients with culture-confirmed pulmonary sporotrichosis. The cytologic findings are presented and discussed in relation to other pulmonary fungal infections and the appropriate stains to be used for their cytologic detection. The clinical symptoms of pulmonary sporotrichosis are nonspecific and may mimic those of tuberculosis or other diseases; the causative organism, Sporothrix schenkii, may remain undetected by routine microbiologic procedures unless there is cause to suspect the diagnosis. Sporotrichosis should be considered in the differential diagnosis when small ovoid intracellular yeast cells (2 microns to 4 microns in length) with an apparent "halo" are observed within macrophages in pulmonary cytology specimens; the recognition of these findings may aid in the diagnosis of this relatively rare disease.

Mites in pulmonary cytology specimens.

Mites were observed in 55 pulmonary specimens examined routinely over a 4.5-yr period. The 55 specimens included 33 spontaneous and 12 aerosol-induced sputum samples, nine bronchial washings, and one fine-needle aspirate. Two specimens contained only fragments of mites; however, in most cases the mites were relatively intact. Many were identified as Tyrophagus sp, but a few were definitely not Tyrophagus sp. Although only a single mite was noted in most instances, eight specimens contained more than one, with the maximum being six. No patient was found to have a mite in more than one specimen. Mite eggs were found in five of the specimens containing mites. One specimen contained adults and a larval form. Mites appeared to be surrounded by acute inflammatory cells in 13 cases. Specimens were processed by the Saccomanno blending technique with smears prepared by pipetting a portion directly onto a slide or by utilizing a cytocentrifuge. No mites were found in nonpulmonary specimens. Both a seasonal variation and an interobserver variability were found. Mites often resemble specimen contaminants, such as food, and may be overlooked on routine screening. Mites are known to represent or produce a respiratory allergen, but the significance of their presence in pulmonary cytology specimens has not been determined.

Crystals occurring in pulmonary cytology specimens. Association with Aspergillus infection.

Birefringent needlelike crystals in rosette or wheat-sheaf-like arrangements were found in pulmonary cytology specimens from 11 of 65 patients who had either sputum cultures positive for Aspergillus or histologically confirmed pulmonary aspergilloma. No crystals were found in specimens from 60 control patients with and without known fungal disease. The crystals were most often associated with A. niger infection (45.4%), followed by A. flavus (16%). Crystals were also observed in one case of A. fumigatus infection and in one case in which the species was not determined. In two cases, crystals were found more than one year before sputum cultures became positive; in one of these patients, a fungus ball was not identified by X ray until five years after the first appearance of the crystals in the sputum. Sixty-four percent of the patients with crystals also showed moderate to severe cytologic atypia. The crystals are thought to be calcium oxalate. We conclude that the presence of birefringent needlelike crystals with rosette or wheat-sheaf-like arrangements in pulmonary cytology specimens is a reliable marker for the presence of Aspergillus infection, which may be detected before cultures are positive or a fungus ball is evident on X ray.

Comparison of sputum and lung asbestos body counts in former asbestos workers.

The asbestos (ferruginous) body content of lung tissues was compared to the number of asbestos bodies (AB) in sputum from 6 former amosite asbestos workers. Lung tissue was obtained at autopsy (5 subjects) and lobectomy (1 subject), and AB content was determined by a digestion-concentration technique. The sputum obtained was collected by both spontaneous cough (a pooled, 3-day specimen) and aerosolized hypertonic saline induction. Four slides treated with Papanicolaou's stain were prepared from each cytologic specimen and scored from 0 to 3 + on the basis of the total number of AB counted on the 4 slides. A significant correlation (P < 0.02) was found between the number of AB per gram of lung tissue and the percentage of sputum specimens from each worker than were positive for AB. Asbestos bodies were identified in 46% of sputum specimens from the 4 workers whose lungs contained 900 or more AB/g of lung tissue.

Statistical comparison of aerosol-induced and spontaneous sputum specimens in the Tyler Asbestos Workers Program.

More than 10,000 sputum specimens, including both aerosol-induced and three-day pooled spontaneous specimens, were processed within a five-year period from a surveillance program of a cohort of 858 former asbestos workers and 188 controls. During the first two years, slides were prepared by the "pick and smear" method; thereafter, the Saccomanno technique was used. Each specimen was examined for both the degree of cellular atypia and the number of ferruginous bodies. Data analysis revealed that the number of ferruginous bodies in the spontaneous specimens showed a higher correlation with chest X rays and pulmonary function tests than did those in aerosol-induced specimens. Consequently, a study was undertaken to compare the results of all aerosol-induced and spontaneous cough specimens prepared by the pick and smear and Saccomanno methods. Matched pairs of specimens from the same individuals were also compared. More specimens with atypias were obtained from the spontaneous sputa using the Saccomanno technique. More ferruginous bodies were found in the aerosol-induced specimens using the pick and smear method. The pick and smear method was more effective for detecting ferruginous bodies in both induced and spontaneous specimens.

The clinical significance of ferruginous bodies in sputa.

The clinical significance of ferruginous bodies in sputa was examined in a study of 674 former asbestos workers. Data from occupational histories and smoking behavior questionnaires, chest radiographs, spirometric measurements and counts of ferruginous bodies were obtained as part of a five-year surveillance program. Statistical analysis demonstrated that ferruginous bodies found in the sputa were significantly related to radiographic findings of interstitial pulmonary disease and pleural fibrosis and to spirometric findings of restrictive lung disease. Age and cigarette smoking were also found to be related to the presence of ferruginous bodies.

Ferruginous bodies in sputa of former asbestos workers.

Routine cytopathologic examinations were performed at six-month intervals on sputum specimens from 628 former asbestos workers and 138 control patients. The occurrence of ferruginous bodies in sputa is found to increase as a logarithmic function of the length of occupational exposure to asbestos in workdays. No significant association is found between the occurrence of ferruginous bodies and the worker's age, smoking history, degree of cellular epithelial atypia, or time since last exposure. We conclude that the presence of ferruginous bodies in sputa is evidence of probable significant occupational exposure to asbestos dust. Their absence does not indicate lack of exposure. We can also conclude that routine cytopathology procedures are sufficient for the detection of ferruginous bodies in sputa.