Retrospective evaluation of gynecologic cytodiagnosis. II. Interlaboratory reproducibility as shown in rescreening large consecutive samples of reported cases.

Two laboratories exchanged and rescreened a large sample of cases with cervicovaginal smears they had consecutively accessioned to examine the reproducibility of gynecologic cytodiagnosis under optimum conditions. At least a "working agreement" (diagnoses within +/- 1 category on a ten-category scale) was achieved in diagnoses of normal, benign reaction and squamous abnormality (from minimal dysplasia though invasive cancer) in 18,859 cases (96.8%), of endometrial abnormality in 21 cases (42%) and of "unsatisfactory" in 99 cases (20.7%). Larger differences occurred in greater than or equal to 30% of cases except in the categories of "normal" and "benign reaction," reaching a maximum of 82% for moderate dysplasia. Reexamining 382 cases decreased disagreement by category to the 20% to 65% range only in the five categories of dysplasia plus carcinoma in situ. Agreement was not predicated on the presence of endocervical cells or squamous metaplasia; the basis for "unsatisfactory" calls was not uniform. Comparison of the laboratories' diagnoses with referee diagnoses or, on 178 cases, with tissue diagnoses also demonstrated differences in diagnostic criteria.

Retrospective evaluation of gynecologic cytodiagnosis. I. Reproducibility using an experimental diagnostic scale.

A system for the exchange of specimens between two independent laboratories ("A" and "B") was designed to enable them to review a consecutive series of one another's gynecologic smears. Another cytopathologist "refereed" diagnoses differing beyond agreed limits and examined all smears called carcinoma in situ or a more severe lesion by either laboratory. A ten-numeral gradient nomenclature scale was developed to code all cytodiagnoses for data processing. This analysis of the reproducibility of the experimental diagnostic system is based on all participants' examination of 100 selected specimens, half contributed by reviewing laboratory A and half by reviewing laboratory B, with the target value of each specimen established by the contributor. Intralaboratory reproducibility, expressed by a correlation coefficient (r), ranged from 0.78 to 0.93. After adjusting for straightforward squamous-cell lesions, r ranged from 0.88 to 0.91. Intralaboratory agreement +/- 1 scalar category, defined as a "working consensus," ranged from 77% to 84%. Interlaboratory correlation ranged from r = 0.74 to 0.94. Adjustment for squamous-cell lesions exerted less effect than it had on intralaboratory reproducibility. Interlaboratory agreement within one category ranged from 58% (laboratory B reviewing specimens contributed by laboratory A) to 92% (referee examining specimens contributed by laboratory B). Initially, laboratory A tended to classify a given specimen higher on the scale than did laboratory B and the referee, but as the study progressed, both laboratories tended to converge toward a more congruent diagnostic position, with referee greater than A greater than B. The level of discrimination attained by all participants validates use of this system to compare a larger series of diagnoses.

Recognition of a second serogroup of Legionella longbeachae.

A strain of Legionella longbeachae (Tucker 1) that was isolated from the postmortem lung tissue of a pneumonia patient was serologically distinct from four other strains of L. longbeachae. The recognition of a second serogroup of L. longbeachae represents the first reported instance of serogroup diversity within a species of Legionella other than L. pneumophila. The disease caused by the Tucker 1 strain does not seem to be readily distinguishable from that of pneumonia caused by other legionellae.

Comparison of written-visual and microslide-screening test measures in cytotechnology.

A study was conducted to ascertain the relationships among different competence assessment methods for cytotechnologists. Of primary concern was the extent to which examinations using multiple choice questions, color plate photomicrographs and filmstrip formats measure abilities in common with or predict performance levels on a practical, job-sample-microslide examination format in gynecologic cytopathology. The majority of cytotechnologists who volunteered for these tests were trained in schools of cytotechnology approved by the Committee on Allied Health Education and Accreditation (CAHEA) of the American Medical Association (AMA). School training, certification by the International Academy of Cytology (IAC) and by the American Society of Clinical Pathologists (ASCP) and participation in current continuing education activities were all associated with relatively higher test scores. The filmstrips format was least consistent in reflecting these distinctions. Overall, participants' scores in each test correlated to varying degrees with their scores on all other tests. Combinations of certain formats appear to have limited potential as performance evaluation methods; some of their drawbacks are presented.

Rapid demonstration of Legionella pneumophila in unembedded tissue. An adaptation of the Giménez stain.

The Giménez stain, originally developed for demonstrating rickettsiae, readily stained the Legionnaires' disease bacterium (Legionella pneumophila) in frozen tissue sections and smears of fresh or formalin-fixed lung tissue from patients who had confirmed Legionnaires' disease. With the Giménez procedure, the bacterium stained bright red against a blue-green background. The tissue Gram procedures also stained L. pneumophila in frozen sections and smears, but the staining reaction was weak, and these stains were neither as sensitive nor a consistent as the Giménez procedure.

Pathogenesis of acute Legionnaires' disease pneumonia.

Immunofluorescent study of the lungs in cases of fatal suspected acute Legionnaires' disease enabled confirmation of the presence of Legionella pneumophila. In addition, probable pathogenetic mechanisms that had not been as clearly visualized by light microscopy became apparent: the retrograde involvement of the larger bronchioles and proximal airways, invasion of the interstitium, extension to pleura, and lymphatic and hematogenous spread. Organisms were demonstrated to occur in the liver and spleen of one patient. The development of technics for the earliest possible diagnostic verification of Legionnaires' disease, with specimens obtained as untraumatically as practical from selected sites and screened by specific immunofluorescent microscopic examination, should contribute to patient survival.

Fatal pneumonia caused by Legionella pneumophila, serogroup 3: demonstration of the bacilli in extrathoracic organs.

An immunosuppressed patient with malignant lymphoma died of acute pneumonia caused by Legionella pneumophila. Bacilli of serogroup 3 were detected in areas of pnemonia, in a mediastinal lymph node, and in the liver and spleen by direct immunofluorescence done on tissue obtained at autopsy. That the extrathoracic fluorescent material represented intact bacteria rather than antigenic fragments or antigen-antibody complexes was confirmed by finding intracellular bacilli in the liver by electron microscopy. To our knowledge, this case represents the first example of fatal disease attributed to serogroup 3 L. pneumophila and the first case in which L. pneumophila has been demonstrated in extrathoracic organs.

"Pittsburgh pneumonia agent": a bacterium phenotypically similar to Legionella pneumophila and identical to the TATLOCK bacterium.

The "Pittsburgh pneumonia agent," isolated by Pasculle and co-workers from human lung tissue, has been cultured on artificial media and characterized. The "Pittsburgh" bacterium and the TATLOCK and HEBA bacteria have identical cultural, biochemical, and antigenic characteristics. They also have the same cellular fatty-acid composition, and DNA relatedness indicates that they belong to the same species.

A Legionella-like bacterium related to WIGA in a fatal case of pneumonia.

An unusual bacterium serologically related to a "rickettsia-like agent," designated previously as WIGA, was seen in lung tissue from a patient who died of pneumonia of unknown cause. A fluorescent antibody conjugate prepared with the WIGA organism, isolated in 1959, was used to stain the lung tissue. Enormous numbers of fluorescent bacteria in the lungs of this patient confirm the pathogenicity of this unusual bacterium.

Effect of various histological fixatives on fluorescent antibody detection of Legionnaires disease bacteria.

Human lung tissue containing the Legionnaires disease bacterium was fixed in seven different histological fixatives, processed, and embedded in paraffin. Deparaffined sections from each were stained by fluorescent antibody and by Dieterle silver impregnation. With the fluorescent antibody stain, the Legionnaires disease bacterium could be detected in tissues prepared with any of the fixatures, but the Dieterle silver impregnation was not satisfactory on Zenker-fixed tissues.

Ultrastructure of the Legionnaires' disease bacterium. A study using transmission electron microscopy.

The Legionnaires' disease (LD) bacterium appeared ultrastructurally identical in human lung, egg yolk membrane, and artificial media, seen as a blunt or tapering rod measuring 0.3 to 0.9 micron in diameter and greater than or equal to 2.0 micron long. Greatly elongated forms were commonly found in cultures and yold sac membranes after 5 to 7 days of growth but were only rarely seen in human lung. The LD bacterium was clearly prokaryotic. Prominent features included electron-lucent nucleoids interspersed among areas of well-defined ribosomes; cleanly circumscribed cytoplasmic vacuoles or granular inclusions; and a double envelope enclosure, each portion consisting of a triple-layered "unit" membrane, approximately 75 A wide. Division always occurred as a pinching, nonseptate process typical of bacteria with a double, gram-negative type of envelope. No definite structure was seen in the periplasmic space that might represent the peptidoglycan layer. These features of the LD bacterium confirm earlier reports of the gram-negative staining reaction of organisms obtained from cultures and preliminary evidence of their gram-negative ultrastructure. We found no unique features that would aid in the ultrastructural differentiation of the LD bacterium from other small gram-negative bacilli.

Pathologic findings in guinea pigs inoculated intraperitoneally with the Legionnaires' disease bacterium.

Tissues from guinea pigs inoculated intraperitoneally with the Legionnaires' disease (LD) bacterium were studied with light, immunofluorescent, and electron microscopy. The principal gross lesion was diffuse peritonitis of varying severity. Microscopically, the peritonitis of covered by a mixed inflammatory infiltrate of macrophages, neutrophils, fibrin, and cellular debris. Foci of inflammation and necrosis were consistently observed in the splenic parenchyma, and similar lesions were often found in the lungs, liver, lymph nodes, pancreas, heart, and other organs. Numerous LD bacteria were seen in the peritoneal exudate; fewer were found in disseminated lesions. In electron micrographs, the highest concentrations were seen in macrophages, with fewer organisms present in neutrophils or extracellular spaces. Although the lung is the primary organ konwn to be affected by Legionnaires' disease in humans, our findings indicate that the LD bacterium is capable of dissemination.

Pulmonary sequelae of acute Legionnaires' disease pneumonia.

Patients with acute Legionnaires' disease (LD) pneumonia may have persistent chronic pulmonary changes, as shown by the histologic appearance of specimens of lung from patients who had survived and autopsy specimens from patients who died after a protracted clinical course. Acute pneumonia was not seen in these lungs, and LD organisms could not be identified by the direct fluorescent antibody technique or the Dieterle silver impregnation strain; instead, there was organizing pneumonia with various degrees of interstitial inflammation and fibrosis. The LD pneumonia may fail to resolve, and the lung parenchyma in areas of previous acute inflammation is not restored to normal in some patients.

Ultrastructure of the agent of Legionnaires' disease in the human lung.

This report confirms the gram-negative ultrastructural characteristics of the Legionnaires' disease organism by direct examination of pulmonary tissue from six confirmed cases--two from the original Philadelphia epidemic of 1976 and four from more recent sporadic cases. All microorganisms seen in all six lungs were identical ultrastructurally and were predominantely within intra-alveolar macrophages, as previously observed by light microscopy. They appeared as short, blunt rods that were clearly prokaryotic; i.e., they had diffuse electron-lucent nucleoid areas interspersed among areas of well-defined ribosomes, a pinching nonseptic division, and enclosure within a double envelope consisting of two three-layer "unit" membranes, each approximately 75 A wide. This structure, together with a pinching division, is typical of gram-negative bacteria. The Legionnaires' disease organism multiples both intracellularly and extracellularly in tissue and has no unique ultrastructural features that would aid in its specific identification. These findings are compared with recent reports describing the ultrastructure of what was considered to be the Legionnaires' disease organism in yolk sac and culture medium, and in one human lung.

Legionnaires' disease. Pathological and historical aspects of a 'new' disease.

Autopsy tissues and protocols from 26 epidemiologically defined fatal cases of Legionnaires' disease occurring during the 1976 Philadelphia outbreak were reviewed. Consistent pathologic features were limited to the lung, where an acute pneumonia characterized by intra-alveolar exudation of neutrophils, macrophages, and fibrin was observed. An etiologic agent common to most of the victims of Legionnaires' disease was identified within the pneumonic process by application of the Dieterle silver impregnation stain. In some cases, other pulmonary histologic findings were noted, chiefly acute diffuse alveolar damage. However, the importance of acute diffuse alveolar damage is not understood.

Rescreening as a quality control procedure in cytopathology.

Many cytopathologists regard the 10% rescreening regulation of the Center for Disease Control and Social Security Administration as an unsatisfactory procedure for internal monitoring of cytoscreener performance. The principal deficiencies actually lie in the lack of specified performance criteria and an associated decision rule. By specifying these mission elements of a monitoring strategy are obtained as attribute acceptance sampling plans. The acceptance sampling approach to monitoring cytoscreener performance by rescreening "negative" slides id discussed.