Manual method for liquid-based cytology: a demonstration using 1,000 gynecological cytologies collected directly to vial and prepared by a smear-slide technique.

We report on the formulation and use of an alcoholic-agar additive solution generally useful for rapid, inexpensive liquid-based cytology slide preparation. Gynecological cytology specimens were collected from 1,000 women. Two hundred fifty aliquots each of CytoRich, CytoRich Red (Tripath Imaging, Inc., Burlington, NC), Preservcyt (Cytyc Corp., Boxborough, MA), and DINA*TRANS (DINA*CYT Corp., Portland, OR) fixatives were used for this study. Fixed cell suspensions from 1,000 women, seen consecutively by a general obstetrics and gynecology practice, were vortex-mixed and transferred into a premeasured amount of alcoholic-agar in test tubes. Test tubes were conventionally centrifuged, cells were trapped in a spontaneously formed agar-gel, and the supernatant solutions were decanted. Vortex-mixing cell buttons caused a rapid gel-to-sol transition, affording viscous cell suspensions that were applied to slides, smeared, and stained using Papanicolaou stains. Slides showed unclumped, monolayered, uniform, random cell-spreads. All of the fixatives afforded crisp presentation of normal and abnormal cells. There was an about 3-fold increase in HSIL+ (0.7-1.8%) and LSIL diagnoses (1.3-4.4%), and a 45% reduction in ASCUS diagnoses (3.3-1.8%), as compared to our cytology laboratory's previous year's patients' statistics with a concurrent 0.2% unsatisfactory rate, due solely to inadequate sampling. This manual method makes liquid-based cytology inexpensive and does not require specialized preparative devices.

Specialized preparative devices are not needed for liquid-based, thin-layer cytology: an alternate manual method using a metastable alcoholic gel.

The blending and use of a metastable alcoholic gel is described. The gel creates durable, uniform suspensions of cells and small aggregates of cells. It allows the random, uniform transfer of virtually any liquid-fixed cytology specimens to a glass slide without the use of specialized devices, proprietary disposables, or adhesive-coated glass slides. The method is inexpensive and can be performed in a timely fashion, not impacting laboratory turnaround times or requiring the specimen to be processed by other laboratories. It empowers cytology laboratories of any size or level of complexity to make slides from readily available commercial cytology fixatives. It allows the total contents of liquid-based cytology specimen vials to be examined, providing a "cytological method" for quality assuring liquid-based cytology samples.

Performance characteristics of the Indiana University Medical Center endometrial sampler (Tao Brush) in an outpatient office setting, first year's outcomes: recognizing histological patterns in cytology preparations of endometrial brushings.

Following successful hysterectomy-controlled trials, Tao brush endometrial cytology with liquid fixation was offered to clinicians for office use. This report recounts our first year's cytology and correlative histology outcomes. One hundred thirteen cases were accrued. Correlative tissue examinations comprising Pipelle (Prodimed, Neuilly-en-Thelle, France) biopsy, hysteroscopy and biopsy, dilatation and curettage, and hysterectomy were available at this institution for 59 cases. In 42 cases, cytology diagnoses could be compared to histology diagnoses. Twenty-five of 63 normal brushings were followed up. Fourteen were normal. Eleven Pipelle biopsies of cytologically atrophic endometrium were quantitatively limited and insufficient for diagnosis. Thirty-seven cases were abnormal, and 15 of these showed nuclear anaplasia. Twenty-eight of the abnormal cases were followed up. All correlative tissue examinations confirmed an abnormality. All 15 cases with nuclear anaplasia showed significant histopathology comprising atypical endometrial hyperplasia, endometrial intraepithelial neoplasia (EIN), endometrial intraepithelial carcinoma (EIC), and invasive adenocarcinoma. There were 13 inadequate endometrial brushings. Three cases had insufficient cellular material. The remaining 10 cases were cellular but were chiefly cervical/endocervical samples. Two of the cellular cases resulted from clinicians failing to replace the protective sheath over the brush bristles before removing the Tao brush from the endometrial cavity. The remaining 11 cases resulted from inaccessibility of the uterine cavity due to a tight or stenotic cervix. The Tao brush is a reliable uterine sampling device that performs well as a diagnostic tool for outpatient assessment of the endometrium of women with patent cervices. An advantage of endometrial cytology is that it accurately represents atrophic endometrium, and it is an effective case-finding tool for EIN and EIC. Women with tight or stenotic cervices are poor candidates for endometrial brushing, and may experience pain if the procedure is attempted. Diagn. Cytopathol. 2000;22:186-195.

Liquid-based cervical-cell collection with brushes and wooden spatulas: a comparison of 100 conventional smears from high-risk women to liquid-fixed cytocentrifuge slides, demonstrating a cost-effective, alternative monolayer slide preparation method.

Our purpose was to demonstrate that 17.5-mm liquid-based cytocentrifuge circle-slides made using a Hettich (Andreas Hettich Co., Tuttlingen, Germany) cytocentrifuge are at least as efficacious as conventional smears for detecting cervical abnormalities. One hundred conventional smears were collected with cytobrushes and wooden spatulas from high-risk women. Both devices were then placed into CytoRich Red (AutoCyte, Inc., Burlington, NC). Cells were concentrated from CytoRich Red by centrifugation and suspended in CytoRich Yellow. Two 17.5-mm circle-slides were produced, compared to each other in order to test reproducibility of diagnoses between slides, and compared to conventional slides. Sixty-five normals, three ASCUS, three LSIL, and two HSIL matched. Overall, cytocentrifugation yielded 27 additional findings among 25 cases. Also, it downgraded one conventional ASCUS to normal (immature metaplasia and chronic inflammation) and one conventional HSIL to normal (transitional cell metaplasia). Cytocentrifugation of 14 conventional normals afforded four ASCUS and 10 LSIL. Cytocentrifugation of 11 conventional ASCUS afforded nine LSIL and two HSIL (one with AIS). One HSIL + AIS was found with a conventional HSIL. Cytobrushes and wooden spatulas can be used to collect material for liquid-based cervico-vaginal cytology if they are placed into CytoRich Red. Hettich cytocentrifuge slides were more reliable than conventional smears in presenting well-dispersed, sharply imaged cells, affording greater diagnostic sensitivity and certainty. Compared to previous split-sample studies, three different outcomes were noted: 1) the endocervical component was consistently well-represented; 2) AIS was seen more often in liquid-based slides; and 3) the liquid-based slide did not underdiagnose its conventional companion. The liquid-based preparations downgraded one ASCUS to normal and one conventional HSIL to transitional-cell metaplasia. Histology correlation of these two cases agreed with the liquid-based cytology diagnoses, which validates their specificity. These improvements are ascribed to differences in fixation and processing. The low cost, ease of operation, and reusable chambers of the Hettich cytocentrifuge make it a cost-effective liquid-based cytology instrument, especially for small to intermediate-size laboratories. The claims that this paper makes must be proven by additional studies, and test implementation needs to be scrutinized by appropriate regulatory agencies whose standards may vary from country to country. Diagn. Cytopathol. 2000;22:86-91.

Rare epithelial sheets with "cancer-like" nuclei presenting against a background of cytologically normal-appearing endometrial epithelium in endometrial brush samplings: establishing a differential diagnosis.

Liquid-fixed Tao brush samples showing small quantities (less than 10%) of endometrial epithelial sheets with cancer-like nuclei that are admixed with benign endometrium raise a concern about false-positive cytology interpretations. This paper details 7 cases along with the outcomes of three prior papers that touch on the differential diagnosis of "small amounts of atypical epithelium with cancer-like nuclei." Liquid-fixed, cytocentrifuge-processed Tao brush endometrial samples from 7 women showing "small amounts of atypical epithelium with cancer-like nuclei" were followed up by hysterectomy. Clinical presentations, diagnostic tests, surgical procedures, and tissue outcomes are detailed. Four women had hyperplastic polyps (three with focal atypical complex hyperplasia, and one with focal atypical simple hyperplasia). One had endometrial microcarcinoma. One had p53-positive endometrial intraepithelial carcinoma (EIC). One had endometrial intraepithelial neoplasia (EIN). The differential diagnosis of the index cytological finding, "small quantities of endometrial epithelial sheets with cancer-like nuclei admixed with benign endometrium," includes hyperplastic polyp, EIC, microcarcinoma/EIN, and carcinoma metastatic to the endometrium. Combining endometrial brushing, endovaginal sonography/sonohysterography, and selective immunostaining may be useful for both detecting and sorting out these lesions. Diagn. Cytopathol. 1999;21:378-386.

Endocervical cell collection using cytobrush, liquid-fixation, and cytocentrifugation: A feasibility study using 455 hysterectomy specimens.

Differential uterine curettage may be performed for the evaluation of abnormal uterine bleeding. The purpose of this study was to test the feasibility of substituting cytological methods for differential uterine curettage to evaluate the entire glandular axis of the uterus and the transformation zone. In order to simulate differential uterine sampling, the endocervix was brushed in 455 uteri from which an endometrial cytology sample had been previously collected. Twenty-five percent of the brushes' cell-sample was examined using two 17.5 mm circle-slides prepared with a Hettich cytocentrifuge. The resulting slides were homogeneous and cellular. They were compared to tissue sections of matched hysterectomy specimens. Endometrial adenocarcinoma was overrepresented in the endocervical cytology preparations. This was ascribed either to prior endometrial collection with "drop-down contamination" of the endocervix and/or to entry of the cytobrush into the endometrial cavity. Otherwise, cytological and histological diagnoses were comparable for squamous and endocervical-adenomatous lesions. Under the conditions of this simulation, endometrial and endocervical brushings appeared to complement each other as case-finding techniques for evaluating abnormal uterine bleeding. Diagn. Cytopathol. 1999;21:419-426.

Histology correlation with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) cytology diagnoses: An argument to ensure ASCUS follow-up that is as aggressive as that for LSIL.

The purpose of the present study was to compare histology outcomes of cytological atypical squamous cells of undetermined significance (ASCUS) to the histology outcomes of cytological low-grade squamous intraepithelial lesion (LSIL). Cases with a cytology diagnosis of ASCUS and LSIL that had correlative histology obtained within 3 mo of the cervico-vaginal smear were accrued from the files of the Cytology and Histology Departments of Mercy Hospital Medical Center (Des Moines, IA). All of the cytology cases were examined by a cytotechnologist, a cytopathologist, and the histopathologist who signed out the biopsy material. The laboratory's benign: ASCUS: LSIL: HSIL + ratio was 94.0%: 3.8%: 1.5%: 0.7% for the time period of this analysis; its ASCUS:SIL proportion was 1.76. Histology correlations to 249 ASCUS cases showed 45 (18%) CIN2s (cervical intraepithelial neoplasm 2) or CIN3s, 134 (54%) CIN1s, and 70 (28%) nonneoplastic biopsies. Ten of 24 (42%) ASCUS cases that showed histological CIN3 and seven of 21 (33%) ASCUS cases that showed histological CIN2 contained atypical immature squamous metaplastic cells, while the remainder showed squamous cells with mature, superficial/intermediate-type cytoplasm. Histology correlations to 576 LSIL cases showed 96 (17%) CIN2s or CIN3s, 397 (69%) CIN1s, and 83 (14%) nonneoplastic biopsies. One hundred forty-six (59%) ASCUS and 424 (74%) LSILs had histological koilocytosis with nuclear atypia (KA); the sole histological finding of KA was seen among 76 (30.5%) ASCUS and 146 (25.3%) LSIL cases. The majority of cytological ASCUS and LSIL cases selected by clinicians for biopsy have histological CIN and a substantial minority show CIN2 and CIN3, which, in the case of ASCUS, is frequently associated with atypical immature squamous metaplastic cells. There is a high prevalence of histological KA among ASCUS and LSIL cases. Diagn. Cytopathol. 21:292-295, 1999.

Endometrial brush cytology of advanced postmenopausal endometrium: does endometrial intraepithelial neoplasia exist in the absence of hyperplasia?

Postmenopausal uterine bleeding is an indication to sample the endometrium for diagnostic purposes. The endometrial brush cytologies of 20 advanced postmenopausal women collected at the time of hysterectomy in order to benchmark the expected morphology of postmenopausal endometrial brushings were reviewed. No women had symptoms or gross findings of primary endomyometrial disease. Endometrium was collected at the surgical pathology laboratory using the Tao Brush and CytoRich Fixative System. After formalin fixation of the uterus, the entire endometrium was embedded for routine histology. Sixteen endometrial brushings and matched endometrial sections showed endometrial atrophy, one brushing showed many ciliated epithelial cells, and three brushings showed focal (less than 10%) epithelial-cell atypia. In two atypias, abnormal endometrial epithelial-cell sheets contained enlarged, clear nuclei with nuclear notches and grooves resembling papillary thyroid cancer. One case showed no histological counterpart to this finding. The other case showed thickening of the pericornual fundic endometrium with cystic glands. The third case with epithelial atypia showed abnormal endometrial-cell sheets with nuclei resembling atypical hyperplasia or type I endometrial adenocarcinoma; corresponding endometrial tissue sections showed rare, irregular glands and back-to-back gland clusters with equivalent nuclear features. Atypical epithelium may be found in atrophic uteri in the absence of gross endometrial thickening. This may be a common event related either to de novo intraepithelial dysplasia in a noncycling endometrium or to hyperplasia that has partly regressed with estradiol withdrawal. This study shows that, in addition to endometrial intraepithelial carcinoma (EIC), isolated atypical glands with morphological and immunohistochemical features of atypical hyperplasia or type I endometrial adenocarcinoma may be found in grossly normal advanced postmenopausal endometrium of asymptomatic patients. This atypical epithelium is readily apparent in endometrial brush preparations, but requires serial sectioning of the endometrium to be demonstrated histologically. We have not established the natural history of this lesion, and in the absence of EIC or gross endometrial thickening indicative of atypical hyperplasia, we do not know whether this degree of epithelial atypia should be an indication for hysterectomy.

Endometrial intraepithelial carcinoma diagnosed by brush cytology and p53 immunostaining, and confirmed by hysterectomy.

Endometrial intraepithelial carcinoma (EIC) is associated with tumors of high nuclear grade and usually is seen in endometria without complex atypical hyperplasia. Tumor cells oftentimes show an accumulation of mutant p53 protein. This report describes a case of EIC detected by endometrial brush cytology and p53 immunostaining, and confirmed by hysterectomy.

Ciliated cell adenocarcinoma of the endometrium diagnosed by endometrial brush cytology and confirmed by hysterectomy: a case report detailing a highly efficient cytology collection and processing technique.

A case of ciliated endometrioid adenocarcinoma of the endometrium, diagnosed by endometrial brush cytology and confirmed by histology, is reported. Features are those of outspoken endometrial adenocarcinoma presenting in a postmenopausal woman with abnormal uterine bleeding. Distinctive characteristics include ciliated neoplastic cells, intracytoplasmic ciliary cysts, complex epithelial stratification, and muscular invasion by malignant ciliated cells in the matched hysterectomy specimen. This report illustrates the merit of linking an easy-to-use liquid fixative with brush-sampling of the endometrium.

Liquid fixation of endometrial brush cytology ensures a well-preserved, representative cell sample with frequent tissue correlation.

Endometrium was obtained by brushing 656 hysterectomy specimens with a MedScand cytobrush (Hollywood, FL) in order to evaluate linking an easy-to-use liquid fixative with brush-sampling of the endometrium, and to determine the fixative's operating characteristics when applied to brush-sampling the endometrium. Liquid-fixed brush-samples yielded 4 to 6 slides per case, and any one slide accurately represented the case diagnosis. Tissue fragments were incidentally collected by the brush in 524 cases (79.9%), were least often obtained from senile and weakly proliferative endometria, and were always obtained from high-grade endometrial hyperplasias and carcinomas. With tissue fragments obtained by brush-sampling, the diagnosis matched that of the hysterectomy endometrium. Cytology alone separated benign endometrium from high-grade atypical hyperplasia and carcinoma, showing that clinical decision-making can be based on endometrial brush cytology collected into a liquid fixative. Furthermore, the addition of tissue-fragment histology increased the diagnostic accuracy of the endometrial brushing procedure to 92.5% overall and 100% for high-grade atypical hyperplasias and carcinomas. At this juncture, we advocate endometrial brushing as a sensitive and generally specific case-finding technique; and perhaps as experience teaches us to distinguish low-grade nonatypical hyperplasias from purely physiological changes of the endometrium, it may become accepted as a definitive diagnostic method. The advantages of examining a liquid-fixed suspension are that 1) because these preparations are homogeneous from slide to slide, with any one slide affording the same diagnosis as any other slide from the same case, the number of slides examined may be greatly reduced, and 2) tissue fragments can be used to quality-control the cytology diagnosis in the majority of cases, especially in cases of endometrial hyperplasia and carcinoma.

Hepatocellular injury induced by chronic low-dose CCl4 in spontaneous and renal hypertensive rats: a correlation to the reversal of experimental rat hypertensive models.

Renal hypertension has been reduced to normal levels in the rat with subcutaneous injections of carbon tetrachloride (CCl4). Previous light microscopic evaluation of liver sections demonstrated varying degrees of hepatocellular and hepatic parenchymal injury secondary to CCl4 administration. In normotensive rat populations, saline injections have been shown not to cause hypertension, and the treatment of normal rats with subcutaneous CCl4 did not change their blood pressure over that of noninjected controls. In this study we compare the extent of blood pressure reduction with the degree of CCl4-induced liver injury in the renal-induced, spontaneous hypertensive rat (SHR), and normotensive rat. Statistically significant blood pressure reduction followed CCl4 injection of renal (p less than .01) and SHR (p less than .001) hypertensive animals. The morphological liver injury appeared most sensitively reflected in hepatocellular nuclear atypia. The SHR animals were the most resistant to CCl4 liver damage by chronic CCl4 treatment. There was no measurable effect of CCl4 treatment on the animals as assessed by their activity, rate of development and weight gain. The degree of blood pressure reduction due to CCl4 treatment in the SHR animals was similar to that previously reported in DOCA and renal-induced hypertensive animals.

Aspiration biopsy of the prostate gland: a brief review of collection, fixation, and pattern recognition with special attention to benign and malignant prostatic epithelium.

The preparation and use of an alternate collection device for aspiration biopsy of the prostate is presented. The device is inexpensive and can be constructed from readily available office materials. Its use in conjunction with a liquid-phase collection of cellular products of aspiration biopsy by a backwash technique is described. Following this, a brief discussion of pattern recognition in cytological preparations of benign and malignant prostate epithelial aggregates is presented and illustrated.

Aspiration biopsy of the prostate gland.

Aspiration biopsy is a rapid and relatively painless technique for evaluating an abnormal prostate gland. It can be performed on outpatients without anesthesia and is easily tolerated. It sensitivity in detecting cancer is at least equivalent to that of core biopsies, in part because several areas of the prostate may be sampled in a relatively atraumatic fashion. Few complications are encountered. Adequate performance of aspiration biopsy of the prostate requires experience and diligence, both by the urologist, in obtaining adequate specimens, and by the processing laboratory and pathologist, in interpreting these specimens. It is hoped that aspiration biopsy will lead to the early diagnosis of prostatic cancer, effecting lower morbidity, reduced hospital cost, greater ease in performing radical prostatectomy, and more accurate techniques for predicting the prognosis in affected patients.

Prostatitis and aspiration biopsy cytology of prostate.

Aspiration biopsy of the prostate is contraindicated when active prostatitis is present. However, we have found prostatitis in 45.2 percent of 250 consecutive aspiration biopsy specimens, confirming that urologists have difficulty in distinguishing prostatitis from carcinoma on a clinical basis. The epithelial changes accompanying prostatitis also may be confused with carcinoma. We review the microscopic features which distinguish prostatitis from carcinoma. Because 14.1 percent of the patients with prostatitis in this study also had carcinoma, repeat follow-up biopsy is necessary if suspicion of carcinoma persists after adequate therapy for prostatitis.

Is cytology capable of adequately grading prostate carcinoma? Matched series of 50 cases comparing cytologic and histologic pattern diagnoses.

A perceived disadvantage of fine-needle aspiration biopsy of the prostate is that aspiration biopsies cannot be correlated with current tissue-grading systems. We compared patterns of cellular arrangement among 50 cases of prostate carcinoma studied in histologic and cytologic specimens obtained simultaneously. Cell patterns were independently scored in a semiquantitative fashion on both histologic and cytologic material, and "predicted Gleason scores" were assigned to the cytologic specimens. When carcinomas were classified as well differentiated (Gleason score 2 to 4), moderately differentiated (Gleason score 5 to 7), or poorly differentiated (Gleason score 8 to 10), there was 84 per cent agreement between histology and cytology. In no instance was there overlap between well-differentiated and poorly differentiated adenocarcinoma in the matched specimens. We believe that it is possible to accurately predict tissue patterns of prostate carcinoma and to estimate Gleason scores in aspiration biopsy specimens.

Divergent differentiation in neoplasms. Pathologic, biologic, and clinical considerations.

In this rather brief review, we have illustrated and discussed selected examples of dual or multiple differentiation within certain neoplasms. In so doing, we have attempted to present those cases which highlight common diagnostic problems and which have important biologic and clinical implications. We have shown how tumors arising in many organs such as the lung, gastrointestinal tract, and thyroid can simultaneously manifest combined endocrine-nonendocrine differentiation, and we have addressed this phenomenon in relation to the "parent cell of origin" theory. We have emphasized that while many tumors do in fact resemble the tissues in which they arise, the possession of a particular phenotype, such as endocrine, does not necessarily imply that the tumor in question was derived from an endocrine progenitor cell. To reinforce the concept that the histomorphologic, biochemical, and functional properties of tumor cells reflect ongoing differentiation events rather than a static situation, we have presented examples of tumors in which changes in phenotype have been observed during the course of disease, e.g., small cell carcinomas of the lung and prostate, as well as tumors in which divergent differentiation reflects similar lines of differentiation occurring in normal tissues, e.g., certain neurogenic tumors. Finally we have considered important clinical implications of mixed differentiation and changing phenotype and their impact on the pathologist.

Pathologic changes in murine leishmaniasis (Leishmania donovani) with special reference to the dynamics of granuloma formation in the liver.

The histopathologic changes in liver, spleen, and bone marrow of BALB/c mice infected for 6 months with Leishmania donovani are described. Granulomas were the most important histologic lesions found; and the dynamics of their formation, collagen deposition, and resolution in the liver were studied. The number of hepatic granulomas increased until the eighth week and then decreased steadily. In contrast to the liver granulomas, those of spleen and bone marrow do not mature or show collagen deposition. Actual granuloma counts in the liver support the idea that mature granulomas revert to poorly formed ones and finally resolve without scarring. BALB/c mice are suitable for the study of the dynamics of granuloma formation and resolution and the survival of L donovani in an in vivo system. The relevance of these changes to the pathology of L donovani infection in man are discussed.