Lambrecht P. Kok

PENILE STUDIES AND HETEROSEXUAL PARTNERS - PENISCOPY, CYTOLOGY, HISTOLOGY, AND IMMUNOCYTOCHEMISTRY

The male partners of 20 women with human papilloma virus (HPV) infection (subtypes 6, 11, 16, and 18) were studied. These men were unsuspecting and allowed investigation “for scientific reasons” in the context of the abnormal findings in their female partners. Peniscopy (study of the penis with a colposcope) was abnormal in 17 cases. In the cytologic‐brush preparations of the urethra, hyperkeratosis (visualized with the modified Papanicolaou technique) occurred in all cases, but koilocytosis in only one. Both urothelial cells and squamous cells in the urethral samples were positive for HPV, as shown in the immunocytochemistry. In the histologic sections taken from flat lesions there was hyperkeratosis, dyskeratosis, and abnormal nuclear maturation. The authors concluded that, using simple techniques, subtle disturbances in maturation of nuclei and cytoplasm can be established in penile epithelium of these sexual partners. Similar changes are established in other HPV lesions. It seems likely that a significant proportion of the male population is infected in countries were HPV is an important factor in cervical carcinogenesis.

MICROWAVE-STIMULATED STAINING OF PLASTIC EMBEDDED BONE-MARROW SECTIONS WITH THE ROMANOWSKY-GIEMSA STAIN - IMPROVED STAINING PATTERNS

Staining plastic sections with the Romanowsky-Giemsa method is both time-consuming and difficult. This paper reports how the staining time can be reduced to 25 min using microwave irradiation of the staining solution. It is shown that staining results depend on the fixative used, staining temperature, dye concentration and pH of the staining solution as well as on several parameters of the microwave irradiation technique. The staining patterns are improved when compared with those obtained by conventional staining of plastic sections. The colors are more brilliant and greater contrasts are observed. Basophilia, polychromasia, and orthochromasia accompanying red cell maturation are more pronounced. For white cell maturation the initial appearance of specific granules (neutrophil, basophil, and eosinophil) is more evident. Thus, cell classification is easily accomplished using the described technique. It is suggested that microwave-stimulated staining be considered for routine use.

Theory and practice of combining coagulant fixation and microwave histoprocessing

The German, F. Blum, introduced formalin as a fixative in 1893. Formalin rapidly became popular for hardening and preserving gross human and animal specimens. As a result, microscopy for diagnostic pathology by combining paraffin embedding and formalin fixation was developed. Alcohol-based fixatives have coagulation of proteins as their main preservative effect. Because there is no cross-linking, immunostaining is not compromised, and DNA and RNA is not damaged. Ethyl alcohol was used by Dutch scientists of the 18th century, but was replaced by the cheaper formalin. Addition of low molecular weight polyethylene glycol (PEG) optimized the coagulant fixative, Kryofix. The polyethylene glycol prevents excessive hardening and enhances the speed of coagulation of proteins. Kryofix was used on a large scale for skin biopsies in Leiden between 1987 and 2001. DNA preservation by the formulated coagulant fixative, BoonFix, is related to the concentration of ethyl alcohol, PEG and acetic acid. BoonFix has been used since 2004 in Leiden for over 40,000 diagnostic skin biopsies and more than 100,000 cervical samples. A literature review and three decades of experience with coagulant, formalin-free fixatives in pathology suggest that when health authorities realize that formalin invalidates expensive tests, it might eventually be eliminated legislatively from diagnostic pathology. Finally, coagulant fixation is optimal for microwave histoprocessing where ethyl alcohol is followed by isopropanol.

Human papillomavirus testing as a cytology gold standard: comparing Surinam with the Netherlands.

Polymerase chain reaction to detect high-risk human papillomavirus has been suggested as a gold standard for cytology. The Netherlands and Surinam were prospectively compared in regard to the proportions of Negative, Atypical Squamous Cells of Undetermined Significance, and Squamous Intraepithelial Lesion smears that had detectable high-risk human papillomavirus. For the Netherlands, 14 600 negative, 270 Atypical Squamous Cells of Undetermined Significance and 120 Squamous Intraepithelial Lesion smears were evaluated by polymerase chain reaction. For Surinam, 150 negative, 50 Atypical Squamous Cells of Undetermined Significance, and 150 Squamous Intraepithelial Lesion smears were evaluated by polymerase chain reaction. In all, 4% of Dutch and 80% of Surinamese negative smears had detectable high-risk human papillomavirus (χ2=1313, P<0.00001). In total, 41.9% of Dutch and 84% of Surinamese Atypical Squamous Cells of Undetermined Significance smears had detectable high-risk human papillomavirus (χ2=28, P<0.00001). Totally, 67.5% of Dutch and 94% of Surinamese SIL smears had detectable high-risk human papillomavirus (χ2=30, P<0.00001). The Negative: Atypical Squamous Cells of Undetermined Significance odds ratio was 0.058 for the Netherlands and 0.762 for Surinam (χ2homog=31, P<0.00001). The Negative: Squamous Intraepithelial Lesion odds ratio was 0.020 for the Netherlands and 0.255 for Surinam (χ2homog=31, P<0.00001). The Atypical Squamous Cells of Undetermined Significance: Squamous Intraepithelial Lesion odds ratio was 0.347 for the Netherlands and 0.335 for Surinam (χ2homog=0.005, P>0.75). Human papillomavirus DNA testing may not be a suitable gold standard in general because its use would make specificity and sensitivity prevalence-dependent. A new statistic, the percent of Negative pap smears with detectable high-risk human papillomavirus, is posited, which may be important if human papillomavirus DNA testing is used clinically.

STAINING OF THE NUCLEOLUS WITH PROTEIN AND RNA STAINS FOR AUTOMATIC-MEASUREMENT OF NUCLEOLAR SIZE IN PARAFFIN SECTIONS

In this paper two staining methods for automatic assessment of nucleolar profile area in paraffin sections of breast carcinomas using the IBAS 2000 image analyzer are reported. In the first method, the nucleolar proteins are stained with phloxine B, in the second the nucleolar RNA is stained with methyl green-pyronin. Both methods are fully reproducible. Statistically, the profile areas observed for the same patient by the two methods were found not to differ significantly. Because of their rigidity and their size in relation to section thickness, nucleoli are particularly attractive cell structures for quantitation, for instance in studies of variables that might be useful in establishing the prognosis for carcinoma patients.

Population-based cervical screening with a 5-year interval in The Netherlands. Stabilization of the incidence of squamous cell carcinoma and its precursor lesions in the screened population.

OBJECTIVE To evaluate the outcome of population-based cervical screening at 5-year intervals. STUDY DESIGN Results from the west region of the Netherlands (population 2 million) were used. The 1995-2000 round was compared with the first 2 years of the second (2001-2002). All results were prospectively collected in a central database. Positive cytologies and histoscores per 1,000 screened for preinvasive squamous cervical intraepithelial neoplasia (CIN) lesions and invasive squamous cell carcinoma were calculated. RESULTS In the first round, 378,081 women were screened; in the second round, 100,561 women were screened. In both rounds the youngest screenees had the highest cytoscores. Cytoscores in the first round did not differ significantly from those in the second. The histoscore for CIN 1 and 2 was 1.42 per 1,000 in the first round and 1.18 per 1,000 (NS, P < .01) in the second. The histoscore for CIN 3 was 2.07 per 1,000 in the first round and 2.13 per 1,000 (NS, P < .01) in the second. Histoscores for invasive squamous cell carcinoma remained virtually the same (0.16 per 1,000 in the first, 0.14 per 1,000 in the second round). CONCLUSION Population-based screening at 5-year intervals in the Netherlands may result in stabilization of positive cytology and of the incidence of CIN and (histologic) invasive squamous cell carcinoma. The program seems more cost effective than that of 2 decades ago, with a screening interval of 3 years.

Building and using a PACS in pathology and cytology

Applications of Picture Archiving and Communication Systems (PACS) in Pathology and Cytology are currently hardly used in the diagnostic process. Here we describe a system that, together with existing equipment, stores data from cervix smears, and aids the physician in the diagnostic process. The system comprises of a scanning system for the specimen and a multi-level storage system partly on disk and partly on optical media. The system adds additional benefits in the diagnostic process while at the same time provides a safe long-term storage and archive of the data. Due to the open nature and the cost-effectiveness of this solution applications of this system can be found not only in cervical cytology but also in pathology or other parts of medicine where image processing or storage is a major issue.