Archival Smears Research Articles

Cytological features of signet‐ring cell carcinoma of the lung: Comparison with the goblet‐cell‐type adenocarcinoma of the lung

Signet-ring cell carcinoma (SRCC) and goblet-cell-type adenocarcinoma (GCA) are mucin-producing lung adenocarcinomas. Primary SRCC shows an aggressive clinical course, whereas GCA shows infrequent distant metastasis, but more frequent intrapulmonary metastases resembling lobar pneumonia. To distinguish SRCC from GCA, this study investigated the respective cytological features of these lesions. We selected 10 cases each of SRCC and GCA from the archival imprint smears. We assessed them for the following 10 cytological features. Necrosis/debris was observed in 60% of the SRCC and 90% of the GCA. A mucinous background was observed in 10% of the SRCC and 90% of the GCA. Significant inflammation was observed in none of the SRCC and 80% of the GCA. Stromal cluster was observed in 30% of the SRCC and 70% of the GCA. Nuclear overlapping was observed in 50% of the SRCC and in all of the GCA. Single tumor cells were observed in 80% of the SRCC and 10% of the GCA. Honeycomb-like cluster was observed in none of the SRCC and 80% of the GCA. Prominent nucleolus was observed in 50% of the SRCC and 40% of the GCA. Nuclear membrane irregularity was observed in 70% of SRCC and 60% of the GCA. Nuclear pleomorphism was observed in all of the SRCC and none of the GCA. The cytological features of SRCC were the presence of single tumor cells and nuclear pleomorphism, whereas that of GCA were the presence of abundant mucin and significant inflammation in the background, and a honeycomb-like cluster.

Expression of Cytokeratin 19 and Protein p63 in Fine Needle Aspiration Biopsy of Papillary Thyroid Carcinoma

To analyze the immunocytochemical distribution of CK19 and p63 on archival cytologic smears of 27 papillary thyroid carcinomas (PTCs), 22 benign thyroid lesions and 5 malignant non-PTC lesions.Archival cytologic smears of 27 papillary carcinomas, 22 benign thyroid lesions and 5 malignant nonpapillary carcinomas were processed for immunocytochemical detection of CK19 and p63, and results were compared.CK19 showed strong cytoplasmic staining in 22/27 fine needle aspiration biopsies (FNABs) of PTCs, in 5 benign lesions and in 4 malignant lesions of the control group. p63 Positivity was present in FNABs of 20/27 PTC and in 1 FNAB of nodular hyperplasia. Eighteen FNABs of PTC (66.6%) showed both strong CK19 staining and p63-positive cells, whereas none of the control cases showed coexpression of CK19 and p63.Coexistence of strong CK19 positivity and p63-positive cells can enhance the cytologic diagnosis of PTC.

Digital Image Analysis of Mean AgNOR Area Objectively Discriminates between Reactive Plasmacytosis and Multiple Myeloma in Cytologic Smears from Bone Marrow Aspirates

Objective: To differentiate between reactive plasmacytosis (RP) and multiple myeloma (MM) in bone marrow aspirates by assessment of silver-stainable nucleolar organizer regions (AgNORs). Methods: Archival cytologic smears (n = 69) from bone marrow aspirates were retrospectively investigated. Twenty cases had RP and 49 cases MM (24 low-grade, 22 intermediate-grade and 3 high-grade MM). Diagnoses were confirmed by evaluation of the corresponding bone marrow trephine biopsies. Cytologic smears were subjected to AgNOR staining using a standardized silver-staining protocol and assessed by two independent clinical histopathologists using digital image analysis. AgNOR count and area were analyzed in 100 plasma cell nuclei per sample. Results: The mean AgNOR area was found to be significantly (p < 0.001) higher in low-grade MM (mean: 23.4 µm²; range: 19.2–26.7 µm²) than in RP cases (mean: 6.2 µm²; range: 3.8–8.4 µm²). In contrast, mean AgNOR count failed to disclose significant differences between low-grade MM (mean: 5.36; range: 1–7) and RP (mean: 3.68; range: 1–6). Conclusion: Digital image analysis of the mean AgNOR area might be used as a diagnostically useful adjunct to aid objectively in the challenging differentiation between RP and MM in bone marrow aspirates.

Cervical cytology biobanking: quality of DNA from archival cervical Pap-stained smears

Aims:Cervical cytology biobanking is a feasible concept in cervical pathology and could be an indispensable tool for fundamental and applied molecular biological research. PCR is a powerful molecular technique that...

High-risk human papillomavirus is present in cytologically false-negative smears: an analysis of “normal” smears preceding CIN2/3

Aims:Cervical screening, currently performed by cervical cytology, depends on the timely detection of malignant lesions for its success. The presence of high-risk human papillomavirus (hrHPV) is associated with an increased...

Does HPV-status 6–12 months after treatment of high grade dysplasia in the uterine cervix predict long term recurrence?

Background Women once treated for high grade cervical dysplasia have a high long term risk for developing new dysplasia or cancer. Objectives To investigate if human papilloma virus (HPV)-negativity after treatment of cervical dysplasia reduces the need for frequent long term follow up. Design Case/control study based on archival smears. Methods Women with cervical intraepithelial neoplasi (CIN)2-3, treated for dysplasia and with recurrence of CIN2+ more than 2 years after treatment were compared with controls without recurrence, matched for age and date of treatment. High risk-HPV-DNA were analysed with PCR from two archival smears per woman. Mean follow up time was 14.6 years. Results 24% (45/189) of cases and 11% (43/378) of controls were HPV-positive in any of two smears. Odds ratio (OR) = 2.5 (1.6–3.8). Conclusion HPV-status 6–12 months after treatment of high grade dysplasia is of limited value for the design of long term follow up.

Nucleic Acid Amplification of Mycobacterium tuberculosis Complex DNA from Archival Fine Needle Aspiration Smear Scrapings vs. Fresh Fine Needle Aspirates of Tuberculous Lymphadenitis

To assess the efficacy of the nucleic acid amplification (NAA) technique for Mycobacterium tuberculosis (MTB) complex from archival fine needle aspirate (FNA) smear scrapings of confirmed cases of extrapulmonary tuberculosis (EPTB) for a retrospective diagnosis of EPTB as compared to NAA from fresh FNA material from the same cases.Smear scrapings from 51 cases; 33 cases of tuberculous lymphadenitis (from patients who had undergone NAA 1 year before for MTB from fresh FNA material); 13 negative controls from nontuberculous, archival FNA smears; and 5 known acid-fast bacilli (AFB)-positive sputum smears, were subjected to NAA using the IS6110 primer sequence of M tuberculosis. Ziehl-Neelsen staining was done in all the smears.Of the 33 cases of tuberculous lymphadenitis, 15 (45.4%) were AFB positive and 18 (64.5%) AFB negative. MTB NAA was positive in 73.3% (11 of 15 AFB-positive cases) in the freshly aspirated material and was observed in 60% (9 of 15 AFB-positive cases) when done on DNA extracted from the archival smear scrapings of the same cases. Similarly, in the 18 AFB-negative cases, MTB NAA positivity was 72.2% (13 of 18) on fresh material and 44.4% (8 of 18) on archival smear scrapings from the same AFB-negative cases. Overall NAA positivity was 51.5% for archival smear scrapings as compared to 71% for fresh FNA of the same cases.Low NAA sensitivity of MTB DNA in archival material of known tuberculous cases limits the routine use of NAA based retrospective molecular diagnosis of MTB complex.

HER-2/neu Evaluation by Fluorescence in Situ Hybridization on Destained Cytologic Smears from Primary and Metastatic Breast Cancer

To evaluate HER-2/neu amplification by fluorescence in situ hybridization (FISH) (HER-2/neu by FISH) on archival cytologic smears stained with May-Grünwald-Giemsa (MGG) stain.Cytologic specimens from 69 breast cancer lesions (48 primary and 21 metastatic), stained with MGG stain for routine diagnostic cytology, were destained and subjected to HER-2/neu by FISH. Fifteen of the 69 samples were also evaluated by FISH on paired fresh smears.HER-2/neu by FISH was successfully assayed in 25 of the 48 primary tumors and in 15 of the 21 metastatic lesions, corresponding to an overall feasibility of 58%. These cases had been archived between 1 month and 10 years prior to FISH analysis. Eight of the 25 primary and 5 of the 15 metastatic tumors were amplified. In 15 of the 40 evaluable cases, HER-2/neu was also assessed on the corresponding fresh smears: 8 tumors were amplified and 7 unamplified on both destained MGG and fresh smears.HER-2/neu can be detected by FISH on routinely MGG-stained cytologic slides. This approach allows HER-2/neu evaluation whenever histologic sections or fresh cytologic material are not available. In these cases, HER-2/neu assessment on destained cytologic smears plays a role in the selection of targeted therapy.

P63 immunostaining in destained bronchoscopic cytological specimens

The p53 homologous squamous stem-cell regulatory protein p63 is expressed in squamous carcinomas but is not characteristically detected in small-cell carcinomas (SCCs). A panel of thyroid transcription factor (TTF) 1 and p63 has been shown to be useful in distinguishing SCCs from poorly differentiated squamous carcinoma of the lung (PDSLC) in small biopsies and cytological cell blocks. Because tumor samples frequently are limited to cytological smears, we attempted to detect p63 in destained slides from a spectrum of pulmonary malignancies. Archival alcohol-fixed smears from 60 cases of cytologically diagnosed malignancies in bronchoscopically (n = 59) or fine-needle aspiration-obtained specimens (n = 1) were destained in acid alcohol, postfixed in 10% formalin, subjected to citrate-based antigen retrieval, and immunostained by exposure to anti-p63 monoclonal antibody 4A4, followed by reagents from a streptavidin-biotin immunoperoxidase kit, and diaminobenzidine as the chromogen. Postfixation in 10% formalin was found to be necessary for immunostaining. Normal ciliated and goblet cells were p63 negative, but reserve cells were p63 positive. All cases of squamous-cell carcinoma were positive for p63. Of 10 tumor samples originally diagnosed as SCC, only 6 samples were p63 negative and 4 samples exhibited positive staining. However, proper interpretation of the immunohistochemical (IHC) staining pattern and careful scrutiny of the cytological features and biopsy specimens in three of four cases led us to reclassify three cases into PDSLC. All adenocarcinomas (ACAs; n = 12), large-cell carcinomas (n = 4), and metastatic ACAs (n = 5) were p63 negative. Positive staining was seen in 9/16 tumors designated as non-SCCs; these tumors were not classified further into distinct histological categories.p63 staining in destained slides may be of value in facilitating the differential diagnosis between PDSLC and SCC. Criteria for conservative interpretation of results are discussed and include examination of reserve cells and ciliated cells on the same slide as internal positive and negative controls.

Significance of MiB-1 staining in smears with atypical glandular cells.

MiB-1 immunostaining may facilitate recognition of developing adenocarcinoma in cervical smear screening. In a retrospective analysis of prospectively collected data of 170 patients with atypical endocervical glandular cells and with repeat smears, archival Papanicolaou-stained smears were restained for MiB-1 and classified for the presence of preneoplastic changes of MiB-1 positive epithelial fragments. The results of classification based on MiB-1 positive epithelial fragments corresponded "roughly" with cytomorphological diagnoses. Of the 38 patients in which the primary smear was found to be MiB-1-positive, 12 of these persisted in the repeat smear. Thirty-eight of the 132 repeat smears of patients originally negative for MiB-1 pathology were positive. Of the total 50 MiB-1 positive repeat smears, four showed adenocarcinoma in situ on cytomorphological grounds. MiB-1 staining enhances detection of (pre)neoplastic changes. This approach does not destroy the morphology of the original smear and can be applied to routine material.

Type-specific associations of human papillomavirus load with risk of developing cervical carcinoma in situ.

We have previously shown that high human papillomavirus (HPV) 16 load in Papanicolaou smears negative for dysplasia is strongly associated with risk for carcinoma in situ (CIS) of the cervix. Here we study the amount of HPV DNA for some of the most frequent high-risk HPV types as determinants of progression to cervical CIS. Real-time PCR is used to estimate the normalized viral load of HPV 16, 18, 31, 33, 35, 39, 45, 52, 58 and 67 in 457 cases of cervical CIS and 552 matched population controls. A total of 2,747 archival Pap smears from gynecologic health examinations, collected over a period of up to 26 years, were analyzed to assess viral load during the infection history. Cervical smear samples differ widely in amount of DNA, underscoring the need for normalization of HPV load to number of cells in the sample. The risk of developing cervical CIS increases with higher viral load for most of the HPV types studied. The range of copy numbers per cell does not differ between HPV types but the odds ratio for CIS in the percentile with highest viral load is substantially higher for HPV 16 (OR = 36.9; 95% CI = 8.9-153.2) than for HPV 31 (OR = 3.2; 95% CI = 1.1-9.1) or HPV 18/45 (OR = 2.6; 95% CI = 1.0-6.4). Therefore, HPV viral load may be predictive of future risk of cervical CIS at a stage when smears are negative for squamous abnormalities, but differences between HPV types need closer attention.

Optimal fixation conditions for immunocytochemical analysis of estrogen receptor in cytologic specimens of breast carcinoma.

The techniques for immunostaining estrogen receptor (ER) in cytologic specimens have varied, as have the detection rates. The authors compared various fixation methods for their effect on ER detection in cytologic smears of breast carcinoma. Smears were prepared by gently scraping the cut surfaces of 47 resected breast carcinoma specimens and placing immediately in 1 of the following conditions: 1) a sequence of 10% formalin-methanol-acetone fixatives at -20 degrees C (Abbott method); 2) 10% formalin at room temperature; and 3) Carnoy's fixative at room temperature and then Papanicolaou stained (Carnoy's-Pap). Destaining of Carnoy's-Pap smears (Carnoy's-dPap) was initially attempted before ER staining. One set of smears was also air-dried for 3 minutes before using the Abbott method. Smears and corresponding tissue sections were immunostained with anti-ER antibody 6F11 using a similar protocol except for antigen retrieval, which was not initially applied on cytologic slides. All the ER-negative smears that had been fixed with 10% formalin or Carnoy's-Pap were restained after antigen retrieval. Agreement between cytologic and histologic findings was expressed by both concordance and the kappa coefficient. ER detection in smears processed with the Abbott method correlated best with findings from tissue samples, with an overall correlation of 91.5% (kappa = 0.80). Findings from air-dried smears were less optimal (concordance, 84.4%; kappa = 0.65), followed by Carnoy's-Pap (concordance, 71.4%; kappa = 0.45), formalin (concordance, 31%; kappa = 0.05), and Carnoy's-dPap (concordance, 29.4%; kappa = 0.04). Antigen retrieval converted most of the ER-negative smears to positive (18 of 20 smears in formalin and 6 of 8 smears in Carnoy's-Pap), leading to a final concordance of 93% and kappa = 0.83 for both conditions. Antigen retrieval also led to stronger staining intensity without causing false positivity. Antigen retrieval was found to greatly improve ER immunodetectability and staining intensity in formalin-fixed and Carnoy's-Pap smears. The former may offer an alternative to the Abbott method because of its easiness and the latter can be reliably used in archival Pap-stained smears for retrospective analysis of ER. Air-drying, destaining Pap smear, and fixation in formalin or Carnoy's-Pap without antigen retrieval are not recommended.

A retrospective review on atypical glandular cells of undetermined significance (agus) using the Bethesda 2001 classification

Objectives The Bethesda system for reporting cervicovaginal cytologic diagnoses was recently revised in 2001. Pathologists are required to report not only whether the smear favors neoplastic changes, but also the origin of the abnormal cells. In this study, archival smears were reviewed to evaluate the usefulness of the new classification. Methods Smears having atypical glandular cells taken between January 1995 and December 1997 were reviewed and subclassified according to the revised Bethesda classification. Case records were then reviewed and cases with discrepancies between the cytological evaluation and corresponding final histological diagnoses were further reviewed. Results There were 138 smears reviewed. The mean age of the patients was 47 (range, 18 to 78). Thirty-four smears favored neoplasia and 104 favored “NOS” (“not otherwise specified”). Sixty smears favored endocervical origin and 78 endometrial origin. Forty-three patients (31%) had significant pathologies, including 12 (8.7%) patients with high-grade CIN, 2 (1.4%) with low-grade CIN, 5 (3.6%) with HPV infection, 7 (5.1%) with carcinoma of the corpus, 1 (0.7%) with cervical adenocarcinoma in situ, 4 (2.9%) with adenocarcinoma of the cervix, 3 (2.2%) with endometrial hyperplasia, and 5 (3.6%) with carcinoma of the ovary. Two (1.4%) patients had double primary female genital malignancies and 2 patients (1.4%) had extragenital malignancies. Significant correlation was found between smears “favor neoplasia” and a final diagnosis with significant pathology (χ 2 test, P < 0.05). Significant association was found between AGC favored endocervical origin and a final diagnosis with cervical diseases (χ 2 test, P < 0.05). Four of the 43 patients who had significant pathologies had lesions found during their subsequent visits and all of them had cervical smears classified as AGC “favor neoplasia”. Conclusions AGC found on cervical smears are an indication for early and intensive investigation.

Fine‐needle aspiration cytology and immunocytochemistry in the diagnosis of 24 gastrointestinal stromal tumors: A quick, reliable diagnostic method

The diagnosis of gastrointestinal stromal tumors (GISTs) is generally established on histopathologic examination of surgical specimens. Fine-needle aspiration (FNA), performed under the guidance of ultrasound or computed tomography, is being used with increasing frequency in an attempt to diagnose primary and/or metastatic GISTs before surgery. The present study was undertaken to characterize the cytological appearance of these tumors and to assess the role of cytology, together with immunocytochemistry (ICC), in the diagnosis of GISTs. Twenty-four GISTs diagnosed by FNA cytology at our institution have been reviewed. Immunocytochemical studies with c-kit and CD34 were performed in all cases on current or archival Papanicolaou-stained smears. All cases stained with c-kit, and 19 reacted with CD34. Cytomorphology and immunocytochemical characteristics are discussed. Our results confirm the utility of FNA together with ICC in the diagnosis of primary and/or metastatic GISTs.

Usefulness of Silver Intensification of Immunostaining for Cytologic Diagnosis of Primary Melanoma of the Female Genital Organs

To improve the procedure for diagnosing vaginal melanoma with cytopathologic analysis of HMB-45. The study examined silver intensification of immunostaining of HMB-45 in nine cases of primary melanoma of the vagina and vulva using archival Papanicolaou-stained smears. All nine samples showed positive staining for HMB-45. Five cases showed intensive staining, two moderate and two weak. The positive staining was black in the cytoplasm of melanoma cells but was detected in neither the background nor normal squamous cells. Though destaining of Papanicolaou stain was not performed before immunostaining, the positivity of immunostaining was easily judged. After morphologic observation, immunocytochemical study of HMB-45 is possible even though time has passed since the cytologic specimen was obtained. When there is a suspicion of amelanotic melanoma or scantily pigmented melanoma of the vagina and vulva, cytogenesis with HMB-45 is helpful, especially because it involves little invasion.

Archival cervical smears: a versatile resource for molecular investigations.

Archival cervical smears: a versatile resource for molecular investigations Archival cervical smears represent a huge resource of pathological specimens. This, together with long clinical follow-up data, makes archival smears the most valuable resource for cervical cancer research. Despite this huge potential, only a few molecular investigations have been carried out based on archival smears. It has been shown that archival smears can be used for amplification of genomic sequences by polymerase chain reaction (PCR). However, it is unknown whether PCR can be applied to minute dyskaryotic cells microdissected from archival cervical smears and whether these archival materials are suitable for reverse transcription PCR (RT-PCR). To address these issues, we prepared DNA and RNA samples from dyskaryotic cells microdissected from archival cervical smears with a storage time of 11 years and systematically tested the extent that these materials can be used for PCR-based molecular investigations at both DNA and RNA levels. Our results showed that a crude DNA preparation simply by proteinase K digestion was suitable for PCR amplification of genomic sequences. By targeting the amplified genomic sequence to 250 bp or less, most if not all archival smears could be used for PCR and are therefore suitable for screening gene mutations and loss of heterozygosity, human papillomavirus typing, etc. Purified DNA samples from microdissected dyskaryotic cells were adequate for restriction enzyme digestion and could be used for a PCR-based clonality analysis of the androgen receptor gene. Finally, RNA samples extracted from dyskaryotic cells microdissected from archival smears were adequate for RT-PCR as long as a gene-specific primer was used for the RT reaction and the target sequence was restricted to 150 bp or less. In summary, our results demonstrated that archival cervical smears are suitable for a range of molecular investigations at both DNA and RNA levels. The potential gain of knowledge on cervical cancer by the molecular study of archival smears is immense.

Retrospective Study of Chlamydia trachomatis Using the Polymerase Chain Reaction on Archival Papanicolaou-Stained Cytologic Smears

To detect chlamydial DNA on archived Papanicolaou-stained (Pap) smears using the polymerase chain reaction (PCR) technique.A PCR assay was designed to identify chlamydial DNA using consensus sequences unique to the genus Chlamydia in the 16S rRNA gene. This assay produced a 109 base pair product containing a single Pvu II restriction site. One hundred cervicovaginal Pap smears from a teen clinic population were processed for DNA isolation and PCR. Amplifiable DNA was isolated from 93 of the 100 cases as determined by a human growth hormone gene. These specimens were subjected to chlamydial PCR.PCR analysis of the 93 samples yielded 6 that were positive for the chlamydial 16S rRNA sequence. The six positive chlamydial amplicons were purified and subjected to Pvu II restriction enzyme analysis to validate their identity. The analysis confirmed the identity of the products, as a single Pvu II restriction site resulted in 41 base pair and 68 base pair products, as predicted.PCR testing for Chlamydia trachomatis can be performed on DNA isolated from archival Pap smears. Using this methodology, 6.5% of young women in our teen clinic population were positive for chlamydial DNA.

Reassessment of sperm morphology of archival semen smears from the period 1980--1994.

Several reports have suggested that sperm counts of normal men have declined in many geographical regions during the last decades. Deterioration of sperm morphology has also been reported in some studies covering long sample collecting periods. The original semen analysis data of our semen laboratory from the period 1980--1994 showed a significant decline in the proportion of spermatozoa with normal morphology. The finding was, however, questioned because of changes in sperm morphology assessment criteria during the study period. In the present study 1745 smears were re-analysed to cover evenly the whole study period. The samples were examined in random order by using strict assessment criteria. Multiple linear regression analysis of the re-analysed data showed no effect of the year of sample delivery on sperm morphology between the years 1980 and 1994. However, there was a significant decline in the proportion of normal spermatozoa with later year of men's birth.

New Data from Old Trypanosomatid Preparations

Assessing the diversity of the Trypanosomatidae is difficult because of the relatively small number of species that can be cultured. This same problem thwarts efforts to identify the hosts and insect vectors of Phytomonas, a genus of parasites of plants that includes species responsible for devastating epiphytotics of economically important plantations. Here, Myrna Serrano, Marta Teixeira and Erney Camargo review the studies that have led to the development of a PCR-based technique for processing insect and plant juices fixed on glass slides. The method overcomes the need for cultivation, facilitates field collections and also permits the molecular examination of archival smears of Phytomonas. In principle, the method can be adapted to any trypanosomatid as well as to any fastidious parasitic or free-living organism.

Molecular assessment of post-BMT chimerism using various biologic specimens and automated DNA sizing technology.

Highly polymorphic microsatellite markers provide useful genetic markers for detection of complete or mixed chimerism in patients after allogeneic BMT (allo-BMT). We report application of automated DNA sizing technology for detection of post-BMT chimerism using fresh peripheral blood, BM, or archival blood smears and various DNA isolation techniques. Donors' and recipients' DNA was amplified with fluorescent PCR primers specific for short tandem repeat (STR) marker loci: FGA, VWA, TH01, F13A1, D21S11. Chimerism was assessed in 14 recipients after allo-BMT. A complete chimerism was detected in 10 patients, in 3 patients we observed fluctuations of chimerism status, and mixed chimerism was assessed in 1 patient. We show that DNA from different types of biologic specimens (whole peripheral blood, BM suspension, archival blood smears), prepared according to the various isolation techniques (salting-out method, phenol chloroform extraction, Chelex procedure) and amplified with fluorescent PCR primers for microsatellite markers, enable identification of chimerism status following allo-BMT in children.