Role of unstained smears in determining sample adequacy

OBJECTIVE: To determine adequacy with air-dried, unstained smears. STUDY DESIGN: The study was conducted on a total of 70 cases. The cases were divided into 2 groups. Group I consisted of all 70 and was analyzed by a consultant, observer A. Group II consisted of 41 cases from group I. In addition to observer A, a junior resident with 3 months' experience in pathology (observer B), analyzed the slides independently. The results were compared with those on stained smears. RESULTS: When correlated with stained smears, in group I, 55 of 58 (94.8%) cases were reported as adequate, and 11 of 12 cases (91.7%) were labeled inadequate. All were proven correct. In group II, stained smears confirmed that 33 of 35 (94.3%) were labeled adequate by observer A and 33 of 36 (91.7%) by observer B. Stained smears did not confirm 1 of 6 (16.6%) cases labeled inadequate by observer A and 1 of 5 (20%) cases by observer B. CONCLUSION: Unstained smear examination is an effective technique for determining adequacy. An inexperienced practitioner can perform it as well.     

Air-dried smears for optimal diagnostic immunocytochemistry

OBJECTIVE: To exploit formalin-postfixed, air-dried smears for diagnostic immunocytochemistry (ICC). STUDY DESIGN: A series of 144 cases of diagnostic fine needle cytology samples in which air-dried, supplementary smears were available was used to exploit postfixation in the process of antigenic stabilization and rescue for immunocytochemical staining. RESULTS: Postfixation with formalin and with a formalin/ethanol solution gave comparable results as far as recovery and immunocytochemical detection of most monoclonal and polyclonal antibodies. The visualization of the antibody reactions was often superior to that obtained with wet-fixed slides, probably due to the interaction of slow cell dehydration with their consequent optimal flattening observed with formalin postfixation after short rehydration in physiologic saline. CONCLUSION: Although wet fixation of cytopathologic slides in 95% ethanol represents a common standard for ICC, the usage of formalin-postfixed air-dried smears proved reliable and efficient for antigenic rescue and may enter routine usage in cytopathology laboratories. Moreover, in some instances, the visual evaluation of results was easier in the larger, well-flattened cells obtained in air-dried cells.     

The Fahraeus effect in sheet flows with a gap thickness of 4 to 36 microns

We measured the Fahraeus effect of blood flowing in a sheet flow model formed with two glass slides. The number of red blood cells in the sheet flow was counted to determine a sheet hematocrit Hs and the discharge hematocrit Hd was measured from blood collection. For a Hd in the range of 3 to 30 percent, we find that Hs/Hd is about .83 for a gap of 4.1 microns. When the discharge hematocrit is 30 percent, the ratio decreases to .66 as the gap approaches 7 microns and then increases as the gap becomes thicker. The results indicate that the hematocrit ratio for a gap thicker than 4.1 microns is an increasing function of the discharge hematocrit. The value of Hs/Hd found for the sheet flow models and its dependence on Hd are comparable to those of circular tubes when their diameter equals the gap thickness.     

Studies on the manufacture of hematological and cytological specimens using the centrifugation technic]

Centrifugal preparation (Cp) represents a method of creating haematological parameters which, in addition to a qualitative improvement of representing single cells, is suitable to produce reproducible haematological smear preparations. In qualitative and quantitative respect centrifugal preparations will reflect the intravasal conditions better than the common blood smear technique. Further advantages are the concentration of cellular elements and in connection with it the opportunity of finding cells rarely present. With a small amount of work centrifugal preparations enable counting values to be established as well as cytological and cytochemical findings from the venous blood to be obtained. As a result of the test it turned out that the common glass slides cannot be replaced by plastic foils ones without certain disadvantages appearing. First investigations of attempting to represent cells in culture medium as well as analyzing liquors and processing other biological liquids with the help of the centrifugal preparation technique brought unsatisfactory results. Centrifugal preparation provides the possibility of performing pre-programmed haematological examinations beginning with blood collection and ending up in the evaluation by a computer.     

Sputum cytology for the diagnosis of lung cancer. Comparison of smear and modified cell block methods

OBJECTIVE: To compare the sputum smear cytology and cell block methods for specimen adequacy, cytology quality and diagnostic accuracy in the diagnosis of lung cancer. STUDY DESIGN: We assessed 2,524 sputum specimens from 768 patients. The specimens were prepared as smears and cell blocks for cytopathologic examination between March 1, 1992, and December 31, 1998. The smear and cell block slides were evaluated both separately and together, and the results were compared with radiologic and histopathologic diagnoses. RESULTS: The sensitivity of the smear method was 69.4% and specificity was 99.5%. The sensitivity of the cell block method was 84.4% and specificity, 100%. The sensitivity of the smear and cell block together was 87.6% and specificity, 99.5%. CONCLUSION: The cell block method increases the sensitivity and specificity of sputum cytology, and when smear and cell block slides are evaluated together, sensitivity reaches its highest value. Therefore, application of smear and cell block methods together seems most useful in the diagnosis of lung cancer.     

Restoration of broken cytology slides and creation of multiple slides from a single smear preparation.

A technique was developed for restoring broken cytology slides so that they are close to their original condition and for making multiple slides from a single smear preparation. The method is applicable to both cytologic preparations and histologic sections. In this study the fragmented smear preparation was treated with Pro-Texx, which penetrated, impregnated and solidified the full thickness of the pieces of the smear, enabling them to be lifted from the pieces of the broken slide. The removed pieces of the smear preparation were reassembled onto a new slide, which was then restained and coverslipped. In preparing multiple teaching slides, the treated smear preparation was divided as planned, with each portion mounted onto a separate slide, which was then restained and coverslipped. Ten other fine needle aspiration cases with broken slides have been restored, and more teaching slides were prepared from a single smear preparation using the same technique. All were equally successful. This technique provides an excellent method of smear transfer in cases of broken slides and creation of multiple slides from a single smear preparation for cytology teaching. This is particularly useful for unusual cases.     

Detection of the HTLV-I gene on cytologic smear slides

OBJECTIVE: To apply the polymerase chain reaction (PCR) for detection of the HTLV-I gene from cytologic smear slides. STUDY DESIGN: Samples were from seven cases of serum anti-ATL antibody (ATLA)-positive T-cell lymphoma and three from ATLA-negative T-cell lymphoma. Six of the seven ATLA-positive cases were confirmed to be ATLL by Southern blotting. From the seventh case a fresh sample for blotting could not obtained. DNA was extracted from the cytologic smear slides of all 10 cases; they had been stained with Papanicolaou or May-Giemsa stain, digested with proteinase K and precipitated with phenol and ethanol. The target sequence in the pX region of the HTLV-I gene was amplified by PCR. RESULTS: All seven ATLA-positive cases, including one that had not yet been confirmed by Southern blotting, showed a single band, as predicted, while the three ATLA-negative cases showed no band. CONCLUSION: If cytologic smear slides are available but a fresh sample is not, the PCR method should provide evidence that the virus is present since in our study sufficient DNA templates were successfully extracted from the stained cytologic smear slides for detection of the virus.     

Reproducibility of coccolith morphometry: Evaluation of spraying and smear slide preparation techniques

It is of great importance to assess the internal accuracy and reproducibility of coccolith morphometry, and to understand any systematic differences between various sample preparation techniques, so that data obtained with different methods can be adequately compared. Here, we performed a comparative study between two techniques regularly used to prepare nannofossil microscope slides, the standard smear slide and spraying methods. With each technique, ten replicate slides were prepared and morphometric measurements were carried out on the coccolith genus Calcidiscus as well as full assemblage counts to determine the reproducibility of coccolith size measurements and relative species abundances for each method.For either method, two significant sources of variance are related to the morphometric data, the variance between replicate slides and the variance within slides. Thus, in order to reduce the total variance of the estimate of mean coccolith size, it is beneficial to increase the number of replicate slides as well as the number of measurements on each slide. If the aim of the morphometric study is to address the mean size of a coccolith taxon or species complex (a group of closely related sub-species), one could rely on either preparation technique, since no significant difference in (log-normalised) mean size between the two tested methodologies was found. Likewise, the 10th percentiles are statistically equal for both methods. However, the 90th percentile values are significantly larger in the sprayed slides than in the smear slides, indicating that the spraying method may either favour larger coccoliths or better resolve the full extent of size variability present in the sample. Future tests are needed to investigate whether, and if so, how, size-fractionation may occur when using the spraying method. Nevertheless, the spraying method is preferred based on the reproducibility of proportion estimates since no significant difference between replicate samples was observed, in stark contrast to the smear slide series with 3–4 times higher variance. It appears that information gained from one sprayed slide requires counting at least three replicate smear slides.     

Staining The Fibrin Network In Blood Smears

The following procedure is described: Prepare smears of fresh blood on clean slides and place them immediately in a moist chamber to prevent them from drying. Permit them to remain in the chamber long enough for the blood to coagulate - 6 to 10 minutes. Transfer the slides to distilled water to hemolyze the red blood corpuscles, leaving them in the water until all traces of the red color have disappeared from the smear, when the fibrin network appears as a whitish film on the slide. Stain the network for about 8 minutes in carbol crystal violet (dissolve 0.4 g. of crystal violet in 10 ml. of 95% ethyl alcohol and mix with 100 ml. of distilled water in which is dissolved 1 g. of phenol). Thoroughly dehydrate and clear die fibrin network by passing the slides through three changes of pure dioxane. Mount in euparal and cover with a cover glass.     

Comparison of Carnoy's solution and 96% ethyl alcohol fixation in bloody Pap smears

OBJECTIVE: To compare 2 methods of fixation in bloody Pap smears with Carnoy's solution and 96% ethyl alcohol. STUDY DESIGN: After observation of contact bleeding, 2 samples were prepared from cervical cells with conventional Pap smear. One sample was fixed in 96% ethyl alcohol and another sample was fixed in Carnoy's solution. RESULTS: Of 450 slides, 410 were selected for study. In study of cell adequacy, diagnosis of squamous cells and glandular cells was better in Carnoy's-fixed slides. Blood contamination of slides was reduced in Carnoy's-fixed slides (13.85% vs. 49.51%), and clearance of slides was increased in Carnoy's-fixed slides. Diagnosis of inflammatory cells and pathogenic microorganisms in was increased in Carnoy's-fixed slides, but no difference was seen in diagnosis of epithelial cell and glandular cell abnormalities. CONCLUSION: Carnoy's solution can be used as an effective fixative in bloody smears in conventional Pap tests.     

Accuracy of thin-layer cytology in patients undergoing cervical cone biopsy

OBJECTIVE: To compare the accuracy of thin-layer cytology with Autocyte PREP (TriPath Imaging Inc., Burlington, North Carolina, U.S.A.) with conventional smears in 500 women undergoing cervical cone biopsy. STUDY DESIGN: The study was performed among 500 consecutive women presenting for cone biopsy for high grade cervical intraepithelial neoplasia (CIN) on biopsy in 350 (70%) and discrepant cytology/colpohistology in 150 (30%). Before performing a cone biopsy, two cervical samples were collected for conventional smears and thin-layer cytologic slides, with randomization of the order. Conventional smears were stained and diagnosed at Pasteur Cerba, while thin-layer cytologic slides were processed at a local TriPath office (Meylan, France) and sent in a masked fashion for screening at Pasteur Cerba. Any slides initially read as normal were reviewed again and reported without knowledge of the other cytologic or cone biopsy data. The final cytologic diagnoses for the two methods were compared with histopathology of the cone biopsy. RESULTS: The conventional smear was unsatisfactory in 58 (11.6%) of cases, while there were 4 (0.8%) unsatisfactory thin-layer cytologic slides (P < .001). Endocervical cells were missing from 31 (6.2%) of conventional smears and 34 (6.8%) of thin-layer cytologic slides. For the pooled data, sensitivities of conventional smear and thin layer for detecting high grade CIN (0.82% and 0.86%, respectively) were similar as were specificities (0.40% and 0.43%, respectively). When first samples were compared, the sensitivities of the conventional smear and thin layer for high grade CIN were 0.79% and 0.89%, respectively (P = .02), with corresponding specificities of 0.41% and 0.36% (P < .01). CONCLUSION: When controlled for sample order, the sensitivity of thin-layer cytology for detecting high grade CIN was significantly higher than that of conventional smears in patients with previous abnormal cytology, but at the expense of specificity.     

Fine needle aspiration biopsy of the breast. Value of nuclear morphometry after different sampling methods

OBJECTIVE: To study the potential of nuclear morphometry in supporting the interpretation of fine needle aspiration biopsy (FNAB) samples of the breast fixed in 50% ethanol and centrifuged on slides. STUDY DESIGN: Computerized morphometry was used to outline the nuclei of breast epithelial cells in breast cancer, fibroadenoma and fibrocystic disease. The diagnoses were histologically confirmed. We applied 2 different sampling methods (measurements done on cell groups and on free cells). RESULTS: The mean nuclear area of cell groups of malignant samples (23) varied from 42 to 125 microns 2, in fibroadenomas from 30 to 50 microns 2 and in fibrocystic disease from 26 to 57 microns 2. The mean nuclear area of free cells varied as follows: cancer, 66-181 microns 2; fibroadenoma, 33-70 microns 2; fibrocystic disease, 35-60 microns 2. Apocrine metaplasia was excluded from comparison on a morphologic basis. CONCLUSION: The study suggests that if the mean nuclear area of cell groups is < 42 microns 2, the lesion is probably benign; if > 57 microns 2, and apocrine metaplasia is excluded, malignancy should be considered. The differential diagnosis between carcinoma and fibroadenoma could be based on free cells: mean area of free cell nuclei < or = 65 microns 2 suggested a benign lesion, and of > or = 71 microns 2 suggested a malignant lesion. Morphometric nuclear size features (exemplified by nuclear area) appeared efficient in distinguishing between malignant and benign lesions when measured from free cells and cell groups.     

Application of MIRU–VNTR on smear slides: a shortcut for detection of polyclonal infections in tuberculosis patients

Mixed (polyclonal) infections are one of the main problems in tuberculosis (TB) management. The best available method for detecting polyclonal infections in TB is mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU–VNTR). According to multiple studies, MIRU–VNTR method can be applied to detect TB-related polyclonal infections in sputum samples or cultures. Setup of MIRU–VNTR on smear slides can be an efficient approach, regardless of the limitations of cultures and sputum samples in many laboratories. The present study aimed at investigating the diagnostic potential of MIRU–VNTR on smear slides in detecting mixed infections. Ziehl–Neelsen-stained microscopic slides were prepared from 14 clinical specimens. For amplifying 24 MIRU–VNTR loci, PCR assay was performed on the smear slides, clinical specimens, and cultures. Based on the 24-locus MIRU–VNTR analysis, polyclonal infections were reported in 42.85% of smear slides, while the corresponding rate was estimated at 57.1% (8/14) in the clinical samples. In the corresponding cultures, the rate of mixed infection was 7.14% (1/14). Use of smear slides can be a safe option for transferring clinical specimens between environmental and reference laboratories. Considering their significant impact on TB treatment, it is essential to diagnose mixed infections in low-resource countries with a high prevalence of mixed infections. The present findings show that direct MIRU–VNTR on smear slides can be conveniently used for the detection of mixed infections.

     

Lymphonodular cryptococcosis diagnosed by fine needle aspiration cytology in hyper-IgM syndrome. A case report

BACKGROUND: Most cases of cryptococcosis are diagnosed when signs of meningitis have appeared. We report a case of lymphonodular cryptococcosis that was diagnosed by fine needle aspiration cytology (FNAC), excisional biopsy of a cervical lymph node and culture of aspirated material. CASE: An 11-year-old boy presented with a history of fever and enlarged bilateral cervical lymph nodes of two weeks' duration. Past medical history included immunoglobulin replacement for hyper-IgM syndrome for the previous eight years. FNAC smears from a cervical lymph node showed numerous yeasts of various sizes, ranging from 5 to 15 microns in diameter, located in the cytoplasm of multinucleated giant cells and in the background. In air-dried, Diff-Quik-stained slides, the yeasts stained blue and were surrounded by clear halos. Aspirated material collected in the syringe was cultured, and Cryptococcus neoformans was isolated. CONCLUSION: This case report suggests that a combination of FNAC and culture is a simple and useful method of diagnosing fungal infections. Early diagnosis by FNAC makes possible the early initiation of treatment.     

Higher diagnostic accuracy with the ThinPrep method in a simulated intraoperative environment

Objective:  To compare the accuracy of intraoperative fine needle aspiration cytology samples prepared by the ThinPrep method to conventional cytological methods. Specimen adequacy and turn around time (TAT) were also assessed.
Methods:  Fifty consecutive fresh tumours submitted for histological analysis were aspirated and each prepared as follows: (i) direct smear with H&E stain, (ii) direct smear with Pap stain, (iii) ThinPrep slide with H&E stain, and (iv) ThinPrep slide with Pap stain. The slides were randomly distributed to three cytopathologists for interpretation. The quality of the preparation, the diagnosis and the time needed for interpretation were recorded.
Results:  Accuracy was measured as the percentage of absolute agreement between the cytological and the histopathological diagnoses of the lesions. Histologically, there were 43 malignant and six benign lesions and one atypical lipoma. The TAT began when the slides/cytolyte specimens arrived at the lab and ended with the pathologist's diagnosis.
Conclusions:  In terms of accuracy and specimen adequacy, ThinPrep slides with Pap stain is the best procedure. This advantage however is offset by the longer testing time.     

Ag-staining of nucleolus organizer regions of chromosomes after A-,C-, G-, or R-banding procedures.

Metaphase chromosome preparations were made from leukocyte cultures of normal individuals. The cells were fixed in methanol:acetic acid (3:1 v/v), then dropped on cold, wet slides which were air-dried before storage at 4 degrees C. The slides were stained to identify the chromosomes by one of the following procedures: (1) Quinacrine. Slides were stained for 10 min in quinacrine mustard solution, rinsed in running tap water for 2 min, and mounted in Tris-maleat buffer, pH 5.6.     

Sensitivity studies of AutoPap System Location-Guided Screening of cervical-vaginal cytologic smears.

OBJECTIVE: To present the results of a study that assessed the efficacy of a cervical cytology screening method utilizing the AutoPap System with Location-Guided Screening (AutoPap LGS) software for detecting abnormal Papanicolaou smear slides. STUDY DESIGN: Two hundred cases of abnormal cervical and vaginal smears were selected from the recent archives of the Taipei Institute of Pathology. For each abnormal slide, a matched "within normal limit" slide was included in the study. The slides were processed on the AutoPap Primary Screening System to select slides for Review or No Review and identify areas of the Review slides for human review and diagnosis (AutoPap LGS). The effectiveness of AutoPap LGS for detecting abnormal Papanicolaou smear slides was evaluated at multiple No Review rates. RESULTS: The AutoPap LGS demonstrated statistically superior sensitivity over current laboratory practice for the identification of abnormal slides. Assessing the potential benefit of the AutoPap LGS using a projection method, it is expected that the AutoPap LGS would detect an additional 52 low grade squamous intraepithelial lesion and 13 high grade squamous intraepithelial lesion cases missed by current laboratory practice in a population of 2,860 cases. CONCLUSION: The effectiveness of AutoPap LGS was demonstrated by its statistically superior performance in the detection of missed abnormal slides as compared to current laboratory practice at the Taipei Institute of Pathology.     

Decidual cells in the human ovary at term: II. Morphometric analysis of cytoplasmic processes and organelles

Morphometric analysis of human ovarian decidual cells was performed with a Videoplan computer, and mean values were established for the area and perimeter of cellular processes and organelles. Two-hundred forty electron micrographs representing 160 cells were analyzed. The mean decidual cell area was 218.7 microns2, of which 34.5 microns2 was occupied by the nucleus (15.8% of the cytoplasmic area); the nucleus contained 1.74 micron2 of nucleolar material (0.8%). The endoplasmic reticulum occupied 13.63 microns2 (6.2%). Mitochondria occupied 7.3 microns2 (3.3%) and the Golgi network 5.49 microns2 (2.5%). Decidual secretory bodies occupied 0.91 micron2 (0.42%) and cytoplasmic processes 1.89 micron2 (0.94%). The remainder of the cytoplasm, containing inclusions and cytoskeleton, represented 71% of the cell area. Perimeter measurements indicated an average decidual cell was surrounded by 87.8 microns of plasma membrane. The mean nuclear membrane measured 28.3 microns (representing 32.3% of the plasma membrane, pm, or 4.1% of total cellular membranes, cm). Outer mitochondrial membranes measured 156.6 microns (178% pm, 23.5% cm); endoplasmic reticulum membranes measured 350.3 microns (400% pm, 52.6% cm); Golgi membrane measured 30.77 microns (35% pm; 4.5% cm) and membrane surrounding secretory bodies measured 9.8 microns (11.2% pm; 1.4% cm). A mean of 280 secretory bodies per ovarian decidual cell was calculated. The plasma membranes of evaginated cytoplasmic processes represented 22.3% of the total pm (19.6 microns or 2.9% cm). A mean of seven such processes was observed per 87.8 microns of plasma membrane (160/cell). These morphometric data provide a baseline for comparisons of human ovarian decidual cells with uterine decidua, in vivo and in vitro, as well as with decidual cells of other species.     

Localization and morphometric analysis of masticatory muscle afferent neurons in the nucleus of the mesencephalic root of the trigeminal nerve in the cat

Injections of horseradish peroxidase (HRP) were made into the ipsilateral temporal muscle and contralateral masseter muscle of 10 cats in order to identify and characterize neurons in the nucleus of the mesencephalic root of the trigeminal nerve that innervate muscle receptors in the orofacial periphery. Neurons labelled by HRP injections and unlabelled cells from 5 control cats were measured with a computer-based image analyzer, and their position was mapped on a stereotaxic graph. Cells that innervate the masseter and temporal muscles were identified throughout the rostrocaudal extent of the nucleus. There was no indication of a somatotopic pattern nor of a specific segregation within the nucleus for cells innervating muscle receptors. The nucleus contained small, rounded unipolar neurons located primarily in the dorsal border of the periaqueductal gray (PAG) matter in the rostral part of the nucleus and larger oval unipolar neurons which were scattered throughout the nucleus, but were predominant in the pontine portion of the nucleus. HRP injections labelled both large and small cells, as well as occasional multipolar cells. The last-mentioned tended to be located in the lateral margins of the PAG. The mean geometric values obtained for the control group were: area 552.7 microns2 perimeter 110.3 microns; maximum diameter 36.0 microns. and diameter of an equivalent circle 26.1 microns. The mean values of the labelled neurons were: area 606.6 microns2; perimeter 100.1 microns; maximum diameter 36.0 microns, and diameter of an equivalent circle 27.2 microns.     

A novel method of sputum processing for cytologic diagnosis of lung cancer

OBJECTIVE: To compare the diagnostic sensitivity and cytologic findings of the sputum-processing method with those of the traditional sputum smear method. STUDY DESIGN: From May to December 2001, 300 consecutive sputum samples were collected from 168 patients suspected of having lung cancer in the Chest Department, Taipei Veterans General Hospital. After the sputum smear method, the remaining sputum material was processed by homogenization, filtration and fixation. All the slides were stained with Papanicolaou stain and reviewed by 2 cytologists. RESULTS: Of the 300 sputum samples, 141 from 79 patients were finally diagnosed as lung malignancies. The mean number of sputum samples was 1.78 per patient. Among the 79 patients, 46 had peripheral lung malignancies (58.2%). The overall diagnostic sensitivities of sputum smear and sputum-processing methods were 29% and 31%, respectively (P = .776). Tumor location and cell types did not change the difference significantly; however, among patients with small cell lung cancer, there was a higher detection rate with the sputum-processing method (50% vs. 20%, P = .350). Eight patients with negative results with the sputum smear had positive results with the sputum-processing method (8 of 79 = 10.1%). Microscopic morphologic differences between the 2 methods were described. CONCLUSION: There was no significant difference in diagnostic sensitivity between the sputum smear and sputum-processing methods. However, the sputum-processing method may play a role in patients with small cell lung carcinoma.