Immunostaining of estrogen receptor,progesterone receptor,MIB1 antigen,and c-erbB-2 oncoprotein in cytologic specimens: A simplified method with formalin fixation

An effective but simple fixation protocol for the immunocytochemical staining of cytologic smears for estrogen and progesterone receptors, the Ki-67 antigen (using MIB1 antibody), andc-erbB-2protein is described. One hundred twenty-seven smears from a variety of malignant and benign breast lesions showed good preservation of antigenicity when subjected to the following fixation protocol: Freshly made smears were air-dried for 20 min to 14 h at 22°C before immersing in 10% buffered formalin for 2–14 h. Immunostaining followed microwave-stimulated epitope retrieval. There was strong concordance of staining with corresponding tissue sections in 15 cases of malignant tumors (ER: r = 0.7381; PR: r = 0.6684; MIB1: r = 0.7234). Immunostaining staining, when delayed for 5–10 days in about half the smears, showed no noticeable difference in reactivity, attesting to effective storage of the formalin-fixed smears at room temperature. Diagn. Cytopathol. 17:127–133, 1997. © 1997 Wiley-Liss, Inc.     

Double Fixation with Paraformaldehyde and Acetone for Optimal Immunocytochemical Demonstration of DNA Polymerase α

Abstract

Because the fixation procedure is a crucial step for immunocytochemical staining of DNA polymerase α, we sought an optimal fixation for demonstration of the enzyme using HeLa S3 cells. Double fixtion with paraformaldehyde and acetone produced intense stainability when slides were dipped in a 4% paraformaldehyde solution for 10 min, then in acetone for 5 rnin at 4°C. In contrast, fixation with ethanol or methanol resulted in considerable reduction of immunoreactivity. Single fixation with paraformaldehyde or acetone yielded intermediate stainability. Poor stainability also resulted in decrease of positive cells. (The J Histotechnol 12:285, 1989)     

Methodological aspects of the immunostaining of proliferating cell nuclear antigen (PCNA) in cytospin preparations of MCF-7 cell line

Cytospins of a human breast cancer cell line (MCF-7) were studied for the expression of PCNA, a cell cycle-related protein, using a variety of fixation and immunostaining procedures. The best fixative for PCNA was found to be buffered formaldehyde solution at 4°C followed by methanol at 20°C, whereas alcoholic fixatives decreased greatly the PCNA immunoreactivity. Air-drying procedures of cytospins prior to and after fixation greatly undermined the PCNA immunostaining. A modified immunoperoxidase method provided a stronger staining of the PCNA-reactive cells than the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. PCNA immunoreactivity could be maintained up to 2 mo, putting slides in methanol at ? 2°C. In conclusion, our report indicates that PCNA is a labile antigen, which may critically be affected by temperature and air-drying procedures. © 1994 Wiley-Liss, Inc.     

Rehydration of air-dried smears with normal saline. Application in fine-needle aspiration cytologic examination

In fine-needle aspiration cytologic examination, nuclear features are often better assessed in hematoxylin and eosin (H and E) or Papanicolaou- (Pap) stained than Romanowsky-stained smears. However, both H and E and Pap stains require the use of immediately wet-fixed smears for cytomorphologic preservation. Some degree of air drying is usually inevitable. Placing air-dried smears in normal saline for 30 seconds before fixation in 95% alcohol is found to be a simple means of rehydrating the cells. The quality of the rehydrated smears is superior or identical to that of the immediately wet-fixed smears, provided that the period of drying does not exceed 30 minutes.     

Commercial laboratory practice evaluation of air-dried/rehydrated cervicovaginal smears vs. traditionally-fixed smears

Air-drying artifacts remain a significant problem in the interpretation of cervicovaginal cytologic smears. One historical, and little-used, method to combat these artifacts is to have smears submitted solely air-dried, subsequently rehydrated, fixed, and then stained as usual. Reported here is a 1992–1993 retrospective matched provider study of 6,788 air-dried/rehydrated smears and 1,691 traditionally-fixed smears. No significant differences either in the percentage of abnormalities (8.6% vs. 8.2%) or the degree of abnormality (class II, 6.9%/6.9%; class III, 1.7%/1.3%; class IV, .01%/.06%; and class V, .01%/0%) were seen between the two techniques. Cytology-biopsy correlation remained in the 98–99% range for three large providers switching from air-dried to traditionally-fixed smears. These findings strengthen our belief that the air-dried/rehydrated technique is a viable alternative to combat the usual “air-dried” artifact problem. Diagn. Cytopathol. 16:174–176, 1997. © 1997 Wiley-Liss, Inc.     

Ultrafast Papanicolaou stain and cell-transfer technique enhance cytologic diagnosis of Hodgkin lymphoma

Diagnosis of Hodgkin lymphoma (HL) by fine-needle aspiration (FNA) is often hampered by aspirated blood that camouflage scattered Hodgkin cells and Reed-Sternberg (HRS) cells, and the absence of HRS cells in the smears submitted for immunophenotyping. The objective of this study was to develop a simple protocol to overcome these problems. The visibility of HRS cells in Diff-Quik, traditional, and Ultrafast Papanicolaou (UFP) stains in FNA smears were compared in 73 cases of HL. HRS cells were found to be most visible in UFP because of the hemolysis of aspirated blood and the highlighting of HRS cells by the red-staining nucleoli. UFP-stained smears containing HRS cells were subsequently immunophenotyped via the cell-transfer technique. UFP staining was found to have no deleterious effect on the immunoreactivity of cellular CD15 and CD30 antigens of HRS cells. This simple protocol enhances the cytologic diagnosis of HL, feasible even with a single smear.     

Practical immunocytochemical identification of human blood cells

A practical immunocytochemical method of demonstrating surface antigens of human blood cells on air-dried smears or other cytologic preparations has been developed. This method uses monoclonal antibodies as the primary antibodies and calf intestinal alkaline phosphatase as the enzymatic indicator. Combined staining with cytochemical stains for myeloperoxidase or nonspecific esterase on the same slide is also possible when needed. These methods are very useful for accurate identification of human blood cells on the commonly available clinical specimens and are very helpful in the diagnosis and classification of various hematologic neoplasms, including chronic lymphocytic leukemias, acute leukemias, and related diseases.     

Application of nucleolar organizer region staining technique to air-dried blood smears]

Silver staining of nucleolar organizer regions (NOR) was applied to air-dried peripheral and bone marrow smears of normal subjects and leukemic patients. Specimens were fixed in buffered acetone formalin. Even in smears kept for 2 years at room temperature, the stainability of Ag-NOR was well preserved. By dipping Giemsa-stained smears in 5% trichloracetic acid and then placing them in methanol for 5 minutes, the stain was leached out. After the dye had been removed, the smears were clearly stained by a Ag-NOR staining technique. The mean number of Ag-NOR per nucleus of mature granulocytes and mononuclear cells in normal peripheral bloods was 0.59 and 1.43 respectively. The mean number of Ag-NOR per nucleus of peripheral and bone marrow leukemic cells from patients with acute leukemia and chronic myelocytic leukemia in blastic crisis was 2.32 and 2.66 respectively. On the other hand, the mean number of Ag-NOR per nucleus of peripheral leukemic cells from patients with chronic lymphocytic leukemia was 1.48. These results suggest that acute leukemia cells possess a more active proliferating potential. The Ag-NOR staining technique is very simple and might be useful for investigation of hematologic cells.     

Clear cell follicular adenoma of the thyroid: A case report

A case of clear cell follicular adenoma of the thyroid is presented. The patient presented with a single, hyperactive nodule in the right lobe. The cytologic features include cellular smears with numerous disrupted cells and a granular background. The cytoplasm was abundant, pale grayblue vacuolated or granulated, but not clear. Thyreoglobulin was demonstrated both histologically and ultrastructurally, confirming the follicular-cell derivation of the tumor. Ultrastructurally, the cytoplasm was filled with empty, membrane bound vacuoles. The clear cell change might represent an artifact of formalin fixation and/or the paraffin embedding procedure. Diagn Cytopathol 1996;15:124–126. © 1996 Wiley-Liss, Inc.     

ABC-immunoperoxidase staining of cytologic preparations: improvement of specificity.

Immunoperoxidase (IP) methods perfected on formalin-fixed, paraffin-embedded tissues (FFPE) and then applied to aspirate smears may result in high background staining and a significant number of false-positive results. This is especially true if polyclonal primary antibodies are used or if aspirates and fluids contain a high interstitial or serum protein content. Because of a recurring problem with antiserum to alpha-fetoprotein (AFP), AFP was selected as the primary test antibody with which to evaluate our avidin-biotin complex (ABC)-IP method. The same method, with diaminobenzidine (DAB) or aminoethylcarbazole (AEC) chromogens, was performed on six types of cytologic preparations of a fresh liver specimen. The liver did not stain for AFP in FFPE and frozen tissue; therefore, it could be used to evaluate potential false-positive staining of direct touch imprints, washed aspirate smears, and cytospins that were both air-dried and alcohol-carbowax-fixed. Initial chromogen incubation times were standardized to give identical results on AFP-positive fixed hepatoma and fetal liver controls. Cytologic preparations immunostained with ABC-IP with AEC chromogen resulted in varying background and hepatocyte staining. In comparison, the ABC-IP method using DAB chromogen resulted in no false-positive results and a clean background. The ABC-IP method with AEC standardized for sensitivity on a fixed tissue control required a markedly shortened chromogen incubation time to preclude significant false-positive staining of cytology specimens. It appears that use of AEC chromogen for this antibody with incubation time standardized on a FFPE tissue control and then applied to cytologic preparations also amplifies nonspecific and background staining, contributing difficulty in assessing a true-positive result.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diagnostic utility of MIC-2 immunocytochemical staining in the differential diagnosis of small blue cell tumors

Ewing's sarcoma (ES) and peripheral neuroectodermal tumor (PNET) are considered in the differential diagnosis of small round blue cell tumors of infancy and childhood which includes neuroblastoma, rhabdomyosarcoma and malignant lymphoma. Fine-needle aspiration diagnosis of these neoplasms can be particularly difficult when the neoplasms are composed of poorly differentiated cells or fail to produce a stroma. MIC-2 is a highly sensitive and specific marker for the PNET/ES group of neoplasms and has been studied extensively in surgical pathology. Other small blue cell neoplasms including rhabdomyosarcoma, blastemal Wilm's tumor, and lymphoblastic lymphoma have also shown positivity, but the staining reactions are usually weak and focal. The utility of this marker in the differential of small blue cell neoplasms in cytologic material has not been examined. Twenty cases of small blue cell neoplasms obtained by fine-needle aspiration (FNA) were studied. MIC-2 antibody was applied retrospectively to formalin-fixed cell block material and destained alcohol-fixed and air-dried cytologic preparations. These cases include primitive neuroectodermal tumor (five cases), Ewing's sarcoma (two cases), neuroblastoma (four cases), Wilms's tumor (four cases), lymphoblastic lymphoma (two cases), and small-cell carcinoma (three cases). The cases were judged positive when the majority of the cells showed cytoplasmic staining. Diffuse cytoplasmic staining was observed in all seven cases of PNET/ES. Staining could be seen on the destained air-dried smears (three cases), fixed smears (two cases), or the cell block material (two cases). None of the other 13 small blue cell neoplasms showed positive staining. We conclude that MIC-2 is a sensitive and specific marker for the PNET/ES group of neoplasms in specimens from formalin-fixed cell block, air-dried, and alcohol-fixed cytologic material and is useful in the differential diagnosis of small blue cell tumors. Diagn. Cytopathol. 1998;19:410–416. © 1998 Wiley-Liss, Inc.     

Methanol as an alternative fixative for cytological smears

Ninety-five percent (95%) ethanol is the standard cytological fixative used in many laboratories. Commercially available ethanol is expensive and not freely available in some institutions. Methanol, a tissue dehydrant, is also known to be a cytological fixative. However its efficacy has not been assessed or documented in the literature. One hundred and eight consecutive fine needle aspiration biopsies (FNAB) of thyroid performed at the Department of Pathology, Faculty of Medicine, Colombo were included in a study to assess the efficacy of methanol as a cytological fixative. Aspirated material was smeared on at least 2 slides, one fixed in ethanol and the other in methanol, and stained with haematoxylin and eosin (H&E). The 2 smears were separately assessed for preservation of colloid and cells (nuclei and cytoplasm), as determined by the staining quality with the H&E stain. A score was given for each smear and the final scores for ethanol and methanol were statistically compared. The evaporation rates for ethanol and methanol were calculated. The total score for preservation of colloid was 294/300 (98%) for methanol and 291/300 (97%) for ethanol (p = 0.4). The total score for preservation of cells (nuclear and cytoplasmic) was 276/279 (98.9%) for methanol and 274/279 (98.2%) for ethanol (p = 0.7). The evaporation rates per 100 ml when the bottles used for fixation were kept closed and open per 24 hours were 1 and 37 for methanol and 0 and 17 for ethanol. Literature search did not show inhalational side effects of methanol in humans under standard laboratory conditions. We conclude that methanol is as effective as ethanol for fixation of smears and cheaper.     

The influence of the wet-fixed Papanicolaou and the air-dried Giemsa techniques on nuclear parameters in breast cancer cytology: a cytomorphometric study

The influence of fixation and staining on morphometric nuclear parameters in cytologic samples of breast lesions has been investigated with respect to the wet-fix and the air-dry techniques. Aspiration biopsy material from 55 benign and malignant breast lesions was smeared onto slides and stained with a routine Papanicolaou technique (after wet fixation) or with the Azur B-eosin Y stain (after air drying). Tumors were grouped according to both histological grade and lymph node status. Nuclear area, diameter, and perimeter were measured from each specimen with an image analyzer and correlated to grade and stage of the disease. All nuclear parameters were significantly smaller in wet-fixed slides. The coefficient of variation was smaller in air-dried samples, which allowed better discrimination between different groups. Nuclear perimeter had the highest discriminative power. Unintentional air drying in wet-fixed smears increased the coefficient of variation for all nuclear parameters. Air drying of cytologic smears is recommended for morphometric investigations if chromatin texture is of minor importance.     

Avidin-biotin amplification procedures for the detection of human terminal deoxynucleotidyl transferase in cell smears

The authors have developed fluorescent avidin and avidin-peroxidase assays to detect human terminal deoxynucleotidyl transferase (TdT) in cell smears. These assays used specifically purified rabbit anti-calf TdT antibody, biotinylated goat anti-rabbit IgG antibody, and either fluorescein isothiocyanate-avidin or avidin-biotinylated horseradish peroxidase complex. The use of phosphate-buffered saline rather than TRIS-buffered saline or borate-buffered saline was essential to obtain optimal results in the fluorescent avidin assay. Fixation of cells with either 0.5% paraformaldehyde or 10% neutral buffered formalin was found to be superior to either no fixation or fixation with cold methanol or cold 0.1% glutaraldehyde in ethanol. The sensitivities of the fluorescent avidin and avidin-peroxidase assays were shown to be identical, based on staining intensities and percentages of positive cells when both assays were performed on cells from 14 patients with acute lymphoblastic leukemia (ALL), and 5 patients with lymphoblastic lymphoma (LL).     

Immunofluorescence assay for human immunodeficiency virus antibody: investigation of cell fixation for virus inactivation and antigen preservation.

Four cell fixation procedures were investigated for their abilities to inactivate human immunodeficiency virus (HIV) and preserve its antigenicity for antibody detection by immunofluorescence in MOLT-4-T4 cells. Air-dried cell smears were fixed in cold acetone, in acetone-methanol (1:1), in acetone-methanol (1:1) followed by 70% ethanol and then methanol, or in paraformaldehyde-acetone. Acetone alone did not inactivate cell-associated HIV, but the other three procedures did. HIV inactivation was achieved by storage of acetone-fixed cells at -70 degrees for 40 days. Antigenicity was measured by immunofluorescence assay titrations of selected human sera, a cerebrospinal fluid, and a gp41 monoclonal antibody. Acetone provided the best fixation as measured by fluorescence intensity and antibody titers. The other fixation methods all yielded weaker fluorescence signals and/or decreased titers. Acetone fixation and storage for 40 days at -70 degrees C provides safe and accurate immunofluorescence assay reagents.     

Morphological diagnosis of parvovirus B19 infection. A cytopathic effect easily recognized in air-dried, formalin-fixed bone marrow smears stained with hematoxylin-eosin or Wright-Giemsa.

Readily identifiable intranuclear inclusions characterize parvovirus B19 cytopathic effect in formalin-fixed, paraffin-embedded material. Such inclusions are not apparent in air-dried smears of bone marrow aspirates. Brief formalin fixation of bone marrow smears, followed by either hematoxylin-eosin or Wright-Giemsa staining, permitted easy detection of parvovirus B19 inclusions in material from a renal transplant recipient with parvovirus B19 infection documented serologically and by electron microscopy. Formalin fixation of bone marrow and peripheral blood smears before hematoxylin-eosin or Wright-Giemsa staining may simplify the morphological diagnosis of parvovirus B19 infection.     

Effects of various fixatives and temperature on the quality of glycogen demonstration in the brain and liver tissues

The visualization of glycogen deposits in cells and tissues is important for studying glycogen metabolism as well as diagnosis of glycogen storage diseases. Evidence suggests that the demonstration of glycogen can better be enhanced by factors such the choice of fixative and temperature during fixation. Here, we assessed efficacy of neutral buffered formalin (NBF), alcoholic formalin (AF) and paraformaldehyde (PFA) at 4 °C, 37 °C and 40 °C using Periodic Acid Schiff's staining method. Each liver specimen was fixed in NBF and AF while the brain tissues were fixed in NBF, AF and PFA. We found that there was a better PAS staining intensity with the liver tissues fixed in AF compared with NBF. Also, there was no difference in the quality of the staining for tissues fixed in AF at 37 °C, 4 °C and 40 °C, but fixation with NBF at 4 °C gave the best staining quality when compared with 40 °C and 37 °C. Furthermore, hippocampal tissues fixed in AF showed better quality of PAS staining compared with NBF and PFA. A significant increase in staining intensity was observed for PFA when compared with NBF. Superior staining intensity for PAS was observed at 4 °C for hippocampal tissues fixed with NBF, AF and PFA. Taken together our results show that AF at a temperature of 4 °C gave the best result. Hence, glycogen demonstration can better be enhanced by the choice of fixative and temperature during fixation.     

PCR amplification of DNA obtained from archived hematoxylin-eosin– and giemsa-stained breast cancer aspirates

The study of DNA abnormalities in fine-needle aspiration (FNA) specimens for breast cancer could be helpful in improving the capacity of diagnosis and specially to obtain prognostic or predictive information. The aim of the present study was to verify whether it is possible to perform molecular analysis in slides of breast cancer FNA specimens, previously stained by hematoxylin-eosin (H&E) and Giemsa, stored at least for 3 years. For this purpose, 10 cases of FNA obtained from breast cancer patients diagnosed between 1993 and 1994 in our institute were used. Five cytologic smears were alcohol-fixed and stained with H&E. The other five were air-fixed and Giemsa stained. DNA was isolated from the cytologic smears and amplified by using radioactive polymerase chain reaction (PCR) aimed at BAT-26 marker. Our results demonstrate that archived stained smears prepared for cytologic examinations can be used for molecular analyses by using a PCR amplification method. DNA could be isolated and PCR amplified independently of the prior fixation and staining procedures. So, we conclude that the application of molecular biology techniques to the existing archival smears may become a valuable tool to detect genetic changes in samples from breast cancer aspirates. Diagn. Cytopathol. 1998;19:395–397. © 1998 Wiley-Liss, Inc.     

Paranuclear blue inclusions: An aid in the cytopathologic diagnosis of primary and metastatic pulmonary small-cell carcinoma

Accurate diagnosis of small-cell carcinoma of the lung (SCLC) is clinically important because of the therapeutic implications. SCLC must be distinguished from non-small-cell carcinoma (NSCLC) and lymphoma. Paranuclear blue inclusions (PBIs) were recently described as a feature of metastatic SCLC on air-dried Wright-stained bone marrow aspirate smears. To determine the utility of PBIs in distinguishing SCLC from NSCLC and lymphoma, we evaluated air-dried Diff-Quik-stained smears from 103 fine-needle aspiration (FNA) specimens and 14 touch imprint specimens. PBIs were identified in 24 (89%) of 27 cases of SCLC, in 6 (9%) of 64 non-small-cell carcinomas (P < 0.00001), and in two (8%) of the 26 lymphoma cases (P < 0.00001). No PBIs were seen on any of the alcohol-fixed Papanicolaou or hematoxylin-eosin (H&E) stained smears examined. In conclusion, PBIs appear to be a feature of SCLC on air-dried cytologic material stained with Romanowsky type stains. In the presence of cytologic features of SCLC, the identification of PBIs provides a useful diagnostic feature for diferentiating between SCLC and NSCLC carcinomas, and between SCLC and lymphomas in FNA specimens and touch imprints from surgical specimens. © 1994 Wiley-Liss, Inc.     

KP1 Immunohistochemical Demonstration of Macrophages in Colorectal Tissues: Effects of Different Fixatives and Different Fixation Times

Abstract

The effects of 10 fixatives and formalin fixation time were assessed on the immunoreactivity of KP1 (CD68 antigens) in colorectal tissues in paraffin embedded sections. A panel of fixatives including B5, periodate-lysine-paraformaldehydedichromate, periodate-lysine-paraformaldehyde, 4% paraformaldehyde (PF), zinc formalin, formol dichromate, 10% neutral buffered formalin (NBF), Bouin fluid, Carnoy fluid, and acetone were used to determine an optimal fixative. Another set of colorectal tissues were fixed in 10% NBF progressively at intervals of 1,2,4, and 20 hr; 2,7, and 14 days; and 4 wk to investigate the formalin-induced detrimental effect on CD68 antigens of macrophages.

The best result was found after fixation in B5. Short fixation in NBF for 4 hr with trypsinization also gave satisfactory results. There was a distinct decrease in staining intensity after 20 hr exposure to NBF, and the reactivities were totally abolished after 2 days. Trypsinization did enhance the staining intensity significantly with no discrimination of fixatives. (The J Histotechnol 17:329, 1994)