Evidence for false aneuploid peaks in flow cytometric analysis of paraffin-embedded tissue

It has recently been shown that bimodal histograms with false aneuploid peaks may be obtained by DNA flow cytometry from histologically normal tissue allowed to autolyze. To investigate if such peaks can be generated from surgically excised archival tissue, 198 paraffin blocks from 179 patients containing histologically normal spleen (n = 65), liver (n = 26), thyroid (n = 32), pancreas (n = 19), salivary gland (n = 49), or lymph node tissue (n = 7), obtained from the archives of two university pathology departments, were analyzed for nuclear DNA content. The great majority (n = 160, 83.8%) of the 191 interpretable histograms had a single symmetrical G1 peak; and 8 histograms, all produced from liver tissue had a tetraploid pattern. A slight or a prominent repeatable deviation in the G1 peak outline was present in 14 (7.3%) cases. A peak resembling an aneuploid G1 peak with a DNA index (DI) ranging from 1.14 to 1.38 was repeatedly produced from 9 (4.7%) blocks containing histologically normal or inflamed splenic (n = 3), pancreatic (n = 3), liver (n = 1), thyroid (n = 1), or lymph node (n = 1) tissue. The three abnormal peaks produced from pancreatic tissue were rounded in shape and resembled closely the ones that can be obtained from autolytic pancreatic tissue, and the six remaining extra peaks were all fused with the "diploid" peak. In conclusion, false peaks, probably caused by degradation of the nuclear contents during formalin fixation or before it, may rarely be obtained from surgical paraffin-embedded samples.     

Comparison of fresh, ethanol-preserved, and paraffin-embedded samples in DNA flow cytometry

Fresh, ethanol-preserved, and formalin-fixed and paraffin-embedded samples taken from the same part of 15 human tumors, and from one normal spleen and one pancreas were analyzed for nuclear DNA content by flow cytometry. The coefficient of variation (CV) values of the G1 peaks were smaller in the fresh than in the other samples (P less than 0.001). The DNA ploidy of the tumors was the same in all types of samples. The DNA indices (DIs) measured from either ethanol-preserved or formalin-fixed tissue correlated strongly with those obtained from fresh tissue (P less than 0.001), although they tended to be somewhat smaller in the fresh samples. The S-phase fractions measured from all types of samples were of the same order of magnitude in most cases (P less than 0.001). Uninterpretable histograms were most often obtained from fresh samples. Identical DI values and rather constant S-phase fractions were obtained from ethanol-preserved samples stored at 4 degrees C for up to 5 months. It is concluded that all three types of samples are suitable for the determination of DNA ploidy, DI, and S-phase fraction and that 50% ethanol is suitable for long-term preservation of flow cytometric samples.     

Different opinions on classification of DNA histograms produced from paraffin-embedded tissue

Flow cytometric DNA ploidy determination has been regarded as an objective prognostic parameter in several types of human cancer. To test whether DNA histograms are similarly interpreted, a series of flow cytometric DNA histograms was posted to six investigators working in the field for independent classification. The histograms were produced from paraffin-embedded adrenal adenomas or non-neoplastic tissue and had several different patterns. Only 44% of the histograms were similarly classified by all investigators, and 85% by five of the six participants, when DNA ploidy was evaluated. Different criteria for tetraploidy existed, and also some uncertainty in classifying peridiploid and small aneuploid peaks. It is concluded that lack of consensus on histogram classification may result in widely varying percentages of DNA aneuploid tumors found even if the data are similar. Until general agreement is reached on the definition of DNA aneuploidy and its subclasses, classification of DNA histograms is variable and subjective.     

Comparative studies of nuclear DNA content in benign and malignant thyroid lesions

Currently available data suggest that DNA aneuploidy is associated with aggressive behavior of and unfavorable prognosis in several malignant human tumors as compared with diploid malignancies. However, the diagnostic and prognostic importance of flow cytometric DNA measurements in the case of thyroid neoplasms remains controversial. Therefore, the aim of our study was to evaluate utility of DNA index (DI) and proliferative index (PI) in distinguishing benign from malignant thyroid lesions taking into account the possible influence of intra-tumor heterogeneity and tissue preparation mode on DNA flow-cytometry measurements. A retrospective study was performed on 71 paraffin-embedded specimens from 57 patients with benign and malignant thyroid pathologies: 13 colloid goitres, 12 parenchymatous goitres, 19 adenomas and 13 carcinomas. In 14 of 57 cases two separate specimens taken from different areas of the same lesion were analysed and DNA parameters were compared. Additionally, flow cytometry DNA analysis was parallelly performed on 3 adjacent but differently processed tissue sections (fresh, formalin-fixed and paraffin-embedded) taken from each of 26 surgically excised thyroid lesions. DNA content was also analysed in both fresh and formalin-fixed twin specimens of normal pig thyroid glands (N = 6). We demonstrated that all tumors diagnosed as thyroid carcinomas were associated with abnormal nuclear DNA content although aneuploidy was not found specific to malignant thyroid tumors. Aneuploid samples of benign thyroid lesions exhibited higher proliferative activity, expressed as mean PI values, than diploid ones. In carcinomas the mean PI values were significantly higher than in benign lesions, independently whether they concerned aneuploid or diploid tissues. Considering intra-tumor heterogeneity, the flow cytometric DNA parameters can be assumed as reproducible despite differences in the mode of tissue fixation and preparation for analysis.     

The resolution of aneuploid DNA stem lines by flow cytometry: limitations imposed by the coefficient of variation and the percentage of aneuploid nuclei

Factors important in the resolution of cell sub-populations with differing DNA contents were investigated using an EPICS C flow cytometer. Software is available for the EPICS C which permits data from any two histograms to be superimposed or added together before display. Samples of fresh and archival thyroid tissue, stained with propidium iodide, were analysed on the flow cytometer and the peak channel number noted. The photomultiplier (PMT) voltage was increased and the sample analysed again producing a second histogram with a higher peak channel number. The two histograms were added together to simulate a cell suspension with two sub-populations with a different DNA content. By systematically altering the PMT voltage and the number of nuclei included in each analysis, it was possible to examine the importance of DNA index and the percentage of tumor cells with an aneuploid DNA content for both fresh and paraffin-embedded thyroid nuclei. The crucial importance of achieving a low coefficient of variation (CV) was demonstrated and consequently the reservations that pertain when archival material is studied, particularly in tumours where DNA aneuploidy is frequently expressed with a low DNA index.     

Cytologic criteria of cystic papillary carcinoma of the thyroid

OBJECTIVE: To establish differential cytologic criteria between benign and malignant thyroid cysts. STUDY DESIGN: The study was a retrospective, transverse, analytic, comparative one of 3 groups of patients with nonfunctional thyroid nodules subjected to fine needle aspiration biopsy (FNAB) and surgical resection of the lesions, with histologic study as the diagnostic gold standard. Fifteen cases of cystic papillary carcinomas (group 1) with initial false negative diagnoses, 42 goiters accompanied by cystic degeneration (group 2) and 15 noncystic papillary carcinomas (group 3) were studied. Independent variables were age and sex; dependent variables were the presence of tridimensional fragments, papillae, anisonucleosis, nuclear bars, pseudoinclusions, powdery chromatin, cytoplasmic vacuoles, metaplastic cytoplasm, psammoma bodies, autolysis, multinucleated giant cells, spindle cells, colloid, monolayered laminae and macrophages in FNAB specimens. Statistical analysis was performed by central tendency measures and the chi 2 test. RESULTS: The chi 2 test revealed a statistically significant difference between group 2 and the groups with papillary carcinoma based on the presence of tridimensional fragments, anisonucleosis, nuclear bars, pseudoinclusions, powdery chromatin, cytoplasmic vacuoles, metaplastic cytoplasm and autolysis. CONCLUSION: The above cytologic characteristics must be searched for systematically in the FNAB of every cystic lesion of the thyroid to rule out the presence of cystic papillary thyroid carcinoma and to decrease the rate of false negative results.     

Methodologic aspects of nuclear DNA assessment of gliomas with astrocytic and/or oligodendrocytic differentiation. Correlation of image and flow cytometric studies on paraffin-embedded specimens

A total of 239 samples from paraffin-embedded, formalin-fixed astrocytic and/or oligodendrocytic gliomas from 111 patients were deparaffinized and disaggregated for image cytometric (ICM) and flow cytometric (FCM) DNA assessments. Each measurement technique produced evaluable histograms in about 85% of the samples analyzed. In the 10% that could not be analyzed by FCM, the background counts were too high and the coefficients of variation were too broad for precise evaluation. The failures with ICM were due to a shortage of Feulgen-stained tumor cell nuclei after the deparaffinization and disaggregation procedures. The results obtained were identical in 77% of the samples evaluable by both methods and practically identical (i.e., euploid versus aneuploid) in an additional 18%. The reasons for completely divergent DNA ploidy patterns in 5% of the samples could not be clarified. About 80% of the histopathologically highly malignant gliomas were found to consist of neoplastic cells with an aneuploid or tetraploid nuclear DNA distribution pattern. The results show that cytometric DNA assessments can be reliably performed on paraffin-embedded specimens of gliomas with astrocytic and/or oligodendrocytic differentiation by means of FCM and ICM on deparaffinized and disaggregated specimens.     

Fine needle aspiration biopsy of Hashimoto's thyroiditis. Sources of diagnostic error.

OBJECTIVE: To determine the accuracy of cytologic interpretation in the diagnosis of Hashimoto's thyroiditis (HT). STUDY DESIGN: At Ottawa Hospital from 1987 to 1994, 1,638 fine needle aspiration biopsies (FNABs) from thyroid were performed. HT was suggested in 184 FNAB samples taken from 157 patients. Of the 184 aspirates diagnosed with HT, 39 had corresponding surgical specimens taken from 31 patients. A retrospective review of these FNABs and surgical pathology slides formed the basis of this study. RESULTS: In 27 (69%) aspirates, HT was diagnosed on both the FNAB and surgical specimens. In 10 of 27 FNABs an associated lesion was not sampled by FNAB. In four of these 10 aspirates some of the cellular features of HT were misinterpreted, and the possibility of an associated neoplasm could not be ruled out. This resulted in four false positive diagnoses. In 12 (31%) FNABs from nine patients, the cytologic diagnosis of HT was not confirmed histologically. These cases included five Hürthle cell adenomas and one case each of follicular adenoma, nodular goiter, macrofollicular adenoma and malignant lymphoma. This resulted in five false negative diagnoses. CONCLUSION: These results support the value of FNAB in the diagnosis of HT. The presence of hyperplastic follicular cells on FNAB samples from HT may mimic a follicular neoplasm and result in a false positive interpretation. Adequate sampling of the thyroid is important, particularly when there is an associated lesion. The diagnosis of lymphocytic thyroiditis should not be made when only a few lymphocytes are present. Finally, pleomorphic Hürthle cells may be present in aspirates from Hürthle cell neoplasms and underdiagnosed as HT, especially when they are associated with a few lymphocytes.     

Methodologic aspects of DNA assessment by means of image cytometry in tumors of the salivary glands. A comparison between the results obtained using sections and cytospin preparations from the same paraffin-embedded specimens

The image cytometric nuclear DNA assessments on paraffin-embedded tissue sections and on Cytospin preparations of disaggregated specimens from the same cases were compared in 98 salivary gland tumors, including 21 acinic cell carcinomas, 29 mucoepidermoid carcinomas, 21 adenocarcinomas and 27 adenoid cystic carcinomas. The histogram type (diploid, tetraploid or aneuploid) and the number of cells with DNA values greater than 2.5c (expressed in relative units) were considered as variables in the correlation. A high correlation between the results in different specimens was found in acinic cell carcinomas, mucoepidermoid carcinomas and adenocarcinomas; the histogram type and the number of cells with DNA values greater than 2.5c were essentially the same between specimen types in these three tumor entities. The cases of adenoid cystic carcinomas showed a considerably lower degree of correlation: in 8 of the 27 cases, the Cytospin preparations yielded diploid histograms, while the tissue sections yielded aneuploid histograms. The number of cells with DNA values greater than 2.5c was notably lower in the Cytospin preparations from adenoid cystic carcinoma; the reasons for this exceptional behavior of the cells of adenoid cystic carcinoma are discussed. These findings demonstrate that paraffin-embedded specimens of different tumor entities, even from the same organ, can be affected differently by disaggregation procedures. While retrospective studies on disaggregated paraffin-embedded specimens can yield reliable results, comparative assessments using both DNA analysis techniques, as in this study, should be performed before a large number of cases is evaluated.     

Simplified and versatile method for isolation of high-quality RNA from pancreas

Isolation of high-quality RNA from pancreas is challenging because the organ contains large quantities of RNases and undergoes autolysis upon harvest. Here we present a simplified perfusion method of the pancreas using an RNase inhibitor. The technique consistently yields high-quality RNA from stored pancreas samples suitable for molecular biology applications, including quantitative RT-PCR. Yields are comparable to RNA isolated from pancreas immediately, but superior to RNA isolated from stored samples that were snap-frozen or immersed in an RNase inhibitor solution. In addition, when compared to the previously reported in situ ductal perfusion technique, our method does not cause histological artifacts.     

Nuclear DNA cytophotometry in prostate carcinoma

Eighty patients with prostate carcinoma underwent fine-needle aspiration biopsy for cytologic grading and DNA-single-cell fluorescence photometry before and at 6-month intervals after endocrine treatment. The histograms of DNA values showed single peaks and bimodal and scattered distributions which correlated to the different tumor grades before therapy. The DNA values were significantly different from the controls with benign prostatic hypertrophy. After start of therapy, regressive changes of the DNA-histograms were increases of diploid and hypodiploid DNA values and disappearance of secondary peaks. Progressive changes were increased scattering of DNA values and appearance of secondary peaks. Progressive changes in the histograms were closely related to clinical remission and stable disease, but related poorly to clinical progression. The survival correlated with the pretherapeutic DNA-histograms and with the DNA-median, third-quartile, and maximum parameters.     

Comparison of DNA ploidy in routine fine needle aspiration biopsy samples and paraffin-embedded tissue samples

The nuclear DNA content was determined by flow cytometry (FCM) from unfixed fine needle aspiration (FNA) biopsy samples of 31 human tumors, and from the same tumors after their excision, fixation with formalin and embedding in paraffin. The ploidy of the histograms was the same in 29 (94%) of the 31 cases. The disagreement in two cases may be explained by clonal heterogeneity of the tumors. The DNA index of the aneuploid cases was identical in fresh and fixed samples. The coefficient of variation of the diploid peaks (P less than .001) and the mean percentage of S-phase cells (P = .06) were larger in the fixed samples. It is concluded that routine FNA biopsy is a practical and reliable method for collecting cells for FCM DNA ploidy determination.     

DNA quantitation of distal bile duct carcinoma measured by image and flow cytometry

OBJECTIVE: To evaluate discrepancies between flow cytometry (FCM) and image cytometry (ICM), ploidy incidence and relation between DNA ploidies and survival in distal bile duct carcinomas (DBDCs). STUDY DESIGN: Forty-four archival tumor samples from patients with DBDC who underwent subtotal pancreatoduodenectomy from 1985 to 1996 were examined for DNA ploidy using FCM and ICM. RESULTS: Overall, 59% (26/44) of the tumors were aneuploid by at least one of the two techniques. We detected more cases of aneuploidy with ICM than FCM in formalin-fixed, paraffin-embedded DBDCs, 62% (21/34) versus 33% (13/40), respectively. When results could be compared, moderate strength of agreement (kappa = .45) was demonstrated. No correlation was found between DNA ploidy by FCM, ICM or combined FCM-ICM and survival time (P = .80, P = .35, and P = .54, respectively). CONCLUSION: Approximately 59% of DNA histograms contained aneuploid cell populations. Although ICM, as compared to FCM, is more sensitive in assessing the ploidy status of DBDC, both methods were complementary. Most discrepancies between FCM and ICM were due to the dilution of aneuploid populations by non-neoplastic diploid cells. DNA ploidy assessment in DBDC did not offer the possibility of improving the ability to predict survival.     

Semiautomation of preparation of fixed paraffin-embedded tissue for DNA analysis

The usual manual preparation of single-cell suspensions from fixed paraffin-embedded tissue sections for flow cytometric (FCM) DNA ploidy analysis is a time-consuming, labor-intensive technique that requires 70 minutes to deparaffinize and rehydrate 50 microns sections as the initial step. Manual deparaffinization was compared with two semiautomated methods using an automatic slide stainer with either a 70-minute or 35-minute schedule. Samples from 6 normal tissues and 21 tumors (13 diploid and 8 aneuploid) were prepared using all three methods and analyzed by FCM. The mean cell counts in all samples were over 10(6). The DNA indices for the three samples prepared from a given tissue showed no significant differences. Using the 70-minute automation schedule, no aneuploid peaks were lost, and the ratio of G0G1 normal cells to aneuploid tumor cells was maintained. The automation of deparaffinization can thus provide a significant reduction in the labor need to produce single-cell suspensions for FCM; it can be especially helpful when handling large numbers of tumors. At the same time, the automated procedure decreases the exposure to hazardous chemicals and lowers the chance of losing tissue.     

Dependence of DNA-histograms on the sampling techniques in fine needle aspirates of the breast.

48 fine needle aspiration biopsy (FNAB) samples from 25 breast cancer cases, originally used for cytodiagnosis were subjected to DNA cytometry. There were air dried smears stained with the MGG method, and samples stained with HE or PAP stain after 50% ethanol fixation and cytocentrifugation. Different sampling strategies were applied. Four methods were tested: method 1: cell groups measured, method 2: all cells measured, method 3: free cells measured, and method 4: atypical free cells measured. Method 4 showed most often DNA aneuploid histogram patterns, sampling method 1 had the highest number of DNA diploid histogram patterns. Diagnostic approaches may benefit from a sampling method detecting the hiding aneuploid cell population. Grading of neoplasm could potentially benefit from other approaches.     

Comparison of automated and manual techniques for analysis of DNA frequency distributions in bladder washings

Quantitative methods for interpretation of flow cytometry DNA histograms are required for the widespread clinical use of this technology. The usefulness of a histogram analysis technique in this setting requires that it be operator independent, easy to implement in a clinical laboratory, and provide high sensitivity to the desired information. Additionally, the technique must be tolerant of the relatively low signal-to-noise ratios often found in DNA distributions obtained from clinical samples. Among the factors that have been used to assess the malignant potential of tumors are the presence of an aneuploid population, the proportion of hyperdiploid cells, the width of the G1 peak, the DNA index, and the fraction of cells in S. A computer-based method has been developed for extraction of the above-mentioned features from DNA histograms. The program detects peaks in the histogram and uses straight-line fits to the cumulative frequency distribution to define cell population bounds. A test set of 44 histograms compiled from bladder irrigation specimens obtained from patients with a present or past history of transitional cell carcinoma (TCC) was analyzed by five collaborating laboratories forming a Network sponsored by the National Cancer Institute (NCI). This test set was used to evaluate the performance of the computer-based method by comparing results with those of four expert observers. In this preliminary analysis, perfect agreement was found in the detection of aneuploid cell populations by all observers and the computer-based method. Correlation of percent hyperdiploid cell fraction was also excellent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometric DNA analysis of corneal epithelium

We have modified an existing technique in order to perform DNA analysis by flow cytometry (FCM) of corneal epithelium from the mouse, rat, chicken, rabbit, and human. This protocol permitted an investigation of human corneal scrapings from several categories: normal, aphakic bullous keratopathy (ABK), keratoconus (KC), Fuch's dystrophy, edema, epithelial dysplasia, and lipid degeneration. No abnormal characteristic cell-kinetic profile was detected when averaged DNA histograms were compared statistically between the normal and either ABK, KC, edema, or Fuch's dystrophy groups. Abnormal DNA histograms were recorded for cell samples that were taken 1) from three individuals who had epithelial dysplasia and 2) from one individual diagnosed with lipid degeneration. The former condition was characterized by histograms that had a subpopulation of cells with an aneuploid amount of DNA or had higher than normal percentages of cells in the S and G2 + M phases of the cell cycle. Corneal cells from the patient who had lipid degeneration had an abnormally high percentage of cells in the G2 + M phases of the cell cycle. The availability of accurate DNA flow cytometric analysis of corneal epithelium allows further studies on this issue from both experimental and clinical situations.     

Flow cytometric DNA measurements in human thyroid tumors

By means of flow cytometry (FCM), DNA distribution pattern and the fraction of cells in the various phases of the cell cycle were studied in 52 samples of normal thyroid tissues, follicular adenomas, follicular carcinomas, medullary carcinoma and fibrosarcomas. In the normal thyroid tissues and follicular adenomas DNA diploid cell populations only were found. Among 20 follicular carcinomas in 13 cases (65%) together with the DNA diploid cells, DNA aneuploid cell lines were also observed. S-phase fraction in follicular adenomas is higher than in the normal thyroid tissues and lower than those in thyroid carcinomas. The percentage of S-phase cells in DNA aneuploid populations is significantly higher (S = 19 +/- 9.3%) than in the diploid cell lines (S = 3.7 +/- 2.6%). DNA aneuploid cell populations were predominantly observed in carcinomas with a high degree of morphological anaplasia.     

A practical graphical method for estimating the fraction of cells in S in DNA histograms from clinical tumor samples containing aneuploid cell populations

A graphical method for the analysis of unperturbed DNA histograms is presented in which the area of the normalized histogram subtended by the fraction of cells in S is represented by a trapezoid whose dimensions are dependent on features common to all such histograms. The technique takes measurement variability into account. This method was applied to a variety of synthetic DNA histograms. Overall, calculated values for the fraction of cells in S correlated well with actual values. This method was applied to 36 diploid cases of non-Hodgkin lymphoma; results correlated well with those obtained by a computer-based method. The results of the graphical-method were also highly reproducible between different observers. The graphical method can be used in the presence of aneuploid cell populations. Techniques for calculating S fractions in the presence of aneuploidy in clinical samples are described. These techniques were applied to synthetic histograms of mixed diploid and aneuploid populations. Calculated values correlated well with actual values.