Correction of autoradiographic grain count in respect to precisely calculated background

Summary It was endeavored to find a criterion for significantly labeled cells in quantitative autoradiography. Measurements of the autoradiographic background were performed and it was found that: 1. the value of the background over the non-proliferating epithelial cells from an animal injected with ³H-thymidine is higher than over the same cells from animals not injected with an isotope, 2. the value of the background in emulsion over the tissue specimen is higher than away from the specimen. Therefore, one should take into account the background over the tissue. Nomograms are shown for quick evaluation of the percentage of cells labeled with 1, 2, 3 or 4 grains, which should be disregarded as due to the background. To obtain this percentage for a given experiment its appropriate parameters: the labeling index, the mean grain count over the cell, the standard deviation of the grain count distribution and the background grain count distribution should be taken into account.     

Factors which Affect Efficiency of Autoradiography with Tritiated Thymidine

The overall efficiency of autoradiography with tritium-labeled thymidine has been found to be influenced by the following conditions: (1) exposure in an atmosphere of CO₂ and the use of the stripping-film technique, both of which increase the autoradiographic efficiency when compared to exposure in air or to dip-coating technique; (2) latent image fading, which increases with increasing exposure. Up to 2 wk of exposure, however, this disadvantage is counterbalanced by the fading of the mechanical background produced during stripping or dip-coating; (3) the thickness of the inert coating interposed between the labeled locus and the sensitized emulsion. A layer of inert coating can be obtained that will arrest all beta particles from tritium, while having no effect on more energetic emitters like C¹⁴; (4) the amount of tritiated thymidine given, with relatively large amounts producing an increase in the mean grain count per labeled cell but not in the percentage of cells identifiable as labeled; and (5) the type of fixative and the staining procedure used. Feulgen stain reduces the mean grain count in cells labeled with radioactive thymidine, while fixation with acetic alcohol (1:3) reduces the grain count in cells labeled with precursors of ribonucleic acid.     

Split-sample analysis of discarded cells from liquid-based Pap smear sampling devices

OBJECTIVE: To examine cells that were retained on sampling devices used to collect ThinPrep (Cytyc Corp., Boxborough, Massachusetts, U.S.A) Pap smears in order to evaluate both the number and significance of cells that are routinely discarded with these devices after liquid-based specimens are collected. STUDY DESIGN: One hundred Pap smears from 100 women were prospectively procured after gynecologic Pap smears were collected for the ThinPrep Pap test. The sampling end of the collection devices was cut off and placed in a vial that contained SUREPATH preservative fluid (TriPath Imaging, Inc., Burlington, North Carolina, U.S.A). The residual cell samples were processed using the SurePath PREPSTAIN slide processor (TriPath). A single liquid-based slide was prepared from the sampling devices from each of the 100 specimens collected. The slides produced from the discarded devices were reviewed for the following: squamous cells, endocervical component, epithelial cell abnormalities and miscellaneous findings. The slides prepared from the "throw-away" (TA) material were subsequently compared with the primary ThinPrep Pap smear slide. RESULTS: Twenty-five percent of the TA samples had an equal or greater number of squamous cells per high-power microscopic field when compared to the primary ThinPrep slide, with 8% of the TA slides demonstrating greater overall cellularity. An endocervical component was present on 27 of 66 cervical samples (40.9%). Three of five cases (60%) interpreted as atypical squamous cells of undetermined significance had similar cells on the TA slides. Two cases of atypical glandular cells of undetermined significance had no abnormal cells on the TA slides. Twelve of 14 cases (85.71%) of low grade squamous intraepithelial lesion contained similar cells on the TA slides. Two of four cases (50%) of high grade squamous intraepithelial lesion also had similar abnormal cells on the TA slides. Miscellaneous findings included 1 case of benign endometrial cells and 4 Candida infections present on both preparations, along with 1 case of Trichomonas vaginalis organisms present on the ThinPrep slide only. In 1 specimen, several multinucleated histiocytic giant cells were present only on the TA slide. CONCLUSIONS: Specimens prepared from TA collecting devices used for the ThinPrep Pap test are less sensitive than the primary specimen for the detection of cervical lesions. This is in contrast to split-sample studies involving ThinPrep and conventional smears. Our study documented the presence of normal and abnormal cells discarded from ThinPrep sampling devices in a high percentage of cases. Discarded abnormal cells on the TA slides were, however, few when compared to the primary specimen, with only 1 exception involving a high grade lesion.     

Objective analysis of stallion sperm motility

An image-analysis system utilizing a microcomputer and CellSoft computer-assisted semen analysis software package was evaluated to assess stallion sperm motility characteristics. Analyses were performed at 37°C on a 6 μl drop of diluted semen placed on a glass slide and covered with an 18 mm² coverslip. Four groups of 25 cells each per slide, four slides per ejaculate and four ejaculates from each of three stallions were analyzed in a nested model. The percentage of motile sperm cells, mean velocity (μm/sec), mean linearity, and mean angular head displacement (μm) were measured. Statistical analysis of variance components showed that within ejaculates, more variation was accounted for in the differences among groups of 25 cells than among slides. Predicted standard deviations calculated for combinations of slides and groups of cells showed that a combination of two slides from which a total of 400 cells were analyzed resulted in a mean intra-assay coefficient of variation (CV) of 5.7% for the four measured variables. The following are individual coefficients of variation: percentage of motile cells (7.8%), mean velocity (6.4%), mean linearity (1.9%) and mean angular head displacement (6.6%). When ejaculate differences were included in the model and predicted standard deviations were calculated for a single ejaculate, the mean inter-assay CV was 9.2%. Mean velocity (6.4%) and mean linearity (4.7%) were more repeatable among ejaculates than either the percentage of motile sperm (14.4%) or angular head displacement (11.2%). It was concluded that this system is precise enough to determine differences in motility characteristics of stallion semen samples.     

The preparation of additional smears from a cervical scrape: impact on the rate of detection of cervical neoplasia

The preparation of additional smears from a cervical scrape: impact on the rate of detection of cervical neoplasia It has been known for some time that only a proportion of the cells on the smear-taking device is transferred to the slide. This can give rise to errors in reporting although the smear may have been taken correctly. This study was undertaken to identify a quick and simple method of improving the accuracy of the Papanicolaou test. A conventional smear and five additional smears were obtained from 62 women attending a Genito-Urinary Medicine clinic. The cell content of the conventional smears and the additional smears was compared. Dyskaryotic cells were detected both in the conventional smear and in the first and second additional smears from 22 women. Dyskaryotic cells were detected in the first and second additional smears only in five women. Thus, the conventional smear failed to detect biopsy-confirmed cervical abnormality in these women. A cell count of the first additional smear in the five cases where the conventional smear was negative showed that they contained, on average, 310 dyskaryotic cells. The preparation of one additional cervical smear per cervical scrape could significantly increase the accuracy of the cervical smear test by 11% (P=0.025, McNemar's test).     

Monocyte kinetics: observations after pulse labeling

Because monocytes and their precursors cannot be recognized with certainty in tissues, an approach to the study of monocyte kinetics was made through examination of the peripheral blood. Injection of a single pulse of tritiated thymidine into rats resulted in the appearance of labeled monocytes identified as circulating peroxidase-positive mononuclear cells. The increase in the percent of labeled cells and in the mean grain count per cell followed a course described by a mathematical model with a generation time of 21 hours and a DNA synthesis time of 12.5 hours. The generation and synthesis times appear to be very uniform for the monocyte so that the phasing of cells represented by the uptake of label could be followed for more than two generations, a property not shared by neutrophils or lymphocytes. Monocytes appear in the circulation within eight hours of DNA synthesis.     

The effect of reducing the number of cells scored on the performance of the in vivo rat liver UDS assay

The most labour-intensive feature of the in vivo rat liver UDS assay is the scoring of hepatocyte autoradiograms by microscope. Even with image analyser and computer equipment the scoring phase of a full study might require half of the technical effort applied. Practice recommended by guidelines has been to score 50 cells/slide and two slides per animal. Now sufficient data have been accumulated, an evaluation was made to observe whether this procedure was necessary. An analysis of the accumulated UDS database in our laboratory was made to determine the sources of variability of mean net nuclear grain count, [N - C]. It was observed that the two largest components of variation in negative control animal mean [N - C]. were between-day and interanimal variability. The contribution from sampling error during slide scoring was relatively small. Theoretical calculations showed that the greater sampling error derived from scoring 30 rather than 50 cells/slide would result in only a marginal increase in total assay variation. To test this, 30 cells/slide were randomly selected from the 50 cells scored originally in negative control animals in each of 18 studies over an 18-month period. It was confirmed that reducing the number of cells had a negligible effect on the variation of negative control animal mean [N - C] values. Furthermore, analysis of data from 10 more studies confirmed that within-study variation would be essentially unaffected by scoring 30 cells/slide. The use of 30 rather than 50 cells per slide (a total of 60 cells per animal) has therefore been adopted for all current studies and scoring procedures modified to avoid operator bias during the selection of a smaller number of cells.     

The fate of epidermal colcemid-arrested mitoses

This study reports the fate of hairless mouse epidermal basal cells arrested in mitosis by a traditional stathmokinetic dose of 0.15 mg Colcemid. Epidermal basal cells in the S phase were labeled with 30 microCi (3H)TdR i.p. After 1 h, four animals from a cage of eight mice were given 0.15 mg Colcemid (Fluka) in 0.5 ml saline, and the other four mice were given saline only. Groups of eight mice (four experimental, four controls) were sacrificed 4, 9, 13, 21 and 25 h after (3H)TdR injection (i.e. 3, 8, 12, 16, 20 and 24 h after Colcemid). The following cell kinetic parameters were determined: the number of labeled basal and suprabasal cells, the mean grain count of the labeled cells, the specific activity, the mitotic count, the number of labeled mitoses, the fraction of labeled mitoses curve and the fraction of cells in S and in G2 as determined by flow cytometry. "Labeled paired twins", i.e. adjoining labeled cells with approximately the same grain count, were also scored. All the results taken together support the conclusion that cells labeled with (3H)TdR and arrested 1 h later with 0.15 mg Colcemid go through at least one subsequent cell division and thereafter some of them move out into the suprabasal layer at a normal rate. Hence, after this dose of Colcemid, cells arrested in mitosis for some hours do not die, and the Colcemid treatment does not seem to produce hyperploid cells. The study confirms the usefulness of this dose of Colcemid as a convenient tool for cell kinetic studies.     

The red blood cells in growing guinea pigs. (Cavia cobaya). II. Communication: erythropoiesis and red blood cells in the postnatal development period

Bone-marrow smears of 175 guinea pigs aged 1-27 days and venous blood samples of 351 animals aged 1-25 days were prepared for cell counting. A significant increase of erythroblasts were found between life day 1 and 2; normoblasts increased in number synchronously with a decrease of erythroblasts after the 5th day. The percentage of the erythroid bone marrow increased from 10 to 14 during the developmental period. Beyond the perinatal period the red blood picture is characterized by the following changes: a decrease of erythrocyte count, hematocrit, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin; a constant mean corpuscular hemoglobin concentration; an increase of the reticulocyte count. The decrease of the red cell count is compensated by a decreasing oxygen affinity attained by an important increase of 2,3-DPG. Nevertheless, the stimulus for a raising erythropoiesis remains constant which can be shown by the growing percentage of erythroid cells and reticulocytes. The difference between the human postnatal development and that of the guinea pig becomes obvious. Cell counts in dependence of body masses in postnatally growing guinea pigs, veil the perinatal finding of the increase in erythrocytes up to the 5th day and the decrease of the mean corpuscular volume after the 3rd day.     

The life span of red blood cells in the amphibian larvae, Rana catesbeiana

Nearly one hundred amphibian tadpoles were made anemic by phenylhydrazine injection. During the recovery period radioactive thymidine was incorporated into the DNA of the nucleated amphibian red blood cells. Over the next 135 days blood was drawn and smears of circulating red blood cells were prepared. The blood smears from each tadpole were autoradiographed and percentage of labeled nuclei (PLN) determined. The average life span of tadpole red blood cells was calculated from the change in PLN, and is about 100 days. It is concluded that during the transition from tadpole to frog, the tadpole red blood cell life span must be drastically shortened to account for the hemoglobin transition observed during amphibian metamorphosis.     

In vitro differential effects of sodium selenite on the growth of human hepatoma cells and human embryonic liver cells

The effects of various concentrations of Na₂SeO₃ on human hepatoma cells and human embryonic liver cells was investigated in vitro. For human hepatoma cells, mitotic index and cell count decreased with increasing selenium concentrations. At 1 μg/mL Na₂SeO₃, mitotic activity of human hepatoma cells were partially arrested. In human embryonic liver cells continuously treated with Na₂SeO₃, (1 μg/mL) cell count of the treated group decreased only by d 7; mitotic index, labeled index, and mean silver grain number per 50 labeled nuclei were the same as in the control group on exposure to 1, 3, and 5 μg/mL for up to 72 h. In mixed cultures of human hepatoma and embryonic liver cells treated with 3 and 5 μg/mL of Na₂SeO₃ for 24 h, hepatoma cells showed vacuolated cytoplasms, distorted nuclei, condensed chromatin, and even pyknosis, whereas the embryonic liver cells retained a normal morphology under the same treatment.     

Bone marrow cell differential counts obtained by multidimensional flow cytometry.

Five-dimensional flow cytometric analysis of normal bone marrow aspirates was utilized to determine the frequency of neutrophils, eosinophils, monocytes, lymphocytes, nucleated erythrocytes, reticulocytes, platelets, and a cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils. Each of these bone marrow cell populations had unique features with respect to forward light scatter, orthogonal light scatter, and staining with Thiazole-Orange, LDS-751, and CD45 labeled with Phycoerythrin (PE). The identity of the cell populations was verified by sorting each of the cell populations and subsequent light microscopic examination of the cells. The frequencies of the nucleated bone marrow cell subpopulations of 50 normal donors were for neutrophils, mean 72.3%; SD +/- 5.1; 95% limits, 70.9-73.8%; eosinophils, mean 1.8%; SD +/- 1.3; 95% limits, 1.4-2.1%; monocytes, mean, 2.8%; SD +/- 1.2; 95% limits, 2.5-3.1%; lymphocytes, mean 12.1%; SD +/- 3.6; 95% limits 11.1-13.2%; nucleated erythrocytes, mean 8.9%; SD +/- 3.9; 95% limits, 7.8-10.1%; and the cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils, mean 1.6%; SD +/- 1.2; 95% limits, 1.3-1.9%. The percentage of reticulocytes in bone marrow aspirates from 50 normal donors correlated with the reticulocyte frequency in the peripheral blood of these donors. However, the mean frequency of reticulocytes was significantly greater (p < 0.0001) in bone marrow (mean 2.19%; SD +/- 0.88) than in peripheral blood (mean 1.71%; SD +/- 0.88). The technique could discriminate between immature and mature reticulocytes based on the brighter staining with both Thiazole-Orange and LDS-751 of the immature reticulocytes. This was confirmed by cell sorting of both reticulocyte populations, which revealed larger clumps of New Methylene Blue staining material in the brighter Thiazole-Orange and LDS-751 stained reticulocytes. The immature reticulocytes were present in normal bone marrow, but not in normal peripheral blood. As expected, a significantly greater frequency of nucleated cells was found in bone marrow aspirates (mean 0.85%; SD +/- 0.59) than in peripheral blood (mean 0.20%; SD +/- 0.11). The frequency of platelets was significantly lower in bone marrow (mean 1.24%; SD +/- 0.69) than in peripheral blood (mean 2.94%, SD +/- 1.14). Flow cytometric bone marrow analysis can provide clinical laboratories with a technique that generates quantitative bone marrow cell differentials and potentially can reduce the need for light microscopic examination of bone marrow smears.     

Proliferative activity in endometrial hyperplasia and adenocarcinoma

Studies on the proliferative activity of cells in endometrial hyperplasia and adenocarcinoma were performed using techniques detecting Proliferating Cell Nuclear Antigen (PCNA) and Nucleolar Organizer Regions (NORs). PCNA expression was defined as the percentage of nuclei showing reactivity in 200 cells per sample. The mean AgNOR count per cell was calculated following the analysis of at least 100 nuclei per sample at a magnification of x 400. Student-t test was used for the statistical analysis. The results obtained indicate that the evaluation of cell proliferative activity expressed by AgNOR count and PCNA index can help in the distinction between atypical hyperplasia and well-differentiated adenocarcinoma, and thus can serve as a useful pathological criterion.     

Influence of different cell extraction methods on cytometric features.

The purpose of this study was to investigate the influence of different cell extraction procedures on relative nuclear DNA content (IOD), nuclear area, and texture features of Feulgen-stained nuclei. In imprints and smears of fine-needle aspirates and suspensions from one human liver specimen, 50 diploid, 50 tetraploid, and 25 octaploid nuclei were measured from each slide. In addition, for DNA measurements, the progressive mean of IOD and tetraploid/diploid and octaploid/diploid ratios was calculated. The results show that the progressive mean of the IOD is constant after measuring 25-30 nuclei. For the three types of specimens, the IOD of diploid nuclei varied slightly. The average coefficient of variation was about 5% for the fine-needle aspirates, imprints, and suspensions. For all tissue sampling methods, the 99% confidence limits of the tetraploid/diploid ratio and octaploid/diploid ratio were within the range of 1.9-2.1 and 3.7-4.3, respectively. The nuclear area and most of the texture features showed a significant difference (p less than 0.01) between the three sampling methods in all nuclear populations. In conclusion, different tissue sampling methods have little or no effect on DNA-related IOD measurements, whereas the outcome of nuclear area and texture features is very dependent of the cell extraction procedure.     

Cellular cohesiveness in benign and malignant thyroid follicular tumours varies significantly,but the difference is not useful in diagnosis of individual cases

T. Rozkos, A. Ryska, J. Cap, F. Sobande and J. Laco
Cellular cohesiveness in benign and malignant thyroid follicular tumours varies significantly, but the difference is not useful in diagnosis of individual cases Introduction: The aim of our study was to search for new, readily available and statistically reliable cytological markers for differentiating benign and malignant follicular thyroid neoplasms pre‐operatively. Methods: Cohesiveness of tumour cells in cytology slides from a series of 58 follicular tumours diagnosed between 1998 and 2004 inclusive was studied, including 48 follicular adenomas, and eight minimally invasive and two widely invasive follicular carcinomas. Photomicrographs of the cytology slides were taken and the digital images were analysed using computer image analysis software. We evaluated the relative proportions of cells arranged in groups of various sizes. The cohesiveness of the cells in cytological smears was then correlated with the immunohistochemical expression of E‐cadherin in corresponding histological slides. Results: Cases from 15 men (26%) and 43 women (74%) with a mean age of 50 years (range, 19–79) were analysed. In follicular adenomas and carcinomas, respectively, isolated cells were seen in 16.8% and 24.7% (P = 0.028), groups of two to five cells in 9.7% and 11.5% (P = 0.145) and groups of more than five cells in 73.5% and 63.8% (P = 0.041). The mean cell count in groups with more than five cells was 46.5 and 27.0 in adenomas and carcinomas, respectively (P < 0.001). Cell cohesiveness, either as percentage of cells in groups of more than five (R² = 0.026) or as mean cell count per group of more than five (R² = 0.005), was not found to be dependent on the expression of E‐cadherin. Using a threshold of 13% isolated tumour cells in cytological smears, follicular adenomas and carcinomas could be distinguished with 90% sensitivity and 41% specificity. Conclusions: Although we demonstrated a statistically significant difference in cell cohesion between follicular adenomas and carcinomas, these could not be distinguished in the clinical setting by evaluation of the percentage of isolated cells in cytological smears because the specificity was too low. The absence of correlation of cellular cohesiveness with E‐cadherin expression indicates that other factors are probably responsible for the loss of cohesiveness observed in follicular thyroid malignancy.     

Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours

U.S. Choi and D.Y. Kim Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours Objective: To investigate whether Diff‐Quik stained fine needle aspirate smears can be used to evaluate Ki‐67 expression by immunocytochemistry. Methods: Both cytological and histological samples were obtained from 24 dogs with spontaneously developed mammary gland tumours. The cytological and histological specimens were examined by Diff‐Quik and H&E stains, respectively. After examination, both samples were immunostained using the same Ki‐67 antibody. The % Ki‐67 values were calculated based on the percentage of positively stained tumour cells per 500 and 1000 tumour cells in cytology and histology specimens, respectively. Results: Ki‐67 staining was successful in 17/24 smears (71%) and 19/23 sections (83%). The correlation coefficient between the percentage of Ki‐67‐positive cells in cytological smears and in the histological sections was 0.677 (P < 0.01). These values were significantly different between histologically benign and malignant tumour groups both in cytology and histology samples (P < 0.001). The threshold value of the percentage of Ki‐67‐positive cells for distinguishing benign from malignant tumours was set at 4.85% with 90.9% sensitivity and 92.3% specificity by Receiver Operating Characteristic (ROC) curve using histopathology as the gold standard. Conclusion: Diff‐Quik‐stained cytology smears can be used to detect the presence of Ki‐67 antigen when histology sections are not available.     

ANALYSIS OF TRITIUM INCORPORATION INTO INDIVIDUAL CELLS BY AUTORADIOGRAPHY OF SQUASH PREPARATIONS

The relation between tritium content of individual cells and grain count obtained in autoradiographs of squashed cells was investigated. The tissues used were root meristems of Tradescantia paludosa and intestinal epithelium of the mouse. The relation between grain count and tritium content is affected by self-absorption which depends on the thickness of the labeled cell. Therefore, squashed preparations were sectioned to determine the uniformity of thickness of nuclei. In a preparation of mouse cells, thicknesses were 1.18 ± 0.35 µ, and in a preparation of Tradescantia cells, 2.97 ± 0.35 µ. The effects of similar and larger variations in thickness upon grain count were studied in material squashed with different pressures; no marked correlation was found. The lack of correlation is explained by the geometric relation between labeled nuclei and the emulsion. By counting grains and directly measuring tritium content in a glass proportional counting tube in the same preparation, the yield of grains per disintegration was measured in Tradescantia cells and found to be 1 grain for 10.9 disintegrations with AR 10 autoradiographic film and 1 grain for 19.3 disintegrations for NTB nuclear track liquid emulsion. Latent image fading may pose a problem with long exposures; the conditions of its occurrence are as yet not well known.     

Estimating the Median Generation Time of Proliferating Cell Systems in Steady State

For proliferating cell systems in which the usual "labeled mitoses" method cannot be used to estimate generation times, an alternative scheme is derived. The method presented here is based on observation (by autoradiography) of the median grain count of labeled interphase cells following a pulse of labeled DNA precursor. It is shown that the median generation time of the labeled cells will be equal to the time required for the median grain count to halve, starting from the time when half the labeled cells have completed their first division. This starting time is determined from observation of the first wave of labeled mitoses. The procedure was designed to minimize error resulting from such factors as radiation damage, label reutilization, and the use of a nonzero grain counting threshold. The method is applied to the analysis of two cases of acute leukemia in man.     

Fully automated and fast image analysis of autoradiographs with a TAS-Leitz. Determination of size, Feulgen fluorescence and grain counts of individual nuclei and their evaluation by a simplified cluster analysis

A fully automatic analysis system based on television image analysis was developed to measure simultaneously three parameters in individual nuclei of microscopic autoradiographs prepared from mouse jejunal crypt cell squashes and ascites tumor cell smears: size, Feulgen fluorescence and reflection from silver grains. A dark light camera with an image intensified silicon tube (RCA-ISIT), an automatic scanning stage and an autofocus device were fitted to a Leitz-TAS microscope. The camera permitted localization of Feulgen stained nuclei and measurement of area and light intensity by means of incident of light fluorescence in the red. After automatic changes of the Opak-illuminator silver grains were determined by means of polarized incident light reflected from the grains in the blue. A 25 X oil objective (aperture 0.75) yielded sufficient resolution for measurements. The nadir between the proportions of labeled and unlabeled nuclei was calculated from the data of one specimen on a PDP-computer using a new algorithm based on the minimal variance of the logarithm of reflected light per nucleus. Labeling indices determined by visual grain counting and by automatic analysis of the autoradiographs were well correlated (r = 0.87 to 0.92). Visual grain counts/nucleus and reflected light/nucleus correlated well when individual nuclei were compared (r = 0.92 to 0.97) or means of labeled nuclei of various specimens prepared during a 5 year period (r = 0.90 to 0.93). Quenching of nuclear Feulgen fluorescence was minimal. The optimal labeling range is 30-100 grain counts/nucleus. The time interval between measurements of two specimens was 25 min for a squash of approximately 350 crypt cells within a 3 mm X 3 mm field, and 20 min for a meandering scan with 1,000 ascites tumor cells.