Manual Counting Method Research Articles

Comparison of sperm counting chambers used by participants in a national andrology proficiency testing program

Proficiency testing (PT) is a widely recognized system to ensure quality testing among clinical laboratories. The American Association of Bioanalysts (AAB) offers an andrology sperm count PT module, and reports results according to the type of counting chamber used by the participating laboratory. Several types of sperm counting chambers are commercially available, and it is difficult for the andrology laboratory to know which is the most advantageous to use. In this report, we have examined the results and variation associated with the use of various sperm counting chambers. PT samples, two challenges per event, were mailed to participating laboratories in the spring and fall of 2001, 2002 and 2003 (total of 6 events and 12 challenges). Suspensions of fixed human sperm were provided to participating laboratories in two concentrations: approximately 16 sperm million/ml and 55 million sperm/ml. Laboratories were instructed to perform sperm counting procedures on the survey samples using the lab’s routine methodology, and to report the results, as well the methodology and type of counting chamber used, to the AAB. The number of participating laboratories over the 6 events ranged from 470–519. The vast majority of laboratories used manual procedures (83.4%) compared with computer assisted semen analysis (CASA; 16.6%). The most common counting chamber used by laboratories performing manual procedures was the hemacytometer (47.0%), followed by the Makler, Micro Cell and Cell Vu chambers (34.6%, 13.6% and 4.8%, respectively). Of those laboratories using CASA procedures, 56.5% used the Makler chamber while the remainder used the Micro Cell chamber (43.5%). In order to determine which counting chamber tended to “overestimate” or “underestimate” sperm counts, the grand mean sperm count was calculated for all participants within each challenge, and these values were compared with the mean results for each counting chamber group. Manual cell counts using the Cell Vu chamber tended to overestimate sperm counts the greatest (mean ± SE; 119.6 ± 12.7%) while CASA cell counts using the Micro Cell chamber tended to underestimate the most (75.3 ± 1.6%). Interestingly, cell counts using the Makler for both the manual and CASA methods came closest to the grand mean (101.8 ± 4.21% and 98.8 ± 2.6%, respectively). Overall, a wide range of variation was noted in the reported results. Coefficients of variation (CV) ranged from a low of 20.4% to a high of 392.9%. Manual counts using the hemacytometer yielded the highest mean CV (124.2 ± 26.0%) while manual counts using the Makler chamber demonstrated the lowest mean CV (32.0 ± 1.7%). These data indicate that the majority of laboratories participating in the AAB andrology PT program performed manual counting methods and used the hematytometer for counting sperm. However, this procedure was also associated with the greatest variation. Although disposable counting chambers, such as the Cell Vu and Micro Cell, have become widely used, and may be more convenient compared with other chambers, manual counting methods using the Makler chamber appeared to yield the most reliable and consistent results.

An evaluation of the utility of performing body fluid counts on the coulter LH 750.

Performing a manual body fluid count is a tedious, time-consuming, and frequently imprecise process for any clinical laboratory. The ability to perform many of these counts with an automated hematology analyzer has the real potential of making a major impact on laboratory precision and productivity. The manual chamber count is fraught with many variables and is often very technologist/technique dependent, often leading to inaccuracies in test results. Our laboratory undertook a series of studies designed to evaluate the capability of the Coulter LH 750 hematology analyzer to supplement our current manual method in the performance of body fluid analysis. First, we established the performance of our current manual counting method, the gold standard against which LH 750 performance would be judged. We looked at the precision of manual cell counting by having 4 technologists perform manual counts on each of 35 spinal, synovial, peritoneal, and other fluids with white blood cell (WBC) and/or red blood cell (RBC) counts of greater than 0.3 x 10(9) /L and 0.03 x 10(12) /L, respectively. Our results support earlier reports that the variability in counts among technologists using the manual chamber cell count methods is often very significant. After each sample was manually counted, it was analyzed on the Coulter LH 750, and the results were compared with those of our current manual method. Results showed good correlation between the manual and LH 750 methods. Next, we conducted a series of separate tests to evaluate the stability of different cellular elements (WBCs and RBCs) in each of these body fluid types. The study consisted of 2 sets of 4--3-mL samples of each fluid type--which were analyzed on the LH 750 immediately on receipt and after 1 hour, 4 hours, 8 hours, 16 hours, and 24 hours. The findings suggested varying degrees of stability that were dependent on fluid type and initial cell concentration. Finally, we looked at whether it is necessary to perform background counts before analyzing each body fluid sample and the impact of automating body fluid counts on our workload.

Histologic modification of cutaneous aging by caloric restriction in rats

Problem: To investigate whether caloric restriction can modify age-induced intrinsic changes of the skin in Fischer 344 rats. Methods: Fischer 344 rats were procured from the National Institute of Aging and 3 groups of ad libitum (AL) and calorie-restricted (CR)rats were set up for study: young (4 months), adult (1 year), and old (24 months+). Samples of the ventral skin were subjected to histological processing and paraffin microtomy. Readings were obtained in blinded fashion, and standard manual methods of ocular grid morphometry and stereological point counting were applied. The parameters assessed were the number of epidermal nuclei, depth of epidermis, dermis, and adipose layer (μm), and percentage area of dermis occupied by collagen bundles, elastic fibers, pilosebaceous units, and capillaries. Using SPSS 11.0 and Excel statistical software the data were analyzed by 2-factor analysis of variance (general linear model-univariate procedure) to test the null hypotheses about the effects of age, diet, and age-diet interaction effects on the dependent variables, ie, various skin parameters. Results: Both age and diet had significant effects on the epidermal component of the skin. Diet effect was seen in the dermal thickness (F9.26,P.005) with larger values in aging AL animals. Dermal adipose tissue showed pronounced effects of calorie restriction (F5.89,P.021). Elastic and collagen fibers manifested tri-phasic age effect in both nutritional groups with higher values in adult animals. There was no noticeable change in the percentage area of pilosebaceous units. Conclusion: This model shows alterations of skin histomorphology with age, some of which can be modulated with caloric restriction. Significance: Microscopic studies are useful for investigating the effects of chemotherapeutic agents on the skin in suitable age-controlled laboratory animals. The use of CR animals may elucidate whether dietary control can modify the response to such therapies. Support: None reported.

Flow cytometric characterization of cell populations in bronchoalveolar lavage and bronchial brushings from patients with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a serious, chronic inflammatory disease of the airway associated with cigarette smoking. Leucocytes are involved in the inflammatory process in the airways in COPD. There is a need for accurate characterization of cellular populations in bronchoalveolar lavage (BAL) due to variation in the predominant cell types reported, which were investigated mostly with manual counting techniques. Bronchial brushings and BAL were obtained from human subjects undergoing fiber optic bronchoscopy. Flow cytometry was applied to identify various cell types. Quenching of autofluorescence of BAL-derived alveolar macrophages was achieved with eta-octyl beta-D-galactopyranoside and crystal violet. Comparisons of cell counts obtained with flow cytometric and manual counting methods were performed. Correlation analysis showed that manual cell counting methods overestimated the percentage of macrophages when compared with flow cytometric methods (R2 = 0.54). There was also a small tendency by manual counting to underestimate the percentage of lymphocytes and neutrophils. Using flow cytometry, the percentage and absolute numbers of alveolar macrophages and lymphocytes in BAL were not significantly different between patients with COPD and control subjects. The percentage and absolute numbers of neutrophils were higher in BAL from patients with moderate to severe COPD. This novel flow cytometric assay for identification of various cell types from heterogenous samples of BAL and bronchial brushing will allow further investigation of cell characteristics, such as cytokine production and receptor expression, and an accurate evaluation of apoptosis for different cell types and provide a rationale for urgently required effective treatments for COPD.

Medication event monitoring system (MEMS®): The asthma clinical research network (ACRN) experience

Rationale The eDEM™ Monitor, a MEMS® device, was introduced into the “Improving Asthma Control Trial (IMPACT)”, a 12-month, multi-center, study to monitor adherence to a twice-daily oral dosing regimen in mild asthmatics. The objective was to establish study standards and compare eDEM's use in monitoring adherence versus manual pill count. Methods The ACRN established standard procedures, including certification requirements, prior to initiating IMPACT. eDEM determined adherence to a twice-daily oral tablet dosing regimen prescibed inconjunction with inhaler use, administered in a double-blind, double-dummy fashion in 225 randomized subjects (mean age=33 yrs). Agreement between the two methods of measuring adherence was assessed by the concordance correlation coefficient at specific visits, and an overall concordance correlation coefficient that combined the data from these visits and adjusted for the repeated measurements within each subject. Results The average adherence for both eDEM and pill count was consistently above 90%. When evaluating the visits separately, the concordance correlation coefficient to measure agreement between the two methods ranged from 0.92 to 0.74. For all visits combined, with adjustment for the correlation within each subject, the estimated overall concordance correlation coefficient between eDEM and pill count adherence was 0.91. Conclusions The excellent agreement between the adherence measured via eDEM and the manual pill count method supports the reliability of this device for patient monitoring in clinical trials.

Qualitative and Quantitative Measurement of Human Brain Activity Using Pixel Subtraction Algorithm

Purpose: To develop an automated quantification program, which is called FALBA (Functional & Anatomical Labeling of Brain Activation), and to provide information on the brain centers, brain activity (%) and hemispheric lateralization index on the basis of a brain activation map obtained from functional MR imaging. Materials and Methods: The 3-dimensional activation MR images were processed by a statistical parametric mapping program (SPM99, The Wellcome Department of Cognitive Neurology, University College London, UK) and MRIcro software (www.mricro.com). The 3-dimensional images were first converted into 2-dimensional sectional images, and then overlapped with the corresponding T1-weighted images. Then, the image dataset was extended to -59 mm to 83 mm with a 2 mm slice-gap, giving 73 axial images. By using a pixel subtraction method, the differences in the R, G, B values between the T1-weighted images and the activation images were extracted, in order to produce black & white (B/W) differentiation images, in which each pixel is represented by 24-bit R, G, B true colors. Subsequently, another pixel differentiation method was applied to two template images, namely one functional and one anatomical index image, in order to generate functional and anatomical differentiation images containing regional brain activation information based on the Brodmann's and anatomical areas, respectively. In addition, the regional brain lateralization indices were automatically determined, in order to evaluate the hemispheric predominance, with the positive (+) and negative (-) indices showing left and right predominance, respectively. Results: The manual counting method currently used is time consuming and has limited accuracy and reliability in the case of the activated cerebrocortical regions. The FALBA program we developed was 240 times faster than the manual counting method: -10 hours for manual accounting and -2.5 minutes for the FALBA program using a Pentium IV processor. Compared with the FALBA program, the manual quantification method showed an average error of 0.3340.007 (%). Thus, the manual counting method gave less accurate quantitative information on brain activation than the FALBA program. Conclusion: The FALBA program is capable of providing accurate quantitative results, including the identification of the brain activation region and lateralization index with respect to the functional and anatomical areas. Also, the processing time was dramatically shortened in comparison with the manual counting method.

Interobserver Reproducibility of MIB-1 Labeling Index in Astrocytic Tumors Using Different Counting Methods

MIB-1 labeling index has been used as a complementary method to differentiate better and worse prognostic groups of astrocytic tumors. However, its reproducibility has been doubted because of the interlaboratory and interobserver variability. We studied the interobserver reproducibility of four manual counting methods of MIB-1 labeling index, including direct counting, area fraction, line intersection with a graticule, and line intersection without a graticule to estimate the total number of the cells. Using line intersection estimate, either with or without a graticule, yielded a worse result. Those MIB-1 labeling indices of astrocytomas were overlapping with those of anaplastic astrocytomas and glioblastomas. Besides, the intraclass correlation coefficient was smaller, and the coefficient of variation was greater than that attained when using a graticule and counting the total number of cells either directly or by area-fraction estimate. The level of interobserver agreement using an MIB-1 labeling index cutoff value of 11.0 was moderate to substantial when applying the line-intersection estimate, and it was almost perfect when applying the direct counting or the area-fraction estimate. The interobserver reproducibility and agreement of MIB-1 labeling index in astrocytic tumors were different depending on the counting methods. Direct counting with a graticule yielded the best result. Before establishing reference standards for staining and counting, the reproducibility of MIB-1 labeling index should be verified.

Automated counting of phytoplankton by pattern recognition: a comparison with a manual counting method

Computer-based image analysis and pattern recognition methods were used to construct a system able automatically to identify, count and measure selected groups of phytoplankton. An image analysis algorithm was employed to isolate and measure objects from digitized images of a phytoplankton sample. The measurements obtained were used to identify selected groups of phytoplankton by a combination of artificial neural networks and simple rule-based procedures. The system was trained and tested using samples of lake water covering an annual growth, cycle from Lough Neagh in Northern Ireland. Total volume estimates were obtained for the four major phytoplankton species, using both the automated sjstem and a manual counting method. Estimates of total cell volume obtained from the automated system were within 10% of those derived by manual analysis of the same cells. The automated system produced total cell volume estimates dose to those obtained from manual analysis of different aliquots of the same water sample. Variation between successive counts of the same water sample was higher with the automated system than with the manual counting method. Limitations and possible improvements to the technology are discussed.

Blood basophil numbers in chronic ordinary urticaria and healthy controls: diurnal variation, influence of loratadine and prednisolone and relationship to disease activity.

The basopenia of chronic urticaria relates to histamine releasing autoantibodies in the serum of patients with autoimmune urticaria. This reduction in circulating basophils may be due to active recruitment into weals. If so, it might be expected that numbers in blood would be reduced when urticaria is active and increased after treatment. The primary aim of this study was to look at diurnal variation of basophil numbers in patients with chronic ordinary urticaria (not physical or vasculitic) in relation to disease activity and the effect of treatment with antihistamines and corticosteroids, and to compare the results with healthy controls. A secondary aim was to compare a standard manual counting method with automated basophil counts and to look at numbers of other circulating leucocytes that might be relevant to urticaria pathogenesis. Manual basophil counts using a toluidine blue stain and automated 5-part differentials (Coulter Gen. S) were performed at 4-hourly intervals from 08.00 to 20.00 in 10 healthy controls (six women, age 24 to 63 years) and seven chronic urticaria patients (five women, 24 to 50 years). All chronic urticaria patients had severe daily or almost daily urticaria. Only one of six chronic urticaria sera showed in vitro basophil histamine releasing activity. Counts were performed without treatment, after a week of taking loratadine 10 mg daily and after 3 days of adding prednisolone at 0.6 mg/kg/day (maximum 40 mg). Daily urticarial activity scores (UAS) were derived from weal numbers and itch, maximum 7. There was no significant overall diurnal variation of basophil numbers in healthy controls or chronic urticaria patients. Mean (SE) manually counted basophil were higher in healthy controls than chronic urticaria (43.4/ microL (2.1) vs. 4.4 (0.8), P < 0.001). Basophil counts were reduced in healthy controls on steroids (19.2 (1.9), P < 0.001) but increased in chronic urticaria (8.9 (1.9), P < 0.001). Loratadine did not influence them. UAS fell on treatment (3.3 (0.4) baseline, 1.4 (0.5) on loratadine and 0.5 (0.2) on prednisolone with loratadine, P < 0.001). There was a negative linear correlation between basophil numbers and UAS in untreated chronic urticaria patients (P = 0.001, Spearman rank correlation). Manual and automated basophil counts showed poor agreement. Lymphocyte numbers were lower in chronic urticaria than healthy controls. Neutrophils increased whereas lymphocytes and eosinophils decreased in all subjects on prednisolone. They were unaffected by loratadine. The results are consistent with the hypothesis that circulating basophils may be recruited from blood into urticarial weals during disease activity. Automated counts are not suitable for assessing basophil numbers in chronic urticaria. The relevance of reduced lymphocyte numbers in chronic urticaria needs to be explored.

Validating Automated PMN Counts on Ascitic Fluid

Spontaneous bacterial peritonitis (SBP) is a frequent complication of cirrhosis, occurring in 10% to 30% of hospitalized patients with ascites; the SBP mortality rate is about 25%. SBP diagnosis requires an ascitic fluid polymorphonuclear (PMN) cell count of >250 cells/mm3. To date, light microscopy and manual counting methods, which are laborious and time consuming and often are given low priority, have been used. Waits of 24 hours for results of manual PMN counts …

Interpretation of atypical patterns encountered when using a flow cytometry-based method to detect residual leukocytes in leukoreduced red blood cell components.

Universal leukoreduction of blood components is becoming the standard of care. Flow cytometry methods are being used for quality control of the leukoreduction process. We provide an atlas of atypical flow cytograms generated by a commercial LeucoCOUNT assay that was used to enumerate residual leukocytes in leukoreduced red blood cell components. Numeric results are derived from a flow cytogram generated by the assay. Three types of atypical flow cytogram patterns were observed during process validation or routine quality control of leukoreduced red blood cell components. (a) Fixation artifact: Fixation of control or test samples can alter the staining intensity compared with fresh cells. (b) "Rain" pattern: Flow cytometry methods count slightly damaged leukocytes not removed during leukoreduction. Slightly damaged leukocytes appear on a flow cytogram like "rain" falling from a well-defined "cloud" of intact residual leukocytes. Discrepancies between automated flow cytometry results and subjective manual counting methods can occur. (c) Autofluorescence-debris pattern: Cell debris and age-related changes in the sample can cause shifts in the fluorescence staining pattern, resulting in erroneous test results. Review of flow cytograms is essential for accurate reporting of flow cytometry-based methods for enumerating residual leukocytes in leukoreduced blood components.

A comparative study of MIB-1 staining indices of gliomas measured by NIH Image analysis program and conventional manual cell counting method

To evaluate the NIH Image measured MIB-1 staining index (SI) as a useful tool for the determination of proliferative activity of gliomas, MIB-1 immunohistochemistry was performed on paraffin sections to estimate the proliferative activity of 50 gliomas. Five to eight RGB images at 200 magnifications were acquired per sample in a Macintosh computer using a RD-175 digital camera and Adope Photoshop program, followed by conversion of the RGB image to B (blue subtracted) and R (red subtracted) images and finally automatic cell counting of more than 1000 cells per sample by the NIH Image analysis program. The mean MIB-1 staining indices by both NIH count and visually assessed (manual) count respectively were 12.7% ±9.9% (mean ± standard deviation) and 12.6%±9.9% in all gliomas (n = 50), 2.9%± 0.6% and 2.3%± 0.6% in grade I and II astrocytomas (n = 9), 10.7%± 8.9% and 11.1%±9.0% in grade III gliomas (n=17), 16.1%±9.1% and 16.3%±9.1% In glioblastoma multiformes (n = 20), 26.0%±4.9% and 24.5% ± 2.6% in other glial tumors (n = 4). These paired values were assumed to be identical, since the p value obtained by paired t-test in all gliomas was 0.905. We conclude that the MIB-1 staining index measured by NIH Image is reliable and universal. [Neurol Res 2000; 22: 495-500]

Eosinophil chemotaxis by chemokines: a study by a simple photometric assay.

The effects of a panel of 15 chemokines on eosinophil chemotaxis were studied by a new photometric assay which is both less tedious and less laborious than the conventional manual counting methods. Approximately 40 chemokines have been identified to date, but there is little information on the eosinophil migration-inducing ability of chemokines other than CC chemokine receptor (CCR) 3 ligands. Eosinophil migration was measured by the Boyden chamber technique with a 96-well multiwell chamber and polycarbonate membrane filter. Eosinophil migration was assessed by determination of the eosinophil peroxidase (EPO) activity, and photometric measurement was performed with a microtiter plate reader. The assay was sensitive enough to detect 200 eosinophils, and the time required was within 4 h. CCR3 ligands, i.e., regulated on activation normal T-cell expressed and secreted (RANTES), eotaxin, eotaxin-2, and monocyte chemoattractant protein (MCP)-3, induced significant migration, while other chemokines showed no significant migration-inducing ability. Although the chemotaxis induction by these chemokines was efficiently inhibited by anti-CCR3 mAb, anti-CCR1 mAb failed to show any inhibitory effects. The photometric assay is suitable for analyzing a large number of samples. CCR3 ligands are the most important chemokines inducing eosinophil chemotaxis; thus, CCR3 represents a possible therapeutic target for the treatment of allergic diseases.

Neuroendocrine Differentiation in Prostatic Carcinoma During Hormonal Treatment

Objectives. Neuroendocrine differentiation (NED) is a common feature in adenocarcinoma of the prostate. Several studies suggest that NED may have a major impact on cancer progression as neuroendocrine (NE) secretory products have been shown to possess growth stimulatory effects. NED has also been proposed to constitute part of the mechanism by which a prostate cancer cell progresses toward androgen independence as NE tumor cells have been demonstrated to be devoid of androgen receptor immunoreactivity. In this retrospective study, we evaluated NED status in prostate cancer specimens from patients undergoing androgen ablation therapy. Methods. The degree of NED in transurethral resection of the prostate (TURP) samples from 53 patients with prostate cancer was investigated by immunocytochemistry using polyclonal rabbit immunoglobin G (IgG) against chromogranin A (CgA). Changes in NED with time were determined by a manual semiquantitative cell counting method. Results. During androgen withdrawal therapy, 21 tumors (40%) displayed increased NED concomitant with histopathologic tumor progression, whereas 29 carcinomas (55%) showed no change in NED status. However, a majority of the histopathologically unchanged tumors displayed marked NED at the first TURP and an increase in NED was by definition not possible. In only 3 cases (5%) was a decrease in NED observed with time. Conclusions. Androgen ablation therapy may be a contributing factor to the increase in NED of prostatic adenocarcinoma with time, and our findings imply that androgen withdrawal therapy enhances the selection and progression of NED, androgen-independent tumor cells.

Use of a 96-well plate reader to evaluate proliferation of equine satellite cell clones in vitro

We have adapted a methylene blue staining assay to measure proliferation of equine satellite cell clones in a 96-well format. This technique allows rapid and accurate measurement of proliferating satellite cells which is a considerable enhancement over manual counting methods. Methylene blue is incorporated into the nuclei and intracellular matrix of satellite cells and then released into the aqueous phase. Absorbance of stained cells is read at 620 nm and correlates with increasing numbers of cells (range tested from 1 × 103 to 4 × 104). This method was used to determine the response of equine satellite cells to FGF, both human recombinant and bovine, and to IGF-1. This format is very efficient in measuring and comparing the proliferation of equine satellite cells. However, fusion of cells to form multinucleated myotubes cannot be assayed using this method because it lacks the sensitivity and specificity to differentiate multinucleated from mononucleated cells, and to detect expression of myogenic proteins. The assay could be accurately applied from 0 to 144 hours, before significant fusion and differentiation takes place. Using this assay will reduce analysis time to quantitate the proliferation response of equine satellite cells to different growth factors.

Reticulocyte counts in laboratory animals using a manual and a flow cytometric procedure (sysmex R-3000™)

An evaluation of the flow cytometric procedure for reticulocyte counting was performed, together with a comparison to a reference manual counting method. An automated reticulocyte analyser, the Sysmex R-3000™ (Flow cytometric auramine O methods) was used to enumerate the absolute reticulocyte counts and the percentage of reticulocytes in blood obtained from rats, dogs and monkeys. Simultaneously, a maturation distribution was obtained by subdividing the reticulocytes into three maturation classes according to their fluorescence intensity. The reticulocyte counts and the maturation distributions can be determined rapidly and easily. The coefficients of variation (CV) for the within-day precision study in the reticulocyte counts of the three species were found to be 9.7%–20.5% for the micro-scopic method and 4.4%–9.4% for the flow cytometric method. A very good correlation was observed between the microscopic count and flow cytometric methods (r=0.958). In conclusion, because of high reproducibility and good agreement with the manual counting method for these species, flow cytometric analysis of reticulocytes is an attractive alternative procedure to micro-scopic enumeration in toxicological studies.

Automatic volumetric capillary cytometry for counting white cells in white cell‐reduced plateletpheresis components

As the benefits of white cell (WBC)-reduced blood components become increasingly apparent, the need has arisen for a simple, automated WBC-counting technique that is sensitive to low WBC concentrations. Automated volumetric capillary cytometry was evaluated for its ability to quantify residual WBCs in WBC-reduced plateletpheresis components. The volumetric capillary cytometry system evaluated uses a laser to excite fluorescent dye-labeled nucleated cells. The number of nucleated cells per microliter is reported. Four studies were performed: linearity, precision of results near the value of 5 x 10(6) WBCs per unit, the limit of detection, and correlation to the Nageotte manual counting method. Assay values correlated to expected values (range, 0-125 WBC/microliter) with an r2 > 0.99. In the range of 5 x 10(5) WBCs per unit the CV was 8.5 percent, and concentration differences of 0.15 log10 were detectable. The limit of detection was 1.0 WBCs per microliter (95% upper confidence limit). The assay correlated to the Nageotte method with an r2 of 0.98, slope of 1.0, and y-intercept of 2.0 WBCs per microliter. Assay results were 10 to 15 percent higher than Nageotte results, in samples with values near 5 x 10(6) WBCs per unit. Technician time per sample was 2 to 3 minutes. Volumetric capillary cytometry is precise and sensitive to small differences in WBC concentration in the range of clinical interest. The device provides an efficient new method for quality assurance and control of WBC-reduced plateletpheresis products.

A CITATION ANALYSIS OF LIS SERIAL LITERATURE PUBLISHED IN DENMARK 1957–1986

The focus of the citation analysis reported is the information exchange between the Danish library‐information profession and LIS communities in other countries. Consideration is given to the diffusion of ideas and innovations from foreign countries into the Danish LIS world. Citation evidence is also used to shed light on structural characteristics of the LIS periodical literature and other communication media and some of the communication patterns characterising the LIS field in Denmark. The raw material for the citation analysis was gathered by the manual citation counting method and not drawn from computerised citation databases. The fact that a surprisingly large proportion of the references cited by Danish LIS authors belong to the so‐called ‘hidden’ category — denoting cited references embedded in the text of journal papers — is noted as a key finding. The observation on the considerable number of ‘hidden’ citations is developed further. Journals and books (monographs) are the publication formats most frequently relied on by LIS authors. It was found that the majority of the citations are to relatively recent materials. Next to Danish material, publications in English and produced in the United States and in Great Britain are those most heavily relied on by the Danish LIS community. Ranking of journals by number of citations shows that a very small number of journals accounts for the majority of journal citations. On the whole, the works cited point to a definite interest in public libraries and issues relating to the planning, structure and legislation of public libraries. Works on research and academic libraries and on theoretical aspects of LIS did not attract the same amount of citations.

A Computerized Method for Determination of Microvascular Density

Currently accepted stereological methods for vascular density measurements involve manual counting of labeled vessels with a grid image overlay and determination of vessel-grid intersections. This method both is tedious and may be prone to error; therefore, our laboratory has developed a method for computer-automated determination of microvascular density using digital image processing techniques. An image of the microvasculature is acquired using computer videomicroscopy. The image is processed in three general steps, involving (1) background correction, (2) thresholding of the gray level image to create a binary image, and (3) processing of the binary image using erosion, dilation, and skeletonization algorithms. Testing of this procedure was performed on 328 typical images of skeletal muscle tissue sections taken from the hindlimb of Sprague-Dawley rats and quantitated by both traditional measures and our new computer method. Results from this comparison reveal that the automated vessel counting is highly correlated ( r 2 = 0.71) and decreased analysis time from 15 min/image to 30 sec/image when compared with manual counting methods. Our computer-based method also appears to be superior to the traditional method due to the unbiased and nonsubjective nature of determining vessel-grid intersections.

Reticulocyte counts in canine and rat blood made by flow cytometry

Absolute and percentage reticulocyte counts for sequestrated blood samples from healthy Wistar rats (n = 132) and beagles (n = 64) were made by thiazole orange and flow cytometry. The flow cytometric method showed good agreement with a manual counting method for both species, and the precision data for both of these counting methods were similar.