Electronic Cell Counter Research Articles

Ethylenediaminetetraacetic Acid-Dependent Pseudothrombocytopenia in a Cardiac Surgical Patient

Ethylenediaminetetraacetic Acid-Dependent Pseudothrombocytopenia in a Cardiac Surgical Patient

Evaluation of a haematological analyser (Sysmex F-800) with equine blood.

A semiautomatic electronic blood cell counter (Sysmex F-800) was evaluated with equine blood, according to the protocol of the International Committee for Standardization in Haematology (ICSH, 1984). The precision and overall reproducibility were acceptable for all the parameters studied except for the platelet count, in which a coefficient of variation of 18.8% and 21.7% was obtained for within and between batch precision and 26.76% for overall reproducibility. Carry-over for the haematocrit value and platelet count was unsatisfactory, thus the use of a blank diluent sample between different analyses was necessary. Linearity of the analyser was acceptable in the range studied. A total of 100 equine blood samples were studied with the Sysmex F-800 and the manual method. The correlations between both Methods were acceptable for all the parameters. In the storage study, the red blood cell count and the haemoglobin concentration were the parameters with longest stability (72 h) at 4 degrees C and 25 degrees C. Platelet count decreased significantly at 3 h post-extraction in those samples stored at 4 degrees C. During the evaluation period, Sysmex F-800 was easy to handle and it is well suited for routine equine blood cell analysis.

Evaluation of a hematology analyzer with canine and feline blood.

A semiautomatic electronic blood cell counter (Sysmex F-800:Toa Medical Electronics Europa Gmbh, Hamburg, Germany) was evaluated using canine and feline blood, following the International Committee for Standardization in Hematology protocol (ICSH, 1984). Precision and overall reproducibility were acceptable for all the parameters studied except for the feline platelet count, in which overlapping of erythrocyte and platelet populations prohibited determination of an accurate platelet count. Since carry-over from canine hematocrit values and platelet counts and from feline hematocrit values was unsatisfactory, the use of a blank diluent sample between different analyses was necessary. Linearity of the analyzer was acceptable in the studied range. Thirty canine and feline blood samples were analyzed using the Sysmex F-800 and a manual method. Correlations between both methods were acceptable for all the parameters, except for feline platelet count and erythrocyte indices for both species. In the storage study, red blood cell count and hemoglobin concentration were the parameters with the longest stability (72 hours at 4 degrees C and 25 degrees C) in both species. A statistically significant increase in MCV was obtained at 12 hours post-extraction in canine samples stored at 25 degrees C and at 24 hours in refrigerated samples. Feline leucocyte counts showed a downward trend at 12 hours post-extraction at both temperatures. Canine platelet count decreased significantly at 6 hours post-extraction in samples stored at 4 degrees C. During the evaluation period, Sysmex F-800 was user friendly and appeared well suited for routine canine and feline blood cell analysis.

Cell Counting

AbstractThis unit presents protocols for counting cells using either a hemacytometer or electronically using a Coulter counter. Cell counting with a hemacytometer permits effective discrimination of live from dead cells using trypan blue exclusion. In addition, the procedure is less subject to errors arising from cell clumping or size heterogeneity. Counting cells is more quickly and easily performed using an electronic counter, but live‐dead discrimination is unreliable. Cell populations containing large numbers of dead cells and/or cell clumps are difficult to count accurately. In addition, electronic counting requires resetting of the instrument for cell populations of different sizes; heterogeneous populations can give rise to inaccurate counts, and resting and activated cells may require counting at separate settings. In general, electronic cell counting is best performed on fresh peripheral blood cells.

Monitoring Cell Growth

This appendix provides two protocols for monitoring cell growth. Counting cells using a hemacytometer is tedious but it allows one to effectively distinguish live cells from dead cells (using Trypan Blue exclusion). In addition, this procedure is less subject to errors due to cell clumping or heterogeneity of cell size. The use of an electronic cell counter is quicker and easier than counting cells using a hemacytometer. However, an electronic cell counter as currently constructed does not distinguish live from dead cells in a reliable fashion and is subject to error due to the presence of cell clumps. Overall, the electronic cell counter is best reserved for repetitive and rapid counting of fresh peripheral blood cells and should be used with caution when counting cell populations derived from tissues.

Effect of Freezing on Somatic Cell Count of Quarter Milk Samples as Determined by a Fossomatic Electronic Cell Counter

The objective of this study was to determine the effect of freezing and the duration of freezing on somatic cell count (SCC) in milk samples from quarters with no signs of clinical mastitis. Effect was determined by a fluoro-opto-electronic cell counter. Samples were frozen in a commercial freezer at approximately –20°C. The milk samples were thawed at 1, 2, 3, 4, 7, 14, or 28 d. Another batch of samples was frozen 1 d, thawed, frozen again for 1 d, and then thawed. The SCC was determined on the fresh samples and after any one of the freezing periods. On average, the SCC were lower after all freezing periods than they were before freezing. However, the difference in SCC before and after freezing was small and relatively constant on the natural logarithm scale. The decrease in SCC was larger with longer freezing periods. When SCC thresholds of 200,000, 250,000, and 500,000 cells/ml were used to predict infection, freezing appeared to have little impact on the sensitivity and specificity of diagnostic parameters. Therefore, SCC, as determined by a fluoro-opto-electronic cell counter, determined for frozen quarter milk samples can be used in mastitis control and research programs.

Feline ocular epithelial response to growth factors in vitro

Abstract Objective To examine the proliferative abilities of growth factors known to participate in wound healing on feline lens, iris pigment, ciliary, and retinal pigment epithelium cultured in vitro. Animals 8 clinically normal cats. Procedure Iris pigment, lens, ciliary, and retinal pigment epithelia of normal eyes of cats were isolated and cultured. Morphologic characteristics of primary cell cultures were studied by light and electron microscopy. Subcultures of epithelial cells were exposed to media supplemented with 0.5% fetal bovine serum plus various combinations of insulin and/or growth factors, including transforming growth factor-α, epidermal growth factor, acidic fibroblast growth factor, and basic fibroblast growth factor. Growth promoting effects were evaluated by counting with an electronic cell counter. Results Cells retained many of the morphologic characteristics of in vivo cells. Cell proliferation assays indicated that transforming growth factor-α stimulated lens and ciliary epithelial cell growth, and epidermal growth factor enhanced lens and iris pigment epithelial cell growth. Acidic fibroblast growth factor had proliferative effects on lens, iris pigment, and ciliary epithelium. Basic fibroblast growth factor was the most potent stimulator of all mitogens used, and caused substantial proliferation in all cell types. Insulin alone stimulated lens and ciliary epithelial proliferation but, combined with other growth factors, had a synergistic effect with those causing cell proliferation, except acidic fibroblast growth factor with iris pigment epithelium. Conclusion Morphologic studies support the argument that pigment-producing cells are involved in feline ocular sarcoma. Growth factor studies indicated that ciliary epithelium has the most profound proliferative effect of all growth factors used. These data may help guide future studies in determining the cell of origin for feline ocular sarcoma. (Am J Vet Res 1996;57:1748–1752)

Analysis of novel apolipoprotein B mutations using a modified U937 cell line LDL binding assay

The U937 myelomonocyte proliferation assay can be used to detect patients with familial defective apolipoprotein B-100 (FDB). Previous studies have employed electronic cell counting to assess cell proliferation. We simplified the assay using 3H-thymidine incorporation DNA analysis to measure cell growth. We tested the modified method by analyzing the effects of different concentrations of native low density lipoproteins (LDL), methylated LDL, as well as LDLs obtained from patients with FDB on cell growth. Methylation of LDL to various degrees reduced cell proliferation correspondingly, and LDLs obtained from FDB patients decreased cell growth confirming that the modified method was able to detect binding defective species of LDL. We applied this method to analyze three novel apoB polymorphisms recently characterized in this laboratory (apoB His1896→Arg, apoB Asn1887 → Ser, apoB Ala4454 → Thr, which did not significantly alter U937 cell proliferation. Our results show that this simplified assay can be used for screening for LDL variants with defective binding.

α-thalassaemia and β-thalassaemia traits: biological difference based on red cell indices and zinc protoporphyrin

α-thalassaemia and β-thalassaemia traits are two commonly encountered haemoglobinopathies in South East Asia. Both present with hypochromia and microcytosis. The use of modern electronic or optical cell counters which measure accurately the red cell parameters has allowed for an initial quick screening for the presence of thalassaemia. Seven red cell parameters were measured by the Technicon H1 cell analyser—red cell count, Hb, MCV, MCH, red cell distribution width (RDW) and haemoglobin distribution width (HDW). Discriminant analysis of these parameters and zinc protoprophyin (ZP) indicates that in healthy individuals, α-thalassaemia can be differentiated from β-thalassaemia with an accuracy of 86% when analysed as a group. In hospitalized patients the accuracy dropped to 71% due to biases of concomitant illness. The two most important parameters for indicating differences between α- and β-thalassaemia are MCV and ZP. The results confirm further that though α- and β-thalassaemia have the same phenotypic expression they differ in their biology.

Batch cultivation ofMethylosinus trichosporium OB3b: V. Characterization of poly-β-hydroxybutyrate production under methane-dependent growth conditions

Methanotrophs have promising applications in the bioremediation of chlorinated hydrocarbons and in the production of a biopolymer, poly-beta-hydroxybutyrate (PHB). Batch bioreactor culture conditions were studied for the accumulation of PHB by methane-grown Methylosinus trichosporium OB3b, and to evaluate the effect of PHB on the bacterial capacity to degrade trichloroethylene (TCE), a common groundwater contaminant. The PHB content of the washed and lyophilized cells was measured by gas chromatography (GC), after hydrochloric acid (HCl) propanolysis. A differential GC-based assay was developed for the monomer and the polymer of beta-hydroxybutyrate utilizing 1% and 10% HCl (v/v) reaction mixtures, respectively. During bioreactor growth in a Cu-deficient modified Higgins' medium, the cells accumulated PHB upon depletion of nitrate. A biomass yield of 3.2 g dry wt/L and a PHB accumulation of approximately 10% (w/w) were reached after 140 to 160 h, without adversely affecting the propene or TCE epoxidation specific rate given by whole cells containing soluble methane monooxygenase (sMMO). The TCE biotransformation capacity ( approximately 0.25 mg TCE oxidized/mg dry cell wt) of resting cells containing approximately 10% PHB was consistently approximately 1.6-fold greater than that of cells containing only approximately 2% PHB. Higher levels (>10%) of accumulated PHB did not enhance this biotransformation capacity further. By replacing the bioreactor inlet air + CO(2) mixture with pure O(2) at approximately 85 h of batch operation, a PHB accumulation of approximately 45% was achieved after 160 h, but the whole-cell sMMO activity was markedly decreased. In contrast, cells grown in a 10 muM Cu-supplemented Higgins' nitrate minimal salts medium (particulate MMO formation) accumulated up to 50% PHB in only 120 h, coupled with a very high biomass yield of 18 g dry cell wt/L. High PHB accumulations above approximately 20% by both the -Cu and the +Cu grown cells resulted in a decreased ratio of the electronic cell count to the absorbance at 660 nm, which is commonly used to monitor bacterial growth. (c) 1996 John Wiley & Sons, Inc.

Marked interference of hyperglycemia in measurements of mean (red) cell volume by Technicon H analyzers

Severe hyperglycemia can result in falsely high results for mean cell (erythrocyte) volume (MCV), which will also cause false results for erythrocyte indices calculated on the basis of MCV. Falsely high MCV results were obtained with the Technicon H1 and H2 analyzers and (to a lesser extent) with the Coulter T660. The H analyzers were more susceptible to this interference than was the Coulter T660. This difference in sensitivity of MCV to hyperglycemia can be explained by the use of sodium dodecyl sulfate in the Technicon erythrocyte diluent and by differences in incubation times. In severe hyperglycemia, results for MCV, mean cell hemoglobin concentration, and hematocrit obtained from electronic cell counters, especially Technicon H systems, are unreliable.

A Complete Platelet System for Metabolic Studies: Pure, Defined, and Viable over 8 Hours

We have been using a human platelet preparation to investigate various aspects of cellular metabolism. These experiments require a pure, quantitated platelet preparation that is viable for up to 8 h. The purity of the preparation has previously been characterized and we have now assessed its viability over 8 h and developed a simple spectrophotometric counting method. This method of counting results in estimations that are within 6% of those from an electronic cell counter (Coulter). Pure platelets stored in a Hepes physiological saline were resuspended periodically in autologous plasma to assess function over 24 h. The platelets consumed oxygen, changed shape, aggregated, and released dense granule ATP at all times. There was a slow and linear decrease in the various processes with time, ranging from 1.7% per hour for oxygen consumption to 3.8% per hour for percentage aggregation. The preparation is therefore not stable, but the changes with time are small and defined and it represents a useful system for the study of platelet and cell biology.

Laminin promotes differentiation, adhesion and proliferation of cell cultures derived from human acoustic nerve schwannoma.

The influence of laminin on cell cultures derived from unilateral acoustic nerve schwannomas was investigated. Cell cultures were initiated from 12 schwannomas, removed via the enlarged middle cranial fossa approach. Tumor tissue was dispersed by collagenase treatment and cells seeded in uncoated or laminin-coated culture dishes. Confluent cultures were immunocytochemically characterized with antibodies against S-100, CD 68, factor VIII-related antigen and type IV collagen. Cell adhesion in response to different doses of laminin was evaluated with an electronic cell counter. The effect of laminin on cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxy-uridine (BRDU) into cellular DNA. Cells cultured on laminin as substrate appeared more differentiated with long, fusiform, cytoplasmic processes. Cultured cells stained positive for S-100, not for factor VIII-related antigen or CD 68. Only cells cultured on laminin deposited a dense extracellular network of type IV collagen. When laminin was added to the culture medium, cell attachment and proliferation was stimulated in a dose dependent manner. Maximal stimulation of both was observed with a laminin concentration of 50 micrograms/ml, which induced a nearly 2-fold increase in cell attachment and an approximately 66% increase in DNA content. Since laminin is a major component of the extracellular matrix in schwannomas, the possibility exists that laminin is also mitogenic for human neoplastic Schwann cells in situ.

Electronic cell counting to measure total cell numbers in bronchoalveolar lavage fluid.

Cell counting of bronchoalveolar lavage (BAL) fluid is performed manually in routine practice. This has both methodological and inherent errors; however, the accuracy and suitability of automated counting devices have been questioned. In this study, a Coulter Counter D Industrial model was calibrated and then used to measure the total cell count in unprocessed bronchoalveolar lavage fluid, and compared to a standard manual method. Bronchoalveolar lavage was performed on 34 patients undergoing routine bronchoscopy. An aliquot of unprocessed bronchoalveolar lavage fluid was taken for all counting procedures. Manual counts were performed blind by two experienced independent observers using improved Neubauer chambers. Electronic counting measured 1 ml aliquots suspended in 10 and 20 ml Isoton counting 0.5 and 1 ml duplicates. The correlation coefficients between electronic and manual counts were good. The coefficients of repeatability of electronic counts, for repeat counts, both on the same dilution (Intra-Coulter: 0.1 x 10(5) cells.ml-1) and different dilutions (Interdilution-Coulter: 0.48 x 10(5) cells.ml-1), were superior compared to those for repeat manual counts by the same observer (1.03 x 10(5) cells.ml-1), and counts between observers (1.82 x 10(5) cells.ml-1). This method offers a quick, precise and simple method for counting cells in unprocessed bronchoalveolar lavage fluid, which is both less labour intensive and subjective than manual counting.

Normative hematologic and serum biochemical values for adult and infant rhesus monkeys(Macaca mulatta)in a controlled laboratory environment

During the conduct of a long-term toxicity/reproduction study using rhesus monkeys (Macaca mulatta), periodic hematologic and serum biochemical analyses were undertaken on blood obtained from 15 untreated (vehicle control) adult female monkeys over a period of 3 yr, 20 untreated adult males over a period of 6 yr, and 9 infants (5 male, 4 female, whose dams were the vehicle control females) from 12 wk through 2 yr of age. All samples were obtained by femoral puncture. To facilitate handling, ketamine hydrochloride (Ket.HCl) was administered to the males, and for some of the later infant samplings. Complete blood cell counts were performed using electronic cell counters, while reticulocyte counts and leukocyte differentials were assessed manually. The serum biochemistry analyses were performed with automated analyzers. These data were obtained as part of a health monitoring program, with medians and 2.5 to 97.5 percentiles for each parameter indicated. Some monitored parameters were found to be affected by the type of equipment used and by the use of Ket.HCl.

Screening for familial defective apolipoprotein B-100 with improved U937 monocyte proliferation assay

Frostegård et al. (J Lipid Res 1990;31:37-44) demonstrated that the proliferation of the human monocyte cell line U937 is critically dependent on the uptake of low-density lipoprotein (LDL) via the apo B, E (LDL) receptor, a characteristic that was used to detect patients with familial defective apolipoprotein B-100 (FDB). Here we applied this principle to develop a simple and reproducible assay for the detection of patients with functionally defective LDL. We added serum to U937 cells in cholesterol-free incubation medium and determined the increase in cell number after a 72-h incubation at 37 degrees C by using an electronic cell counter. Sera from 10 normolipidemic individuals and from 34 patients with type IIa hyperlipoproteinemia stimulated the growth of U937 cells in proportion to the exogenous cholesterol concentration (r = 0.83, P < 0.001) and the LDL-cholesterol concentration (r = 0.81, P < 0.001). However, sera from 16 patients with FDB stimulated less cell proliferation than did sera from patients with type IIa hyperlipoproteinemia with equal LDL-cholesterol concentrations. With a 15% reduction in growth as the cutoff value, this test had a sensitivity and specificity for diagnosis of FDB of 87.5% and 100%, respectively. The improved U937 monocyte proliferation assay can be used for screening hypercholesterolemic patients to detect individuals with functionally defective LDL.

Cell behaviour on polymer surfaces with different functional groups

Surfaces with differently chargeable functional groups were prepared on low density polyethylene sheets by corona discharge treatment, followed by graft copolymerization of acrylic acid (−COOH, negatively chargeable) and a subsequent substitution reaction of carboxylic acid groups to hydroxyl (−CH 2OH, neutral) or amide (−CONH 2, neutral) groups. The amide groups grafted on the surface were further converted to amine groups (−CH 2NH 2, positively chargeable). The prepared surfaces were characterized by measuring the water contact angle, electron spectroscopy for Chemical analysis and Fourier-transform infrared spectroscopy in the attenuated total reflectance mode. It was observed that the wettability of the different functional group-grafted surfaces largely increases compared with the control surface but is not much affected by the kind of functional groups grafted. The interaction of Chinese hamster ovary cells with the functional group-grafted surfaces was investigated. The cells adhered and grown on the surfaces were counted using an electronic cell counter and observed by a scanning electron microscope. The surface grafted with amine groups was best for cell adhesion, spreading and growth probably owing to the positively chargeable character in aqueous cell culture medium. For surfaces grafted with neutral functional groups, the surface grafted with hydroxyl groups showed better cell spreading than that grafted with amide groups. Biomaterials (1994) 15, (9) 705–711

Prevalence of High Somatic Cell Counts in Bulk Tank Goat Milk

Higher SCC have been observed in goat milk than in cow milk. This study evaluated the prevalence of high SCC and determined whether increased neutrophils were a factor in high SCC. Bulk tank goat milk samples were collected from commercial dairy goat herds in California, Arkansas, Michigan, and Wisconsin. Total SCC were determined using a Fossomatic Electronic Cell Counter and microscopically using pyronin Y-methyl green stain. Differential counts were determined using Wright's-stained cytospin centrifuge preparations. Average SCC were high (6.12 log10 cells/ml; geometric mean, 1.32×106 cells/ml). The average of the Fossomatic and direct microscopic SCC indicated that 8.6% of the producers had SCC <.75×106 cells/ml and that 34.5% were <1×l06 cells/ml. The current goat milk SCC standard is 1×106 cells/ml; the cow milk SCC standard will be .75×106 cells/ml in 1993. Milk samples contained 9.9% macrophages, 2.8% lymphocytes, and 87% neutrophils. These data indicate that high SCC were prevalent and that increased neutrophils contributed to high SCC in the bulk tank goat milk.

Validation of a simple method to count very low white cell concentrations in filtered red cells or platelets.

The increased performance of white cell (WBC) filters makes it difficult to count precisely the number of residual WBCs. Concentrations as low as 0.01 WBC per microL cannot be determined with electronic cell counters, conventional hemocytometers, or the flow cytometric techniques currently being used. This article describes a simple, manual method using a Nageotte hemocytometer with a large-volume chamber (50 microL) to count the number of WBCs contained in red cell (RBC) suspensions (preparations A, B, and C) and in platelet suspensions (preparation D) diluted 1 in 10 pure, or concentrated two fold. To validate the method, several reference ranges, prepared by successively adding mononuclear cells to a suspension of pure RBCs or platelets, were used. Among the different series, validation ranges varied from 0.2 to 12 to 0.01 to 0.5 WBCs per microL and correlation coefficients ranged from 0.929 to 0.996. To determine the limit of accurate detection, accuracy tests (n = 160) were carried out by two experienced operators on samples with WBC concentrations of about 5, 10, and 120 times the concentration at the theoretical limit of detection (1 WBC/chamber). No significant difference was observed in the various types of preparations (A, B, C, D) in the tests performed by the two operators. However, intra-assay coefficients of variation were 18, 9.5, and 2.2 percent, respectively, at WBC concentrations of 5, 10, and 120 times that at the theoretical limit of detection. These observations show that a limit of accurate detection (10%) seems to be reached when 10 cells are observed in a Nageotte hemocytometer.(ABSTRACT TRUNCATED AT 250 WORDS)

An optical method for the determination of platelet count in platelet samples contaminated with red blood cells

We present a simple photometric method to determine the total concentration of platelets present in a sample independently of red blood cell concentration. Standard optical density curves for platelet samples ranging in concentration from 0 to 1.5×109 cells/ml and contaminated with red blood cells ranging in concentration from 0 to 0.03×109 cells/ml are determined. A study of the absorbance spectra of red blood cells and platelets suggests that by calculating the absorbance difference between two wavelengths, an estimate of red blood cell concentration can be made. Then, in the second step of this two-step method, the individual absorbance measurements at the two wavelengths are matched to the standard values determined previously to derive an estimate of platelet concentration. In a trial of 62 unknown platelet samples contaminated with red blood cells, the standard deviation for the error in platelet count was 0.16×109 cells/ml with a mean difference of 0.011×109 platelets/ml. We conclude that our method may be useful in laboratories not equipped with electronic cell counters as well as in applications such as the development of noninvasive measurements of platelet concentration in platelet transfusion packs.