Coulter Counter use in the enumeration of muscle and fat stem cells

Although the manual counting chamber (hemacytometer) is the gold standard for counting cells, this method is subject to great variability due to the human factor. The automated cell counter Coulter Counter) canenumerate cells in less time and with greater accuracy than the hemacytometer by removing many of the steps in which errors are made. While the Coulter Counter (and others of its type) has been used for many years in the cell culture field, there have been few studies to validate its use with specific cell types. We conducted several experiments in which we assessed the accuracy of the Coulter Counter over counts made with a hemacytometer as well as validated its use for the counting of satellite cells and preadipocytes.     

ADCC analysis with the Coulter counter: a highly sensitive, ultramicro assay suitable for clinical and experimental application

Antibody-dependent cellular cytotoxicity (ADCC) is an in vitro immune mechanism implicated in several in vivo phenomena such as transplant rejection, tumor immunity and parasite elimination. We developed a method for detecting ADCC using the Coulter Counter and the Coulter Channelyzer that circumvents many of the disadvantages associated with existing assays for ADCC. Effector mononuclear cells were incubated with chicken red blood cell (CRBC) targets and anti-target antibody for 1-1, 5h. Killing was quantified by the Coulter Counter on the basis of size differences between effector and target cell nuclei. Using a 4 microliter total volume we were able to detect cytotoxic levels of 55% when as few as 5000 effector cells were incubated with an equal number of target cells. This method for the detection of ADCC may be suitable for clinical and research application.     

Display and analysis of cell size distributions with a Coulter Counter interfaced to an Apple II microcomputer

A model ZBI Coulter Counter was interfaced to an Apple II microcomputer via analog and digital interfaces. It was possible to obtain a graphic display of cell size versus channel number (1000 channels), integration of selected channels, plots, listings, and storage of the data on floppy disk, using a convenient menu selection enabling operators unfamiliar with computer technology to master the system quickly. Examples are presented of calibration of the Coulter Counter and its use for rapid and precise analysis of E rosettes.     

Advantages of a new anticoagulant in routine hematology on the Coulter Counter S-Plus STKR analyzer

The authors report the advantages of a new anticoagulant-antiaggregant mixture that avoids the deleterious effects of ethylenediaminotetraacetic acid (EDTA) on mean platelet volume and also prevents EDTA-induced platelet clumping. It is suitable for routine cell counting and sizing with the Coulter Counter S-Plus STKR. The values of the common hematologic parameters agree well with those from EDTA-treated samples and are stable for at least eight hours after sampling.     

Assay method for Vibrio cholerae and Escherichia coli enterotoxins by automated counting of floating chinese hamster ovary cells in culture medium.

As Chinese hamster ovary (CHO) cells on plastic proliferate, many cells float off into the medium instead of piling up after they form a monolayer. Fewer cells were floating in the medium when CHO cells were incubated with cholera toxin at a concentration as low as 10 pg/ml. The toxin increased the adhesiveness of the cells forming confluent monolayers so that the floating cells accumulated on the adherent monolayers. On the basis of this finding, a simple, quantitative assay method for cholera and Escherichia coli enterotoxins was devised by cultivating CHO cells in a Linbro multidish and counting the cells in the medium with a Coulter Counter. The method was sensitive enough to detect toxins in 100- to 200-fold-diluted culture media of toxigenic E. coli strains. Little or no activity was detected by this method in the culture medium of nontoxigenic E. coli.     

Rapid screening for significant bacteriuria using a Coulter Counter.

A Coulter Counter was evaluated for detecting significant bacteriuria. Simple urinary particle counts showed agreement in 883 of the 956 (92.4%) urine specimens examined compared to a standard culture procedure. They also provided a semiquantitative estimate of bacterial numbers in specimens diagnosed as infected by culture. Sample preparation before counting was minimal, and results were obtained within two to four minutes.     

A parallel comparison of three automated multichannel blood cell counting systems for analysis of blood of laboratory animals

A parallel evaluation was performed on three automated haematology analysers, the Sysmex K-4500 and Coulter Counter S-PLUS STKR impedance analysers, and the Techinicon H*1E flow cytometry analyser. The same blood samples from animals from three different species were analysed on the same day. An analytical -comparison of haemograms from healthy monkeys, dogs, rats and phlebotomised rats was made from paired blood samples anticoagulated with dipotassium ethylenediaminetetra-acetate. Each instrument was calibrated with commercially available material based on human blood products. The precision of each system was good, but whereas the Coulter Counter S-PLUS STKR had better precision for white blood cell counts, platelet counts showed greater variability. When compared to spun packed cell volume (PCV), or to each of the other instruments, the Coulter system consistently gave lower haematocrit values. The magnitude of relative bias of spun PCV values was about −7% in monkey, −5% in dogs and −10% in rats. It was deemed necessary with this instrument to make adjustments to the calibration, especially for rats. The Coulter also gave consistently higher white blood cells (WBC) counts in monkeys, and lower platelet counts in rats compared to the other two instruments. The biases may be due to inherent physical differences between the analytical methods and/or the calibration techniques. With few exceptions, each instrument provides reliable results for all major animal species encountered in routine experimental and toxicological haematology.     

Diameters of erythrocytes of different ages measured by scanning electron-microscopy

Contradictory reports exist about the age-dependent changes of erythrocyte diameters. All the investigations were carried out using Coulter Counter, by which the erythrocytes are sucked through a glass capillary. The contact with a glass surface causes the cell to deform instantly. This explains the lack of accuracy of the measurements subsequently taken.We measured the size of red cells of different ages by scanning electron-microscopy, thus avoiding deformations. We found that the average diameter decreases with the age of the red cell. The mean diameter was 7.77 μm in young cells, 7.34 μm in middle-aged and 7.00 μm in old cells.     

The mean red cell volume in long distance runners

Summary Red cell indices were determined in 6 well trained runners before and after a 100 km race, and Coulter Counter (CC) determinations compared with calculated values derived from centrifuged hematocrit (ctrf), red cell count (CC) and hemoglobin measurements. The following changes were observed immediately after the race, as compared to values 3 days before: MCV(ctrf) decreased by 4.9% (p<0.001), MCV(CC) increased by 1.9% (p<0.05), MCHC(ctrf) increased by 4% and MCHC(CC) decreased by 3%. The increase in MCV(CC) suggests that intraerythrocyte osmolality was increased, this probably leading to swelling of the cells induced by a shift of water from the diluting Coulter Counter solution into the red cells prior to the MCV measurement. The decrease in MCV(ctrf) immediately after the race was not correlated with the increase in plasma osmolality. This suggests that plasma osmolality alone was not the key factor for regulation of red cell volume. The changes in MCV(ctrf), which contributed to a surprising stability of the hematocrit value and plasma volume, might represent a physiological principle for the maintenance of a favourable blood viscosity.     

Comparison of four leukocyte differential methods with the National Committee for Clinical Laboratory Standards (NCCLS) reference method

The authors compared four leukocyte differential counting methods with the National Committee for Clinical Laboratory Standards Reference Leukocyte Differential Method H20-T to determine the clinical sensitivity of the methods. The three-part differential performed by the Coulter Counter Model S-Plus IV and the Toa E-5000, when combined with instrument flags and defined laboratory checking limits for red blood cell and platelet values, are safe and efficacious screening methods for the presence of morphologic abnormalities. The Geometric Data Hematrak 590 proved comparable in clinical sensitivity to a random 100-cell eye-count differential.     

A multicenter evaluation of the Iris iQ200 automated urine microscopy analyzer body fluids module and comparison with hemacytometer cell counts

Manual hemacytometer cell counting of body fluids is labor-intensive and requires skilled testing personnel. We performed a multicenter evaluation of the Iris iQ200 automated microscopy analyzer Body Fluids Module (Iris Diagnostics, Chatsworth, CA) and compared 350 iQ200 body fluid cell counts with manual hemacytometer cell counts. Within-run imprecision, expressed as coefficient of variation (CV), ranged from 2.6% to 5.9% for RBC counts between 875 and 475 x 10(6)/L and from 4.2% to 6.5% for nucleated cell counts between 820 and 590 x 10(6)/L. The lower limits of detection, based on a CV of 20% or less, were 30 and 35 x 10(6)/L for RBCs and nucleated cells, respectively. There was very good agreement between automated iQ200 and manual body fluid cell counts based on slopes and r2 values. The iQ200 has satisfactory performance for enumerating RBCs and nucleated cells in most body fluids, with the exception of cerebrospinal fluid specimens that contain low cell numbers.     

Micro coulter counters with platinum black electroplated electrodes for human blood cell sensing

We demonstrated a novel micro Coulter counter featuring platinum-black electrodes for human blood cell counting application. Two designs of micro Coulter counter were fabricated using two distinct technologies: integrated parylene and soft lithography. Platinum-black enhanced detection in the intermediate frequency range (∼100 Hz to 7 MHz), which is the operation frequency suitable for sensing the cells flowing by the electrodes. A detailed theoretical modeling of the sensing mechanism has been performed for the design of the electrodes, and electrical impedance spectra measurements confirmed the theoretical model. The surface morphology and roughness of the platinum black electroplated surface were characterized by SEM and AFM measurements. Polystyrene beads of various sizes were initially used to validate the operation of the devices, and using excitation frequency of 10 kHz, the signal magnitude was found to be correlated with the volume of the individual bead. Human blood cell sensing was successfully demonstrated with diluted whole blood and leukocyte rich plasma under the same excitation frequency. The histogram of impedance magnitude of the cells matched well with volume distributions of erythrocytes and leukocytes measured by conventional counting techniques. Micro Coulter counters have the advantages of small foot-print, low sample volume, and reduced cost of operation. Further development of the devices can lead to the development of a highly-sensitive and high-throughput handheld blood counting system for point-of-care applications.     

Observations on the use of the Coulter model D electronic cell counter in clinical haematology

The use of the Coulter electronic cell counting machine (model D) in a routine pathological laboratory is described, as are also the technical details involved.     

Zur Volumenbestimmung von Leukocyten

Zusammenfassung Volumenbestimmungen von Leukocyten mit dem Coulter Counter (Modell B) zeigen eine rasche Schrumpfung der Leukocytenvolumina unter dem Einfluß des Hämolysemittels Saponin, weswegen keine absoluten Größenangaben der Leucocyten möglich sind. Im Phasenkontrastmikroskop läßt sich diese Wirkung des Hämolysemittels nicht beobachten. Die mit dem Coulter Counter gewonnenen Ergebnisse beruhen deshalb vermutlich lediglich auf einer Änderung der elektrischen Leitfähigkeit der Leukocytenmembranen durch Saponin.

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Summary The volumina of leukocytes with the Coulter Counter (Modell B) undergo a rapid shrinkage under the influence of saponin. Therefore no absolute values for the size of leukocytes are available. The influence of the haemolytic substance saponin cannot be observed with the phasecontrast microscope. In consequence the measurements with the Coulter Counter in a suspension of saponin are originated probably only by changing the electric conductibility of the membranes of the leukocytes.

     

The red cell osmometer. A useful inaccuracy in measurement of mean corpuscular volume in hyperosmolar states.

The Coulter S Counter gives a value for mean corpuscular volume which does not necessarily reflect red cell size in vivo. This phenomenon may be used in early diagnosis and hence prevention of the 'hyperosmolar syndrome'.     

Assay of immune cytolysis of lymphocytes and tumour cells by automatic determination of cell volume distribution.

Immune cytolysis (lysis) of cells due to the action of antibody in the presence of complement is usually substantiated by the uptake of vital dye by the cells, or by the escape of radiolabel from the cells. Immune cytolysis has now been assayed by determination of cell volume distribution with a Coulter multi-channel particle size analyser used in conjunction with a Coulter counter. For Ehrlich ascites and sarcoma-180 cells, volume degradation corresponding to vital staining was obtained only if trypsin (final concentration 625 microgram/ml) was added immediately after the usual 1 h incubation period for cells, antibody and complement. For L1210 leukaemia cells, trypsin was added at 0 degrees just 1 min before Coulter evaluation, to avoid potentiation of antibody-mediated cell lysis by trypsin. Immune cytolysis of mouse thymic, splenic and lymph node lymphocytes required addition of pronase (final concentration 625 microgram/ml) at 0 degrees for further disruption of antibody-damaged cells, prior to determination of cell volume distribution in the Coulter equipment. Scanning electron micrographs of L1210 cells undergoing immune cytolysis illustrated the changes in cell volume recorded by the Coulter apparatus. This new method for determination of immune cytolysis provides detailed information about the volume distribution of target cells, which permits detection of subtle changes and gives insight into the process of cytolysis. It is not intended to displace other procedures in routine use, except that complete automation of the present method is possible in future.     

A new method for semi-automated quantitation of E-rosettes using a particle size analyser (Coulter Channelyzer).

Visual counts of the number of SRBC attached to individual lymphocytes in E-rosette preparations from 3 healthy young adults were made by 3 experienced observers: from these data the size distribution profile of particles in each E-rosette preparation was calculated. In parallel experiments the size distribution profile was measured directly with a particle size analyser (Coulter Counter and Channelyzer): in every case the observed profile lay within the 95% confidence band around the reconstructed profiles. It was concluded that the Coulter Channelyzer profile is an accurate measurement of the E-rosette size distribution and that particle sizing could provide a reasonable basis for semi-automated tests for E-rosettes. A mathematical model has been developed by which the percentage of lymphocytes forming E-rosettes, the distribution of number of attached SRBC and the average avidity of the lymphocyte receptor for SRBC could be deduced from the size-distribution profiles. The method has a high degree of precision, and a good correlation (P less than 0.01) was obtained when the percentage of E-rosettes (defined for this purpose as having three or more attached SRBC) obtained from the new method was compared with results from standard light microscopy.     

Permanent slide preparations of T lymphocyte-sheep red blood cell rosettes

Rosette formation between sheep erythrocytes (SRBC) and human thymus-derived lymphocytes (T cells) is used to monitor T cells in various human diseases. Rosettes are usually counted in a hemacytometer immediately after preparation. This paper reports a technique for permanently fixing and staining rosettes for sereial and comparative studies, a procedure which has probably been less well standardized, less reproducibly performed, and less widely used than many investigators appreciate. The technique described has the advantages of providing distinct morphological identification of the rosette-forming cell and it produces a permanent mount for future reference.     

Ploidy patterns in hepatic tumors induced by Mirex

The effects of chronic exposure to dietary Mirex was investigated in rat livers over a 13 month period. The distribution of ploidy (diploid and tetraploid nuclei) in nodular and nonnodular areas was analyzed in a Coulter Counter fitted with a Channelizer. The nodules, such as adenomas and carcinomas, were identified in histologic sections obtained from companion samples, part of which was used for analysis of nuclear ploidy. The carcinogen disturbed the distribution of nuclei in the ploidy classes, selectively reducing the number of tetraploid cells. This reduction in tetraploid cells corresponded to the nature of the tumor, the most significant effect being noted in hepatocellular carcinomas.     

Monocyte counting: discrepancies in results obtained with different automated instruments.

To determine the accuracy of several methods for measuring the monocyte count, the results obtained by a number of different automated cell counters were analysed. Considerable discrepancies occurred for monocyte counts obtained in normal blood among the counters. The results of a visual monocyte count on a total of 800 leucocytes were used as the reference method. The technique of measuring the monocyte count by using dual staining with monoclonal antibodies CD45 and CD14 provided the closest agreement with the reference method. Six other automated counting systems were assessed. Two of these systems (Coulter VCS and Technicon H1) gave results, which, although under-estimating monocytosis, correlated well with the results obtained by the reference technique. A third system (Toa Sysmex NE-8000) gave unreliable results. Three of the automated systems evaluated measured a "third population"--that is, monocytes together with other leucocytes. One of these systems (Ortho ELT 1500), overestimated the count, as expected, but correlated well with the reference method. The second of these "third population counters" (Coulter S Plus IV) correlated moderately well with the reference monocytosis, while the Toa Sysmex E-5000 correlated poorly. It is clear that problems exist in the evaluation of different instruments for counting monocytes. An accurate and reliable reference method is a pre-requisite to evaluate this aspect of cell counters. As the visual method is too cumbersome a different reference method would be useful. Based on the results of this study, it is suggested that the technique using fluorescence labelled monoclonal antibodies should be regarded as an acceptable alternative.