Automated cell counter

Aperture impedance counters are flow cytometers, as are all current automated cell counters. How cytometers measure cells as the cells flow hydraulically, one at a time, through sensing zones. 

Other laboratory equipment and reagents visible light microscope, hemacytometer, Trypan blue solution, laboratory balance, refrigerated centrifuge, household bleach 10% solution, automated cell counter (Coulter Z1 Counter, Beckman-Coulter, Miami, FL), flow cytometer (EPICS Elite II, Beckman-Coulter). 

Artefactual pseudothrombocytopenia is typically an error of automated cell counters. When platelet clumps are mistaken for leukocytes this can result in pseudoleukocytosis (30,36). Edetic acid can also cause agglutination of granulocytes, and confusing findings such as combined pseudothrombocytopenia, pseudoneutropenia, and pseudolymphocytosis can occur (28). 

The increasing sophistication of automated cell counters has resulted in virtual replacement of many manual determinations, such as the white blood cell (WBC) differential (Hunt, 2004). Because the instrument is evaluating thousands of cells individually, as opposed to the traditional 100 used to perform a manual differential, the instrument differential is almost always more accurate than the manual differential. However, notable exceptions... 

Cell counter. Countess automated cell counter (Invitrogen, Carlsbad, CA). 

Automated Cell Counter Instrument that uses electric impedance or optical light scatter to rapidly count blood cells. 

Harvest the cells from a sufficient number of tissue culture flaslcs. Wash the cells and resuspend in cold 1% BSA/PBS. If necessary break up clumps of cells by repeated pipetting or by syringing them through a 23G needle. Measure the cell concentration with a haemocytometer or automated cell counter and dilute to a final concentration of 4 x 10 /ml in cold 1% BSA/PBS. 

Since its invention in the early 1950 s [13], the Coulter principle has been so widely accepted in the field of medical technology that presently over 98% of automated cell counters incorporate the Coulter principle. Besides counting blood cells for which the Coulter Principle was originally invented, this method can be used to coimt and size any particulate material that can be suspended in an electrolyte solution. During the past fifty years, the method has been utilized to characterize thousands of different industrial particulate materials. Over 7000 references to the uses of various COULTER counter models have been documented [14]. 

Cell counting chamber or automated cell counter. 

Automated cell counter, type Countess (Invitrogen) or TC20 (Bio-Rad) or equivalent. 

For cell count consistency between screens, we use an automated cell counter such as the Invitrogen Countess or the Bio-Rad TC20. Both count cells and determine their viability using trypan blue. Alternatively a hemocytometer can be used. 

Class II devices are those for which general controls alone are insufficient to provide reasonable assurance of safety and effectiveness and for which sufficient information is available to establish special controls to provide this assurance. Special controls may include performance standards, postmarket surveillance, and guidance for analytical/clinical data. Examples of class II IVDs are automated differential cell counters, fetal hemoglobin test systems, sickle-cell tests, and Toxoplasma gondii serological reagents. 

The cell volume can be calculated from cell diameters measured either manually in cells in suspension on microscopic slides or automatically in a flow cytometer. Also certain types of cell counters provide automated information on cell diameter [14,18,19,21,23]. 

Buffy coat (white blood cell fraction) obtained from Red Cross must be kept in cold storage until use. Take an aliquot of the buffy coat and dilute with HBSS to determine cell concentration. Either a hemacytometer or an automated particle counter can be used for cell count (see Note 3). 

Resuspend the cell pellet in labeling buffer (about 5 mL final total volume) and proceed to count recovered cells in triplicate (see Note 7). An automated particle counter is highly recommended because it is possible to set size gates for cancer cells, erythrocytes, and leukocytes. 

Use CEM, Molt 4/clone 8, or C8166 cells grown in cell cultures that were subcultured or supplied with fresh culture medium ( 10 mL fresh medium added to a 15-20 mL cell culture) 1 d before initiation of the experiment. Pellet the cells (10 min at 1200 rpm ( 300g), room temperature), resuspend in a few milliliters of culture medium, count the cell number by an automated Coulter counter (Analis, Gent, Belgium), and adjust the cell number by adding an appropriate volume of culture medium in order to obtain a cell density of 500,000 cells/mL. 

In addition to the automated devices and processing units that were developed primarily to automate chemistry and immunoassay that are described above, a variety of other instruments and processes have been automiated and used in the clinical laboratory. They include urine analyzers, flow cytometers, hematology cell counters, nucleic add analyzers, microtiter plate systems, point-of-care analyzers, and remotely located systems. 

The first commercially successful automated blood cell counter, the Model A Coulter Counter, was introduced in 1956. The Model A counted cells by using electrical impedance properties, and the Coulter Counter quickly became the instrument of choice for counting red and white blood cells. When it was later demonstrated that cell volume was roughly proportional to the electrical impedance signal amplitude, the Coulter Counter was modified to provide MCV as the mean of the individually measured red cell volumes. Impedance counters calculated HCT as (RBC x MCV)/10 and, by the early 1970s, these instruments also counted platelets. By the mid-1970s, impedance counters combined with photometric hemoglobinometers to produce CBCs. 

Hematocrit Measurement of the volume of packed red cells in a blood specimen by centrifugation. The procedure is performed using a tube with graduated markings or with automated blood cell counters. It is used as an indicator of erythrocyte status in disease. For example, anemia shows a low hematocrit, polycythemia, high values, [nih]... 

Aperture impedance and most other automated counters measure MCV and RBC independently, in contrast to the manual methods where MCV and MCH accuracies depend on hemocytometer red cell count accuracy. 

Classification is traditionally by examination of a well prepared and Romanowsky stained blood film although, increasingly, red cell indices are routinely provided by automated counters. These observations are given clinical relevance by their association with the most common causes in what is called the etio-logic approach. In addition it is also possible to relate cytomorphology to disturbances in the functional capacity of the erythron so that anaemia is seen to result from impaired production of red cells or their shortened survival (Table 1). 

Automation of cell counting (total cells) is possible with electronic counters, especially for non-clumping single suspension cells. A method that is widely used for total cell numbers is counting cell nuclei after dissolving the cytoplasm. This is particularly useful for large clumps of cells, where cells are inaccessible (e.g. in matrices) or where cells are difficult to trypsinize off substrates (e.g. microcarriers). If determination of viability is the prime consideration, then the most accurate method is to count the cells that have the ability to divide by the mitotic index method. 

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