CYTOMETERS — devices for calculation of uniform elements of blood, urine and cerebrospinal liquid, and also microorganisms. It is offered To. page the fr. physiologist L. Ch. Malassez in 1874.
To. pages represent a thick slide plate with deepening, at the bottom to-rogo the calculating grid is engraved; over deepening impose polished cover glass. Constant height To. the page is provided with dense grinding of cover and subject glasses before formation of iridescent Newtonian rings (a strip of an interference).
Structural elements of all types of grids are big and small squares. Grids of various types — Thomas, Byurker, Predtechensky, Türk, Neybauer, Goryaev, Fuchs — Rosenthal, etc. — differ in various grouping of big and small squares.
The known sizes — height of the camera, the area of the grid and its divisions and cultivation taken for a blood analysis — allow to calculate quantity of uniform elements in a certain volume (1 mkl) blood (or other environment).
Distinguish opened and closed To. page. In the closed camera cover glass is ground in after its filling, and at the same time air traps can get to it. Such To. page. (Toma — Tseyssa with Thomas's grid, Dungera with a special grid) are inconvenient in work and are not used.
Open cameras (fig. 1) are filled after grinding of cover glass. They have two grids on one slide plate. Plates with the engraved grids are delimited by fillets one from another, and also from other part of a slide plate. Existence of fillets gives the chance to regulate filling of cameras. Some cameras are supplied with the metal clips fixing cover glass.
Opened To. page S. P. Alferov in 1883, then in 1905 for the first time described Byurker (To. Burker). Open cytometers of Klyucharev, Gausser and Levi, Gelber, Glaubermann are known. In the USSR cytometers of Goryaev and Fuchs — Rosenthal are widely used. The camera Goryaeva with Goryaev's (fig. 2) grid has volume 0,9 mkl, the area of a grid of 9 mm 2 . The grid consists of 225 big squares; from them 100 — empty, 25 — are divided everyone into 16 small squares, 100 — are divided by strips.
The cytometer of Fuchs — Rosenthal with Fuchs's grid — Rosenthal (fig. 3) has volume 3,2 mkl, the area of a grid of 16 mm 2 , it consists of 256 big squares (the squares divided by strips do not consider).
A technique of work
Before work microscope slide K. the page and polished cover glass are washed under a stream of mains water and dry wiped. Then densely grind in cover glass to the camera (before emergence of iridescent Newtonian rings because only provided that volume To. page it is constant).
Contents of a test tube before filling of the camera several times are mixed, then the melted-off end of a glass rod selected from a test tube, inclining it, the drop of blood is applied on a slide plate at the edge of cover glass. If one drop of blood is not enough for full filling To. villages, add still a drop. If blood is taken in the mixer, then the first drops from a capillary of the mixer release, and To. pages fill with a drop from an ampoule of the mixer. The remains of liquid from a slide plate delete with a gauze tampon. Calculation is begun in 3 min. after filling of the camera (during this time there is sedimentation of uniform elements of blood) under a microscope at small increase (a lens X 8, an eyepiece of X 10 or X 15) and the darkened field of vision (to the covered diaphragm or at a little lowered condenser). The cells lying in a square (fig. 4) are subject to the account. The cells crossed by the parties of squares consider as follows: if more than a half of a cell is in a square, then it is considered if out of — it is not considered. The cells crossed by lines precisely in the middle consider on two adjacent, right and upper, lines of squares and do not consider on two others. During the use of the corresponding parting solutions erythrocytes, leukocytes, thrombocytes, eosinophils, basophiles and reticulocytes can be counted in Goryaev's camera.
Erythrocytes consider in 80 small squares, i.e. in 5 big squares located on diagonal. Calculation is carried out on a formula:
X = a*4000*200/80,
where and — the quantity of erythrocytes counted in 80 small squares, 80 — quantity of the counted small squares, 200 — extent of cultivation of blood, 4000 — a multiplier for obtaining maintenance of erythrocytes in 1 mkl blood (the volume of a small square 1/4000 mkl). Practically the quantity of erythrocytes counted in 5 big squares is multiplied on 10 000.
Leukocytes count in 1600 small squares (in 100 big squares). Calculation is carried out on a formula:
X = a * 4000 * 20/1600,
where and — the quantity of leukocytes counted in 1600 small squares, 1600 — quantity of the counted small squares, 20 — extent of cultivation of blood, 4000 — a multiplier for obtaining maintenance of leukocytes in 1 mkl blood. Practically the quantity of leukocytes counted in 1600 small squares is multiplied on 50.
Thrombocytes count in 400 small squares (25 big squares on the diagonal of a grid). The isotonic solutions of sodium chloride, various solutions preserving thrombocytes and hemolyzing erythrocytes can be reactants for cultivation.
Consider thrombocytes under a usual microscope and by means of the phase and contrast device (see. Phase-contrast microscopy ) for their more accurate identification. After cultivation the test tube with blood is left for 25 — 30 min. for hemolysis of erythrocytes. Then contents of a test tube are repeatedly mixed and filled To. pages, to-ruyu place for 5 min. in a moist chamber (e.g., Petri dishes with wet cotton wool) for sedimentation of thrombocytes. Calculation is carried out on a formula:
X = a*4000*200/400,
where and — quantity of thrombocytes, counted in 400 small squares, 200 — extent of cultivation of blood, 400 — number of the counted small squares, 4000 — a multiplier for obtaining maintenance of thrombocytes in 1 mkl blood. Practically the quantity of thrombocytes counted in 400 small squares is multiplied for 2000.
Quantity of basophiles and eosinophils count in Goryaev's camera in 1600 small squares, as well as leukocytes. Almost counted quantity of basophiles and eosinophils is multiplied on 50.
Reticulocytes count in Goryaev's camera in 80 small squares as erythrocytes. Almost counted quantity of reticulocytes is multiplied on 10 000.
Definition of total quantity of cells in cerebrospinal liquid (see) is better to make in Fuchs's camera — Rosenthal (because of a small number of leukocytes in cerebrospinal liquid). Count on all grid (256 big squares) under small increase in a microscope (an eyepiece of X 15, a lens x 8).
At very large number of cells calculation of a half of a grid is allowed (with the subsequent multiplication of result by 2). Calculation is carried out on a formula:
X = a*11/3,2*10,
where and — the quantity of cells counted in 256 squares, 11/10 — extent of cultivation, 3,2 — the volume of the camera in mkl. Practically at calculation in Fuchs's camera — Rosenthal number of leukocytes divide on 3.
In Goryaev's camera of a cell of cerebrospinal liquid consider not less than 3 times (also all area), every time filling the camera again, then take an arithmetic average. Calculation is carried out on a formula:
X = a*11/0,9*10,
where and — an arithmetic average the quantity of cells counted on all grid, 11/10 — extent of cultivation, 0,9 — the volume of the camera in mkl. Practically the number of the counted leukocytes multiply on 1,2.
Quantitative definition of uniform elements in urine carries out in Fuchs's cameras — Rosenthal, Goryaev: calculation of erythrocytes and leukocytes at average increase in a microscope, calculation of cylinders — at small. Extent of cultivation of urine depends on a method of a research (see. Urine ).
Calculation in To. page it is not quite exact. The error of a method makes from 10 to 20% depending on quantity of the counted elements.
Leaving and storage conditions
It is necessary to protect cameras from pollution and hit of dust on a grid. After work the camera and cover glass are washed under a stream of mains water and it is careful, but carefully wipe a pure napkin (it is possible gauze). Then paper and put away in a box.
See also Blood .
Bibliography: The reference book on clinical laboratory methods of a research, under the editorship of E. A. Kost, page 22, M., 1975.
D. N. Ishmukhametov