Patent GB-1346836-A (GB)
Title: Automatic apparatus for determining the percentage population of particulates in a medium
Inventor: GRONER WARREN
IPC: G01N1/31
Filed: 1971-10-21
Abstract: 1346836 Determination of leukocytes TECHNICON INSTRUMENTS CORP 21 Oct 1971 [30 Oct 1970] 48928/71 Heading B1X [Also in Divisions Gl and G4] In an apparatus for determining the population of each type of leukocyt (white cells) in a blood sample, blood samples aspirated into a tubing system 3 flow as a wash liquid segmented steam into three paths 15, 17, 19 and then into four paths AI, AII, AIII, AIV. Each path has fed into it reagents to (a) dye a particular group of leukocytes; (b) disolve away the erythrocytes and (c) to stain the remaining membranes so that their refractive index is equal to that of the blood plasma. Each path thus delivers to a nozzle 157 a stream of fluid in which only the selected leucocytes are visible. The streams of fluid from nozzles 157 flow into the centre of four counting cells 65, 83, 101, 129 respectively while a pressurized sheathing liquid from inlet 169 fills the rest of the cell. Suitable synchronising means is provided (not shown) to ensure that individual parts of one blood sample arrive at the counting cells simultaneously and they are drawn through at the same speed by a constant pressure source by pipes 177. The counting cells each include a light source 183 and photocells for detecting direct and scattered light and feeding circuits which calculate the numbers of each type of leukocyte (see Divisions G1 and G4). In the common part of paths AI and AII the blood sample at 15 is first fixed by a 10% formalin solution buffered with sodium phosphate to pH 7.0, segmented by air at 33 and is then mixed with acetic acid from 41 which acts as a lysing agent to remove the red blood cells. The samples pass through heating and cooling bath as indicated in the figure. The sample is then divided between the two paths AI and AII. In path AI the sample is first mixed with a substrate of hydrogen peroxide, at 49, and a dark dye, at 51, comprising a solution of 4-chloro-1-napthol in 2.2'-dioxyethanol, buffered to pH 4.5 again segmented by air at 55. This stains both eosinophils and neutrophils. Propylene glycol is then added at 60 to match the red cell membrances. In path All the sample is first mixed with hydrogen peroxide at 67 and an acid dye, at 69, similar to the dark dye above but buffered to pH 3.5 and at room temperature. This stains only the eosinophils. Propylene glycol is then added at 77 as before.  In path AIII the sample is first mixed with a lysing agent and dye, at 85, comprising .1% neutral red and 1% Saponin buffered to pH 5.5 which stains only the basophils. It is then fixed by a formalin solution at 91 and match again with propylene glycol at 95. In path AIV the sample is first fixed by a solution of 20% formalin buffered with phosphate to pH 6, at 103. Then the stream is mixed with a substrate of naphthol butyrate at 111 and a coupling reagent hexazonium pararosanilin, at 115 which stains only the monocytes. Acetic acid at 119 and propylene glycol at 123 are added to lyse the red cells and match the membranes as before.  The large lyphocytes which are not stained are separately counted in path AI by detecting different sizes of pulses from the photocells.