Coulter counter

The Model A's aperture was the shaft hole of a ring jewel, a disc of synthetic ruby or sapphire of the kind made in Switzerland as bearings for mechanical watches, cemented over a larger hole in a glass tube.^[11] Jewels from several suppliers had bores of very uniform diameter, and those without chips at the orifice gave pulses consistent in quality. The hole described in Coulter's 1956 paper was 100 µm wide and 67 µm long, and the Model S later used 100 µm apertures with a length of 0.75 diameters.^[2][5] Beckman Coulter's standard apertures now run from 10 µm to 2,000 µm, and one aperture measures particles from 2% to 60% of its diameter.^[12]

A vacuum raises mercury in a reservoir. When the reservoir is opened to the air the mercury returns through a bulb in the glass manometer and touches a start electrode, then a stop electrode, and the suspension drawn through the aperture between those two contacts is the count volume. For the 50 µm and 100 µm apertures it was 500 µL, accurate to 0.25%, and the mercury column that drove it was 0.127 m high, equal to 1.74 m of water.^[11] Joseph R. Coulter Jr. said later: "It was the manometer that made the counter work. It was simple, it was easy to control, and it kept working."^[11]

In the Model A the pulses were amplified for display on an oscilloscope tube, and those above a level set by a threshold control were added up on three decade counters and a mechanical counter. The prototype's electronics unit held 33 vacuum tubes plus the oscilloscope tube.^[11][2] A count of about 50,000 cells took about 15 seconds, a rate of about 3,300 cells a second, and a manual count of 500 cells took about 20 minutes. According to Graham, the automated count had one tenth of the error of a count by an expert technologist and took 1.25% of the time.^[2][a]

Later models added size bins and automation. The Model B held the aperture current constant and used two interlocked thresholds to count one size bin at a time, and the Model H plotter assembled a 25-bin size distribution from a run of about 100 seconds. An experimental twelve-bin Model C needed 350 vacuum tubes, more power than clinical laboratories usually had.^[13] The transistorized Model F projected an image of the aperture on a screen in place of a microscope.^[14][5] The Model S of late 1968 diluted the blood, split it between a white-cell bath and a red-cell bath, counted each through three apertures, discarded the aperture whose count disagreed with the other two, and printed seven parameters.^[5]

Coulter was born in Little Rock, Arkansas, in 1913. In 1939 he went to the Far East as a sales and service engineer for X-ray equipment, and in hospitals there he saw technicians count blood cells by eye on hemocytometers. In Graham's account the atomic bombings of Hiroshima and Nagasaki in August 1945 made the need plain: counts that were rapid and accurate, and repeatable at close intervals for whole populations, to follow the recovery of bone marrow after radiation exposure.^[4] A manual count was uncertain by about 16% for red cells and 21% for white cells. In drafts of a proposal for Navy support, Coulter wrote of "an accurate, simple, and rapid means of obtaining red blood cell counts" for use in the event of atomic attack.^[7]

The brothers' first approach was optical. In 1934 Andrew Moldavan had proposed counting blood cells photo-electrically as they flowed through a capillary on a microscope stage.^[15] After buying a house in Chicago in April 1947, Wallace and Joseph experimented in the basement and found the author's criticisms of the method justified, and by late 1947 a capillary tube cut to its minimum length still gave no counts of single cells.^[7]

Around Christmas 1947, according to Graham, Coulter was reconsidering a method he had read about as an electrical engineering student at the Georgia School of Technology, for calculating the resistance of a suspension of particles whose conductivity differs from that of the liquid. Graham cites Maxwell's Treatise on Electricity and Magnetism for it. In July 1948 Coulter obtained a reprint of a short note that used a conductivity cell to show red cells to be poor conductors, and on July 26 he wrote the first statement of the principle: two chambers joined by one small aperture, as short as possible, with an electrode in each.^[16]

Arcs through mica gave apertures of erratic size. On October 16, 1948, Coulter heated the tip of a sharpened needle, burned a hole in a cellophane wrapper from a pack of Joseph's cigarettes, bound the wrapper to a glass tube with rubber bands, and showed that cells in his diluted blood changed an electric current flowing through the hole. "It didn't hold up long," he said later, "but we were able to count some cells."^[7][16] Two weeks later he repeated the experiment with a hole 76 µm wide in cellulose acetate film 22 µm thick. The blood was diluted several thousand times in 0.9% sodium chloride, the pulses on the oscilloscope lasted about 1 ms, and a microscope showed the cells crossing as the pulses appeared.^[16]

Patent attorneys to whom Coulter described the idea did not grasp it, and he was told repeatedly, "You can't patent a hole." A former colleague introduced a patent attorney who did understand. An examiner who shared the doubt surmised that a patent might be allowed on sensing particles in a constricted current path if the application included more than an axial current through an aperture, and the application as filed described current paths transverse to the flow, apertures that were not cylinders, and an insulated needle swept through a stationary suspension. It was filed on August 27, 1949, and the patent, US 2,656,508, was granted on October 20, 1953.^[16][17]

Coulter sent a proposal to the Office of Naval Research on April 30, 1951. The office's reply of May 16 recorded the work as "To Supply Blood Cell Counter", and partial funding came under contract NONR-1054(00).^[7][11] An estimate put the hydrostatic head needed for a few thousand cells a second at about 1.8 m, so the brothers tried motor-driven syringe pumps before settling on vacuum and the manometer.^[11] A prototype Model A went to the Navy office near the end of 1953.^[11] The first production counters were assembled in the basement of a Chicago apartment building on North Kenmore Avenue, in an unfinished area by the furnace, by the company's first employee, a technician who, Wallace recalled, built them faster than he could sell them and lent him his car for sales calls.^[2]

About 20 Model A counters had been placed by June 1957. Coulter Electronics, Inc., was incorporated on April 14, 1958, with Joseph as president; more than 1,500 counters had been placed by February 1961, and the company moved to Hialeah, Florida, in December 1961.^[2][18] The Model S followed in late 1968, and an advertisement five months after its first one claimed 1,000 installations in two years. It did not separate white cells into types or count platelets.^[5]

Beckman Instruments acquired Coulter Corporation in 1997, creating Beckman Coulter, and Danaher acquired Beckman Coulter in 2011.^[19] Joseph died on November 27, 1995, and Wallace on August 7, 1998.^[20]

Before automation a complete blood count meant a technician, a hemocytometer, a microscope and 15 to 30 minutes. The Model S made the red cell count, white cell count, hemoglobin, hematocrit and three red cell indices a routine printout, and Graham counted at least 6,500 DxH 800 analyzers installed by 2018, each able to run 100 samples an hour through Coulter apertures.^[5][4] Automated analyzers measure cells by electrical impedance, by optical analysis, or by both. Instruments of the 1980s such as Coulter's VCS transducers added a second, high-frequency measurement that responds to the interior of a cell.^[6]

Beckman Coulter lists microorganisms, cell types, pharmaceuticals, colorants, foods, abrasives, explosives, minerals and metals among the materials measured with the principle, and it gives upper size limits that depend on the material, about 500 µm for sand and about 75 µm for tungsten carbide.^[12]

In 1965 M. J. Fulwyler described a device that measured cell volume in a Coulter aperture, enclosed each cell in a droplet charged according to the volume sensed, and deflected the droplet in an electrostatic field into a collection vessel. It separated mixtures of mouse and human red cells, and hamster ovary cells that passed through it kept growing at their normal rate.^[22]

In 1970 DeBlois and Bean counted polystyrene spheres 90 nm across with single submicron pores etched in plastic sheet.^[23] In 1994 a paper on counting polymers in a single ion channel opened with a description of the Coulter counter, and in 1996 single strands of DNA were driven through a protein pore.^[24][25]