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The Coulter Principle: Foundation of an Industry

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The Coulter Principle: Foundation of an Industry Special Feature The Coulter Principle: Foundation of an Industry Marshall Don. Graham Beckman Coulter, Inc. THE FIFTIETH ANNIVERSARY OF THE COULTER hopes of patenting his method for automating the Keywords: PRINCIPLE erythrocyte count.1 For each specimen, a technician Coulter Principle, spent some 30 tedious minutes at a microscope hematological Introduced in the mid-1950s, the Coulter Principle counting the red cells in a standard chamber, the instrumentation, became the foundation of an industry responding to result being only rarely repeatable. Automating such automated the need for automated cell-counting instruments. counts had tantalized Wallace (Fig. 1) since he had blood-cell counts, The industry developed in three acts, as Wallace H. read Moldavan’s 1934 note proposing photoelectric particle analysis, Coulter and his brother Joseph R. Coulter, Jr., counting of cells in a suspension as it flowed through cytometry developed the simple idea of passing cells through a capillary tube mounted on a microscope.2 Over a a sensing aperture. In Act I, Wallace’s desire to decade had elapsed before Wallace discovered automate the routine erythrocyte count led to a a better method, and a second decade was to pass simple idea, the definition of the Coulter Prin- before he was prepared to announce it. ciple, its patenting, its acceptance by the National Institutes of Health, and its description at a national conference. In Act II, the Coulter brothers ad- ACT I dressed the practicalities of a commercial instru- ment and of a business organization to support its After gathering technical experience elsewhere, manufacture and sale. In Act III, a broad research Wallace returned to Chicago in 1946 and was soon effort developed regarding volumetric errors origi- joined in his quest by his brother, Joseph R. Coulter, nating in functional characteristics of the sensing Jr. (Fig. 2). As he later reminisced about their work aperture, and the brothers’ growing organization in a basement laboratory:3 ‘‘The approach originally found solutions permitting introduction of increas- was to count blood cells going down a capillary ingly automated hematology analyzers. Today the tube, passing through a light beam, like counting industry thrives, with several participants. people going down a corridor, but we weren’t get- ting very good signals. We asked the question, ÔIs INTRODUCTION there some way, besides modulating a light beam, to generate electrical pulses by the passage of a cell?Õ ‘‘You can’t patent a hole.’’ With that opinion, Although we didn’t know it at the time, blood cells several attorneys dismissed Wallace H. Coulter’s are insulators—so we arrived at a solution by modu- lating an electric current instead of a light beam.’’ Correspondence: M. Don. Graham, Ph.D., Beckman Coulter, Inc., In a characteristically simple solution, by 1947 Kentucky Technical Facility, 115 Patton Court, Nicholasville, Wallace had reduced the capillary tube to its mini- KY 40356; Phone or automatic Fax: +1.859.885.5007; 1 E-mail: [email protected] or [email protected] mum length: ‘‘When we started we didn’t have JALA 2003;8:72–81. much money, so we made an aperture by making Copyright c 2003 by The Association for Laboratory Automation a small hole with a hot needle in a piece of cellophane 1535-5535/2003/$30.00 + 0 from a cigarette package. It didn’t hold up long, but doi:10.1016/S1535-5535(03)00023-6 we were able to count some cells.’’ Held onto the end 72 JALA December 2003 Special Feature Figure 1. Wallace H. Coulter encountered the routine erythro- Figure 2. Joseph R. Coulter, Jr., after his Army service, joined his cyte count during his visits to hospitals for General Electric X-Ray brother Wallace in a small basement laboratory and helped solve Corporation and began experiments toward automating it after many difficulties while incorporating the Coulter Principle into a returning to Chicago in 1946. In 1947, he discovered one form of practical automated instrument. In 1958, he helped found Coulter the Coulter Principle. Electronics, Inc. of a glass tube by a rubber band, the punctured cellophane pension flow was described, as well as apertures having non- separated two electrodes connected to a source of electric circular cross sections. An insulated needle sweeping past current while cells suspended in ionic medium were passed particles in a stationary suspension was another example, through it simultaneously with the current. A cell’s displace- a particle’s presence being signaled by a pulse in the current ment of liquid equal to its own volume within the aperture between the moving needle and a second electrode contacting was proportionally signaled by a voltage pulse between the the conductive suspending medium. The new Coulter Princi- electrodes passing the current through the aperture. ple being thus defined, the seminal patent was issued 50 years The brothers had found electrical contrasts between cells ago on October 20, 1953, with the preferred embodiment being and the suspending medium that were some 10 times the ones a pinpoint aperture formed in the lower wall of a test tube.4 obtained photoelectrically, the resultant voltage pulses Meanwhile, the Coulter brothers had continued develop- readily permitting an accurate count of the cells in the ment, with emphasis on automation where feasible. With volume of cell suspension passing through the aperture. This volunteer help from Walter Hogg, Joseph’s friend from discovery had prompted Wallace’s unexpectedly prolonged Army days, the brothers assembled an experimental in- search for an attorney willing to prepare a patent applica- strument under contract to the Office of Naval Research: tion. Finally, in 1948, he was introduced to Irving Silverman a mechanical metering system moved a controlled volume of who recognized the new method’s significant potential, and cell suspension through the aperture while an interface unit in August 1949, a patent application was filed. provided it an electric current and sensed resultant signal But the patent examiner also doubted that a hole could be pulses via a voltage amplifier having an adjustable threshold. patented. Fortunately, he also surmised that if an application Signal pulses above threshold triggered a pulse counter were to include examples other than an axial current and (Berkeley Scientific Model 410; Beckman Instrument, [then] sensing path through an aperture, a patent might be obtained Richmond, CA) mounted atop the interface unit. Repeated on the principle of sensing particles in a constricted current runs of a sample at successively increasing thresholds path.1 An analogous path transverse to the aperture sus- permitted manual recording of a cumulative distribution of JALA December 2003 73 Special Feature cell size. The need to detect partial occlusion of the aperture were built; the interface electronics and oscilloscope were soon became evident, so an oscilloscope was added to allow integrated into a single compact console also including de- simultaneous monitoring of the signal pulses and the cade counters to accumulate the voltage pulses. In 1953, threshold setting. these prototypes went to the National Institutes of Health Experience also emphasized the need to meter a precise (NIH) for evaluation, and in May 1956, a patent application volume of cell suspension through the aperture. Together, was filed on the manometer metering system.6 the Coulter brothers invented an elegant solution based on The Coulter Principle was formally announced on a mercury manometer (Fig. 3). As Joseph once remarked:5 October 3, 1956 in Wallace’s sole technical paper:7 ‘‘In the ‘‘It was the manometer that made the counter work. It was new counter individual cells are caused to move through simple, it was easy to control, and it kept working.’’ Com- a small constricted electric current path in the suspending bined with a microscope focused on the aperture, this meter- fluid and detection is based upon differences in electrical ing system formed the basis of a sample stand that would conductivity between the cell and the suspending fluid. The remain essentially unchanged for over 30 years. In 1952, the constricted current path is analogous to a light beam of small first two instruments incorporating the new sample stand dimensions in an optical system. In passing thru the small current path in the fluid the individual blood cell changes the electrical resistance in the circuit, and causes a change in the voltage drop appearing across the current path. The electric current path of small dimensions and the flow of cell bearing fluid thru the path is provided for with a very simple structure. The boundary of the current path is the bore of a submerged orifice of small dimensions in the wall of an insulated vessel. ’’ In Figure 3, this submerged orifice is the central hole in aperture wafer A. Shortly thereafter, the two NIH evaluations were pub- lished.8,9 Both noted the improved accuracy, efficiency, and convenience of the Coulter method for counting erythro- cytes. The journal issue containing one evaluation8 also con- tained the first advertisement for the new Coulter CounterÒ. The second evaluation9 attributed skewness in the size dis- tribution to cell coincidence and presented a manual method for correcting the count for coincidence loss. It also included preliminary data on leukocyte counting. Figure 3. Functional schematic of the Coulter sample stand, co- The feasibility of automating significant hematological invented by the Coulter brothers.6 When stopcock F is opened, tasks had been demonstrated, but the work of commercial- the mercury in the manometer reservoir R is drawn upward by ization yet lay ahead. Reliable mounting of the aperture had a small vacuum pump connected to P, and the resulting pressure proven difficult.10 The mercury used in the manometer meter- head causes movement of mercury column J after the stopcock is ing the suspension through the aperture was a concern, closed, drawing sample suspension E from the sample vessel emphasizing an advantage of the mechanical system used in through the hole in aperture wafer A into sample tube B.
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