US2369577A - Method and apparatus for counting blood corpuscles - Google Patents

Description

Feb. 13, 1945. J. KIELLAND METHOD AND APPARATUS FOR COUNTING BLOOD CORPUSCLES Filed May 12, 1941 D 5 WM Mn TL @N T WK N V 3 a mm R 0 a N W P in in: 5::

Patented Feb-13, 1945 METHOD AND APPARATUS ron BLOOD conruscms comma Jan Kielland, Bergen, Norway; vested in the Alien Property Custodian Application May 12, 1941, Serial No. 393,133 In Norway and Germany July 3, 1939 1 Claim. (Cl. 235-92) This invention relates to the counting of blood corpuscies and comprehends within its scope an improved process and apparatus for counting either red or white blood corpuscles present in a sample of blood. More particularly the invention comprises a method and apparatus for causing blood corpuscles to pass successively one after another past a given point at which they may be counted.

The standard practice of the art for counting blood corpuscles has involved the dilution of blood with a suitable diluting liquid in an appropriate proportion, and thereafter placing a drop of the diluted blood in a counting chamber of known height having engraved in its bottom surface a network of unit areas of known size. The number of blood corpuscles on one or more of the unit areas is then counted by an operator with the aid of a microscope. From the known dimensions of the apparatus the number of corpuscles per cubic mm. is calculated. Variations have been made in this procedure, but in general all methods, so far as I am aware, involve the counting by an operator of the number of corpuscles in a unit area. Such methods involve an error of v15%.

In accordance with my invention a sample of blood is first diluted in a known proportion with a suitable dilution liquid, and thereafter the blood corpuscles while suspended in the dilution liquid are caused to pass successively one after another through a confined space and are counted while passing through this confined space. The blood corpuscles may be caused to move through the confined space and past the counting point by applying pressure to the liquid prior to its passing therethrough, or by the application of a reduced pressure at the outlet end of the confined space. The just recited method may be advantageously carried out by the use of the apparatus of the invention, which includes a vessel for diluted blood and connected therewith a channel member so dimensioned in cross section as to cause the blood corpuscles which are to be counted to pass therethrough and to pass the counting point in the channel individually and successively. At least a part of the oppositely disposed walls of the channel member must be of transparent material in order to permit the counting of the corpuscles passing successively through the channel. In practice it has been found advantageous to make the entire apparatus of a transparent material such as glass. The counting is with particular advantage accomplished by a counting device dependent for operation on the photoelectric principle either with or without a microscope.

The process of the invention provides a simpler, more rapid and more precise method of counting blood corpuscles than those heretofore employed. The apparatus is not complicated, and, if a counting device embodying a photo-electric cell in conjunction with a recording and counting arrangement is employed, the operation of the apparatus becomes automatic with an attendant increase in precision. In fact if the proper manipulative precautions are taken and the apparatus is properly proportioned, a precision approximating may be attained either by the use of automatic means or by counting manually with the aid of a microscope. In actual practice a precision approximating 100% is seldom required, so that the dimensions of the apparatus and the manipulation thereof may be less carefully controlled, and the result will still be more exact than that obtained by previously known methods and apparatus.

In an apparatus designed to secure the greatest possible precision, the channel member is dimensioned on the basis 0! the size and shape of the blood corpuscles to be counted. Red blood corpuscles, which are plastic, are when stationary biconcave discs with a normal average diameter of 7.5a and a thickness of 2 to 3 In blood diseases the diameter may vary from 4 to 13;; and the thickness may vary up to about 5a. The white blood corpuscles are approximately spherical and have a diameter ranging from about 6 to 22 All red blood corpuscles, even the largest, may because of their plasticity pass through a channel having a height of about 5;; and a width of about 10 The smallest of the red blood corpuscles may be caused to pass through such a channel individually by increasing the dilution of the blood sample. It has been shown by tests, however, that the red corpuscles in a suitably diluted blood sample will pass successively one after another through a channel considerably larger than the one just described. For example, a tube having a diameter of 20 1. may be successfully employed as the channel member. Standard calibrated tubes of this dimension are readily available at low cost, thus making possible in practice the use of a new tube for each test. Ordinarily in a typical sample of blood there is only about one white corpuscle to every thousand red ones, and even in extreme cases the proportion is less than ten white blood corpuscles for each thousand reds. Because of this fact a count of the usually desired precision will not be harmed by one or two white blood corpuscles passing through the channel, when a channel of sufliciently large dimensions is being used, and being erroneously counted together with the red corpuscles. In general the counting of approximately five hundred red corpuscles will provide a result of sufflcient accuracy and the number of white corpuscles involved is thus negligible.

Best results are obtained by preventing the inlet of the counting passage from being clogged by the larger white corpuscles. This may be ac complished in several ways, among which may be mentioned the following. The diluted blood is first passed through a channel having both a width and height of at least 24a, and thereafter through a channel of the same width but having a height of about a. By this expedient the one or more white corpuscles occurring in an average sample being counted will be stopped at the entrance to the shallow channel, while a suflicient number of red corpuscles to give a. precise count will continue through the shallow channel.

Appropriate means may be provided at either the inlet or outlet end of the channel for causing the diluted blood sample to pass through the counting channel at a desired rate. For example. means may be provided for increasing the pressur in a vessel connected with the inlet end of the channel. Such means may operate by increasing the air pressure over the diluted blood sample in the vessel. If the vessel is closed, the increased pressure may be produced by means of the application of heat. Alternatively a reduced pressure may be supplied in the outlet end of the channel or in a vessel connected thereto. In a particularly effective and simple modification of the apparatus of the invention, the channel member is closed at one end by being sealed. into a bulb member. If the opposite end of the channel is immersed in a diluted sample of blood, the blood will rise in the channel member due to capillary action. This action may be expedited by preheating the bulb and letting it cool while the channel is being filled. The length of the channel will then contain a predetermined definite quantity of blood. If now heat is applied to the bulb, the blood sample will be forced outwards through the counting channel, and the blood corpuscles may be counted during their outward passage, or the counting operation may take place while the blood sample is filling the channel.

In order to explain in greater detail appropriate apparatus for carrying out certain embodiments of my invention, reference will be had to the accompanying drawing in which:

Fig. 1 includes a diagrammatic perspective view of an apparatus having a counting channel connected at each end to vessels for the diluted blood. and a diagrammatic plan view of a photoelectric countin means associated with the counting channel.

Fig. 2 is an axial section of an alternative structur in which the counting channel member is sealed to a bulb member.

In accordance with one embodiment of my invention illustrated in Fig. l, the lower end of a vessel I is connected to a channel 2 which is a narrowing passage, either progressively as ill-ustrated or in a stepwise decreasing cross section. Channel 2 is integrally connected with a counting channel member 3 having a width A and a height B, the said dimensions in at least a portion of the length of channel 3 being such that the cross counting mechanism as illustrated in Fig. 1.

section of the interior passage will permit the blood corpuscles which are to be counted to pass individually and successively through the passage. As here illustrated the dimensions of the channel member 3 are substantially uniform throughout its length. As previously indicated the dimensions A and B may with advantage be made to equal approximately 1011 and 5# respectively in an apparatus in which the highest precision in counting the red corpuscles is desired, but the member 3 may also be of larger dimension, such as for example a tube having a diameter of 20a. The counting channel 3 opens into and is integrally connected with a channel member 5, which increases in cross sectional dimension and provides a connection to a second vessel 6. In the application of the process in such an apparatus, a diluting liquid is with advantage added to container l in sufllcient quantity so that it passes through and fills the three channel members and vessel 6 to a height equivalent to that in vessel l. A measured quantity of blood is then introduced into the liquid in container I and is caused to pass through the channel together with a dilution liquid, for example, by the application of air pressure admitted to the container l by means of pipe 1 or by the reduction of pressure in container 6 by exhausting air through pipe 6'. The channel member 3 is made of a transparent material, at least in its central section. Opposed to this transparent sect on one may employ a microscope and count the blood corpuscles passing beneath the field. The microscope may be replaced by a source of light 4 which transmits light through the counting channel 3 against a light sensitive surface I, for xample, a selenium plate. The light sensitive member I is cormected in an electrical circuit comprising a source of electrical current 8, an amplifier 9 and a counting apparatu l0. Such an arrangement counts the number of corpuscles passing through the channel by virtue of the interruption of current flow through the circuit, and automatically registers the number of interruptions in the counting mechanism. A lens may with advantage b placed between the counting channel and the light sensitive surface, in which case and also in the case where a microscope is employed, it is advisable to surround the counting channel with an immersion oil in order to reduce the refraction of the light rays.

In an apparatus such as that diagrammatically represented in Fig. 2, the channel member 3 is closed at one end by being sealed into a bulb II, which may be warmed by a source of heat l2. As previously described a device of this sort is operated by immersing the end of the channel 3 in a sample of blood 11, whereby the blood will rise in the channel 3 due to capillary action and possibly also by the aid of the cooling of the bulb, if this is preheated. The corpuscles may be counted with the aid of a microscope as they pass through in the direction of the bulb, or by means of a photoelectric cell associated with a ternatively the diluted blood sample may be permitted to pass into the channel and then be expelled therefrom by means of heating the bulb,

the counting being done as the liquid passes outwardly through channel 3.

In accordance with a further embodiment of my invention, blood corpuscles may be precisely counted by means of apparatus such as is disclosed in Figs. 1 and 2 under conditions such that the counting mechanism is sensitive to only the form of corpuscle to be counted. In such a method of operation a channel member having a diameter at least as large as the largest white corpuscle is employed. The red corpuscles are made colorless, for example by means of acetic acid, while the visibility of the White corpuscles is increased by the application of an appropriate dye. Under such conditions only the colored white corpuscles will register in the counting mechanism.

I claim:

Apparatus for counting blood corpuscles which comprises in combination a capillary channel member having at least a portion of its opposing walls of transparent material, and having a pas- JAN KIELLAND.

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