2. Cell counting
posted : 2009.12.09
It was in the spring of 1948 that I was surprised to find a noticeable error in the cell counting at Research Laboratory of Keio University. It was difficult to obtain any experimental equipment or instruments in those chaotic days of Japan following the end of the World War II, when even gaining a mouthful of daily food was the serious concern of the people.
Neither a pipette for the cell counting nor a hemocytometer available at that time had the accuracy as those of today. When measuring microsomes such as erythrocytes, we must consider Poisson Distribution, in addition to the instrumental error mentioned above and also "human error" being caused by naked eyes.
A most curious fact as it appeared to me was the fact itself that a cubic millimeter of the blood contains h to S million corpuscles. For what purpose did Mother Nature has created such small particles as 7 microns or higher micron order in their sizes. It will be an interesting problem to begin with which I may refer later.
I suggested an improvement of the cell counting method and tried to develop it by myself. However, there were no reference books for the purpose. I used to fancy and ruminated only over the failure. Finally, two methods came up to my mind worth enough to be tested. One was to use reflection of the cell images, project the images in hemocytometer, and plot them by the aid of optical or electronic instrument. Another was to dye erythrocytes and perform the cell counting by the colorimetric comparison.
The first method would have been easier if the optical and electronic sciences have been developed as those of today, but at that time tremendous amount of money and time was required to obtain the apparatus.
Nihon Kogaku Optical Industry was trying to develop a projector called "Epidiascope". However, it was good for optical use, but not suitable for the purpose of the cell counting. The application of electron beam was considered also, but it required highly complicated equipment and was not suitable for my experiment. Later, I heard the electron beam was developed in England. However, same as the science of RADAR, it was not known by the Japanese people until end of the World War II.
As the result, I tried the second method. While I was studying I learned the use of red filter which was suggested by Dr. Takashi Azuma of Toshiba Laboratory. When the red filter was applied in the colorimeter, influence of the hemoglobin became negligible. At the beginning it was difficult to obtain colorimeter with the red filter. Later, I developed a phctoelectric-colorixrxeter and spectrophotometer by the assistance of Professor Yoshio Suga, Applied Physics, Tokyo University.
Those hand-made tools were composed of galvanometer, photoelectric cell, dry battery, etc. and were primitive, but to me they were most reliable apparatus for the purpose at that time.The readers may remember that most tools for emeriments were very difficult to obtain in Japan during the period of l9b5 to 1951.
In order to operate the photoelectric-colorimeter, setting of the cuvette in accurate position was essential to obtain accurate result. I tried one way and another, and finally decided to use a fixed cuvette having two holes, one for inlet and the other for outlet. When positions of the cuvette, the funnel, and large injector were stabilized, the apparatus served properly for the purpose.
The cell counting was nephelometric although we called it colorimetric and when the position of the cuvette becomes out of order even slightly the beam scattered, and the total number of the cells varied each time. The stabilization of the cuvette led me to discover a phenomenon of "Streaming Transparency", the detail of which will be stated later.
I learned the truth of the proverb "The poverty is the mother of invention" from my own real experience.
At that time I could not obtain even an accurate red glass filter. Therefore, I planned to transform flat erythrocytes into spheric form, so to apply a strong light on the surface of the erythrocytes, reflect, and enlarge the image of the erythrocytes and observe them. It is a well known fact that the erythrocytes in a hypertonic salt solution turn into some confetti-like shape. And also that pathologic erythrocytes have spheric shape too.
From above fact it is easy to consider that there will be very little difference between the pathologic erythrocytes and the normal erythrocytes under this spectroscopic test since the effect of hemoglobin is neglible.
In short the erythrocytes in hypertonic salt solution change their color to pale white and some peaked and embroidered parts of the confettilike erythrocytes are so vague and indistinct because their sizes are less than the light wave length. In this portion there may occur deflection and/or reflection of the ight.
In l.5 to 2.5% salt solution the erythrocytes become spheric shape, in 3% or more concentrated solution they change into a little pressed or flat shape, and in l0% solution they turn into leaf-like flat shape as shown on Fig 2.[Not given]
When a beam of light was passed through the particles of4 to 8 microns in diameter, a peculiar phenomenon was observed. I noticed the size of the particle has very little effect optically, and it was one of the reason why I changed the erythrocytes into spheric shape. The sizes of spheric erythrocytes became appropchnately 5 to 6 microns in diameter. Finally 3% salt solution was selected for the purpose of the cell counting experiment.
Diluted the blood cells to 200 or 400 times in volume by the 3% salt solution, using the red filter, passed beams of light through the mixture, checked the (T) transmissivity of light and computed the number of the erythrocytes.
Mixing the erythrocytes with the diluted solution in the special cuvette as shown in Fig 1[Not given], I observed the change in the transmissivity of light passing through. The solution was kept still for one or two minutes after mixing in the cuvette, then the rate of transmissivity increased rapidly by 2 -3, as shown on Fig 5[Not given].
This change of transmissivity indigates the decrease of number of erythrocytes approximately 25 -40x104. In general, I can say that the more spherical the shape of erythrocytes the less change of the transmissivity of light will occur, and that the more flat they are the more change of the transmissivity of light will occur.
The transmissidty of light and the shape of the erythrocytes in various diluted salt solution were observed and the result is shown on Fig 2[Not given] through Fig 5[Not given]. This result shows that flat erythrocytes increase when the color index of patient is low, and decrease when color index of
patient is high.
Later, Professor K. Kuroda has discovered the similar phenomenon by other means and he gave it a term called "Streaming Transparency".
when the erytlmocytes were falling dorm or prrecipitating in the salt solution, I noticed that the cells were approaching gradually as if they were attracting one another, and then bouncing and separating again when they met once. This phenomenon was quite interesting to me and made me suspect if they have some static electric charges.