Flow analyzer and system for analysis of fluids with particles

The method and apparatus for analyzing particles and particularly blood cells by conveying the particles along a broad shallow path where the path is magnified and converted into a series of still frame images with multiple particles in individual images. The still frame images are manipulated and combined as digital images to generate measures of the blood cells in the overall fluid sample giving different selected measures, such as, particle count, particle count of different kinds of cells, cell area, and cell count of different predetermined categories of cell type.

BACKGROUND OF INVENTION 
Substantial advances have been made in automating the job of counting blood 
cells in a serum sample. The most well-known instrument for performing 
blood counts is the so-called Coulter counter in which blood cells are 
passed in single file through an orifice and detected and counted by the 
manner in which they change the electric properties at the orifice. Up 
until the present time, however, there has been no automated equipment 
available for analyzing and evaluating the multiple cells, such as, normal 
cells, target cells, sickle cells, etc. which may be found in a flowing 
stream of a given blood sample. Thus, where multiple cell information of 
this type is desired, the standard commercial way of obtaining it is by 
preparing a microscope slide with the cells fixed on an image plane and 
having a human operator or pattern recognition machine count statistically 
significant numbers of the cells as the cells are observed one-at-a-time 
on the slide through a microscope. 
Some attempts have been made in recent years to provide optical analysis of 
particles flowing in a flow stream. For instance, Kay, et al., Journal of 
Histochemistry and Cytochemistry, Volume 27, page 329 (1979) shows a 
Coulter type orifice for moving cells in single file with the cells 
magnified on a vidicon. Additionally, Kachel, et al., Journal of 
Histochemistry and Cytochemistry, Volume 27, page 335, shows a device for 
moving cells in single file through a microscopic area where they are 
photographed. While these workers have done some work in automating 
particle analysis in single file no successful work has been reported 
where automated particle analysis was accomplished in a flowing stream 
without the requirement of arranging the particles into a single file 
stream. See for instance Flow Cytometry and Sorting Melaned et al, John 
Wiley & Sons 1979, Chapter 1. 
SUMMARY OF THE INVENTION 
In accordance with this invention we have provided a method and apparatus 
for moving a fluid sample, preferably a blood sample, through a controlled 
flow path where the particles in the sample are confined to a shallow but 
broad imaging area which is on the order of the depth of the particles and 
many times wider than the particles. The stream of particles in the 
imaging area is magnified and a series of still frame images of the 
particles are prepared and then algebraically combined to generate 
measures of the cell content of the original flow stream. In this way more 
than one particle can be examined in a single field, and different 
particles can be optically distinguished with a number of important 
advantages. For instance, two cells flowing together can be optically 
recognized whereas a Coulter counter could recognize them as a single 
double-sized cell. 
Preferably, the still frame images of the flow stream are provided by 
imaging the magnified image of the stream on a CCD (charge coupled device) 
camera from which still frame images are taken and analyzed in digital 
form. In this way the still frame images may be enhanced with the digital 
image enhancement techniques which have been developed for satellite 
pictures and the individual frames may be analyzed to provide data on 
individual cells, such as, size, cross-sectional area, shape, (circular 
cell, target cell, sickle cell, etc.), optical density, hemoglobin content 
on the cell basis, etc. Not only can individual cells be analyzed and 
optically sorted in this way, but additionally when the cells are so 
analyzed and sorted different types of cells may be individually counted 
to give automatically and at a single pass, the number of normal red cells 
per volume of sample, the number of target red cells per cc of sample, the 
number of sickled red cells per cc of sample, the number of white cells, 
the number of platelets, etc. per cc. of sample. 
Thus, once a series of still frame images is prepared in digital form in 
accordance with this invention, a wide variety of very sophisticated 
information can be obtained about the particles in the series of images 
depending upon the complexity of computer equipment and software which may 
be used for analysis of the images. 
Preferably information derived from still frame images is combined 
algebraically to provide composite information reflecting the content of 
the multiple still frame images and/or predetermined reference images, and 
the composite information thus obtained may be used in a variety of ways. 
Thus, in simple systems the information may be printed out, for instance, 
to advise a hematologist about composite measurements made from a blood 
sample. In more complex systems, the composite measurements may be used by 
process control, such as pressure in a homogenizer, temperature in a 
crystallizer, or nutrient feed rate in a microbial culture where the 
system monitors particle size or number. 
Thus, it will be noted that the invention may be used for analysis of a 
variety of optically perceptible particles moving in a stream, both 
biological particles, such as cells in blood or cells, bacteria, casts and 
crystals in urine or particles in gas analyzers, etc., and the output of 
these measurements may be employed for process control, such as dispensing 
nutrients into a stream containing microorganisms as mentioned above, the 
control of the growth of polymers and crystals, etc. 
The information which is provided may be correlated readily to the original 
volume of blood sample from which the still frame images are made by a 
variety of methods to calibrate the results for both particle size and 
concentration. For instance calibration may be accomplished by adding 
calibrator particles to the original blood sample in a known concentration 
so that the calibrator particles may be counted independently of the 
normal blood cells to provide volume calibration for the normal blood 
cells. Alternatively, calibration may be accomplished by providing a 
cross-hatch of fixed dimension in the field of view. 
The flow stream of particles to be magnified and imaged is preferably 
provided in a flow chamber which moves the particles in a stream which is 
approximately the thickness of the thickest particles and many times wider 
than the widest particles, for instance, more than one hundred times as 
wide as the widest particles. The flow chamber might be designed to create 
turbulent flow to permit asymetric particles to be viewed from multiple 
directions. On the other hand the flow chamber may be designed to orient 
the particles. 
Preferably the flow stream in the imaging area has a cross-sectional area 
of minimum shear which is not substantially larger than the minimum 
cross-sectional area of the particles whereby the particles are aligned in 
the flow stream with their minimum cross-sectional area extended 
transverse to their direction of flow. The term "minimum shear" is used 
herein to mean "minimum velocity gradient" so that a particle moving in 
the stream tends to align itself with the direction of the stream much as 
a log floating down a river will align itself with the direction of flow 
where there is a flow gradient.

Referring now in detail to the drawings, and particularly to FIG. 1, the 
apparatus shown therein includes a body 10 containing a flow chamber 
having an inlet 12 for a blood sample and an outlet 14 with a passageway 
16 extending between them past an imaging area 18. The passageway 16 has 
an inlet with a conduit 20 adapted to be connected to a volume of saline 
solution 22. As illustrated in FIGS. 2 and 3, the inlet 12 for the blood 
sample has a needle 24 in the passageway 16 downstream from the conduit 20 
with the needle 24 connected to a container 26 adapted to hold the blood 
sample to be analyzed. 
The cross-sectional area of the passageway 16 becomes progressively smaller 
as the passageway extends from the blood inlet 12 to the outlet 14 while 
at the same time the passageway 16 becomes much shallower and much wider. 
Thus, as illustrated in FIGS. 2 and 3 the passageway 16 has a width and 
depth of about 5,000 microns at the blood inlet 12 and a width and depth 
of about 500 microns at midpoint 28, and a depth of 100 microns with a 
width exceeding 5,000 microns at the examination area 18. 
It will be appreciated that the flow stream through the examination area 18 
is many times deeper than the largest cell which have a maximum dimension 
of about 20 microns, but with the flow passageway shaped in this way the 
blood stream entering through the opening 12 is confined to a stable flow 
path of minimum shear in the examination area 18, and the disc-like cells 
are oriented in that area with their maximum cross-sectional area visible 
in the plane of FIG. 2. The flow characteristics in the passageway 16 may 
be controlled by adjusting the fluid pressure in containers 22 and 26 
either automatically or by adjusting the static heights thereof. 
A microscope 30 is focused on the examination area 18 and the examination 
area 18 is illuminated from below by a strobe light 32 which is preferably 
a U.S. Scientific Instrument Corporation Model 3018 containing a 2UP1.5 
lamp. The output of the microscope 30 is focused on a CCD camera 34 which 
is preferably a CCD camera model number TC1160BD manufactured by RCA. The 
output of the CCD camera is converted to a series of still frame images, 
and suitable electronic processors are employed for evaluating those 
images. One processor which may be employed is the processor marketed as 
Image Analysis System Model C-1285 by Hamamatsu Systems, Inc., Waltham, 
Mass. Preferably the output of the CCD camera is connected to an 
electronic processor 36 which is illustrated in greater detail in FIG. 4 
and includes a black and white television monitor 38 and a frame grabber 
40 which stores still frame images of the subject viewed by the CCD 
camera. The frame grabber is preferably a Model FG08 frame grabber made by 
the Matrox Corporation of Montreal, the output of which is supplied to a 
video refresh memory 42 Model RGB 256 made by Matrox Corporation which are 
both coupled to the multibus 44 of the central processing unit 46 which is 
preferably an Intel 80/20 computer. The multibus is also coupled to a 48K 
random access memory 48 of Electronic Solutions, Inc., and a 16K dual port 
random access memory 50 Model RM 117 of Data Cube Corporation. The output 
of the video refresh memory is also coupled to a color monitor 52 which 
may be used to provide digitally enhanced video images of individual still 
frames for human examination. 
The second output of the dual port ram 50 is connected to a multibus 54 
which is connected to an Applied Micro Devices central processing unit 56, 
a 48K random access memory of Electronic Solutions, Inc. 58 and removable 
storage in the form of a floppy disc controller 60, such as an Advanced 
Micro Devices Model 8/8 and two units of Shugart floppy disc storage 62. 
A wide variety of programming may be employed for processing pictures with 
the apparatus of FIG. 4 depending upon the particular task which user 
wishes to perform. 
As mentioned above, the programming of the Hamamatsu System 1285 may be 
employed. Preferably, however, the programming is performed as follows: 
The tasks are first divided into those which must address each pixel in a 
given image and those which only address a small subset of the total. 
Since much time will be spent in the first class of tasks, they are 
programmed in assembly language on the interface processor 46 (the Intel 
80/20 in FIG. 4). The output of these operations are then transferred to 
the host machine 56 via the dual ported ram 50. On the host side almost 
all of the necessary programming is more suitably done in a high level 
language such as Pascal (BASIC or FORTRAN could be in principal be used 
also). The types of tasks that are done in the assembly language includes 
greyscale transformations, convolutions, and greyscale histogram 
calculations. The types of tasks done on the host side include overall 
control of the other devices, identification and segmentation of object of 
interest in the field of view, calculation of parameters associated with 
objects thus found, and formating the output of results. Another way of 
considering this separation of tasks in this fashion is that tasks which 
must be performed at speeds great compared to a human operator are done in 
assembly. Tasks which are either complicated or which can operate at less 
than the maximum speed can be programmed in the higher language. Objects 
are found in a field of view primarily by setting a greyscale window 
function for values known to be characteristic of the desired object. 
These values can be established by prior knowledge or by well-known 
histogram techniques. When a pixel belonging to an object has been located 
in the field of view, an edge tracing program is invoked to outline the 
whole object associated with that pixel. Once the edge has been found, 
then many relevant parameters such as location, area, integrated optical 
density, and various moments can easily be calculated. Probability of 
membership in previously defined subgroups can be determined from these 
derived parameters by means of standard decision theory. Definitions of 
blood cell morphoplogy classifications are established by trained 
observers. These definitions are then used as the basis of the selected 
algorithms. Accuracy of the method is determined by comparison of machine 
results with those of trained observers examining the same samples. Output 
of the results can be programmed to be any of a variety of formats. 
Histograms, line plots, and tabular summaries are available for particular 
needs.