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Description  |
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FIELD OF THE INVENTION
The present invention relates to the measurement of blood cell indices and,
more particularly, to apparatus and methods for in vivo quantitative
measurement of red and white blood cell indices.
BACKGROUND OF THE INVENTION
The red blood cell count, white blood cell count and the determination of
blood cell indices are the most frequent of all clinical laboratory tests
performed on patients in doctors' offices, clinics and hospitals in the
United States and throughout the world. These tests provide essential
information as to the health characteristics of individuals of all age
groups.
Processes for evaluating and testing blood which are in widespread use in
the art today uniformly require a sample of blood to be removed from the
body for testing and analysis. The procedure for taking blood, known as
phlebotomy, ordinarily involves the removal of blood from capillary or
peripherial blood (usually in infants) and venous blood. Only certain
licensed persons are permitted by law to carry out this procedure. These
include nurses, laboratory technicians, physicians, and other specially
trained persons.
In infant phlebotomies, the sample is ordinarily taken from the palmar
surfaces of the tip of a finger or the plantar surfaces of the great toe
or heel. The site is first rubbed vigorously with a gauze pad moistened
with seventy-percent alcohol to remove dirt and debris and to increase
blood circulation. Once the skin has dried, a puncture 2-3 mm deep is made
with a blade or lancet. After the sample has been collected, slight
pressure is applied to the area of the puncture with a gauze pad. This
procedure is usually painful and frightening to the infant which results
in further difficulties in effecting the phlebotomy.
The most common form of adult patient phlebotomy involves the removal of
venous blood through puncture of a vein in the patient's arm. This
procedure is also somewhat painful and is viewed with apprehension by most
patients. Veins must be carefully inspected, particularly with those
patients who have already had numerous punctures. The patient's life may
depend on vein patency, and care must be taken to preserve these vessels.
Hematomas or ecchymoses are usually evidence of the operator's poor
technique or judgment.
The equipment necessary for this procedure ordinarily includes a syringe
and a needle of the appropriate diameter. This needle must be carefully
inspected as a blunt or bent tip will damage the patient's vein and often
lead to failure to collect blood from the vein. A tourniquet is also used
in the procedure in order to make the veins more prominent and help to
eliminate blind probing.
The procedure involves making the patient comfortable, preferably having
them sit in a chair with the patient's arm accessible to the operator. The
tourniquet is then applied and the skin surrounding the target site is
cleaned with a seventy-percent alcohol solution and permitted to dry. Once
the appropriate vein has been located, the needle is pushed into the vein
with a single direct puncture of both the skin and the vein. The
tourniquet is then loosened and the desired amount of blood is obtained.
The operator must be aware of the patient at all times as the trauma of
venipuncture can cause the patient to faint. After the procedure is
completed the operator must also insure that the patient's condition is
satisfactory before he is dismissed.
Complications of venipunctures do occur and include a measurable increase
in the concentration of blood cells when the tourniquet is applied for
periods greater than sixty seconds. Failure of the blood to enter the
syringe may also occur. This may result from excessive pull on the plunger
of the syringe which can cause the vein to collapse. The piercing of the
outer coat of the vein without entering the lumen can also account for
failure. These complications are occasionally followed by hematoma
formation. When this occurs, the needle must be withdrawn and the
procedure must be carried out on the other arm. Late complications include
possible thrombosis of the vein due to trauma, especially following many
venipunctures at the same site. Finally, where non-disposable or
contaminated needles are used, the transmission of contagious diseases
such as serum hepatitis, etc. may be effected.
Blood removed from the patient's body is usually transported to a clinical
laboratory where it is tested and the results are sent back to the
requesting physician. In most doctors' offices in the Untied States today,
such tests are performed at central laboratories and the results are
usually available the following day. In addition to the delay, there is
also a substantial cost for such testing because of the logistics involved
in specimen collection, transport, laboratory accessioning, skilled and
licensed personnel required for the removal procedure and testing of the
specimens, reagents utilized and expensive instrumentation employed.
Further disadvantages of this in vitro method include the numerous sources
of error introduced into the analysis of the specimen as a result of in
vitro changes in the blood sample. For example, in blood kept at room
temperature, swelling of the red blood cells between six and twenty-four
hours raises the hematocrit and mean corpuscular volume (MCV) and lowers
the mean corpuscular hemoglobin concentration and the red blood cell
sedimentation rate.
Current in vitro testing requires strict adherence to procedure. Before
taking a sample from a tube of venous blood for hematologic determination,
it is important that the blood be mixed thoroughly. If the tube has been
standing, this requires at least sixty inversions of the tube or two
minutes on a mechanical rotator; less than this leads to unacceptable
deterioration in precision.
The most common tests performed on blood samples taken from patients are
the hematocrit (Hct), or the hemoglobin (Hb), which are often used
interchangably, depending upon the individual preference of the treating
physician. They are used to determine anemia, to monitor conditions in
which the blood loss occurs, chronic diseases, drug reactions, allergies,
and the course of therapy.
The Hct of a sample of blood is defined as the ratio of the volume of
erythrocytes (red blood cells) to that of the whole blood. It is expressed
as a percentage or, preferably, as a decimal fraction. The units (L/L) are
implied. The venous hematocrit agrees closely with the hematocrit obtained
from a skin puncture; both are greater than the total body hematocrit.
Hemoglobin, the main component of the red blood cell, is a conjugated
protein that serves as a vehicle for the transportation of oxygen and
CO.sub.2, throughout the body. When fully saturated, each gram of
hemoglobin holds 1.34 ml of oxygen. The red cell mass of the adult
contains approximately 600 g of hemoglobin, capable of carrying 800 ml of
oxygen. The main function of hemoglobin is to transport oxygen from the
lungs, where oxygen tension is high, to the tissues, where it is low. As
used in this application the term hemoglobin (Hb) refers to the
concentration of the iron-containing protein pigment found in red blood
cells.
The Hct and Hb are often provided along with the total red blood cell count
(RBC) which is usually expressed in the form of a concentration--cells per
unit volume of blood. Once these three values are known (Hct, Hb and RBC),
three red blood cell indices are calculated. These indices are
particularly useful in the morphologic characterization of anemias. These
values include the mean cell volume (MCV) which is the average volume of
red blood cells and is calculated from the Hct and the RBC. Utilizing the
formula:
MCV=Hct.times.1,000/RBC (in millions per u)
The mean cell hemoglobin (MCH) may also be calculated and is the content of
Hb in the average red blood cell; it is calculated from the Hb
concentration and the RBC utilizing the following formula:
##EQU1##
Another index calculable from the Hb and Hct is the mean cell hemoglobin
concentration (MCHC). This index is the average concentration of Hb in a
given volume of packed red blood cells. It is calculated using the
following formula:
##EQU2##
Other characteristics of red blood cells which are available utilizing
today's testing methods include values for the variability of the MCV
about a mean value and estimates of abnormality in red blood cell
morphology.
The above described indices are discussed in much greater detail in John
Bernard Henry, M.D., Clinical Diagnosis And Management By Laboratory
Methods, Part IV (17th edition 1984).
Modern clinical laboratory instrumentation has been built to make these
primary analyses simultaneously in vitro on blood samples removed from the
patient and the calculated indices are readily produced by these
instruments. The calculated indices are often the preferred data on which
physicians base their conclusions about a patient's condition.
A large number of testing methods, instrumentation, and techniques have
been used in measuring and approximating values for Hct, Hb and RBC. The
most common method used to determine the Hct (the ratio of packed red
blood cells to volume of whole blood) involves centrifugation wherein a
given blood sample is placed into a centrifuge for five minutes at
approximately 10,000 to 12,000 g. The volume is then calculated by
measuring the level of the red blood cells as a ratio of the total volume.
Sources of error in this method include insuring that the sample is subject
to adequate centrifugal force for a sufficient duration so that the red
cells may be packed and give an accurate reading. In addition, the final
value must be corrected for trapped plasma present within the packed red
blood cells. Technical errors in this method include failing to mix the
blood adequately before sampling, improper reading of the level of cells
to plasma, and irregularity of the inside diameter of the specimen tubes.
Methods used in the art to determine the Hb in a sample of blood include
the cyanmethemoglobin method, the oxyhemoglobin method and the method of
measuring iron content of the sample. Of the above three methods, the
first (the cyanmethoglobin method) is recommended by the International
Committee for Standardization in Hematology. That method involves diluting
a sample of blood in a solution of potassium ferricyanide and potassium
cyanide. The potassium ferricyanide oxidizes hemoglobins and potassium
cyanide provides cyanide ions to form hemiglobincyanide which has a broad
absorption maximum at a wavelength of approximately 540 nm. The absorbence
of the overall solution can then be measured in a photometer or
spectrophotometer at 540 nm and compared with that of a standard
hemiglobincyanide solution.
The oxyhemoglobin method is not widely used, however it does yield
reproducible results. The main disadvantage however is the lack of a
stable standard with which to compare the results. This method involves
the creation of a 1:251 dilution of blood in 0.007 N NH.sub.4 OH utilizing
distilled water. This solution is then shaken to insure proper mixing and
oxygenation of the hemoglobin. The solution is read in a photometer with a
green filter with a 0.007 N ammonium hydroxide solution used as a
standard.
The last method listed above involves a procedure whereby Hb may be
measured by determining the iron content of the whole blood. The
non-hemoglobin iron in blood is negligible compared to hemoglobin iron,
however, the iron must first be separated from the hemoglobin, usually by
acid or by ashing. It is then either titrated with TiCl.sub.3 or complexed
with a reagent to develop color that can be measured photometrically since
the iron content of hemoglobin is given as 0.347 percent, the
concentration of hemoglobin in blood is calculated by dividing the iron
concentration by 3.47.
Numerous sources of error are present in the above methods including those
of the sample, the method, the equipment, and/or the operator. Errors
inherent in the sample include improper venipuncture technique which may
introduce hemo concentration, which will make hemoglobin concentration and
cell counts too high. The photometer used for determining Hb must be
calibrated in the laboratory before its initial use and must be re-checked
frequently. The wavelength settings, filters and meter readings require
constant monitoring.
The RBC, or red blood cell count may be determined by a number of different
cell counting procedures. Any of these procedures includes three steps:
dilution of the blood, sampling the diluted suspension into a measured
volume, and counting the cells in that volume. Optical and electronic
equipment have been developed to perform red blood cell counting, inter
alia, in flowing systems in vitro. This equipment can also separately
measure Hb by a chemical method in a separate analytic channel. The most
widely used of such instruments is manufactured by Coulter Diagnostics of
Hialeah, Florida, wherein red blood cells are passed through a glass tube
with an aperture such that single cells can be detected based on a
decrease in the voltage between electrodes positioned in a constricted
portion of the tube. This measurement of voltage decrease yields an
indirect value for RBC. The MCH is then computed utilizing the
relationship:
MCH=Hb/RBC
then the Hct is calculated from the relationship:
Hct=MCV.times.RBC
after MCV is indirectly derived from the mean height of the voltage pulses
formed during the red blood cell count.
Another instrument, made by Technicon Instrument Co., of Tarrytown, N.Y.,
and Fisher Scientific Co., of Pittsburgh, Pa., generates a value for RBC
by using a system wherein red blood cells flowing in glass tubes are
counted by deflection (scattering) of a beam of light that is directed to
an opposing photo multiplier. Hb is measured by a chemical method in a
separate channel of the instrument. The above-described instruments and
methods are all characterized by their in-vitro analysis of specimen of
blood withdrawn from the patient's body without the use of image analysis
and yielding indirect (calculated or estimated) values for red blood cell
indices.
Image analysis has been utilized to examine stained smears of blood fixed
to glass slides with computer-controlled microscopes. Examples of such
instruments include the "Hematrak" instruments manufactured by the
Geometric Data Corp., Wayne, Pa. These instruments utilize pattern
recognition software which identifies each of the major classes of white
blood cells according to size, shape, staining and other morphologic
characteristics. They also identify red blood cells for their morphology.
The imaging system includes a microscope with an automated scanning stage
and an automatic focusing objective. Filters split the light transmitted
through the stained cell into red, blue, and green portions of the
spectrum. These allow the staining properties of the portions of the cells
to be characterized. These instruments, however, do not produce any
quantitative measures of red blood cells. These, and similar instruments,
are also characterized by their in-vitro analysis of stained blood, fixed
to slides for analysis. None perform quantitative analysis of red blood
cell parameters.
Instruments which utilize flowing systems in conjunction with image
analysis are exemplified by the International Remote Imaging Systems
(IRIS) instruments which are designed to analyze in-vitro stream of the
specimen flowing through glass tubing. This system utilizes a flow
analyzer and system described in U.S. Pat. No. 4,338,024, issued to Bolz,
et al., and assigned to IRIS. In that system, urine specimens are
aspirated through a flow cell which is designed to orient cells and
particles in one plane. Moving particles and cells are then photographed
by a high-speed camera and counted by a microprocessor programmed to
identify and classify the different particles and cells, including red
blood cells. No quantitative analysis of red blood cell parameters is
performed and the spectral analysis utilized is by transmission, rather
than reflectance spectrophotometry.
A video computer system for measuring the lineal density of red blood cells
and capillaries in vivo is described in an article by C. G. Ellis, et al.
in Microvascular Research 27, Pages 1-13 (1984). This system utilizes a
computerized frame-by-frame analysis of video images in order to perform
continuous measurements of lineal density based on the spatial-average of
blood opacity over a selected length of capillary. This method does not
attempt to measure or even detect individual RBCs, but rather is directed
to the measurement of light intensities along the centerline of the image
of a capillary such that the number of RBCs in a given length of capillary
is inversely related to he average light intensity over that length. A
light intensity profile is determined utilizing a video analyzer which,
for each frame, measured the light intensity values along a given length
of capillary, first in the absence of RBCs to determine the "background
light intensities" and then as the flow of RBCs is reestablished. A plot
of mean opacity versus RBC for a particular capillary segment is then
plotted and thus a value for RBC is estimated given a particular measured
mean opacity. This system does not measure Hb, Hct or any of the other
indices directly.
OBJECTS AND STATEMENT OF THE INVENTION
It is therefore an object of the present invention to provide an apparatus
for in vivo analysis of red and white blood cell indices which eliminates
the requirement that a specimen of blood be removed from the patient.
It is another object of the present invention to provide an apparatus for
analysis of red and white blood cell indices which eliminates the pain and
discomfort inflicted on the patient.
It is a further object of the present invention to eliminate the errors
inherent in phlebotomy procedures.
It is another object of the present invention to provide an apparatus which
eliminates the errors present in current in vitro analysis of red and
white blood cell specimens by eliminating the need for chemical
interactions, reagents, controlled materials, standards or calibrators.
It is another object of the present invention to provide an apparatus which
is convenient to use and which provides immediate, accurate data on a
patient without the need for a separate testing laboratory.
It is a further object of the present invention to provide a method of in
vivo measurement of red and white blood cell indices quantitatively
through the use of image analysis and reflectance spectrometry. These
objects and others are accomplished by the present invention and
particular embodiments of this invention are described herein below.
In one advantageous embodiment of an apparatus employing the present
invention, a variant of a conventional ophthalmoscope is used to visualize
and capture images of red and white blood cells in capillaries, small
arteries or veins of the mucus membranes which line the inner surface of
the eyelids and the exposed surface of the eye outside the iris (known as
the conjunctiva), retina or other accessible sites on the mucus membranes.
In these areas, capillaries are present whose visible appearance under
magnification shows a continuous stream of blood. The opthalmoscope is
used to identify and capture images of this blood flow in capillaries
which are sized to allow the flow of only one or a few blood cells at a
time to pass a given point. These images are captured from the lumen of
the capillary, enlarged and fed into an appropriate computerized image
analysis system. The images of numerous cells, potentially from many
different capillaries, are capable of analysis by the system.
The computerized image analysis system directly measures characteristics of
red blood cells including the boundaries of the individual red blood cell
and, utilizing algorithms which relate area to volume, directly computes
the volume of that particular cell. This measurement and calculation is
carried out on a large number of red blood cell images captured by the
ophthalmoscope which, when averaged, yield a direct measured value for
mean cell volume (MCV) without the need for establishing or estimating the
hematocrit (Hct) or red blood cell number (RBC).
The mean cell hemoglobin concentration (MCHC) is determined simultaneously
with the direct measurement of MCV by measuring the intensity of red color
(from the iron-containing protein pigment) which is attributable to that
pigment. That intensity relates to a given concentration of hemoglobin
within the cell. This measurement of intensity can be done either by
incorporating a spectroscopic photodetector which selects and measures the
wavelength peak maxima characteristics of hemoglobin or by incorporating a
filter in an optical chopper that absorbs the wavelengths characteristic
of hemoglobin to yield a value by subtraction spectral analysis. The
volume of the captured red cell undergoing image analysis is known from
given algorithms relating area to volume and the intensity is measured as
indicated above. Together they yield a hemoglobin concentration for that
particular red blood cell. As these measurements are carried out on a
large number of red blood cells the mean cell hemoglobin concentration
(MCHC) can be readily determined. From these two direct measurements, i.e.
MCHC and MCV, the mean cell hemoglobin (MCH) can be readily calculated
using the algorithm
MCH=MCV.times.MCHC
From the above indices, the Hb and RBC can be easily calculated.
While a relative value for the Hct can be measured, that measurement will
not represent the central hematocrit as yielded by in vitro
instrumentation in use in the art today. This difference stems from the
fact that the present apparatus measures the Hct within the capillary
itself as a proportion of the space within the still image occupied by red
blood cells as opposed to the space which is not occupied by red blood
cells. The central Hct, in contrast, represents the ratio of the volume of
red blood cells to that of the whole blood as taken from a specimen
removed from a vein of the patient and subject to centrifugation. These
values can be easily correlated, however, through empirical analysis by
comparing the direct measurement of the Hct from a specimen taken from the
patient with the measured Hct derived from the indices measured above.
This comparison will yield a conversion factor which can equate the two
values. By this method the actual Hct can also be calculated from the
measurements outlined above.
White blood cells (WBC) are approximately the same size or larger than RBC
and are of sufficient size to be differentiated from the noncellular
plasma. The white blood cell count can be readily determined using the
same apparatus. The WBC in normal healthy individuals has a broad range of
values with an average normal being 7,500 WBC per cubic millimeter. Given
the fact that the RBC count also varies widely among normal healthy people
with an average normal count being approximately 5,000,000 RBCs per cubic
millimeter, an average normal healthy individual would have one WBC for
every 667 RBCs in the circulation.
The apparatus of the present invention captures images of both white and
red cells that pass through capillaries or venules. Those images are
subjected to analyses their respective sizes, volumes and hemoglobin
content. This last indice is done by subtraction reflectance spectroscopy.
This also serves to distinguish between the WBCs and the RBCs since an
unstained RBC is approximately 1/3 hemoglobin and exhibits a distinctive
red color. WBCs in contrast do not contain hemoglobin. Therefore they have
no red color and show no difference in the reflected spectral properties.
By keeping track of the ratio of WBCs with respect to the RBCs and
accumulating a statistically reliable number, the actual ratio of WBCs to
RBCs is determined. Furthermore, since the apparatus computes a RBC count,
the WBC count is calculated using that ratio (RBC/WBC).
The results of computations for the directly measured parameters MCV, Hct
and MCHC, and the calculated parameters MCH, Hb, WBC and RBC can then be
printed on an appropriate report form which includes patient
identification and demographics previously entered into the apparatus
through separate input means, for example a keyboard. A visual display of
histograms of these parameters can also be reviewed and printed as
required. These results, including the numerical values and histogram
displays, are retained in the computer memory and may be transmitted "on
line" to a larger laboratory computer or maintained in memory as desired.
The foregoing and other objects, features and advantages of the present
invention will become apparent from the description of preferred
embodiments in connection with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic block diagram of an apparatus in accordance with one
embodiment of the present invention;
FIG. 2 is a schematic block diagram of video analysis components of one
embodiment of the present invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Referring now to the drawings and, in particular, to FIGS. 1-2, there is
depicted one embodiment of an apparatus in accordance with the present
invention. FIG. 1 shows a schematic block diagram of one preferred
embodiment including a focusable light source and visual image receiver 20
for capturing images of the red blood cells in conjunctival capillaries of
the eye 22. This focusable light source and visual image receiver
(FLS-VIR) is preferably a conventional ophthalmoscope or slit lamp similar
to those manufactured by Welch-Allyn of Skaneateles Falls, N.Y., or Zeiss
Co. Either of these conventional FLS-VIR devices can be equipped with
additional magnification capability to make them adaptable for use with
the present invention. Since red blood cells average 7.4 microns in
diameter and have an average volume of 87 .+-.5 cubic microns, some
magnification is necessary for accurate analysis. This magnification falls
within the range of from 150X to about 650X depending upon the analysis
equipment used.
Conventional ophthalmoscopes provide approximately ten-fold magnification
of the focused image. This is sufficient for selection of the field for
testing and for focusing of the image however increased magnification is
necessary for subsequent image analysis and reflectance spectroscopy.
Slit lamps provide approximately a forty-fold magnification to the viewer.
This magnification is sufficient for visualizing individual red and white
blood cells flowing within the capillaries however subsequent image
analysis and spectrophotometry would require further magnification. Both
ophthalmoscopes and slit lamps have substantially more components and
capabilities than are required for the present invention. For this reason,
the instrument necessary for image analysis and reflectance spectroscopy
can be added to pre-existing components or integrated with a simplified
version of an ophthalmoscope which includes only the light source,
focusing capability and the image receiver.
Sufficient light must be available to permit ordinary visualization of the
small blood vessels of the conjunctiva, including the capillaries,
arterioles and venules. The conjunctiva will be referred to herein in
further descriptions however it is important to note that other sites can
also provide satisfactory images of red cells in vivo according to the
present invention. The conjunctival capillaries are preferred because they
are readily accessible and provide an excellent contrast between the red
blood cells in the capillaries and the white background of the
conjunctiva.
The FLS-VIR 20 is adapted to allow for subsequent image splitting, and
transmission to an optical enlarger 24 for enlargement and processing.
Transmission of these images is preferably through fiber optic
transmission lines, however, any appropriate transmission medium is
acceptable.
Subsequent processing and transmission of the images captured and magnified
by the FLS-VIR can include for example slit lamp cameras and/or video
camera attachments similar to those used for operating room
ophthalmoscopes, micro-surgical devices and television ophthalmoscopy.
In one particularly advantageous embodiment of the present invention, the
elements of the FLS-VIR can be mounted in a rigid assembly wherein the
patient's head is maintained in a fixed cradle similar to current slit
lamp fixtures. Utilizing this arrangement, the image can be viewed
directly by the physician and also split to a side viewing port for
subsequent computerized image analysis.
The image splitter discussed above diverts a portion of the image
visualized by the operator and subjects that image to further analysis.
The image splitting can be accomplished by several well-known techniques
utilizing various optical devices. These devices include partially
reflective mirrors and interspersed fiber optical strands.
Instrumentation for this subsequent analysis includes an image magnifier
24, a light chopper and filter 26 and a video camera scanner or projector
28. The images produced directly on the pickup tube of an ordinary
vidicon-type television camera would be approximately 9.4.times.12.5 mm
and require approximately 1.0 foot candles. This image can be displayed on
a printer/screen 31 either prior or subsequent to analysis by computer 30.
Alternatively, the split image of the blood cells can be passed through the
beam splitter enlarging optics 26 and filtered either before or after
being transmitted by fiber optics transmission lines to a television
pick-up tube. Video signals of sufficient quality for image analysis are
produced by silicon diode vidicons that have a 5-10X sensitivity of
standard vidicons, with light levels of 0.1 to 0.2 foot candles at the
base plate of the tube. A phosphor screen-fiber optic device may be
employed, such as is used to obtain images from transmission
electron-microscopy (TEM), with a sensitive TV screen also equipped with
fiber optics. Weak signals may be enhanced through electronic
amplification and/or computer processing of the signals that reach the
vidicon so that accurate image analysis can be performed.
In each case, the captured image is presented to a video camera which
subsequently transforms the image into electronic information to be
subsequently analyzed by computer 30.
Hb analysis is performed by interposing an optical chopper shown generally
at 26 in line with the image transmission. The chopper consists of a
conventional motor driven wheel 29 having a filter slot with an
appropriate filter 27. That filter 27 is designed to absorb the specific
energy in the red portion of the spectrum corresponding to the maxima of
absorption of the hemoglobin of red blood cells. This maxima is generally
in the range of about 650 nm. The filter should have a broad absorption
band width on either side of the maxima in order to remove the light
characteristic of hemoglobin from the subsequent image and image
processing. This light characteristic results during the very short
instant when the rotating wheel interposes the filter between the primary
image of the red cell that has been illuminated with white light and a
subsequent image or transformed data representing the earlier image. The
instantaneous image of each red blood cell analyzed by the instrument will
be presented both with and without the intensity of red light that is
entirely attributable to the iron-containing protein pigment of
hemoglobin. The hemoglobin concentration of each red blood cell will then
be determined by subtraction of the signal generated by the pigment from
the background in the same cell and calculated according to Beers' Law as
modified by the characteristics that change in the case of reflectance vs
transmission spectroscopy and may be confirmed empirically. Utilizing this
technique, a computer receiving this data performs subtraction reflectance
spectrometry to determine the hemoglobin concentration of each individual
red blood cell analyzed.
The Optical Image Receiver Enhancer-Enlarger (OIREE) functions to receive,
encode, and, in one version, to enlarge the image and make it suitable for
visual analysis and display by other components of the instrument. The
receiving component of the OIREE is, in one embodiment, a high speed video
camera with a very rapid shutter sufficient to allow the capture of
instantaneous images of red blood cells. The shutter speed should be slow
enough to allow the optical chopper described above to provide an image
with and without red filtering, and yet fast enough to preserve discrete
cell margins without blurring. Ordinary vidicons, silicon diode vidicons
with high sensitivity or such vidicons as the "15-3 precision scanner"
used by Bausch & Lomb in the Omnicon 5000, can be employed. These vidicons
may be combined advantageously with software to accommodate and correct
for non-uniform distributions of light falling on the vidicon so as to
make the signals readily analyzable by the image analysis components of
the apparatus.
Optical enhancement can be achieved by several established techniques. The
primary function of this component is enlargement. The unenhanced image
can be enlarged through conventional optics by means of a series of lenses
as in a telescope or microscope. Alternatively, the image that has been
received directly into a video camera can be enlarged by the action of the
video camera. This enlarged image is then operated upon by image
enhancement programs to detect, clarify and sharpen boundaries between red
blood cells and their surroundings, and to enhance color characteristics.
This component may, alternatively, be positioned in line before, after, or
among several others of the instruments depending upon the preferred
configuration.
The enlarged image from the OIREE is preferably projected on to a video
screen. The raster of this screen can provide subsequent enlargement and
enhancement functions where the OIREE has not yet operated upon the
enlarged image of the red blood cell and is essentially functioning only
as a video camera projection.
Alternatively, the video camera can record the continuously generated
captured images on to a recording medium such as video tape, which can
then be played into the video projector at varying speeds and used to
obtain images for subsequent analysis by Computer 30.
As detailed in FIG. 2, a series of light images 34 of flowing red and white
blood cells are produced on the photo sensitive face of a special vidicon
camera tube 36 by an optical system which includes an image splitter, a
light chopper-filter, enlarging optics and a signal amplifier. The
brightness forming the image at each point on the screen is converted into
electrical voltages by repeatedly scanning the image with an exploring
spot formed by the electron beam of the camera tube. This spot generates
an electrical video signal which indicates the brightness at each of its
instantaneous positions. The video signal 38 is amplified in a
conventional video amplifier 40 and may, optionally, be combined with a
video display (not shown). That signal determines the brightness of a
reproducing spot formed by the display tubes' electron beam. The
reproducing spot moves over the latter screen synchronously and is
coordinated with the exploring spot. The reproducing spot reconstructs the
brightness distribution of the image that was received on the vidicon
camera face with the possibility of enlargement and/or amplification of
signal intensity. The amplified video signal 41 is then subject to
correction for "shading" and sensitivity adjustment. The corrected
amplified video signal 44 is then encoded 46 for subsequent translation
into critical information.
Conversion of the signal to digital values is accomplished by a high
resolution digitizer 48 and occurs in real time. These values are
transmitted to a computer through a data base and memory register. The
video signal that actually produces the video display is generated from
this memory. The system may also be provided with a separate input means
54 such as a keyboard so that patient and specimen information can be
included in the display and analysis.
Alternatively, charge coupled devices can be substituted for the
conventional optics described above. Since charge coupled devices have
very short focal lengths, as little as 3 mm they permit the ac | | |
