U.S. patent number 4,500,509 [Application Number 06/356,578] was granted by the patent office on 1985-02-19 for metachromatic dye sorption and fluorescent light emmisive means for differential determination of developmental stages of neutrophilic granulocytic cells and other leukocytes.
Invention is credited to Lawrence Kass.
United States Patent |
4,500,509 |
Kass |
* February 19, 1985 |
Metachromatic dye sorption and fluorescent light emmisive means for
differential determination of developmental stages of neutrophilic
granulocytic cells and other leukocytes
Abstract
Differentiation, identification and enumeration of dye
responsive human blood cells essential to diagnosis and prognosis
of certain diseases is established by preparation of the reaction
product of a human blood specimen of the patient in an aqueous
fixative-free environment at blood temperatures with selected
basic, cationic quaternary dyes, particularly basic orange #21. The
so-prepared specimen becomes instantly responsive to emissive wave
energy source exposure to stimulate reproducible fluorescent light
emission from all of said dye responsive cells. The quantum of said
fluorescent light energy emitted provides individual sets of data
response relative to hue, value, chroma and intensity to permit
differentiation, identification and enumeration of all of said dye
responsive components in said blood specimen. By use of a bi-modal
wave energy system involving additional use of white light wave
energy absorbance, independent sets of data can provide both
confirmatory and ancillary information.
Inventors: |
Kass; Lawrence (Toledo,
OH) |
[*] Notice: |
The portion of the term of this patent
subsequent to August 23, 2000 has been disclaimed. |
Family
ID: |
23402036 |
Appl.
No.: |
06/356,578 |
Filed: |
March 9, 1982 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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242662 |
Mar 11, 1981 |
4400370 |
|
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129680 |
Mar 12, 1980 |
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Current U.S.
Class: |
435/34;
435/39 |
Current CPC
Class: |
G01N
21/6428 (20130101); G01N 33/5094 (20130101); G01N
21/6458 (20130101); G01N 2001/305 (20130101) |
Current International
Class: |
G01N
21/64 (20060101); G01N 33/50 (20060101); G01N
1/30 (20060101); G01N 031/00 (); G01N 001/00 () |
Field of
Search: |
;424/3 ;8/644,657
;250/461.2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Gurr, Synth. Dyes in Biol. Med. & Chem., Acd. Press, NY, 1971,
pp. 54, 55, 90, 117, 147-149, 395, 805. .
Conn's Biol. Stains, Williams & Wilkins, Baltimore, 9th ed.,
1977, pp. 43, 368, 404-407, 415-428. .
Gray, The Ency. of Microscop. & Microtech., Van
Nostrand-Reinhold Co., N.Y., 1973, pp. 398-399, 469, 551, 563.
.
Humason, Animal Tissue Tech., W. H. Freeman & Co., San
Francisco, 1972, pp. 128-132. .
Hallberg, Acta Med. Scand., Suppl., vol. 180, 1946, pp. 7-15. .
Pilot, Use of Base Fluids for Counting Eosinophils, U. of Ill., May
23, 1950, pp. 870-871. .
Kodak Adv., Sci. Amer., May 1976, p. 49. .
Ruddell, J. Invertebr. Pathol., vol. 31, 1978, pp. 313-323. .
Williams, J. of Lab. & Clin. Med., vol. VIII, Oct. 1922-Sep.
1923, pp. 250-253. .
Ruddell, Chem. Abs., vol. 89, 1978, Ab. No. 89:54252q; Chem. Sub.
Index, p. 2791cs. .
Simpson, Stain Tech., vol. 45, No. 5, 1970, pp. 221-223. .
MacConaill, Ireland J. Med. Sci., Jun. 1964, pp. 243-250. .
Sabin, Bull. Johns Hopkins U., vol. XXXIV, No. 391, Sep. 1923, pp.
277-288. .
Spiridonovitch, The Anatomical Record, vol. 26, Jan-May 1924, pp.
367-373. .
Moore, PSEBM, vol. 82, 1953, pp. 601-603..
|
Primary Examiner: Warden; Robert J.
Assistant Examiner: McCowin; K. S.
Attorney, Agent or Firm: Fay & Sharpe
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. application Ser.
No. 242,662 filed Mar. 11, 1981, Pat. No. 4,400,370, entitled
"Metachromatic Dye Sorption Means for Differential Determination of
Developmental Stages of Neutrophilic Granulocytic Cells and Other
Leukocytes" which in turn is a continuation-in-part of the patent
application U.S. Ser. No. 129,680 filed Mar. 12, 1980, entitled
"Individual Leukocyte Determination by Means of Differential
Metachromatic Dye Sorption".
Claims
Having described the best mode presently known to practice the
disclosed invention, what is claimed is:
1. In analysis of human blood cells present in a donor specimen in
a fixative-free aqueous environment under influence of a
fluorescent light emissive energy source, the improvement in
identification, enumeration and study of monocytes present in said
specimen which comprises staining said specimen with an aqueous
solution of a basic, cationic, quaternary organic dyestuff selected
from the group consisting of basic orange #21, basic red #13, basic
red #36, basic red #49, basic violet #7, basic violet #15, basic
violet #16 basic violet #36, basic violet #39 basic violet #40 and
carboeyanine; K-5 subjecting said dye stained specimen to emissive
wave energy to thereby stimulate fluorescence of said dye exposed
cells, emitted fluorescence light providing means for
differentiation of monocytes present in the specimen from all other
blood cells.
2. In analysis of human blood leukocytes and lymphocytes and the
developmental stages of neutrophilic granulocytic cells present in
a donor specimen in a fixative-free aqueous environment under
influence of a fluorescent light emissive energy source, the
improvement in differentiation of each of the individual cell
species present into recognizable enumeratable cell species which
comprises staining said specimen with an aqueous solution of basic
orange #21, subjectingg said dye-stained specimen to emissive wave
energy stimulating fluorescence of said dye exposed cells, thereby
effecting means for differentiation of each of the cells present in
the specimen into cell species identifiable each one from the
others.
3. The method of claim 2, where the fluorescent light emissive
energy stimulation originates, at least in part, from the coherent
light of a laser beam.
4. The method of claim 2, wherein the essential blood cell
differentiation method is effected by means of an automatic
leukocyte counting means capable of bi-modal wave energy impact on
the dyed microscopic field comprising the cells being subjected to
analysis and means for observing the said same field under each
modal impact means.
5. A method of supervital analysis of human normal or pathologic
blood cells present in a donor specimen in an aqueous fixative free
environment which comprises exposing said specimen (a) to the
dyestuff basic orange #21, (b) stimulating fluorescence of each of
the stained species present in said specimen by impact of wave
energy and differentiating each of the said blood cells one from
the other by the presence or absence of characteristic fluorescent
color and pattern of the nucleus and cytoplasm and by the number,
size, arrangement, pattern and fluorescent color or quanta of light
energy intensities emitted by the granules present, their size,
number and locus of the granules in the cytoplasm, if present.
6. A comparative method of supervital analysis of human normal or
pathologic blood cells present in a donor specimen including one or
more of the following species; myeloblasts, promyelocytes,
myelocytes, metamyelocytes platelets, bands, neutrophils,
eosinophils, lymphocytes, basophils, monocytes, B-lymphocytes and
T-lymphocytes, each one thereof present having been made optically
identifiable one from the other by separate selective exposure both
to white light absorbance and fluorescent light emission thereby
verifying identification of species from the foregoing listed cells
present by optical patterns characteristic of said species and by
the different quanta of energy reflected and/or emitted from the
nucleus, cytoplasm and granules of said blood cells as observed
under the foregoing bi-modal light effects.
7. A method of microscopic analysis under bi-modal light sources as
described in claim 6, where one healthy normal donor specimen is
examined comparatively with a second donor specimen having a
suspected pathological origin to provide diagnostic
information.
8. A method of fixative-free, supravital human blood cell analysis
whereby optical differentiation, comparison and enumeration are
made possible of T-lymphocytes and B-lymphocytes present in a donor
specimen which comprises metachromatic staining of said specimen in
a fixative-free aqueous environment with basic orange #21 dye,
subjecting said dyed specimen to a fluorescent light stimulating
energy source and thereby differentiating T-cells present from
B-cells by a cluster of bright yellow fluorescent granules in the
cytoplasm of the T-cells, which may show some fluorescent dull
green coloration of the nucleus, and B-cells present whose
cytoplasm is essentially free from any fluorescent granules and/or
fluorescent color but the nucleus of which does exhibit dull green
fluorescence thereby providing required identification.
9. A method of identifying, differentiating and enumerating dye
responsive cells in a human blood specimen which comprises staining
the specimen in a fixative-free aqueous environment which basic
orange #21 dye, subjecting the dyed specimen to fluorescent light
emissive wave energy to stimulate fluorescent light emissions from
said dye responsive cells, each of said cells emitting quanta of
fluorescent light energy of hue, value, chroma and intensity which
provides identifying means for differentiation and enumeration of
each one of said dye responsive cells.
10. The method of claim 9 wherein the dye responsive cells present
in said human blood specimen include one or more of the following
species: myeloblasts, promyelocytes, myelocytes, metamyelocytes,
platelets, bands, lymphocytes including B-cells and T-cells,
basophils, neutrophils, eosinophils or monocytes.
11. A method of supravital human blood analysis whereby optical
differentiation, enumeration and comparison of each of the
individuals in a granulocytic myeloid series including, when
present; promyelocytes, myelocytes, metamyelocytes, bands or
neutrophils is achieved by metachromatically staining at least one
supravital, fixative free human blood specimen fraction by contact
in an aqueous environment with a basic orange #21 dye solution,
thereby differentiating the promyelocytes by fluorescent bright
yellow primary granules on one side area of the cytoplasm;
myelocytes by a relatively even distribution of bright yellow
primary granules throughout the cytoplasm; metamyelocytes by a
smaller mass of fluorescent bright yellow primary granules in the
smaller crescent opposite an unstained clear protrusion of
cytoplasm inwardly into the nucleus; the bands by a relatively
small cluster of primary bright yellow fluorescent granules in a
dominant field of cytoplasm and generally bifurcating the mass of
the nucleus; and the neutrophils by a less distinct cytoplasm
exhibiting a deep green fluorescence in which the rare granules
present fluoresce bright yellow, each member of the foregoing
series having an apparently decreasing proportionate cell volume of
the nucleus remaining substantially unstained, the first three
members of the series having a generally larger over all cell
volume cell size than the last two members of the series.
12. In analysis of human blood cells present in a donor specimen in
a fixative-free aqueous environment under influence of a
fluorescent light emissive energy source, the improvement in
identification, enumeration and study of each one or more of the
individual cell species present in said specimen; said blood cell
species present from the group consisting of myeloblasts,
promyelocytes, myelocytes, metamyelocytes, platelets, bands,
neutrophils, eosinophils, lymphocytes, basophils, monocytes,
B-lymphocytes and T-lymphocytes; which comprises staining said
specimen with an aqueous solution of basic orange #21, subjecting
said dye-stained specimen to emissive wave energy stimulating
fluorescence of said dye exposed cells, thereby effecting means for
differentiation of each one of the aforementioned cell species
present in the blood specimen from all other cells.
13. The method of supravital human blood cell analysis of claim 12
wherein a band cell containing specimen is subjected to a
fluorescent light stimulating energy source, whereby said band
cells are differentiated from all other blood-related cells by a
relatively small cluster of bright yellow fluorescent granules in
portions of the cytoplasm which in pattern substantially bifurcates
the central portion of the substantially color free nucleus.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
In a parent application and a first continuation-in-part
application thereof, discovery of a broad class of dyestuffs were
disclosed which, under visible light range absorbance in a
supravital blood analysis, provided notable advantage in the field
of cytology.
This application is a continuation-in-part of U.S. Ser. 242,662
filed Mar. 11, 1981, and is related to the same subject matter as
the first above Parent case which provides an improved method for
optical differentiation of the five individual white blood cell
species by use of a class of basic quaternary metachromatic dyes
which were found to supravitally stain each of the species within a
temperature range.
The subject matter of the first continuation-in-part application
relates basically to a similar area of determination but is founded
on the discovery that certain specific sub-classes of the dyestuffs
broadly useful for differentiation, identification and enumeration
of human blood cell leukocytes were also singularly useful as well
in differential determination of developmental stages of
neutrophilic granulocytic cells. The foregoing inventions were
reduced to practice using light waves of the same wave length as
present in ordinary daylight herein sometimes call white light
spectrum.
Since reduction to practice of the first continuation-in-part
application, U.S. Ser. 242,662 of Mar. 11, 1981, microscopic
examinations of a supravitally dyed field has been made available
which permits the observer to practice the general method of
supravital human blood analyses as originally disclosed using
"white" light (an electromagnetic energy form of radiation having a
wave length of from about 4000 to 7700 angstroms), but now enlarged
to include fluorescent light emissions. In the Parent and the
Continuations white light spectra passing through the supravitally
dyed specimen field is absorbed. Fluorescent light is related to
light emissions and may be caused by the flow of some form of
energy into the emitting body. Emission spectra of fluorescence
results from the flow of energy being absorbed and light being
emitted in characteristic frequency.
Fluorescent light emission for the purposes herein in the
fluorescent microscope may be ultra-violet, violet and sometimes
blue radiations. Mercury vapor light is commonly used. However,
early experimentation points to the value of a single, coherent
beam of light, as from laser technology, to be of use as well in
some instances. It is known that in flow cytometry apparatus laser
beams have been used to excite single cells exposed to acridine
orange dye in cell sorters. Instruments can look at a single cell,
determine its specific patterns and measure the intensity of the
fluorescent colors. However, acridine orange has serious
limitations being both pH and temperature dependant. Acridine
orange does not exhibit useful metachromasia under an absorbtive
light mode. Additionally, the dye tends to diffuse out and is not
useful in supravital examination of (living) cells, e.g., it is
concentration dependant. In the presently disclosed methods, laser
light stimulation of prepared microscopic cells dyed with basic
orange #21, for example, provides unique means for identification,
differentiation and enumeration of blood cells and other tissues
capable of manufacturing, transferring or storing blood cells.
Laser light does not appear to excite or bring out a new quality of
chromasia or interfere with metachromasia, so important a factor in
the use of the dyestuffs of this invention. It can be focused with
high power density within one cell making them more distinct and
the measurement more precise. The colors emitted appear to be
unchanged by the form of energy causing the sorbed dyes of this
invention to fluoresce.
Using the specific groups of dyes herein disclosed and claimed, but
particularly basic orange #21 which is unique with fluorescent
means, and also as hereinbefore disclosed in the Parent case and
the first Continuation-in-part also unique with white light
absorbance, the art of cytology is immeasurably advanced.
Dyes originally disclosed in the Parent application have been
broadly classed as methines, polymethines and cyanine dyes. Dyes
broadly within the class include carbocyanines, merocyanines,
azacyanines, oxanols, etc. However, so very few of this broad class
have been found to be metachromatic and useful, particularly in the
present field of use where they must also be metachromatic under
fluorescent conditions as well as white light absorbance.
2. Description of the Prior Art
Ehrlich made biological elements more readily and easily recognized
under microscopic examination and for photographic observation by
use of dye stains (aniline dyes) to identify certain white blood
cells. Ehrlich was the first to note that some dyes were
metachromatic, observing that the staining of the cell or
components such as granules of leukocytes causes the cell to take
on a color different than that of the stain in solution or expected
color from the stain. Basophils, for example, were observed to take
on a color different from the stain. Other histological specimens
other than blood cells have also been reported to stain in a
plurality of identifiably different colors.
A review of the state of the art indicates it is almost universal
practice, before staining (which presently uses a plurality of
chemically differing dyestuffs in admixture) to employ a fixative
procedure which may require up to an half hour treatment before the
biological specimen is subjected to dye stain. Fixatives are
generally preservatives and denaturants that often interfere with
the sensitivity of the dye sorption. Illustratively, fixatives
include formaldehyde both as liquid and vapor, absolute alcohols
(methyl), picroformal, etc. Very often living cells do not stain
using vital dyes and fixatives have been essential to staining the
specimens. Cytochemistry includes considerable information on
techniques developed to assure reproducible staining of blood
cells. Many essential additives are normally unstable and
deteriorate rapidly, thus making cellular identification difficult
and in some instances unreliable. Dr. Thomas E. Necheles has
observed in relation to leukocyte analysis that this "system has
undergone little or no change in fifty years."
Dye staining does serve, however, as a means of discernment of
otherwise undiscernable detail of conferring a color reaction on
cells and their stainable components; metabolic, functional or
pathological.
United States hospitals began leukocyte counting in the early
1900's, using the count as indicia as to whether emergency surgery
was necessary, for example. In the U.S. alone, more than half a
million differential counts are performed every day, most of them
by manual methods. It is important that total white cell counts and
differential cell counts be performed and reported without delay.
Time is of essence and providing required analysis more rapidly is
a desideratum.
The value of leukocyte counting having been established, the demand
for rapid blood analysis has developed so that beginning about 1950
with the work of Mellors and Papincolaou (1952) development of
automated differential leukocyte counting instrumentation means had
developed into a plurality of intruments by 1980. The CYDAK unit
was early used to investigate the feasability of blood cell
classification which pointed up the importance of specialized
staining procedures and features were extracted from optical
density histograms of each cell image. The procedure established
that cells could be differentiated into four of the five classes of
leukocytes, namely; neutrophils, eosinophils, lymphocytes and
monocytes. Young (1969) published results on an automated
classification of five cell classes and Bacus in 1971 extended the
differentiation.
However, it is understood that automated differential systems
presently rely upon multiple dye usage and dye degradation systems
or indirect fluorescent measurement using fluorescent dyes.
In the prior art staining of blood it has been observed that it is
practice to use two or more stains in combination (Romanowski,
Giemsa and Wright stains). These methods are difficult in practice
to provide quality control. The methods require standardization in
preparation of each dye stain component as well as in the method of
specimen staining. In development of successful automated leukocyte
counters, reproducibility of staining is even more important to
verifiable analysis.
LARC stainer (used in commercial automated differential leukocyte
counter) is reported (Mogler 1973) to be a mixture of some ten
thiazine dyes, oesin Y and 2.sup.1, 4.sup.1, 5.sup.1
tribromofluorescein (P. N. Marshall). Present art stains most often
are in fixative alcoholic solutions and employ two or more stains
in combination. Accurate analysis of vital blood staining is made
most difficult. With the difficulty presented in the controlled
oxidation of methylene blue essential to Romanowski stains, for
example, the problems of quality control of the added ten
individually different dye stains as are used in combination become
awesome.
It has been recognized in the art that the widespread
standardization and adoption of a limited number of stains would
ensure greater accuracy and reproducibility in cytological studies.
Serious introduction of artifacts have been observed by use of
fixatives and cause difficulty in interpretation and
misinterpretation and leukocyte differentiation and enumeration. pH
adjustments, heavy metal cations have been reported to prevent
cytochemical tests from working in the expected manner. Some dyes,
particularly azo dyes, are noted to demonstrate non-specific
precipitation around cells; other degenerative changes in fixed
blood samples include vacuoles, clover-leafing of nuclei,
distortion cell shapes and smudges and interference with ideal
staining. The importance of performing differential counts on as
near living cells in the shortest possible time in order to obtain
optimally useful and valuable blood cell analyses has been
recognized. Alcoholic dye solutions interfere with supravital
staining. So far as is known, freshly prepared water soluble stains
exhibit a minimum denaturant effect upon supravital blood during
examination. All dyestuffs are more or less toxic to the blood
cells, but some are more so that others. It is material that the
cells under examination remain living as long as possible. Rapidity
of staining obviously shortens the exposure time, thus allowing
greater opportunity to examine leukocyte cells before all vitality
is lost. Automated differential leukocyte counting in less minutes
is sought for.
Studies and review of the prior art of performing microscopic blood
analyses and disease diagnosis has indicated it is not unusual for
pathologists to warm the dye and the blood specimen to body
temperatures (about 37.degree. C.) before contact. Dr. Sabin had a
"warm box" to insure temperature control.
It has also been noted that some dyes used in the prior art are
quite temperature sensitive. The literature reports that cresylecht
violet is not an operative stain above 30.degree. C. It is
considered important for the purposes of this method as disclosed
herein that the dyestuff be useful to stain leukocytes at
temperatures as high as 37.degree. C. and no difficulty has been
observed with the select dyes to temperatures of about 40.degree.
C.
In the Parent application, a relatively small number of
metachromatic dyestuffs are disclosed as useful in identification
of one or more species of leukocyte. Identification and
differentiation was specifically related to polymophonuclear
leukocytes (neutrophils), eosinophils, basophils, lymphocytes
generally, and monocytes. A uniting commonality observed was that
all of the dyes found to be operative for the purposes of the
Parent application metachromatically stained monocytes
differentially from others in the above group.
The unusual qualities of the dye basic orange #21 (CI #48035 and
Spectral Curve 7) were observed in relation to the eosinophils,
basophils, and monocytes, but as the B-cells are few in number they
were initially overlooked. It was initially observed in the Parent
case that optical differentiation between mature and immature
neutrophils appeared potential in that the mature granules were
different in chroma from the immature granules which were more red
and orange in comparison. As this group, including myeloblasts,
promyelocytes, myelocytes, metamyelocytes and bands are not always
present in all blood specimens or present in significant numbers as
is often the case with T-lymphocytes (or T-cells) and B-lymphocytes
(or B-cells) they were not then all specifically identified as
being metachromatically and differentially stained by basic orange
#21.
Subsequent to completion of the work supportive of the Parent
application, continuing research on the use of this unique dye in
similar blood donor studies established that it was reproducibly
possible, using this selected basic cationic dye of the methine,
polymethine and quinoline class to distinguish through
metachromatic response certain lymphocytes. It is also possible
further to identify at least ten recognized granulocytes and
lymphocytic cells established in the art to be of vital interest to
the health sciences Platelets, also identified as thrombocytes, can
also be identified and enumerated.
Further, this differentiation was immediate, it required no complex
biochemistry or arduous pre-treatment of the blood specimens.
Additionally, it was noted the dye exhibited minimum toxicity.
Micro spectrophotometric measurements were made with an aperture
small enough to measure the color in the granules of supravitally
stained leukocyte granulocytic cells. No other part of the cell
entered into the measurements to any extent were found to provide
extinction coefficients of the colors of the different leukocyte
species which were consistently different and were often of an
order of differences in hue, value or chroma of the order of 50
nanometers. These were recognizable peaks, consistent over many
cells. It is understood that differences of the order of 5 nm are
significant in microspectrophotometric measurements if the
differences are consistent and reproducible.
Among the immature granulocytic cells immediately identifiable and
distinguishable one from the other are myeloblasts and cells of the
myeloid series, namely; promyelocytes, myelocytes and
metamyelocytes. These are believed to be and are generally
understood to be precursors of the polymorphonuclear leukocytes or
neutrophils, which are also stained metachromatically so as to be
readily and easily distinguished, identified and enumerated by the
supravital blood analyses made possible by the advances disclosed
herein.
As disclosed in the parent application, it is also practical at the
same time to distinguish neutrophils, eosinophils, basophils,
lymphocytes and monocytes from each other and from the foregoing
precursors should they all be present in a specific blood sample
under microspectrophotometric analyses.
Additionally, it has also been found that this unique dye provides
an optically different pattern of color as well as a different
density of each color of granule in bands, neutrophils and
leukocytes. Thus, this quality of leukocyte cell can also be
uniquely separated by optical differentiation from the other
immature cells identified above. The differentiations in color,
color arrangement and color density are also of such a degree of
magnitude of difference that both human counting manually of all
the above individually named cells can be accomplished, both with
white light spectra (absorption) and fluorescence (emission).
Sources of fluorescent emission energies are, for example; mercury
vapor lights, tungsten-halogen sources, lasers, deuterium sources,
zenon, etc.
Evidence available also indicates automatic differential counting
equipment will develop based upon and to be accommodated by
differences due to the presence or absence of color and the
physical patterns established in the nucleus and by the relative
number, size, arrangement or pattern and hue, value and chroma
(color) and color density due to the number of granules in the
cytoplasm. The duality of colors under bimodal light exposures
provides a double check on observation and a means of discovery of
differentials in cell structure.
Almost unbelievably, but also demonstrated in the basic research
thus far completed, is the further ability to differentiate
B-lymphocytes or B-cells from T-lymphocytes or T-cells. Again, it
is possible to spectrally identify each of these important
lymphocytes, one from the other, qualitatively and quantitatively
using the same dyestuff in the same supravital, fixative free
analysis as well as to distinguish and enumerate immature and
mature cells including bands. T-lymphocytes have been observed and
identified in lymph node tissues and other tissues associated with
white blood cell metabolism by means of the fluorescent mode of
observation.
Earlier discovery of the capacity of basic organe #21 to
differentiate, in addition to those cells disclosed in the Parent
application, myeloblasts and blood cells of the myeloid series as
well as bands and T-lymphocytes and B-lymphocytes extends the
original potential field of usefulness of the dye unexpectedly
beyond the capacity recognized in the Parent application.
Supravital blood specimen fractions of fluids associated with
healthy tissue or tissue suspected of abnormality such as plasma,
lymph, serum, etc., containing one or more of the above cells after
metachromatic staining may be examined microscopically under this
bi-modal energy system herein described, or by use of either light
source alone and thus differentiate each species of cell indicated
above permitting enumeration and comparative study.
The present advance in the art, coupled with the parent disclosure
establishes unparalleled advance in hematology, cytology and
immunology and the ability to plan and conduct researches in an
unlimited area of human health. Need for costly reactants,
invaluable research time and more accurate data assembly have been
thereby measurably advanced.
The art of diagnosis of disease has a new horizon beyond the
present limits with the finding of fluorescent responses using a
limited few of the basic quaternary cationic chemical class that
has produced the unique dyes of this disclosure.
Initial observations made frist in the parent application (U.S.
Ser. 129,680, filed Mar. 12, 1980) using a Zeiss fluorescent
microscope revealed that carbocyanine K-5, a methine-polymethine
class of dye, to be metachromatic both under white light absorbance
and emitted fluorescence. Later examination of a large group of
dyes within the above chromophore classification including reds and
violets (listed below) and basic orange 21 all were found to
supravitally dye monocytes to exhibit this dually metachromatic or
bi-modal light response under the Zeiss fluorescent microscope.
Pursuing observation that basic orange #21 had shown not only
metachromasia under white light spectra, but also under conditions
of fluorescence stimulated continued research establishing that
basic orange #21 is operable under fluorescent light sources to
produce substantially the same patterns of cell geometry and
arrangement in the same cells as were disclosed in the
Continuation-in-part identification above. However, the fluorescent
colors were not of the same color response as with white light
sources, though the geometric patterns were entirely corraborated.
Parallel examinations of the same prepared slide in a large number
of instances of both normal leukocytes and those of patients with
various stages of diseased conditions both under normal white light
wave lengths, as was the subject matter of the parent application
(U.S. Ser. No. 129,680), and under fluorescent light, as here,
produced a remarkable demonstration of repeated leukocyte
identifications and confirmations under both white light wave
lengths and under fluorescent light wave lengths but with
identifying colors of both different in bi-modal means of
observation in hue, value and chroma and of varying visible light
intensity, as well.
Studies carried forward using species of methine and polymethine
dyes in leukocyte cell identifications continued to confirm unusual
properties of certain dyes in this class, particularly basic orange
#21, basic red #13, basic red #36, basic red #49, basic violet
#7,15, #16, #36, #39 and #40. All of the just identified dyes were
found to be metachromatic under white light wave lengths and under
fluorescent emission and all instantly stained monocytes
characteristically and metachromatically under the bi-modal means
here described. Further tests determined that all of the foregoing
dyestuffs were quite unusual in that they are also metachromatic in
conjunction with certain biological specimens under fluorescent
light and were also metachromatically fluorescent when used as
supravital to stain monocytes.
Samples of dyes located in a world-wide search total about 2,000 in
number and have been subjected to testing. Many of these dyes are
no longer available from or through known dye sources.
Specific studies of basic orange #21 further reveal it to be unique
among the dyes numbered above. Basic orange #21 is the only dye
presently known which exhibits a bi-modal function for
identification of all the biological blood cells named below. This
unique dye functions under both white light spectrum and
fluorescent light spectrum stimuli in a metachromatically different
identification of each individual ones of the following leukocytes
including the developmental stages of neutrophilic, granulocytic
cells. Rapid supravital staining with aqueous solutions of basic
orange #21 makes possible optical differentiation, identification
and enumeration under fluorescent light spectra, for each one of
the following cells; that is, with one prepared microscopic field
and with either manual, sequential or simultaneous examination
stereo-optical devices under white light spectra and fluorescent
spectra stimulus. Two different but characteristically distinctive
color patterns become available and each can be checked against the
other to confirm identification of neutrophils, eosinophils,
basophils, monocytes, lymphocytes, promyelocytes, myelocytes,
metamyelocytes, bands and B-cells as well as T-cells! Time has not
permitted exhaustive study of possible limitations of more advanced
computer operated high technology devices where simultaneous
readings of bi-ocular screens image both white and fluorescent
light projections from a single specimen field as possible.
Optical devices are known which permit both simultaneous and
sequential bi-modal analyses of apparatus useful for simultaneous
measurement of absorption and fluorescence. (See page 144, J.
Membrane Biology 33, 141-183 [1977].COPYRGT.. Springer-Verlag, New
York, Inc., 1977). Thus it is not unknown to subject a dyed
biological specimen to observation under the stimulus of a bi-modal
light source.
It is further a matter of record that fluorescence alone is often
an easier, faster and more versatile light source in stimulating
microscopic differentiation, but it is also known that this light
source may introduce complications in calibration and accuracy due
to artifact vulnerability. However, by making possible sequential
use of both white light wave length and fluorescent light wave
length in a comparative observation of a single biological test
specimen dyed with a dyestuff that is not only metachromatic under
white light spectra but metachromatic under fluorescent light
spectra makes possible observation of both similarities and
differences existant in the characteristic known components of
leukocytes as to their nuclei primary granules, secondary granules,
etc. Unique aspects of cell structure of eosinophils have been
observed which assist in their differentiation, identification and
enumeration.
The granules of eosinophils specifically show strong fluorescence
only around the periphery of the granule in a "case" or "shell"
pattern. As far as ascertained, this border or peripheral
fluorescent pattern or shell uniquely identifies eosinophils. Such
a structural indicia of a blood cell has not heretofor been known
to have been described. The observation suggests that white blood
cells are releated tissues upon further comparative studies under
both fluorescence and white light spectra will be found to reveal
further avenues of discovery and stimulate novel studies through
newly observed apparent structural differentiation not even
suspected to exist heretofore.
Another illustration of promise is a noted difference in the degree
of intensity of nuclear fluorescence among various T-cells
(T-lymphocytes). It is anticipated that these observed differences
in fluorescent light emission provide clues to identification of
T-cell sub-sets, e.g. supressor and killer cells. While it is
recognized that there is an observable significant difference in
T-cells under bi-modal light illumination, the significance of the
differences noted is not presently understood.
Present vital interest in immune studies suggest a locus of
practical interest and application of the bi-modal observations
possible with the supravital analytical methods hereby introduced.
Observations of leukocytes and their developmental stages and the
recognition of structural differences of the biochemistry and
biophysics through bimodal light observations will lead to deeper
understanding of their order and the disorders of disease.
A study of the chemical structures of basic orange #21 and base
orange #22 was conducted upon finding the first to be most unusual
and the second inoperative for the purposes herein.
The only differences to be observed are that the indolyl radical of
each basic orange varies only by a change in the methyl group from
a 2 position in basic orange #21 to a 1 position in basic orange
#22. The methyl group is substituted on a carbon in basic group #21
and on a nitrogen group in basic orange 22. The 2 position in basic
orange #22 has a phenyl group in place of the methyl group of basic
orange #21 at the 2 position in the structure.
Prior art references indicate that it was not unusual in supravital
analyses to employ three concentrations of dye in three
preparations of slides in such analyses as are an essential check
on results. With basic orange #21, the color differentials are so
separated and the colors so exceptionally vivid that one can
readily distinguish primary from secondary granules, instantly,
with one dye and one slide, and with either white light spectra or
fluorescent light spectra.
SUMMARY OF THE INVENTION
The present invention advances the art of cytology by providing a
single basic quaternary cationic organic dyestuff of the methine
and polymethine series which is selectively metachromatically
sorbed by one or more peripheral blood cell leukocytes which
provides unusual improvement in identification and differentiation
between immature and mature members of the various species of the
myeloid series and the mature white blood cells under either or
both white light absorbance or fluorescent light stimulus.
Heretofore, cytochemical means and complex stains had to be used
for blood cell differentiation, often requiring an hour or more of
tedious preparation to prepare and microscopically analyze by
differentiation and enumeration of a single species of known
leukocytes.
In practice, it now is practical to differentially stain and
identify with one single pure dye (others may be permissibly
combined for specific studies) in a simple aqueous contact with a
peripheral venous blood sample or fraction thereof, including
leukocyte enriched specimens thereof, each one of the following
species or types of precursor cells, white blood cells, platelets,
tissues closely associated with blood cells, etc., may be
accurately and readily identified. These species include
myeloblasts, promyelocytes, myelocytes, metamyelocytes, bands,
neutrophils, eosinophils, basophils, B-lymphocytes, T-lymphocytes
and monocytes. Platelets can also be identified and counted, and
also shown metachromasia in their granules.
Each of the above species of leukocytes when subjected to
supravital analyses after treatment with basic orange #21, is
differentiated under both white light spectra and fluorescent light
spectra by an unusual metachromatic response of the cells to the
dye and to the light source to which the prepared field is
exposed.
Under the bi-modal light sources in each instance of quality of
light souce employed, each one of the individual species of blood
related cells recited can be differentiated from its neighbors,
each species can be counted, the total count of any species present
determined, each species can be studies as to its morphology, and
many determinations made of great value to the health sciences.
Fundamentally, each of the above named white blood cells or
leukocytes differentially sorb light, either white light or
fluorescent light, from the same pure metachromatic dyestuff,
depending upon the quality of the dye, the species of leukocyte,
and the dye reception by elements of the specific cells present in
the specimen fraction analyzed.
In the absence of fixatives, the basic dye of this invention is
sorbed metachromatically so that each one class, type or species of
leukocyte reflects a characteristic light spectra or color
different from every other class, type or species of leukocyte
present in the sample. The strikingly vivid metachromasia under
both white light and fluorescent light of the single orange
dyestuff of this invention is unique and remarkable. Each species
of the series including myeloblasts, promyelocytes, myelocytes,
metamyelocytes, bands, neutrophils, eosinophils, basophils,
B-lymphocytes, T-lymphocytes and monocytes so sorbs the single
metachromatic stain as to reflect a distinguishing light spectra or
color in the visible light range and another and different
distinguishing light spectra or color when exposed to a fluorescent
light range. Combinations of the dyes of this invention in
combinations of others of the same chemical class exhibiting
similar metachromatic behavior under bi-modal light sources is not
precluded.
DETAILED DESCRIPTION OF THE INVENTION
This invention is a continuation-in-part of the parent application
U.S. Ser. 129,680 filed Mar. 12, 1980, which was based upon the
discovery of a group of unique metachromatic dyes which could be
used singly, but oftentimes in combination, to identify and
distinguish five species of white blood cells, namely; neutrophils,
eosinophils, basophils, lymphocytes and monocytes, one from the
other, when present in a human blood specimen. This specification
is also a further continuation-in-part of U.S. Ser. 242,662 of Mar.
11, 1981.
This invention provides further development of the discovery of the
capacity of the dye known as basic orange #21 which used alone in
an aqueous medium using supravital blood analysis technique on a
fixative-free specimen or fraction to stain in an unusual and
distinctive metachromatic manner the previously identified series
of human blood cells and platelets, but under fluorescent
light.
This individual species identification each one from the other
under fluorescent light reflects a remarkable order of
differential.
Basic orange #21 is identified by the Color Index number 48035, by
its chemical structures and the spectral curves which are part of
the Parent cases and the public record.
While it is not intended to be bound by theory, it is well known
that almost any foreign additive has a tendency to denature
proteinaceous materials. Heretofore, use of fixatives in
preparation of blood samples for staining has been universal
practice. Experience has indicated that fixing interferes with the
co-operation between the metachromaticity of the cell and the
metachromatic quality of the dyes of this invention. Troublesome
artifacts in the field are also avoided by the simple expedient of
supravital, fixative free, specimen use using a supravital
technique. By use of the bi-modal light source technique herein,
identification of the individual cells hereinbefore identified can
be checked out eliminating artifact confusion entirely.
In the practice of this invention, staining is sufficiently
instantaneous so that at normal blood temperatures (37.degree. C.)
the cytologist does not have to wait or resort to fine
cytochemistry practices before cell dye development occurs and
spectral differentiation between the previously enumerated family
members of leukocytes, before beginning his microscopic studies,
either manually or by automated differential leukocyte counting
systems.
Types of fixative free blood samples that can be used:
1. Anticoagulated (E.D.T.A., citrated, heparin) whole blood.
2. Suspensions of leukocytes obtained by dextran and/or gravity
sedimentation of anticoagulated whole blood.
3. Samples of whole blood treated with hypotonic solution lyse red
blood cells, leaving primarily white blood cells and platelets
behind.
4. Samples of other body fluids, like spinal fluid or pleural or
ascitic fluid, as well as samples of joint fluid where white blood
cells are of interest.
The present invention does not specifically provide for an
automatic differential leukocyte counting systems under either
white light spectra or fluorescent light spectra. Such means of
analysis have been under in-depth examination and may be near
commercialization.
The College of American Pathologists Conference in Aspen, Colo.,
August 1975, has published a series of papers delivered at that
time in a collection entitled "Differential Leukocyte Counting".
These reports provide development and "State of the Art" interest
in automatic differential blood cell counting computers. Attention
is directed to U.S. Pat. Nos. 3,916,205 and 4,146,604 (Kleinerman)
where certain fluorescent dyes are used in particular combinations
for automatic differentiation of certain leukocytes and other blood
cells based on fluorescent light response. These references are
deemed pertinent to the subject matter and ends of this disclosure.
It is to be additionally noted that Kleinerman relies upon cell
fixation, customary in microscopic studies of leukocytes and appear
to rely upon fluorescent dyes which are not required to be or are
metachromatic as well as fluoroescently active in the presence of
leukocyte cells.
The prior art indicates several levels of discrimination in the
performance of leukocyte differential counting. Basic or primary is
differentiation between polymorphonuclear cells and "mononuclear"
cells. On an intermediate level, the differentiation of polymorphs
into neutrophils, eosinphils and basophils and the separation of
"mononuclear" between monocytes and lymphocytes is said to be
possible in principle.
An apparent third level of difficulty involving differentiation of
neutrophils into immature and mature forms and the division of
lymphocytes into normal and reactive types was originally
recognized and mentioned in the parent application. The present
continuation-in-part application as far as is presently known
provides the only method of differentiating and confirming by
observation, employing only one single pure dyestuff the blasts,
myeloid series, bands, polymorphonuclear leukocytes (neutrophils),
eosinophils, basophils, B-cells and T-cells as well as monocytes
with a single dye and single specimen fraction and a selection of
light sources, under both white light and/or fluorescent light
stimuli.
The present state of the art in automated differential leukocyte
counters is understood to be in an advanced state of development
insofar as the use of white light and/or fluoroescent light and a
simple aqueous dye is concerned. Manual differentials with
preliminary complexity presently appear principally relied upon.
Automated differential counters are understood to be of two general
classes or groups: 1. pattern recognition systems and 2.
cytochemical differentiation systems. It is understood that
staining methods of the prior art have been used with greater or
less success and machine operators can monitor the operation on a
cell-by-cell basis.
Present cytochemical systems, while precise, have yet to develop
satisfactory calibrators and require highly qualified operators.
The advantages of being able to observe a single microscopic field
under bi-modal light control would appear advantageous.
In a brief survey of the prior art fluorescent dye and fluorescent
light source methods of the prior art, the following points are of
record. 1. At least two light sources are essential including
violet and ultraviolet light; 2. A third light source appears
needed as well. 3. The system is understood to require a plurality
of fluorescent dye stains to identify and differentiate the species
of leukocyte. 4. The system requires alcohol-fixed blood smears. 5.
The system requires staining times of the order of ten minutes and
rinsing time of one minute followed by a drying procedure. 6. There
appears to be a decreasing order of fluorescence intensity from (a)
eosinophils to (b) neutrophils to (c) monocytes to (d) lymphocytes.
(Basophils identification is not reported). 7. In a flow cytometer
tube system, the blood cells are fixed with formaldehyde and
stained with three different stains. 8. Detected leukocyte
fluorescences are differentially counted and classified by means of
ratios of fluorescent light. 9. One patentee discloses
identification of only four of the five leukocyte species. 10.
Three fluorescent dyestuffs are specified which must be combined to
produce a "single dye" composition which combination of dyes
appears essential to the operation or method, not merely
advantageous.
In the parent application ordinary white light was used to
illuminate the microscopic field which is an electromagnetic energy
form of radiation having the capacity to cause the sensation of
vision in light radiation of from about 4000 to 7700 angstroms wave
length. In the Parent case the selected portion of the visible
light spectra passing through the dye-leukocyte medium in the field
was absorbed. The term "fluorescence" is understood to be related
to emissivity. Fluorescence is believed to be caused by emission of
electromagnetic radiation by the selective dye-leukocyte emitting
and which emissions cease when the energy input ceases. Present
experience indicates the source of electromagnetic energy causing
metachromatic emissions from and differentiating the basic orange
#21 leukocyte dyed cells as described herein is not critical. It
may be of a mercury vapor lamp, as commonly used in fluorescent
microscopes; or it has been found satisfactory functional when the
energy form is a very selective beam as from a laser. The term
"absorption" appears to be applied to white light spectra, and
"fluorescence" in the case of the latter emissions.
The methods disclosed are based on a supravital technique. There is
possible a continuous monitoring system in hospital diagnosis and
treatment where continuous critical white blood cell observation
directly on the patient would be a desired end such as within the
potential of the bi-modal methods disclosed.
The term supravital stain and supravital staining does not preclude
the possibility of continuous perfusion through a shunt circuit
from the blood vessels of living organisms and continuous
monitoring of all possible white blood cells as they are passed
through a specialized by-pass tube for observation and count using
a coherent laser light as the energy source for fluorescence of
each individual blood cell as it is observed under cell cytometry
and as it is individually illumined (or made emissive of
fluorescent light) as it passes a point of focus in the
microscopically enlarged field.
Based on limitations inherent in panoptically stained specimens,
over the past several decades a number of cytochemical tests have
been devised to more precisely distinguish one type of blood cell
from another. In general these tests are designed to detect
increased amount of one type of substance in a particular cell
compared to another, or to detect a substance(s) within a
characteristic cellular organelle in one cell compared to another.
For example, activity of nonspecific esterase is unusually high in
monocytes and this activity appears to be particularly sensitive to
inhibition by sodium fluoride. Likewise, identification of
granulocytic cells depend for the most part upon demonstration of
properties of lysosomes. For these purposes, detection of
myeloperoxidase and specific esterase activities have been useful
as cytochemical tests. Lysosomal granules of eosinophils contain
myeloperoxidase that is resistant to inhibition by sodium cyanide,
and granules of basophils stain metachromatically with a variety of
dyes, due in part to their high content of cationic substances like
heparin.
As will be noted herein, monocytes show a non-staining nuclear
reaction but are identified by cytoplasmic and granular color
differentiation with a few other unusual dyes disclosed herein
which are also metachromatic under both white light and fluorescent
light.
The supravital stain technique utilizing living blood cells and
their differential affinities for supravital staining of these
cells with dilute aqueous fixative free dyes avoid artifacts that
often occur with conventional fixatives. Using both white light and
fluorescent light substantially eliminates the confusion caused by
artifacts. The vital staining technique and bi-modal light sources
provided herein define a more accurate reflection of cellular
localization of the dye (e.g. lysosomes, fibrillar structures,
nuclear chromatin) than use of one light quality alone. Continued
experience with the basic orange #21 dye and improvements in the
automated technology of differential blood cell counting under
bi-modal light sources in conjunction with supravital fixative-free
staining of peripheral blood leukocytes introduces a further
important addition to the field of cytochemistry.
It is convenient at this point to refer to FIG. I as an aid in
understanding leukocyte identification and differentiation as
provided by the advance in the art herein.
Limitation to black and white illustration of FIG. I and the fact
that the essential representations involve three dimensional
objects and color variation and fluorescence all of which are
identified only poorly by the grossness of language and black and
white illustration and of limited spatial dimensions is an
unfortunate loss of letters patent printed in black and white.
All blood cells appear to originate from undifferentiated stem
cells called mesenchymal cells. Immediate descendants of the stem
cells are called blasts, and the specific myeloblasts are
understood to be progenitors of the leukocytes differentiated and
made identifiable and enumeratable by their supravital analyses
under either white light absorbance or fluorescent light emission
or both, either simultaneously or sequentially when exposed to
basic orange #21 in a fixative free aqueous environment.
Myeloblasts are identified herein by the absence of granules or
lysosomes which characteristically identify the three descendant
cells of the myeloid series by their metachromatic color sorbtion
or fluorescence. The three descendant cells, namely; promyelocytes,
myelocytes and metamyelocytes are each separately identified by the
metachromatic and differential color and color distribution as will
be described.
Promyelocytes are readily identified by the following manual or
automatic observations. They are generally largest in size of the
myeloid series shown. The oval nucleus N-1 is not stained by basic
orange #21 under absorbance nor by fluorescence and is relatively a
larger part of the total granulocytic cell. Under absorbance the
cytoplasm C is closely packed with large numbers of relatively
small primary granules of an orange-red color (magenta) 1, and a
few scattered violet granules 2 generally distributed amongst a
large mass of orange-red primary granules. Under fluorescent
emissivity the primary granules or lysosomes 1 fluoresce brightly
yellow and the cytoplasm fluoresces a duller green.
Myelocytes also possess a non-stained ovate nucleus N-2 of slightly
reduced area (volume). The outstanding distinguishing fact is the
definite development in the myelocyte of larger secondary yellow
granules 4 in a generally thickening crescent of the immature
myelocyte cell. Dual imaginary lines a-a.sup.1 bracket and
delineate the increasing number of larger secondary yellow granules
4 of the myelocytes from the decreasing number of smaller
orange-red or magenta primary granules 6. One can observe that
myelocytes are distinguished from promyelocytes by the noticeable
isolated component of developing secondary yellow granules 4. These
absorbent (white light) magenta granules also fluoresce brightly
yellow under fluorescent emissivity, and the developing secondary
(larger) granules fluoresce a pale green.
Metamyelocytes like the two cells above (N.sub.1 and N.sub.2) also
have a non-stained nucleus N-3 which begins to exhibit developing
lobular pattern as distinct from the priorly described ovate form
of the first cell in the myeloid series. The diminishing mass of
smaller, primary, orange-red or magenta granules 6 now becomes a
minor proportionate area of the total area of the observed
cytoplasm C-2 of the cell. Larger secondary yellow granules 8
appear to displace a significant central portion of the previously
ovate unstained nucleus N-3, which defines the change intended by
the verbal expression--from ovate to lobular--. Fluorescent
response patterns are consistently similar and recognizable. The
secondary cells now have a larger size than in the myelocytes.
Bands are progressively distinctive and have been set apart from
the first three cells described showing metachromatic staining of
the granules and which are members of the myeloid series.
So far as presently known, bands have not been heretofore
distinguished from all other leukocytes by the metachromasia of any
dye.
Bands are distinguished from all other leukocytes by an unstained
lobular formed nucleus N-5 which, along with the overall band size
has noticeably decreased in area (volume) as compared with the
prior leukocyte cells of the myeloid series. Additionally the
lobular form of the unstained nucleus N-5 has become more
bifurcated by further inward growth of the cytoplasm C-6. Growth by
number increase of secondary yellow granules 12 in the cytoplasm
has displaced all but a very small cluster of remaining primary,
smaller, orange-red granules 10. Note that the cluster of red
granules 10 are specifically located in the inward protrusion or
movement of the cytoplasm C-6 tending to segment the nucleus N-5.
Larger, secondary yellow granules 12 have succeeded to take over
the cytoplasm C-6 except for this characteristic contrasting color
group of bands. The important point of separation of bands is the
small cluster of red granules 10 in cytoplasm C-6 at 10.
This outstanding point of differentiation of bands from all other
leukocytes is suggested as extremely useful in development of
automated equipment adapted to perceive the small primary
orange-red granules 10 surrounded by a large preponderance of
secondary yellow larger granules 12.
Like in absorbance, bands under fluorescence show a small cluster
of yellow fluorescent granules 10 in cytoplasm C-6 in the above
characteristic cell pattern with fluorescent pale green secondary
granules 12 also preponderant.
Ability to easily, rapidly and certainly distinguish, identify and
enumerate bands from all other leukocytes with but a single
dyestuff under a bi-modal wave energy stimulus as described above
is beyond expectation and beyond any known theory of band
function.
Neutrophils are mature white blood cells and were known to be
recognizable from the five main classes of white blood cells of
interest in the Parent application. Further detail of interest has
now been established.
Neutrophils, eosinophils and basophils, in the absence of color
reproductions as in the drawings, are all of relatively similar
physical configuration. Using basic orange #21 as the sole
supravital dye in a fixative free environment, neutrophils are
identified by secondary granules 16 in the cytoplasm C-8 which are
mainly yellow. The nucleus N-8 is not stained and is generally
segmented. The large secondary yellow granules 16 constitute the
major area (mass) of the cytoplasm C-8. Under fluorescent stimulus
the neutrophils are primarily identified, as above, by the
characteristic tri-lobed (segmented) nucleus N-8 and the dull green
fluorescence of the cytoplasm C-8.
Eosinophils under absorbed white light spectra also possess a
segmented unstained N-10, but the large secondary granules 18 are
differentiated from the neutrophils and basophils by their orange
color which is the characterizing and main feature of the cytoplasm
C-10.
Fluorescent excitation disclosed a most interesting and
characterizing development with eosinophils. The general structure
disclosing bisected nucleus N-10 is common with bi-modal light
examination. However, the secondary granules show a bright yellow
outline or "shell" fluorescence about the periphery of the
secondary granules 18 which has not been heretofor recognized. This
unique "marking" noted with eosinophils under emissive
(fluorescent) stimulus provides a means of checking observations
made under the earlier absorbance examination as well as suggesting
a phenomena of interest as to the structure of the secondary
granules themselves not known heretofor. Under both sources of
energy input observation the general structures are otherwise
confirmatory.
Basophils segmented nucleus N-12 is also unstained by basic orange
#21 under absorbed white light spectra but the secondary granules
20 differ from the granules of the neutrophils 18 by
metachromatically staining basophils granules 20 a bright crimson
color having a faint blue tinge or undertone.
Fluorescent stimulus of the basophils also provides substantially a
pattern structurally similar to that of absorbent light. However,
the fluorescence of the secondary granules 20 provides an unusually
brilliant lemon yellow fluorescent light emission therefrom.
Note in the indicated cell lineage that each of the leukocytes
shown in the FIGURE, neutrophils, eosinophils and basophils
actually are derived from their specific precursor bands. The
FIGURE as drawn does not include this progression in detail.
Promyelocytes are indicated as precursors of monocytes.
B-lymphocytes and T-lymphocytes have essential oval nuclei N-14 and
N-16, respectively. Each of which nucleus N-14 and N-16 under
absorbent light stains a similar yellow. The cytoplasm C-14 and
C-16 in each case remains unstained. One characteristic of the
cytoplasm C-16 of T-lymphocytes clearly differentiates
T-lymphocytes from B-lymphocytes. This is the presence of the small
cluster of red granules 22 in the cytoplasm C-16 of
T-lymphocytes.
Observing the same specimens as above under the influence of a
mercury vapor light source, as illustrative, the T-cells small
cluster of granules 22 in the cytoplasm 16 fluoresce bright yellow
and the nuclei N-16 have been observed to be most often
substantially unstained but have been noted to exhibit a dull green
fluorescence under the same examinations. The pattern under both
methods of observation is quite comparative and confirmatory. In
B-cells, the nucleus has been consistently a dull fluorescent
green.
Observations under both qualities of energy stimulation, B-cells
vary by a yellow color of the nucleus under absorption and a dull
green fluorescence under emissive light conditions. In both aspects
of bi-modal light energy examination there have been no cluster of
secondary granules in the cytoplasms of B-cells observable. This
pattern is decisive.
As noted in the Parent application, monocytes have been unusual in
that under white light observation all of the dyes originally found
metachromatically active of the basic quaternary dyes disclosed
therein, and under the more recent studies utilizing fluorescent
light emissive energies, monocytes have consistently acted both
metachromatic as well as fluorescent, not only with basic orange
#21, but with several other basic red and basic violet cyanine
colors priorly disclosed herein.
With basic orange #21, the monocytes do not stain as to their
generally ovate nucleus N-18. However, the cytoplasm C-18 acquires
a pink cast in which are discernable a scattered, relatively small
number of crimson and pink granules 24 differing in hue, value and
chroma sufficiently from the "pink" cast of the cytoplasm C-18 to
be clearly optically differentiated from cytoplasm C-18 which is of
a generally similar pink coloration.
Under fluorescent electromagnetic wave energy stimulus, all the
indicated members of the class of cyanine or methine and
polymethine dyes referred to herein cause fluorescent emissions
from so dyed monocytes. With basic orange #21, the few granules 24
in the cytoplasm C-18 are a bright fluorescent yellow. Named basic
violets and basic reds that stain monocyte nuclei metachromatically
by absorption also fluoresce metachromatically by emissive
energy.
Identification and enumeration of monocytes has been simplified by
discovery as also described in the parent application with the set
of unusual dyes therein disclosed. The standard fluoride sensitive
non-specific esterase reaction cytochemically used for monocyte
identification often requres an hour or more to complete and
requires accurate cytochemical manipulation to be successful. With
any one of the disclosed dyes, the dyeing of buffy coat suspension,
whole blood or separated fractions and examination can be performed
simply, without chemical adjustments, in the order of minutes.
Staining of monocytes by the present application method using only
basic orange #21 is instantaneous as to the cytoplasm, and the
granules in the cytoplasm.
It should be noted that the metachromatic dyes may ultimately stain
cells to a point where bi-modal identification are lost. Thus, the
color differences reported in this disclosure may be lost, or
diminished to a great degree if analyses are not promptly
performed. As practical staining occurs almost immediately in all
known instances, no extended waiting period for maximum
differentials is, however, necessary.
In the prior art identification and differentiation of monocytes
has been accomplished by time-consuming and complex cytochemical
treatment of the cells involving non-esterase reaction, fixed cell
preparation, hexazotization, pH adjustment and dye staining with
multiple dyestuffs requiring about sixty minutes to accomplish what
can be done with any one of the dyes of the Parent application
under either white light spectra or fluorescent spectra, if
desired, in less than a minute by a simple dye and blood sample
contact with an aqueous system. With the specific dye of the
present process as herein disclosed, there is also instantaneous
and preferential staining of the cytoplasm of monocytes.
The unique cyanine dyes named herein are prepared for the proposed
end use and for the purposes of this invention in filtered aqueous
solution at approximately 1% concentration of the pure basic orange
#21, as illustrative, in distilled water. The dye concentration is
not particularly critical but permits some variation. It is
preferred that aqueous solutions be used while fresh and that toxic
additives not be included. Interference with the metachromatic
reaction between dyestuff and the specific type or class of
leukocyte may be totally inhibited by the presence of any of the
known classical fixatives.
The definitive language "supravital" as used herein is an important
limitation. It is applied to the original blood sample and is
applied to living cells freshly removed from a living organism, or
one freshly sacrificed, or equivalent. As the term is used here it
is intended to exclude all "fixatives" but permits use of
anti-coagulants (heparin, E.D.T.A., etc.). The blood cells may also
be removed from bone marrow, urine and other biological specimens
containing them, including as illustrative, lymphatic tissue and
spleen.
The general practice of this invention is illustrated by the
following.
A 1% solution in distilled water is made up of the selected basic
quaternary cationic dye. Filter off all non-dissolved solids. Most
useful is basic orange #21. If practice indicates it necessary, one
or more of the subject dyes can be blended together as aqueous
solutions if for a specific end.
The aqueous solution of the selected, previously identified, single
pure dye (or one can employ combinations of one or more of the pure
dyes as are disclosed in Tables I and II, as illustrated in Table
III of the Parent application) is solubilized to produce a simple
aqueous dye solution. (Consideration of various volumetric
proportions of the aqueous dye solution, and various strengths of
aqueous dye solutions may provide optimum conditions for various
specific cytological analyses.) Some experimentation may lead to
specific combinations having particular advantage and is
contemplated by but beyond the scope of this disclosure.
Blood samples may be made available from various sources but fresh
samples of venous blood from which erythrocytes have been removed
(centrifugation, hypotonic lysis, gravity sedimentation, density
gradient sedimentation, etc.,) or the sample may be a plasma
enriched with white blood cells by known physicochemical techniques
have been principally used. Tissues having cells (as described
above) present may also be dyed and subjected to bi-modal light
stimulus for study.
It is preferred to combine the aqueous dye and blood sample, both
as freshly prepared, at the temperature of normal blood or body
(about 36.degree.-40.degree. C.) where favorable to the analyses
planned. More rapid and sharper staining at the higher temperature
is generally obtained. Basic orange #21 does not appear temperature
sensitive.
Dye and blood solutions work well when combined volumetrically at a
ratio of about 1:4. Gently agitate the mixture for several seconds
and examine a drop of the mixture immediately as a wet mount using
a glass coverslip under a light microscope or automated
differential leukocyte counting device, if available. Other means
of contact between the dye and blood cells including using known
media, illustratively gelatin, emulsions, etc., impregnated with
the dye at about 1% dye concentration. Fixing the sample seriously
interferes with the unusual metachromatic co-action of the
dyestuffs of this invention with the leukocytes.
Basic orange #21 dye makes possible examination under white light
alone, under fluorescent light alone, or both either
simultaneously, singularly or in sequence depending upon equipment
available and to distinguish the following one from the other if
they are present together: myeloblasts, promyelocytes,
metamyelocytes, myelocytes, bands, neutrophils, eosinophils,
basophils, B-lymphocytes, T-lymphocytes and monocytes. Outline of
the means of differentiation for enumeration and other study have
been priorly developed in the Figure. The foregoing technique aids
in identification.
The examples included herein as illustrative will assist one
skilled in the art to appreciate the potential of the novel methods
proposed. Not the least of the advantages of the method(s) are
leukocyte counts (total), leukocyte counts of species, diagnosis of
diseases, particularly leukemias, and the monitoring of patients
receiving a variety of critical treatments, illustratively,
chemotherapy, radiation therapy, ACTH, etc.
Identification and enumeration of one, selections or all of the
species of leukocytes is often critically important in diagnosis
and treatment of disease.
The examples which follow the detailed description of the invention
are intended to illustrate the utility of the invention and its
practice. Obviously, they are not exhaustive nor are they to be
considered limiting.
EXAMPLE 1
(Basic Orange #21)
[An extensive series of methine, polymethine, quinoid and/or
carbocyanine dyes were obtained based upon the initial discovery
that basic orange #21 was found to be unusually metachromatic in
relation to differential staining of white blood cells. Only basic
orange #21 of over 2000 dyestuffs was found to exhibit the unusual
metachromasia under white light and fluorescence as herein
illustrated.]
50 ml. of peripheral blood was obtained from normal individuals in
a series of heparinized tubes.
Duplicate tubes were admixed with 10 ml. of a 1% solution of basic
orange #21 dyestuff in distilled water at about 37.degree. C. The
temperature has not been observed to be critical with this dye
use.
Under microscopic study eosinophils originally described as having
brown granules are now more accurately described here as having
large orange granules in the cytoplasm under absorbance and
fluoresce a bright yellow in a shell pattern with a non-stained
lobular nucleus under both lights. Basophils were bi-modally
observed to have a similar geometric configuration, but the
granules under absorbance were bright crimson in mass tone with a
faint blue tinge and fluoresce a bright yellow. The nucleus was
also lobular, and not stained under bi-modular examination.
Monocytes were clearly differentiated by non-stained oval nucleus
and a pink cytoplasm containing a few crimson and pink granules.
Under fluorescent light the granules are bright yellow. Non-stained
may include very pale casts depending somewhat on the elapsed time
before reading the slide.
Quite remarkably, the differential staining of both mature and
immature neutrophils was more specific than any dyestuff heretofore
observed in our studies or reported elsewhere in the prior art.
Under absorbance mature neutrophils were spectrally identifiable by
the few mainly yellow larger granules in the cytoplasm and the
lobate unstained nucleus. Under florescence, the cytoplasm was a
dull green fluorescense and the granules were not as visible.
EXAMPLE 2
T-cell rich and B-cell rich suspensions of human blood were
prepared from 50 ml. specimens of peripheral blood obtained from
normal individuals in heparinized Vacutainer tubes by passage of
Ficoll-Hypaque enriched fractions through micro-columns of nylon
mesh gauze. T-cell rich and B-cell rich suspensions were eluted
from the columns using controlled temperature conditions with
selected different buffers as is known in the prior art.
Fractions of these recovered suspensions were subjected to
immunologic analyses using T-cell rosetting for T-cells and surface
immuno-globulin detection for B-cells. One drop of a 1% aqueous
solution of basic orange #21 dye was incorporated into 5 drops of
the recovered suspension in separate test tubes. Each tube
contained approximately 2.times.10.sup.6 lymphocytes. Lymphocytes
identified as T-cells by virtue of T-cell rosette formation were
microscopically observed under white light spectra to contain small
groups or clusters of 5 to 10 or more red granules. Under the
fluorescent mode, the nucleus in some instances were unstained, or
a dull green fluorescence, and the same clusters of granules
fluoresced a bright yellow. Lymphocytes identified as B-cells by
immunological methods exhibited only a rare red stained granule in
the cytoplasm, most of the B-lymphocytes had no red granules
present in the cytoplasm. The nucleus was oval and under absorbance
stained yellow in both T-cells and B-cells. Under the fluorescent
mode the nuclei of B-lymphocytes were a dull fluorescent green and
the cytoplasm was unaffected.
Use of basic orange #21 provides a new, rapid method for
identifying, differentiating and counting T and B lymphocytes or
cells bi-modally or under fluorescent stimulus alone. Present
methods involve use of unstable biological reagents, (sheep cells),
require radioisotopic techniques which are costly and time
demanding, require control of many variables including temperature,
pH of incubation media, etc., which no longer appear necessary.
EXAMPLE 3
A 48 year old woman patient terminally ill with breast cancer
developed septicemia and a high fever shortly before death. At that
crucial time her white blood cell count rose to 45,000 per
mm.sup.3. By use of the standard Wright's stain, it was established
that 60% of the peripheral blood leukocytes were bands having a
characteristic unsegmented nucleus.
Specimens of the patient's blood was stained with a 1% aqueous
solution of basic orange #21 as a supravital stain without
fixatives.
Bands were identified by the consistent presence of a small cluster
of red staining (primary) granules amidst a larger number of larger
(secondary) granules which were metachromatically stained yellow as
well as the typical unsegmented nucleus. Fluorescent light caused
the primary granules in the cluster to exhibit a yellow
fluorescence.
Thus it was possible to make positive identification,
differentiation and enumeration of the band forms on the basis of
their cytoplasmic maturation and color differentiated primary vs.
secondary granulation under absorbance, and the yellow fluorescence
of the cluster of primary granules.
EXAMPLE 4
A 24 year old man developed fatigue, and was found on physical
examination to have an enlarged spleen. Laboratory values included
a white blood cell count of 15,000/mm.sup.3, and approximately 75%
of the cells were atypical lymphocytes. On further immunologic
testing, these were found to be T-cells. The patient had a positive
mono spot test, and the diagnosis of infectious mononucleosis was
made. Using basic orange #21 as a supravital stain, most of the
lymphocytes contained a cluster of metachromatically staining red
granules in the cytoplasm very near the unstained nucleus. Using
fluorescence, these same granules fluoresced bright yellow with a
dull fluorescently green nucleus.
EXAMPLE 5
A 75 year old man developed enlarged lymph nodes in the neck and
groin, as well as an enlarged spleen. Laboratory studies disclosed
anemia, and white blood cell count of 80,000/mm.sup.3.
Approximately 90% of these cells were lymphocytes, and on bone
marrow examination, the marrow was largely replaced by similar
appearing lymphocytes. The diagnosis of chronic lymphocytic
leukemia was made. On immunologic testing, the lymphocytes were B
cells. Under white light using basic orange #21 as a supravital
stain, no granules were seen. Using fluorescence, no granules could
be visuallized in the cytoplasm of the lymphocytes.
EXAMPLE 6
A 33 year old man developed weakness and fatigue, and was found to
have a markedly enlarged spleen. Laboratory examination revealed a
white blood cell count of 800,000/mm.sup.3 along with a mild anemia
but a normal platelet count. On a standard Wright's stain of the
peripheral blood, all stages of maturation of leukocytes were seen,
with predominance of promyelocytes, myelocytes and metamyelocytes.
Numerous eosinophils, basophils, and nucleated erythrocytes were
seen. On the basis of bone marrow and cytogenetic evaluations, the
diagnosis of chronic granulocytic leukemia was made.
With basic orange #21 used supravitally with white light, the
expected metachromatic differentiation of leukocytes was seen. With
fluorescent light, the primary granules that stained
metachromatically red appeared as bright yellow to yellow green
fluorescence. Cells that contained mainly secondary granules (e.g.
neutrophils) showed bright yellow color using white light, but dull
green fluorescence using fluorescent light. Basophils showed bright
yellow fluorescence of their granules, and the same granules
appeared bright red using white light. In eosinophils, the granules
stained bright orange using white light, but using fluorescent
light, the granules showed fluorescence only around their
periphery, in a "shell" fluorescent pattern.
EXAMPLE 7
A 25 year old woman with bronchial asthma had a routine blood count
as part of her physical examination. On the leukocyte differential,
approximately 40% eosinophils were found. Using basic orange #21
supravitally with white light, the granules of eosinophils stained
orange. Using fluorescent light, the same granules showed a "shell"
fluorescence.
EXAMPLE 8
A 62 year old woman with diabetes mellitus and terminal carcinoma
of the colon developed anemia and leukocytosis shortly before
death. The white blood cell count was 35,000/mm.sup.3, and on
Wright's stain showed numerous myelocytes and promyelocytes. Using
basic orange #21 as a supravital stain with white light, the
metachromatically staining red or magenta colored granules of
promyelocytes and myelocytes exhibited a bright yellow
fluorescence. As expected, these granules were more numerous in
promyelocytes than in myelocytes.
EXAMPLE 9
In a 47 year old male with acute lymphoblastic leukemia, the white
blood cell count was 10,000/mm.sup.3 containing 80% leukemic
lymphoblasts (PAS positive and terminal deoxynucleatidyl
transferase passive). With basic orange #21 used supravitally,
these leukemic lymphoblasts showed faint yellow nuclear staining
with white light, and little or no nuclear fluorescence using
fluorescent light.
Leukemic blasts from several patients with acute leukemia show a
substially diminished fluorescence compared to normal leukocytes.
Thus differences between normal and leukemic cells are
differentiated.
DEVELOPMENT OF THE INVENTION
In the foregoing specification and examples there has been emphasis
on the importance of the advances here disclosed in application to
automated differential leukocyte computing devices. It is not known
presently that "off the shelf" commercial equipment is directly
useful without some modification for advantageous use of the
bi-modal light method herein disclosed or for sole use of
fluorescent light which has been employed in the prior art.
It is known, however, that at least three pieces of analytical
equipment based on emission of fluorescent light are available
using, in some instances, prior art dyestuffs which are not
metachromatically fluorescent in nature. It is understood these
devises are capable of determining patterns of light illuminated
cells, of determining sizes of special patterns defined by
fluorescent color differences and that various coherent light
lasers are used, in whole or in part, for identification and
enumeration of so differentiated cells. Those known in the prior
art include Epic V (Coulter), FACS (Becton-Dickinson) and
Cytofluorograph (Ortho).
Those skilled in the art and working in the field of medical
technology are aware of the importance of rapid, accurate
determination of the various differential leukocyte counts for a
variety of ends. It has been estimated that in the United States
each day a half million differential leukocyte counts are
performed, most of them by manual techniques at an annual cost of
over 750 million dollars.
Such counts, whether manual or automated, have a fundamental
requirement of identification, spectral differentiation,
enumeration and diagnostic aid in practice of medicine. The
foregoing advance in these fundamentals will no doubt give rise to
advances in ancillary automated equipment as herein indicated.
Blood counts as are of concern herein, whether manual or automated,
are vital aids in examination and determination of the nature of
disease. Fevers of unexplained origin; whether viral or
non-pyogenic infection, pyogenic involving appendix, gall-bladder,
fallopian tubes; prognosis of patients with various diseases in
various stages; malignancies including Hodgkins disease; pulmnonary
disease; surveillance of patient treatment with adrenocortical
steroids; various kinds of acute and chronic leukemias,
differentiation in diagnosis, between aseptic infarction of bone
and osteomyelitis; bacterial infections and many other medical
questions are aided in diagnosis, prognosis and treatment by
accurate leukocyte counting, analysis and cytological study.
As used herein, the term metachromatic has relation to not only to
the peculiar and unusual quality of the dyestuffs disclosed but to
the quality of the various components, illustratively nucleus and
cytoplasm, of each of the individual species of leukocytes which
metachromatically co-react with something akin to synergism to
produce the differentiation in spectral response which makes the
described advances in cytochemistry possible. In essence, each
white blood cell species sorbs (or fails to sorb) a single
metachromatic dyestuff in some unusual and unique manner so that
each dye-sorbed cell reflects an individual and different light
spectra under ordinary white light stimulus and absorbance and
fluorescent emissions. Oddly the dyes herein disclosed are
metachromatic under both white light and fluorescent emission.
It has been well known that certain granular leukocytes have
different affinities for various dyes, that is, basophils have
affinity for basic dyes, eosinophils have affinity for acid dyes
and that neutrophils do not stain intensely with either acid or
basic dyes, no morphological features or chemical behavior is
suggested that makes possible the specificity of basic orange #21
in making such sharp distinctions as here found between
T-lymphocytes and B-lymphocytes and the unusual distinction in
bands, under bi-modal light stimulus as disclosed above.
This is the more arcane when it is found that basic orange #22,
which varies as to its structural chemistry only in two secondary
group position variations as discussed earlier, is totally
inoperative for the uses as set out herein.
Identification, differentiation and enumeration of monocytes has
valuable diagnostic significance. Increased numbers of monocytes in
the blood may indicate the presence of active tuberculosis,
septicemia or blood poisoning and lymphomas like Hodgkins disease
in diagnosis. Increased number of monocytes in the blood of persons
recovering from hypoplastic or aplastic anemia may herald a
favorable prognosis for the patient. Rapid and accurate microscopic
analyses of monocytes under two different types of light quality
favors extended application of a valuable technique.
Detection, identification and enumeration of polymorphonuclear
leukocytes (neutrophils) are critical parameters in all blood
evaluations. They are especially vital in the diagnosis of acute
infections like pneumonia or peritonitis where the number of
neutrophils are increased. They are important in monitoring
patients receiving chemotherapy and for radiation therapy.
Decreased numbers can occur in overwhelming infection, as a
manifestation of drug toxicity, hyperactivity of the spleen and in
acute leukemia.
If the absolute neutrophil count falls below 1000 mm.sup.3, the
risk of infection increases sharply. The specific dye of this
invention instantly stains the granules or lysosomes which are
characteristic identifying structures of polymorphonuclear
leukocytes or neutrophils under either one or both described light
sources.
Eosinophils are involved in allergic reactions, as are the
basophils. Lymphocytes are involved in inflammation and to a
greater extent in immune reactions and response to antigens
(foreign bodies). Eosinophils are here instantly identified by the
large orange granules in the cytoplasm, and the lobular
configuration of the unstained nucleus under light absorption and
the unusual shell fluorescence when the dyed blood or tissue
specimen is stimulated to fluoresce.
Eosinophil counts are used in following the medical administration
of adrenocorticotrophic hormone ACTH in the treatment of clinical
conditions. Prior methods introduced confusing artifacts and
indefinite forms confusingly similar to the eosinophils. Accuracy
of the blood cell count with the prior art decreases with the time
between blood sample preparation and completion of the count.
Multiple dyes are essentially used. Acid and base staining is often
required. The prior art dyes used tend to crystallize out of
solution on standing.
Lymphocytes, specifically identifiable as a class with blue borrel,
are known to be related to inflammation and immunity. They are
increased in number in the blood of persons with chronic lymphatic
leukemia and in persons with pertussis (whooping cough). The count
may be decreased in patients undergoing chemotherapy and
radiotherapy, in patients with lymphoma and various types of
hereditary immunological deficiencies.
Basophils have a cytoplasm which contains large granules that are
rich in cationic substances like heparin, serotonin and histamine.
They are involved, for example, in allergic reactions.
The principal advance in this continuation-in-part application has
been the discovery that there is presently one unique dye which
differentially stains lymphocytes, previously stained only as a
class by blue borrel at lower temperatures which can be used in a
single pure form for both manual and automatic identification and
under both white light spectra and fluorescent spectra to give two
modes of checking out a given microscopic cell field in the study
of each indicated species.
In prior art studies of T-cells and B-cells their differentiation
has involved more complex biochemical preparations and procedures
including: 1. Acid phosphatase (enzymes); 2. Non-specific esterase;
3. Fragments of human immunoglobulines (Ig G); 4. Non-metachromatic
fluorescent dyes, and 5. SRBC--sheep red blood cell preparations
having a useful life of about 14 days, it is the basis for rosette
formation for identification of T-cells, and is temperature
dependant.
T-lymphocytes are found in 60-80% in peripheral blood and 85-90% in
the thoracic duct. These cells are known to be related to allograft
rejections and both T-cells and B-cells are important
considerations in immunology and pathology. B-cells are fewer in
number and are from 10-30% in germinal center and medullary
cords.
Drug addicts show significant reduction in T-cells.
The vast majority of congenital immunological disorders have
relation to T-cell and B-cell systems. Neoplastic diseases are also
known to involve the immunogenetic system and patients are
recognized to have abnormalities of T-lymphocyte and B-lymphocyte
systems. Adult lymphomas are most often of B-cell origin. Childhood
lymphomas, on the other hand, bear T-cell markers believed to
relate to the more active thymus in early childhood.
Chronic lymphocytic leukemia is a B-cell related, leukemic cells
are B-cells and none are known to have T-cell clusters. From the
foregoing brief notation, the importance of ready identification,
comparison and enumeration of these cells has far reaching
significance.
The term metachromatic believed first used by Ehrlich (1897)
describes a stain which changes apparent color when sorbed by
certain cells. The dye is said to exhibit metachromasia and has
been observed as a property of relatively few pure dyes, chiefly
basic cationic dyes including methines, polymethines and
carbocyanines which color tissue elements in a different color.
Metachromasia is also defined as the assumption of different color
spectra by different substances when stained by the same dye.
Fluorescent metachromasia is very rare. In cytology as here,
metachromatic granules or other cell elements are those which
assume a color different from that of the dye used to stain
them.
Inherent in the above discussion of the terms metachromatic and
metachromasia, two factors are involved. One is the biological cell
(and its specialized part), which has been called "metachromatic"
or "chromotropic" and is a quality or character of the biological
cell specimen, and the other is the quality of the dye. Very few
dyes possess whatever quality is essential to stimulate
structure(s) within a cell to exhibit metachromasia under
absorbance as well as fluorescent modes. Conn (9th Edition) reports
"pure dyes showing this reaction are few in number". Few reports
found indicate that the phenomena has heretofore involved more than
two distinct color spectra. In one instance "a light green-blue
nuclear stain with a violet metachromasia for cartilage" was
reported. However, with stains being normally applied to fixed
tissues whose chemical and physical nature is altered by the usual
prestaining preparatory procedures, essential cooperation between
the character of the natural biological structures within a cell
specimen (may be thereby altered and rendered not sensitive to what
might otherwise react) so that dye sorption does not occur.
Fluorescent dyestuffs are well known. In fluorescence, more light
energy is emitted over select narrow frequencies than is absorbed
in these select frequencies, although the total light reflected is
not more than the total light absorbed. Fluorescent metachromasia
is known (certain dyes in the acridine chromophore class may be
metachromatically fluorescent). The term was so used in the Parent
application to conform with the term as found in Conn (9th
Edition), and Gurr's in "Synthetic Dyes in Biology, Medicine and
Chemistry" and Gurr's "Rational use of Dyes in Biology" where no
references to "Fluorescent metachromasia" have been noted. In this
continuation-in-part application it has been found that a few
unusual dyes disclosed in the Parent application are dually
metachromatic and these are specific to this disclosure.
Of greatest interest and of most promise as a class are the
methine, polymethine, quinoline and carbocyanine dyes where the so
identified chromophoric group bridges between other chromophoric
groups in the cationic class. Often, for example, a methine or
polymethine group is found to bridge between one or more quinoline
bearing chromophores.
Subsequent to the filing of the Parent application where basic
orange #21, a polymethine dye, was first found to be promising for
the purposes of white blood cell classification, samples of other
basic orange dyes, identified in the Color Index, namely; basic
oranges numbered 22, 27, 42, 44 and 46 were found and checked for
metachromasia. The foregoing were dyes reported to be in the
polymethine class. Basic oranges 24, 25, 26 and 28 were also
tested. The latter were not polymethines. Of all the basic oranges
tested including methine and polymethine basic oranges, only basic
orange #21 dye was of utility for the purposes herein
disclosed.
Basic red 13 and basic violet 16, of the methine and polymethine
chromophore class as originally disclosed in the Parent case have
been established as bi-modally metachromatically valuable for
monocyte blood cell identification hereunder. The search has been
extended to cover similarly identified and available methine and
polymethine reds and violets of the Color Index.
Basic reds 14, 15, 27, 37, 68 and 102 upon trail testing were found
to lack that quality of metachromasia essential to stain monocytes
and/or other leukocytes (by absorbance).
However, basic reds 36 and 49 (all polymethines) established as
useful for differential metachromatic staining of white blood cells
in the first continuation-in-part application also have use here.
Basic violet (dyes) 7, 15, 16, 39 and 40 (again all polymethines)
were also found useful and operative. However, basic violet 14, not
classed as a polymethine in the Color Index, developed no
metachromasia in dyeing white blood cells under absorbance or
emissive energy.
* * * * *