Detection of reticulocytes, RNA or DNA

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention generally relates to the detection and enumeration of 
reticulocytes in a blood sample. More particularly, the present invention 
relates to a dye which is suitable for staining ribonucleic acid polymers 
(RNA) and deoxyribonucleic acid (DNA) and is particularly suitable for 
staining reticulocytes. The invention further relates to a fluorescent 
composition. 
2. Description of the Prior Art. 
In many cases, there is a need to detect RNA or RNA containing substances. 
Thus, for example, reticulocytes are a substance known to contain RNA, and 
detection and enumeration of reticulocytes in a blood sample are of value 
to clinicians. The reticulocyte count of a blood sample is used as an 
indicator of erythropoietic activity, has diagnostic and prognostic value 
in acute hemorrhage and hemolytic anemia, and is a measure response to 
iron, vitamin B.sub.12 and folic acid therapy. As known in the art, 
reticulocytes are precursors to mature red blood cells, and hence the term 
reticulocyte embraces the evolution and development of the cell whereby a 
mature red blood cell is generated. 
In the past, reticulocytes in a blood sample have been determined by both 
manual and automated methods by using appropriate stains such as new 
methylene blue (NMB), brilliant cresyl blue (BCB), acridine orange and 
pyronin Y. 
Vital staining with the dye new methylene blue is considered to be the 
reference method for reticulocyte determinations, and in use this dye 
precipitates RNA. The method is manual, requires counting large numbers 
(for example, 500 to 1,000) of cells with a microscope, is slow, tedious, 
and subject to errors. New methylene blue is nonfluorescent and true 
precipitated RNA is often difficult to differentiate from precipitated 
stain. 
Acridine orange has had some use in staining reticulocytes by both manual 
and automated procedures. Acridine orange also precipitates RNA, and this 
prevents quantitative estimates of RNA content because of potential 
quenching. Moreover, acridine orange does not lead to a diffuse 
fluorescent distribution of stained cells. Age profiles of the cells 
(based on RNA content being proportional to fluorescence) are not 
reliable. Acridine orange has a great affinity for the plastic tubing in 
flow cytometers which leads to increased background and lengthy procedures 
for removing the dye from the flow cytometer tubing. In addition, acridine 
orange stained cells are difficult to separate from the autofluorescent 
red cell peak, and the reticulocyte count is usually lower than that 
obtained with new methylene blue. 
The use of pyronin Y requires prior fixation of the erythrocytes with 
formalin, is cumbersome, time consuming, and generally yields poor 
results. Moreover, pyronin Y has a very low quantum efficiency, leading to 
very low fluorescent signals. 
U.S. patent application Ser. No. 460,144 filed Jan. 24, 1983, now U.S. Pat. 
No. 4,571,388 relates to a method for detecting reticulocytes utilizing 
thioflavin T as a dye for staining reticulocytes. 
A dye for staining reticulocytes preferably has the following properties: 
1. The dye should not fluoresce in the absence of RNA. 
2. The dye should have a good fluorescent quantum yield. 
3. The dye must be able to penetrate the membrane of cells containing RNA. 
4. The dye should preferably have an excitation peak at about 488 nm. 
None of the aforementioned known dyes for RNA and reticulocytes have all of 
the desirable features described hereinabove. 
Accordingly, there is a need for providing a dye better suited for staining 
reticulocytes so as to provide a procedure for accurately determining 
reticulocytes in a blood sample. 
In accordance with one aspect of the present invention, there is provided 
an improvement in a process for detecting reticulocytes wherein the 
reticulocytes are stained with a dye having the following formula: 
##STR2## 
Wherein: X=O,S, Se, or C (CH.sub.3).sub.n ; R.sub.1 =alkyl having from 1-6 
carbons; 
R.sub.2 =alkyl having from 1-6 carbons; 
R.sub.3 =fused benzene, alkyl (having 1-6 carbons, methoxy or is hydrogen; 
R.sub.4 =alkyl having 1-6 carbons, methoxy or is hydrogen; and 
n=zero or an integer from 1-6 
In accordance with another aspect of the present invention, reticulocytes 
are detected in a flow cytometer after the reticulocytes have been stained 
with the dye of the invention. 
In accordance with a further aspect of the present invention, there is 
provided a composition comprised of reticulocytes stained with the dye of 
the invention. 
The dye of the invention differs structually from thioflavin T. Thioflavin 
T has the following structure: 
##STR3## 
For convenience in the description hereinbelow and to describe a preferred 
embodiment of the invention, the dye of the invention for staining 
reticulocytes where R.sub.1 .dbd.R.sub.2 .dbd.CH.sub.3 ; R.sub.3 
.dbd.R.sub.4 .dbd.H, X.dbd.S and n=0 is referred to as "thiazole orange". 
Applicant has found that thiazole orange is an effective dye for staining 
reticulocytes. The use of thiazole orange offers the further advantage 
that thiazole orange when unbound to ribonucleic acid provides little or 
no fluorescence, whereas thiazole orange when bound to ribonucleic acid in 
the reticulocytes is fluorescent. Thiazole orange can be excited at 488 nm 
whereas thioflavin T is excited at a maximum of about 455 nm. 
In accordance with the present invention, when staining reticulocytes in a 
blood sample, the dyes of the invention may be employed as an aqueous 
solution, and in particular as an isotonic saline solution, which may 
contain a minor amount of methanol. The blood sample, which may be whole 
blood or a blood fraction, is stained with the dye by mixing the blood 
sample with the solution of thiazole orange. It has been found that by 
using thiazole orange as the stain, it is possible to detect and enumerate 
reticulocytes in a whole blood sample. 
The dyes of the invention exhibit a strong absorption peak (unbound) in the 
range of from about 470 nm to about 600 nm; however, in the unbound state, 
the dye does not provide either a detectable excitation or emission peak. 
When thiazole orange stains the RNA in the reticulocytes, the optical 
properties thereof change dramatically. In particular, the absorption 
curve shifts to a longer wavelength, and the dye now exhibits strong 
fluorescence. For thiazole orange, the excitation maximum is at about 510 
nm, and the emission maximum is at about 530 nm, giving a Stokes shift of 
about 20 nm. As a result of the excitation peak of the bound dye being in 
the order of about 510 nm, in using the automatic flow cytometer, the 
light source may be a mercury lamp which has an energy line at about 485 
nm or an argon ion laser which has strong emission at 488 nm. The lack of 
fluorescence of the dye when not bound to nucleic acid provides low 
backgrounds and allows an operator to select a fluorescent threshold (or 
"gates") for an automatic flow cytometer by simply running an unstained 
control. Although excitation may be effected at other wavelengths, the 
thiazole orange stained reticulocytes are preferably excited at a 
wavelength of from about 460 nm to about 520 nm. 
The dyes of the invention do not precipitate RNA, and as a result, the 
stained reticulocyte cells maintain a relatively homogeneous distribution 
of intracellular RNA, whereby there is a nearly linear relationship 
between the fluorescent signal measured for an individual reticulocyte and 
its RNA content. Clinically, this provides the physician with additional 
information beyond the reticulocyte count in that RNA content is a 
function of reticulocyte age. Accordingly, by using a dye of the 
invention, a clinician has the ability to do reticulocyte age profiles as 
well as simple reticulocyte counts. 
The use of dyes of the invention for staining reticulocytes in a blood 
sample offers the further advantage that the fluorescent signals from the 
stained reticulocytes are well separated from those of the mature 
erythrocytes, whereby results can be directly read in an automatic low 
cytometer without extensive data manipulation. 
Reticulocytes, RNA or DNA stained with a dye of the invention, although 
preferably enumerated in an automatic flow cytometer, can also be counted 
by a manual procedure or automated microscopy. 
Automatic flow cytometers are well known in the art, and the present 
invention is not limited to the use of any particular flow cytometer. 
Thus, for example, thiazole orange stained reticulocytes may be detected 
and enumerated in the FACS 440.TM. flow cytometer or the FACS Analyzer.TM. 
flow cytometer, both sold by Becton Dickinson and Company. In using such 
automatic flow cytometers, fluorescent gates are set by use of an 
unstained control sample, and the fluorescent gates are then used on the 
stained sample. 
The use of an automatic flow cytometer for detection and enumeration of 
reticulocytes stained with thiazole orange provides results which closely 
correlate with results obtained by a known standard method for enumerating 
reticulocytes which uses methylene blue or acridine orange. 
The use of reticulocytes stained with thiazole orange in an automatic flow 
cytometer is particularly advantageous in that there are low fluorescent 
backgrounds and fluorescent gates may be easily selected by use of an 
unstained control. Moreover, there is no precipitation of intracellular 
reticulocyte RNA, whereby the cells need not be fixed. In addition, there 
is a linear relationship between the fluorescent signal for an individual 
reticulocyte, which provides information as to reticulocyte age. 
Still another advantage of the present invention is that thiazole orange 
stained reticulocytes can be used in an automatic flow cytometer having 
lower sensitivities, e.g., one may use a mercury arc lamp as opposed to an 
argon laser. 
Although Flow Cytometry and Sorting, pages 457-58 Edited by Melamed et al., 
John Wiley & Sons, describes the use of both acridine orange and 
thioflavin T for staining RNA of living cells, this publication does not 
disclose the use of the dyes of the invention as a stain for reticulocyte 
detection and enumeration in an automatic flow cytometer.

The following example illustrates various features of the present invention 
but is not intended to in any way limit the scope of the invention as set 
forth in the claims. 
EXAMPLE 1 
A dye of the invention wherein R.sub.1 and R.sub.2 .dbd.CH.sub.3, X.dbd.S 
n=0 and R.sub.3 and R.sub.4 are hydrogen (thiazole orange) was used in a 
procedure for reticulocyte staining. 
A 1 mg/ml stock solution of the dye in methanol was prepared. A 1:8,000 
dilution of the stock solution in pH 7.2 phosphate buffered saline was 
made, 5 uL of whole blood was mixed into 1 mL of the diluted dye solution. 
The sample was analyzed on a FACS 440.TM. flow cytometer with an 
excitation wavelength of 488 nm and a 530/30 nm bandpass filter. 
FIGS. 1-6 show FACS data for reticulocyte analysis of normal and anemic 
blood using thiazole orange of this example. FIG. 1 shows a fluorescence 
histogram (FIG. 1B) and fluorescence vs. forward scatter (FIG. 1A) for 
normal, unstained blood. FIG. 2 shows a fluorescence histogram (FIG. 2B) 
and fluorescence vs. forward scatter (FIG. 2A) for normal blood stained 
with thiazole orange. FIG. 3 shows a fluorescence histogram (FIG. 3B) and 
fluorescence vs. forward scatter (FIG. 3A) for unstained anemic blood 
(17.3% reticulocytes by new methylene blue assay). FIG. 4 shows a 
fluorescence histogram (FIG. 4B) and fluorescence vs. forward scatter 
(FIG. 4A) for the anemic blood after staining. FIG. 5 shows samples of 
anemic blood (8.4% reticulocytes by new methlyene blue assay) stained with 
thiazole orange of this example for varying lengths of time: 30 minutes 
(FIG. 5A), 70 minutes (FIG. 5B) and 7 hours (FIG. 5C). analysis with new 
methylene blue and flow cytometry analysis using thiazole orange. 
Thiazole orange is shown to be fluorescent only when bound to nucleic acid 
polymers. FIG. 7 shows fluorescence emission spectra of thiazole orange 
with DNA, RNA and in phosphate buffered saline (PBS) which was free of 
nucleic acids. 
A solution of thiazole orange in DMSO was prepared (5.times.10.sup.-3 M). 
The solution was diluted into PBS (5.times.10.sup.-5 M). A cuvette 
containing 30 uL of the thiazole orange solution, 90 uL of DNA solution (1 
mg/mL) and 2.88 mL of PBS was mixed and the fluorescence emission spectrum 
measured on a Perkin-Elmer MPF-2A fluorescence spectrophotometer. The 
excitation wavelength was 500 nm and the emission was measured from 510 to 
610 nm. A cuvette containing 30 uL of the thiazole orange solution, 50 uL 
of RNA (1 mg/mL) and 2.92 mL PBS was mixed and the fluorescence emission 
spectrum measured in the same way as described above. A cuvette containing 
30 uL of the thiazole orange solution and 2.97 mL PBS was mixed and the 
fluorescence emission spectrum measured. 
EXAMPLE 2 
A dye wherein R.sub.1 .dbd.R.sub.2 .dbd.CH.sub.3,R.sub.3 .dbd.fused 
benzene, R.sub.4 .dbd.H, X.dbd.S and n=0 (thiazole red), was shown to be 
fluorescent only in the presence of nucleic acid polymers. FIG. 8 shows 
fluorescence emission spectra of thiazole red with DNA, RNA and in PBS. 
The spectra were obtained in the same way as described in Example 1 except 
the excitation wavelength was 520 nm and the emission spectra were 
measured from 530 to 630 nm. The structure of thiazole red is: 
##STR4## 
EXAMPLE 3 A dye wherein R.sub.1 .dbd.R.sub.2 .dbd.CH.sub.3, R.sub.3 
.dbd.R.sub.4 .dbd.H, X=0 and n=0 (methyl oxazole yellow), was shown to be 
fluorescent only when bound to nucleic acid polymers. FIG. 9 shows 
fluorescence emission spectra of methyl oxazole yellow with DNA, RNA and 
in PBS. The spectra were obtained in the same way as described in Example 
1 except the excitation wavelength was 480 nm and the emission spectra 
were measured from 490 to 590 nm. The structure of methyl oxazole yellow 
is: 
##STR5## 
EXAMPLE 4 
A dye wherein R.sub.1 .dbd.R.sub.2 .dbd.CH.sub.3, R.sub.3 .dbd.R.sub.4 
.dbd.H, X.dbd.S and n=1 (thiazole blue), was shown to be fluorescent only 
in the presence of nucleic acid polymers. FIG. 10 shows fluorescence 
emission spectra of thiazole blue with DNA, RNA and in PBS. The spectra 
were obtained in the same way as described in Example 1 except the 
excitation wavelength was 630 and the emission spectra were measured from 
640 to 740 nm. 
EXAMPLE 5 
A dye wherein R.sub.1 .dbd.R.sub.2 .dbd.CH.sub.2 CO.sub.2 H, R.sub.3 
.dbd.R.sub.4 .dbd.H, X.dbd.S and n=0 (thiazole orange dicarboxylate), was 
shown to be fluorescent only when bound to nucleic acid polymers. FIG. 11 
shows fluorescence emission spectra of thiazole orange dicarboxylate with 
DNA, RNA and in PBS. The spectra were obtained in the same way as 
described in Example 1. 
EXAMPLE 6 
A dye wherein R.sub.1 .dbd.R.sub.2 .dbd.CH.sub.3, R.sub.3 .dbd.R.sub.4 
.dbd.OCH.sub.3, X.dbd.S and n=0 (dimethoxy thiazole orange), was shown to 
be fluorescent only when bound to nucleic acid polymers. FIG. 12 shows 
fluorescence emission spectra of dimethoxy thiazole orange with DNA, RNA 
and in PBS The spectra were obtained in the same way as described in 
Example 1 except the excitation wavelength was 510 nm and the emission 
spectra were measured from 520 to 620 nm. The structure of dimethoxy 
thiazole orange is: 
##STR6## 
Numerous modifications and variations of the present invention are possible 
in light of the above teachings, and, therefore, are within the scope of 
the appended claims. The invention may be practiced otherwise than as 
particularly described.