Dry preparation for reticulocyte staining

A dry stain for reticulocyte staining comprising a mixture of new methylene blue N dye, potassium acetate, and an anti-blood-clotting agent. The stain is prepared in a concentrated aqueous solution, filtered, dispensed volumetrically into tubes, and dried. The correct quantity of dye per tube is determined spectrophotometrically, and the proper ratio of dye to potassium acetate is determined by pH measurements. For staining reticulocytes, whole blood is added to the tube and mixed with the dry stain. Smears are then prepared on slides in a conventional manner for microscopic examination.

BACKGROUND OF THE INVENTION 
This invention relates to a dry stain which can be mixed with whole blood 
to prepare a microscope slide, and to a method of preparing the stain. 
The determination of reticulocyte count as a percentage of total red cells 
is an extremely useful and important procedure in clinical hematology. A 
number of techniques have been used for staining blood for such analysis. 
Most of these techniques involve the use of a liquid stain preparation 
which is mixed with the blood. 
Three techniques for reticulocyte staining are described in Staining 
Procedures (3rd Ed.) edited by George Clark, published for the Biological 
Stain Commission by the Williams and Wilkens Company, Baltimore, Maryland 
(pp. 128-130). These techniques are described in the following 
publications: (1) Brecher, G., New Methylene Blue as a Reticulocyte Stain, 
American Journal of Clinical Pathology, Vol. 19, pp. 895-896 (1949), (2) 
Robertson, O. H., The Effects of Experimental Plethora on Blood 
Production, Journal of Experimental Medicine, Vol. 26, pp. 221-237 (1917), 
and (3) Cunningham, R. S., A Method for Permanent Staining of Reticulated 
Red Cells, Archives of Internal Medicine, Vol. 26, pp. 405-409 (1920). 
The technique described by Brecher uses a solution of 0.5% new methylene 
blue N and 1.6% potassium oxalate in water, and this solution is mixed 
with blood in equal volumes. After 10 to 15 minutes a drop is placed on a 
slide and a smear is made in a normal manner. The Brecher formula, which 
is presently the most widely used, has been favored because of the sharper 
reticulum it produces and because of the consistency of dye purity from 
batch to batch. One of the problems associated with the Brecher stain is 
that precipitate forms in it continuously over a long period of time, and 
therefore, in order to avoid slide artifacts which might interfere with 
the count, the stain must be filtered each time it is used. If the stain 
is dried, precipitates form when it is redissolved. Even if the insoluble 
matter is removed directly prior to drying, more precipitate forms 
immediately when the stain is dissolved in the blood. This precipitate is 
probably an insoluble salt of the divalent anions and the new methylene 
blue N dye. 
The Robertson publication describes a wet procedure using brilliant cresyl 
blue. A saturated aqueous stock solution of the stain is prepared in 0.85% 
NaCl. The stock is diluted 80-180 times, and 20 volumes of the dilute 
solution are mixed with one volume of blood. The counts are made of fresh 
preparations sealed with vasoline to prevent drying. The technique 
reported by Cunningham also uses brilliant cresyl blue followed by 
Wright's stain. Using a solution of 0.3% alcoholic brilliant cresyl blue, 
a drop is dried on a slide. A drop of blood is placed on the stain, mixed, 
spread and dried. The smear is then counterstained by the normal technique 
with Wright's stain. 
One of the problems with liquid stains is that insoluble granules form 
continuously in the solution. Since such a precipitate can produce 
artifacts on the slide which would interfere with the reticulocyte count, 
the stain must be filtered each time it is used to remove this residue. 
Following filtration, the stain must be volumetrically dispensed into the 
blood that is to be analyzed. These are time consuming, incovenient and 
messy operations for the modern clinical laboratory. 
SUMMARY OF THE INVENTION 
It is therefore an object of the present invention to provide a 
reticulocyte stain that can be prepared in dried form. A further object is 
to provide such a stain which can be mixed directly with whole blood 
without forming precipitates which can cause artifact problems. Still 
another object is to provide a dry stain which can be employed in a 
reticulocyte staining technique which is cleaner and simpler than 
techniques employing conventional liquid stains. 
Briefly, the stain of the present invention comprises a mixture of new 
methylene blue N dye, a salt of a strong base and a weak acid of a 
monovalent anion, and an amount of an anticoagulent effective to prevent 
the occurrence of blood clotting. 
The stain is preferably stored in tubes into which a predetermined volume 
of blood is to be added. The stain stock solution comprising the dye, salt 
and anticoagulant is dispensed into the tubes and then dried. As used 
herein, the word tube means test tube or other suitable container, the 
shape of which is immaterial.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The dry stain preparation which is described herein can be mixed directly 
with blood for reticulocyte staining. The stain is prepared in a 
disposable stain tube which can be stored under refrigeration until such 
time that a predetermined volume V.sub.b of blood is added thereto for 
staining. All volumes referred to herein are measured in ml. 
The salt employed in the manufacture of the stain of the present invention 
increases the pH of the stain into a favorable range around neutrality. A 
pH value in the range of 5-9 is acceptable, but outside that range the 
staining is affected, becoming lighter at lower pH values and darker at 
higher pH values. 
Incorporated in the dry stain of the present invention is a dye known in 
the art as new methylene blue N which is readily available in powdered 
form. This dye is also known as C. I. No. 52030, which number is taken 
from the text Colour Index edited by F. M. Rowe, 2nd Edition, published by 
the Society of Dyers and Colourists, Bradford, England and The American 
Association of Textile Chemists, Lowell, Massachusetts (1956-1958, 
supplement 1963). To utilize this dye in the method of this invention, a 
concentrated aqueous solution thereof referred to as the dye stock 
solution is prepared and filtered or centrifuged to remove insolubles. 
A concentrated aqueous solution of a salt of a strong base and a weak acid 
of a monovalent anion is also prepared. The salt is employed in the 
manufacture of the stain of the present invention to increase the pH of 
the stain into a favorable range around neutrality. A pH value in the 
range of 5-9 is acceptable, but outside that range the staining is 
affected, stained cells becoming lighter at lower pH values and darker at 
higher pH values. By strong base is meant a compound such as sodium 
hydroxide, potassium hydroxide or the like. By a weak acid of a monovalent 
anion is meant acetic acid, propionic acid, butanoic acid or the like. 
Preferred salts are sodium acetate and potassium acetate. The two 
previously prepared aqueous solutions, i.e., the dye and salt solutions, 
are mixed in such a ratio as to produce the desired pH at the final 
dilution. This pH value is determined by diluting the contents of a single 
tube, which aliquot size will be determined hereinbelow, with a volume of 
deionized water equal to the volume of blood to be added to the stain tube 
and reading the pH by standard techniques. Although values of pH from 5 to 
9 are acceptable, values near 7 are generally preferred. However, other 
factors such as the effect of the salt on blood coagulation may cause the 
optimal pH to vary from values near 7. 
The aliquot size of the mixed solution is determined as follows. A sample 
volume V.sub.s of the mixed solution of dye and salt is diluted to 500 ml 
in 0.005 molar, pH 7 phosphate buffer. Using a spectrophotometer, an 
absorbance spectrum is run from 550 to 650 nm. Since the spectral 
absorbance is sensitive to temperature, it is necessary to make spectral 
measurements at a constant temperature, e.g. 22.degree. C. In addition, 
the solutions may be bleached by excessive exposure to light, so 
appropriate precautions should be taken. As shown in FIG. 1 an aliquot 
size corresponding to 50 .mu.l of 10% w/v new methylene blue N dye gives 
peak absorbance readings in the 0.5 to 1.0 range when the measurement is 
performed as herein described. Letting D be the average of the absorbances 
at the two absorbance maxima for a 1 cm light path-length, the aliquot 
volume V.sub.a is given by 
EQU V.sub.a =KV.sub.b V.sub.s /D (1) 
where K is a constant which may be chosen to correspond to the depth of 
staining desired. For light staining, K is equal to 0.38, and for dark 
staining, K is equal to 0.75. An intermediate value of K of about 0.53 is 
preferred. Improved results may be obtained by taking two 
spectrophotometer readings and averaging the results. 
A sufficient amount of an anticoagulant is added to the stock solution to 
prevent blood clotting or platelet aggregation upon mixture of the 
solution with whole blood. The exact amount of anticoagulant required will 
depend somewhat upon the choice of salt in the stock solution. A preferred 
anticoagulant is ethylene diamine tetracetate anticoagulant (EDTA) which 
is well known in the art as an anticoagulant for use in the collection of 
blood samples. 
At this point in the process the following control checks can be made on 
the stain solution. An amount V.sub.t of stain stock solution is mixed 
with 0.005 M pH 7 phosphate buffer. The volume of said buffer employed is 
500 ml for each ml of blood for which the stain tube is designed. Thus. if 
a volume V.sub.t of stain stock solution sufficient to stain 1.0 ml of 
blood is to be added to each stain tube, then a volume V.sub.t of stain 
stock solution is dispensed into 500 ml of said buffer solution. A clean, 
dry 1 cm diameter cuvette is filled with the resultant solution and placed 
in a spectrophotometer. The average of the 530 nm and 630 nm peaks is 
determined in the aforementioned manner. This average optical absorbance 
should be within the range of 0.38 and 0.75, the preferred value being 
0.53. 
The pH of the stain stock solution can be determined in the following 
manner. An amount of distilled water is added to a volume V.sub.t of stain 
stock solution to form a total volume equal to the volume V.sub.b. The 
measured pH should be substantially equal to the predetermined pH value. 
Since the volume V.sub.t is usually much smaller than V.sub.b, it is often 
sufficient to add a volume V.sub.t of stain stock solution to a volume 
V.sub.b of distilled water to make this pH check. 
An amount of the resultant mixture suitable for mixing with the 
predetermined amount V.sub.b of blood is dispensed into a small test tube 
or similar container referred to herein as a stain tube. The solution is 
evaporated to dryness in the stain tube by any convenient method; however, 
excessive heating which could cause degradation of the dye should be 
avoided. Such evaporation methods as freeze drying and air evaporation may 
be employed. A small amount of heat may be employed during evaporation. 
The tubes may then be stored in a sealed container or individually 
stoppered. When properly sealed, the resultant dry stain may be stored at 
2.degree.-6.degree. C. for at least one year. 
The dry stain is utilized in the following manner. It has been found to be 
convenient to employ 1.0 ml samples of blood, and if this volume is 
employed, the term V.sub.b disappears from equation 1. The blood is added 
to the tube containing the stain, and the tube is capped and placed on an 
aliquot mixer for 15 minutes. Smears are then prepared immediately and 
observed under oil immersion. At the intermediate concentration, wherein 
the value of K in equation 1 is taken to be 0.53 and the pH value of the 
mixture of the dye stock solution and the salt solution is in the central 
region of the preferred range, the red cells appear grey to greenish-grey. 
The reticulum stands out sharply as dark blue-black strands. With the 
lower stain concentrations, the red cell staining becomes more reddish, 
taking on a clay color, while at higher concentrations, their color is 
blue. The reticulum staining does not appear to change significantly in 
either case but always remains very dark and distinct. If the staining is 
too light, it becomes difficult to distinguish cells, whereas dark 
staining reduces the contrast between the reticulum and background. 
A preferred stain, which gives excellent reticulocyte staining, comprises 
new methylene blue N dye, potassium acetate and EDTA. In accordance with 
the above described method of forming the dry stain, the dye and salt 
should be present in relative amounts which are effective, when diluted 
with a volume V.sub.b pure deionized water, to form a solution, the pH of 
which is between 5 and 9. The EDTA should be present in an amount that is 
effective to prevent the occurrence of blood clotting. This preferred 
composition comprises 30-70 wt.% new methylene blue N, 15-35 wt.% 
potassium acetate and 15-35 wt.% EDTA, the EDTA preferably being present 
in an amount equal to the amount of potassium acetate. 
Since no precipitation on initial mixing of the salt and dye occurs, it may 
be advantageous in a manufacturing operation to eliminate the step of 
mixing the solutions outside the tubes and instead to add aliquots of the 
two solutions directly to the tubes. By using solutions of the highest 
concentrations, the volume of liquid to be evaporated will be kept to a 
minimum. 
SPECIFIC EXAMPLE 
The lyophilized stain of the present invention is particularly adaptable 
for storage in small test tubes in amounts necessary to stain a single 
blood sample, which in the following example is taken to be 1.0 ml whole 
blood. The following is a typical example of a method of forming such 
lyophilized stain. 
A dye stock solution is prepared by dissolving new methylene blue N (C.I. 
No. 52030) in distilled water at a concentration of 10%, i.e., 1 gm dye 
per 10 ml distilled water. Enough reagent for about 20 tubes of 
lyophilized stain can be obtained from one ml of dye stock. The dye stock 
solution is then filtered through course filter paper to remove 
insolubles. A volume of 50 .mu.l of dye stock is diluted in 500 ml 0.005 M 
pH 7 phosphate buffer. A clean, dry 1 cm diameter cuvette is filled with 
the resultant solution and placed in a Beckman Model 25 spectrophotometer. 
The cuvette should not be prerinsed with the solution since it will 
slightly stain the cuvette, thereby producing an erroneous result. During 
this process, unnecessary exposure to light should be avoided. As 
illustrated by curve 10 of FIG. 1, the spectrophotometer is employed to 
obtain an absorbance spectrum (in optical density units) over the range of 
550 nm-650 nm at 22.degree. C. A second 50 .mu.1 sample of dye stock is 
diluted in phosphate buffer as described above and a second absorbance 
spectrum is obtained on the same graph as illustrated by curve 12 in FIG. 
1. The base line or zero absorbance curve is represented by line 14. 
the average absorbance D of the two spectra is obtained in the following 
manner. Curves 10 and 12 have absorbance peaks at 590 nm and 631 nm. The 
absorbance values of curves 10 and 12 at the 590 nm peak are 0.489 and 
0.474, these values being corrected to 0.476 and 0.461 after subtracting 
the base line value of 0.013 from the actual readings. The average 
absorbance value of the 590 nm peak is therefore 0.468. Similarly, the 
average absorbance of the 631 nm peak is obtained by subtracting the base 
line value of 0.022 from the two absorbance readings of 0.560 and 0.530 to 
obtain absorbance values of 0.528 and 0.508 for curves 10 and 12, 
respectively. The average absorbance of the 631 nm peak is therefore 
0.518. The average absorbance D of the 590 nm peak and the 631 nm peak is 
determined to be 0.493. 
The volume V.sub.a of dye stock required per tube of lyophilized stain is 
calculated from equation 1. The intermediate value of 0.53 is employed for 
the constant K. The sample volume V.sub.s of dye stock employed in the 
determination of the value of absorbance D was 50 .mu.1; therefore, the 
value V.sub.s to be substituted into equation 1 is 0.05. For an average 
absorbance D of 0.493, the aliquot volume is therefore determined to be 
0.053 ml to prepare an amount of lyophilized dye to stain 1 ml of blood. 
The stain stock solution, which is the final reagent to be added to the 
tube just prior to drying, is prepared in the following manner. Potassium 
acetate is selected as the salt to raise the pH of the stain into a 
favorable range around neutrality. Since the presence of potassium acetate 
increases the tendency for blood to coagulate, the lowest amount of 
potassium acetate was selected to safely prevent the pH level from 
dropping below the minimum acceptable value of 5.0. Referring to FIG. 2, 
it can be seen that for concentrations of potassium acetate around 0.0025 
grams per cc in a solution of 0.5 grams per 100 cc of new methylene blue N 
stain, the pH value is about 6 and that the pH value drops sharply at 
lower concentrations of potassium acetate. The slightly higher pH value of 
6.1 was therefore chosen for the pH of the final stain stock solution, 
thereby necessitating a concentration of potassium acetate of 0.0028 grams 
per cc of the 0.5% w/v new methylene blue N stain. It is noted that the 
stated concentration of new methylene blue N stain solution is about that 
of one aliquot volume V.sub.a diluted to 1.0 cc. The total amount of stain 
stock solution will therefore require x grams of potassium acetate where x 
is determined by the equation 
EQU x=0.0028 v.sub.d /V.sub.a (2) 
where V.sub.d is the total amount of dye stock solution in ml which has 
been prepared. Since V.sub.d is 500 ml and V.sub.a has been determined to 
be 0.053 ml then x can be determined to be 26.42 grams. 
As stated previously, the presence of potassium acetate increases the 
tendency for blood to coagulate; therefore, more EDTA is required than if 
potassium acetate were not employed. It has been found that an amount of 
EDTA in the final stain stock solution equivalent to the amount of 
potassium acetate will provide the necessary anticoagulation effect. 
Therefore, 26.42 grams of EDTA are employed. 
Because of the dispensing equipment employed in the filling of the stain 
tubes, a minimum amount of the stain stock solution had to be dispensed in 
each tube to insure accuracy. Therefore, the potassium acetate and EDTA 
were dissolved in a volume V.sub.s distilled water in ml which was mixed 
with the dye stock solution to form the final stain stock solution. The 
volume V.sub.s is determined by the equation 
##EQU1## 
where V.sub.t is the desired volume of stain stock to be dispensed per 
tube in ml as determined by the dispensing equipment, viz. 0.2 ml. Since 
V.sub.d is 500 ml and V.sub.a is 0.053 ml, the volume V.sub.s is 1,386.8 
ml. 
The 26.42 grams of potassium acetate and 26.42 grams of EDTA are added to 
the 1,386.8 ml of distilled water where these additives dissolve rapidly 
at room temperature. The resultant solution is mixed with the 500 ml of 
dye stock solution to form the final reagent to be added to the tubes. 
Insolubles are removed from this stain stock solution by filtration with 
coarse filter paper. Prior to dispensing this stain solution, the 
following control checks are made . 
Since 0.2 ml of the stain stock solution is to be dispensed into each stain 
tube, the same amount of that solution is diluted in 500 ml of 0.005 M pH 
7 phosphate buffer in a clean, dry 1 cm cuvette which is placed into a 
spectrophotometer. Using the method described above in conjunction with 
FIG. 1, the average absorbance value of the two peaks should be 
0.53.+-.0.02 optical density units. 
If the optical density is within the prescribed range, the stain stock 
solution pH is then determined. A single tube volume of 0.2 ml of stain 
stock solution is diluted in 1 ml of distilled water. The pH value of the 
resultant solution should be within the range of 6.1.+-.0.3. 
If the above quality checks have been met, the stain stock solution is 
ready for dispensing into the stain tubes. A volume of 0.2 ml of stain 
stock solution is dispensed into each tube. A precision of .+-.1% should 
be maintained during the filling of the tubes. All of the filled tubes are 
then placed in a refrigerator where the temperature is gradually decreased 
to -50.degree. C. over a time period of two hours. The refrigerator is 
then evacuated, and the temperature is then gradually increased to ambient 
temperature during a two hour time period. The chamber remains evacuated 
for 2-24 hours to insure removal of the water component of the dispensed 
stain stock solution. The tubes are then individually corked as soon as 
possible after they are removed from the lyophilization chamber to avoid 
the possibility of moisture from the air penetrating the dry stain. The 
cork must fit well enough to prevent moisture from reaching the dry stain. 
The tubes can then be stored at 2.degree.-6.degree. C. for at least one 
year prior to use. 
Although the present invention has been described with respect to details 
of certain embodiments thereof, it is not intended that such details be 
limitations upon the scope of the invention except insofar as set forth in 
the following claims.