Multi-purpose blood diluent for use in electronic blood analysis instrumentation

A multi-purpose blood diluent especially suitable for use in electronic enumeration and sizing of blood cells, determination of hemoglobin concentration and their collective indices and platelet parameters in a single blood sample by means of suitable electronic instrumentation. The diluent is characterized as azide-free and capable of affording accurate, reproducible hematological test results from the said suitable electronic instrumentation. The diluent employs a chelating agent which functions as a bacteriostat and fungistat agent to prevent adverse growth of micro-organisms and a compatible stabilizing agent to maintain desired morphology of red blood cells and an inorganic metallic sulfate for suppressing turbidity caused by abnormal globulins and/or high leukocyte counts which are encountered in certain blood samples. Because of the capability for use of the diluent in platelet determinations by the instrumentation, the diluent further is free of detergent action which would cause bubbles capable of interfering with the enumeration of platelets by the instrumentation. A modified and adjusted embodiment of the diluent enables its use in veterinary laboratory testing applications.

BACKGROUND OF THE INVENTION 
This invention concerns a blood diluent especially suitable for use in 
electronic enumeration and sizing of blood cells, determination of 
hemoglobin and their collective indices and platelet parameters in a 
single blood cell sample by means of suitable electronic instrumentation. 
The diluent comprises a stable water solution of chemical salts providing 
an electrolytic solution to which a blood sample can be added so as to 
dilute the larger number of red blood cells, white blood cells, platelets 
and other blood components and enable the desired parameter of these blood 
components to be measured, counted and evaluated. 
The invention also concerns the use of a modified embodiment of the diluent 
in veterinary laboratory testing applications. 
It is a common medical diagnostic procedure to analyze and test a blood 
sample of a patient in order to make certain classic determinations with 
respect to the blood sample. This procedure is an important tool for the 
physician. Six characteristically important parameters are referred to as 
red blood cell count (RBC), the hematocrit (HCT), the hemoglobin (HGB), 
the mean corpuscular volume (HCV), the mean corpuscular hemoglobin (MCH), 
and the mean corpuscular hemoglobin concentration (MCHC). A seventh 
important determination is white blood cell count (WBC). An instrument 
which will accept a patient's blood sample and process the blood sample 
automatically and continuously to provide the parameters or determinations 
enumerated is described and claimed in U.S. Pat. No. 3,549,994. Said Pat. 
No. 3,549,994 provides acceptable definitions of said parameters and 
illuminates the problems to be solved in the handling of the blood sample 
as it is drawn through the fluid system of said patented apparatus. 
Coulter Electronics, Inc. of Hialeah, Florida also manufactures and sells 
other blood cell counting and analyzing instruments which are less 
sophisticated than the apparatus of said U.S. Pat. No. 3,549,994, but 
which are operated to determine red blood cell and white blood cell count, 
hemoglobin concentration and their collective indices such as HCT, MCV, 
MCH and MCHC. The multi-purpose diluent embodying the invention is 
suitable for use with such instrumentation as well, where problems 
attendant the successful handling of the blood sample are the same. In 
other words, this blood diluent is compatible with other electronic 
particle analysis instruments utilizing the so-called Coulter principle. 
Such instruments may be referred to herein, at times, selectively by the 
registered trademark "COULTER COUNTER.RTM." owned by Coulter Electronics, 
Inc. 
In U.S. Pat. No. 3,962,125 issued for the invention "Multi-Purpose Diluent 
For Use In Blood Analysis By Electronic Instrumentation Of The Coulter 
Type", there is described and claimed a diluent especially suitable for 
eliminating the problems attendant the use of prior blood diluents which 
use sodium azide as the effective bacteriostatic agent. U.S. Pat. No. 
3,962,125 illuminates further the characteristics of red blood cells which 
must be taken into account in order to provide a practical and suitable 
diluent. 
The diluent of U.S. Pat. No. 3,962,125 did not focus on suitability thereof 
for use in making platelet parameter determinations. At the time of the 
invention of said U.S. Pat. No. 3,962,125, Coulter Electronics, Inc. had 
marketed an instrument known as the Model "S" and Model "S"-Sr. which did 
not have the capability for making platelet determinations. Consequently, 
the diluent of U.S. Pat. No. 3,962,125 was not concerned with the critical 
factor of bubble formation by reason of detergent action thereof which 
would cause bubbles capable of interfering with the enumeration of 
platelets by electronic instrumentation capable of making such 
determinations. Since that time, Coulter Electronics, Inc. has introduced 
electronic instrumentation known as the Model "S"-Plus which does have 
platelet determination capability and in connection with the multi-purpose 
diluent embodying the invention is especially suitable for dilution of 
blood samples which are drawn through such instruments during its 
operation. 
SUMMARY OF THE INVENTION 
The invention provides a multi-purpose, electrolytic solution for use in 
hematological enumeration of blood cells, the determination of hemoglobin 
concentration and other important parameters of a blood sample and 
platelet determinations by means of automated electronic particle analysis 
apparatus of the type manufactured by Coulter Electronics, Inc. of 
Hialeah, Florida and marketed as its Model "S"-Plus. The solution is 
osmotically balanced for mean cell volume stability, is azide free and 
contains a suitable stabilizer agent for maintaining the desired 
morphology of red blood cells. The solution contains a metallic sulfate 
for suppressing turbidity caused by abnormal globulins and/or high 
leukocyte counts which are encountered in certain blood samples and is 
characterized by being free of detergent action which would cause bubbles 
capable of interfering with the enumeration of platelets by the electronic 
instrumentation. 
The invention also includes a modified and adjusted embodiment of the 
diluent solution which enables it to be used in veterinary medicine 
laboratory testing applications. 
The invention provides a blood diluent which can be used successfully with 
the electronic instrumentation of Coulter Electronics, Inc. and which is 
compatible with their present calibration for making such hematological 
analysis. 
The blood diluent embodying the invention successfully substitutes a 
non-toxic and more compatible bacteriostatic agent for sodium azide 
without diminution in effectiveness. Further, the diluent is osmotically 
balanced and unreactive so as to eliminate any interference with the 
required chemical conversion of hemoglobin materials to cyanmethemoglobin 
for proper hemoglobin concentration determinations contemplated by the 
apparatus in question. 
Further, the blood diluent embodying the invention is free of detergent 
action which would contribute to formation of bubbles in the 
instrumentation which would interfere with making the platelet 
determinations desired for the instrument.

DESCRIPTION OF PREFERRED FORMULATION 
An example of the multi-purpose blood diluent embodying the invention is as 
follows: 
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Ingredient Approximate Amounts 
______________________________________ 
Sodium chloride anhydrous 
5 gms/L 
Sodium dihydrogen phosphate . H.sub.2 O 
0.2 gms/L 
Disodium phosphate . 7 H.sub.2 O 
2.0 gms/L 
Sodium sulfate anhydrous 
10 gms/L 
8-Quinolinol (8-hydroxyquinoline) 
0.145 gms/L 
Procaine hydrochloride anhydrous 
0.125 gms/L 
Distilled H.sub.2 O Sufficient quantities 
to produce 1 liter 
______________________________________ 
The diluent specified above was adjusted to a pH of 7.2 to 7.6 by a 
suitable buffering agent which constituted the phosphate salt. The 
osmolality of the blood diluent was maintained at 316 to 330 
milliosmoles/Kg. 8-hydroxyquinoline citrate can be substituted for 
8-hydroxyquinoline with the additional advantage of more ready 
dissolution. The amount of the citrate ingredient used would be 0.145 
gms/L. Likewise, potassium dihydrogen phosphate and dipotassium phosphate 
can be substituted, respectively, for the sodium phosphate salts. 
The desired characteristic of osmotic balance is procured through the use 
of sodium chloride. The diluent is totally unreactive and osmotically 
balanced for mean cell volume with use of procaine hydrochloride and 
phosphate salts for buffering effectiveness. The procaine hydrochloride 
serves to stabilize the red blood cell volume. Sodium sulfate serves to 
eliminate turbidity due to elevated white blood cell counts which affects 
hemoglobin determinations. 
The bacteriostatic agent which replaces the sodium azide is 8-quinolinol or 
8-quinolinol citrate. The bacteriostatic agent has low toxicity and hence 
completely eliminates the problem of possible adverse toxic effect 
supplicable to laboratory technicians as would be the case with the use of 
sodium azide. Also, 8-quinolinol or 8-quinoline citrate does not form any 
known hazardous substances with copper or lead commonly used in water 
drainage systems. Acceptable substitutes were found to be 
8-hydroxyquinaldine (2-methyl-8-quinolinol), 8-hydroxyquinoline-5-sulfonic 
acid-dihydrate and 8-hydroxy-5-nitroquinoline, all being from the family 
of hydroxyquinolines. 
It has been known to use the procaine hydrochloride alone in larger amounts 
in diluent reagents of this type. However, such larger amounts resulted in 
undesirable hemolysis. We believe that the use of 8-quinolinol as a 
chelating agent in the presence of procaine hydrochloride for maintaining 
red cell morphology was not known or expected to be effective as a blood 
diluent used in this environment. These ingredients were found to be most 
compatible in the subject diluent and performed their respective functions 
without interference one with the other. 
Procaine hydrochloride is known generally to act as an anesthetic. Other 
chemical compounds which were found to be compatible with the family of 
hydroxyquinoline compounds were methohexital sodium (sodium 
a-dl-l-methyl-5-allyl-5-(1-methyl-2-pentynyl) barbituate, mepivacaine 
hydrochloride (1-methyl-2,6'-pipecoloxylidide monohydrochloride) and 
lidocaine hydrochloride. These compounds also are known generally to act 
as anesthetics. The precise quantities of each used may vary as dictated 
by their chemical formulation. 
Further, the blood diluent embodying the invention is suitable for use in 
diluting blood samples for making platelet determinations. In its activity 
to suppress growth of microorganisms, no small bubbles are produced as is 
the case with use of other bacteriostatic agents. Such small bubbles where 
produced in an electronic instrument for making platelet determinations 
could result in the bubbles being misinterpreted as small particles, such 
as platelets. Consequently, by reason of the fact that the blood diluent 
embodying the invention does not generate small bubbles during its 
bacteriostatic and fungistatic activities, the blood diluent is especially 
useful in electronic instruments such as the COULTER COUNTER.RTM. Model 
"S"-Plus now available on the market. 
Another advantage of the blood diluent by reason of its capability of being 
used in electronic instrumentation is that it is not corrosive with 
respect to segmenting and transferring valves found in the COULTER 
COUNTER.RTM. Model "S"-Plus instrument. There is produced a blood diluent 
embodying the invention which is an electrolyte capable of conducting 
current, which stabilizes the red blood cells so that their cubic volume 
can be accurately measured, which has no adverse effect on white blood 
cells and can function as an electrolyte for counting white blood cells 
and platelets by electronic methodology, has no adverse effect on blood 
platelets and does not interfere with the conversion of hemoglobin to the 
cyanmethemoglobin form in which hemoglobin is measured. 
Preparation of the diluent does not require any special procedures or any 
special order of addition of ingredients to the water. Consequently, the 
invention does not concern any methodology in formulation of the diluent. 
The mixture of ingredients is done mechanically by moderate stirring over 
suitable periods of time. The solution is then filtered through a 0.2 
micron filter and storable in suitable containers directly. Consequently, 
the cost of manufacture is modest and advantageous because it does not 
involve critical and expensive procedures. 
Although preferred formulation has been specified above, the range of pH 
and osmolality may be broadened for useful purposes. Thus, the pH range 
may be maintained from between a pH of 7.2-7.6. Likewise, the useful range 
of osmolality may be between 316 and 330 milliosmoles. This can be 
accomplished by varying the amount of active ingredients used for the 
purpose as specified herein. 
The blood diluent embodying the invention provides for advantages which are 
additional to and not found in the blood diluent described and claimed in 
U.S. Pat. No. 3,962,125 and is especially adapted for use with a blood 
sample which is passed through an electronic particle analyzer of the 
general type manufactured by Coulter Electronics, Inc. of Hialeah, Fla. 
Long term stability studies of the diluent invention reveal no microbial 
growth and no detergent action giving rise to interfering bubbles was 
encountered. No harmful effects on components of the instruments were 
encountered. 
The diluent embodying the invention can be adapted for use in veterinary 
medicine as follows: 
(a) quantity of sodium chloride anhydrous is reduced to 3.75 gms/liter. 
(b) pH is adjusted to a pH of between 7.0 and 7.6 by a suitable buffering 
agent, such as, a phosphate salt. 
(c) osmolality is maintained at between 285-300 milliosmoles/kg.