Multiple analysis hematology reference control reagent and method of making the same

A single diagnostic test reagent for use as a multiple-analysis hematology reference control for monitoring the precision and accuracy of measurements or determinations of red blood cell, white blood cell and platelet blood cell counting, hemoglobin content, hematocrit, mean cell volume and mean platelet volume determination, red cell distribution width and platelet distribution width determination, and determination of mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and thrombocytocrit. The control reagent includes ingredients for monitoring platelet parameters also notwithstanding that potential interfering effects of red blood cells normally observed in blood specimens also are present in the control reagent. The control reagent is suitable for use in automated or semi-automated hematology instruments and by users of manual methodologies. The invention includes a method of making said control reagent.

BACKGROUND OF THE INVENTION 
This invention concerns a stable hematology reference control reagent 
suitable for use in common medical diagnostic procedures to analyze and 
test a patient's blood sample for making classic determinations with 
respect to the blood sample and more particularly, provides a novel 
diagnostic test control reagent which includes ingredients providing the 
control reagent with the capability of monitoring the desired platelet 
parameters of the patient's blood sample as well as the seven classic 
determinations or parameters with respect to the blood sample. In other 
words, the invention provides a single hematology reference control 
reagent which can be used for monitoring the precision and accuracy of the 
classic hematology measurements or determinations, i.e., red blood cell 
count (RBC), white blood cell count (WBC), hematocrit (HCT), the 
hemoglobin (HGB), the mean corpuscular hemoglobin (MCH), the mean 
corpuscular volume (MCV) and the mean corpuscular hemoglobin concentration 
(MHC) and as a control also for monitoring the accuracy and precision of 
the measurement and determination of platelet parameters such as platelet 
count, mean platelet volume, platelet volume fraction (thrombocytocrit) 
and platelet distribution width using the same whole blood product that is 
conventionally used for the control for the previously mentioned classical 
hematology parameters. 
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 diagnostic 
tool for the physician. As a result of modern technological advances, 
there have been automated instruments developed which will accept a 
patient's blood sample and process the sample automatically and 
continuously to provide the parameters or determinations described above 
as the seven classic parameters of a blood sample. An instrument which 
will accept a patient's blood sample and process same automaticlly and 
continuously to provide these seven classic parameters or determinations 
enumerated is described and claimed in U.S. Pat. No. 3,549,994. Said U.S. 
Pat. No. 3,549,994 provides acceptable definitions of such parameters and 
illuminates the problems to be solved in handling of the blood sample as 
it is drawn through the fluid system of said patented apparatus. 
Coulter Electronics, Inc. of Hialeah, Fla., the assignee of this patent 
application, 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 counts, hemoglobin concentration and their 
collected indices such as HCT, MCV, MCH and MCHC. 
In the use of such an instrument which may be referred to herein, at times, 
by the registered trademark "COULTER COUNTER" owned by Coulter 
Electronics, Inc., there is required to be employed a multi-purpose 
diluent comprising an electrolyte which enables electronic measurements to 
be made by the COULTER COUNTER.RTM. instrument. A suitable multi-purpose 
diluent for use in blood analysis by an electronic instrument such as the 
COULTER COUNTER.RTM. is described and claimed in U.S. Pat. No. 3,962,125. 
A suitable reagent for determining leukocytes and hemoglobin in the blood 
sample by means of a high speed automated hematology instrument such as 
the COULTER COUNTER.RTM.is described in U.S. Pat. No. 3,874,852 issued 
Apr. 1, 1975. The reagent described and claimed in said patent is a lysing 
reagent for converting hemoglobin to a chromogen for making the desired 
determinations. 
Coulter Electronics, Inc. has heretofore provided a hematology reference 
control for accuracy in electronic estimation of blood cell values capable 
of functioning with the diluent and lysing reagents discussed above. One 
such reference control has been sold by Coulter Electronics under its 
registered Trademark 4C which comprises a modified whole blood hematology 
reference control prepared from fresh human blood. Fixed erythrocytes were 
added to simulate leukocytes. This reference control had seven known blood 
values which were stable for a desired period of time. The reference 
control was prepared for use with the COULTER COUNTER.RTM. and accessory 
mean corpuscular volume hematacrit computers and hemoglobinominators. When 
used with the blood diluent identified above, it served as a check on 
accuracy of dilution, red blood cell counts, and MCV-Hematocrit computer 
calibration. After addition of a lysing reagent, the reference control 
served as a check on white blood cell counts. Thus, the so-called 4C.RTM. 
hematology reference control was utilized in the COULTER COUNTER.RTM. for 
electronic estimation of red and white blood cells, hematocrit, mean 
corpuscular volume, hemoglobin, mean corpuscular hemoglobin and mean 
corpuscular hemoglobin concentration. Other hematology reference controls 
also were available for use with the COULTER COUNTER.RTM. which comprise 
stabilized human red blood cell suspensions such as the product known as 
Baker Haem-C.RTM. of J. P. Baker Chemical Corporation or the product known 
as CH-60.RTM. of the Dade Reagent Company of Hialeah, Fla. However, none 
of these mentioned hematology control reagents were capable of being used 
for testing accuracy of measurements of platelet parameters. Consequently, 
for making such determinations, a separate control had to be employed for 
monitoring the platelet parameters. In other words, a separate control had 
to be employed for making the platelet determinations and the hematology 
control reagents for making the seven classical measurements or parameter 
determinations could not be employed for platelet control functions. 
Further, prior art platelet counting has been inadequate because of the 
lack of stable platelet controls containing other blood components, 
namely, red blood cells. Thus, the increasing availability of 
instrumentation for distinguishing platelets from red blood cells and 
automatically performing platelet counts has created a great need for 
stable control products which contain both platelets and red blood cells. 
The single hematology control of this invention includes the capability of 
monitoring platelet parameters as well as the seven classic parameters of 
hematology measurements. This hematology control has the advantage in that 
it eliminates the need for a separate control material for monitoring 
platelet parameters and provides a more meaningful determination of 
platelet parameters in that the potential interfering red blood cells, red 
blood cell metabolites, etc., normally observed in patient blood specimens 
are present in the control material of the invention. This is especially 
important for platelet counts performed with manual methodologies using 
light microscopy or phase microscopy, where great skills on the part of 
laboratory technologists is required to adequately differentiate blood 
platelets from red blood cells and other cellular materials. The subject 
invention also provides a means for monitoring these measurements using 
manual methodology or automated or semi-automated hematology instruments 
for hematology measurements along with a means for testing accuracy of 
platelet count, mean platelet volume, platelet volume fraction 
(thrombocytocrit) and platelet distribution based on the use in the 
reference control of the present invention of the same whole blood product 
which is conventionally used for control of the classic seven hematology 
parameters. 
SUMMARY OF THE INVENTION 
This invention consists of a stable whole blood control containing 
platelets prepared by adding freshly procured human blood platelets or 
animal blood platelets either natively drawn or preserved through chemical 
or physical fixation to a conventional, commercially available whole blood 
hematology control, such as said 4C.RTM., Baker Haem-C.RTM. or Dade 
CH-60.RTM.. Such heretofore conventional, commercially available whole 
blood hematology control reagents are conditioned in accordance with the 
invention by adding sodium chloride and urea. The sodium chloride and urea 
may be added directly to the whole blood control or may be dissolved first 
in water and then added to the whole blood control. The components are 
present and/or are added in proportion such that the values for WBC, RBC, 
HGB, HCT, MCV, MCH, MCHC, red cell distribution width, platelet count, 
mean platelet volume, thrombocytocrit and platelet volume width 
distribution approximate those found in normally occurring human blood 
specimens and which can be measured by using a variety of known manual or 
automatic instrumentation methodologies. The suspension medium can 
comprise an artificial medium, human or animal plasma or animal protein 
solutions. 
The 4C.RTM. product of Coulter Electronics, Inc. is a stable reference 
control which was especially suitable for use with the COULTER 
COUNTER.RTM. instrument when establishing standards of quality control for 
the performance of the instrument. A correctly calibrated and functioning 
instrument is utilized to provide the seven measured or calculated 
parameters within a range of expected results when the 4C.RTM. is used. 
Also, the invention encompasses the disclosed method of making the stable 
reference control. 
DETAILED DESCRIPTION OF THE INVENTION 
This invention utilizes a combination of urea and sodium chloride as (1) a 
stabilizing agent for use with whole blood controls that include platelets 
and (2) an inhibitor of the detrimental effects of animal and human red 
blood cell metabolites, by-products, and chemicals inherent to the red 
blood cell and white blood cell on animal and human platelets. 
Urea alone previously has been employed as a stabilizing agent in pure 
suspensions of human blood platelets prepared as a control material to be 
used in connection with performing platelet counts. The human blood 
platelets prepared in this manner have been essentially in pure 
suspensions; no residual red blood cells or white blood cells have been 
allowed to be present in such controls. The function of the added urea in 
such control suspensions simply has been to prevent the gradual 
disintegration of the platelets during subsequent storage over a period of 
time equaling several months. The overall effect has been to provide a 
suspension of platelets that remains relatively constant (.+-.20%) in the 
number of blood platelets per unit volume. If the urea were not added, the 
number of particles per unit volume would gradually increase as the 
platelets began to disintegrate usually within several days. 
Heretofore, red blood cells and white blood cells have not been allowed to 
be present in platelet controls. The reason is believed to be that these 
cells almost immediately cause platelets to aggregate and disintegrate, 
thereby destroying the effectiveness of the mixed control material. In 
addition, urea in high concentrations was known to be detrimental to the 
stability and integrity of red blood cells, thereby apparently precluding 
its use as a preservative in suspensions containing red blood cells. As a 
result, prior known platelet controls have consisted simply of pure 
suspensions of human platelets in the presence of urea as a preservative, 
no red blood cells or other formed elements of the blood having been 
allowed to be present, thereby diminishing their effectiveness as control 
materials. 
Until the present invention, there have been no known successful 
combinations of whole blood reference controls that contain preserved 
human platelets stabilized with two molar urea. In fact, present art 
teaches that when two molar urea employed in this invention is added to 
human blood cells, the red blood cells are destroyed (Owen, J. D., 
"Computer Simulated Urea Reflection Coefficients In Human Red Blood 
Cells," Biochem. et Biophys. Acta 443, 306-310, 1976; Saunders, A. M., 
Scott, F., "Hematologic Automation By Use Of Continuous Flow Systems," J. 
Histochem. Cytochem. 22, 707-710, 1974). 
We have discovered that urea in the presence of sodium chloride serves to 
prevent the deleterious effect of autologous red blood cells and white 
blood cells on blood platelets in the suspension. Consequently, we provide 
a stable blood platelet suspension that contains red blood cells and 
approximates the composition of human blood. There is provided a single 
stable suspension which contains both blood platelets and red blood cells, 
made possible through the use of a combination of urea and sodium chloride 
which is added to previously stabilized human red blood cell suspensions, 
such as Coulter 4C.RTM., Baker Haem-C.RTM., or Dade CH-60.RTM., which can 
easily tolerate the otherwise detrimental effects of urea. The combination 
of urea, saline and the previously preserved red blood cell suspension is 
itself uniquely stable and provides a compatible medium in which either 
native blood platelets or blood platelets that have been fixed using 
formaldehyde, glutaraldehyde, osmic acid or other chemical fixative agents 
can coexist in stable form. 
Another derived benefit of this invention is that the combination of urea, 
saline and previously fixed red blood cell suspensions provides an 
environment in which the cell volume of individual blood platelets remain 
unchanged. By use of a combination blood platelet and red blood cell 
control suspension, it is now possible for laboratories to monitor the 
accuracy of platelet count, mean platelet volume (MPV) and platelet volume 
distribution (PDW) measurements, so that effective patient measurements 
may be performed on a routine basis. 
A whole blood control containing platelets according to the invention was 
prepared by mixing conventional, commercially available whole blood 
control products such as Coulter 4C.RTM., Baker Haem-C.RTM. or Dade 
CH-60.RTM. with freshly collected or chemically preserved human blood 
platelets or animal blood platelets and a mixture of urea and sodium 
chloride. After stabilizing for several days, the suspension was found to 
have stable values for the hematology parameters WBC, RBC, HGB, MCV, HCT, 
MCH, MCHC, red cell distribution width, platelet count, mean platelet 
volume, thrombocytocrit and platelet volume distribution width. The 
detrimental effects of the red blood cells and white cell analogs present 
in the whole blood controls used as substrate was prevented by the 
addition of sodium chloride and urea and the material could be used as a 
stable control for several months.

METHOD OF PREING CONTROL REAGENT 
The first step, in one mode of preparing a whole blood platelet control of 
this invention was to procure a volume of conventionally manufactured 
whole blood hematology reference control material such as Coulter 4C.RTM., 
Baker Haem-C.RTM. or Dade CH-60.RTM., or any other commercially 
manufactured or other suitable whole blood hematology control material 
comprising treated erythrocytes in an artificial medium. Then human or 
animal blood platelets were procured through venipuncture or other 
bleeding procedure of the donor human or animal subject. The human or 
animal blood platelets can then be used in any manner whatsoever either as 
freshly obtained or after any of the chemical or physical fixative 
procedures have been applied to the platelets. 
The next step was to prepare a solution or urea and sodium chloride in 
water or other vehicle for subsequent mixing with the whole blood control 
and the platelets. In the solution, the urea and sodium chloride are added 
in the following relationship, generally, to 1000 ml of medium, e.g., 
water, 
Urea--120 gms 
Sodium Chloride--9 gms 
Variations in urea used in one liter of medium range between 100 and 140 
gms and sodium chloride between 8 and 10 gms. The urea and sodium chloride 
were either mixed with each other and subsequently added to the control 
suspension or were dissolved in approximately one liter of medium, e.g., 
water, (for example,) and the resulting solution added at that time to the 
control material in amounts to be discussed hereinafter. A salt equivalent 
to sodium chloride may be utilized. 
The final step was to simply mix the commercially or individually prepared 
whole blood hematology reference control, the untreated, unstabilized or 
previously stabilized platelet suspension and the solution of urea and 
sodium chloride together. After mixing for approximately 30 minutes, and 
allowing the cell suspension to stabilize for approximately 48 hours, the 
control material was stable for complete hematoloty parameters, as stated. 
A selective suspension of red blood cells and platelets is realized. The 
suspension medium can be artificial, or a human or animal plasma or human 
or animal protein solutions. 
The following examples show how suspensions of treated red blood cells, 
platelets and solutions of sodium chloride and urea can be mixed together 
to provide the whole blood control of the present invention, as described: 
(1) A solution containing 24 grams of urea and 1.8 grams of sodium chloride 
in 200 ml of distilled water is added to 1000 ml of a treated whole blood 
suspension such as Coulter 4C.RTM.. 
A solution containing 2.4 grams of urea and 0.18 grams of sodium chloride 
in 20 ml of distilled water is added to 100 ml of a human platelet 
suspension. 
Each resulting suspension is mixed for several minutes and then treated red 
blood cell suspension and platelet suspension, both containing urea, are 
mixed with each other thoroughly for approximately thirty minutes and 
allowed to subsequently stabilize for approximately 48 hours to provide 
the reference control of this invention. 
(2) 20.0 grams of urea and 1.5 grams of sodium chloride are directly added 
to 1000 ml of a treated whole blood suspension such as Coulter 4C.RTM.. 
100 ml of a human platelet suspension is then directly added to the 
treated whole blood control suspension containing urea and sodium 
chloride. The suspension is mixed for approximately 30 minutes and allowed 
to stabilize for 48 hours to provide the reference control of this 
invention. It will be noted from this embodiment that it is not required 
to solublize the urea and sodium chloride prior to admixture with the 
treated whole blood suspension. 
(3) A solution containing 26.6 grams of urea and 1.98 grams of sodium 
chloride in 220 ml of distilled water is directly added to 1000 ml of a 
treated whole blood control suspension such as Coulter 4C.RTM.. 100 ml of 
a human platelet suspension is then added. The resulting suspension is 
mixed for approximately 30 minutes and allowed to stabilize for 48 hours 
to provide the reference control of this invention. 
In the reference control of the present invention, urea is present in the 
relationship of between about 16.7 to about 23.3 grams in approximately 
one liter of the reference control of the invention. 
Also, in the reference control of the invention, sodium chloride is added, 
in relation to the urea concentration, i.e., 1.33 to 1.67 grams sodium 
chloride to each liter of reference control containing the aforementioned 
16.7 to 23.3 grams of urea. 
It is to be understood that this added amount of sodium chloride is 
additional to the residual salinity of the treated whole blood suspension 
or platelet suspension to which same is added. It will further be 
understood that the significant factor in accordance with the invention, 
is the relationship between the urea and the added sodium chloride, not 
the total salinity of the reference control. 
It is this relationship between the urea and the added sodium chloride that 
enables the preparation of a stable reference control containing at least 
both red blood cells and platelets. 
The following will be noted with respect to the effect and importance of 
added sodium chloride. Most starting materials used in the present 
suspension, stabilized blood controls and platelet suspensions contain 
previously added sodium chloride. The invention describes adding a mixture 
of urea and sodium chloride to these materials so that they may co-exist. 
The purpose of the added sodium chloride is to maintain the osmotic and 
ionic balance when urea is added. 
One should consider that there exists an independence of cell count 
(platelets, red blood cells and white blood cells) and cell 
characteristics (mean cell volume, and cell morphology) from chemical 
composition of the reference control of the invention. The chemical 
composition of the reference control of the invention consisting of 
previously stabilized red blood cells and fixed red blood cells, 
platelets, and added urea and added sodium chloride, is independent of the 
desired cell count and cell characteristics. For example, the invention 
allows approximately 100 ml of a suspension containing either very many 
platelets or very few platelets to be added to approximately 1000 ml of an 
existing whole blood control such as Coulter 4C.RTM. having a high or low 
red blood count in white blood count value, in the high or low mean cell 
line value so that the resulting approximate volume of 1100 ml of cell 
suspension has relatively few or many platelets, red blood cells and white 
blood cells, depending upon the levels desired. 
As used in the art and in this specification, the terms "stabilized" and 
"treated" as applied to blood cells, are synonymous and are contrasted 
with the term "fixed cells." Both the initial starting materials described 
in this invention (the previously stabilized cell control and the 
platelets) can consist of either stabilized or treated cells, chemically 
fixed cells or native untreated cells. 
For example, Coulter 4C.RTM. consists of stabilized (treated) red blood 
cells and chemically fixed red blood cells. 
Stabilized (treated) cells consist of human or non-human cells to which 
various preservatives have been added to prolong their life. 
Chemically fixed cells consist of human or non-human cells which have been 
chemically hardened, i.e., tanned, usually by chemicals such as 
glutaraldehyde, formaldehyde, osmic acid or uranyl acetate. 
Stabilized cells are generally susceptible to hemolysis or disruption in 
the presence of surfactants or low osmolality, while chemically fixed 
cells retain their morphology under such environments. 
As explained, the hematology reference control embodying the invention was 
suitable for use in making manual determinations as well. Persons skilled 
in the art know the methodologies used for manual determinations of RBC, 
WBC, HGB and HCT using such control reagents. It is believed unnecessary 
to explain in detail these manual methodologies, it being sufficient to 
appreciate that the reference control of the invention can be used both 
for instrumentation and manual methodologies, as described herein.