AU525932B2 - Hematology reference control reagent - Google Patents

Description

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'4 TAUSTRALLA PATFNTS ACT 1O52

APPLICATION FOR A PATENT (CONVENTION OR

4 8067/79 21 We ggpLTERL..LECTRONICS, INC., of' 590 West 20th Street, Hialeah, Florida 33010, Insert full name(s) United States of America and address(es) of declarant

hereby apply for the grant of a Patent for an invention entitled "MULTIPLE ANALYSIS HEMATOLOGY REFERENCE CONTROL, REAGENT AND METHOD OF MAKING TO~ SAM""-

Delete which is described in the accompanying complete specification. the words whi Insert full name of The application is a Convention application and is based on the application(s) for patent applicant Company. or similar protection made e *00

in United States of Amnerica *0 0

0 400 4 on 15th June, 1978 underNo. 915,761 Insert country and C~000 date of filing of each inUnited States of America plication on which prior is based. 0 00

0 0 on 6th June, 1979 undorNo. 44841 Insert full name of ap 00 cant in each basic 0 S' 00

~000 ,~pPC:WON C-CPTED AND A~DV~T

ALLOW ED 000 are not applicable. 00 04 0 The address for service is caie of DAVIES COLLISON, Patent Attorneys, of 04 I Little Collins Street, Melbourne, in the State of Victoria, Commonwealth of Australia. Insert full :.amefs) *04 and address(es) of actual inventor(s). O

*0 4. Dated this 7 th day of June, 19 79 040*

I

0440 044

4 State manner in whic 0 (a member of the firm of DAVIES applicant(s) derive ti COLLIS0ON for and on behalf of -the from actual inventor(: Applicant)

To: THE COMMISSIONER OF PATENTS Davies Collison, Melbourne and Canberra.

Sigature(s) o declarant (No attestation or othb signature is required.~

B3ACKGROUND OF THSE INVENTION analyze

make This invention concerns a stable hematology c blood S

DECLARATION IN SUPPORT OF CONVENTION APPLICATION4 FOR A PATENT OR PATENT OF~ ADD.ITON 4806 7/79 (The declaration shall be made by the applicant, or, if the applicant is a body corpor. ate, by a person authorized by the body corporate to make the declarationon its behalf.)

In support of the Convention Application made for a Patent for an invention entitled MULTIM.; ANALYSIS HEMATOLOGY''REFERENCE CONTROL REAGENT AND METHOD OF MAKING THE SAME Insert full name(s) and address(es) of I Joseph Rubin, of declarant We- 590 West 20th Street, Hialeah, .Florida 33010, United States of America

do solemnly and sincerely declare as follows- Delete the words wbich 1. thie pplet are ntt applicable. Ieaf pft Mlro adto1 Insert full name of cr I am authorized by COULTER ELECTRONICS, INC. applicant Company.

the applicant.__... for the paten to make this declaration on its behalf.

2. TAwbqjcr n lat~on S.as (fined by Section 141 of the Act Wa made Insert country and in Amer ie on the.15tIIh june date of filing of each ap- plication on which priority is based. Jlam T.,.IIutciss on Insert fNil name of appli- by cant in each basic on the. iht ,Iule 19..9 mpplication. Htchjis son q~

Delete the words which are not applicable. of the invention referrej to in the basic

Insert full :.ame~s) Alan H. Runck, Douglas Armstrong and and address(es) of James T. Hitchisson, residing at: 7215 Miami actual inventor(s). Lakes Drive, Miami Lakes, Florida 33014 USA- 3520 N.W, 99th Avenue, Coral Springs, Florida 33015. U9A;and *660 Falcon Ave., Miami SpringS, Florida 33166, ISA,resp. -49- the actual inventor,.-.S of the invention and the facts upon which the applicant.. are is entiled to make the application are as followvs:-

State manner in wich bAssignment Oated: June 12, 197s, *hereby applicant(s) derive title the said actual dnventors .assigned their rights from actual inventor(s). to the applicant.

4. The basic applicaition. referred to in paragraph 2 of this Declaration wereI~

the first application... made in a Convention country in respect of the invention the. subject of the application.

Declared at Hialeah, this 23rd day of May, 979 Florida d, arn(5). of Sign=ure (No attestation or other signature is required.) Jose h Ru in, Vice Presidett I.

It is a common med1ical diagnostic procedure to comnplis. analyze and test a blood sample of a patient in ordor to measurei make certain classic determinations with respect to the A suita] blood sample. This procedure is an important diagnostic by an e~

(12) AUSTRALIAN PATENT ABRIDGMENT (19) AU (11) AU-B-48 067/79

(54) MULTIPLE ANALYSIS HEMATOLOGY REFERENCE CONTROL REAGENT AND METHOD OF MAKING THE SAME

(71) COULTER ELECTRONICS, INC. (21) 48 067/79 525 932 (22) 14.6.79 (24) 15.6.78 (31) 915761 (32) 15.6.78 (33) US 44841 6.15.79 US

(43) 20.12.79 (44) 9.12.82 (51) 2 GO1N 33/16 A61B 5/14 (72) ALAN H. RUNCK, DOUGLAS ARMSTRONG AND JAMES T. HUTCHISSON (74) DM (57) Claim 1. A multiple analysis hematology reference control comprising a suspension of red blood cells and platelets in a saline medium which includes urea, said urea being present in at least a concentration normally detrimental to stability and integrity of red blood cells, said saline medium including added sodium chloride or a salt equivalent thereof in an amount sufficient to prevent the urea from causing deleterious effects such that red blood cell and platelet parameters are generally stable and approximate those occurring in mammalian blood specimens, wherein said control is capable of being used for testing the accuracy and precision of classical red blood cell and platelet parameters.

46* 2

*116/64/13

sodium 20.0 grams of urea and 1.5 qrams of a treated whole chloride are directly added to 1000 ml of 100 ml of a human blood suspension such as Coulter 4C. -11 1. -1A 1-A 4- -P-Qa=4- m.A

C OM M ON WEALTH OF

PATENTS ACT 1952 COMPLETE SPECIFICATION 52 59 AUSTRALIA

3

(Original)

FOR OFFICE USE

48067/79 Class Int. Class

Application Number: Lodged:

Complete Specification Lodged: Accepted: Published: T\ ~g Priority:

Related Art: Ne,

f4 6 <S1

"'Name of Applicant: COULTER ELECTRONICS, INC. 4 1

S,, Address of Applicant: 590 West 20th Street, Hialeah, Florida 33010, United States of America

Actual Inventor(s): ALAN H. RUNCK, DOUGLAS ARMSTRONG and JAMES T. HUTCHISSON

Address for Service: DAVIES COLLISON, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Complete specification for the invention entitled: "MULTIPLE ANALYSIS HEMATOLOGY REFERENCE CONTROL REAGENT AND METHOD OF MAKING THE SAME"

The following statement is a full description of this invention, including the best method of performing it known to us

1

C i r i

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,

o provides a novel diagnostic test control reagent which

includes ingredients providing the control reagent with the

Scapability 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, 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 (MCHC) and as a control

also for monitoring the accuracy and precision of the

mieasurement 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 technolog-

ical advances, there have been automated instruments

1' 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 automatically and continuously to provide these

seven classic parameters or determinations enumerated is

described and claimed in U.S. Patent No. 3,549,994. Said U.S. Patent 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, Florida, the

assignee of this patent application, also manufacturers and

sells other blood cell counting and analyzing instruments

which are less sophisticated than the apparatus of said

U.S. Patent 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

Scomp ising an electrolyte which enables electronic

measurements to be made by the COULTER COUNTER instrument.

A suitable multi-purpose diluent for use in blood analysis

by an electronic instrument such as the COULTER COUNTEI

is described and claimed in U.S. Patent 3,962,125.

SA 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

Sis described in U.S. Patent 3,874,852 issued April 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 comprised 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 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 4- "hematology reference

control was utilized in tne COULTER COUNTERV for electronic estimation, of red and white blood cells, hematocrit, mean corpuscular volume, hemoglobin, mean corpuscular hemo- globin and mean corpuscular hemoglobin concentration.

Other hematology reference controls also were available for

use with the COULTER COUNTE' which comprise stabilized

human red blood cell suspensions such as the product known

as Baker Haem- of J. P. Baker Chemical Corporation or the

product known as CH-60" of the Dade Reagent Company of

0 Hialeah, Florida. However, none of these mentioned hema-

J tology 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 oi 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 automat-

ically 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 S 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 S invention. This is especially important for platelet counts

C> performed with manual methodologies using light microscopy

or phase microscopy, where great skills on the part of laboratory technologists is required to adequately differe-

ntiate 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.

SUMMAY OF THE IN VE1TION

This invention consists of a stable whole blood control and sodixL ,containing platelets prepared by adding freshly procured human-_ whole blc blood platelets or animal blood platelets either natively drawn inhibitox or preserved through chemical or physical fixation to a red blooc conventional, commercially available whole blood hematology cD inherent I. control, such as said 4CS Baker. Haem-C or Dade animal ax Such heretofore conventional, commercially available whole blood

hematology control reagents are conditioned in accordance with M C Do stabilizi 00 the invention by adding sodium chloride and urea. The 1444 10 plateletE sodium chloride and urea may be added directly to the whole connectic blood control or may be dissolved first in water and then added blood plz to the whole blood control. The components are present and/or ally in I are added in proportion such that the values for WBC, RBC, HGB, white blc HCT, MCV, .MCH, MCHC, red cell distribution width, platelet controls. count, mean platelet volume, thrombocytocrit and platelet suspensic volume width distribution approximate those found in normally integrat: occurring human blood specimens and which can be measured by over a PE using a variety of known manual or automatic instrumentation 44,. effect hz 4* 4

methodologies. The suspension medium can comprise a artificial remains j medium) human or animal plasma or animal protein solutions. plateletc The 4C&product of Coulter Electronics, Inc. is a number o: stable reference control which was especially suitable for as the p' Y* use with the COULTER COUNTER@instrument when establishing several 4 standards of quality control for the performance of the

instrument. A correctly calibrated and functioning have not instrument is utilized to provide the seven measured or The reas( calculated parameters within a range of expected results when immediat the 4C® is used. disinteg Also, the invention encompasses the disclosed meh1 pf mixed co ~je he stable reference control.

A) preparing a stabilized suspension of human or animal blood urea a

platelets; volume

B) procuring a conventional whole blood hematology reference al

DETAILED DESCRIPTION OF TH1 INVENTION

This invention utilizes a combination of urea

and sodium chloride as a stabilizing agent for use with

whole blood controls, that include platelets and an

inhibitor of the detrimental effects of animal and human

red blood cell metabolites, by-products, and chemicals

o inherent to the red blood cell and white blood cell on

t- animal and human platelets.

Urea alone previously has been employed as a 0 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 essenti-

ally 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 dis-

integration 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 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

Soncentrations 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

a of human platelets in the presence of urea as a preserv-

S ative, no red blood cells or other formed elements of the

blood having been allowed to be present, thereby diminishing 0. 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, "Computer Simulated Urea Reflection Coeffir

cients in Human Red Blood Cells", Biochem, et Biophys Acta

443, 306-310, 1976; Saunders, A. 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

-9-

/l-Y i, I blood cell suspensions, such as Coulter 44 Baker Haem-c,

Sor1 Dade CH-60d, which can easily tolerate the otherwise pres detrimental effects of urea. The combination of urea, pr" was saline and the previously preserved red blood cell suspension

is itself uniquely stable and provides a compatible medium urea for in which either native blood platelets or blood platelets

Sthat have been fixed using formaldehyde, glutaraldehyde-

ing osmic acid or other chemical fixative agents can coexist '0 00 in stable form.

T0 Another derived benefit of this invention is

that the combination of urea, saline and previously fixed

d red blood cell suspensions provides an environment in which

the cell volume of individual blood platelets remain un-

o changed. By use of a combination blood platelet and red

blood cell control suspension, it is now possible for laboratories to monitor the accure-ay of platelet count, mean

a 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 accord-

to the invention was prepared by mixi.ng conventional, of commercially available whole blood control products such as

Coulter 4CO, Baker Haem-d or Dade CH-60.with freshly

collected or chemically preserved human blood platelets or

animal blood platelets and a mixture of urea and sodium

chloride. After stabilizing fpr several days, the suspension ns 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

d 30 and platelet volume distribution width. The detrimental

-10- E 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

005 for several months. qO*

-lOa-

~ETHOD M OF PEARN CONTROLREGN ori The first step,.in one mode of preparing a whole cont blood platelet control of this invention was to procure a stab volume of conventionally manufactured whole blood hematology reference control material such as Coulter rD ~gR) Baker Haem-d 'Jor Dade CH-6& or any other commercially r Sfor manufactured or other suitable whole blood hematologyfo

control material comprising treated erythrocytes in an L00 ive c.0 artificial medium. Then human or animal blood platelets were procured through venipuncture or other bleeding 1 h anix ~0 procedure of the donor human or animal subject. The human or animal blood platelets can then be used in any tree manner whatsoever either as freshly obtained or after any chl c of the chemical or physical fixative procedures have been who] applied to the platelets.

11 The next step was to prepare a solution of urea 1. and sodium chloride in water or other vehicle for subse- isZ quent mixing with the wh'le blood control and the platelets. s uci In the solution, the urea and sodium chloride are added

in the following relationship, generally, to 1000 ml of graii medium, e.g. water, addE Urea 120 gins Sodium Chloride 9 gins mini Variations in urea used in one liter of medium range between 25 pla 100 and 140 gins and sodium chloride, between 8 and 10 gins. eac] The urea and sodium chloride were either mixed with each all( other and subsequently added to the control suspension or to were dissolved in approximately one liter of medium, e.g.

water, 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

cm 30 minutes, and allowing the cell suspension to stabilize

for approximately 48 hours, the control material was stable for complete hematology parameters, as stated. A selec-

ive 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 ard solutions of sodium

chloride and urea can be mixed together to provide the

whole blood control of the present invention, as described:

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.

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 the 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.

-12-

j

ND 46* 2

*116/64/13

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. 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 t" minutes and allowed to stabilize for 48 hours to provide

O the reference control of this invention. It will be o0 t 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.

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. 100 ml of a human

platelet suspension is then added. The resulting suspen-

sion 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 ;3.3 grams in approximately one liter of the

control of the invention.

Also, in the reference control of the invention,

sodium chloride is added, in relation to the urea concen-

tration, 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 cf 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

S chloride, not the total salinity of the reference control.

o It is this relationship between the urea and

Co 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, stabi- lized blood controls and platelec 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 m'l of a suspension containin~g 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 having

.14a

L

*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

Ro 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 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 add- ed 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 susceptable to

hemolysis or disruption in the presence of surfactants or

low osmolality, while chemically fixed cells retain their

morphology under such environments. NOTE: Throughout the specification and claims, the term "saline medium" means a solution of sodium chloride or

chemical salt equivalent thereof.

control. was utiiiLzea in tne CUUiL'VJR CUUNTERk-a tor eiectronic

i T

A

Claims (8)

TECLAIMS D)EFININtG THE INVENTION ARE AS FOLLOWS:

1. A multiple analysis hematology reference control comprisin~g a suspension of red blood cells and platelets in a saline medium which includes urea, said urea being present in at least a concentration normally detrimental to stability and integrity of red blood cells, said saline medium including added sodium chloride or a salt equivalent thereof in an amount sufficient to prevent the urea from causing deleterious effects such that red blood cell and platelet parameters are genera .y stable and approximate A 00

*000 those occurring in mammalian blood specimens, wherein said control is capable of being used for testing the accuracy and precision of classical red blood cell and platelet parameters.

2. A reference control as described in claim 1 in which 1(0 A' A *l A said medium is an artificial one.

3. A reference control as described in claim 1 in which C. (lOW said medium is a human or animal blood plasma.

4. A reference control as described in claim 1 in which said medium is a human or animal protein solution.

A reference control as described in claim 1 in which said platelets are of human or animal origin.

6. A reference control as described in claim 1 in which urea is present in the relationship of between 16.7 to 23.3 grams in approximately 1 liter of the reference control.

7. A reference control as described in claim 6 in which sodium chloride is added in the relationship of between 1. 33 and 1. 67 grams to approximately one liter of the reference control.

8. A multiple analysis hematology reference control com- prising a single suspension of red blood cells and platelets, said suspension including urea and sodium chloride in a 16

v~i suspension medium which enables use of the control for monitoring the accuracy and precision of measurements or determinations of classical hematology parameters and platelet parameters of blood sample, said urea being present in at least a concentration normally detrimental to stability and integrity of red blood cells, said urea concentration not causing deleterious effects such that red blood cell and platelet parameters are generally stable and approximate those occurring in mammalian blood specimens, wherein said reference control is capable of being used for testing the accuracy and precision of classical red blood cell and platelet parameters.

9. A reference control as described in Claim 8 in which

said medium is an artificial one.

A reference control as described in claim 8 in which

said medium is a human or animal blood plasma.

11. A reference control as described in claim 8 in which

said medium is a human or animal protein solution.

12. A reference control as described in claim 8 in which said

platelets are of human or animal origin.

13. A reference control as des'cribed in claim 8 in which the

urea is present in the ratio of between 16.7 and 23.3 grams in

approximately one liter of the reference control.

14. A reference control as described in claim 13 in which

sodium chloride is added in the relationship of between 1.33

and 1.67 grams in approximately one liter of said reference

control.

A method of making a single hematological reference

control for monitoring the accuracy of measurements of both

conventional hematology parameters and platelet parameters

comprising;

1~

A) preparing a :stabilized suspension of human or animal blood

platelets;

B) procuring a conventional whole blood hematology reference

control comprising erythrcytes in a suspension medium;

C) mixing the stabilized platelet suspension and the reference

control with a mixture of urea and sodium chloride or the like salt

selected to be compatible with the hematology components of

the mixture for a preselected period of time; and

D) allowing the resultant suspension to stabilize for

approximately 48 hours,

said urea being present in at least a concentration

normally detrimental to stability and integrity of erythrocytes,

said urea concentration not causing deleterious effects such that

erythrocyte and platelet parameters are generally stable and

approximate those occuring in mammalian blood specimens.

16. The method as described in claim 15 in which the urea

and chloride salt are mixed together and added to the control

of paragraph initially.

17. The method as described in claim 15 in which the.

urea and chloride salt are dissolved in a reoenabol volume of

water and then added to the control of paragraph

18.. The method as described in claim 15 in which a suspension

medium is employed which is artificial or of a natural biological

character.

IT L

S19. The method as described in claim 15 in which the

urea and chloride salt are dissolved in a reanble

volume of water and then added to the control of paragraph

and then the platelet suspension of paragraph added

thereto.

The method as described in claim .15 in which the

the urea and chloride salt are added respectively to the

control of paragraph and to the platelet suspension of

paragraph and the resulting suspensions admixed.

21. The method as described in claim 20 in which the

urea and chloride salt are solublized before addition to

said control of paragraph and platelet suspension of

*paragraph

22. The step or features disclosed *sy an

ncombinati on thereof

Dated this 6th day of June, 1979

COULTER ELECTRONICS, INC.

by its Patent Attorneys

DAVIES COLLISON.

i .fMM g H l it i J