CA1249208A - Hematology control compositions for three populations of leukocytes; and methods for their preparation and use in whole blood control systems - Google Patents

Abstract

ABSTRACT

This invention primarily is directed to a hematology reference control solution, the three separate white cell control portions thereof consisting of three types of fixed red cells of determined size distribution for checking the operation of a particle analyzing instrument, including its predetermined lower and upper threshold settings for each class or subclass of leukocytes.
For preparing a human granulocyte analogue, nurse shark erythrocytes are altered and fixed in a chilled solution to simulate in number, size and distribution the granulocytes in human whole blood.
Such a granulocyte analogue is useful as a free-standing reference control, for determination of human granulocytes only, or can be comingled with reference controls for lymphocytes and mononuclear cells for use with multi-parameter instruments to give a trimodal leukocyte reference control, which itself can be included in a multiple-analysis hematology reference control, having components for also determining red blood cell and platelet parameters.

Description

This invention rel~t~8 to h~atology contr~71 compositions, and methods for their w e in a refere~ce BtB~dArd fo~ partic~e ~n~ly~i~
instrumentation of the COULTER~ type. More p~reicul~rly9 this inventi~n relate~ to ~ three-co~ponent By8tem for 8imulating the three ~ajor component8 of human leukocyte8, n~mely lymphocytes9 mononuclear c211~ and gr~nulocyte8, and optionally sub-tivisions thereof, ~hich i~
compatible with kn~wn red blood cell and platelet control~.
~ononuclear cells ~re Bingle nucleated blood cells which include monocytes and numerous mature ~nd immature forms of ly~phocyte~ and immature myelocytes and erythrocytic blood cells. The ~odified COULTER COUNTER Model S Plus c~n delini~te mature lymphocytes and polymorphic granulocytes from the general group of mononucl~ar cell~
found in circulating blood under normal end patho10gic~1 condition6 The6e circulating mononuclear blood cells include promyelocytes, ~yelocytes and b1ase cells as ~ell a~ monocytes. Since monocytes sre the moet prevalent mononucle~r cell popul~tion under normal hemopoietic condition8, the 90 to 160 fL ~egion can be referred to as the ~onocyte rexion.
The use of electronic particle counters in hematology is well known. V. S. Patent 2,656,508 di8clo8e8 n basic app~ratu~ utilizing the Coulter principle for this purpose. U. S. Patent 3,757,2l3 contains a de8cription of 8ever~1 such deYices ~hich incorporate threshold circuitry. Thre8hold circuitry excludes random amplitude cignal6 caused by noise and backg~ound debris of inconsequential particle~ in the suspen6ion. It may ~180 be used to limit tbe ~ize range of the particles counted. Adju~table threshold circuit6 with dials marked off in mathematically related dial settings are in co~mon use.
Although this disclosure will be directed primarily to embodiments involving the use of electronic particle counters of the COULTER type, it 6hould be understood t'hat the particle controls herein disclosed, and their method8 of u8e described herein, find w;de Qpplication with particle counters generally. Accordingly, the term "electronic particle counter" should be understood to include, in additioo to COULTER COUNTER~ in8truments, any other type of particle counter which discri~in~te8 between p~reicl~8 of ~arioua di~es by the use of electronic di~eriminator circuito ("th~e~hold6") which recpond electronically eo eignalB indiCAtiVe of particle ~ , mn3s or particle volume. ~OULTER ~nd COUITER COUNTE~ ~re Regi~tered Trsdemarks of Coulter ~lectronics, Inc.
Calibration check techniques for red blood c~ll (erythrocyte) 3 white blood cell (leukocyte) and platelet ~thrombocyte) counts ~nd physical attribute~ ~re ~ell developed. The material used for checking calibration, hereinafter c~lled a c~ntrol, al80 can be used to calibrate a hematology in6trument. The techniqueA for using a control generally in~olve counting known popul~tions of particle6 suspended in a liquid vehicle iD the control preparation, whish usually is diluted aub~tantially with a diluent psior to counting.
Heretofore9 however, no control had been developed for use with three sub-groups of leukocyee8, namely, lymphocyte8, mononuclesr cell~ and granulocy~es, since the equipment for 2utomatic co~nting of these sub-g~oupg hsd not been developed It is evident that a control product ~u8t accurately indicate on a comparative ~a8is wh~t ~ te8t ~ample of fresh blood constieutes ~ith regard to the determinstion~ in question. It is further evident how important it i6 for the control product to 8imul2te fre~h blood, since blood components, ~uch a5 red blood cells, can hemolyz~ 810wly and undergo changes in si~e snd shape ~ithin hours after removal from a blood donor. Similarly, white blood cells suffer degenerative changes.
Quality control long has been a nece6sary and routine procedure in clinical hematology. Accuracy in the counting of red and white blood cella and in the making of hematocrit a~d hemoglobin determinations of the patient'~ serum is dependent, in part, upon the use of adequate control 5tandard8. Thus, the accuracy of the manual technique of particle counting, such as by the clas6ical method of microscopy, can be checked by giving the technician a so-called "blind" sample, or control standard, containing a known concentration of particles for compari~on with the unknown samples for which ~etermination has to be made. With the numerous types of automatic equipment for particle counting now available, quality control by the use of control standards is likewise necessary since the possibility of malfunctioning of the instrument i6 ever present. Consequently, the importance of accurate and reliable checks on hematological determinations that may be used in the diagnosis of disease is clear.
The traditional method of maintaining a quality control program for automatic particle counting equipment has consisted of providing fresh human blood as a whole blood standard. However this fresh blood is usable for only one day. Consequently, durable blood products were developed which did not require fresh human blood.
In U. S. Patent 4,485,175, an automatic method is described for differential determination of three populations of leukocytes using a COUl.TER COU~TER Model S Plus automated blood counter. Accordingly there is now a need for a reliable check of threshold calibration and additional operational performances for electronic particle counters typified by the COULTER COUNTER analyzers now marketed. Operators then can identify and document -the volume ranges between which the three populations of leukocytes are to be counted routinely. There is further a need in the art for a reliable method for demonstrating instrument stability over a prolonged period of use.
Human lymphocytes, mononuclear cells and granulocytes have a specific size distribution ran~e and after stabilization (for example with a fixative, such as formaldehyde~, their responsiveness in diluents may not permit proper size discrimination. This would result in an inability to evaluate proper instrument operation. Both the upper and lower size limits for each population of leukocytes must be represented in the reference control material. In addition, the mean cell volume of each leukocyte population in the reference control material should be very close to that of normal h~mman blood. When upper and lower size limits and mean cell volume are thus specified, it becomes a virtual necessity for the volume distribution histogram of the control material to approximate the normal distribution of the fresh human cells. This volume distribution must remain relatively constant regardless of the total white cell count and the range of ratio6 for all three p~pul~tion~ representing abnormal low, ~ormal and abnormal high condition~ for human leukocytes. Therefore, it is necessary that the preservation process used in the ~anufacture of the reference control 6uspension doe~ not cause significant shrinking or ~welling of the cells. Al80 one mu6t be ~ure that aging of the reference control does not result in deterior~tion o the volume distribution histogram characteristics or other parameters. A fur~her requirement for the leukocyte component in a whole blood reference control for ~ulti-parameter instruments is that the cells must not be completely lysed by the lytic reagent.
~ith the increasing use of automated device~ capable of performing multiple hematological determinations and with the introduction of techniques of automated cell counting, the so1ution to the problem lies not in the pur~uit of more effective way8 oF
st~bilizing "real" human leukocytes, but in ~ub~tituting a surrogate whi~h ~atisfies the specifications against which the product is made.
Therefore, animal cells which could be converted into a u~eful control or calibrator were sought.
~merous ci~ations to prior ~rt are set forth with some expla~atioll in this application and reference is especially made to the following:

U~ S. Patent6 4,179~398; 4,213,876; 4,264,470; 4,299,726;
4,3~9,490; 4,405,719 and U. S. Patent 4,485,175.
A novel feature of this invention is the preparation of a stable, reproducible hematology control for three population6 of white blood cells. In the three-component leukocyte sy&tem of this invention, methods are given for preparing simulated lymphocytes from mammals, for example human erythrocytes, 6imulated mononuclear cells from avian, for example turkey erythrocytes, and simulated granulocyte~
from the erythrocytes of fishes, for example the nur~e shark (~inglymostoma cirratum~. In addition, a novel method i6 included for the preparation of altered red blood cells of a predetermined size ~ ~ f.~. ~ f ~

within limit~, by control of the ~ge of the red blood cell before tresCment ~nd ~he tem~er~ture 8nd concentr~tion of tbe fi~ing 2~ent.
It ha8 been di~covered th~t tbe nur8e eh~rk (Gingly~oseoma cirrstum~ has erythrocytes (red blood cell~) th~t nor~lly f~ll into a size range which i8 ~ htly l~rge~ th~n human gr&n~locyte~ ~nd that these erytbrocyte5 cnn be ~lte~ed Bnd fi~ed 80 aC to be similar in volum~ distribution to the volume distribution of hu~an ~r~nulocytes in whole blood, in order to be useful ~8 a human gr~nulocyte reference control. The granulocyte ~nalogue 80 prepared ia stable and reproducible for use a8 a atand alone reference c~ntrol, or in trimodal white blood cell composition6 which al80 contain al~ered and fixed turkeg ~ed blood cell8 to simulate human mononuclesr cell~, and altered and fi~ed h~man red blood cella t~ si~ulate human lymphocyte~. The white blood cell ~n810~ue8 ~180 s~n be included in a single multiple-analysi6 hem~tology reference control which determines not only ~hite blood cell component~, but al50 red blood cell ant platelet parameter&. The r3tio and total cell count for the three leukocyte populationg C8n be adjusted to repre8ent pathological as ~ell 38 ~orm~l conditions in hu~an blood. These composition~ are 2U us2ful likewise in control ~nd c91ibr9tor compositions particularly for ~utomated particle analy8i8 in~trU~ents employing the Couleer principle.
The cells treated by the methods disclosed herein and ~he compositioDs including auch cellfi provide ~n excellent system of checks and balances 80 necess~ry in hematological deter~inations.
By way of example, illustr~tive embodiments of the invention now will be described with reference to the accompanying drawings in which:
FIGU~ES lA and 1~ compar~tively show the leukocyte distribution histograms of a sa~ple made on the CO~LTER COUNTER Model S Plus automated blood cell counter.
FlGURE lA aho~s 9 histogram developed ~rom a normal blood sample.
FIGUUE lB shows a histogram developed from the hematology control system and method of the pre~ent invention.

~2'~ J~

The invention hss sufficient flexability to allow the preparation of lymphocyte, mononuclear cell and granulocyte populations which may be larger or smaller th~n those shown in FIGURE lB. The changes in size then can reflect abnormal white blood cell size distributions, S which are characteristic of human pathological conditions.
~ ny system for automated differential counting of human leukocytes, which distinguishes three populations of leukocytes from other cells in the blood on the basis of characteristic size range and volume distribution, requires that the reference control material used as such closely simulate the si~e range and volume distribution characteristics of the respective cells in normal human blood. The problem is to find methods which accurately will produce cells of a given size in reproducible quantities sufficient to be available, as needed for use in controls for automated counting equipment.
l~.S. Patent 4,485,175, describes a method and reagent system for three-volume differentia] determination of lymphocyte, mononuclear cell and granulocyte populations of leukocytes, using one type of a modified COULTER COUNTER Model S Plus instrument now sold as the Model S Plus IV diff and Model S Plus V. ~ollowing ehe procedure of this invention, the three classes of leukocytes are counted in femtoliters (fL) approximately ~s follows:
lymphocytes 35 to g0 ~ 3 fL
mononuclear cells 90 to 160 + 3 fL
granulocytes 160 to 450 _ 3 fL
It is understood that the leukocytes which are classified and counted in the range designated as "mononuclear cells" include monocytes.
The procedure of this invention allows the treated red blood cells from different sources to match a plurality of threshold settings between about 25 fL and about 700 fL for many types of blood counting instruments. The other COULTER COUNTER instruments, with which this invention can be used, are of the Model S and Model S Plus types. The COULTER COUNTER Model S counts leukocytes from 25 fL to infinity. Other types of COULTER COIJNT~R Model S Plus instruments c~unt ly~phocyte3 from ~bout 45 fL to ~bout 99 fL ~nd count mononuclear and granulocytec fro~ abo~t 99 ~L to infini~y 4 Altho~gh methoda are kno~n for prep~rin~ control compositions for automatic countin8 instrumentation, whe~ ~11 leukocytes ~re counted ~8 a Bingle~ whole populeeion, no ~ethodB h~ve been described for the preparativn of blood cell control~ for the differenti~l counting of i~di~idual component~ of the leukocyte population. It i8 ~ecesB~ry to prepcse ~n ~nalogue for each of the major leu~ocyte components including lymphocyte~, mononuclesr cell8 and granulocytes in order to check the ~hreshold settings of electronic particle counter~.
Previou~ly avflil~ble reference control products for checking the performsnce char~cteristics of psrticle analy6i~ inatrument~ i~ whole blood oontrol~ ~ere designed to evsluate total ~hite cell couot. The only requirement was a stable non-ly~ible component ~hich hsd a majority of particles, aboYe a mini~um threshold, for example 35 to 45 fL. ~o~ever, the cur~ent multiple white blood cell populatio~
analysi~ requires particle~ of specific size incre~ent~. Furthermore, there i5 a need for such a reference control whi~h can be used over a long period of time ~ithout substantiAl cnange occurring in the 2D referenc2 Yalues Gbtained therefrom.
U. S. Patent 3,873,467 disclose8 a control composition for ~hite blood cells uaing red blood cells stabilized by ~eans of sub~tituting for the bl~od plas~a, ~ stabilizing media and fixed ~wollen red blood cells having increased mean cell volume to sub8titute for all of the leukocytes present. ~ieh swelling to approximately 50X greater volu~e, the ~pecific graYity of the cell is reduced to be in the range of 1.06 to 1.0~.
In the present invention, fixed human red blood cells substitute only for tl-e smaller lymphocyte portion of the ~hite blood cells, and there i6 commi~gled with these simulated lymphocyte6, larger fixed animal red blood cell6, representing the two larger sizes oE the white blood cells, namely the mononuclesr cella and the granulocyte~. In this manner all three populnt;on6 of the white blood cell~ are identified. ln all instance6, the red blood cells from thre~ 6pecies of vertebratea are fixed 80 that they ~ill not be ly&ed when ~r~ f ~

determinin~ the white blood cell p~r~meter8 in the a~e blood c~ntrol s~mpleO In sddieion, the count for eJch of the three popul~tion~ m~y be varied in propostion t~ one ~noth~r uithout eff~ct;ng a ~ajor chift in the original ~olume distribution.
The primary ~ethod~ in this inv~ntion for controllin~ the ~ize of the lymphocyte analogue9 are proper Belection of the size of the non-fixed human red blood cell~, the o~molality of the fixing æolution ~nd the variation in fixation temperstuTe. Shrinking or expanaivn of the cells by manipulating their 08motiC en~ironment prior to fixation has limita~ions d~e to criticality of the fixation proce~ required to maint~in ~tability of the alter~d cells during the lysin~ of the untreated human red blood cells in the mixture.
Al~o, it ia necess~ry to treat cuch red blood cells in ~ manner ~hich allows them to be ~hrunken or ~olle~ ~ithout exce~ive eell associatiGn or hemolysi to approximate the si~e needed.
m e pres2nt invention embodies ~ compositi~n prepared by mixing a suspension of fixed hu~an red blood cells to 3imulate hum~n ly~phocytes, fixed red blood oell8 fro~ turkeys to sim~late human ~ononuclear cells> and a ~u8pen8ion of ~tabilized red ~lood cells from 2Q nurse 3harks to 8i~ulate human granulocyte8, all as~e~bled in ~ liquid media and in such proportions ~B to pro~ide a si~gle composition to simulate human white cellB (See FIGURE lB). Thi8 leu~ocyte analogue composition i~ then comingled 6rith human red blood cell~ to be ly~ed~
and stabilized platelets or platelet ~nalogue6 to provide a cingle ~ultiple-analy~ia refe~ence control. The ratio of the simulated white blood cell population c~n be adjusted to represent abnormal lo~, normal and abnormal high conditions witho~lt affecting the size distribution of each type of sim~lated ~hite blood cell. Modification of the preparative step8 will allow the production of particle~ which represent different types of ~hite blood cells which ~re characteristic of abnormal hum~n white blood cell condition~.
In a preferred embodiment of this invention, the media i~
prepared according to the teachings of ~. S. P~tents 4,299,726 and 4,358,394; and the platelet~ are ~tabilized according to any one of the ~ethod6 described in U. S. Patents 4,264,470, 4,339,490, or 4,405,719; all ~f the~e patent8 are ~ssigned to ~oulter Elect~onics, Inc.
Thi~ i~vention inc1ude~ the ~election ~nd specific trest~ent step~ for re~ blood cell~ fro~ one or ~ore ~nim~l ~ourcec. ~enerally, it i~ not pos~ible to 8hrink or ~well red blood cell~ more thsn about 30~ to 50X total. ~herefore, it i8 nece~sary to ~tart ~ith cells ~hich ~pproximate each population aB sho~n iD FIGU~S lA ~nd lB. The essen~e of the pre~ent invention lie8 in the diseovery ~h~t red blood cells fro~ the so~rce6 ~tated abo~e have suitcble characteri~tica to 0 allow them to be shrunken or 8wollen ~nd fixed ~ithout exce3sive cell ~ssociation ~nd hemolysi8 to ~pproximate the size of the corresponding human blood cell ~ith a n~rsow range of mean cell volume.
ln the oll~ctin~ step~ the red blood cells are sufipended in an antico~gulant, 0uch R8 ~n alkali met~l 9alt of ethylenediaminetetraaCetiC ~cid (EDTA) di~solved in 8 physiologis~l ~ali~e solutioD (sodiu~ chloride)0 It i~ enYi~ioned that other anticoagulant8 a~d ~alt8 will do, as long as they ~o not c~use undue hemolysis or cell ~ssociation.
~ith regard to EDT~, we h~e discovered th~t the ~nticoagulant will sffect the size of the blood cell as a functi~n of concentration ~nd the ti~P of exposure. Fgr ex~mpl~, ~ells suspended in 2.5 mM to lO~M EDTA will ret~in the same 8ize for up to 24 hours af~er collection. At a concentration gre~ter than 10 ~M ~DTA, for example 20 ~M, the cell9 ~ill begin to 8well in les6 than 24 hour~. After ~4 hours, the size o~ the cell increases ~ith the concentration of EDTA
ss well a8 the exposure time. The mainten~nce or increase in cell Bize a6 a function of EDTA concentration and time of exposure, can be preserved by fixation. It i8 assumed that EDTA can significantly disrupt cell membrane structure and thi9 disruption becomes magni~ied as a function of time. Thu~, it is pos~ible to manipulate cell size by controlling the concentration of anticoagulant and time of exposure prior to fi~tion.
In order to use universal prepar~tive procedureB for all white blood cell analogue populations, it is preferred to use red blood cells which must be shru~k prior to fixation. At the time of fix~tion, it io imporC~nt to ~aint8in hypertonicity to prevent 3ny s~elling of the cells. ~he effecti~e fin~l concentr~tion 3f the s~lt 301ution should be in a rAnge ~hich iB equ~l to 5nd up to four ti~e~
greAter than thse of nor~al ~e~um, depe~ding on the degree of shrinking required to si~ulate a specific white blood cell population.
The addition of a fi~ing agent add~ to the tonicity ~os~olaliey) of the fi~in8 solution.
Fixing of the shrunken cell~ ia i~poxtsnt to toughen the cell membranea and to prevent biodegr~tion of the membranes. Thi8 i~
accomplished by contscting tbe suspension of the cells ~ith a 801ution of an organic sldehyde, including moDoaldehydes ~uch as formaldehyde, or di~ldehydes such a8 glutaraldellyde. Glutaraldehyde i6 the preferred sldehyde, ~ince it resct~ ~o~e ~peedily than formuldehyde Glutasaldehyde can be added in higher concentrations than the fiDal concentratio~, BO long a8 th~ fin~l concentration thereof i8 in ~he range of about O.lX to l.OX. In accordance with the 2bove value range ex~mple, b~t u~ing formaldehyde in plac~ of glutarsldehyde, the final concPntration of fonmaldehyde is 2.5X to 10%; the preferred concentrstion i8 5Xo The only practical limitation~ on selection of an appropriate aldehyde ~nd cvncentr~tion thereof are elimination of undue cell ~ssoriation and hemolysis and potential unde~irhble electroly~ic effectc. The specific conditioos recomme~ded for each of the leukocyte componeots are given in each of the examples 6ub~equently set forthO
It has been discovered that the smalle6t size of red blood cells with the nnrrowe~t distribution width are obtained with any kind of blood using fresh cells, or cells aged for not more than four dAys after phlebotomy, ~ith a low concentration of the anticoagulent, used for collecting the frefih blood. A higher concentration of EDTA will result in larger non-fi~ed as well as fixed sells, wh;ch might not fit the criteria for the particular size range ~ought.
In a preferred embodiment~ the blood cell~ are added to a chilled hypertonic salt solution containing glutaraldehyde. The reduced temperature has been shown to provide a qualitatively different cell as ~ea~ured on a sizing apparatus 8uch as a COVLTER COVNTER analyzer.

A qualitative di~ference includes a lower mean cell volume compared to fixing at room temperature. This difference, however, might not be evident until treatment of the fixed cell with a lysing agent as LYSE
S~ II reagent in ISOTON~ II diluent. LYSE S and ISOTON are Regi6tered Trademarks of Coulter Electronics, Inc.
~ fter fixation, the cells are allowed to settle by gravity, are separated from the liquid phase, then are washed with a phosphate buffered saline solution and placed in a storage solution.
Since all three of the leukocyte components ~re to be combined into a single reference control for use with the known lysing agent for the red blood cells, the formulation for the diluent is the same for measuring all three components of the leukocyte analogue. The preferred formula for this diluent, as well as the ~ormula for the lysing agent to be used for lysing the untreated red blood cells to be added later to the white blood cell assembly, are given below:
DILUENT PREFERRED RANGE
1. Procaine hydrochloride 0.11 g/L 0.05 to 0.25 g/L

2. ~-(2-acetamido)imino- 1.40 g/L 1.0 to 2.5 g/L
diacetic acid (ADA)

3. Dimethylolurea 1.0 g/L 0.5 to 2.5 g/L

4. q.s. to 1 liter with distilled water LYSING AGENT
1. Dodecyltrimethyl- 35 g/L 20 to 55 g/L
ammonium chloride-50% solution 2. Tetradecyltrimethyl- 3.7 g/L 2 to 6 g/L
ammonium bromide 3. Potassium cyanide 300 mg/L 125 to 1250 mg/L
4. q.8. to 1 liter with distilled water The media for the hematological reference controls having stability up to six months includes lactose, fungicides and antibiotics, and supplementary agents such as purine nucleosides, bile salt, and cholic acid derivatives, phenothiazine compounds and the salts thereof having antihistamine properties, and 4-aminoben~oic acid esters and derivatives and their salts having anesthetic properties, P~ 3 or combin~tion~ ~hereof. Such medi~ ore ~ully de~cribed in U. S.
PaCenes 4,~13,~76; 4,299,~26, 8nt 4,~58,394.
The follo~ing ~pecific e~a~ple i~ di~closed in U. S. Patent 4,299,726:
St~bili~ing ~edia for Conferring Long Term St~bilisy on Red Blood Cell~-P~eferred Formu1aeion, Approximate Amounts Liter For~ul~tion 1. D;stilIed w~ter 500. ~1 2. Propyl paraben 0.3 to 1.0 ~m 3. Hethyl par~ben 0.5 ~o 1.0 gm 4. Proc~ine hydrochloride 0.1 to 0.5 gm

5. Deoxycholic acid 0.1 to 0.9 gm

6. Lac~o~e 10.0 to 50.0 gm

7. Actidione 0.1 to 0.6 8m

8. Tri~odium citrate dihydr~te 3.0 to 8.0 ~m

9. Citric acid monohydrate 0.3 to 0.9 g~

10. SodiuR dihydrogen pho~phate monohydrate 0.8 to 2.5 gm

11. Phenergau hydroehl~ride 0.1 to 1.0 gm

12. Colistimethate, sodium 0.2 to-0.9 gm

13. Penicilli~ G., sodiu~ ~.5 to x 106 _3 ~ 106 units

14. Rsnamyc;n ~ulfate 0.2 to 0.8

15. Neomycin ~ulfate 0.2 to 1.0 g~

16. 5' AMP 0.4 to 1.0 gm

17. ~denine 0.2 to 0.8 gm

18. Inosine 0.4 to 1.0 gm

19. Dihydro~treptomycin sulfate 0.2 to 1.0 gm

20. Tetracycline hydrochloride 0.2 to 1.0 gm

21. 30Z Bovine albumin 100 - 350 ml

22. q.~. to 1 liter with difiti11ed water For preparing the Iymphocyte an~logue, human erythrocytes are fixed with glutaraldehyde, following the procedure detailed below in Example I, and then included in a predetermined amount in refere~ce controla and calibrator~, together with the mononucleAr cell analogue (ExAmple 2) ~nd the granulocyte analogue (Example 3~, or in whole ~v~ J~3 blood controls ~ont~ining, in 3ddieion to the lynpbocyt~ ~nalogue~, washed red blo~d c~ , or w~3hed red blood cell~ and platelet~.
F~e~h human red blood ce11s mu~t be ~hed to re~o~e dono~
apecific plaam~ protein~. Thi~ ~ill reduc~ the prob~bility of cell S ~gglutin2tion when mi~ing red cell~ from multiple blood cell donors.
Although mo8t fi~tion occur~ withi~ t~o hour~, ~ore time i~
required for the red blood cell~ to be tots11y re~istant to the u~ual ~ed blood ~ell lytic agents employed in COULT~ COUNT~R Dutomated hematology instrumenta. With cnreful selection of the hu~an ~ed blood 10 ce11P ~od their d;lution in g ~tandard pho~phate buffered ~olution, the length of time for fixation ~ith gluc~r~ldehyde will be optima1 bet~een 4~ a~d 72 hour~. Les~ than 48 hours of fi~ation m~y reault in partia11y fi~ed red blood ce11 with ~ mean cell volu~e 1ess th~
that for a nor~l human lymphocyte. P~rti~l fi~ation ~an be 15 determined by detecting a shift to the left (drop in cell volume) in a parti~lly fixed population after the addition of 10Z by volume of LYS~
S II reagent in ISOTON-II diluent. The addition of a lytic agen~ to diluent de~ipned fo~ the Model S Plu~ COULTER COUNTXR series ~e.g.
~odel S P1u~ II, III, ~nd IV) will 8ho~ si~il~r re~ult~. The 20 formulation for these diluents and lyPing ~gents are disclo~ed hereinafter. A totally fi~ed human red blood cell (e.g. 48 hour fixation) ~ho~s a slight or no ~hift to the left (i.e _ decrea~e in apparent volume) in the particle Bize distribution. Over-fi~ation of red blood cells with glutaraldehyde i~ noted after 72 to 96 hours by a 25 drop in particle si~e which i6 independent of the effect of above mentioned red blood cell lytic agent~ on 48 hour fixed hu~nn red blood cell~. The de~ired amount of fi~ation will produce cells which nre counted in the range of about 35 to 90 f 3 fL using ~ COULl'ER COVNTER
Model S Plus type analyzer.
Human mononuclear cell8 are leukocyte8 ~hich are intermediste in ~ize between lymphocyte~ and granulocyee~, and are counted in the size range, or monocyte region of between 90 and 160 - 3 fL. These cells are larger than fixed humnn red blood cell~.
Avian red blood cells are larger than fixed human red blood 35 cell~, nnd close to the size of human mononuclear Cell8. For the 1~

purpoae of thi~ inYentiC~ it ha~ been found that ~owl red blood cells auch ~B ~oo~e, chicken, duek, and prefernbly tur~ey red blood cell~, clo~ely ~atch the si&e ~nd ~hape of human mononucl~ar ~ell~, and lend themael~e3 to the 2ldeh~de at~bilization proce~. The~e stabili2ed 5 "~;mul~ted" monocyte c~ provide a ~ati0factory ~ub~titute for human mononucledr cella in our control COmpO~itiOD, A proce~ for prep~ring a ~ononucle~r cell snnlogu¢ from turkey red blood cells ia detailed in ~x~mple 2.
Hum~n granulocytes repreBent the lar8est aize of the ~hree leukocyte populaeion6 now counted by the COULT~R COUNTER Model S Plu~
automsted cell counter. Ibe grAnulocytes ~re counted in the size range between about 160 and 450 ~ 3 fL.
It has n~ been di~covered that proper treatment of the red blood cells of the nurse ~h~rk (Gingiymo~toma cirratu~) re~ult~ in ~ si~e Aimilar to human granulocyte8. These erythrocyte~ generally 3ho~
excellent suspen~ion ~tabili~y, highly reproducible volume di6tribution characteri6tic8 and are ~v~ ble on ~ commerci~l Rcale.
~hen fixed with a cross-linking agent 8uch afi glutarnldehyde t~ese erythrocyte~ ~ill be electronically ~imil~r to the hu~an granulocy~e ~hite blood cells.
Other non-humnn vertebrates ~hich are kno~ to have red blood cells i~ th~ de~ired 8ize r~nge for human grnnulocyte~ include other fi~bes, partic~lnrly member~ of the ~hark fnmily and reptiles ~uch a~
alligators. All of the9e non-human vertebratn~ have nucleated red blood cells which could be used. ~o~ever, consid~rations, auch as availability in quantity at reasonable expen6e, must be considered.
Red blood cell6 of the nurse 8hark sre rendily nvailable in quantity.
The red blood cel1s of alligato~s tend to be less readily available, because alligators are pre6ently a "prot~cted" 6pecies by government authoritieD.
The cells of both ~lligator6 and nurse shark~ sre nucleated, bu~
the presence of a nucleus is not essential for their use a3 a substitute for human white blood cells for the purposes of this invention. The presence of the nucleus in the shark red blood cell is not detriment~lJ inas1~uch as the cell8 are stabilized ~ith a cross-linking agent, ~uch ~a glut~raldehyde, which pre~ents ehe cell membrane and cytopl~m fro~ being 8tripped from the nucleud during ly~i8. The principle of differential 8naly0i~ of f~esh hu~an ~hite blood cella i~ b~ed in p~rt on the time of cytopl~s~ic membrane 5 destruction, which i8 apecific for each type of ~hite blood cell popul~tion.
Shrinking or e~pandin~ of red blood cell~ by ~anipulating their osmotic environment prior to fi~ation ha~ limitetiona due to criticality of the fi~tion proce88 required to ~aint~in stsbility of the altered cells. Generally, one cannot ahrink or swell erythrocyte~
more than ~boue 30% to 50~ for this purpo~e. Therefore, ie i8 necesasry to ~t~rt with ~nimal erythrocytea ~hieh are clo~e in size ~o ~hat ~;11 be needed ~in811y for use a8 a human granulocyte ~urrogateO
The ueeful~esa of nur~e shsrk erythrocytes ns surrogate human lS granulocytes ic limited by the nece8~ity to ~hrink them to ~ithin the granulocyte size ~nge. ~po~ure of erythrocytes to hypo- or hyperosmotic environments has the principal effect of chan&ing the mean corpu~cular volume, slightly inc~easing or dPcrea~ing the width~
of the si~e diat~ibution hi8tograms, but c~using only a trivial effect on sy~metry of the size distrihution histogrA~.
A standardized ~nd st~bili~ed red blood cell composition from the nur~e shark provide~ a 8uitable ~eference control ~hich iB u6eful in the enumeration of humsn gr~nulocytes by automated ~eans using in~truments opersting under the Coulter priDciple, or by variou~
micro~copic techniques, such as illumination or pha~e contrast methods.
In accordance with thi5 invention, nurse shark erythrocytes are altered and stabilized to 9imulate a human granulocyte analogue with re6pect to volume in femtoliters (fL) and count as 1 X 103 per microliter (uL). These fixed shark cells are stable for more than four months, based on volume distribution and count. The product is designed to behzve in a manner ~hich a8 closely as possible simulates fresh human granulocytes. In addition, the product i~ de6igned to possess a feature not found in fresh normal ~ranulocytes, that is 8 ~ ;J `~ 3 r;..J~

high level of aeability of t~e par~met~rs ~e~0ured by eh~ cell counter3 in ~hich it io used.
Example 3 iB a ~pecific exa~ple of preerred r~a~ene~ ~od techniques for treatin~ the sh8rk cella, it bein8 under~t4~d th~t the formulations ar~ only illuBtrative. The reagent0 ~nd/or tecbniques de~cribed can ~180 be ~p~lie~ble to red blood cellc from ~nim~13 other ~h~n sharks. Other ingredients ~nd proportion~ c8n be employed, in ~ccordnnce ~ith this diaclosure. However, the ~ize of ehe cell i~ a non-lytic ~olution, ~8 in ~ buffer, may not Yisually 8how the entire effect of the treat~ent employed in this invention. The proces~
requires the control of:
1. the concentrati~n of the antico~gulant when collectiD~ the blood cell~;
2. the age of the anticoag-ulated non-fixed cell~;
3. ~he concentration of cells during fixation;
4. the o~molality of the fixing solution;
5. the te~perature of the- fixing solution; and 6. the time of expo~ur~ to ~ cold hyperosmotic ~olution prior to fixation.
It has beeo discovered that the 8malles~ si~e of red blood cell6 with the narrowe6t di~tribution ~idth are obtained u~ing fresh cell~, or cells s~ed for not mo~e than four days after phlebQtomy, with a lo~
concentration of EDTA. VBing ~ higher concentratioo of EDT~ on cells aged more than 24 hours ~ill result in larger non-fi~ed a~ wPI1 ~s fixed cells which might not f;t the 160 to 45~ ~ 3 femtoliter criteria fo~ human granulocytes.
Maintaining the fixing solution at O to 10C~ preferably 4C, prior to and after the additioD of chilled anticoagulnted blood also will result in a smaller blood cell for use in this invention.
Nurse shar~ cellR treated by the above method are highly stable when used in the re~erence control compo6ition of this invention.
The simulated granulocytes can be used in a stand alone granulocyte control, or in a composite and unitary control ~tandard which measures other blood parameters. More particularly, thi~ novel granulocyte control i8 used in combination with compatible controls for other leukocyt~ component~ ~uch e~ lymphocyte~ ~nd ~ononuclear cell~, to furni~3h a leukocyte control, ~hich in turo i~ used in combinstion ~ieh reference control~ for red blood C2118 ~nd platele~
psr~meter2.
The following ~xamples 1 to 3 8ive detailed instr~ctions for one ~ethod of preparin~ e~ch of the three component~ of the leukocyte reference contr~l. Ex~mple 4 sho~s an a~sembly of the three leukocyte populations. These procedures ~re controlled carefully throughout i~
order to make ~ure th~t the ce11s 8re not damaged and thst the fixed cell~ become substantially totally re~i~tant to the U8U~l red blood cell Iytic reagent. It will be underatood thst the formul~tion~ are only illustr~tive, and other ingredients and proportions mAy be employed, in accordance ~ith this di6closure.
E~AMP~E 1 LYMPHOCYTE AN~LOGUE FROH ~UMAN RED B~O~D C~LLS
The following i8 ~ ~pecific example ~ preferred rèagents and recom~ended specific procedural steps for treating human red blood çell~ to obtain 8 normal s;~ed lymphocyte analogueO It ~ill be understood that the fo~mul~tions and the procedures only are illustrative and th~t other ingredieDtB3 proportion6 ~nd procedureR
can be employed, in accordance with ~he di~closure~ in tbis lnvent ion .
ANTICOAGULAN$S FOR COLLLCTION OF WHOLE BL~OD
One or ~ore of the following antico~gulanta can be used, as deter~ined by those skilled in the art.
1. Stflndard ACD (~cid-citrate-dextrose) 2. Standsrd CPD (citrate-pho6phate-dextrose) 3. Di~odium EDTA (ethylenediamine tetraacetate), 2 mg/ml of blood.
STABILIZING ~EDIA (LITER FORMULATION), a~ ~bove ~et forth ~nd with reference to U. S. Patent 4,299,726.
FRESH BLOOD WASH SOLUTION AND WASHING AND RESUSPENDING SOLUTION FOR
FIXED CELLS (WRS) (~HOSPHATE BUFFERED SALI~E - LITER FORMULATIO~) 1. Sodium pho~phate monoba6ic 0.2 g 2. Sodium pho6phate diba6ic .7H20 2.0 g ~,, jd~ $ F.

3. Sodium a~ide 0.1 g 4. Sodi~D chloride 9,4 g 5. q.8. to 1 liter ~ith distilled w~ter pH 7.3 to 7.5 osmolality 320 to 34Q ~O~m¦kg.
~RYTHROCYTE DILUTING SOLUTION
1. Sodium chloride 6.96 g 2. Pota~ciu~ chloride 0,30 g 3. Sodium pho~phate monobanic 1.31 g 4. Sodium pho~phste diba3ic .7H2010.35 8 5. q.~. to 1 liter ~ith distilled water pH 7.3 to 7.5 oj~nolality 3~0 to 340 mOsm/kg.
PR~CEDUR~ -15 1. Select human red blood cells having a mean cell volume range of about 81 to 89 fL and red çell di~tribution width of lesc tha~ 18 Wash the pAcked hu~an red blood cells with the Fre6h Blood Ws~h SIDlUtioD.
~0 2A Dilute the ~aahed packed cells ~ith the ~rythrocyte Diluting Solution to a coun~ of 1 ~ 106~uL.
3. Prepare ~ glutsr~ldehyde fixaeive re~gent having ~ glutaraldehyde content ok about 1.0% to 10% by sdding a co~mercial ~5~
glutaraldehyde product to the Erythrocyte Diluting Solution. The preferre~ concentration i8 5Z.
4. Add a measured amount of the fixati~e of step 3 to the washed red blood cell suspension to obtain a final glutaraldehyde concentration of 0.1% to l.OX., and mix for about 20 minutes.
Tran~fer to sealed contsinera ~hich are rolled ~lowly for 1~ to 7 hour6.
5. Cen~rifuge the fixed cel1s at about 400 RCF for about 5 minutes.
Remove the supernatent fluid, wash cells aeveral times ~ith the Fresh Blood Wash Solution (WKS), then resu9pend in the Washing and ResuRpending Solution (WRS).

J~

6. Determine the mean cell volume to ~ake c~ta;n th~t fi~ation iD
complete. P~rtiAl fi~tio~ ~ill BhO~ ~ d~op in ~olu~e ~ter the addition of 10X LYSE S lI re~gent in ISOT~ II diluent previou~ly de~cribed. ~ totally fixed red blood cell ~hows le than ~ 5 fL eh~nge in sppar~nt volume.
7. For a st~nd ~lo~e Iy~phocyte control, re~uRpend the wa~hed fixed cells in the re~uapendi~g ~olution ~nd ~dju~t the concentrDtiou to ~imulatP that of human lymphocyte~ in normal human blood.
8. For multiple he~otological control~, resuspend the ~aahed fi~ed cells in the mediA for the multiple parameter hematology control, the cell count being appropriate to measure lymphocytea, 9. The fixed cells can ~e ~tor~d for a time period of up to about 8iX
~DO t~
In~~ccordance ~ith th~ a~oY2 example, but u~ing ~ormaldehyde in pl~ce of glutaraldehyde, the fin&l concentration of for~aldehyde i8 2.5~ to 10%. The tlme required for fisation ~it~ formaldehyde i~
~ longer than ~ith glutaraldehyde.
In accordance with the above example, but Rtarting ~ith other ~ype~ of mam3ali~ red blood cells, comparable re~ult~ are obt~ined.
E~!LE 2 MONONVCLEAR C~LL A~A~OGUE FROM TURXEY RED ~LOOD CELLS
The follo~ing i~ a specific example of preferred reag~nta a~d recommPnded apecific procedur~l ~tep8 for treating turkey red blood cell~ to obtain the mononuclear cell an~logue. It ia understood that the leukDcytes which are cla8Bified and counted in the ~bove designated range a8 mononuclear c~lls i~cludes monocyte3. It will be underatood that the formulation8 and the procedures are only illustrative and that other ingredienta, proportions and procedurea may be employed, in accordance ~ith the dificlosures in this in~eDtiOn~
ANTICO~GUI~NTS F0R COLLECTION OF WHOLE BLOOD
One or more of the following anticoagulant~ can be used in ~uitable quantity, as determined by one skilled in the art.
1. Standard ACD (acid-citrate-dextrose) ~o~ J~

2. 5t~ndard CPD (citr~te-pho~ph~te-de~tro~e~
3. Disodium ED~A tethylenediamine tets~acetate), 2 ~g/~l of blood.
STABILIZING MEDIA (LITER FORMULATION), a8 above ~et for~h and with S reference to U. S. P~tent 4,299,726.
TURKEY ERYTHROCYTE WASHING AND DILUTING SOLUTION(I~WDS~LlTER
~ORMULATION) 1. Sodium pho~phate monobasic 1.31 2. Sodi~ phosphate diba~ic 10,35 g 3. Sodium chloride 2.50 g 4. Potassiu~ chloride 0,3 g 5. q.8. to 1 liter with dist;lled water p~ 7.3 ~ 0.1 08molality 200 ~ 10 ~O~m/kg.
15 ~ASHI~G AND ~ES~SPENDING SOL~TION FOR Fl~ED CELLS (WRS), as ~et forth iD Exampl~ 1 -PROCEDURE
1. Centriiuge turkey fresh whole blood ~t 700 RCF fnr 10 minutes st ambient temperature. Remove the ~upernatent alon~ with the buffy coat, being careful ~ot to disturb the packed red blood cell~.
2. ~ash turkey red blood cells two times with 4 to 10 ~olume~ ~f TurkPy Eryehrocyte Washi~ ~nd Diluting Solution (TEWDS).
3. Dilute ~ith Turkey Erythrocyte Washing and Diluting Solution (TEWDS~ ~nd measure out a 2 ml sample for determin~tion of red blood cell count ~0.33 ~ 106/uL) and mean cell volume ~pproximately 155 fL) evaluation.
4. Prepare ~ glutsrnldehyde fixing reagent having a glutnraldehyde cont~nt of about 1.0 to 10.0% by adding a co~mercial 25X
glutaraldehyde product to the Turkey EryChrocyte ~ashing and Diluting Solution (TE~DS). The preferred concentration i6 5%.
. Add 1 volume of 1.0 to 10.0X glutaraldehyde ixative ~olution to 9 volume3 of the washed red cell suspension, and mix thoroughly for about 3 to 4 minutes. Transfer to 8ealed container~ which are rolled alo~ly for 20 to 28 hours.

3~*ll~
~1 . C~ntrifug~ the fixed cell3 ~t ~bout 400 RCF for 5 ~inutea. Remove ~he 8upe~0atent flui~ and wa~h ~e~er81 time~ ~ith the va~h ~olutioo (~Rs3.
7. ~or a st~nd alone moDonuGlear c~11 cont~ol, re8ucpend the ~hed fixed cell~ in the resu~pend;ng ~olution snd ~djust the concentration to si~ulate the number o~ monon~clear cell~ in normsl huDsn blood.
B. For multiple bemotological controls re~u8pe~d the ~sched fi~ed cell~ in the *t~bilizing media, a~ ~bove aet forth, for the multiple eontrol in the sppropriate concentration to measure ~ononuclear cella.
9. The fixed cell8 c~ b~ stored for ~ time period of up to abo~t 8i~ ~onths~
~;MPLE 3 GRANULOCYTE A~ALOGUE FROM BED BLODD CELLS OF THE NURSE SHARK
The follo~ing i8 a specific example of preferred Feagents ~nd reco~mended specific proced~r~1 step8 for ~re~ting red blood cells of the ~urse sh~rk (Ginglymo8tom~ cirratum) to obtain the ~ranulocyte analogue. It ~ill be under8tood that the formul~tion~ ~nd the procedures are only illustrsti~e, snd that other ingredie~ts, proportiona ~nd procedure8 ~ay be e~ployed, in ~ccordance ~ith the disclosure~ in this in~ention.
A~TICOAGULA~TS FOR COLL~CTIO~ OF WHOLE ~LOOD (LIT~R FOR~ULATIOM~
1. DisDdiu~ EDTA (ethylenedi~mine tetraacetate) 16.~ g 2. Sodium chloride 17 g 3. Uren 21 g 4. q.8. to 1 lter with distilled water The shark ordinnrily h~s a high concentration of urea in its blood which help5 to maint8in osmotic equilibrium of the red blood cella. ~or this reason ure8 i8 added to the 6ubsssembly in order to mimir the resulting osmotic conditions.
STABILIZING MEDIA (LlTER FORMULATION) 9 as above set forth.
WASHI~G AND RESUSPENDING SOLUTION POR FIXED CELLS ~WRS~, aB ~bove set forth.

~f3$

SaARR C~LL FI~ING SOLVTION (LITER FORMULATIO~
1. ~odium chloride 100 g . q.s. to 1 liter with di~tilled ~ter 3. Osmolality 3000 4 100 ~O~m/kg FINAL FlXING REAGENT (LITER ~ORMVLATION) 1. Shark fi~ing solutioD 0.9~0 L
2. Glut~raldehyde 25I solutioD 0.040 L
3. Packet ~hark red blood çell3 0.020 L
PROCEDURE
1. Collect fresh whole blood ~rom a nur8e 8hark in ~n ethylenediamine tetrsacetic acid (EDTA) anticoagul~nt 801ution hnving a final concentr~tion of 2.5 to 30 mM (typic~lly 5 mM~. Thi~ i8 ~
filtered ~DT~ ~olqtion h~ving an o~ol~lity of 1030 ~ 30 ~O~m/kg and a ~ of 7.0 4 1.
2. Centrifuge the anticoagulated whole blood mixture within 24 hours of phlebotomy; remove the supernat~nt fluid and the bufy coat cont~ining the ~hite blGo~ cell~, being careful not to disturb the packed red blood cell~. The glutaraldehyde fixstion processing of blood can take place ~ithin four day6 if it i8 ~tored in lefi~ th~n 20 mM EDTA; nnd fixatio~ an t3k~ place in 1e~8 than ~ne day if stosed i~ 20 to 30 m~ of EDTA.
3. Prepare ~Dd chill to ~bout O to 10 C ~ glut~r~ldeh~de fixing solution which ha8 a gluearaldehyde concent~tion of 1.0~, and an o~molality of 1000 to 4000 mO8/kg (p~referred concentr~tion i8 3000 4 100 mOs/kg3. The p~ range is 4.5 to 8; preferred p~ i8 4.0 ~ .5.
4. Centriuge; within 60 minutes after centrifugation add the non-washed pscked red blood cells, with mixing, to the chilled fixing solution (see Final Fixing ~esgent) to give ~
glutaraldehyde concentration of 1.0%. Continue the fixing operstion for 8 minimum of 6 hours at O to 10 C and, if needed, for up to 4B hours.
5. Wash the fixed cell~ two or three ti~es with approximately 5 volume~ of phosphate buffered saline solution.

23 6. Store cell~ in the ~a~hin8 ~nd Re~u5pending Solution for ~ ti~e period up to 60 d~y~.
7. For R ~tand ~lone granulocyte control, re~uepend the ~a~hed fi~ed cell~ in the resuspending colution, and ~dju~t the cell concentrstion to simulate that of gr~nulocyte~ in nor~al hu~
blood.
8. For multiple hemotologic~l control0, re8u~pend ehe ~s~hed fi~ed cells in the st~billzing media, a~ abDve ~et forth, for the multiple control in tbe appropriate concentration to mea~ure granulocytes.
9. The fixed cells can be ~tored for u time period of up to about 8iX
months.
In accordance ~ith the abo~e procedure end techniques~ but ~ub~tit~ting for the ~hark red blood cells the red blood cells from other sources, si~ilar re~ult8 are obt9ined to give modified cells useful for other purposes in addition to simul~te granulocyte~, EXAMPLE 4 -_ ln a sub-assembly for determi~ing the total white blood cell content o a normal human blood 8ample, the followin~ quantitie~ of the individual component~ are employed:
Stock Solution 0.0146 L Ex~mple 1 lymphocytes 3.5 ~ 10~/uL
0.0105 L ~xample 2 mononuclear Cell8 1.8 ~ 106/UL
0.975 L Exæmple 3 granulocytes 0.4 ~ 10~/uL
0.0004 L diluent pho~phate buffered saline Thi6 sub-assembly can be stored for up to about ~ix months.
It has been found that untreated human red blood cells, stnbilized by su~pension in media described earlier, can ~atisfactorily provide the red blood cell component of the subject composition. The untreated red blood cells are hemolyzed readily in the prior operational step for the white blood cell count ~nd ~ub~equent hemoglobin determination.
Suspenaions of untreated humun red blood cells, aimulated ~hite blood cells, and stabilized or 8imulated platelets ure mixed in such proportion that the finul red blood cell, white blood cell and

24 platelet counto, 8C ~ell ~ he~o~lobin cont~nt and hematocrit f~ll in the r~nge con~ider~d norm~l for hum~n ~lood.
Stabili~ed platelet~ are furni~hed by methodo known in the ~rt.
Useful ~ethod~ includ~:
1. A co~bination of iodo~ceta~ide and an iminodi~cetic ~cid or salt thereof, together with a compatible bacteriostatic ~gent io an ~queous ~olution ~hich i8 ~aintained at a precelected r~nge of p~ and oamolality as i~ described in U. S. Patent 4,405,719.
2. A fi~tive-at~bilizing compo~ition cont~ining ~
glutar~ldehyde concentration of 0.1~ to 5~ ~nd ~ non-ionic surfact~nt ~hich i8 a mi~ture of ethoxylate~ of certain i~omeric linear slcohols, as is more fully de~cribed in U. S. P~tent 4,389,490.
3. A hum~n platelet ~nalo~ue eomprising go~t erythrocyte~
stsbilized, combined and blendéd a8 necessary ts have a size r~nge ~nd volume distributioo close to that of human platelets9 ~ de~cribed in ~. S. Patent 4,264,470.
Where the platelets ~re used as controls for procedures that include unfi~ed red blood cell~ which are to be ly~ed9 it i~ necessary to u~e fi~ed cells a8 a leukocyte ~nalogue. The~e c~n be prepared, for exa~ple, by the ~ethod de~cribed in U. S. Pntent 4,179,398.
The values for each of the he~tological parameters can be ~aried to represent ~bnonmsl low and ~bnonm~l high conditions_ me white blood cell count in nonmal blood ifi 5~000 to 11,000 per ~icroliter IUL~ ~ith ~ lymphocyte ~alue of 20 to 40%, mononuclear cell value o~
less than 10% and a granulocyte value of 60 to 80%. The normal range in human blood for red blood cella i8 4,000,000 to 5,000,000 cells per microliter. The normal hemoglobin value i8 12 to 16 grams/100 ml.
The ter~ "hematocrit" i8 defined as the ratio of volume of packed red blood cells to the volume of ~hole blood. The normal ratio in hwmans i~ about 45~. The mean corpu~cular ~olume i8 the ratio of the volume of packed red blood cells in ml per liter of blood to red blood cell~
in million~ per microliter. me mean corpuscular hemoglobin concentration i8 an index indicating the mean or average ~eight of hemoglobin per 100 ml of packed red blood cells in terms of percent.

as The ~e~n corpu~cul~r hemo~l~bin i~ the r~tig of he~o~lobi~ ~ont20t, in gr~m~ p~r liter, to red blood cells, io ~illion3 per microliter.
A control product IllU8t ~ceurately indic~te on ~ compsrlltive baE~i~
what a test ~nple o fsesh ~hole blos~d constitutes with regard to ~11 the above determination3. It i8 evident ho~ impo~tant it i~ for the control product to simulate nor~al humsn cell~ in An anticoagul2nt.
Thi~ in~ention primarily io directed to a hematology reference control aolution, the ~hite cell control portions thereof con~is~ing of three types of fixed red blood ~ells of determined 6i~e distribution for checking the predetermined lo~er and upper thre~hold setting~ for each class or 8ubclesB of leukocyteB ~ aB Aet eleCtriCBIly iD the p~rt;cle snalyzing iD~tru~ent. The upper and lower electronic threshold setting for eacb of the cla8~e~ of leukocytes are ~hecked b~
the relationship of the p~rticle co~t8-of each of the di~crimiDator regions compared to kno~n value~. Each type of simulated leukocyte ~use maintai~ ~ize independent of total cell count and independent of it~ proportionality to oDe or ~ore type~ of simulated leuko~yte populations.
The sell~ treated by the ~ethod disclosed herein provide ~o 20 excellent 8y8tem of checks and balances ~o aece~sary in hematologieal dete~minations.
~ hile in the foregoin~ 8pecification, a detail~d de~cripti~n of the inveotion has been set do~n for the purpose of illu~tration, many variations in the detail8 herein given may be made by tbo~e akilled in khe art without departing from the Bpirit and scope of the in~ention.

Claims (30)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A hematology reference control fluid suspension for determining multiple hematology parameters in an automatic cell counting and sizing instrument, said reference control comprising a predetermined number of treated red blood cells to be counted in at least two of the following approximate size ranges:
35 to 90 ? 3 fL
90 to 160 ? 3 fL
160 to 450 ? 3 fL
in a compatible aqueous and substantially isotonic stabilizing media, said cells in each size range functioning as a substitute for one of three white cell sub-populations in human blood, namely lymphocytes, monocytes or granulocytes.

2. The reference control of claim 1 wherein said treated red blood cells include those which have a volume which is measured by said instrument in the size range of 160 to 450 ? 3 fL, are derived from non-human verterbrates selected from the group consisting of reptiles and fish, and function to simulate human granulocytes.

3. The reference control of claim 1 wherein said stabilizing media includes lactose, fungicides and antibiotics, and supplementary agents selected from the group consisting of purine nucleosides, bile salts and cholic acid derivatives, phenothiazine compounds and their salts having antihistamine properties, and 4-aminobenzoic acid ester derivatives and their salts having local anesthetic properties.

4. The reference control of any one of claims 1, 2 or 3 wherein said treated red blood cells include those which have a volume which is measured by said instrument in the size range of 35 fL to 90 ? 3 fL, are derived from mammalian blood and function to simulate human lymphocytes.

5. The reference control of any one of claims 1, 2 or 3 wherein said treated red blood cells include those which have a volume which is measured by said instrument in the size range of 90 fL to 160 ? 3 fL, are derived from fowl and function to simulate human mononuclear cells.

6. The reference control of any one of claims 1, 2 or 3 which has a shelf life of for about up to 6 months.

7. The reference control of claim 1 which further includes a platelet control composition.

8. The reference control of claim 7 in which said platelet control composition includes a suspension of platelets stabilized by treatment with a fixative-stabilizing composition containing glutaraldehyde in a concentration of 0.1% to 5% and a non-ionic surfactant which is a mixture of ethoxylates of isomeric linear alcohols.

9. The reference control of claim 7 in which said platelet control composition includes a suspension of platelets stabilized by treatment with a composition comprising iodoacetamide and one or more compounds selected from the group consisting of an iminodiacetic acid and a buffer salt thereof at a preselected range of pH and osmolality.

10. The reference control of any one of claims 7, 8 or 9 in which said platelet control composition includes a suspension of a human platelet analogue derived from goat erythrocytes.

11. A hematology reference control fluid suspension for use with an automatic blood cell counting instrument, said reference control comprising a predetermined number of treated red blood cells which function to simulate human granulocytes and are derived from non-human vertebrates selected from the group consisting of reptiles and fish.

12. The reference control of claims 2 or 11 wherein said reptiles are alligators (Alligator mississipiensis).

13. The reference control of claims 2 or 11 wherein said fish are nurse sharks (Ginglymostoma cirratum).

14. A stand alone hematology reference control fluid suspension for use with an automatic blood cell counting instrument, said reference control comprising a predetermined number of treated red blood cells of the nurse shark which function to simulate human granulocytes.

15. The reference control of claims 11 or 14 further including a predetermined number of treated red blood cells which function to simulate human mononuclear cells and are derived from fowl.

16. The reference control of claims 11 or 14 further including a predetermined number of treated red blood cells which function to simulate human lymphocytes and are derived from mammals.

17. The reference control of any one of claims 1, 11 or 14 which further includes a suspension of stabilized red blood cells in an aqueous media for hemoglobin determination, said red blood cells retaining their ability to respond to a lysing agent by stromatolization.

18. The reference control of claims 11 or 14 which further includes a platelet control composition.

19. A hematology reference control comprising at least two types of simulated leukocytes, wherein each type of simulated leukocyte maintains size independent of total cell count and independent of its proportionality to one or more types of simulated leukocyte populations.

20. A hematology reference control fluid suspension for determining multiple hematology parameters in an automatic cell counting and sizing instrument, said reference control comprising:
a suspension of stabilized human red blood cells in an aqueous media for hemoglobin determination, said human blood cells retaining their ability to respond to a lysing agent by stromatolization; simulated white blood cells for determining lymphocytes, monocytes and granulocytes; and stabilized or simulated platelet cells; all said cells being in such proportions that the final parameters for red blood cells, white blood cells and platelet cells fall in a predetermined range expected for specified human normal and diseased conditions.

21. A stand alone hematology reference control fluid suspension for use with an automatic blood cell counting instrument, said reference control comprising a predetermined number of treated red blood cells which function to simulate human mononuclear cells and are derived from turkeys.

22. In a process for making hematology reference control compositions, the improvement which comprises collecting blood in an anticoagulant at a predetermined concentration, centrifuging the collected blood to obtain packed red blood cells, and reacting the packed red blood cells with a chilled fixing solution within about 4 days after phlebotomy.

23. The process of claim 22 wherein the blood is derived from non-human vertebrates selected from the group consisting of reptiles and fish.

24. The process of claim 22 the improvement wherein said chilled fixing solution has a temperature of about 0 to 10°C.

25. The process of any one of claims 22, 23 or 24 wherein said anticoagulant is selected from the group consisting of ethylenediaminetetracetic acid and its buffer salts.

26. The process of any one of claims 22, 23 or 24 wherein the concentration of said anticoagulant is between 2.5mM and 30mM.

27. The process of any one of claims 22, 23 or 24 wherein said fixing solution is glutaraldehyde.

28. The process of any one of claims 22, 23 or 24 wherein said fixing solution is formaldehyde.

29. The process of claim 22 wherein the reaction with said fixing soltuion is under hyperosmotic and slightly acid to approximately neutral conditions.

30. The process of claim 29 wherein said hyperosmotic conditions are from about 1000 to 4000 mOsm/kg and the pH is 4.0 to 8Ø