CA2191218A1 - Method and reagent system for the improved determination of white blood cell subpopulations - Google Patents

Abstract

The invention provides a novel lytic reagent composition highly selective in its interactions with the cell membranes of white blood cells and also provides a method of using the reagent S composition in a particle analyzing system to effect a significantly improved white blood cell differential determination. The lytic reagent composition is characterized by an ability to selectively shrink the white blood cells into the increasing size order of lymphocytes, basophils, monocytes, cosinophils and neutrophils, and effect a five component separation of these major subpopulations of white blood cells on the histogram of an automated blood analyzer when used in conjunction with a suitable blood diluent as shown in Figures IA to IC.

Description

2 1 ~ 1 2 1 8 PCTIUS94/06062 METHOD AND REAGENT SYSTEM FOR THE IMPROVED
DETT~RMT~ATION OF WHITE BLOOD t-~T T SUBPOPUL~TTONS
FIFT T) OF THF INVF~TION:
This invention relates to an improved method and Iytic reagent composition for the differentiation and PnllmPrAfinn of at least three subpopulations of white blood cells. When combined with a suitable blood diluent, the lytic reagent ~ posiliol. of the invention is characterized by an ability to ~ lly effect a very clean s~alalic,l~ of white blood cells into t&ee or more subpopulations on a leukocyte volume hictngrAm~ with little or no cellular debris. The Iytic reagent ...",rrJcili.,.) can, in practice, enable the t'lACCifit'Atinn and counting of five subpopulations of white blood cells, in the order of (1) Iyll~ o~yl~, (2) basophils, (3) monocytes, (4) eosinophils and (5) neutrophils. Such data are extremely useful for the detection of abnormal distributions in subpopulations of mature white blood cells and morphnlngirAl abnnrmAIitiPc in a blood sample.
BA~'Kt~ROUND OF T~F INV~l~TION:
There are three major pu~uulatiul~s of cells in the blood ~ .".l" ;~ the platelets, ery&ocytes (red blood cells) and leukocytes (white blood cells).
Within the white blood cell population, many subpopulations exist, five of which are considered prPrl~ ly in hPmAtn~ ical analysis. These white cell subpopulations include monomorphonuclear Iymphocytes, which constitute about 15 - 45% of the white blood cells in a normal mature population, polyll.ùl~l~onuclear basophils (0-3%); monomul~ ,...rl uWl~D~yLe:~ (~0 -10%); polymu.~hul-u~lear eosinophils (~0 - 5%); and polyll-u.l-l n.~. ~lPAr neutrophils, which constitute from about 45-80% of the mature white blood ceLs in a population. In many diseases, there is a clinically ci~nifirAnt alteration in the normal distribution of these mature cell types. In addition, immature or abnormal cell types are often associated with the disease state. Hematological analysis of white blood cells, including white cell ~l~Ull-~ldliUI- and flagging of abnnrmAlitiPc, in rnnjilnrtinn with trA~litinnAI diagnostic tPrhnirlllPc, is thus an ill.~Ulldl~l tool in the diagnosis and treatment of disease.
Prior to the mid-1970's, white blood cell differential analyses were c-nnrlllctPd by manual PyAminAtinn, with a tPrhniriAn viewing blood film slides with the aid of a microscope. Since that time, hPmAtnlngirAI analysis has been AlltomAt~rl~ making its use both widespread and commonplace.
SUIISTITUTE SHEET (RULE 26) WO 9513Z622 2 1 ~ ~ 2 1 8 PCTIUS94/06062 ~

While the mPthnrl~)lngiPc for ~ analysis vary, most often the enumeration and analysis involves ~UIJj~lillg a diluted sample of whole blood to a lytic reagent which ~Llul~laluly~s ar~.therefore ~ ullaL~s the red blood cell population and .cimllltAnpr~usl~modifies the cell ~ .b~ P~ of the more prevalent white cell subpopulations so that the cytoplasm leeches out, causing differential shrinkage of the different cell types and enabling (1;~1, ;...;, .~I;nn and sorting thereof. The size and number of white blood cells in the sample are then detected with the aid of an autûmated analyzer, which typically includes a detector adapted to detect the volume (size) and/or opacity of the white blood cells in the sample by electrical or optical dirrt ~ S. The white blood cells are counted-for a period of time sufficient to gather data for analysis, data points are stored in a memory device and then analyzed in an Al~nrithmir processor. The data can then be displayed in the form of a two flimPncir~nAl or three II;IIIrl~c~ l histogram.
Heretofore, a number of Iysing reagents and reagent systems have been developed which have provided the clinician with the ability to isolate the white cell population from the red cell population of whole blood and to further differentiate the white cell population into smaller subpopulations.
Patents considered ~ s~.llalive of the art in the field include U.S. Patent Nos. 4,286,963; 4,485,175; 4,529,705; 4,745,071; and WO88/07187.
U.S. Patent No. 4,~86,963 to Ledis et al describes a lytic diluent and method for achieving rapid Iysis of red blood cells in whole blood. This diluent enhances the ability of AlltnmAtPCI il~Ll.. rl.l,ll;.l.~ to perform lirf~l~lllial .l~lr,...;~l;nnc of lymphoid and myeloid subpopulations of leukocytes and the uludl~lilaliv~ flPtPrminAtinn of hPmrlglnbin. The lytic diluent employed by Ledis et al is composed of a mixture of at least one quaternary Ammonillm salt and an aryl ~ ci short chain alkanol in buffered aqueous medium (pH 3.5 to 5.0) . The Iytic diluent of this Ledis et al patent is limited in its ability to effect ~ r.r~ lir~n of the leukocyte population into the two principal subpopulations; namely the Iymphoid and myeloid fractions.
U.S. Patent No. 4,485,175, also to Ledis et al, describes a reagent system and method for performance of differential determinations of leukocytes into three subpûpulations utilizing Alltr~mAtPd cell counting equipment, in which the reagent system includes a blood diluent and lytic reagent. The Iytic reagent comprises an aqueous mixture of aliphatic quaternary Alllllllll~illlll compounds, which when added to a diluted blood sample under mild rrlnrlitinng of concentration and at a relatively slow rate causes SUBSTITUTE SHEET (RULE 26) .... . . .. . .. . . .. ..... . .. ...

~ WO 9~/32622 2 1 ~ 1 2 1 8 PCTIUS94/06062 volume mnrlifi~Atinnc to the various subpopulations of white blood cells, enabling a three ~:uI~lluoll~llL s~aIaLiul~.
U. S . Patent No. 4, 529, 705 to Larsen relates to a reagent for combined diluting and Iytic of whole blood cells. The reagent comprises an aliphatic ~LuaL~Il.aly Ammnnillm salt and at least one anion selected from the group consisting of sulfate, carbonate, formate and acetate anions, which are employed for the purpose of ~l~V~I~Lil~g ay,~ aLiull of platelets in the sample and an alkali metal cyanide for lUllVt~ iUII of hemoglobin to a chromagen.
U.S. Patent No. 4,745,071 to Lapicola describes a reagent system for dirr~ llLial blood analysis including a blood diluent and Iytic reagent. The Iytic reagent of the '071 patent comprises an aqueous solution of a single aliphatic quaternary Ammonillm salt and may also contain puLas~i cyanide as a chromagen forming agent. The preferred quaternary Ammnnillm salt is the dodecyl (C12) homolog.
WO88/07187 describes a reagent system enabling differentiation of white blood cells into at least five distinct subpopulations. The PCT
application discloses a reagent system ~ a Iytic reagent and a ~nmrAninl~ reagent for the Iytic reagent referred to as a "quench". The primary functions of the quench are to rehrd the activity of the Iytic reagent and to restore the ionic balance of the sample ~u~se~lu~l-L to its treatment with Iytic reagent.
Generally, the white blood cell ~lirr~ IlLials obtained in accordance with the described t~hniqu.oc fall irlto one of two rAt~gnri.~c. The first category is a three ~uIllluol~ L screening differential resulting in the isolation and qllAntififAtinn of three fractions of white blood cells including a Iymphocyte component, a midregion or monocyte component and a granulocyte (preflnminRntly neutrophilic) component. The granulocyte population usually includes the neutrophils, as well as eosinophils, and possibly basophils, while the mirlr~ginn often contains monocytes, other ,...",I",..rl~Ar cells, basophils and eosinophils. J~:~JalaLiul~ among populations is frequently poor, resulting in low correlation between AlltnmAtf~d and manual differential counts, which is undesirable. Poor s~.~.,.li",~ of subpopulations is further undesirable as it decreases the ability to accurately flag abnnrm~litil~c The second category of white blood cell analyzers is capable of effecting a four or five component separation. Such systems are substantially more SUBSTITUTE SHEET (RULE 26) WO 95132622 2 1 9 ~ 2 1 8 PCTIUS94/06062 complex than the three ~u~ uu~ differential analyzers, resulting in higher costs to the operator for materials and labor. ~A~ri`iJlIlctrAfinn of this type of system is described in WO88/07187, a Pate,;lE;t Cooperation Treaty application to Coulter Electronics.
As will be d,u~ idL~d by persons skilled in this area of technology, a deaner separation between sul",ut,ulaliu.~s, together with the ability to separate up to five subpopulations would result in a qi~nifir~nt uvt~ l in the reliability and accuracy of white blood cell differential analysis. Despite the existence of many different reagents on the market for blood differential analysis, there still exists a need for a system capable of identifying at least three and p~r~ably five distinct subpopulations of white blood cells which is relatively simple to use and ~ ly effects a clean separation of the cells on a leukocyte volume histogram, to increase the ability to flag ~hnnrmAIitiPc A~u~diIl~ly, it is an object of the present invention to provide an improved Iytic reagent composition which sel~.livt:ly modifies blood cell volume by reacting with the cell ~ eb to cause red blood cells to rupture and selectively shrinks the white blood cell population into at least three distinct, highly separated .UIllpO.~I~ when used in combination with a suitable blood diluent.
Another object of the invention is to provide an improved Iytic reagent rnmrnciti~n capable of effecting a five uIll,uull~ ser~r~tinn of white blood cells into the five major S subpopulations on the leukocyte volume histogram of an ~u~ul~-al~:d analyzer.
A still further object of the invention is to provide a flexible blood analysis reagent system that can be optimized for both human and multiple species applications.
Yet another object of the invention is to provide an improved method for ~lltnm~tf~d white blood cell differential analysis.
Further objects and advantages will be appreciated from the following disclosure.
SUMM~RY OF THF T~VF~TION:
These as well as related objects are adlieved in accordance with the present invention, which provides a novel Iytic reagent composition highly selective in its intPr~rt;onC with the cell lll~lllbldllt~S of white blood cells and also provides a method of using the reagent rnmrncitinn in an ~ltnm system to effect a ci~nifit7~ntly improved white blood cell differential SUBSTITUTE SHEET (RULE 26) .. . ... ... . . . . . .. . ....

The Iytic reagent ~ fl~ is 1~ ;I by an ability to 3~ iv~:1y shrink the white blood cells into the increasing size order of Iymphocytes, basophils, monocytes, f-f cinf~philc and neutrophils, and effect a five component cfr~rAnnn of these major subpopulations of white blood cells on the histogram of an ~ d blood analyzer when used in conjunction with a suitable blood diluent The five ffmrfnf-nt S~lua~ iOII
can be analyzed on ~ull~ ially available Allllllll~e~ blood analyzers, such as the Coulter S Plus IV (Coulter Electronics, Hialeah, Florida) by manipulating the threshold values fixed within the device, optionally with the aid of image f-nl~ ", ~,~l techniques, such as those described in my copending patent application U S Serial No 07/650,686, filed on February 5, 1991 In the absence of such manipulations, the Iytic reagent composition of the instant invention can readily be optimized to effect a C;Y~II;r;IAIII1Y
improved three ~UIII,UUl~el~ sc~luOl-l~iul~ on the hictfgr~m of commercially available ,~ d blood analyærs when compared to the reagents conventionally employed on such ill~lUllll~
In addition to effecting an improved white blood cell dirr~ ial, the Iytic reagent ff,mrf,citifln of the present invention has the advantage of being S~rr;fi'-~lly flexible to enable optimi7Atifn for dirr~ ial blood analysis of multiple animal species The Iytic reagent .UIll~osi~io~ of the instant invention ~ c an aqueous solution of active Iytic reagents selected from the group .s;~ Of an admixture of at least one group (A) aliphatic quaternary Alllllll~
compound with at least one group (B) aromatic, heterocyclic or ;~ iUIliC
u,Ua~L~laly Allllll~ l rnmrollnfl an admixture of at least one group (A) aliphatic ulua~lllcuy Ammf~nillm compound with at least one group (C) l~t~lu~y~lic U,ua~l,la.y Ammf~nillm ~u lpoulld; and an admixture of at least fne group (C) h~.u.y.lic llud~llldly A~ with at least one group (B) aromatic, h~ .u.y~lic or zwitterionic U,ua~ .y All~
uul~uul~, wherein the group (A) quaternary Ammflnillm compound is ~:,ul~s~ d by the formula R1 ~ -- R3 X
~4 SUBSTiTUTE SHEET (RULE 26) WO 95/32622 2 ~ 9 1 2 1 8 PCIIUS94/06062 wherein Rl is a long chain aLkyl group r~ from~10 to 20 carbon atoms, R2, R3 _nd R4 are short chain alkyl groups ~..,.~,1;..;.~ from 1 to 6 carbon atoms, and X is an ion selected from the `gr~oup consisting of halide, sulfate, pho~l~L~: and nitrate ions; and /=\
E13C ~ O ~ ;
the group (B) L~Urll~llL~y Ammrmillm compound is l.~l~ .led by the general formula: _ _ (F'2) n R~ ~ ( [CH2 ~o ~)n (X )p (F2)n wherein Rl is a long chain alkyl group ~ , from 8 to 20 carbon atoms, R2 is an alkyl group .. ~-;.. ;.. g 1 to 6 carbon atoms, m is an integer from 1 to 4, A is a member selected from the group cu~ lilLg of:
~~ ; ~? and - 50 3, n is an integer from 0 to 1, such that when n=0, the remaining nitrogen bonds form part of a heterocyclic ring, X is an ion selected from the group consisting of halide, phosphate, sulfate and nitrate ions and p is an integer from 0 to 1.
The group (C) quaternary Ammr-nitlm compound is l~ l-L~d by the formula -- -- _ Rl --N~ X
wherein R1 is a long chain alkyl radical comprising from 10 to 20 carbon atoms, and X is an ion selected from the group consisting of halide, sulfate, rhosrhAt~ and nitrate ions.
The use of an admixture in aqueous solution of at least one of the Llu,ll.-ll,d.y Ammoni.lm compounds of group (A) with at least one of the quaternary Ammrlnillm compounds of group (B), or an admixture of at least SUBS~lTUTE SHEET ~RULE 26) W095/32622 219 1~ 1~ . PCTIUS94/06062 one of the quaternary Ammonillm rnmrQIlnri~ from group (C) with at least one of the uluaL~ aly ~,.,.",.,~ ulllpoul~ds of group (B) or an admixture of at least one of the qud~.. laly .. I~ compounds from group (A) with at least one quaternary Ammnnillm compound from group (C) in a Iytic reagent ~u~ JOailiull which will selectively modify the cell membrane of white blood cells and is capable of effecting a five rnmrnnr-nt sepArAtirm of white blood cells into Iymphocyte, basophil, monocyte, eosinophil and neutrophil subpopulations on a leukocyte hictngrAm.
White blood cell dirL~-ILiaLic,ll on an Al1tnm~te~1 particle analyzer in accordance with the present invention is made possible by the use of the novel Iytic reagent composition of the present invention in combination with a suitable blood diluent. The blood diluent can be any of the blood diluents previously known in the art, and preferably comprises at least one organic buffer and a cell L~ LIal~e stabilizing agent and an antimicrobial agent. The method of the invention comprises (1) sulu~lyil-g a whole blood sample, a volume of blood diluent and a volume of the Iytic reagent wlllluosiLiul~ to a particle analyzing system; (2) rapidly admixing in the particle analyzer the whole blood sample, the blood diluent and the Iytic reagent composition of the present invention, wherein the individual qual~ dly Ammnnillm compounds ulllpoll~llL~ in the lytic reagent .UIll~U~iLio.~ are present in a ratio and quantity sufficient to effect at least a three ~UIllI.lUllt~lll st~,d-dLiu.~ of white blood cells; and (3) analyzing the whole blood sample in the AlltnmAtf~i particle analyzer to dirr~ ial~ and enumerate at least three subpopulations of white blood cells.
BRTFF DE~-RTPTION OF THF DRAWING:
Figures lA to IC are leukocyte volumetric histograms showing the total number of cells versus cell volume for human samples, which were analyzed using the mr-thnrlnlngy and reagent system of the present invention. An image r-nhAn~r-mrnt system of the type described in copending application U.S. Serial No. 07/650,686 was used in the generation of the l i~lu~.d--ls. Each of Figures lA to lC shows a five ~UlllpUI ~III
s~,ua.dLiol~ of white blood cells, on the ill~l~a~i~lg size order of Iymphocytes, basophils, monocytes, eosinophils and neutrophils.
Figure 2-lA is a leukocyte volume histogram obtained in accordance with the present invention, in Example 3, Experiment 1 herein, on a Unipath CELL-DYN(~) 1600 automated analyzer wherein the Iytic reagent was Iytic reagent 1~ dodecylethyl dimethyl Ammnnillm bromide and SUBSTITUTE 5HEET (RULE 26~

WO 95/32622 2 1 9 1 2 1 8 PCTIUS94106062 ~

N-dodecyl-N, N-dimethyl-3 ammino 1-propane sulfonate and the whole blood sample was blood sample A.
Figure 2-2A is a leukocyte volume histogram Qbtained in accordance with the present invention, on a Unipath CELL-D~ 1600 ~ d analyzer in Example 3, Ex~ el l 1, whereir~thè lytic reagent was Iytic reagent rnmrncition 2, ~ULI~I;siIlg dodecylethyldimethyl Ammnnillm bromide and benzyldimethyltetradecyl Annmnnillm chloride and the whole blood sample was blood sample A.
Figure 2-SA is a .~ ,aldL;v~ leukocyte volume histogram obtained for whole blood sample A on a Unipath CELL-DYN~ 1600 t~mAtPd analyzer using the mAnllfA~tllrer's rernmmrn-lPd reagent system.
Figure 2-lB is a leukocyte volume hictngrAm obtained in accordance with the present invention in Example 3 on a Unipath CELL-DYN@) 1600 Alltnm~tPc~ analyzer, Experiment 1 herein, wherein the Iytic reagent was Iytic reagent 1 and the whole blood sample was blood sample B.
Figure 2-2B is a leukocyte volume histogram obtained in accordance with the present invention on a Unipath CELL-DYN(g) 1600 AlltOmAtP~
analyzer in Example 3, F.Yr-PrimPnt 1, wherein the Iytic reagent was Iytic reagent rnmrncitinn 2 and the whole blood sample was blood sample B.
Figure 2-SB is a ~Ulll~Jdlal;Ve leukocyte volume hictngrAm 15 obtained for whole blood sample B on a Unipath CELL-DYN~ 1600 Al1tl7m~trd analyzer using the ...~.~..r, LI~I'S I~ clpd reagent system.
Figure 2-3C is a leukocyte volume histogram obtained in ac.ul~dll~
with the present invention, in Example 3, F.YrPrim~nt 1 herein on a Unipath CELL-DYN~ 1600 AlltrlmAt~l analyzer, wherein the lytic reagent was Iytic reagent ~oIll~cl~iIiull 3 ..""l,.ici,~g, dodecylhrimethyl "".,...~.~;"... chloride dodecyldimethyl (2-phenoxy ethyl) ~.. ,.. ;"", bromide and the whole blood sample was blood sample C
Pigure 2-SC is a ~UIilpdLdL;v~ leukocyte volume histogram obtained for whole blood sample C on a Unipath CELL-DYN~) 1600 Alltnm~tP~ analyzer using the mAn:lfArhIrer's rPrnmmPn~Pd reagent system.
Figure 3A is a leukocyte volume histogram showing the total number of cells versus cell volume for the human blood sample analyzed in Example 3, E~l el;~ l,L 2 herein, using a lytic reagent composition of the present invention rnmrricing~ 1-dodecylpyridinium chloride and benzyldimethyltehradecyl AmmoniIlm chloride in a S Plus II(~ tnm~tpd particle analyzer (Coulter Electronics, Hialeah, Florida).
SUB5TITUTE SHEET (RULE 26) _ _ _ _ . . .... . . ...... . ...... . .. . . .. . . .

WO 95/32622 2 1 g 1~2 ~ 8 PCT/US94/06062 Figure 3B is a ~ iv~ hictrlgrArn showing total number of cells versus cell volume for the same human blood sample analyzed in Example 3, Experiment 2, which was andlyzed on the Coulter S Plus II(~) AlltrmAte~i analyzer using the blood iirr.-L~.,Lial reagent system rerrlmmr-nrl~d by the mAnllfArtllrer.
Figure 4A is a leukocyte volume histogram showing the total number of cells vesus cell volume for the human blood sample analyzed in Example 3, E,~ .æl~ 3 herein, using a Iytic reagent composition of the present invention comprising dodecylethyl- dimethyl Amm~nillm chloride and hexadecylpyridinium chloride in a Coulter S Plus IV(~ automated differential particle analyzer.
Figure 4B is a ~u..lpa.aLiv~ leukocyte volume histogram showing total number of cells versus cell volume for the same human blood sample illustrated in Figure 4A, which was andlyzed on the Coulter S Plus IV~3 àuLulllaL~d particle andlyzer using the blood differential reagent system rer~mml~n-l~d by the mAnllfArhlrer.
DETATT ~n DFS('RTPTION OF TETF INVFI~TION:
The reagent system of the present invention comprises a novel Iytic reagent composition and a suitdble blood diluent which, when combined with a sample of whole blood is capable of effectively removing the red blood cell population and ~ y effecting a cignifirAntly improved ~
of white blood cells on a leukocyte volume hictrlgrAm of an ~ 1 blood andlyzer. The reagent system has the adv~.~Lag~ of being sufficiently flexible for U~Li~ g AlltnmAt~rl white blood cell iirr~-~.,Lials for multiple animal species and is particularly suitable for h~mAt~lrlgirAl analysis of human blood samples. In the following description, the invention is first described in terms of its application in hu}nan blood analysis, after which a description of the manner in which the reagent system can be optimized for multiple species applicdtions is provided.
Th~ Lytic R.oAvent After an extensive i~v~Li~LiulL, the inventors herein have dis.uv~ d thdt the admixture in aqueous solution of aliphatic uud~llldly Ammrmillm compounds with at least one aromatic, heterocyclic or .~.ilL~-iu-Lic ulud~ a~y A------rl~ " detergent results in a lytic reagent that is highly selective for white blood cell mrmhrAnl~c and is, in fact, cdpable of effecting a five component separation of white blood cells. During the SUBSTITUTE SHEET (RULE 26) inv~sti~Ation, it was also Liis~L~vel~d that long chain alkyl pyridinium rnmroun~1c in ~ ." with at least one;al~phatic LludlellL~Lly Allllll~ll;lllll rc~mrUIln~ or at least one aromatic, helelv~y~lic or ~wi~h.iuluc quaternary Amm--nillm c~mro~n~l are highly specific for white blood cell ....... ,1,.. ,~.~. and can be used to improve the ~ iL I~ of white blood cell subpopulations on a volumetric hictngrAm.
The lytic reagent composition of the present invention can thus be described as an aqueous solution of active Iytic reagents selected from the group ~UlLsi:~liLlg of:
(a) an ~.1."; A 1 l l l L- of at least one group (A) aliphatic quaternary Alllllll~ll;ll~ll compound with at least one group (B) aromatic, llelelucy.lic, or zwitterionic LlualL-IILaly ~mmonillm compound;
(b) an admixture of at least one group (A) aliphatic ~udlellLtLIy ..... ,.. ~),.il.. compound with at least one group (C) heterocyclic LAuaLell.~Lly .. -.,~;,.. , ~LJLLL~vuL~d; and (c) an a~luLL~Iule of at least one group (C) heterocyclic Llud~ellL~Lly nllllllllll;lllll compound with at least one group (B) aromatic, llelelu.y.lic, or ~willelLolLic LlualellkLly Ammcnillm uu-~uuL-d.
The aliphatic group (A) Llualell.aly Ammnni11m compound can be yle~ellled by the formuAa:
LRz R1 -- N1 _ ~3 X

wherein Rl is a long chain alkyl group ~rmtAining from 10 to 20, and yLe~eL~ly .~..,I~;..;.~g 12, carbon atoms, R2, R3 and Ra~ are all short chain alkyl groups .~L ., .1~ "-i, .~ from l to 6 carbon atoms, and X is an ion selected from the group consisting of halide, ~ ' o sulfate, phosphate, nitrate ions, and H~C ~ 5, ~
Bxamples of preferred group (A) aliphatic IIUdlelllaly ~IIIIIIIIIII;IJIII salts are described in U.S. Patent No. 4,745,071 to Lapicola, the pertinent portions of which are il~ul~ulaLed by reference, with the dodecyltrimethyl Amm~ni1lm halides and dodecylethyldimethyl ~mm~nillm halides being particularly preferred. Hexadecyltri-methylammonium-p-toluene sulfonate is another preferred group (A) compound.
SUBSTITUTE SHEE~ (RULE 26) .. . . . _ . .. .. .. . ... . ... _ ~

WO 95/32622 = PCT/IJS94106062 The group (B) llual~ aly Ammonillm compound, which is either an aromatic, heterocyclic, or a 2:Wi~l;U'liC quaternary AmmonilIm detergent can be l~ st:llled by the general formula:
(R2)n R1 -- N ~ tCH2 3m A) n (X ) p (~2) n ~ _ wherein Rl is a long chain alkyl group ct~ntAinin~ from 8 to 20, preferably 12 to 16, carbon atoms, R2 is an alkyl group .~ l to 6 carbon atoms, m is an integer from 1 to 4, A is a member selected from the group .. ~ of phenyl ( --~ ), phenoxy t-o--~ ) and sulfonate ( -SO-3) radicals, n is an integer from 0 to 1, such that when n=0, the remaining nitrogen (N) bonds forrn part of a heterocyclic ring, X is an ion selected from the group ~ of halide, pl~o~ aL~, sulfate, and nitrate ions and p is an integer from 0 to 1.
When the group (B) compound is a zwitterionic compound, A is blg a sulfonate radical. Examples of the zwitterionic group (B) . nmrolln~c include N-octyl-N, N-dimethyl-3-ammonio-1-propane sulfonate, N-dodecyl-N, N-dimethyl-3-ammonio-1-propane sulfonate, N-tetradecyl-N, N-dimethyl-3-ammonio-1-propane sulfonate, and N-hexadecyl-N, N-dimethyl-3-ammonio-1-propane sulfonate. Examples of the aromatic group (B) compounds include dodecyldimethyl (2-ph~:--u~y~Lllyl) Amm~nillm bromide, benzyldimethyldodecyl Ammnnillm chloride, benzyldi..l~lllyll~L~adecyl Ammnnillm chloride and benzyldimethylhexadecyl Ammnnillm chloride. In the heterocyclic compounds f~n~lmrAcced by the formula when n=0, the nitrogen may form part of a pyrroline ring, a pyrrolidine ring, â pyridine ring or a quinoline or icoq~linolinf. ring. Preferably, the nitrogen forms part of a pyridine ring. A
particularly preferred group (B) heterocyclic compound is hexadecyl pyridinium chloride.
The group (C) heterocyclic quaternary Ammnnillm compound is a pyridinium compound which can be ~ s~l-Led by the general formula:
R1 -- N~ X
SUBSTITUTE SHEFT (RULE 26) W095/32622 2191218 PCTIUS94/06062 ~

wherein Rl is a long chain alkyl radical ~ y, from 10 to 20, and .dbly 12 carbon atoms and X is an ion select~*om the group consisting of halide, phosphate, nitrate and sulfate ions. Particularly preferred group (C) compounds include 15 dodecyl pyridinium chloride and hexadecyl ~yl;-iilliulll chloride.
The Iytic reagent composition of the present invention is prepared by admixing in water (1) at least one of the group (A) compounds with at least one of the group (B) ~ull-puullds, (2) at least one of the group (A) compounds with at least one of the group (C) compounds; or (3) at least one of the group (C) .~,I..ro",l.~s with at least one of the group (B) .UlllpOuu--ls. Preferably the water is ~IPinni7P~l In order to achieve optimum belJal~liùl~ between ~u~popula~ions of white blood cells, it is important that the ratio of the individual quaternary ammonium compound components of the Iytic reagent composition be controlled. In accordance with the invention, it has been .~ ,..,;"r-cl that optimum se~ArA~i- n can ordinarily be achieved when the ratio of the group (A) compound to the group (B) compound in the Iytic reagent composition ranges from about 5:1 to about 75:1; the ratio of the group (A) compound to group (C) compound ranges from about 0.01:1 to about 99:1, and the ratio of the group (C) compound to group (B) compound ranges from about 5:1 to 75:1, ~ u~Liv~ly.
The optimum ratio of the individual ~UlllpUl~llL~ of the active lytic reagents may vary, rlPpPn~in~ upon, for example, the chemical activity of the CUlll~JUUl~dS selected and the individual instrument to be utilized in the differential analysis. Optimization of the ratio within the ranges provided is considered well within the skill in the art and can be achieved, for example, by varying the relative amounts of the individual quaternary ,IIl.l~r~
compound CUIIII.)U1~121115 in the lytic reagent rnmrrlsitil~n, rr~nrlllrtin~ an analysis on the particular analyzer to be used and rnnfl~lrtin~ an ocular trauma test to ~lPtPrminP the ratio which effects the cleanest separation on the leukocyte hictrlgr~m The tohl rnnrPntrAtirln of the active Iytic ill~ i~lL~ in the lytic reagent ulllpOsi~iul~ will typically be from about 7 to about 370 gms/liter. It should be understood, however, that the . I..~ ;nn of the active Iytic reagents in the rrmrositirm can be altered if the volume of the Iytic reagent composition is modified a~u.dil.y,ly, as long as the ratio of the individual quaternary Ammrmil.m compound is mAintAinPd within the defined limits.
SUBSTITUTE SHEET(~ULE26) ...... ..

*~ WO 95/32622 2 1 9 1 2 1 8 - - - PCT/US94/06062 To form a suitable chromagen for hemoglobin ~ , as is desired for operation of most of the commercially available ~lltt~m~t~l blood analyzers, the Iytic reagent ~UIllpOsiliu-l of the present invention can also beprovided with an alkali metal cyanide, such as potassium cyanide (KCN).
Other ~luv~lag~ forming agents can also be employed. It should be dp~ ciaL~d~ however, that the alkali metal cyanide is an optional ingredient used to accomplish a hemoglobin i.ot~rminAti-~rl and is not critical for achieving a three or a five ~omron-ont C~r~r~tion of white blood cells on a leukocyte volume l i~u~lalll.
A particularly preferred form~ tir~n for the Iytic reagent ~u~ usiliu-- is:
Preferred Tr~f~r~ nt C'~n~ntration (B) benzyldimethyl tetradecyl 6.5 gm/liter Ammnnillm chloride (C) l-dodecyl ~ylidiluulll 89.5 gm/liter chloride KCN 0.75 gm/liter water sufficient for 1 liter The Blood Diluent The Iytic reagent romrncitirm of the present invention is used in ,~"...~ i.... with a suitable blood diluent. The diluent can be essentially any one of the diluents hel~ used in blood differential analysis.
Preferably, the diluent is an isotonic ~u~po~i~iull ~ul--lu.;~i.lg at least one organic buffer, a cell m~mhr~n~ stabilizing means and an antimicrobial agent. Most ~ r~ably, the diluent is an isotonic ~UlllpUsi~iUll ~ at least one organic buffer and a m~)n~ hyde as the cell membrane 5t~hili7in~ means and antimicrobial agent. Al~ -a~iv~ly, a germicidal, such as l-hydlu,~y~uyl;.lille-2-thione, which will not adversely interfere with the ionic strength, osmolality, pH, or other cell volume determining .1.,~".. ~ c. or red or white blood cell Iytic ~h~r~rt.orictir.c of the Iytic reagent, can be employed.
The organic buffers which can be used in the blood diluent include ADA (N-[2-Acetamido]-2-imin~ ri~tir acid; N-[Carba-moylmethyl]
imino~ tic acid), MOPS (3-[N-Morpholino] propanesulfonic acid), PIPES
SUBSTlllJTE SHEET (RULE 26) r WO 9~i/32622 2 1 9 1 2 1 8 PCT/US94/06062 (Piperazine-N,N'-bis[2~th~nr-cl-lfnnic acid]; 1~4-PipPr~7in~flirth~nr-cl-lfnnir acid), HEPES (N-[2- IIydlu..y~:~l,yl]lui~ld~ e-N'-[2-eth~nrclllfnnir acid]), BES[N,N-bis['2-IIylllu,~y~Ll-yl]-2-~minn-~thAn~c~lfnni~ acid; 2-[bis(2-Hydlo~.y~ yl) amino]~th~n~-llFonic acid) BIS-TRIS (bis[2-Hydroxyethyl)imino tris-[l~yllluAylllethyl]mrthRnp~7-bis[2-Hydroxy-ethyl]-amino-2-[llyLllu;~ylll~lllyl]-1,3-lulupal~ediol); TES (N-tris[Hydroxymethyl]methyl-2-dll,il,o~ P~"lfcmir acid; 2-([2-Hydroxy-1, 1-bis(hydlu~yll.~ll-yl)-ethyl]amino)~th~nr-c.llfnni~ acid), TAPSO (3-[N-tris(Hydroxymethyl)meLl,yldll,il~o]-2-hydroxy-pro~n~clllfnnir acid), MES (2-[N-Morpholino]ethAnr-c.llfonic acid), DIPSO (3-[N,N-bis(2-l~ydlu~y~lllyl) amino]-2-hy~,a;.ylul~ lfnnir acid), MOPSO (3-[N-Morpholino]-2-hy~luxylulv~,~"P!~-,lfnni~ acid), TRIS (tris [l~ydlu~yllæLl-yl]
slminnmr-th~n~), and ACES (2-[2-Amino-2-oxoethyl)-amino]ethanesulfonic acid; N-[2-Acetamido]-2-i.l";"c"~ .,lrul~ic acid). ACES is particularly preferred.
In addition to diluting the blood sample for ~llUlll~ld~iUII, the purpose of the diluent system is to suitably stabilize cell size, shape and integrity of all of the blood cellular Lu.l,po.~e."~ both before and after treatment with the Iytic reagent composition, in order to promote the diagnostic accuracy of blood histograms derived from ~lltnmAt~d volume analysis and ~ .;buLiu-l.
To ~rrnmrlich this purpose, a preferred fcrmlll~tlnn of the isotonic diluent is:
I-ly,l~di~lll Effective Most ( nn~ r~ge Pr~f~rred sodium sulfate 7.5-12.5 9.7gm/liter sodium chloride 3.0-5.0 4.0gm/liter organic buffer 0.1 - 4.0 2.0 gm/liter fc~rm~l~lPhyde 0.1-15 1.0 ml/liter water sufficient for one liter The advantages of this diluent are described in my copending patent application filed ~ uu~ly herewith.
The pH of the diluent should be adjusted to about 7.1+1.4, and diluent osmolality adjusted to about 325+50 milliosmoles with sodium chloride.
The described diluent will produce accurate histograms with virtually any of the ~lltnm~trcl or semi-~l-tnm~tr-c~ blood analyzers but will produce ciFnifir~ntly improved three component and five component lu~u~;lallls SUBSmUTE SHEEl- (RULE 26) ~' when used in ~ with the Iytic reagent rnmrncitinn of the present invention.
Thr~ thnd In accordance with the inventive mPthn~lnlngy, a whole blood sample, a volume of diluent and a volume of the Iytic reagent rnmrositinn are supplied to an ~ analyzer and rapidly admixed therein, such that the ratio of the individual quaternary ,.. ".. ;.,.-. components in the Iyticreagent rnmrocitinn are present in a ratio and quantity sufficient to effect at least a three .:olllpol~llL ~ludldLiul, of white blood cells and then using the analyzer to dif~l~l,Lal~ and quantify at least one of the white blood cell subpopulations.
In a preferred embodiment, the Iytic reagent rnmrncition is first admixed with an initial volume of diluent The whole blood sample is then dispensed into the thus prepared Iyse/diluent mixture, with a second volume of diluent. In accordance with this ~mhor~im-~nt, the initial volume of diluent can be varied in order to adjust the Iytic strength of the reagent rnmrr~citinn, while the final volume of diluent can be varied in order to adjust the final dilution ratio.
The final ratio of blood diluent to Iytic reagent composition will typically be from about 5:1 to about 250:1 and the final dilution of the blood will be from about 5 to 1000, generally about 250 parts by volume total diluent and Iytic reagent ~U~ osiLiul, per part by volume of the whole blood sample The quantity of total active Iytic reagents to the volume of blood in the counting cuvette of the analyzer should range from about 0.15 mg total active Iytic reagents/~l whole blood to about 3.2 mg total active Iytic reagents/lll whole blood The pH of the final dilution should be from about 5.25 to about 8.75.
When the pH of the final dilution is below 5.25, the neutrophils will shrink faster than the lymphocytes and all subpopulations will begin to merge, which is highly lln~lPcirAh]r- If the pH exceeds about 8.75, the lymphocytes will fail to shrink properly and will remain too large in relation to the neutrophils, again causing a merger effect.
It should be appreciated that the above values are only examples based upon c~lrrr-ccfl]l ~nrml~lAtionc and reasonable deviations therefrom also will provide useful results in achieving the objects of the invention.
In the automated analyzer, the whole blood/reagent system mixture remains in the lysing chamber for a short but sufficient amount of time for the red blood cells to be ~lullld~uly~d and release their hemoglobin and for SUBSTITUTE SHEET (RULE 26) WO 95/32622 2 1 9 1 2 1 8 PCI~/US94/06062 the active Iytic reagents to act on the cell ll~r~ dl e~ of the white blood cells and cause them to shrink. As will be readily d,u~ idl~d by persons skilled in this area of technology, the incubation times may vary clr-pPnrlin~ on factors such as the strength of the active lytic reagents used in the lytic reagent WlllpUsiliUll and the total final concer~tr`a~ion of the active Iytic reagent inthe sample. As a general guideline, for human blood analysis, the whole blood/reagent system mixture will generally remain in the Iysing chamber for a period of time ranging from about 10 to about 30 seconds. The rlr-tr-rmin~tirln of the precise inr~ ti~n time for a given reagent system in accordance with the present invention can be readily ~ Pd by persons skilled in the art.
The suspension resulting from the foregoing is then passed through sensing apertures in a leukocyte counting bath, wherein the white blood cells are counted and the volume (size) and/or opacity is measured by electrical or optical ~I;rr~ There, the white blood cells are counted for a period of time to gather sufficient data for analysis, typically about 10,000 cells. The data points are stored and analyzed in a l~ n ~ r and visually displayed on a white blood cell histogram such as the one illustrated in Figure 1.
Using an automated blood differential analyzer, including an image PnhAn~rmrnt system described in copending U.S. patent application Serial No. 07/650,686, the inventors herein have ;li~-uvrl~ that the lytic reagent ..""~ ...s described herein are highly selective for the III~IILldllr~ of the various white blood cell subpopulations and are sull.l;sil-gly capable of effecting a volume Sr-p~r~tir~n of the white blood cells into five subpopulations. After treatment with the reagent system, the volumes of the Iymphocytes, basophils, monocytes, eosinophils and neutrophils in the human white blood cell population were found to be in the vicinity of 40-109,110-124,125-165,166-200, and 201-500 r~ , lr~e~Liv~ly.
Although the Iytic reagent compositions of the present invention are capable of effecting a five c:ul~lpol~el~ Srl~n nlir") of white blood cells, the five ~O~ uullrll~ cannot ~Iltr~m~hr~lly be readily visualized from the histograms plotted by cullullrl~ially available ~Iltr~m~tr-rl differential analyzers. A five wlllpullell~ r,n- lir"~ . can, however, be analyzed on such systems by manipulating the threshold values set within the instruments themselves in ~rrlrrl~nrr with the volume inf~rm~h~n and peak locations provided herein. Such manipulations are within the skill in the art and will be readily SUBSTITUTE SHEET (RULE ~6~
.... ... . . . . . ..

2~9;1218 .
WO 9S/32622 PCTIUS~4/06062 d~ ial~d by the mAn1lfArt1lrers of Alltl~mAtPr~ and ~pmiAlltomAt differential analyzers.
When utilized on ~vllul~l ially available differential analyzers in the absence of any threshold manipulations, the Iytic reagent LUIll~!osiliol~s of the present invention are capable of showing an improved three component C~ d~ of white blood cells when compared to the reagent systems typically employed on such analyzers. This aspect of the invention is iIlllctrAtPcl in Example 3 herein, wherein e,~u.~ldly lytic reagent LUllL~osiliu.~s within the scope of the present invention were comparatively tested on commercial differential analyzers of several of the leading ull~ t mAnllfA~tllrers.
In order to optimize for an improved three Lulllp~ elll sepArAtion on other mAn1lfActllrers~ sL u...~ , it is necessary to rlPtPrminP the optimum ratio of the individual u,ual~lllaly Ammnnillm compound ~ulllpul~llL~ as well as the total LUll~t:llLldLiVI~ of active lytic reagents in the lysing ~uu~posiliul~. The inventors herein have found that the preferred method of U~ AI;~I involves the lItili7Atinn of a full factorial e~rPrimPntAI design in accordance with the mPthorl~Irlgy described by Box et al, StAtisti~c For r.. John Wiley & Sons, publishers (1978) (the pertinent portions of which are hereby in-v.~vld~d by reference) in r~mhinAti~n with a visual . ..",rA, ;~c ,- of the resulting l~i~Lvgldllls. The use of experimental design is employed to reduce the number of tests required to riptprminp the optimum ratio and total Lul~enLlaLivll of the active Iytic reagnts for a given U~LIu--,~l~L. Since most .ullul-el idl analyzers are fixed in terms of diluent volumes, blood volume, end point dilution ratios, and inrllh~til-n time, the only variables to be optimized for any given illsL.ull-~l,L are the ratio of individual LudL~Illaly Ammfmillm compound components and total quantity of the active Iytic reagents.
~IIltiqpP~`iPC .~lirAtinnc A particular advantage of the present invention is that the reagent system and mPth~ lngy can be adapted for multispecies applications. Since, however, the physiology of white blood cell membranes can differ ci~nifi~AntIy from species to species, the reagent system must be optimized for each animal species whose blood is to be analyzed. O~I;III;~AI;~III is best effected utilizing the full factorial ~x,u~ ldl design in accordance with the mPthrltlr"hY of Box et al, cited hereinabove.
5UBSTITUTE SHEET (RULE 26) WO 95/32622 2 19 1218 PCT/US~)4/06062 Parameters to be optimuzed include the ratio of the individual quaternary AmmnnilIm compound components in the lytic reagent composition, the total amount of the active lytic reagents per unit volume of whole blood, the strength of the Iytic reagent ~nmro,Sitinn and the in~llh~tinn time prior to~ ",.~I,.ti--n The rull factorial design methodology as u~ilized by the inventors herein is illustrated in Exarnple 4 herein. Using this ms~thnrlnlclgy~ the inventors have optimized a number of different lytic reagent ~ulllposiliul~s for several animal species, including the canine and feline species.
Optimized values for a Iytic reagent II~ Q~ ll comprising 1-dodecylpyridinium chloride (group C) and benzyldimethyltetradecyl Ammt~nillm chloride (group B) are provided in Table 1 below.

Ratio Ratio Ratio Lytic Agent Lyse: Time Species Lytic Agents Blood Sample Diluent (Seconds) Canine 37.1:1 0.58:1 1:24.5 20 Feline 12:1 0.38:1 1:16.9 10 Using the afor~m~ntil~n~d full factorial design m~th~ Iogy, the reagentsystem of the present invention can be optimized for virtually any species of animal.
The present invention will be more clearly Iln~f~rctnod from the following specific examples. These examples are for illustrative purposes and are not to be construed as limiting the spirit and scope of the invention in any way.
F.Y~mple 1 This example illustrates the pI~dIa~iOI~ of a preferred lytic reagent ..."~ of the present invention and the use of the lytic reagent ~uIIl~osiLiol~ in an automated dirr~I~l,Iidl analyzer to effect a five componentseparation of white blood cells from a sample of human whole blood.
SUBSTITUTE 5HEET (RULE 26) WO 95/32622 21 9 1~18 PCT/US94/06062 An example of a preferred Iytic reagent i ,.,..I..,c;l;",~ in accordance with the invention was fi~rm~llAti~il in accordance with the following:
Component Amnunt per liter l-dodecyl IJy~ iulll chlioride 89.5 grams (Group C) benzyldimethyl tetradecyl 6.5 grams Ammnnillm chlioride (Group B) water sufficient to make one liter This lytic reagent ~ iul is suitable for use with diluted blood samples. In this example the blood diluent had the following fnrmlllAtinn Cnmronent Amnunt per liti~r sodium sulfate 9.7 gm/liiter sodium chlioride 4.0 gm/liiter organic buffer 2.0 gm/liiter fnrmAl~ hyde 1.0 ml/liiter water sufficient for one liter A 140 iul sample of the Iytic reagent composition was rapidly dispensed into the counting cuvette of t~ie AlltnmAti~d analyzer with 2.0 ml. of the blood diluent (the initial diluent) using a precise metering device, i.e., a syringe. 20 ~ of whole blood sample was ~a~ iul~ed off using a pipetting tip and rapidly iJiicppnse~l into the counting cuvette with 2.84 ml of the blood diluent (final diluent) using a syringe, to obtain a total diluent volume of 4.84 ml and a final total volume in the counting cuvette of 5.0 ml. The reagents were dispensed at a rate and in such a fashion to accomplish a rapid and thorough mixing of the sample.
- The diluted sample was analyzed for leukocyte volume and enumeration by electronic resistance mea~ s of the sample passing through an orifice. Scanning was i~nn~ ti~' for 10 seconds after addition of the reagent system. An image i~nhAn~ mi~nt apparatus of the type described in copending appliication U.S. Serial No. 07/650,686 was utilized in order to enhance the vicllAli7Atinn of the 5~ effected using the reagent system of the inventiion.
SUBSmUTE SHE~T (RULE 26) WO 9S/32622 2 1 9 1 2 1 8 PCT/U~94/0606Z ~

Three different blood samples, rl~ci~At~d samples 1, 2 and 3 were analyzed in accordance with the foregoing. The resulting hict~grAmC are rl~ `odu~d in Figures L~-IC, L~IJe~Liv~:ly. As illllctr~tP~I in Figures lA-lC, the reagent system of the present invention effecte~a`!five .. ~ S~pArAtl~-n of the five major subpopulations of white blood cells on the increasing size order of lylllpllo~yl~s, basophils, ul~llo~yL~, eosinophils and neutrophils, Liv~ly .
The counts obtained on the ~ i analyzer for each of samples 1, 2 and 3 were then compared to manual counts obtained for each of three samples, the results of which are set forth below.

lympho- baso- mono- eosino- neutro-cytes phils cytes phils phils ManualCount 77 -- 3.4 1.8 17.8 Analyzer 71 1.4 2.7 3.3 21.6 lympho- baso- mono- eosino- neutro-cytes phils cytes phils phils Manual Count 37 1 4.2 3.2 54.6 Analyzer 38.8 1.3 5.6 3.7 50.6 lyrnpho- baso- mono- eosino- neutro-cytes phils cytes phils phils Manual Count 9.8 0.5 11 6.9 71.8 Analyzer 11.7 1.4 10.9 7.9 68.1 SUBSTITUTE SHEET (RULE 26) . . .

WO 95/326Z2 2 1 9 1 2 1 8 PCI/US94tO6062 As can be ~p~.~dalt:d by a skilled clinician or ~ noct~ n~ theseresults display strong correlation not only for the normal sample but also for the two highly abnormal samples. Additional i"r~ including correlation ~.~rl;. i~ and flagging rates is discussed in Example 2~
.
~XAMPL~. 2 Ap~ u~dll~al~ly 220 human blood samples were analyzed using the reagent system and method described in Example 1. Correlation ~oPffi~iPntc with manual microscopic ~ ,.. and flagging rates were ~ ,Pf;
in accordance with the NCCLS standard for Reference Leukocyte Differential Count And Evaluation Of I~ u~ dl Methods. The average results are 5l1mm~ri7Pd in Tables 2 and 3 below.

Cn-rPI7~ti-1n CoPffi- entc Iymphocytes ¦ basophils monocytes eosinophils ¦ n~:ulLu~hils 0.98 1 0.18 0.55 0.48 1 0.95 Flagging Rates Irl~L~ l NP~tive Il~ u~ Positive Reference True Negative FalsePositive Norm~l 84.2% 15.8%
Reference False Negative True Positive Ahnorm~l 4 7% 95.8%
These preliminary data on more than 200 normal and abnormal blood .il.æl s indicate a high degree of correlation between leukocyte population data from the prototype analyzer and the manual 800 cell ditr~l~lllials. Lower correlation fl-Pffi~iPntc for basophils, monocytes and eosinophils are common because the leukocyte population data from the prototype analyzer and the manual 800 cell differentials. Lower correlation coefficients for basophils, monocytes and eosinophils are common because the range of sample variation is narrow.
The flagging rates indicate a high efficiency rate and the ability of the system to flag abnormal samples 95.8 percent of the time without causing excessive flagging ûf normal samples.
5UBSTITUTE SH EET (RULE 263 WO 95132622 2 1 9 12 1 8 PCI'/US94/06062 ~

The typical variation from technician to t~rhniriAn when r~mr7rin~
the manual reference method to itself is reported by Koepke, J. et al, "A
Critical Evaluation Of The Manual/Visual Differential~Leukocyte Counting Method", 1~1~11~ (1985)11:173-186. .~, FXAMPL~ 3 r~
This example illushrates that the lytic reagent system of the present invention, when used in romhinAtinn with a suitable blood diluent, is capable of effecting an improved three rt~mrr~nf~nt S~rArAhon of white blood cells on commercially available R~lt~mAt~i particle analyzers when compared to the reagent systems conventionally employed on such Analyzers In each of the Pxrl~rim~ntc herein, the Rllt ~mAt~d analyzers were run in A~ 1' with the mAnllfArhlrer's il~ U~liUIIs, using either the reagent system of the present invention or the reagent system rrr--mmf~n~ l by the respective m~...,~,.l Illll-la and routinely employed in the du~ull,al~d analyzers.l All other ~alallælt:la were mQ;ntAin~d constant.
F~Prim~nt 1 : .. , .. ~. ...
In this ~x~ , whole blood samples were analyzed on a CELL-DYN(!~i 1600 AlltOmAt~d blood analyzer available from Unipath. The following Iytic reagent compositions were prepared and loaded onto the CELL-DYN~ 1600 AlltOmAtf'd analyzer. The diluent was the same as that described in Example 1.
1. dodecylethyldimethyl A 1111111111 i 11111 bromide 27.6 gm N-dodecyl-N,N-dimethyl-3 ammino 1- 2.76 gm propane sulfonate KCN 0.25 gm water 1000 ml 2. dodecylethyldimethyl Amm-millm bromide 27.6 gm benzyldimethyltetradecyl Amm,~nillm 0.792 gm chloride KCN 0.25 gm water 1000 ml .. . .. . ............ . . .. . ..
I Since the lyse, diluent, and blood volumes are all ~0 pr~lr-tl~rmin~d and preset by the i ~Llull~ ",~ r,~ 1, the operator had no control over t~æse parameters.
SUBSmUTE SHEET (RULE 26 ~ wo ss/32622 Z 1 9 1 2 1 8 P~TrUS94106062 3. dude~yll~ ethyl Allllllllllilllll chloride 27.0 gm dodecyldimethyl (2-phenoxy ethyl) 3.86 gm AmmnniIlm bromide KCN 0.25 gm water 1000 ml Each of the foregoing Iytic reagent ~ (1, 2 and 3, ~e.~iv~ly) was run on the CELL-DYN 1600 in ~ull.~ dLiol~ with the specified blood diluent and whole blood samples A, B and C. The resulting 11;CIII~;IAIII~ are set forth in Figures 2-lA, 2-2A, 2-lB, 2-2B and 2-3C. For l (lllll.A i iCI ll l, the same blood samples were then also run on the Alltnm ~tP~l il~Llulll~ using the reagent system rPrnmmPn-lPd by the mAnllf~rtllrer.
The resulting ~ulllpalALiv~ 1AIIIC are reproduced as Figures 2-SA, 2-SB
and 2-SC, l~ t,e.Lively. As is apparent from a 1'111111 IA I ;C.- 1l l of Figure 2-lA and 2-2A with Figure 2-SA, Figures 2-lB and 2-2B with Figure 2-SB, and Figure 2-3C with Figure 2-SC, in each instance a cleaner s~l lA I A 1 ;l l~ l was effected utilizing the reagent system, including the Iytic reagent LUlllpUsiliL,l~, of the present invention.
FypPrimPnt 2 In this experiment, a sample of whole blood was analyzed on a Coulter S Plus II Alllllll.AI~l analyzer (Coulter Electronics, Inc., Hialeah, Florida), using first a reagent system, including a Iytic reagent rnmrncitinn of the present invention, and then 36 using the Iyse/diluent combination rPcommPn~lPd by Coulter Electronics for use in the S Plus II analyzer, as described in U.S. Patent No. 4,521,518. The Iytic reagent composition of the present invention utilized in this e~ .;",.~"l was .Llllpl;s~d of an admixture of 46.0 grams of l-dodecylpyridinium chloride, 3.3 grams of benzyldimethyl tetradecyl Allllll~llljlllll chloride and 0.6 grams KCN as a chromagen-forming reagent in 1000 ml of water. The diluent was the same as in Example 2.
The Coulter S Plus n analyzer does not effect any special mixing p~ dul~, such as that described in U.S. Patent No. 4,485,175, yet by rnmrArin~ the resulting histograms, it is apparent that the reagent of the present invention is capable of effecting a ~pAIAlinl~ of three populations of white blood cells while the mAnllfArtllrerls reagents on the same analyzer separated only two populations of cells.
SUBSTtTUTE SHEET (RULE 26) WO 95132622 2 ~ ~ 1 2 1 8 PCI/US94/06062 ~

~.
F ; ~I 3 '' ~
In this ~ , a sample of whole blood was analyzed on a Coulter S Plus IV ~.II.. AII~d analyzer (Coulter Electronics, Inc., Hialeah, Florida), using first a reagent system, including a lytic reagent ~ of the present invention, and then using the lyse/diluent cnmhinAtinn l~..lllllll~llrl~rl by Coulter Electronics for use in the S Plus IV analyzer, asdescribed in U.S. Patent No. 4,485,175. The Iytic reagent ~Ulll,UUsi~iol~ of thepresent invention utilized in this e~r-orimPnt was comprised of an admixture of 19.2 grams of dodecylethyldimethyl Al..lllllll;lllll chloride, 1.5 grams of hexadecyl pyridinium chloride and 0.6 grams KCN as a ~luu-l-ag~
forming reagent in 1000 ml of water. The diluent was the same as in Example 2.
The resulting l~ u~ s are set forth in Figures 4A and 4B, with Figure 4A illllctr~tin~ the histogram obtained from the analysis employing the reagent system of the present invention.
As can be seen by a ~ 111 of the histograms obtained in accordance with the present invention, versus those obtained utilizing the reagent systems currently available on the market and 11'111111111 lIf~ for use in the ~u~ ially available ;IIII~ analyzers, the Iytic reagent UIllyuSi~iullS of the present invention, when combined with a suitable blood diluent, yield cleaner three ~ull-luù,~,ll s~FArAtinnC of white blood cells.

This example illustrates the use of full hctorial exy~ l design for ulul;lll~ nn of the reagent system of the present invention for multiple species of animals. The ~lu~ al design is employed in order to reduce the number of tests necessary for U~ iUl~ of the critical pArAm~t~rC for a given species.
In accordance with this ~lU~LlLu~, stock solutions of the Iytic reagents are prepared in known l:U~ iUlls and starting Iyse volume, blood sample volume and total fluid volume are set. For example, for ulul;...;~.llillll of the reagent system including a group (A) aliphatic UlUCl~ ly Ammnnillm compound and a group (B) aromatic, heterocyclic or ,io, ic ulud~ ldly ammonium compound for the feline species, the following values could be set:
SUBSTITUTE SHEET (RULE 26) ~ WO 9S/32622 2 1 ~ 1 2 1 8 ~ . . PCTIUS94106062 Group (A) aliphatic uluate~llaly (stock solution) 140 gm/liter ~mmnnillm compound Group (B) aromatic, h~ lu~ydic or 10 grn/liter LCIiul~ç quaternary ~...,...,..;,., compound (stock solution) Starting Iyse volume 140 ~1 Blood sample volume 20 ~LI
Total fluid volume 5 ml in which the Iyse (A) (111), Iyse (B) (111), blood (111), initial diluent (ml) and final diluent (ml) equals the total fluid volume, in this çase 5 ml.
A starting matrix is then prepared, wherein the volume of the Iytic reagent stock solutions, initial diluent volume and in~lhAtinn time range from low to high. An example of the starting matrix is set forth below.
Table 1 Volume Volume T, -.l,~
Quantity Group (A) Group (B) Initial Time s~rk solu. stnrk ~n11l Dilll~nt (secnn~
nw 10% 10% ,5 ml 10 m 50% 50% 7 .'i ml 20 Higi- 90% 90% 4.5ml 30 The matrix is then run, varying the highs, medium and lows, to arrive at a series of experiments whidh will all then be run on an ~lltorn~t~
partide analyzer using the same blood sample. An optical trauma test or visual i.~l,e.liul. of the resulting histograms is then rnn~ rtP~, and the best results selected. The best values then become the midpoints of the next series of tests.
- An example of a full factorial design matrix is set forth below.
SUBSTITUTE 5HEET (RULE 26) Table 2 Full Factorial Design Run# Volume Volume Initial ~, ~ Time Group (A) Group (B) Dilu~ent~ (seconds) stock stock . ~
12 high high low high 17 mid mid mid mid 3 low high low low 5 low low high low 20 mid mid mid mid 10 high low low high 8 high high high low 9 low low low high 16 high high high high 2 high low low low 19 mid mid mid mid 4 high high low low 18 mid mid mid mid 13 low low high high 6 high low high low low low low low 15 low high high high 11 low high low high 7 low high high low 14 high low high high SUDSTITUTE SHtET(~ULE26) ~ WO9S132622 21~12~8 PCT/US94/06062 For the foregoing ~ , the blood sample volume is fixed at 20 1 of blood and the total volume is 5.0 ml.
The actual values for several of the runs to be rrln~ rtl~ in accordance with the Table 2 matrix are set forth in Table 3 below.
Table 3 Group A Group B Initial Blood Time Final stock stock Diluent Sample (seconds) Diluent solution solution L 14 Ill 14 ,ul 0.5ml 20 111 10 4.452ml (10%) (1.96mg) (0.14mg) M 70 ,~1 70 111 2.5ml 20 111 20 2.34ml (50%) (9.8mg) (0.7mg) H 126 1ll 126 ~1 4.5ml 20 Ill 30 0.228ml (90%) (17.64mg) (1.26mg) RUN#
12 High High Low High 126 111 126 111 0.5ml 20 111 30 4.228ml (17.64mg) (1.26mg) 17 Mid Mid Mid Mid 70 111 70 111 2.5ml 20 111 20 2.34ml (9.8mg) (0.7mg) 3 Low High Low Low 14 ,ul 126 ~1 0.5ml 20 ~Ll 10 4.34 ml (1.96mg) (1.26mg) 5 Low Low High Low 14 ~Ll 14111 4.5ml 20 111 10 0.452ml (1.96mg) (0.14mg) SUBSTITUTE SHEET (RULE 26) WO 95/32622 21~1218 PCTNS94/06062 Each e-~rPrimPnt 1l design matrix should be, I ' ~ as quickly as possible in order to minimize sample age, ~Jl~t lably within about 1/2 hour.
The blood sample should be at least 30 rninutes old prior to starting and should also be less than 24 hours dd.
After ~nmrlPting the rnatrix e~ , t~e,l. .t~ are analyzed visually. The best ~- ~t~-~,- ,.---~ in terms of which blood cell su~y~ulalio~l s~Pr~r~inn are chosen and the data recorded (i.e., %A~ %B, initial diluent (ml) and tirne (sec.). The selected data points are then utilized as the starting criteria for the next matrix. This plU~ U~ is repeated until the data is visually optimized.
Atter the data has been visually optimi7P~, the final matrix is used to ...... P correlation of a manual count with an ~lltnm:lt~d count for each run. At least 35 different blood ~.e. ;...~ from the same animal species should be used at this point in the IJlU~t~dUUt:. The .I;rr~ 1 analysis of each matrix box for all 35 ~l~P ..a'..~ is then compared to the manual I;rr~ l of white blood cell sulJ~ul,uldliul~.
An example of the final matrix is set forth in Table 4 below.
Iable 4 (ly~ o~y~s) Run No.
Specimen Manual 12 17 3 ... 11 7 14 Using the aforementioned illustrated experimental design, the reagent system of the present invention can be optimized for virtually any animal species.
From the above description it is apparent that the objects SUBSTITUTE SHEET (RULE 26) WO 95/32622 219 1218 PCT/US~4/06062 of the present invention have been achieved. While or~y certain bodi~ ls have been set forth, alternative embodiments and various mnrlifil ~fil-nc will be apparent from the above description to those skilled inthe art. These and other all~ iv~s are considered equivalents and within the spirit and scope of the present invention.

SUBSTITUTE SHEET (RULE 26

Claims (10)

Having described the invention, what is claimed is:

1. A lytic reagent composition for the automated differential analysis of white blood cells, comprising;
an aqueous solution of active lytic reagents selected from the group consisting of (a) an admixture of at least one group (A) aliphatic quaternary ammonium compound with at least one group (B) aromatic, heterocyclic or zwitterionic quaternary ammonium compound;
(b) an admixture of at least one group (A) aliphatic quaternary ammonium compound with at least one different group (C) heterocyclic quaternary ammonium compound; and (c) an admixture of at least one group (C) heterocyclic quaternary ammonium compound with at least one group (B) aromatic, heterocyclic of zwitterionic quaternary ammonium compound; wherein the group (A) quaternary ammonium compound is represented by the formula:
X-wherein R1 is a long chain alkyl group containing from 10 to 20 carbon atoms, R2, R3 and R4 are short chain alkyl groups containing from 1 to 6 carbon atoms, and X is an ion selected from the group consisting of halide, sulfate, phosphate nitrate ions, and ;
the group (B) quaternary ammonium compound is represented by the formula:
wherein R1 is a long chain alkyl group comprising from 8 to 20 carbon atoms, R2 is an alkyl group containing 1 to 6 carbon atoms, m is an integer from 1 to 4, A is a radical selected from the group consisting of:
; or - SO-3, n is an integer from 0 to 1, such that when n=0, the remaining (N) bonds form part of a heterocyclic ring, x is an ion selected from the group consistingof halide, phosphate, sulfate and nitrate ions and p is an integer from 0 to 1;
the group (C) quaternary ammonium compound is represented by the formula X-wherein R1 is a long chain alkyl radical comprising from 10 to 20 carbon atoms, and X is an ion selected from the group consisting of halide, sulfate, phosphate and nitrate ions;
wherein the individual quaternary ammonium compound components of said lytic reagent are present in a ratio and quantity sufficient to effect at least a three component differential separation of white blood cells.

2. A lytic reagent composition according to claim 1, wherein the reagent composition comprises an aqueous solution of active lytic reagents comprising at least one aliphatic quaternary compound from group (A) and at least one aromatic, heterocyclic or zwitterionic quaternary ammonium compound from group (B), wherein the ratio of the amount of the group (A) compound to the amount of the group (B) compound in the aqueous solution ranges from about 5:1 to about 75:1.

3. A lytic reagent composition according to claim 1, in admixture with an isotonic blood diluent, said diluent having a predetermined pH and osmolality.

4. A lytic reagent composition according to claim 3, wherein the isotonic diluent comprises at least one organic buffer and a cell membrane stabilizing agent and antimicrobial agent.

5. A method for differentiating white blood cells in a particle analyzer into at least three subpopulations comprising the steps of:
(A) supplying a whole blood sample, a volume of blood diluent and a volume of a lytic reagent composition to a particle analyzing system;
(B) rapidly admixing in the particle analyzing system the whole blood sample, the blood diluent and the lytic reagent composition in a manner sufficient to enable the differentiation of at least three subpopulations of white blood cells; and (C) analyzing the resulting mixture in the particle analyzing system to effect at least a three component differential separation of white blood cells;
wherein the lytic reagent comprises:
an aqueous solution of active lytic reagents selected from the group consisting of (a) an admixture of at least one group (A) aliphatic quaternary ammonium compound with at least one group (B) aromatic, heterocyclic or zwitterionic quaternary ammonium compound;
(b) an admixture of at least one group (A) aliphatic quaternary ammonium compound with at least one group (C) heterocyclic quaternary ammonium compound; and (c) an admixture of at least one group (C) heterocyclic quaternary ammonium compound with at least one group (B) aromatic, heterocyclic or zwitterionic quaternary ammonium compound; wherein the group (A) quaternary ammonium compound is represented by the formula:
X-wherein R1 is a long chain alkyl group containing from 10 to 20 carbon atoms, R2, R3 and R4 are short chain alkyl groups containing from 1 to 6 carbon atoms, and x is an ion selected from the group consisting of halide, sulfate, phosphate nitrate ions, and ;
the group quaternary ammonium compound is represented by the formula:
(X-)p in which R1 is a long chain alkyl group comprising from 8 to 20 carbon atoms, R2 is an alkyl group containing, 1 to 6 carbon atoms, m is an integer from 1 to 4, A is a radical selected from the group consisting of:
; or -SO-3, n is an integer from 0 to 1, such that when n=0, the remaining (N) bonds form part of a heterocyclic ring, x is an ion selected from the group consistingof halide, phosphate, sulfate and nitrate ions and p is an integer from 0 to 1;
the group (C) quaternary ammonium compound is represented by the formula X-wherein R1 is a long chain alkyl radical comprising from 10 to 20 carbon atoms, and X is an ion selected from the group consisting of halide, sulfate, phosphate and nitrate ions;
wherein the individual quaternary ammonium compound components of said lytic reagent are present in a ratio and quantity sufficient to effect at least a three component differential separation of white blood cells.

6. A method according to claim 5, wherein the blood diluent is an isotonic diluent having a predetermined pH and osmolality, said diluent comprising at least one organic buffer, and a cell membrane stabilizing agent and antimicrobial agent.

7. A method according to claim 6, wherein the lytic reagent composition comprises an aqueous solution of active lytic reagents comprising at least one aliphatic compound from group (A) and at least one aromatic, heterocyclic or zwitterionic quaternary ammonium compound from group (B), wherein the ratio of the quantity of the group (A) compound to the quantity of the group (B) compound in the aqueous solution ranges from about 5:1 to about 75:1.

8. A reagent kit for use in the automated or semiautomated differential analysis of white blood cells comprising:
(a) a predetermined volume of a lytic reagent composition, for stromatolyzing red blood cells and selectively shrinking white blood cells into at least three subpopulations; and (b) an isotonic blood diluent, wherein the lytic reagent composition comprises:
an aqueous solution of active lytic reagents selected from the group consisting of (a) an admixture of at least one group (A) aliphatic quaternary ammonium compound with at least one group (B) aromatic, heterocyclic or zwitterionic quaternary ammonium compound;
(b) an admixture of at least one group (A) aliphatic quaternary ammonium compound with at least one group (C) heterocyclic quaternary ammonium compound; and (c) an admixture of at least one group (C) heterocyclic quaternary ammonium compound with at least one different group (B) aromatic, heterocyclic or zwitterionic quaternary ammonium compound; wherein the group (A) quaternary ammonium compound is represented by the formula:
X-wherein R1 is a long chain alkyl group containing from 10 to 20 carbon atoms, R2, R3 and R4 are short chain alkyl groups containing from 1 to 6 carbon atoms, and X is an ion selected from the group consisting of halide, sulfate, phosphate, nitrate ions, and ;
the group (B) quaternary ammonium compound is represented by the formula:
(X-)p wherein R1 is a long chain alkyl group comprising from 8 to 20 carbon atoms, R2 is an alkyl group containing 1 to 6 carbon atoms, m is an integer from 1 to 4, A is a radical selected from the group consisting of:
; or -SO-3, n is an integer from 0 to 1, such that when n=0, the remaining (?) bonds form part of a heterocyclic ring, X is an ion selected from the group consistingof halide, phosphate, sulfate and nitrate ions and p is an integer from 0 to 1;
the group (C) quaternary ammonium compound is represented by the formula X?
wherein R1 is a long chain alkyl radical comprising from 10 to 20 carbon atoms, and X is an ion selected from the group consisting of halide, sulfate, phosphate and nitrate ions;
wherein the individual quaternary ammonium compound components of said lytic reagent are present in a ratio and quantity sufficient to effect at least a three component differential separation of white blood cells;
and the isotonic blood diluent has a predetermined pH and osmolality.

9. A reagent kit according to claim 8, where the isotonic blood diluent comprises at least one organic buffer, and a cell membrane stabilizing and antimicrobial agent.

10. A reagent kit according to claim 9, wherein the reagent composition comprises an aqueous solution of active lytic reagents comprising at least one aliphatic quaternary compound from group (A) and at least one aromatic, heterocyclic or zwitterionic quaternary ammonium compound from group (B), wherein the ratio of the amount of the group (A) compound to the amount of the group (B) compound in the aqueous solution ranges from about 5:1 to about 75:1.