Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~_Z1'~ iS81~3
MULTI-PURPOSE BLOOD DII.UENT AND LYSING AGENT FOR DIFFERENTIAL
DETERMINATION OF LY~PHOID-MYELOID POPULATION OF LEUKOCYTES
Thi3 invention coocern~ a reagent system for volume
differentiation of at least two population of leukocytes including
blood diluent especially suitable for uae in electronic enumeration
and Rizing of blood cells, determinatioo of hemoglobin and their
collective indices and platelet parameter in a jingle blood cell
s2mple by means of suit&ble electronic instrumentation, and a
stromatoly~ing reagent for use therewith.
The diluent comprises a stable water solution of chemical
salts providing an electrolytic solution capsble of conducting current
to which a blood sample can be added 80 as to dilute the Ted blood
cells, white blood cells platelets and other blood components and
enable the deiired parameters of these blood component to be
measured, counted and evaluated.
It is a common medical diagnostic prodedure to analyze and
test a blood sample of a patient in order to make certain classic
determination with respect to the blood sample. This procedure it an
important tool for the physician. Six characteristically important
parameters are referred to a red blood cell count (RBC~, the
hematocrit HOT the hemoglobin (Hgb), the mean corpuscular volume
(MCV), the mean corpuscu1ar hemoglobin (MCH), end the mean ~o~pu3cular
hemoglobin concentration (MCHC). A seventh important determination it
white blood cell count (WBC).
Much effort has been devoted to the development of
satisfactorily automated leukocyte differential systems. However, 9
need exits for rePgent systems which will be easily adaptable to
automatic blood counting instruments. In particular, it i8 desirable
to develop reagents and methods for use with the COULTER COUNTER Model
21us series of automated blood cell counters, manufactured by
Coulter ~lectronic~, Inc. of ~ialeah, Florida, which Jill enable the
cell volume data Rccumulated on a COULT~R CHANNELYZER to diacriminaee:
(1) a lymphoid and ~yeloid population, a described in Canadian
Patent 1,158,967, and ~23 lymphocyte, mo~ocyte and granulocyte
rj 35 population, ~8 deocribed in U.8. Patent 4b4~S,175. Such dots arc
uJeful q8 e ~creenln~ tool Por apottin~ abnormQl leukocyte ratioo.
iZ~S89
Abnormal situstions flagged out by this method give information of
diagnostic significance, and for further study. COULTER, COULTER
COUNTER end CHANN~LYZER are registered trademarks of Coulter
Electronic, Inc.
Separation of normal human leukocyees by volume distribution
was first documented by Gauthier and colleagues, (Gauthier, J., Harel,
P., Belanger, C. and Fray~se, J., Can. Med. Assoc. J. 97, 793, (1967)
and Van Dilla &nd colleagues, (Van Dilla, M. A., Fulwyler, M. J. and
Boone, I. U., Proc. Soc. Esp. Biol. MedO 125, 367, in 1967) as a
possible clinical diagnostir method utilizing the principle of
counting and 3izing developed by Wallace H. Coulter and employed in
CO~LTER COUNTER instruments. These methods were based on the
fundamental property of all living cells to regulate their cell volume
by genetic code information. Each type of cell in the circulating
blood has its own characteristic volume ranging from aa ~mqll as 3 fL
for platelets to more than 450 AL for polymorphonuclear cells (fL
designates 1 X 10-15 1iters, a femtoliter). Advanced COULTER
COUNTER instruments have been de3igned to make use of this volume
differential for the purpose of counting and determining the size
distribution of cell components and to detece and monitor pathological
ate
Electrical sizing of particles in suspension by a COULTER
COUNTER type instrument his been previously described and documented
by many clinical hematology investigators. It is well known that the
form and to of the electrical pulse generated by a particle passing
through a defined electrical field is influenced by several factors
including 3ize, shape and conductance of the particle bein8 counted.
In blood cell preparstions diluted in an isotonic salt solution,
conductivity of the cell membrane it far lower than conductivity of
the diluent, end therefore, blood cells may be considered to be
electrically non-conducting for practical consideration.
Erythrocytes and the typhoid leukocytes unfortunately
overlsp conniderably in cell size, and it id not poa~ible to count one
f in the presence of the other by size discrimination slone.
Traditional practice involves the use of serong reagene that
stromstolyoes k ~rythrocyte~, reducing them to very 3m~11 psrticle3
,.
~24~58~
or causing membrane solubiliz~ltion, and at the same time strips the
cytopls~m from the leukocytes, leaving only the resi~tan~ nuclei to be
counted. Since original cell volume is drastically affected and
reduced to a minimum, only a single population is visible by size
distribution analysis.
The COULTER COURTER Model S Plus automated blood cell
counter i5 designed to dilute a sample of whole blood in an isotonic
diluent, add a stromatolysing reagent, and then begin counting after a
few second. Data typically are collected for a very few seconds for
erythrocyte0, leukocytes and platelets; however, an increase in count
cycl2s of a few second etch can be invoked automatically by the
instrument system to achieve needed statistical accuracy. Thus, a
diluent-lysing reagent system for measuring more than one population
of leukocytes must provide erythrocyte lysing kinetics 3ufficiently
rapid to effect complete stromatolysation of the ~rythrocytes during
the lysing period. Changes in leukocyte volume mu8t be minimal during
the data collection step and ideally should be stable for several
minutes. The reagent system mu8t also preserve the integrity of the
erythrocyte and platelet number and size distribution, the hemoglobin
absorbance curve and the total leukocyte count. Finger stick bloods
should be stable when pre-diluted in the isotonic diluent for at least
two hour.
To achieve an analysis of the relative_populations of
lymphoid and myeloid cells in the blood, the leukocyte volume
histogrAm must show cleanly separated lymphoid and myeloid peaks, with
little erythrocyte debris, allowing volleys very close to the
baseline Integration of each peak will give the relative populations
of the lymphoid and myeloid cells. The lymphoid peak has been
demonstrated to contain lymphocytes flnd variant lymphocytes, Chile the
myeloid peak con~ain~ polymorphonuclear cell band, monocytes,
eo~inophils, basophils and other abnormal cells.
In Canadian Patent 1,024,868, 8 rea8ent formula it included
for composition contsinin~ one quaternary ammoniu~ salt detergent
ant potassium cyanide to be employed as 8 lying end chromagen-forming
re~8en~ for obtaining a total leukocyte count, without r~gsrd for
subpopulAtion~, ant hemoglobin determination in ehe COULTER COUNT
~2~ 9
Model S. Further investigation was required to use a plurality of
quaternary ammonium salts as lysing agent for obtaining the
two-population leukocyte count.
In U. S. Patent 4,286,963, a lytic diluent for the rapid
lysing of red blood cells in whole blood for making a differential
determination of lymphoid/myeloid populations of leukocytes, and alto
measuring hemoglobin by chromagen formation, contain a mixture of an
aqueous saline solution of at least one quaternary ~mmonium Walt
having surface acting properties, and certain additives such as
2-phenoxyethanol.
It is known that two volume distribution analysis is
difficult because, with many reagent systems, the two populations
rapidly move into one xo that there i8 not enough time within which to
make the computation for analysis.
In U. S. Patent 4,485,175, a method and reagent system is
described for three-volume differential determination of lymphocyte,
monocyte and gr~nulocyte popultaion~ of leukocytes.
In U. S. Patent 4,384,971, there is described a cleansing
composition for electronic particle counting apparatus, and method for
its use. This composition includes a non-hemolytic polyoxyethylated
alkylphenol detergent, dimethylolurea and l-hydroxypyridine-2-thione
which it adjusted to predetermined osmolality.
_ The present invention relates to a stromatolysing reagent
and a method for subjecting a whole blood sample Jo a reagent system
to achieve at least a volumetric differentiation of more than one
class of leukocyte popula~ion~. More particularly, the invention i8
directed to a ~tromatolysing rengent which includes an additive which
i8 a non-cationic surfactant.
The reagent ~y~tem employed includeA a multi-purpose
isotonic blood diluen~ that comprises a mixture of organic buffering
means, cell membrane stabilizing mean, and 8 germicidal meansl the
end volume concentration of which ~erve~ Jo ccntrol the lytic kinetic
of the ~trom~tolysing reagent upon the leukocyee~ and achieve a
difEerential volume reduction, Chile stabilizing the traditional
`; 35 hemogr~m psrameters.
12~5~9
The stromatoly~ing reagent best suited to this inventive
method it a mi$ture of an aqueous solution of quaternary a onium
salts having surface active properties and a non-cationic surfactant
additive in a volume concentration range ~hae is effective to give the
desired leukocyte volume histograms. The stromatolysing reagent is
hypotonic and therefore tends to combat the shrinkage of the myeloid
cells, such shrinkage being well known in the art. Data are presented
u3ing standard COULTER COUNTER blood cell analyzing equipment in
conjunction with a COULTE~ CNANN~LYZER instrument and an X-Y plotter.
Ancillary calculating and data handling devices are desirable for
complete automation, but are not essential to performance of the
measurements.
In the preferred coy ercial embodiment utilizing this
invention an alkali metal cyanide i8 added as the chromagen-forming
lS agent. Other chromagen-forming agents, such as Drabkin's reagent,
which contains potassium ferricyanide, potassium cyanide and sodium
bicarbonate also may be used.
The p~e~erred non-cationic surfactant~ which are useful as
additives include the ~onionic surfactants, such as the
polyoxyethylated alkylphenols and the polyethylene glycol phenol
ethers; and the anionic alkali metal salt of a C10 - C14 alkyl
sulfate. The additive serves the primAry purpoee of allowing for the
manufacture of a lying agent that will accurately po3ition the
lymphocyte and myeloid population of the instrument.
According to the invention there i8 provided a
stroma~olysing reagent for use in the determination ox leukocyte
populations in blood, two such populations being lymphoid and myeloid,
the blood being proc2ssed through a blood cell analyzer using the
Coulter Principle of operaeion, which employ a cell sensing zone, the
blood having been first diluted wi h on elecerically conductive
isotonically balanced diluent, wherein said stromatoly~ing re~8ent
comprises an aqueous solution of quaternary ammonium silts having
surface sctive properties, said reagent comprl~ing an additive which
i8 a non-cationic surf~ceant, or mixtures thereof, said sal~z and
additive being present in sufficient ~ount~ for po~itio~i~g the
lymphoid and myeloid populatione rel~tiv~ to one another and relative
';
~;~4~5~9
to a blood cell volume reference point, within the time constraint of
said bLood cell analyzer.
The invention further provides a method for determination of
leukocyte population in blood which is being processed through a
blood cell analyzer using the Coulter Principle of operation which
comprises the steps of:
I. diluting a blood sample with an electronically conductive
isotonically balanced diluent, and
II. stromatolysing with a reagent compris1ng an aqueous solution of
quaternary ammonium salts, having surface active properties, said
reagent including an additive which is a non-cationic surfactant, or
mixtures thereof, and said salts and additive being present in
sufficient amounts for affecting the normal leukocyte volume
distribution during said ~tromatolysation 80 as to cause the volume
distribution measurements by said blood cell analyzer to lie within
predetermined limits, with reference to at least one volume reference
point.
By Jay of example only, illustrative embodiments of the
invention now will be described with reference to the accompanying
drawings in which:
In the figures, the axis is femtoliters and the Y-axi~ i8
nonmalized cell count. In Figures 1, 2 and 3 the population at the
left it lymphoid, and at the right is myeloid. In Figure 4, the three
populations, left to right respectively, are lymphocyte, mononuclear
and granulocyte.
Figure 1 comparatively shows a pair o two-population
histograms developed from the same normal blood u8ing a stromatolysing
reagent having two different amounts of the same additive, 9
polyo~yethyl~ted al~yphenol nonionic surfsctfint, but the same quantity
30 of two qua~ernary ammonium salts, the amount of one salt being
different from the other;
Figure 2 comparatively show a pair of two population
hi~togra~ developed from the same normai blood using a 3tro~atolysing
I; reagent hiving two different amount of the tame additive a8 in Figure
1, and significantly different mount of the sue two quaternary
~mmoniu~ salt than a8 in Figure 1;
I,
~2~5~3~
Figure 3 comparatively shows a trio of two~population
histogram developed from the same normal blood each using a
~tromatolysing reagent, having the same preferred combinat;on of
amounts of the two quaternary am~onium sals, but two having different
S additives and one having no additive; and
Figure 4 comparatively shows a pair of three-population
histograms developed from the tame normal blood using a stromatolysing
reagent having preferred amounts of the two quaternary ammonium salts,
but two different amounts of the same nonionic surfactant.
The introduction of high speed automated hematology
instruments such a the COULTER COUNTER Model S as described in
Canadian Patent 936,018 resulted in a need for a high speed
erythrocyte stromatoly~ing reagent which gives a clear, stable,
reproducible solution. In such an instrument, blood is mixed with
conventional d;luent, to provide a first dilution, and then mixed with
a lysing agent, to provide a second dilution. The mixture remains in
the lysing chamber for a short but sufficient amount of time for the
erythrocytes or red blood cells to be disintegrated (3tromatolysed)
and release their hemoglobin. The lysing agent also converts the
hemoglobin to a chromagen suitable for measurement. The resulting
suspension i8 passed through sensing apertures in a leukocyte counting
bath, wherein the leukocyte~ or white blood cells are counted and
sized electronically. InAsmuch aY the ratio of erythrocytes to
leukocytes in normal blood it in the vicinity of lOOO:ls the
erythrocytes must be reduced rapidly to very small fragments, to avoid
interference with leukocyte counting. At the same time, the
leukocytes must not be destroyed, even though they will shrink in size
to a greater or lever degree.
A quaternary ammonium salt hiving surface active properties
advantageously i3 employed as the stromatolysing agent in the COULTER
blood cell analyzer, with the virtuslly i~atantsneoua destruction of
erythrocyte~ to a level avoiding interference ~i~h leukocy~e sizing
and counting. the quatern~ry ~mmonium salt is included in Aqueous
solueion in a concentration within the overall range of about 0.5 Jo
7 35 10 percene, and preferably bout 1 to 5 percent by uei~h~ of the
' solution end it mixed with blood pre~iou~ly diluted with a diluent,
in a ratio of about 1:10 lying agent volume to diluted sample volume
in the counting bath. It will be understood that a different strength
lying agent may be employed where the initial dilution o the blood
sample differs from that described above, in order to provide the æame
ultimate concentration of reactive lysing agent, or ratio of ly3in~
reagent to whole blood present in the counting bath.
When making a two-volwme separation of leukocytes, according
to the method of Canadian Patent 1,1589967, it had been observed that
the lymphoid-myeloid kilogram obtained after ly~is on the COULTER
10 COUNTER S P1UB instrument consisted of a lymphoid peak at about 5~ to
100 fL, and a myeloid peak, containing monocyte~ and granulocytes
~eosinophils and neutrophils) in the volume range of 100 to 450 fL.
The specific quaternary a~monium compound and its
concentration are selected to provide the necessary hemolytic activity
and solubility of the quaternary ammonium compound. In general, with
increasing number of carbon atoms, the solubility of the compound
decreases, and with increasing number of carbon atoms in the long
chain, the hemolytic activity increases.
The long chain quaternary ammonium compounds useful for
their strong lytic action on erythrocytes are commercial materiels
derived from natural products which may vary somewhat from time to
time in their lysing strength, due particularly to their composition
~ith re~pece to the length of the carbon chain attached to the
nitrogen atom. The differential determination of two or more volumes
of leukocytes, on the other hand it very sensitive to the purity or
strength of the lysing agent, and a reliable separation into at least
two volumes of leukocyte~ can be difficult to attain with the
available commercial products sold on the market.
We have now discovered the unexpected result that thy
addition of a non-c~eionic fiurf~ctant (either nonionic or anionic)
which alone his only minor hemolytic effect, shows a synergistic
interaction with the quat~rn~ry ammonium salts. Thus, ~oderstion or
slowing down of the kinetica ox the react;on to a point where reliable
measurement can be waken it dependent upon the concenErations snd
3S relstive rstios of both quaternary ~m~oniu~ ail and the non-cationic
~urf~ctant, while 3till preaer~ing the total lysing effect of the
~24~
reagent. Addition of the non-cationic surfactant to a solution of the
quaternary ammonium salts appears to modify the lying rate, changing
the lymphocyte volume population only slightly, but changing the
myeloid volume population by a significant amount. Careful selection
of the concentration of the quaternary ammonium salts and of the
non-cstionic ~urfactant avoid excessive shrinkage of the lymphoid
population, resulting in fewer "incomplete" computations. Variations
in the quantities of activity of the commercial quaternary ammonium
salt can easily be offset by varying the concentration of the
additive agent. It i8 therefore more practical to control the effect
of the lytic reagent by varying the concentrations of the non-cationic
surfactant along with minor changes in the conceDtraeion of the
quaternary ammonium salts, than by attempting to obtain precise
control by modifying the content and concentration of the quaternary
a~monium salt alone.
Changes in commercial products in term of purity or
concentraton from lot to lot would require the use of a different
concentrations of each quaternary a onium salt, or mixtures thereof
for each lot used in order for the lysing formula to achieve the same
end results with the instrument system. The cell size distribution
measurement is determined by the instrument system by mean of a
predetermined time "window" in the counting cycle, during which the
_cell lysing process i8 nearly complete and cell volume changes are
proceeding ~lo~ly enough for the instrument to measure numbers and
relative Yolumes of the now differentiated leukocytes. The cells must
al80 fall within the volume range of 45 to 99 fL for lymphoids and 100
to 450 fL fox myeloids. Thu3, the lysing reaction must be made to
occur rapidly enough 80 that the cell differentiation changes are
~ub~tantially complete when the tire 'window" opens and data are
collected. Since the timing cycle psrameters are a part of the
overall operational program of the instrument sy~eem, these are
e~entially predetermined and fixed by the manufacturer at the time of
sale. Any adjus~ment~ in dsta po~iPionin~ nece~sry are therefore
more readily jade by adjustment of the lying agent formulation.
Unexpectedly, it ha been found that thy "fit" of the cell population
data in the me~urement range "window" of the in~trume~t ~y~tem can be
~Z~5~3~
attained readily by a suitable mount of an additive which is a
non-c~tionic surfactant to the strom~tolysing agent in order to
regulate the kinetic of ie* action. Thus fine-tuning of the
equipment is best accomplished by varying the concentration of the
additive agent in the stromatolysing reagent rather than by changes in
the equipment. The improvement of this inveation accordingly relates
to the ability to position, relative to one another and a volume
reference point, the lymphoid and ~yeloid populations within the time
constraints of the instrument. This result is accomplished by more
lQ sensitive control of the r2te of reaction of the stromatolysing agent,
by using an additive which is a non-c~tionic surfactant.
This additive also serves a dual purpose in that it also
acts to prevent or to reduce the amount of protein deposit in the
sensing oriices of the instrument which tends to accumulate from the
cell debris resulting from the blood cell stromatolysation.
Satisfactory performance of any of the counting and sizings functions
of automated hematology instruments using electronic pulses generated
in accordance with the Coulter Principle i8 dependent on maintaining
clean and unimpeded fluid pathways, especially in and near the tensing
apertures. Due to the small size of the aperture and the
inaccessability of much of the fluid path, this ~elf-cleaning feature
assume great importance in such equipment.
Studies mode of the optimum concentrations of the C14
lysing reagent for COULTER COUNTER Model S Plus II for counting two
volumes of leukocytes it about 12 - 14 g/L. At these concentrations
the lymphoid distribution width i8 quite narrow, and the myeloid
distribution i8 po~itionet to the right side of the CHANNELYZER plot.
The concentrations of C12 quat necessary for correct placement of
the lymphocyte popul&tion~ or this concentrstion of C14 is
somewhat more flexible9 but the optimum concentration is about 30-40
g/L of the sctive agent. The lymphoid distribueion will then fit
within the 45-99 fL volume region and have modal volumes between 65-75
fly
The myeloid distribution movea to the right (luger apparent
volume in the presence of lo concentrations of C14 and high
concentrations oÇ C12.
5~9
The myeloid distributions are shifted to the left (smaller
apparent volumes) when exposed to higher concentration of C14 and
less C12.
The effect of using as addition agent the nonionic
polyoxyethylated alkylphenol surfactant or the anionic surfactant
sodium lauryl sulfRte is similar. Both compounds move the lymphoid
; and myeloid populations proportionately to the right (larger apparent
volumes). Sodium lauryl sulfate is more potent in this regard.
Studies using dimethylethylhexadecylammonium bromide in the
lysing agent show comparable results.
The significance of these findings relates to the ability to
position, relative to one another and a volume reference point, the
lymphoid and myeloid populations within the time constraints of the
instrument. By the use of appropriate concentrations of essentially
non-hemolytic surfactants, these distribution can be altered to
achieve the desired apparent volume for accomodating the engineering
aspects of the blood cell analyzing equipment.
A disclosed in U. S. Patent 4,384,971, when making a
three-volume separation of leukocytes, it was discovered and
determined by experimentation that the lymphocytes and monocytes are
more sensitive than the granulocytes to the lytic agents usually
employed. By modifying the kinetic of the lytic method in the
COULTER COULTER S Plu~_instrument to allow a more mild exposure of the
white cells in the blood sample to the lyse reagent, the granuLocytes
were less "shocked", i.e. subject to a lower or low gradient of lytic
shock, and thereby not reduced in size to the extent cnused by prior
reagent ~y~tems and methods. Thi8 i8 accomplished by treating the
diluted blood sample with the stromatolysing re~8ent less rapidly, and
when the stromRtolysing reagent it in lower concentr-ation thin i8
routinely the case. However, approximately the same mount of lyse
rea8ent i3 needed in order to ensure complete ~tromatolysation of the
red blood cells. By modification in the kinetics of the method, the
lymphocyte are reduced in volume to 50 to 100 fL, the ~onocyte~ are
r reduced to 100 to 160 fL, and the grsnulocytes demon~trste a volume
range of 180 to 4SO AL. Consequently, the granulocyte population no
; longer overlap the monocyte population and these populations can be
~2~S~39
enumerated separately. The resulting data must be obtained within a
time frame during which time three distinct populations are prevent.
Thiq was the first time that it had been learned how to
control the kinetics of the action Jo as to obtain this important
result. In the pat, the emphasis has been on the virtually
instantaneouY dPstruction of the red blood cell to a level avoiding
interference with leukocyte estimation. Herein, the empha~i~ i8 on
discerning the differential volumes of the individual classes of white
cells which, unlike the red blood cells, vary in many ways with
respect to morphology and the presence of various nuclear forms,
volume, as well as function, in health and with di3ease.
A simplified general description of the blood cell analyzer
instruments which utilize the Coulter Principle of operation is that
they include two fluid vessels or chambers, each containing a
conductive electrolyte solution. At least two elec~rode3 having
opposite polarity are immersed in the electrolyte solution, with each
fluid compartment having one of the electrodes disposed therein. A
sample of the electrolyte solution, hiving the blood cells suspended
therein, is passed through a constricted fluid path or orifice
interposed between the two fluid compartments. Although the
constricted path can take different forms, in each device such path
defines a sensing zone wherein the presence or absence of a particle
gives rite to a detectable change in electrical characteristics of the
path. For example, relatively poorly conductive blood cells passing
through this path dispLace a volume of electrolyte solution equal to
the cell volume, causing a voltage drop by increasing the path
impedance. The re~iRtance pulse defined by the drops in voleage are
used for particle counting and particle volume determinatiGn.
The COULTER COUNTER Model S Plua series of automated blood
cell counter are designed to dilute sample of wbole blood in an
i80tonic electrically contuct;ve diluent, add a lysing agent, and
8~0rtly thereafter begin counting. The diluent-lysing ~y~tem mutt
provide eryth~ocyte lysing kinetic sufficiently rapid to effect
co~ple~e stromatolysation of the red blood cell (erythrocyte~) during
the lysing perlod. During this time period, the cytoplAsm ia rapidly
removed from the leukocytes) ordinarily leaving only a jingle volume
~Z4~S8~
of the lyse-resistant nuclei to be counted. Since original cell
volume is drastically affected and reduced to a minimum, only a single
leukocyte population may be discerned. It is evident that the data
displayed is related both to the time cycle program provided for the
S instrument and to the kinetics of the lysing reaction, as well as to
the initial engineering design parameters of the equipment at the time
it is marketed. By moderating the speed with which the lysing takes
place in an appropriate manner under predetermined conditions, the
display may show more than one volume of leu~ocytes, as is described
with particularity in U. S. Patent 4,485,175 and Canadian Patent
1,158,967.
A preferred class of the non-cationic surfactants useful for
the purpose of this invention includes the nonionic polyoxyethylated
alkylphenol~ which are manufactured by methods known in the art by
reaction of alkylated phenols with an excess of ethylene oxide to form
alkyl aryl ethers of polyethylene, for example, by the following
reaction:
RC6H40H + x(C~2CH20) --> RC6H40(CH2CH2)X_1 CH2CH2
where R is an alkyl group having 8 to 10 carbon atoms, and x is an
integer 8 to 30.
An especially preferred member of this group is the compound
where R = 9 and = 30. The compound has the following product
characteristics:
Appearance clear, almost water-white liquid
1% solution clear and colorless
solubility in water readily soluble, even in cold water
pH of 1% solution neutral
stability stable to acids, alkalis and metallic ions
cloud point clear at 212C
Results from the use of this compound are shown in
histograms or traces B in Figures 1, 2, and 4.
The following nonionic ~urfactant~ can be used to obtain
comparable result to that shown in histograma B in Figure 1, 2, 3
i 35 and 4:
14
Alkylphenoxypoly(ethyleneoxy)ethanol where alkyl normal or
isomeric O Cg, C10, C12 and poly = 8 to 30 moles
of ethyleneoxy;
Dialkylphenolpoly(ethyleneoxy)ethanol, where alkyl = C8, Cg,
and poly = 8 to 30 moles of ethyleneoxy alkyl aryl polyglycol
ether, where alkyl = Cg, Cg, and poly = 8 to 30 mole of
ethyleneoxy ethoxylated phenol, with no alkyl chain.
Condensates of propylene glycol and propylene oxide with ethylene
oxide giving a poly(oxypropylene) poly(oxyethylene) ethanol
with poly(oxypropylene) 15 to 45 moles and
poly(oxyethylene) = 120 to 180 males
The following anionic surfactants can be used to obtain
results comparable to sodium lauryl sulfate shown in histogram C of
Figure 3:
Alkyl sulfoni~ acid salts (sale = Na, K, NH4; alkyl = octyl to
oc~adecyl);
Alpha-olefin sulfonic arid salts (salt = Na, K, NH4;
alkenyl = ~12-C18);
Alkyl aryl sulfonic acid salts (salt = Na, K, NH4;
aryl = benzene, xylene, toluene);
Alkyl diaryl sulfonic scid salts (salt - Na, R, ~14;
aryl= benzene, xylene, toluene);
_ Sulfosuccinates, pa, K or NH4;
Alkyl and dialkyl sulfosuccinaees;
Alkyl phosphates;
3ranched alkyl sulfates (e.g. 2-ethylhexyl sulfate);
Diethanolamine alkyl sulfate;
Alkyl ether sulfona~es (e.g. sodium lauryl ether sulfonate) with 1
to 12 moles of ethylene oxide;
Alkyl naphthalene sulfonate (e.g. lauryl nnphthalene sulfonate);
Nonylphenoxypoly(ethyleneoxy) ethanol (Na or NB4 sulfate);
Alkoxylated arylphenol phosphate (K, Na);
Alkylphenoxypoly(ethyleneoxy)ethanol sulfaee salts (R, Na).
The above co~position~ are sold it c = erce under a number
of tratemarks.
Preferably, the method of this invention uses in combination:
35~
(A) A multi-purpose isotonically balanced diluent comprising for
example an aqueous solution of:
1. organic buffering means;
2. cell membrane stabilizing means; and
3. germicidal means;
said diluent having a predetermined pi and osmolality; and
(B) a lysing agent which is an aqueous solution of quaternary
ammonium salts having surface active properties.
The quaternary ammonium salts have the formula:
_ _
Rl ~R2 -
/ \ R4 X
R~
where Rl i3 a long chain alkyl radical having l0 to 18 carbon atoms;
R2, R3, R4 are short chain alkyl radicals having 1 to 6 carbon
15 atoms and K i3 a salt forming radical such as Cl-, Bra, PO4
and C~3 SO4. In the more useful combinations, the long chain
alkyl has 12 to 16 carbon atoms, the short chains are methyl or ethyl,
and K is chloride or bromide.
The preferred lysing agent employs a combination of
dodecyltrimethylammonium chloride, with tetradecyltrimethylammonium
bromide. Other quaternary ammonium salt3 that give effective result-
include hexadecyltrimethylammonium bromide or
hexadecyldimethylethylammonium bromide in combination with
25 dodecyltrimethylammoniuD chloride.
To form a suitable shromagen for hemoglobin determination,
a8 i8 desired for operation of a COULTER COUNTER Model S Plus
instrument, there can alto be provided an alkali metal cyanide, such
as potaasium cyanide. Other chromagen-forming agellt~ alao can be
30 employed.
Prior Art Example
Fo11Owing the recommended direction which are no public
knowledge for volume and flow adjuctmenes of COULTER COUNTER Model S
589
16
Plus, in order to produce two-volume tlymphoid-myeloid) populations of
leukocytes, the following ingredients are employed in the
concentrations indicated:
DILUENT CON OENTRATION CONCENTRATION
Canadian Patent 1,158,967 PREFERRED RANGE
1. Procaine O.ll g/L 0.05 to 0.25 g/L
hydrochloride
2. N-(2-acetamido)imino- 1.40 g/L 1.0 to 2.5 g/L
diacetic acid DADA)
3. Dimethylolurea 1.00 g/L 0.5 to 2.5 g/L
4. sodium hydroxide 0.50 g/l
5. sodium sulfate, 0.72 g/L
anhydrou 8
6. sodium chloride 4.50 g/L
7. water sufficient for 1 liter
Experimentation has verified that the amounts of the two
~uaternary ammonium salts and the non-cationic surfactant additive can
lie in relatively wide ranges, but that the amounts of these three
component3 are interdependent. Yet also, within certain practical
limits, the amounts of any two of these three variables can be chosen
at will, with the amount of the third component then becoming defined
rather narrowly. Although extreme limits of each range might work
under some conditions with some blood samples, practical, commercially
useful, ranges for these components are as follows:
dodecyltrimethylammonium chloride 10 to 55 g/L;
tetradecyltrimethylammonium bromide 9 to 20 g/L; polyoxyethylated
alkylphenol 4 to 20 mL/L or polyethylene glycol p-isoalkylphenyl ether
4 to 20 mL/L, or sodium lauryl sulfate 0.5 to 5 g/L; or mixtures of
two or more of these non-cationic surfactants in corresponding amount
to fit within the stated ranges of effectiveness.
EXAMPLE I
Using the same concentration of ingredients for the diluen~
as in the Prior Art Example, but changing the concentration of the
inxredients in the lysing reagent to the amounts shown below, the
~LZ~35~9
17
results are shown in tracing A of Figure l using a COULTER COUNTER
Model S Plus II instrument.
LYSING AGENT CONCENTRATION
l. Dodecyltrimethylammonium chloride 55 g/L
2. Tetradecyltrimethylammonium bromide 9 g/L
3. Potassium cyanide 300 mg/L
4. Water sufficient for l liter
When 12 mL/L of the nonionic polyoxyethylaeed alkylphenol
surfactant described above is added to the above formula, the results
are as shcwn on tracing B of Figure l.
EXAMPLE II
Using the same concentration of ingredients for the diluent
as in the Prior Art Example, but changing the concentration of the
ingredients in the lysing reagent to the amounts shown below, the
15 results are as shown in tracing A of Figure 2 using a COULTER COUNTER
Model S Plus II instrument:
LYSING AGENT CONCENTRATION
1. Dodecyltrimethylammonium chloride 10 g/L
2. Tetradecyltrimethylammonium bromide 20 g/L
3. Potassium cyanide 300 mg/L
4. Water sufficient for l liter
: When_8 mL/L of the nonionic polyoxyethylated slkylphenol
~urfactant described above is added to the above formulQ, the results
are as shown on tracing B of Figure 2.
EXAMPLE III
Using the tame concentration of ingredients for the diluent
a in the Prior Art Example, but changing the concentration of the
in8redients in the lying reagent to the amounts shown below the
results are as shown in tracing A of Figure 3 using a CO~LTER COUNTER
Model S Plus II instrument:
lZ~S89
18
LYSING AGENT CONCENTRATION
1. Dodecyltrimethylammonium chloride 3S g/L
2. Tetradecyltrimethylammonium bromide 13 g/L
3. Potas~iwm cyanide 300 mg/L
4. Water sufficient for 1 liter
When 8 ~L/L of a polyethylene glycol p-isoalkylphenyl ether
i8 added to the above formula, the results are a shown on tracing B
of Figure 3. Addition of 1.25 g/L of the anionic surfactent sodium
lauryl sulfate gives tracing C of Figure 3.
For ease of interpreting Figure 3, not shown is a histogram
representing the preferred mixture of the two quats C12 and
C14 with 8 mL/L of the polyoxyethylated alkylphenol. Such
histogram would appear most like histogram or tracing B in Figure 3.
EXAMPLE IV
Using the same concentration of ingredients for the diluent
as in the Prior Art Example, but with the following formulation of
ingredients for the lysing agent and doubling the volume of the lysing
agent, a three-population histogram was obtained using a COULTER
COUNTER Model S Plus II instrument and following the procedures of
U. S. Patent No. 4,485,175 issued November 27,~ 19~4: . .
LYSING AGENT CONCENTRATION
1. Dodecyltrimethyls~monium chloride 18.8 g/L
2. Tetradecyltrimethylammonium bromide 6.5 g/L
3. Potassium cyanide 150 mg/L
4. Water sufficient for 1 liter
The results are as shown in the tracing A of Figure 4.
When 3 mL/L of the nonionic polyoxyethylated alkylphenol
surfactQnt described above is added to the above formula, the results
are as shown on tracing B of Figure 4.
In each of the ~xample~ I to IV, the tracing A contains no
additive and, for that reason, those lysing agents will not yield cell
populations (neither two nor three populstiona) of leukocytes which
are suitably positioned for appropriate discernment or differentiation
by the sutomated cell snsly~er. Each of theae histograms fail in at
1243589
least one oE the criteria Gf properly positioned cell populations, a
well defined valley at the right side of the lymphoids lymphocytes in
Figure 4), or proper population distribution width. To the contrary,
all other of the produced histograms provide instrument useful
differential data. This i8 true even though Figures 1 and 2 were
generated by near end limits of the amounts of the quaternary ammonium
salt 8 .
While certain representative embodiments and details have
been shown or the purpose of illustrating the invention, various
changes and modifications can be made therein without departing from
the scope of the invention as defined in the appended claims.
