Method and reagent composition for performing leukocyte differential counts on fresh and aged whole blood samples, based on intrinsic peroxidase activity of leukocytes

The present invention provides an improved reagent composition and method to perform white blood cell differential counting and subpopulation analysis using both fresh and aged blood samples with accuracy and precision. The invention is particularly applicable for the analysis of aged blood samples that have been stored at room temperature for over a day, thereby allowing accurate and useful information to be obtained from samples that are normally considered to be suboptimal. The improved reagent composition and method are particularly related to the peroxidase method of white blood cell differential determinations. One aspect of the invention includes an improved aqueous reagent composition for carrying out the peroxidase method of differential counting. Another aspect includes the use of a rinse cycle and rinse solution devoid of hemolytic surfactant to alleviate the adverse effects of rinse carryover and to streamline and economize the analytical process, particularly when the analyses are performed on automated hematology analyzers and flow cytometry systems. The composition and method of the invention provide clinically useful data for the differential analysis of whole blood samples.

FIELD OF THE INVENTION 
The present invention relates to an improved leukocyte (i.e., white blood 
cell) differential method for whole blood samples and reagent compositions 
used in such a method. The leukocyte differentiation method and reagent 
compositions of the invention are based on the measurement of the 
intrinsic peroxidase activity of leukocytes. The method and compositions 
of the invention maintain the precision and accuracy of obtaining a 
leukocyte differential count on both fresh blood samples and on aged blood 
samples which have been stored at room temperature for about 48 hours, or 
in the cold, when the samples are analyzed electro-optically by light 
scatter and absorption flow cytometry. 
BACKGROUND OF THE INVENTION 
The five classes of white blood cells or leukocytes normally found in whole 
blood samples are neutrophils, lymphocytes, monocytes, eosinophils, and 
basophils. To determine the relative proportions of these five normal 
types of white blood cells, as well as to detect the presence and 
concentration of any abnormal cells in a whole blood sample, a medical 
diagnostic procedure is conventionally performed to examine a dried, 
stained smear of blood on a microscope slide. Such a procedure is referred 
to as a differential white blood cell count and is described in Miale, J. 
B., "Laboratory Medicine--Hematology", (1967), C. V. Mosby Company, St. 
Louis, Mo., pp. 822-830, 1126, 1127 and 1130. In addition to the 
above-listed five classes of leukocytes in blood samples, differential 
white blood cell counts also detect and measure large unstained cells 
("LUCs"). LUCs represent a small fraction of white blood cells in normal 
blood samples and comprise such cell types as large lymphocytes, activated 
lymphocytes, plasma cells, blast cells, and peroxidase-negative monocytes, 
neutrophils, and eosinophils. 
Semi- and fully-automated hematology processes and automated flow system 
apparatuses therefor have been developed to ease the burden of 
differential white blood cell counting and blood sample analyses, such as 
described in U.S. Pat. No. 3,741,875 to Ansley et al.; U.S. Pat. No. 
4,099,917 to Kim; and U.S. Pat. Nos. 4,801,549 and 4,978,624 to Cremins et 
at. Such processes and systems use electro-optical and cytochemical 
procedures to specifically detect, identify, quantify, and label 
individual cell types. In addition, manual procedures for determining 
white blood cell differential counts are known in the art; for example, 
see Miale, J. B., "Laboratory Medicine--Hematology", (1967), C. V. Mosby 
Company, St. Louis, Mo. 
U.S. Pat. No. 5,389,549 to Hamaguchi et at. describes methods and reagents 
used for classifying leukocytes, in which the methods involve the 
detection of changes in electrical impedance at high frequency or the 
differences in conductivity between particles and fluid medium, and the 
reagents require one or two component solutions which contain specific 
types of anionic and nonionic polyoxyethylene-based surfactants having 
18-30 repeating oxyethylene units per molecule and which also contain 
hyper- or hypo-osmotic agents and solubilizing agents. The two component 
reagents require both a first liquid diluent fluid and a second lysing 
reagent fluid. 
An earlier procedure for preparing a cell suspension for use in such 
systems comprised treating an uncoagulated blood sample with a surfactant 
for about 1.5 minutes to precondition the red blood cells for lysis; 
thereafter adding a fixative to the cells for about 1 minute while 
maintaining a neutral pH; and then incubating the mixture at about 
58.degree. C. to 60.degree. C. for about 2 minutes to lyse the red blood 
cells and fix the white blood cells, as described in U.S. Pat. No. 
4,099,917 to Kim. 
U.S. Pat. Nos. 4,801,549 and 4,978,624 to Cremins et al. describe a method 
and reagent for the determination of a differential white blood cell count 
which is performed more rapidly, which lyses red blood cells in a whole 
blood sample without damaging the white blood cells, and which causes 
minimal extra-cellular precipitation or clumping of cells. Such 
precipitates or cell clumps generate ambiguities in the cell detection and 
recognition phase(s) of the procedure. The procedure (or "peroxidase 
method") described involves a mixture comprising peroxide (i.e., hydrogen 
peroxide) and a suitable chromogen to stain and differentiate particular 
cell types in the leukocyte class. 
It is imperative in each of these processes that as many red blood cells as 
possible be lysed, since red blood cells outnumber white blood cells by 
about 1000-fold in normal blood. Because of this, even if one percent of 
the red blood cells remains unlysed, it is difficult to achieve an 
accurate and precise white blood cell differential count. 
In the peroxidase method as disclosed in U.S. Pat. Nos. 4,801,549 and 
4,978,624, red blood cells are lysed and white cells are crosslinked or 
"fixed" after a whole blood sample is mixed with a solution comprising 
only one surfactant, a fixative such as paraformaldehyde or formaldehyde, 
a sugar or sugar alcohol, and a buffer to maintain approximately neutral 
pH. Hydrogen peroxide and an electron donor chromogen, such as 
4-chloro-1-naphthol, form a dark-purple-colored precipitate in the 
peroxidase-positive granules located in the cytoplasm of certain white 
cells, namely, neutrophils, eosinophils, and monocytes. The precipitate is 
an insoluble reaction product the formation of which is catalyzed by 
endogenous peroxidase enzymes in the intracellular granules. 
Differentiating the cell types is carded out by electro-optical analysis 
in which cell size and degree of staining are measured (i.e., forward 
angle scatter versus absorbance) on a cell-by-cell basis and plotted in a 
cytogram which is then analyzed to obtain both a total white cell count 
and differential count of the different types of white cells in the 
sample. In addition, the total white blood cell count can be obtained 
independently of the differential count. 
Prior to the improvements and advantages afforded by the present invention, 
an alkaline peroxidase diluent had been described and particularly used in 
an alkaline peroxidase method of white blood cell classification carded 
out on a Technicon H6000.TM. automated analyzer system. The prior alkaline 
peroxidase diluent had major drawbacks, such as instability of the reagent 
components and a consequent short shelf and storage life. In addition, the 
user was required to prepare a homogeneous working solution of the 
alkaline peroxidase diluent in order to carry out the alkaline peroxidase 
method. This was accomplished by the user's having to mix together a 
solution containing a high level (i.e., 4.5%) of sodium dodecyl sulfate 
and a solution containing a high level (i.e., 30%) of Brij.RTM. 35, 
thereby resulting in a working alkaline peroxidase diluent having elevated 
concentrations of the two classes of surfactants. Such user preparation 
not only involved irritant diluent reagent components, but was also 
laborious, and had to be performed a number of times, because the 
resulting homogeneous working alkaline peroxidase diluent was stable for 
only one week. As will become clear, the instability, the short storage 
capacity, and the user-handling problems of the alkaline peroxidase 
diluent, as well as the commercial disadvantages related thereto, have 
been vastly improved upon and overcome by the present invention as 
described herein. 
Also prior to the present invention, a major drawback which hampered the 
accuracy and reliability of cell separation and quantification methods, 
particularly the peroxidase method of leukocyte differential counting, was 
variable rinse carryover in the method. It is known that rinse carryover 
varies from system to system and from analysis to analysis. In particular, 
the accuracy and precision of leukocyte differential analyses based on the 
peroxidase reaction method frequently suffered from the effects of such 
variable rinse carryover in the method. Those skilled in the art have 
assumed that rinse carryover contributes only a volumetric dilution to the 
method and that rinse carryover plays no active or functional role in the 
reaction steps of the method. Indeed, until the inventive discovery of the 
present invention, the skilled practitioner did not realize that rinse 
carryover was more than a simple volumetric effect in blood sample 
analysis, and had no solution to the problems offered by the present 
invention and described herein. 
In addition, there is a need in the art for improved reagents and methods 
for analyzing and extracting useful clinical information from both fresh 
(i.e., less than or equal to eight hours postdraw) blood samples and also 
aged blood samples that may have been stored for up to about 48 hours at 
room temperature. It is also necessary to develop the appropriate reagent 
solutions and compositions comprising components which will alleviate the 
newly-described problems generated by variable rinse carryover and will 
yield accurate and reliable results, especially, but not limited to, in 
the employment of electro-optical analysis of a variety of blood sample 
types, e.g., fresh whole blood samples, aged whole blood samples, abnormal 
whole blood samples (e.g., hospital or patient source), and normal whole 
blood samples (e.g., non-hospital or "healthy" donor source) stored in a 
variety of ways (e.g., in the cold or at room temperature). There is a 
further need in the art for reagent compositions that are very stable, 
have long shelf and storage lives (e.g., greater than one week), and 
require no user or customer preparation or handling prior to their use in 
carrying out leukocyte differential counting methods and obtaining results 
therefrom. It is also necessary to achieve and/or to maintain acceptable 
levels of noise at the origins of the resulting cytograms when carrying 
out differential counts on room temperature-stored or aged blood samples 
using automated analyzers. 
SUMMARY OF THE INVENTION 
The present invention provides an improved reagent composition and method 
for quantifying and differentiating leukocytes in both fresh and aged 
whole blood samples using electro-optical procedures and flow cytometry 
analysis. 
It is an object of the present invention to provide an optimal, improved 
reagent composition and method for use in semi- and fully-automated 
systems to avoid the problems of carryover of unwanted reaction components 
from one method step to another, thus allowing for clean separation and 
quantification of cell types without unacceptable levels of origin noise 
and cellular contamination in the cytograms resulting from the method used 
to achieve a white blood cell differential count. 
It is a further object of the invention to provide a rinse reagent solution 
that does not play any functional role in the reaction steps of the white 
blood cell differential method and which does not participate in the 
reaction chemistry of the method, with particular regard to the peroxidase 
method. 
It is another object of the invention to provide an automated peroxidase 
method and reagent composition which yield precise and accurate results 
with a variety of blood sample types, e.g., aged and fresh blood samples, 
abnormal and normal blood samples, and samples stored in both the cold and 
at room temperature. 
Yet another object of the invention is to provide an improved reagent 
composition for use in the peroxidase method of enumerating and 
distinguishing among white blood cell types in a whole blood sample by 
absorption flow cytometry. 
Still another object of the invention is to provide an optimized and 
improved method and reagent composition for the rapid and efficient 
quantitative measurement of the white blood cell count and subpopulation 
differential of whole blood samples using endogenous peroxidase staining 
in conjunction with automated hematology analysis and flow cytometry. 
Another object of the invention is to provide a reagent preparation which 
improves separation of the eosinophil subpopulation of cells in a white 
blood cell sample. 
It is another object of the invention to provide balanced and stable 
reagent compositions for optimizing the reaction steps of the white cell 
differential counting method involving peroxidase staining of aged whole 
blood samples and for removing and preventing any negative effects of 
rinse carryover in the method. 
Yet another object of the invention is to provide safe and stable reagent 
preparations and methods, wherein the reagent compositions are convenient 
and ready-to-use and require no additional preparation or mixing by the 
user or customer prior to use. The reagent compositions of the invention 
are stable and maintain long storage and shelf lives (e.g., greater than 
one week and at least about one year) at room temperature. A further 
object is to streamline the leukocyte differential counting method and to 
reduce the numbers of reagents that are needed for use in carrying out the 
steps of the method. 
Further objects and advantages afforded by the invention will be apparent 
from the detailed description hereinbelow. 
ABBREVIATIONS AND TERMS 
The following abbreviations and terms are defined or explained for the 
convenience of those skilled in the art and are not intended to limit the 
scope of the invention in any way: 
The lysis of red blood cells in the peroxidase method as performed on 
automated analyzers exemplified herein is defined by puncture of the cell 
membrane, causing most or all of the hemoglobin to leak out of the cell. 
The resulting red cell ghosts are capable of being chemically crosslinked 
or fixed by the fixative present in the composition of the peroxidase 
method. The red cell ghosts can be detected as noise in the origin region 
of a cytogram, which results from the electro-optical detection of cells 
in a sample using flow cytometry. 
Peroxidase is abbreviated as "Px" throughout the instant specification. 
As used herein, the terms aqueous reagent composition, reagent solution, 
and diluent are equivalent. 
R1 is the first reaction phase of the Px method of white blood cell 
differential counting. As explained in greater detail herein, in the R1 
phase of the Px method, a whole blood sample is mixed with a first aqueous 
reagent composition ("the Px R1 reagent composition" or "peroxidase R1 
reagent composition") formulated in accordance with the invention. During 
the R1 phase of the Px method, all or most of the red blood cells in the 
sample are lysed and the white blood cells are chemically crosslinked or 
fixed by the fixative present in the R1 reagent composition. 
Fresh blood samples are blood samples that are used for analysis less than 
or equal to eight hours postdraw. The term "fresh blood sample" is 
synonymous with the term "Day 1 sample" as used herein. 
Aged blood samples are blood samples that have been stored at room 
temperature for up to about forty-eight hours postdraw. The term "aged 
blood sample" is considered to encompass the term "Day 2 blood sample" as 
used herein.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates generally to improved methods and reagent 
compositions for the determination and quantification of white blood cell 
differential counts via a peroxidase (Px) method which relies on the 
measurement of the endogenous peroxidase activity of particular types of 
white blood cells. The invention relates particularly to semi-automated 
and automated flow cytometry analyzers and to improvements in the 
peroxidase method for white blood cell measurement and subpopulation 
determination. 
To assist in the understanding of the present invention, a summary of the 
cellular and molecular events which occur in an automated peroxidase 
method of determining leukocyte differential counts is provided. In the 
first or R1 phase of the peroxidase method, a blood sample is mixed with 
an aqueous first reagent (R1) composition (i.e., an R1 solution or 
diluent), and the mixture is heated to about 70.degree. C. for about 15-20 
seconds. During this interval, the red blood cells lyse, their hemoglobin 
leaks out, and the resulting red cell ghosts are fixed. The white blood 
cells are chemically crosslinked or "fixed" by a fixative compound, such 
as formaldehyde, to provide resistance to lysis during the remainder of 
the reaction. In general, red blood cells are more easily lysed than are 
white blood cells and platelets; however, it is not uncommon for the red 
blood cell ghosts and platelets to become crosslinked under the conditions 
used during the procedure, because lysis and fixation are competing 
processes that occur in the same sample reaction mixture. Thus, the 
peroxidase method requires a fine balance between the lysis of red blood 
cells and the fixation of white blood cells in a blood sample. If the 
lytic strength of the Px R1 reagent composition is too great, both the red 
blood cells and the white blood cells will be damaged. Conversely, if the 
concentration of fixative is too great, all of the cells will be fixed 
before red cell lysis can occur. Nevertheless, a white blood cell 
differential count is attainable with high levels of accuracy and reliable 
determinations of cell types and cell numbers, especially in view of the 
speed and the optimum reaction conditions and reagent formulations for the 
improved peroxidase method as developed in accordance with the invention 
to perform rapid sample analyses that occur using semi- and 
fully-automated hematology systems. 
In whole blood, the ratio of red blood cells to white blood cells is about 
1000:1. Therefore, for optimal performance of the automated peroxidase 
method for whole blood sample analysis, it is clear that essentially all 
of the red cells should be lysed in the sample aliquot early in the 
analysis. Consequently, in the first reaction using the R1 diluent, there 
is a competition between lysis of red cells and crosslinking of white 
cells. If red cells survive the lysis step with intact hemoglobin, they 
will likely interfere in the differential count as they can erroneously be 
detected as lymphocytes because of placement in the lymphocyte area of the 
resulting cytogram. However, it is also crucial that the white blood cells 
in the blood sample aliquot are not attacked or degraded by solution 
components during the R1 phase of the peroxidase method. 
During the second reaction phase (also called "reaction 2" or "R2" herein), 
which follows the R1 phase of the peroxidase method, the substrate 
solutions of hydrogen peroxide and an electron donor, such as 
4-chloro-1-naphthol are added to the R1 reaction mixture. These compounds 
are substrates of endogenous cellular peroxidase, which is differentially 
present in those white blood cell types that "stain", including monocytes, 
neutrophils and eosinophils. Lymphocytes and large unstained cells (LUCs) 
do not contain endogenous peroxidase enzyme and, therefore, are not 
stained in the method. Combinations of red cell ghosts and platelets, 
which also do not contain endogenous peroxidase enzymes, are not stained 
and may contribute to origin noise, which is encountered and accommodated 
for in nearly every method involving whole blood cell analyses. 
Incompletely lysed red cells, which contain some or all of their 
hemoglobin, are detected in the lymphocyte region of the cytograms 
generated from the Px method, and interfere with the method. Platelets and 
platelet clumps are also detected in the lymphocyte region and are an 
additional source of interference. 
In the peroxidase method, "staining" is the result of a complex chemical 
reaction in which the electron donor substrate such as 4-chloro-1-napthol 
is oxidized and polymerized into a deep purple reaction product which is 
trapped within the cells. Granules inside the cells stain purple, thereby 
allowing the cells to be observed in the peroxidase reaction mixture 
("effluent") if examined under the microscope (stained cells look black to 
the naked eye using microscopic examination). On automated hematology 
analyzer systems, such as those exemplified by the H.cndot..TM. systems 
commercially available under the trade designation TECHNICON 
H.cndot.1.TM., H.cndot.2.TM., H.cndot.3.TM., and the like, and sold by the 
assignee hereof, detection is made electro-optically by measuring light 
absorption (due to the purple reaction product) and light scatter (due to 
cell size). 
The present invention provides a single new reagent composition for use in 
the R1 phase of the peroxidase method to improve and optimize the 
performance of the overall peroxidase method, as a result of the presence 
of a newly added component to the reagent composition; the component is a 
hemolytic, nonionic surfactant, e.g., Brij.RTM. 35. The presence of such a 
nonionic surfactant in conjunction with an ionic surfactant in the new 
reagent composition affords a novel Px 1 reagent or diluent which can be 
successfully used with the other reagents in subsequent reaction phases of 
the peroxidase method as outlined above. Those skilled in the art will 
appreciate that in the R1 phase of the Px method, the amount and 
concentration of surfactant is critical--i.e., too much surfactant can 
cause the white blood cells in the sample to be attacked and too little 
surfactant will not lyse the red blood cells in the appropriate manner and 
will thus contribute to high origin noise in the resulting cytograms. 
In an embodiment of the invention, the new R1 reagent composition of the 
invention can also be used successfully when a rinse cycle employing an 
aqueous rinse reagent is also performed in the peroxidase method, provided 
that the rinse solution does not also contain a hemolytic nonionic 
surfactant such as Brij.RTM. 35. In particular, the R1 reagent composition 
of the invention can be used in conjunction with a new rinse reagent 
composition that is formulated to contain only a surfactant of the 
Pluronic.RTM. class, in the complete absence of other types of surfactants 
(i.e., without ionic surfactant, such as SDS, and without nonionic 
surfactant, such as Brij.RTM. 35). 
The reagent composition of the invention solves the following problem which 
is further elucidated in the description and examples provided 
hereinbelow. It was determined by the present inventors in carrying out 
the peroxidase method as currently performed on automated analyzer 
systems, such as those exemplified by the above-mentioned H.cndot..TM. 
systems, that there was routinely a volume of about 8 .mu.L of rinse 
solution left over in the reaction chamber at the end of one sample cycle 
of the peroxidase method. This seemingly small amount of rinse carryover 
became part of the next sample cycle; it was simply assumed by those 
performing the method that this volume did not affect the method in any 
functional way. 
However, it was newly found that when such rinse carryover exceeded a 
volume of about 10 .mu.L, the performance of the peroxidase method 
deteriorated. It was also unexpectedly discovered by the present inventors 
that nonionic surfactant, e.g., Brij.RTM. 35, but not ionic surfactant, 
e.g., SDS, present in the rinse solution (and carded over into the R1 
reagent), actually caused the deterioration of the method. Simply put, and 
as will be described further hereinbelow, the present inventors discovered 
that the Brij.RTM. 35-containing rinse conventionally used in the 
peroxidase method was "delivering" nonionic hemolytic surfactant to the R1 
phase of the method, thus allowing the Brij.RTM. 35 to participate in the 
method in a functional, but undesirable, manner in the method. As a result 
of these findings, the present inventors discovered that the rinse used in 
the peroxidase method actually contributed a required component (i.e., 
nonionic surfactant such as Brij.RTM. 35) to the R1 phase of the Px 
method, and that when the volume of rinse carryover varied from system to 
system, the performance and results of the peroxidase method were 
adversely affected. In accordance with the development of the invention as 
described herein, the present inventors first recognized that rinse 
carryover was not merely an innocuous phenomenon associated with the 
performance of the peroxidase method. 
This knowledge led the present inventors to design the abovementioned 
improved R1 reagent composition that contained both nonionic surfactant 
and ionic surfactant for use in the R1 phase of the peroxidase method. In 
addition, the present inventors further improved the method by removing 
nonionic surfactant such as Brij.RTM. 35 from the rinse solution which 
could also be employed in the method via a rinse cycle. In addition, a new 
rinse solution containing nonhemolytic, nonionic surfactant, e.g., 
Pluronic.RTM., was found to be most suitable for use in conjunction with 
the new R1 reagent composition in the Px method. By removing the lytic 
nonionic surfactant from the rinse solution (or by using nonhemolytic 
Pluronic.RTM. as the sole surfactant in the rinse), and by formulating a 
new Px 1 reagent composition containing both a suitable nonionic 
surfactant and an ionic surfactant, the nonionic surfactant in the R1 
reagent could optimally be delivered at a controlled rate in the Px 
method. Also, variation in rinse carryover volume cannot affect the 
results obtained from the new method. Consequently, in the improved R1 
reagent and peroxidase method, the active nonionic surfactant component is 
present only in the reagent composition used in the R1 phase of the method 
where its functional activity is required for the lysis of red cells. 
Moreover, when a rinse is used in the method, the rinse composition minus 
Brij.RTM. 35 (or containing only nonhemolytic Pluronic.RTM. as the 
surfactant) is optimally devoid of any surfactant that can actively 
participate in the peroxidase method, so that unacceptable or unusable 
results caused by detrimental components of the rinse solution are 
alleviated. 
The occurrence of rinse carryover and its associated problems in automated 
hematology methods are discussed further hereinbelow: in the analysis of 
whole blood samples using automated hematology analyzers, all of the blood 
samples analyzed are mixed with reagent solutions and flow through a 
common hydraulic path (e.g., a channel). The channel is cleaned or rinsed 
between each sample cycle by the introduction of a volume of rinse. In 
this process, the channel is never completely devoid of rinse solution or 
allowed to dry out. As a consequence of the rinsing process, some of the 
rinse solution is left behind and enters the next sample cycle. This 
describes the phenomenon of rinse carryover. 
It is thus clear that rinse carryover may contribute variable amounts of 
reactive components from one sample cycle to the next sample cycle in some 
systems used to perform the peroxidase method. This is particularly 
evident when different systems are compared with each other. The 
system-to-system variation of rinse carryover volume can vary in both 
subtle and more pronounced levels to impact negatively on the results of 
the method performed on an automated analyzer. For example, in the latter 
case, more gross volumes of rinse carryover (e.g., greater than about 10 
.mu.L) cause poor results in the method. In the former case, even more 
subtle system-to-system variation in rinse carryover volumes (e.g., 
volumes such as 7.5 .mu.L, 8.0 .mu.L, 8.3 .mu.L among different systems) 
causes each system to perform slightly differently. Until the invention as 
described, both of these kinds of rinse carryover volume variation 
adversely affected and caused problems in the existing peroxidase 
differential counting method. 
As discussed above, prior to the present invention, it was formerly 
accepted by those in the art that variable rinse carryover was simply a 
benign volumetric carryover that occurred in performing the method. 
However, as demonstrated further herein, the present inventors discovered 
through quantitative measurement of carryover volumes and analysis of the 
reaction components and steps of the method, that rinse carryover (whether 
it occurred more subtly yet more insidiously in the form of limited, but 
different, volumes from system to system, or whether it occurred as a more 
dramatic volume change from system to system) contributed more than mere 
volumetric dilution to the method, caused deviations and flaws in the 
existing method, and impaired the performance of the method by playing an 
active role, rather than an inactive one, in the peroxidase method of 
leukocyte identification and quantification. 
As a particular example, it was found that exceeding a particular level of 
active surfactant in the R1 phase of the method caused distortion of the 
resulting cytogram, which was characterized by damaged eosinophils that 
clustered and rose into the neutrophil area of the cytogram (FIGS. 4A-4F), 
as well as by poorly stained and irregular populations of neutrophils. 
Example 5 demonstrates that rinse solution carryover contributed a 
variable amount of the nonionic surfactant Brij.RTM. 35 to the R1 phase of 
the Px method (involving the lysis of red blood cells and the crosslinking 
of white blood cells). 
Accordingly, the present invention succeeded in alleviating active 
participation by surfactant components of the rinse solution in the Px 
method by removing the surfactant components from the rinse, and also 
freed the method from the effects of variable rinse carryover. This was 
further achieved by devising new formulations of the Px R1 reagent 
composition, with an end result of having "active" surfactant components 
present only in the Px R1 reagent composition (where their delivery is 
controlled and constant in the R1 phase of the Px method) and not in the 
rinse solution (where the delivery of rinse reagent components via the 
sample rinse cycle is variable and less well controlled). Consequently, 
the invention yields accurate results as well as a constant concentration 
of the appropriate type of nonionic surfactant, e.g., Brij.RTM. 35, in the 
overall method. 
Another goal of the development of the reagent composition and improved 
peroxidase method of the invention was to simplify the design of automated 
analyzer systems by reducing the number of different types of rinses that 
are currently used in carrying out the peroxidase method and other 
automated hematology analysis methods. If one rinse reagent was developed 
and was found to be compatible with many different blood analysis methods 
and automated systems, then system design would ultimately be simpler and 
more economical. 
Although the simplest rinse composition could be formulated without a 
hemolytic, nonionic surfactant, such as Brij.RTM. 35, it was also found 
that merely any "Brij.RTM. 35-free" rinse solution was still not 
acceptable for use in the peroxidase method. For example, an aqueous rinse 
solution containing only SDS was determined to be unacceptable for use in 
the method because SDS is subject to crystallization at cold temperatures. 
This behavior of SDS would preclude its use and suitability in the aqueous 
rinse composition which must be able to be stored longterm with no adverse 
effects to its components or to their operativity in the rinse reagent 
composition or the rinse cycle of the method. Thus, it was necessary for 
an acceptable formulation of rinse reagent to be discovered and devised. 
Accordingly, one aspect of the present invention is the formulation of an 
aqueous rinse composition containing only the nonhemolytic surfactant 
Pluronic.RTM. for novel use in the Px method. 
As detailed hereinbelow and in the examples, use of the improved, 
dual-surfactant-containing Px R1 reagent composition, as well as a 
suitable rinse solution when a rinse cycle was carried out, resulted in an 
improved peroxidase method that provided acceptable results when both 
fresh and aged blood samples were analyzed. In contrast, the current Px 
method performed using a conventional R1 reagent composition (i.e., 
formulated in the absence of nonionic surfactant, such as Brij.RTM. 35), 
resulted in unacceptable results in the form of high levels of origin 
noise when aged blood samples were analyzed. It is noted that the 
concentration of the nonionic surfactant used in the new Px R1 reagent 
composition approximates the concentration of the nonionic surfactant 
Brij.RTM. 35 that was discovered to be carded over into the R1 phase of 
the method due to rinse carryover in the current peroxidase method. 
In accordance with the improved Px method of the invention, acceptable 
levels of origin noise in the cytograms of aged blood samples were found 
to be a function of including a suitable nonionic surfactant, e.g., 
Brij.RTM. 35, in the Px R1 reaction mixture (FIGS. 1A-1D and 3A-3D). Use 
of a rinse solution devoid of lytic surfactant (or containing an 
appropriate nonhemolytic surfactant, such as Pluronic.RTM.) was also found 
to achieve useful results from aged blood samples in the peroxidase method 
of leukocyte differential counting and to aid further in the ability to 
yield acceptable levels of origin noise. 
The new R1 reagent composition achieves several advantages for the 
successful performance and results of the peroxidase method. For example, 
as mentioned above, the aqueous R1 reagent composition is formulated to 
contain both nonionic surfactant and ionic surfactant at concentrations 
sufficient to lyse red cells, but not to adversely affect the analysis of 
the white cell populations in the sample. The rinse solution for use in 
the improved leukocyte differential method preferably contains a 
nonhemolytic surfactant that is different from the surfactants that were 
discovered to improve the operativity of the novel R1 diluent of the 
improved peroxidase method. Using the R1 diluent reagent in combination 
with the above-described rinse reagent provides a peroxidase method that 
is independent of the effects of active surfactant transfer due to rinse 
carryover. Thus, although rinse carryover may not be completely removed or 
alleviated, the use of a rinse that is free of nonionic hemolytic 
surfactant (e.g., Brij.RTM. 35) eliminates the adverse or detrimental 
effects of rinse carryover and removes the intersystem variability due to 
rinse carryover in carrying out the method. Further, the novel reagent 
composition of the invention, formulated to contain two surfactants as 
described, showed success in maintaining the accuracy of the differential 
analysis, as well as achieving acceptable levels of origin noise when 
analyzing aged blood samples stored both at room temperature and in the 
cold for at least about 48 hours. 
In accordance with the invention, the improved and novel R1 reagent 
composition, most preferably aqueous, includes two, lyric components which 
are different surfactant types: at least one nonionic surfactant such as a 
long chain alkyl ether polyethoxylate, e.g., the 
polyoxyethylene(2-20)lauryl, cetyl, myristyl, stearyl, and oleyl ethers, 
e.g., Brij.RTM. 35, formulated in combination with at least one ionic 
surfactant, preferably of the class of alkali metal salts of an alkyl 
sulfate having from about 10 to about 16 carbon atoms, e.g., sodium 
dodecyl sulfate (SDS). Suitable ionic surfactants for use in the Px R1 
reagent composition also comprise those of the class of zwitterionic 
sulfobetaines with straight chain alkyl groups having from about 10 to 
about 16 carbon atoms, e.g., tetradecyldimethylammoniopropylsulfonate or 
TDAPS and or dodecyldimethylammoniopropanesulfonate or DDAPS. The combined 
action and appropriate concentrations of the surfactants formulated in the 
Px R1 reagent mixture of the invention result in the appropriate lysis of 
the red blood cells as described herein and the leakage and loss of the 
hemoglobin contents of the lysed red cells. 
The improved Px reagent composition also comprises a fixative or 
crosslinking component (e.g., formaldehyde or paraformaldehyde), a sugar 
or sugar alcohol, a buffer or buffer mixture to maintain the pH of the 
reagent in a neutral or near neutral pH range, an inorganic salt or salts, 
and a chelator of polyvalent metal ions, if necessary or desired. All of 
the components of the improved reagent composition and method, including 
the temperature and reaction time in the R1 lytic phase of the reaction, 
are balanced and optimal for achieving improved results in the method. The 
improvements afforded by the present invention ameliorate the clinical 
usefulness of the results of the method. 
This new, dual-surfactant-containing reagent composition used in the R1 
phase of the peroxidase method is stable and has a long shelf life. For 
example, to test the long-term durability of the reagent composition, the 
new R1 reagent composition was prepared and stored for thirty days at 
60.degree. C. (i.e., under conditions of an accelerated stability test 
which indicates to those skilled in the art that the reagent will 
ultimately be stable for one year or more at room temperature (i.e., about 
22.degree.-30.degree. C.)). When this "long-term" stored reagent was used 
in the peroxidase method as described, the results obtained were still 
acceptable and useful. 
In developing and formulating the reagent composition of the invention, 
test reagents were prepared and assayed using both Day 1 and Day 2 blood 
samples, particularly those aged at room temperature. The chemical 
parameter used to evaluate the suitability of the nonionic surfactants, in 
particular, the polyethoxylates, in the R1 reagent composition of the 
invention is known as the hydrophilic lipophilic balance or HLB value (for 
HLB values and molecular formulae, see Encyclopedia of Surfactants, 
compiled by Michael and Irene Ash, Chemical Publishing Company, New York, 
N.Y., 1980; and McCutcheon's Emulsifiers and Detergents, McCutcheon 
Division, MC Publishing Company, Glen Rock, N.J., 1987). The surfactant 
properties of surfactants are correlated with the HLB value. Thus, in 
accordance with an aspect of the invention, the HLB value of a given 
surfactant serves as a useful predictor for whether or not that surfactant 
will be suitable as a component of the reagent composition of the 
invention. In particular, an appropriate HLB value as described 
hereinbelow indicates that a surfactant will function to improve results 
in the analysis and differential determination of whole blood samples that 
are at least about two days old and stored at room temperature. 
In accordance with another aspect of the invention, a reagent composition 
formulated to contain a nonionic surfactant which possessed an HLB greater 
than about 17.3 to 17.5, indicative of too great a hydrophilicity, was 
unsuitable for improved performance of the method. HLB values of 
surfactants suitable for use in the reagent composition of the invention 
range from about 8.9 to about 17.5, preferably about 9.3 to about 17.3, 
and more preferably about 9.5 or 9.6 to about 16.9. Ideally, the 
surfactant in the reagent composition of the invention should be useful in 
an oil-in-water emulsification application, in which water-insoluble oils 
(i.e., lipids from cell membranes) are "dissolved" by the surfactant 
micelles which are present in the aqueous solvent. In aqueous solution, 
micelles have elliptical or spherical shapes and are groups of surfactant 
molecules (e.g., containing about 100 molecules) in which the polar groups 
face the water solvent and the hydrophobic core is in the interior (M. J. 
Rosen, 1978, "Surfactant and Interfacial Phenomena", Wiley and Sons 
Interscience Publications, New York, N.Y.). HLB values of from about 9.5 
to about 17.5 are indicative of utility in such oil-in-water 
emulsifications. As mentioned above, HLB values greater than about 17.3 to 
17.5 , and more particularly, 17.3, did not improve the performance of the 
method. Tables 9 and 10 present the results of the leukocyte differential 
method of the invention carried out using a variety of different nonionic 
surfactants on an automated analyzer (see Example 7). Similarly, 
surfactants having HLB values less than about 9.3 were also not generally 
useful in the method. 
For the ionic surfactant class of alkali metal salts of an alkyl sulfate 
having from about 10 to about 16 carbon atoms, the preferred alkali metal 
cations for the R1 reagent composition of the invention are sodium, 
potassium, and lithium. More preferred are alkali metal dodecyl sulfates, 
with sodium dodecyl sulfate being most preferred. Examples of 
concentration ranges of the anionic surfactants suitable for use are from 
about 0.030 g/L to about 0.150 g/L, preferably about 0.050 g/L to about 
0.125 g/L, and more preferably about 0.085 g/L to about 0.105 g/L. Also 
suitable for use are the anionic alkyl benzene sodium sulfonates having 
from about 10 to about 18 carbon atoms. Examples of other anionic 
surfactants that are suitable for use in the invention are the 
N-acyl-n-alkyltaurates (for example, R--C(O)N(R')CH.sub.2 CH.sub.2 
SO.sub.3.sup.- M.sup.+, where R.dbd.C.sub.10 H.sub.23 --C.sub.14 H.sub.29 
; R'.dbd.CH.sub.3 or H; and M.sup.+ .dbd.Li.sup.+, Na.sup.+, or K.sup.+). 
Further, zwitterionic surfactants, such as members of the sulfobetaine 
family, which also includes the homologous C.sub.16 and C.sub.12 members, 
e.g., TDAPS (tetradecyldimethylammoniopropanesulfonate), are suitable for 
use in the invention (see Example 8). Other examples of zwitterionic 
surfactants which can be used in the invention are derivatives of cholic 
acid, such as CHAPSO (3-[(3-cholamidopropyl) 
dimethylammonio]-2hydroxy-1-propanesulfonate), and the alkyl N,N-dimethyl 
N-oxides having from about 12 to about 16 carbon atoms, also called the 
N-oxides. A particular but nonlimiting example of an N-oxide is lauryl 
dimethylamine N-oxide (LO), and the like. Surfactants similar to TDAPS, 
but having fewer carbon atoms, for example, C.sub.12, e.g., DDAPS 
(N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), may also be used 
in the peroxidase method and reagent of the invention. 
Families of nonionic surfactants that are suitable for use in the reagent 
composition of the invention are (1) polyoxyethylene alkyl or aryl ethers 
(also termed polyethoxylates), including straight-chain aliphatic 
hydrophobes etherified to polyethylene glycol or polyoxyethylene ethanol, 
e.g., Brij.RTM. 35; (2) branched-chain aliphatic/aromatic (e.g., 
octylphenol) hydrophobes etherified to polyethylene glycol, e.g., Triton 
X.RTM.-100; (3) straight-chain aliphatic/aromatic (e.g., n-nonylphenol) 
hydrophobes etherified to polyethylene glycol, e.g., Igepal.RTM. C0897; 
and (4) straight-chain aliphatic (e.g., carboxylic acid) hydrophobes 
esterified to polyethylene glycol, e.g., Myrj.RTM. 53. Of these four 
families, the ester type is subject to hydrolysis in aqueous solution and 
is expected to be somewhat less stable than the ether types of 
surfactants. 
Examples of nonionic surfactants of the first family include, but are not 
necessarily limited to, polyoxyethylene(4) lauryl ether (Brij.RTM. 30); 
polyoxyethylene(23) lauryl ether (Brij.RTM. 35); polyoxyethylene(2) cetyl 
ether (Brij.RTM. 52); polyoxyethylene(20) cetyl ether (Brij.RTM. 58); 
polyoxyethylene(2) stearyl ether (Brij.RTM. 72); 
polyoxyethylene(10)stearyl ether (Brij.RTM. 76); polyoxyethylene(20) 
stearyl ether (Brij.RTM. 78); polyoxyethylene(2) oleyl ether (Brij.RTM. 
92); polyoxyethylene(10) oleyl ether (Brij.RTM. 96); and 
polyoxyethylene(20) oleyl ether (Brij.RTM. 98); polyoxyethylene(21) 
stearyl ether (Brij.RTM. 721); polyoxyethylene(100) stearyl ether 
(Brij.RTM. 700). Of the Brij.RTM. surfactants, the most preferred is 
Brij.RTM. 35. It is noted that, although suitable for use in the 
composition of the invention, the polyoxyethylene oleyl ethers may be less 
stable for long-term storage, due to the presence of double bonds in their 
molecular structures, which makes them susceptible to oxidation. It will 
also be appreciated by those skilled in the art that the most suitable 
nonionic surfactants will have HLB values in the ranges described, in 
accordance with the invention. Other nonlimiting examples of nonionic 
surfactants of the second family include Triton X.RTM.-100 (non-reduced or 
reduced), Triton.RTM.X-114 non-reduced or reduced), Triton X.RTM.-165, and 
Triton X.RTM.-305 (non-reduced and reduced). The nonionic surfactant 
should be present in the reagent composition of the invention at a 
concentration of from about 0.10 g/L to about 0.20 g/L; more preferred is 
a concentration range from about 0.10 g/L to about 0.16 g/L; and most 
preferred is a concentration range of from about 0.12 g/L to about 0.14 
g/L. 
The sugar or sugar alcohol of the composition include sucrose, fructose, 
dextrose, sorbitol, and mannitol. Dextrose is the preferred sugar to be 
used in the reagent composition. However, sugar alcohols, such as 
sorbitol, are more preferred. Sugar alcohols provide for a more stable 
reagent solution over time, due to the inability of the sugar alcohol to 
be air-oxidized. The sugar or sugar alcohol is ideally present in the 
reagent composition at a concentration of about 110.0 g/L to about 120.0 
g/L, more preferably at 113.0 g/L. If a sugar other than dextrose, or a 
sugar alcohol other than sorbitol is used, the amount used should be 
adjusted so that the alternative sugar or sugar alcohol is present at 
approximately the same concentration (g/L) as dextrose or sorbitol. The 
sugar or sugar alcohol is present in the reagent solution to increase the 
detectability of the lymphocytes over the noise (i.e., the red cell ghosts 
and platelets). Either a sugar or a sugar alcohol may be used, depending 
upon the nature and requisites of the analysis. 
Formaldehyde or paraformaldehyde is used in the reagent solution of the 
invention as a fixative (i.e., chemical crosslinking compound) for the 
white blood cells. If formaldehyde is used, it is present in the solution 
in an amount of from about 50 g/L to about 60 g/L. More preferably, 
formaldehyde is present in a concentration of from about 52 g/L to about 
58 g/L. 
The increased stability of the reagent solution when a sugar alcohol is 
used rather than a reducing sugar like glucose relates to the phenomenon 
of air-oxidation of glucose (but not a sugar alcohol) over time to form 
gluconic acid. See, for example, Nishikido et al., Jap. Kokai Tokyo Koho 
80 40, 606, Chem. Abs., 93:22120d, (1950) and U.S. Pat. No. 4,801,549. The 
presence of gluconic acid lowers the pH of the solution. When the pH falls 
outside of the range of the invention, as discussed herein, the method is 
subject to interference due to the non-lysis of red blood cells in the 
sample. Further, a sugar alcohol which cannot be air-oxidized, may 
chemically combine with formaldehyde to form a polyacetal, thereby 
preventing the oxidation of formaldehyde to formic acid, which, if 
produced, would also lower the pH of the reagent solution (U.S. Pat. No. 
4,801,549). 
An inorganic salt may also be included in the reagent solution. Salts 
suitable for use in the present invention may be alkali metal chloride 
salts such as NaCl, KCl and LiCl. Sodium chloride, NaCl, is a preferred 
salt. Such salt may optionally be present because it may aid in 
discriminating the neutrophils from the eosinophils by causing a 
difference in peroxidase stain intensity using light scatter/absorption 
optics. Other halogen salts (i.e., fluoride, bromide and iodide) 
over-inhibit peroxidase activity of the neutrophils, thereby preventing 
the discrimination of neutrophils from the other unstained white blood 
cells (WBCs). The salt, e.g., NaCl, when used, should preferably be 
present in an amount of from about 6.8 mM to about 10.3 mM (or about 0.4 
g/L to about 0.6 g/L). 
The buffer or mixture of buffers useful in this invention should be those 
suitable for maintaining the pH of the reagent solution at from about 6.8 
to about 8.0, preferably from about 6.9 to about 7.6, more preferably 
about 7.0 to about 7.3. It is noted that when the pH is too low, i.e., 
below about 6.8, red blood cell interference is observed in cytograms. 
Suitable buffers include sodium or potassium phosphates, diethyl malonate, 
3-(N-morpholino) propane sulfonic acid, (MOPS), 
N-2-acetamido-2-aminoethane sulfonic acid (ACES), and 
4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES). Preferred is 
a mixture of Na.sub.2 HPO.sub.4 (sodium phosphate, monobasic) and 
NaH.sub.2 PO.sub.4 (sodium phosphate, dibasic). As indicated, the buffers 
should be present in the reagent solution of this invention in an amount 
suitable to maintain the pH of the solution at approximately neutral 
levels. For instance, when a mixture of Na.sub.2 HPO.sub.4 and NaH.sub.2 
PO.sub.4 is used, the mixture should contain a mole ratio of Na.sub.2 
HPO.sub.4 to NaH.sub.2 PO.sub.4 which is from about 2.04:1 to about 0.81:1 
to produce a series of solutions with a pH range of about 6.9 to about 
7.6, preferably from about 7.0 to 7.3. The buffer concentration of such 
mixture in the reagent solution of this invention is from about 75 mM to 
about 125 mM. Above about 125 mM may cause red blood cell interference in 
the resulting cytograms. 
The reagent solution useful in the practice of this invention is an aqueous 
solution and, preferably, deionized water is used. The solution is 
prepared by combining the ingredients, in admixture, in water. A close 
watch should be maintained on the pH of the solution to ensure that it 
stays within the desired range. Those skilled in the art may also include 
other additives in the reagent solution as desired. In particular, metal 
chelators, such as disodium or trisodium ethylenediamine tetraacetic acid 
(EDTA) and ethylenebis(oxyethylenenitrilo)-tetraacetic acid (EGTA) are 
valuable to include in the reagent composition at a concentration of about 
1 mM to about 5 mM to protect other components in the composition from 
polyvalent metal ion-catalyzed autooxidation. In addition to EDTA, 
disodium, trisodium, and tetrasodium EDTA or EGTA are suitable for use in 
the composition, with disodium EDTA, dihydrate being preferred. For 
instance, formaldehyde, sorbitol, SDS, and Brij.RTM. 35 are all 
susceptible to autooxidation. Polyvalent metal ion catalysts, e.g., 
Cu.sup.+2 and Fe.sup.+3, are frequently present in ppb concentrations in 
water which is used as the formulation solvent ("Polymer Stabilization", 
1972, Ed., W. L. Hawkins, Wiley-Interscience, New York, N.Y.). 
A common pattern characteristic of unsuitable reagent compositions for use 
in the R1 phase of the peroxidase method is exemplified by the following: 
percent origin noise greater than 33; artificially elevated white blood 
cell count; and distorted percent neutrophils and percent lymphocytes. 
Based on the testing of many reagent formulations containing a variety of 
surfactant types and evaluating their performances on both fresh and aged 
blood samples, suitable components of the improved reagent composition and 
method of the invention were determined. 
Table 1 provides an example of the preferred components and optimal 
concentrations and concentration ranges in the dual surfactant-containing 
aqueous reagent composition of the invention. It will be appreciated by 
those in the art that the concentrations and ranges of each of the listed 
reagent components may deviate by about .+-.5% to 10% without adversely 
affecting the composition or its use in the method. In addition, for each 
of the components of the new Px R1 reagent composition as listed in Table 
1, the preferred quantities per liter are provided in parentheses, and are 
not intended to be limiting. 
TABLE 1 
______________________________________ 
Component Qty/L 
______________________________________ 
Nonionic surfactant (e.g., Brij .RTM. 35) 
0.10 g-0.20 g 
(0.12-0.14 g) 
Ionic surfactant (e.g., Sodium 
0.085 g-0.115 g 
Dodecyl Sulfate, SDS or TDAPS) 
(0.105 g) 
Sugar or Sugar alcohol (e.g., 
110 g-120 g 
Sorbitol) (113.0 g) 
NaPhosphate, monobasic 
1.98 g-2.18 g 
(2.08 g) 
NaPhosphate, dibasic 11.30 g-12.5 g 
(11.89 g) 
Inorganic salt (e.g., NaCl, KCl, 
0.4 g-0.6 g 
LiCl) (0.488 g) 
Metal ion chelator (e.g., EDTA; 
0.675 g-0.825 g 
EFTA; or di, tri, or tetrasodium 
(0.750 g) 
EDTA or EGTA) 
Fixative (e.g., formaldehyde, 
50 g-60 g 
37 g/dL) (150 mL) 
Deionized Water, q.s. to 
1.00 L 
pH 6.9-7.6 
(7.0-7.5) 
______________________________________ 
Another aspect of the invention relates to a further improvement of the 
analytical results of the peroxidase method as a consequence of the 
incorporation of a rinse cycle and the use of an appropriate aqueous rinse 
reagent composition in the method. The rinse reagent employed in the rinse 
cycle of the improved method is especially advantageous and useful in 
semi- and fully-automated systems, in particular, the TECHNICON 
H.cndot.1.TM., H.cndot.2.TM., and H.cndot.3.TM. systems, and the like, 
which rapidly perform the peroxidase method of white blood cell 
differential counting. The rinse reagent is the subject of the invention 
described in co-pending U.S. application Ser. No. 08/443,363, filed 
concurrently herewith on May 16, 1995, entitled "Universal Rinse Reagent 
Composition For Use in Hematological Analyses of Whole Blood Samples", and 
assigned to the assignee of the present invention. 
The rinse reagent solution, which can be used in the improved leukocyte 
differential counting method, comprises one or more buffering agents or 
compounds or mixtures thereof, for example, monobasic sodium phosphate and 
dibasic sodium phosphate, to provide a pH and an osmolality which are 
close to physiological values, e.g., pH of about 6.9 to about 7.6, and 
preferably a pH of about 7.0 to about 7.1, and an osmolality value of 
approximately 300 mOsmol/kg; an antimicrobial compound to retard microbial 
growth; a non-hemolytic surfactant, such as the Pluronics.RTM., for 
example, P84, P85, P103, P104, P105, and P123 (P105 is preferred due to 
its nonlytic properties in the amounts used in the rinse solution, has a 
molecular weight of about 3300, and comprises about 50% polyoxyethylene, 
by weight); an alkali metal chloride salt, such as NaCl, KCl, LiCl, and 
the like. Nonlimiting examples of suitable antimicrobials include Proclin 
150 (2-methyl-4-isothiazolin-3-one) and Proclin 300 
(5-chloro-2-methyl-4-isothiazolin-3-one) (Rohm & Haas); Germall 115 
(N,N'-methylenebis[N'-(1-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl]urea) 
(Sutton Laboratories); Dowacil 200 
(1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride) (Dow 
Chemical); and Bronopol (Angus Chemical Company). Proclin 150 is preferred 
for use in the rinse composition. 
A water-soluble antioxidant compound, for example, 3,3'-thiodiproprionic 
acid; 3,3'-dithioacetic acid; Trolox.RTM. (i.e., water-soluble vitamin E, 
Hoffman-LaRoche); BHT, butylated hydroxytoluene or (2, 
6-di-tert-butyl-4-methylphenol); BHA, butylated hydroxyanisole or 
(2-tert-butyl-4-methoxyphenol; and MEHQ (.rho.-methoxyphenol); or mixtures 
thereof, may also be used for stabilization of the nonhemolytic surfactant 
in the rinse reagent composition. 3,3'-thiodiproprionic acid is preferred 
for use in the composition. The final osmolality of the rinse solution is 
from about 285 mOsm/kg to about 305 mOsm/kg. 
In its simplest formulation, the rinse reagent may comprise a phosphate 
buffer (e.g., phosphate buffered saline, pH of about 6.8 to 7.8) and a 
nonionic and nonhemolytic surfactant of the Pluronic.RTM. family or class 
of surfactants. Pluronics.RTM. are block copolymers of polyoxyethylene and 
polyoxypropylene of the structure: (EO).sub.x -(PO).sub.y -(EO).sub.x (see 
Pluronic.RTM. & Tetronic.RTM. Surfactants, BASF Corporation, Parsippany, 
N.J., 1987) and are formed by synthesizing the polypropylene glycol unit, 
(PO).sub.y, by controlled polymerization of propylene oxide. Since 
Pluronics.RTM. can vary from about 950 to about 14000 g/mol in molecular 
weight, and (PO) can comprise from about 10 to 90% by weight, then "y" can 
range from about 1 to 133 units. Next, EO polymeric chains are formed on 
both sides of the poly(PO) unit to yield the Pluronic.RTM. copolymer. 
Those in the art are aware that EO polymerization can be controlled 
symetrically so that "x" is the essentially the same on each side or end 
of the Pluronic.RTM. molecule. Nonlimiting examples of the "x" and "y" 
values for the Pluronics.RTM. suitable for use in the universal rinse 
composition are as follows: "x" is about 1 to 36 units, more preferably 
about 17 to 37, and "y" is preferably about 14 to 48 units. 
Pluronics.RTM. suitable for use in the rinse reagent have % EO values, by 
weight, in the range of about 10 to 80% by weight, with a molecular weight 
range from about 2000 to about 8000 g/mol. More preferred is a % EO in the 
range of about 30 to 70% by weight, with a molecular weight range from 
about 3000 to about 3600. For example, Pluronic.RTM. P105 and P85 have % 
EO values of about 50%, by weight, and Pluronic.RTM. P104 and P84 have % 
EO values of about 40%, by weight. 
Table 2 sets forth an exemplary formulation of the rinse reagent that may 
be used to further improve the peroxidase method and includes the 
preferred amounts of each component to yield the appropriate and operative 
pH and osmolality of the final reagent solution. It will be appreciated by 
those in the art that the concentrations and ranges of each of the listed 
rinse solution components may deviate by about .+-.5% to 10% without 
adversely affecting the composition or its use in the improved method. In 
addition, as mentioned above, for each of the components of the rinse 
reagent composition as listed in Table 2, the preferred quantities per 
liter are provided in parenthesis, and are not intended to be limiting. 
TABLE 2 
______________________________________ 
Component Qty/L 
______________________________________ 
Antimicrobial compound (e.g., 
0.25 mL-0.60 mL 
Proclin 150) (0.40 mL) 
Nonhemolytic nonionic surfactant 
0.50 g-1.5 g 
(e.g., Pluronic .RTM. P105) 
(1.00 g) 
NaPhosphate, monobasic 
0.285 g-0.315 g 
(0.300 g) 
NaPhosphate, dibasic 2.28 g-2.52 g 
(2.40 g) 
Inorganic salt (e.g., NaCl, KCl, or 
7.40 g-8.0 g 
LiCl) (7.70 g) 
Antioxidant compound (e.g., 3,3'- 
0.050 g-0.150 g 
thiodiproprionic Acid) 
(0.100 g) 
Deionized water, q.s. to 
1.00 L 
pH 6.9-7.5 
(7.0-7.3) 
Osmolality (mOsmol/kg) 
.apprxeq.285-305 mOsm 
(300 mOsm) 
______________________________________ 
A significant advantage of the improved reagent in the Px method which also 
utilizes a rinse cycle is that the reagent composition of the invention is 
designed and optimized to eliminate the adverse effects attributable to 
variable rinse carryover in which the surfactant component of the rinse 
plays an undesirable and unacceptable functional role in the method. The 
reagent formulation and method also permit the determination of white 
blood cell differentials using aged blood samples that have been stored 
for up to two days at room temperature, without sacrificing accuracy, 
precision, and reliability of results due to unacceptable origin noise. 
Also, employing the reagent composition formulated in accordance with the 
invention in combination with the rinse solution formulation in accordance 
with the invention, the rinse solution does not participate in the 
peroxidase chemistry of the differential counting method. 
In practicing the method encompassed by this invention, the reagent 
solution is rapidly mixed with the sample to be analyzed to form the 
reaction mixture in the R1 phase of the method. Uniform mixture should 
occur within about 5 seconds of the time the reagent solution and the 
blood sample come into contact with each other. If the two are not mixed 
rapidly and uniformly, a nonuniform level of fixation of the red blood 
cells may occur which prevents lysis of the red blood cells, thereby 
greatly impairing the accuracy of the differential WBC count obtained from 
the practice of the method. 
When mixed, the reagent solution and the blood sample are slightly warmer 
than room temperature (e.g., about 5.degree. C.). The reaction mixture 
(including the blood sample) is then rapidly heated to a temperature of 
from about 62.degree. C. to about 76.degree. C., ideally from about 
70.degree. C. to about 75.degree. C., preferably by injection into the 
appropriate chamber(s) of an automated hematology analyzer maintained at a 
suitably elevated temperature. The heating of the reaction mixture should 
take place within about 15 seconds, preferably within about 20 seconds, 
otherwise red blood cell fixation can occur; fixed red cells will not be 
lysed and will be erroneously detected as lymphocytes, thereby interfering 
with the accuracy of the differential WBC count. 
Immediately thereafter, a staining mixture comprising hydrogen peroxide and 
a suitable chromogen such as 4-chloro-1-naphthol is mixed with the 
reaction mixture. The initial temperature of the staining mixture may be 
room temperature and, ideally but not necessarily, the temperature after 
mixing the staining mixture with the reaction mixture is increased to from 
about 62.degree. C. to about 72.degree. C., preferably from about 
65.degree. C. to about 70.degree. C. in a period of within about 30 
seconds, preferably from about 8 to 15 seconds, in order to stain the 
neutrophils, monocytes, and eosinophils which are peroxidase active. 
In practice, the reaction may proceed as follows: an automated hematology 
analyzer reaction chamber is maintained at a temperature of approximately 
72.degree. C. 12.0 .mu.L of whole blood and 250 .mu.L of the R1 reagent 
composition of the present invention are simultaneously injected into the 
system at room temperature, thereby rapidly mixing the two to form the 
reaction mixture, which is then incubated for up to less than about 30 
seconds, preferably about 16 seconds, during which time the temperature of 
the mixture is increased to from about 62.degree. C. to about 72.degree. 
C. At the end of the incubation period, the red blood cells are optimally 
lysed and the white blood cells are fixed (i.e., crosslinked). 
Immediately thereafter, 125 .mu.L of a chromogen mixture (for example, 70 
g/L of 4-chloro-1-naphthol in oxydiethanol) is simultaneously injected 
with 250 .mu.L of a hydrogen peroxide solution comprising 3.0 g/L hydrogen 
peroxide. Both reagents are initially at room temperature, but due to the 
temperature of the reaction chamber, the staining mixture temperature is 
increased to from about 63.degree. C. to about 69.degree. C. within about 
30 seconds, at which time the peroxidase staining of neutrophils and 
eosinophils is completed. The reaction chambers and system hardware are 
rinsed with the disclosed rinse solution to alleviate carryover of 
reagents and solutions from one sample analysis cycle to the next and to 
avoid skewed results due to the presence of contaminating reagents in the 
reaction chambers. Further particulars relating to the automated reaction 
procedure are described in Example 6. 
Although the reagent compositions and procedure of the subject invention 
are illustrated using automated equipment, it will be readily apparent to 
those skilled in the art that the subject matter of the invention may also 
be applicable to manual methods and to manual methods in combination with 
semi-automated or fully-automated methods. Further, the reagent 
compositions and procedures of the invention have been illustrated using 
whole blood to arrive at the differential WBC count therein. It will be 
appreciated by those skilled in the art that the invention may also be 
employed with stock calibrator, control, and other solutions of blood 
cells which are specifically prepared and may be commercially available to 
calibrate and maintain apparatus accuracy. The term "sample" without other 
modifiers as used herein is specifically intended to include either whole 
blood or other solutions which contain blood cells. 
EXAMPLES 
The following examples are illustrative of the invention. They are 
presented to further facilitate an understanding of the inventive concepts 
and in no way are to be interpreted as limiting the present invention. 
Example 1 
Formulation and testing of a peroxidase R1 diluent reagent composition 
containing only ionic surfactant without nonionic surfactant (e.g., SDS 
alone, no Brij.RTM. 35) 
Experiments were designed and carded out to determine the optimum 
formulation for the peroxidase diluent solution for use in the R1 phase of 
the peroxidase method. 
The initial approach in formulating an acceptable and operative R1 
composition or diluent involved the preparation and testing of reagent 
solutions containing ionic surfactant, i.e., the anionic surfactant SDS, 
in the absence of nonionic surfactant, i.e., Brij.RTM. 35, in the reagent 
composition. R1 reagent solutions were prepared based on increasing the 
SDS concentration from about 0.105 g/L to about 0.17 to 0.20 g/L. Several 
studies were completed to assay the performance of these "test" reagents 
containing high levels of SDS on automated systems at throughputs of 102 
and 120 samples/hour. 
Specifically, using an automated hematology analyzer, test R1 reagent 
solutions which contained from 0.16 to 0.20 g/L of SDS (e.g., 0.16, 0.17, 
0.18, 0.19, and 0.20 g/L) were prepared and tested on Day 1 and Day 2 
blood samples as indicated. The SDS test R1 diluents contained no 
Brij.RTM. 35 (e.g., 0.0 g/L). It is noted that the blood samples used in 
these experimental analyses were collected in Vacutainer.TM. tubes in the 
presence of K.sub.3 EDTA. In addition, in conducting these tests, the 
rinse solution used in the rinse cycle contained no Brij.RTM. 35. Rinse 
solutions were also used which contained the non-hemolytic surfactant 
Pluronic.RTM. P105, as described. The results of these analyses are 
presented in Table 3, which demonstrates that for Day 2 blood samples, 
unacceptable results were obtained with all of the test reagents. The data 
for the % neutrophils and % lymphocytes parameters are presented and 
compared with a standard method in which the rinse solution contained 
Brij.RTM. 35 at a concentration of 3.0 g/L and SDS at a concentration of 
2.0 g/L. For the Day 2 blood samples, the recovery of these two parameters 
approached the standard at 0.20 g/L of SDS. However, using SDS at 
concentrations of between 0.16-0.19 g/L, the accuracy was poor due to 
unacceptable origin noise in the Px method. The wide disparity between 
replicate aspirations for particular samples is noted. 
As shown in FIGS. 1A-1D, when Day 1 blood samples were analyzed in the 
peroxidase method using a standard peroxidase reagent solution comprising 
SDS (0.105 g/L) and no Brij.RTM. 35, and including a rinse cycle employing 
a rinse solution comprising Brij.RTM. 35 at a concentration of 3.0 g/L and 
SDS at a concentration of 2.0 g/L, the cytogram results showed nondiffuse, 
cleanly separated cell populations and minimal origin noise (see FIG. 1A). 
FIG. 1B shows the results of the analysis of Day 2 blood samples using the 
same Px R1 reagent solution and rinse solution as described for the FIG. 
1A results. However, in contrast to the FIG. 1A results, the cytograms of 
the Day 2 blood samples showed diffusion of cell populations and higher 
origin noise. FIG. 1C shows the results of an analysis of Day 2 blood 
samples using an R1 test reagent solution containing only ionic surfactant 
in the form of 0.105 g/L of SDS, and including a rinse cycle employing a 
rinse reagent solution containing the non-hemolytic surfactant 
Pluronic.RTM. P105 in phosphate buffered saline (in the absence of both 
Brij.RTM. 35 and SDS). The results of the FIG. 1C cytogram show 
unacceptable noise at the origin. The same problem of unacceptable origin 
noise also resulted using a test reagent solution containing a higher 
amount of ionic surfactant (i.e., 0.17 g/L of SDS) and no Brij.RTM. to 
analyze Day 2 blood samples (see FIG. 1D), and using the same rinse 
solution described for FIG. 1C. Thus, it was determined that a peroxidase 
R1 composition containing only ionic surfactant (e.g., the anionic 
surfactant SDS) was suboptimal to use in the leukocyte differential method 
as described in conjunction with a "Brij.RTM.-free" rinse. 
TABLE 3 
__________________________________________________________________________ 
Standard Method % Neut and % Lymph in Blood Samples Tested on Day 1 and 
Reference Reference 
Day 2 (i.e., Aged and Stored at Room Temperature) 
Day 1, 
Day 2, 
Day 2*, 
Day 2, 
Day 2, 
Day 2, 
Day 2, 
Samp. 
Std.dagger. 
Std 0.16** 
0.17 0.18 0.19 0.20 
No. % N 
% L 
% N 
% L 
% N 
% L 
% N 
% L 
% N 
% L 
% N 
% L 
% N 
% L 
__________________________________________________________________________ 
1a 61.0 
31.6 
58.6 
30.8 
49.8 
41.6 
51.3 
39.5 
54.4 
35.0 
57.6 
32.6 
57.9 
31.8 
1b 57.1 
31.1 
58.5 
30.5 
48.6 
43.3 
51.1 
39.3 
52.5 
38.7 
56.0 
34.0 
59.8 
30.7 
2a 69.0 
19.4 
68.5 
19.1 
71.2 
20.1 
72.0 
18.8 
73.0 
14.8 
68.3 
22.1 
67.9 
21.7 
2b 69.3 
19.5 
69.7 
19.3 
72.5 
18.1 
69.1 
19.5 
73.2 
14.2 
73.0 
17.5 
74.6 
14.9 
3a 59.4 
26.4 
61.3 
25.2 
49.9 
40.8 
53.9 
35.9 
59.9 
28.5 
60.4 
28.5 
63.8 
25.6 
3b 59.8 
26.5 
62.4 
26.2 
54.6 
35.2 
57.2 
30.7 
59.1 
30.0 
58.8 
29.6 
60.7 
26.5 
4a 60.8 
29.4 
60.9 
28.6 
46.7 
43.7 
50.7 
41.6 
50.4 
39.2 
59.4 
33.0 
58.4 
31.4 
4b 60.2 
28.8 
60.8 
29.1 
38.2 
53.0 
48.1 
41.9 
53.4 
38.1 
57.3 
34.0 
53.8 
35.9 
5a 58.8 
27.9 
60.9 
26.8 
46.9 
43.8 
56.0 
34.4 
52.2 
35.8 
56.1 
34.2 
59.3 
29.3 
5b 59.5 
28.0 
61.4 
25.5 
46.6 
43.3 
52.5 
37.2 
54.9 
33.9 
56.4 
33.9 
61.2 
28.1 
6a 64.1 
26.0 
64.0 
26.1 
57.0 
34.1 
60.5 
31.2 
61.0 
29.0 
65.0 
24.7 
62.3 
27.3 
6b 62.0 
25.8 
64.5 
24.8 
53.5 
39.8 
58.8 
30.2 
59.1 
32.5 
61.4 
29.0 
60.0 
30.7 
7a 61.4 
25.4 
61.6 
26.0 
38.6 
51.6 
70.9 
15.2 
44.4 
43.7 
54.2 
35.3 
57.4 
32.0 
7b 60.2 
26.2 
61.9 
25.0 
65.1 
15.6 
45.7 
44.9 
67.8 
14.4 
57.0 
31.1 
56.6 
31.4 
8a 59.8 
26.5 
64.0 
24.9 
43.2 
48.1 
48.5 
43.2 
54.5 
35.4 
52.1 
39.4 
58.4 
30.9 
8b 60.4 
26.9 
64.6 
25.2 
41.6 
49.8 
46.0 
44.2 
51.1 
40.3 
53.8 
37.2 
58.0 
30.6 
9a 58.2 
28.5 
59.7 
29.6 
68.9 
18.8 
51.6 
0.6 
62.3 
26.0 
58.4 
31.1 
55.3 
35.9 
9b 60.1 
28.4 
58.2 
29.8 
65.6 
21.0 
52.8 
37.6 
52.8 
35.8 
55.6 
30.3 
58.9 
31.0 
10a 59.3 
27.1 
64.7 
25.3 
54.5 
35.8 
58.1 
33.3 
58.5 
32.0 
63.7 
27.2 
60.6 
30.7 
10b 59.0 
26.3 
61.3 
26.7 
49.8 
41.0 
58.5 
32.6 
60.8 
30.8 
61.3 
28.8 
61.6 
27.6 
mean 
61.0 
26.8 
62.4 
26.2 
53.1 
36.9 
55.7 
32.6 
57.8 
31.4 
59.3 
30.7 
60.3 
29.2 
__________________________________________________________________________ 
% N: percent neutrophils in sample; % L: percent lymphocytes in sample. 
.dagger. Std signifies a standard of control R1 diluent containing 0.105 
g/L of SDS and no nonionic surfactant, and a rinse cycle and solution 
containing 3.0 g/L of Brij .RTM. 35 and 2.0 g/L of SDS. 
*Day 2 signifies that blood samples were assayed following about 36-48 
hours at room temperature. 
**0.16, 0.17, 0.18, 0.19, and 0.20 are the concentration of SDS in g/L 
present in the test peroxidase R1 reagent solutions. The rinse contained 
1.0 g/L of Pluronic .RTM. P105 in phosphate buffered saline (PBS). 
The sample set was 10 nonhospital bloods, assayed in duplicate. Virgin 
tubes from the same set of donors were stored for 24 hours at room 
temperature and than assayed as the Day 2 samples. 
Example 2 
Formulation and testing of a peroxidase R1 diluent reagent composition 
containing only nonionic surfactant and no ionic surfactant (e.g., only 
Brij.RTM. 35 and no SDS) 
In addition to the test R1 diluent compositions formulated to contain SDS 
alone, in the absence of nonionic surfactant, as described in Example 1, 
reagent compositions were also designed to test the presence of nonionic 
surfactant, i.e., Brij.RTM. 35, alone, in the absence of ionic surfactant, 
e.g., the anionic surfactant SDS. Accordingly, several test R1 diluent 
reagent compositions which contained Brij.RTM. 35 at concentrations of 0.0 
g/L, 0.14 g/L, 0.28 g/L, and 0.42 g/L were prepared and assayed. For the 
test analyses using only Brij.RTM. 35 in the R1 diluent solution, the 
rinse solution used in the rinse cycle contained the non-hemolytic 
surfactant Pluronic.RTM. P105 as used in the assays described in Example 
1. 
The various Brij.RTM. 35-containing R1 test solutions were formulated as 
follows: a 30.0 g/L stock solution of Brij.RTM. 35 was prepared in 
distilled water. To 50 mL aliquots of the peroxidase R1 composition 
(without nonionic surfactant), the following volumes of Brij.RTM. 35 stock 
solution were added: 0.0 mL of Brij.RTM. 35 stock solution to prepare R1 
diluent containing Brij.RTM. 35 at a final concentration of 0.0 g/L; 0.233 
mL of stock solution to prepare R1 diluent containing Brij.RTM. 35 at a 
final concentration of 0.14 g/L; 0.466 mL of stock solution to prepare R1 
diluent containing Brij.RTM. 35 at a final concentration of 0.28 g/L; and 
0.699 mL of stock solution to prepare R1 diluent containing Brij.RTM. 35 
at a final concentration of 0.42 g/L. The solutions were mixed and stored 
in polypropylene screw-top test tubes. 
For test runs, normal blood samples were collected in Vacutainer.TM. tubes 
and were anticoagulated with K.sub.3 EDTA. Samples were assayed in the 
open tube mode. Unopened samples from the same donor set were stored at 
room temperature overnight and were assayed on day 2 (i.e., Day 2 
samples). Data were collected using an automated hematology analyzer with 
manual aspiration. Standard runs were at 102 samples/hour; test runs were 
at 120 samples/hour. For runs which contained 5 pairs of duplicates, the 
standard deviation, SD, (pooled SD over multiple donors) was calculated 
for the peroxidase channel parameters using the following equation: 
SD=[Sum(d.sup.2)/2N].sup.1/2, where d is the difference between duplicate 
values obtained with a particular reagent, and N is the number of samples 
in the set. 
Table 4 depicts a summary of the numerical data obtained from these 
experiments. The performance of test reagents 1-4 for R1 of the peroxidase 
method was compared with performance using a standard R1 reagent in the 
peroxidase method (i.e., the standard R1 reagent contained SDS at a 
concentration of 0.105 g/L). For Day 1 blood sample analyses, the test 
reagents generally yielded unacceptable results. For reagent 1, (i.e., a 
peroxidase R1 reagent comprising neither Brij.RTM. nor SDS), no 
differential data were obtained as a result of severe interference caused 
by unlysed red cells. For test Px R1 reagents 2 to 4 (i.e., reagent 2: 
Brij.RTM. 35 concentration=0.14 with no SDS; reagent 3: Brij.RTM. 35 
concentration=0.28 with no SDS; reagent 4: Brij.RTM. 35 concentration=0.42 
g/L with no SDS), there were a significant number of accuracy and 
imprecision failures (indicated by asterisks) compared with the method 
specifications. For reagents 1, 2, and 3, the percent Noise was high 
(i.e., &gt;34%). The numerical data were also unacceptable for Day 2 blood 
samples using test reagents comprising only Brij.RTM. 35 and no SDS. Thus, 
the numerical data show that test reagent sets, including peroxidase 
method R1 reagents which contained Brij.RTM. 35 (0.14-0.42 g/L and no 
SDS), yielded unacceptable performance. 
TABLE 4 
__________________________________________________________________________ 
Analyses of Peroxidase R1 Reagent Compositions Containing Higher 
Concentrations of Brij .RTM. 35 and no SDS 
REAGENTS WBCP 
% Neut 
% Lys 
% M % Eos 
% LUC 
% Nois 
__________________________________________________________________________ 
DAY 1 SAMPLES 
Standard 5.27 
56.9 28.5 
8 3.7 2.1 23.71 
0.117 
0.75 0.34 
0.74 
0.38 
0.29 0.6 
1: No SDS 
15.25 
ND ND ND ND ND 89.5 
No Brij .RTM. 
*0.545 0.43 
2: No SDS 
5.36 
*61.9 
*21.7 
*9.7 
3.7 2.1 47.7 
Brij .RTM. : 0.14 g/L 
0.16 
*1.65 
*1.45 
0.82 
0.4 0.28 1.44 
3: No SDS 
5.36 
57.3 28.6 
7.9 3.6 1.9 34 
Brij .RTM. : 0.26 g/L 
0.154 
*1.73 
*1.66 
0.59 
0.22 
*0.55 
2.66 
4: No SDS 
4.77 
*53.2 
*27.3 
*10.1 
3.5 *5.1 19.2 
Brij .RTM. : 0.42 g/L 
*1.569 
*7.47 
*6.11 
*1.83 
*0.57 
*2.52 
4.75 
acc spec 0.15 
1 0.5 0.5 0.2 0.5 N/A 
SD spec 0.16 
1.4 1.1 09 0.5 0.5 
DAY 2 SAMPLES 
Standard 5.19 
60.6 27 7.2 2.6 1.8 29.8 
0.081 
1.43 0.82 
0.49 
0.61 
0.29 3.66 
1: No SDS 
ND ND ND ND ND ND 60.1 
No Brij .RTM. 5.77 
2: No SDS 
6.75 
66.7 16.4 
12.8 
1.8 1.2 44.9 
Brij .RTM. : 0.14 g/L 
0.509 
3.41 3.97 
0.86 
0.19 
0.18 3.82 
3: No SDS 
5.4 57.6 29 9.1 1.9 1.7 33.4 
Brij .RTM. : 0.26 g/L 
0.177 
2.76 1.6 1.18 
0.76 
0.23 2 
4: No SDS 
5.27 
55.4 26.7 
10.7 
1.6 4.6 15.2 
Brij .RTM. : 0.42 g/L 
0.25 
2.57 1.96 
1.15 
0.28 
2.61 2.93 
aged acc spec 
0.32 
2.8 2.2 1.6 1 1 N/A 
__________________________________________________________________________ 
In Table 6, the asterisk indicates that the value obtained exceeded the 
accuracy specification ("acc spec") of the automated analyzer used to 
perform the method. "ND" indicates that no acceptable or useful data were 
obtained. "WBCP" indicates the white blood cell count determined from the 
peroxidase method; "% Neut" indicates percent neutrophils; "% Ly" 
indicates percent lymphocytes; "% M" indicates percent monocytes; "% Eos" 
indicates percent eosinophilis; "LUC" indicates percent large unstained 
cells; and "% Nois" indicates percent origin noise. A representative set 
of cytograms from the Day 1 and Day 2 blood sample analyses described in 
Example 2 is displayed in FIGS. 2A-2J. FIGS. 2A-2E represent Day 1 blood 
sample analyses. FIGS. 2F-J represent Day 2 blood sample analyses. As 
described, blood samples were withdrawn in Vacutainer.TM. tubes containing 
K.sub.3 EDTA. The reagents used in these analysis correspond to the 
resulting cytograms as follows: FIG. 2A (Day 1 blood sample) and FIG. 2F 
(Day 2 blood sample): standard R1 reagent comprising 0.12 g/L Brij.RTM. 35 
and 0.105 g/L SDS; FIG. 2B (Day 1 blood sample) and FIG. 2G (Day 2 blood 
sample): SDS-free test reagent 1 comprising 0.0 g/L Brij.RTM. 35 and 0.0 
g/L SDS; FIG. 2C (Day 1 blood sample) and FIG. 2H (Day 2 blood sample): 
SDS-free test reagent 2 comprising 0.14 g/L Brij.RTM. 35 and 0.0 g/L SDS; 
FIG. 2D (Day 1 blood sample) and FIG. 2I (day 2 blood sample): SDS-free 
test reagent 3 comprising 0.28 g/L Brij.RTM. 35 and 0.0 g/L SDS; and FIG. 
2E (Day 1 blood sample) and FIG. 2J (Day 2 blood sample): SDS-free test 
reagent 4 comprising 0.42 g/L Brij.RTM. 35 and 0.0 g/L SDS. 
In the analyses of day 1 blood samples, the cytograms obtained using a Px 
R1 reagent composition comprising Brij.RTM. 35 at 0.12 g/L and SDS at 
0.105 g/L were acceptable. However, when neither Brij.RTM. nor SDS was 
present (e.g., reagent 1), the result was a gross failure of the method 
and no differential count information was obtained; the cytogram showed a 
very dense noise area and very few white blood cells were detected (FIG. 
2B). The failure of the method using reagent 1 can be attributed to the 
absence of surfactants leading to a large number of unlysed red cells 
which were cross-linked and contributed to the high level of origin noise. 
A possible reason for the high noise level may be that surfactants are 
apparently required to increase the permeability of white cells to the 
peroxidase substrates; in the absence of surfactant(s), there is likely to 
be effective inhibition of the staining reaction which occurs inside the 
white blood cells. 
Reagents 2 (FIG. 2C), 3 (FIG. 2D) and 4 (FIG. 2E) yielded approximately the 
same unacceptable cytogram results as described for reagent 1. In these 
cases, the cytograms revealed no "valley" (i.e., a clear zone between the 
dense zones) between lymphocytes and noise, and revealed diffuse cell 
clusters and a wide "trunk" (i.e., the roughly vertical column which 
includes both noise and lymphocytes). As the concentration of Brij.RTM. 
increased, the density of the trunk lessened, but the diffuseness of the 
cell populations increased. 
In general, Day 2 or aged samples showed deterioration in the cytograms 
compared with the corresponding results for fresh blood. In the analyses 
of such Day 2 samples, cells became leaky so that the cytogram depictions 
of discrete populations of cell became more diffuse. This was especially 
accentuated for neutrophils. As for Day 1 samples, reagent 1 yielded gross 
failure of the method (FIG. 2G). The density of the trunk tended to 
decrease with increasing concentration of Brij.RTM. in the Px R1 reagent 
solution (FIGS. 2H-2J). Reagents 2, 3, and 4 did not show a valley in the 
cytogram and there was strong distortion in the differential counts 
obtained with reagents 2 (FIG. 2H) and 4 (FIG. 2J). 
The results of these experiments demonstrated that test peroxidase R1 
reagent compositions which contained only nonionic surfactant, i.e., 
Brij.RTM. 35 at concentrations of 0.14-0.42 g/L, and no SDS, yielded 
unacceptable numerical data and cytograms when used with a Brij.RTM.-free 
rinse solution in the rinse cycle. Similarly, as determined in Example 1, 
a Px R1 diluent which contained SDS only (0.16-0.20 g/L) and no Brij.RTM. 
35 was also not an effective reagent when used with the rinse solution 
that did not contain Brij.RTM. (e.g., a Brij.RTM.-free rinse). It was thus 
concluded that Px R1 diluent containing either an ionic surfactant or a 
nonionic surfactant will not provide acceptable results in the Px method. 
Indeed, the present discovery showed that both ionic surfactant (e.g., 
SDS) and nonionic surfactant (e.g., Brij.RTM. 35) must be formulated into 
the Px R1 reagent composition in order achieve accurate and precise 
results in the method. The present invention also provided the knowledge 
and finding that such a peroxidase R1 reagent composition was particularly 
important when used in conjunction with a rinse sample cycle comprising a 
rinse solution that contained no nonionic surfactant such as Brij.RTM. 35, 
described in further detail in Example 3 hereinbelow. 
Example 3 
Formulation and testing of a peroxidase R1 diluent reagent composition 
containing both nonionic surfactant and ionic surfactant 
A third type of R1 reagent composition was prepared to test the formulation 
of both nonionic surfactant and ionic surfactant in the peroxidase 
composition and method. To formulate R1 diluents containing dual 
surfactants, Brij.RTM. 35 at concentrations of 0.080 g/L, 0.12 g/L, and 
0.16 g/L was formulated into the R1 diluent composition which also 
contained SDS at 0.105 g/L. The dual-surfactant-containing reagent 
composition was used to test both Day 1 and Day 2 blood samples using the 
peroxidase method of leukocyte differential counting. 
An experiment was performed to test this R1 reagent formulation on a sample 
set of five bloods drawn in Vacutainer.TM. tubes containing K.sub.3 EDTA. 
Both Day 1 and Day 2 samples were assayed with a standard reagent set at 
102 samples/hour on an automated analyzer. In addition, both Day 1 and Day 
2 samples were assayed with test Px R1 reagents containing both 0.105 g/L 
of SDS and 0.080, 0.12, and 0.16 g/L of Brij.RTM. 35 at 120 samples/hour. 
For the Day 2 samples, the standard Px R1 reagent contained 0.105 g/L of 
SDS and no Brij.RTM. 35 and was used with a rinse solution containing the 
surfactant Pluronic.RTM. P105 in phosphate buffered saline for comparison 
with the test R1 reagent formulations. 
Accuracy and precision data were obtained (see Table 5 and FIGS. 3A-3D). 
For Day 1 samples, both precision and accuracy were within method 
specifications for all peroxidase channel parameters. For Day 2 samples, 
accuracy (versus Day 1 samples and standard) was acceptable for the test 
reagents that contained SDS and Brij.RTM. 35 at 0.12 g/L and 0.16 g/L. 
With SDS and 0.080 g/L of Brij.RTM. 35, the % neutrophil count was outside 
the accuracy specification. The above-mentioned standard Px reagent and 
rinse at 120 samples/hour yielded unacceptable accuracy results with the 
familiar pattern of elevated WBCP and % lymphocytes, and depressed % 
neutrophils. 
This experiment illustrates that acceptable precision and accuracy were 
obtained for Day 1 and Day 2 samples with test Px R1 reagent solutions 
containing SDS at a concentration of 0.105 g/L and Brij.RTM. 35 at a 
concentration range of 0.12 g/L to 0.16 g/L. 
FIG. 3A depicts the acceptable results of a leukocyte differential analysis 
on Day 1 blood samples employing the peroxidase method. The R1 reagent 
composition for the analysis shown in FIG. 3A is a standard and contained 
0.105 g/L of SDS; the standard rinse reagent contained 3.0 g/L of 
Brij.RTM. 35 and 2.0 g/L of SDS. FIG. 3B depicts the performance results 
of a test Px method using Day 1 blood samples in which the Px R1 reagent 
composition contained 0.105 g/L of SDS and the rinse reagent contained 2.0 
g/L of SDS and no Brij.RTM. 35. FIG. 3C depicts the performance results of 
a test Px method using Day 2 blood samples in which the Px R1 reagent 
composition contained 0.105 g/L of SDS and the rinse reagent contained 2.0 
g/L of SDS and no Brij.RTM. 35. FIG. 3D depicts the performance results of 
a test Px method using Day 2 blood samples in which the Px R1 reagent 
composition contained both 0.12 g/L of Brij.RTM. 35 and 0.105 g/L of SDS, 
and the rinse reagent contained no SDS and no Brij.RTM. 35, but instead 
contained the rinse reagent composition described herein (Table 2) 
comprising the nonhemolytic surfactant Pluronic.RTM. P105. As can be seen 
from a comparison of FIG. 3C with FIG. 3D, there is a significant decrease 
in the thickness of the noise area when the nonionic surfactant Brij.RTM. 
35 was present in the R1 reagent composition. 
TABLE 5 
__________________________________________________________________________ 
Effect of the Presence of Both Nonionic Surfactant (Brij .RTM. 35) and 
Ionic Surfactant 
(SDS) in the Aqueous Peroxidase R1 Reagent Composition 
Sample.dagger-dbl. 
SDS.dagger. 
Brij .RTM. 35.dagger..dagger. 
WBCP 
% Neut 
% Lymph 
% Mono 
% Eos 
% LUC 
__________________________________________________________________________ 
Day 1 
0.105 
0 6.31 
59.1 30.5 5.4 2.8 1.6 
(Std) 0.10 
1.0 0.8 0.5 0.3 0.4 
Day 1 
0.105 
0.080 6.23 
59.6 30.0 5.4 3.1 1.5 
(Test) 0.04 
0.3 0.4 0.4 0.2 0.1 
Day 1 
0.105 
0.12 6.12 
59.3 29.7 6.1 2.8 1.6 
(Test) 0.05 
0.5 0.3 0.8 0.2 0.2 
Day 1 
0.105 
0.16 6.09 
59.6 29.7 6.2 2.6 1.3 
(Test) 0.05 
1.1 0.7 0.3 0.3 0.1 
Day 2 
0.105 
0 6.46 
59.9 29.2 7.0 *1.6 
1.6 
(Std) 
Day 2 
0.105 
0 *9.53 
*44.0 
*45.6 6.6 *1.3 
1.8 
(Test) 
Day 2 
0.105 
0.080 6.332 
*56.0 
30.7 6.8 *1.7 
1.2 
(Test) 
Day 2 
0.105 
0.12 6.17 
60.0 29.5 7.2 *1.5 
1.3 
(Test) 
Day 2 
0.105 
0.16 6.15 
61.1 28.0 7.6 *1.5 
1.2 
(Test) 
__________________________________________________________________________ 
.dagger-dbl.: Day 1 blood samples were used less than 8 hours postdraw; 
Day 2 blood samples were aged at room temperature for 24 hours. Five 
samples were assayed in duplicate with each reagent. 
*Numerical value exceeded the system specification for aged blood samples 
.dagger.,.dagger..dagger.: Std: Standard, Brij .RTM. 35 and SDS 
concentrations in the R1 Reagent Solution are in gram/L. 
WBCP: White Blood Cell Count in the Peroxidase Method; % Neut: % 
Neutrophils in sample; % Lymph: % Lymphocytes in sample; % Mono: % 
Monocytes in sample; % Eos: % Eosinophils in sample; % LUC: % large 
unstained cells in sample. 
As determined from FIGS. 3A and 3B, for Day 1 blood samples using a Px R1 
reagent composition containing ionic surfactant (i.e., SDS) only, and a 
rinse reagent formulation either with or without Brij.RTM., the origin 
noise was considered to be normal and acceptable. However, a different 
result was observed when Day 2 samples aged at room temperature were 
analyzed. For such aged blood sample analysis, a Px R1 reagent comprising 
0.105 g/L SDS combined with a rinse reagent comprising no Brij.RTM. 35 and 
2.0 g/L of SDS produced a cytogram that revealed unacceptable origin noise 
(FIG. 3C). Interestingly and in accordance with the inventive discovery 
herein, the noise problem for Day 2 samples was ameliorated by the 
addition of nonionic surfactant (e.g., Brij.RTM. 35) to the peroxidase R1 
reagent in the Px method (FIG. 3D). More particularly, the Px R1 reagent 
composition used in FIG. 3D comprised 0.12 g/L Brij.RTM. 35 and 0.105 g/L 
SDS, and the rinse reagent employed contained neither Brij.RTM. nor SDS, 
but did contain the nonhemolytic surfactant Pluronic.RTM. P105. Thus, the 
discoveries of the appropriate concentrations of nonionic and ionic 
surfactants to use in the peroxidase R1 reagent composition, and the 
optimal use of a sample rinse cycle and corresponding rinse reagent 
comprising a nonhemolytic surfactant such as Pluronic.RTM. P105, such that 
the final nonionic surfactant concentration was appropriate for the 
condition and volume of the blood sample, resulted in a significant 
improvement in the performance of peroxidase differential counting method, 
as well as in the accuracy, precision, and acceptability of the results 
obtained therefrom. 
Example 4 
Selection of an optimal concentration range for nonionic surfactant in the 
R1 reagent composition for use in the Px a method employing a sample rinse 
cycle and rinse solution free of lyric nonionic surfactant such as 
Brij.RTM. 35 
Experiments were performed to select an optimal concentration range for the 
nonionic surfactant in a peroxidase R1 reagent composition using Brij.RTM. 
35 as the exemplary nonionic surfactant and using a reagent configuration 
which included a rinse free of nonionic surfactant such as Brij.RTM. 35 
(Table 2). 
The performance data were generated from experiments in which Brij.RTM. 35 
was used at the final concentrations of 0.10, 0.12, and 0.14 g/L on two 
sample sets: 1) 26 non-hospital samples and 2) 14 hospital samples. Data 
were obtained on both Day 1 and Day 2 samples collected and stored in 
Vacutainer.TM. tubes in the presence of K.sub.3 EDTA as anticoagulant. 
Both numerical and cytogram data were considered. 
The Px R1 test reagent solutions containing 0.10, 0.12, and 0.14 g/L of 
Brij.RTM. 35 were generated by the addition of 0.085, 1.02, and 1.19 mL, 
respectively, of 30 g/L of Brij.RTM. 35 into aliquots of the peroxidase R1 
reagent mixture (minus surfactant). A rinse solution containing non-lyric 
Pluronic.RTM. surfactant as described herein was used in the sample rinse 
cycle employed following the performance of the steps of the peroxidase 
method using the various R1 test reagent sets. For the standard or control 
reagent configuration, an R1 diluent containing 0.105 g/L of ionic 
surfactant SDS in the absence of nonionic surfactant was used, and a rinse 
reagent solution comprising both Brij.RTM. 35 and SDS was used. 
Non-hospital blood samples were obtained from presumed normal volunteers, 
and hospital samples were obtained from patients at the Westchester County 
Medical Center, New York. 
Data were collected using an automated hematology analyzer (e.g., the 
TECHNICON H.cndot..TM. series) with manual open-tube aspiration. Duplicate 
Day 1 samples were assayed with each reagent set. Unopened samples from 
the same donor set were stored at room temperature overnight and were 
assayed manually with open-tube aspiration on day 2 (i.e., Day 2 samples). 
Software for the standard runs was at 102 samples/hour (s/h). The test 
samples were run at 120 s/h. The automated system was washed each day 
prior to running samples according to the general maintenance instructions 
set forth in the User's Manual. Peroxidase channel gains were set 
according to the specifications of the analyzer as set forth in the User's 
Manual. System imprecision was determined with the standard configuration 
of software and reagents before and after the test reagents were 
evaluated. One fresh non-hospital blood sample was aspirated ten times, 
and the mean and standard deviation (SD) were determined (automatically by 
the system) for all CBC parameters. The SDs were compared to the system's 
iraprecision specifications for fresh non-hospital blood. This procedure 
was followed on each day of the study. Test method imprecision was 
determined off-line by calculating the SD (pooled SD over multiple donors) 
for the peroxidase channel parameters using the following equation: 
SD=[Sum (d.sup.2)/2N].sup.1/2, where d is the difference between duplicate 
values obtained with a particular reagent, and N is the number of samples 
in the set. 
The effect of Brij.RTM. 35 concentration on the outcome of the peroxidase 
method of leukocyte differential counting (26 non-hospital samples) 
In this experiment, 26 non-hospital samples were tested in the peroxidase 
method using the above-described standard peroxidase R1 reagent and also 
using the test peroxidase method R1 reagents containing Brij.RTM. 35 at 
concentrations of 0.10, 0.12, and 0.14 g/L (see Table 6). For Day 1 blood 
samples, the clinical parameters were insensitive to Brij.RTM. 35 
concentration. The percent monocytes was low versus the accuracy 
specification, and the percent noise decreased as the concentration of 
Brij.RTM. 35 increased. With the exception of % Monocytes, all other 
parameters satisfied the accuracy and precision specifications. 
For Day 2 blood samples, the % Eosinophils was below the accuracy 
specification for aged blood for the standard method and for all three 
test methods. All other clinical parameters were within the acceptable 
limits. The % noise response was similar to that observed for the Day 1 
samples tested. For this data set, essentially the same numerical data 
were obtained for Day 1 and for Day 2 samples over the Brij.RTM. 35 
concentration range of 0.10, 0.12 and 0.14 g/L. Comparison of 0.14 versus 
0.10 g/L of Brij.RTM. showed a benefit in the reduction of % noise: 13% 
and 17% for fresh and aged blood, respectively. 
Brij.RTM. 35 Concentration Variation (14 hospital samples) 
In this experiment, the sample set of 14 hospital blood samples was tested 
with standard peroxidase method reagents and also with test Px R1 reagents 
which contained 0.10, 0.12 and 0.14 g/L of Brij.RTM. 35 (see Table 7). For 
the Day 1 and Day 2 blood samples, essentially all of the test reagents 
satisfied the accuracy specification. 
TABLE 6 
______________________________________ 
Effect of Variable Brij .RTM. 35 Concentration in the Px Method 
(26 Non-Hospital Samples) 
Brij .RTM. 35 % 
(g/L) WBCP % N % L % M % E LUC % Nois 
______________________________________ 
Day 1 Samples 
Std 6.08 58.7 29.1 7 2.7 1.9 21.2 
0.1 1 *1.4 0.5 0.2 0.2 0.6 
0.1 6.12 58.7 29.4 *6.2 2.8 2.2 25 
0.12 0.9 0.9 0.5 0.4 0.2 1.4 
0.12 6.1 58.9 29.5 *6.2 2.8 2.1 22.9 
0.11 1 0.8 0.5 0.3 0.2 1.3 
0.14 6.1 58.6 29.6 *6.4 2.8 2 21.7 
0.12 0.8 0.7 0.6 0.2 0.3 1.4 
Day 2 Samples 
Std *6.48 59.2 29.8 6.9 *1.6 1.7 27.1 
0.18 4.2 4.2 0.6 0.3 5 2.3 
0.1 6.29 57.7 31 7.1 *1.6 1.8 24.9 
0.14 1.5 1.7 0.7 0.2 0.4 1.8 
0.12 6.24 58.6 29.9 7.2 *1.6 1.8 23.9 
0.13 1.2 1.2 0.7 0.2 0.3 2.3 
0.14 6.12 59.8 29.1 7 *1.6 1.7 20.8 
0.12 1.2 1.3 0.8 0.4 0.2 1.8 
Specifications: 
Day 1 SD 0.16 1.4 1.1 0.9 0.5 0.5 none 
Day 1 acc 0.15 1 0.5 0.5 0.2 0.5 none 
Day 2 acc 0.32 2.8 2.2 1.8 1 1 none 
______________________________________ 
The cytograms generated from the hospital and non-hospital data sets were 
examined qualitatively for the following characteristics: noise/lymphocyte 
separation; tightness of neutrophil, lymphocyte, monocyte, eosinophil and 
LUC populations; staining intensity and positioning of the cell 
populations on the cytogram; variation in the floating low threshold 
between the noise zone and the lymphocyte zone, and overall general 
appearance were compared with the cytograms obtained from analyses of 
blood samples using standard reagents in the peroxidase method. The sample 
set assayed in the peroxidase method included both Day 1 and Day 2 
non-hospital samples (11 samples) and hospital samples (14 samples). 
The conclusions deduced from the resulting cytograms were that 0.12 g/L of 
Brij.RTM. 35 in the peroxidase R1 diluent yielded the best overall 
cytogram in terms of the separation of cell populations and acceptable 
origin noise level; 0.10 and 0.14 g/L were less desirable than 0.12 g/L, 
but both were considered to be acceptable for use in the method. For Day 2 
samples, 0.10 g/L of Brij.RTM. 35 in the Px R1 test diluent resulted in a 
slightly higher percent noise than did 0.12 g/L of Brij.RTM. 35 (i.e., 
21.7% versus 16.7% for Day 1 samples, and 18.9% versus 17.7% for Day 2 
samples), but the 0.10 g/L Brij.RTM. 35 concentration was determined to be 
better for the analysis of Day 2 blood (i.e., the cell population areas 
were tighter). Conversely, 0.14 g/L of Brij.RTM. 35 in the R1 test diluent 
resulted in a slightly lower percent noise than did 0.12 g/L of this 
surfactant (i.e., 16.2% versus 16.7% for Day 1 samples); however, the 0.14 
g/L concentration of this surfactant was marginally worse for Day 2 blood 
samples (i.e., 0.14 g/L of Brij.RTM. 35 caused diffuseness in the cell 
populations due to attack of the cells by the higher surfactant 
concentration). The results of the qualitative cytogram inspection were 
consistent with the numerical analyses. 
The data from analogous experiments also demonstrated that five different 
lots of Brij.RTM. 35 tested yielded essentially the same performance data 
for both Day 1 and Day 2 normal and hospital blood samples. Therefore, 
lot-to-lot variation of Brij.RTM. 35 is not expected to be a problem for 
producing reagents containing this nonionic surfactant. Based upon the 
combined numerical and 
TABLE 7 
______________________________________ 
Effect of Brij .RTM. 35 Concentration Variation in the Px R1 
Reagent Composition (14 Hospital Samples) 
Brij .RTM. 35 % 
(g/L) WBCP % N % L % M % E LUC % Nois 
______________________________________ 
Day 1 Samples 
Std 15.15 71.8 16.5 5.2 0.7 5.5 8.6 
0.16 *2.9 *3.0 *1.8 0.1 *2.3 0.4 
0.10 15.12 71.9 16.7 4.7 0.7 5.7 10.9 
*0.18 1.3 0.7 *1.3 0.2 0.5 0.6 
0.12 15.12 71.7 17.0 4.9 0.7 5.4 9.2 
0.13 0.8 1.0 0.5 0.1 0.5 0.5 
0.14 15.23 71.2 *17.6 
5.3 0.6 *4.9 9.1 
*0.19 0.8 1.1 0.5 0.1 2.5 0.7 
Day 2 Samples 
Std 14.92 71.0 17.3 6.2 0.6 4.3 12.8 
0.67 2.3 4.2 1.5 0.1 1.7 1.0 
0.10 15.00 70.8 17.7 5.4 0.5 4.9 11.6 
0.29 0.6 1.3 0.4 0.1 9.3 2.3 
0.12 15.10 70.5 17.3 5.6 0.6 5.4 10.7 
0.37 1.3 1.3 0.5 0.1 0.1 1.0 
0.14 15.23 70.1 17.7 5.8 0.7 5.2 9.8 
0.37 1.5 2.1 2.1 0.1 1.3 1.0 
Specifications: 
Day 1 SD 0.16 1.4 1.1 0.9 0.5 0.5 none 
Day 1 acc 0.15 1 0.5 0.5 0.2 0.5 none 
Day 2 acc 0.32 2.8 2.2 1.8 1 1 none 
______________________________________ 
cytogram results, an optimal concentration range for Brij.RTM. 35 in the 
peroxidase R1 reagent composition was selected to be about 0.10 g/L to 
about 0.15 g/L, more preferably, about 0.11 g/L to about 0.13 g/L. It is 
to be understood that the concentration of nonionic surfactant such as 
Brij.RTM. 35 in the reagent composition can be routinely adjusted by the 
skilled practitioner, depending upon the hematology analysis system 
employed in carrying out the differential counting method. 
Example 5 
Avoidance of the problem of variable rinse carryover by the use of a rinse 
solution containing nonhemolytic surfactant in conjunction with the use of 
the dual-surfactant-containing R1 reagent composition in the peroxidase 
method 
To avoid the problem of variable rinse carryover in performing the 
peroxidase white blood cell differential method on automated analyzers, 
the method of the present invention may be further improved by also 
including a rinse solution that contains nonionic, non-hemolytic 
surfactant different from the nonionic surfactant contained in the R1 
reagent composition of the invention. The rinse solution has been 
described hereinabove and comprises non-hemolytic surfactant such as 
Pluronic.RTM. P105, which is inactive and nonfunctional in the peroxidase 
method, and contributes no more than a slight volume increase to the 
method. Intersample rinsing alleviates the formation of deposits and 
eliminates sample/reagent mixture carryover in the peroxidase reaction 
chambers after multiple sample aspirations when performing the peroxidase 
leukocyte differential counting method on semi- and fully-automated 
hematology analyzers. 
A particular illustration of the problem of rinse carryover is as follows: 
prior to the design of the improved present reagent and method, it was 
found by the present inventors that about 8-10 .mu.L of rinse solution was 
left in the reaction chamber after completion of the intersample rinse 
cycle. When the rinse solution was formulated to contain the nonionic 
surfactant Brij.RTM. 35 in accordance with the present invention, this 
seemingly small volume of rinse (i.e., 8 to 10 .mu.L) was found to be 
responsible for adding an mount of Brij.RTM. 35 to the R1 phase of the 
method that was significant enough to adversely affect the cell separation 
results depicted in the cytogram. For example, when the volume of rinse 
carryover exceeded about 10 .mu.L, the eosinophil population migrated up 
into the neutrophil population of the cytogram, and both monocytes and 
lymphocytes moved down in the cytogram. Moreover, when the volume of rinse 
carryover was about 13.3 .mu.L, the peroxidase method was completely 
degraded by the presence of Brij.RTM. 35. Since the volume of the Px R1 
reagent solution added during the first reaction phase of the method was 
0.25 mL, the calculated Brij.RTM. 35 concentration during the first 
reaction phase of the peroxidase method was about 0.093 g/L to 0.120 g/L, 
which corresponds to a volume of rinse carryover of approximately 8.0 to 
10.0 .mu.L. 
The quantification of the concentration of Brij.RTM. 35 that is transferred 
to the R1 stage of the Px method via rinse carryover was determined during 
the course of these tests and is presented as follows: the rinse solution 
contains 3.0 g/L of Brij.RTM. 35. The nominal rinse carryover volume is 10 
.mu.L, which is equivalent to 30 .mu.g of Brij.RTM. 35. The transfer of 30 
.mu.g of Brij.RTM. 35 to the R1 phase of the peroxidase method (i.e., 250 
.mu.L of peroxidase R1 diluent +12 .mu.L of blood, or .about.7 .mu.L of 
plasma, +10 .mu.L of rinse solution yields a total volume of .about.267 
microliters), in turn, yields a Brij.RTM. 35 concentration of 0.112 g/L 
during the R1 phase of the method. The test peroxidase R1 reagents were 
all formulated to contain essentially the same concentration of Brij.RTM. 
35, which allows the delivery of a constant concentration of this nonionic 
surfactant in the peroxidase method. 
FIGS. 4A-4F demonstrate the effects of variable rinse carryover volume in 
the peroxidase method of the invention using an aqueous rinse composition 
without nonionic surfactant, e.g., Brij.RTM. 35 (FIGS. 4A, 4C, and 4E) and 
an aqueous rinse composition with nonionic surfactant (FIGS. 4B, 4D, and 
4F). In the R1 and R2 phases of the peroxidase method, as shown in FIGS. 
4A-4F, the R1 reagent of the method contained 0.105 g/L of SDS and no 
Brij.RTM. 35. FIGS. 4A and 4B depict cytograms resulting from the 
performance of the peroxidase method and accompanying rinse cycle, with 
rinse solution carryover contributing 7.9 .mu.L to the final volume of the 
peroxidase R1 reagent solution (FIG. 4A: no Brij.RTM. 35 in the rinse 
solution; FIG. 4B: Brij.RTM. 35 present in the rinse solution). FIGS. 4C 
and 4D depict cytograms resulting from the performance of the peroxidase 
method and accompanying rinse cycle, with rinse solution carryover 
contributing 10.1 .mu.L to the final volume of the peroxidase R1 reagent 
solution (FIG. 4C: no Brij.RTM. 35 in the rinse solution; FIG. 4D: 
Brij.RTM. 35 present in the rinse solution). FIGS. 4E and 4F depict 
cytograms resulting from the performance of the peroxidase method and 
accompanying rinse cycle, with rinse solution carryover contributing 13.3 
.mu.L to the final volume of the peroxidase R1 reagent solution (FIG. 4E: 
no Brij.RTM. 35 in the rinse solution; FIG. 4F: Brij.RTM. 35 present in 
the rinse solution). As can be determined from the results, Brij.RTM. 35 
present in the rinse solution and the contribution of rinse carryover 
yield a general deterioration of the cytogram results. This result was 
also observed as rinse carryover volumes increased in the presence of 
Brij.RTM. 35 (FIGS. 4B, 4D, and 4F). 
Using the improved reagent composition and method of the invention, 
carryover was insignificant; in addition, the accuracy and precision of 
the results were highly acceptable. The nonionic surfactant Brij.RTM. 35 
was determined by the present inventors to be the agent, which, if present 
in the rinse composition or solution and if carried over in variable 
amounts into the peroxidase method, caused unacceptable cytogram results, 
particularly pronounced in the degradation of the white cell cluster in 
the cytogram. 
As demonstrated herein, the use of nonhemolytic surfactants such as the 
Pluronics.RTM. (e.g., Pluronic.RTM. P105) in the rinse solution in the 
peroxidase method, and the formulation of a new and improved R1 diluent 
reagent composition of the invention alleviated the effect of variable 
rinse carryover, maintained an acceptable level of origin noise, and 
allowed acceptable results to be obtained in the Px method using Day 2 
blood samples. Thus, in accordance with the invention, an R1 reagent 
composition which contains both an ionic surfactant (e.g., SDS) and a 
nonionic surfactant (e.g., the polyethoxylate Brij.RTM. 35) was found by 
experiment to be useful with blood samples that had been aged for 24 hours 
or longer at room temperature. Further, in accordance with the invention, 
the use of a rinse solution containing surfactants like Pluronics.RTM. 
also improved the results obtained from the differential leukocyte 
differential counting method as described herein. 
Example 6 
Automated analysis of the leukocyte differential counting method using the 
R1 reagent composition and peroxidase method 
This example describes the determination of a rapid (i.e., 120 samples/hour 
versus 60 or 102 samples/hour) white blood cell differential analysis 
using an automated hematology analyzer and the improved method and reagent 
of the invention. It will be clear to those having skill in the art that 
various analyzers and systems may be used and afforded the benefits 
described herein using the methods and reagents in accordance with the 
invention. 
Using a whole blood sample, in the R1 reaction phase of the method, 12 
.mu.L of sample was delivered with 250 .mu.L of dual-surfactant-containing 
R1 reagent diluent. About nineteen seconds later, 250 .mu.L of a hydrogen 
peroxide (3.0 g/L)-containing diluent and 125 .mu.L of a 
4-chloro-alpha-naphthol (70 g/L)-containing diluent were added. This is 
considered the second reaction phase (R2). About thirteen seconds after 
the addition of the chromogen-containing reagent, the effluent was passed 
through an electro-optical detection system and a cytogram was prepared. 
Size and degree of white blood cell staining were measured (forward angle 
scatter versus absorbance) on a cell by cell basis and plotted on a 
cytogram. The cytogram was analyzed by computer to obtain the total white 
blood cell count and the subpopulation differential of neutrophils, 
eosinophils, monocytes, lymphocytes, and large unstained cells. The 
figures illustrate cytograms obtained from the electro-optical detection 
system of an automated hematology analyzer used to carry out the method of 
the invention on an automated apparatus. The cytograms reveal the types of 
leukocytes (i.e., WBCs) that are differentiated by the reagent and method 
of the invention: 1) lymphocytes; 2) monocytes; 3) neutrophils; 4) 
eosinophils; 5) origin noise resulting from red cell ghosts and platelets, 
and 6) LUCs (see FIG. 1A). 
The final dilution of whole blood was 1:53, and the total reaction time was 
about 30-32 seconds. A general and nonlimiting example of the thermal 
profile of the peroxidase method performed on an automated analyzer 
follows: In the R1 phase of the method, the blood sample and the R1 
reagent diluent enter the peroxidase chamber which is preset at about 
69.degree. C. .+-.2.degree. C. During the R1 reaction phase, which has a 
duration of about 15 to 20 seconds, the temperature increases to about 
65.degree. C. to about 75.degree. C., or to about 65.degree. C. to about 
70.degree. C. Thereafter, the substrate reagents are added in the second 
reaction phase of the method (i.e., R2); the R2 temperature is about 
50.degree. C. to about 65.degree. C. and has a duration of about 5 to 8 
seconds during which time the temperature is increased to about 73.degree. 
C. .+-.2.degree. C., which is the final temperature at the completion of 
the peroxidase method. The rinse solution was used in the rinse cycle 
after completion of the R2 phase of the peroxidase method. Sample rinse 
cycles are needed to prevent sample carryover from one sample cycle to 
another and to prevent buildup in the hydraulics of the automated 
hematology analyzer systems. 
Example 7 
Analysis of various nonionic surfactants for suitability in the R1 reagent 
composition and method of the invention 
To determine the types of nonionic polyethoxylate surfactants suitable for 
use in the improved reagent composition and peroxidase method of the 
invention, the following experiments were performed. 
Peroxidase R1 test reagent compositions for use in the first reaction phase 
of the method were prepared as follows: 200 .mu.L of 3.0 g/L solutions of 
the various polyethoxylates in distilled water were added to 50.0 mL of 
the phase 1 reagent composition as described, and placed in 60 mL 
polypropylene screw-top centrifuge tubes. The surfactant solutions were 
prepared by adding 3.0 g of polyethoxylate to 97.0 mL of distilled water 
and stirring with heat until the solution began to boil. After cooling to 
room temperature for about 30 minutes, 200 .mu.L of the solution were 
added to the other components of the peroxidase R1 reagent composition to 
make the R1 test reagents. The polyethoxylates as tested herein are 
commercially available from various sources; for example, the Brij.RTM. 
surfactants were obtained from ICI and Ruger; the Macol surfactants were 
obtained from Mazer, the Sipionic surfactants were obtained from Alcolac; 
the Triton X.RTM. surfactants were obtained from Sigma, Union Carbide, or 
Rohm&Haas; IgepalCO897 was obtained from GAF; Pluronic.RTM. P105 was 
obtained from BASF; Surfonic N31.5 was obtained from Huntsman. For the 
ionic surfactants, TDAPS was obtained from Boehfinger-Mannheim and TTAB 
was obtained from Sigma. 
As shown in Table 8, the following families or classes of polyethoxylates 
were used: 
TABLE 8 
______________________________________ 
Surfactant HLB Value Hydrophobe Hydrophile 
______________________________________ 
1) Straight-chain hydrophobe etherified to polyethylene glycol 
Brij .RTM. 52 
5.3 C.sub.16 H.sub.33 O 
POE.sub.2 
Brij .RTM. 30 
9.7 C.sub.12 H.sub.25 O 
POE.sub.4 
Brij .RTM. 76 
12.4 C.sub.18 H.sub.37 O 
POE.sub.10 
Macol .RTM. TD12 
14.5 C.sub.13 H.sub.27 O 
POE.sub.12 
Siponic .RTM. L12 
14.6 C.sub.12 H.sub.25 O 
POE.sub.12 
Brij .RTM. 78 
15.3 C.sub.18 H.sub.37 O 
POE.sub.20 
Brij .RTM. 58 
15.7 C.sub.16 H.sub.37 O 
POE.sub.20 
Siponic .RTM. E15 
16.9 C.sub.16 H.sub.33 O/C.sub.18 H.sub.37 O 
POE.sub.32 
Brij .RTM. 35 
16.9 C.sub.12 H.sub.25 O 
POE.sub.23 
2) Branched-chain octylphenyl hydrophobe etherified to 
polyethylene glycol 
TritonX .RTM. 
13.5 C.sub.8 H.sub.17 OC.sub.6 H.sub.4 O 
POE.sub.9.5 
100 
Triton X .RTM. 
15.8 C.sub.8 H.sub.17 OC.sub.4 O POE.sub.16 
165* 
Triton X .RTM. 
17.3 C.sub.8 H.sub.17 OC.sub.4 O POE.sub.30 
305* 
Triton X .RTM. 
17.9 C.sub.8 H.sub.17 OC.sub.4 O POE.sub.40 
405* 
3) Straight-chain nonylphenyl hydrophobe etherified to 
polyethylene glycol 
Igepal .RTM. 
17.8 C.sub.9 H.sub.19 OC.sub.6 H.sub.4 O 
POE.sub.40 
CO897* 
4) Straight-chain fatty acid esterified to polyethylene glycol 
Myrj .RTM. 53 
17.9 C.sub.17 H.sub.35 CO.sub.2 
POE.sub.50 
______________________________________ 
*The raw material was a 70% (w/w) solution in water; therefore, 4.30 g of 
surfactant solution was mixed with 95.7 g of distilled water to yield a 
final concentration of 3.00 percent (w/w) of surfactant in the reagent 
composition of the first reaction phase of the peroxidase method. 
Ten blood samples per reagent set were collected from normal donors in 
Vacutainer.RTM. tubes and anticoagulated with K.sub.3 EDTA. Data were 
collected on an automated analyzer with manual open-tube aspiration. 
Duplicate Day 1 blood samples were aspirated with each reagent set. 
Unopened samples from the same donor set were stored at room temperature 
overnight and assayed manually on day 2 (Day 2 samples). Software for the 
standard runs occurred at 102 samples/hour; test samples and reagents were 
run at a throughput of 120 samples/hour. The system was washed each day 
prior to running samples according to the operating instructions, and 
peroxidase channel gains were set according to the operating instructions 
for the respective automated system being used. 
System imprecision was determined with the standard configuration of 
software and reagents both before and after the test reagents were 
evaluated. One Day 1 blood sample was aspirated ten times, and the mean 
and standard deviations were determined (automatically by the system) for 
all parameters. The standard deviations were compared to the system's 
imprecision specifications determined for fresh blood. This procedure was 
followed each day of the study. 
To determine test method imprecision, 10 samples were aspirated in 
duplicate. Imprecision was estimated off-line by calculating the standard 
deviation, SD, (pooled SD over multiple donors) for peroxidase channel 
parameters with the following equation: SD=[Sum(d.sup.2)/2N].sup.1/2, 
where d is the difference between duplicate values obtained with a 
particular reagent, and N is the number of samples in a ten sample set. 
The test Px R1 reagents were evaluated in two sets. For each set, a 
standard peroxidase method (e.g., performed on the TECHNICON H.cndot.3.TM. 
automated analyzer) was included as the reference. The reference values 
were defined as Day 1 mean values obtained in the absence of the improved 
method and reagents of the invention. Accuracy ("acc") for Day 1 and Day 2 
sample analysis was determined versus the reference; the accuracy criteria 
for Day 1 and Day 2 aged blood samples are different. In addition, 
imprecision was determined only for Day 1 blood samples (see Table 9). 
It was noted that Brij.RTM. 52 had an HLB of 5.3 (see Table 10), is 
therefore hydrophobic, and is usually utilized for water-in-oil 
emulsification applications. The automated peroxidase method performed on 
the H.cndot.3.TM. automated analyzer utilizes oil-in-water emulsification 
(i.e., the lipid material of red cell and platelet membranes is 
"dissolved" by surfactant micelles) in the aqueous environment of the 
peroxidase effluent). 
TABLE 9 
__________________________________________________________________________ 
Leukocyte Differential Analyses Performed on Day 1 and Day 2 Blood 
Samples Using Different Nonionic Surfactants 
Hydrophile 
Structure 
POE units 
Reagent WBCP 
% NEUT 
% LYMP 
% MONO 
% EOS 
% LUC 
% NOISE 
HLB 
Hydrohobe 
n 
__________________________________________________________________________ 
= 
Day 1 Blood Samples 
Std 6.35 
61.0 26.9 6.6 2.6 2.2 17.8 
sd 0.09 
0.6 0.8 0.6 0.2 0.4 0.6 
Brij .RTM. 58 
6.40 
60.7 27.3 6.6 2.5 2.2 19.5 15.7 
C.sub.16 H.sub.33 
20 
sd 0.10 
1.0 1.0 0.3 0.4 0.2 1.3 
Ig. .RTM. CO897 
6.46 
60.7 27.3 6.5 2.7 2.1 28.0 17.8 
C.sub.9 H.sub.19 
40 
sd 0.10 
1.3 1.1 0.4 0.3 0.5 1.2 
Triton X .RTM. 
6.32 
61.0 27.1 6.6 2.7 2.0 18.9 13.5 
C.sub.8 H.sub.17 
9.5 
100 
sd 0.10 
1.2 0.9 0.7 0.2 0.2 1.0 
Siponic .RTM. 
6.44 
60.5 27.2 6.9 2.7 2.0 23.1 16,.9 
C.sub.16 H.sub.33 
**32 
E15 C.sub.18 H.sub.37 
sd 0.14 
1.3 1.0 0.3 0.3 0.3 1.4 
Triton X .RTM. 400 
6.49 
61.1 27.4 6.0 2.6 2.1 27.1 17.9 
C.sub.8 H.sub.17 
40 
sd 0.12 
1.1 1.1 0.3 0.5 1.0 1.0 
Brij .RTM. 76 
6.50 
60.8 27.1 6.4 2.7 2.3 24.3 12.4 
C.sub.18 H.sub.37 
10 
sd 0.11 
0.9 0.7 0.5 0.2 0.2 0.9 
Myrj .RTM. 53 
6.48 
60.1 27.3 6.1 28 2.1 24.6 17.9 
C.sub.17 H.sub.35 
CO.sub.2 
50 
sd 0.09 
1.2 1.0 0.5 0.3 0.2 0.9 
acc spec 
0.15 
1.0 0.5 0.5 0.2 0.5 none N/A 
sd spec 0.16 
1.4 1.1 0.9 0.5 0.5 none N/A 
Day 2 Blood Samples 
Std 6.62 
59.6 29.7 6.3 1.8 1.7 24.1 
Brij .RTM. 58 
6.62 
60.5 28.5 7.1 1.6 1.7 14.3 15.7 
Ig .RTM. CO897 
*9.79 
*54.4 *33.7 7.3 *1.3 2.3 33.0 17.8 
Triton X .RTM. 100 
6.43 
62.7 26.7 6.9 1.7 1.5 15.4 13.5 
Siponic .RTM. 
6.65 
60.8 28.7 6.6 1.7 1.6 17.7 16.9 
E15 
Triton X .RTM. 
*10.77 
57.1 *31.2 7.2 *1.2 2.3 33.3 17.9 
400 
Brij .RTM. 76 
6.68 
60.8 28.8 6.5 1.7 1.7 23.3 12.4 
Myrj .RTM. 53 
*8.04 
58.8 *30.5 6.7 *1.2 2.0 31.8 17.9 
acc spec 
Day 2 0.32 
2.8 2.2 1.8 1.0 1.0 none N/A 
__________________________________________________________________________ 
*exceeded method specification on H.cndot. .TM. analyzer; **calculation 
based on C.sub.17 H.sub.35 ; sd: mean standard deviation 
TABLE 10 
__________________________________________________________________________ 
Leukocyte Differential Analyses Performed on Day 1 and Day 2 Blood 
Samples Using Different Nonionic Surfactants 
Hydrophile 
Structure 
POE units 
Reagent WBCP 
% NEUT 
% LYMP 
% MONO 
% EOS 
% LUC 
% NOIS 
HLB 
hydrophobe 
n 
__________________________________________________________________________ 
= 
Day 1 Blood Samples 
Std 7.02 
62.4 25.2 6.5 3.1 2.0 20.3 
sd 0.14 
0.8 0.7 0.5 0.2 0.2 0.5 
Brij .RTM. 52 
*7.20 
62.4 25.5 6.0 3.1 2.4 32.4 5.3 
C.sub.16 H.sub.33 
2 
sd 0.09 
1.0 0.8 0.6 0.2 0.4 2.0 
Brij .RTM. 35 
6.97 
62.1 25.9 6.0 3.2 2.1 22.5 16.9 
C.sub.12 H.sub.25 
23 
sd 0.15 
0.7 0.6 0.5 0.3 0.3 0.8 
Siponic .RTM. 
6.97 
61.9 25.7 6.5 3.1 2.3 19.4 14.6 
C.sub.12 H.sub.25 
12 
L12 
sd 0.15 
0.7 0.6 0.4 0.3 0.3 1.4 
Triton X .RTM. 165 
7.04 
62.1 25.7 6.1 3.2 2.2 23.0 15.8 
C.sub.8 H.sub.17 
16 
sd 0.09 
0.7 0.9 0.7 0.2 0.3 0.7 
Triton X .RTM. 
7.04 
62.4 25.4 6.0 3.2 2.3 27.9 17.3 
C.sub.8 H.sub.17 
30 
300 
sd 0.12 
1.0 0.8 0.9 0.3 0.3 1.3 
Brij .RTM. 30 
7.05 
61.5 26.1 6.5 3.1 2.1 21.0 9.7 
C.sub.12 H.sub.25 
4 
sd 0.10 
0.9 0.7 0.5 0.3 0.1 0.9 
Macol .RTM. TD12 
7.01 
62.3 25.4 6.4 3.0 2.0 19.0 14.5 
C.sub.13 H.sub.27 
12 
sd 0.15 
0.9 0.7 0.5 0.3 0.1 0.9 
Brij .RTM. 78 
7.10 
61.4 25.6 6.9 3.0 2.2 23.4 15.3 
C.sub.18 H.sub.37 
20 
sd 0.16 
1.2 1.0 0.6 0.3 0.3 1.2 
accy spec 
0.15 
1 0.5 0.5 0.2 0.5 none N/A 
sd spc 0.16 
1.4 1.1 0.9 0.5 0.5 none N/A 
Day 2 Blood Samples 
Std 7.16 
6.3 25.8 7.4 2.2 1.7 22.4 
Brij .RTM. 52 
*13.10 
*47.8 *33.2 7.6 *1.0 3.0 35.6 5.3 
Brij .RTM. 35 
7.10 
62.5 25.8 7.3 2.2 1.8 19.6 16.9 
Siponic .RTM. 
6.95 
62.7 25.1 7.9 2.2 1.8 14.5 14.5 
L12 
Triton X .RTM. 165 
7.07 
62.6 25.7 7.2 2.3 1.7 21.9 15.8 
Triton X .RTM. 300 
*8.90 
60.8 27.0 7.8 2.3 1.8 35.8 17.3 
Brij .RTM. 30 
7.04 
62.6 26.0 7.2 2.1 1.8 17.0 9.7 
Macol .RTM. TD1 
6.90 
63.9 24.3 7.5 2.2 1.8 13.3 14.5 
Brij .RTM. 78 
7.03 
63.8 25.1 6.9 2.2 1.7 17.3 15.3 
acc spec 
aged 0.32 
2.8 2.2 1.8 1.0 1.0 none N/A 
__________________________________________________________________________ 
*exceeded method specification on H.cndot..TM. analyzer; sd: mean standar 
deviation 
In general, the peroxidase effluent comprises the following components: 
0.25 mL of the Px R1 reagent solution, Px 1; 0.125 mL of the 
chromogen-containing reagent solution, Px 2; 0.25 mL of the 
hydrogen-peroxide-containing reagent solution, Px 3; and 12 .mu.L of blood 
sample. Px 1 and Px 3 (3.0 g/L aqueous hydrogen peroxide) are aqueous 
solutions, while Px 2 is a solution of 4-chloro-naphthol dissolved in 
diethylene glycol (a non-aqueous but water-miscible solvent). 
Surfactants with HLBs in the range of 3-6 are recommended for water-in-oil 
emulsification. In contrast, the recommended HLB range for oil-in-water 
emulsifications are about 8-18 (M. J. Rosen, 1978, Surfactant and 
Interfacial Phenomena, Wiley-Interscience, pp. 243-244). 
Triton X.RTM.-305 was shown to be highly hydrophilic as it has an HLB of 
17.3 and was suitable for oil-in-water emulsification. However, the 
suboptimal performance of this surfactant may be a consequence of its 
extreme hydrophilicity under the conditions of the analysis. As a 
consequence of their numerous peroxidase studies, the present inventors 
have observed that red cells become more resistant to lysis after storage 
at room temperature, due to biochemical changes which occur during storage 
of the sample. The accuracy and reliability of the newly described methods 
and reagents are especially important in view of the deterioration of 
blood samples with time and the need to perform assays on such samples and 
to obtain adequate results in spite of the less-than-optimal aged sample 
conditions. 
Of the peroxidase R1 reagent compositions containing ionic surfactant plus 
the following nonionic surfactants: Brij.RTM. 58, Igepal.RTM. CO897, 
Triton X.RTM.-100, Siponic.RTM. E15, Triton X.RTM.-405, Brij.RTM. 76- and 
Myrj.RTM. 53, all performed accurately and acceptably in the analysis of 
Day 1 blood samples. However, on Day 2 samples, the Px R1 test reagents 
containing Igepal.RTM. CO897, Triton X.RTM.-405, and Myrj.RTM. 53 appeared 
to yield unacceptable numerical and cytogram results (see Tables 11 and 
12). These polyethoxylates had HLB values of 17.8, 17.9 and 17.9, 
respectively, and mean % noise values of 33.0, 33.3 and 31.8, 
respectively. In addition, other common features in the cytograms 
resulting from the use of these surfactants were elevated WBCP and 
distorted % Neutrophils and % Lymphocytes. As indicated, Myrj.RTM. 53 is a 
carboxylic ester of stearic acid, which is likely to be less stable in 
aqueous solution due to hydrolysis. The abbreviations used in Tables 9 and 
10 are: WBCP: % total white blood cells; %NEUT: % neutrophils; LYMPH: % 
lymphocytes; % MONO: % monocytes; % EOS: % eosinophils; % LUC: % large 
unstained cells; % NOIS: % origin noise following the performance of the 
peroxidase method; HLB: hydrophilic lipophilic balance value. 
Without being bound in any way by theory, the possible function(s) of 
surfactant in the R1 reagent composition used in the peroxidase method may 
include one or more of the following: (1) membrane penetration leading to 
lysis of red cells and platelets, (2) complexation of water-insoluble 
4-chloronapthol, and transport of this substrate into the cells where 
staining occurs and (3) emulsification of red-cellular debris thereby 
reducing buildup in the peroxidase channel. 
In the representative cytograms depicted in the accompanying figures, the 
width and darkness (related to the number of cells) of the 
noise/lymphocyte "trunk" at the origin increased with increasing percent 
noise. In general, cytograms obtained with aged blood samples exhibit 
neutrophils which have fallen below the boundaries of the neutrophil 
population in fresh blood. There is also a tendency for populations to 
spread out in aged blood. The results from this example showed that the 
cytograms were comparable among the various families of polyethoxylate 
surfactants tested (see Table 8, Example 7). In particular, however, the 
surfactants which derived from surfactant families 1 and 2 (e.g., straight 
chain or branched octylphenyl hydrophobe etherified to polyethylene 
glycol), described hereinabove, and having an HLB in the range of about 
9.6 to about 16.9 performed in an acceptable manner in the analyses of 
both Day 1 and Day 2 blood samples. These results are consistent with the 
generally-recommended surfactant HLB range of about 8-18 for oil-in-water 
emulsifications. In automated peroxidase methods such as that performed on 
the exemplary H.cndot.3.TM. system, red cell membrane debris is lipid 
("oil") material which is emulsified by surfactant micelles in an aqueous 
environment; hence, the operative mode for these types of analyses is 
oil-in-water emulsification. 
Example 8 
Analysis of surfactants having low HLB values and assessment of 
Pluronics.RTM. for suitability in the Px R1 reagent composition and 
peroxidase method of leukocyte differential counting 
Experiments were performed to test the potential utility and effectiveness 
of polyethoxylate surfactants having a lower HLB range (.about.8) 
formulated into Px R1 reagent solutions containing 0.105 g/L of SDS. 
Accordingly, surfactants having HLB values from 5.3 to 17.9 were tested. 
The performance of the test R1 reagent compositions, as a part of a reagent 
set, was determined with a sample set comprised of five Day 1 non-hospital 
blood samples and five Day 2 non-hospital samples that were assayed after 
storage at room temperature. Performance was judged versus current 
automated analyzer specifications for accuracy and precision. The 
following parameters were monitored: WBCP, % neutrophils (% N or Neut), % 
lymphocytes (% Ly), % monocytes (% M), % eosinophils (% Eos), % large 
unstained cells (% LUCs) and peroxidase noise (% Noise). 
Test Px R1 reagents containing SDS, at 0.105 g/L, and the following other 
surfactants, e.g., Brij.RTM. 35, Macol.RTM. NP4, Surfonic.RTM. N31.5 , 
Pluronic.RTM. P105, Macol.RTM. TD3, and Triton X.RTM. 35, were prepared 
according to the procedure described in Example 7. The exemplary test 
surfactants and their corresponding HLB values are presented: 
______________________________________ 
Surfactant HLB Value 
______________________________________ 
Brij .RTM. 35 16.9 
Macol .RTM. NP4 8.9 
Surfonic .RTM. N31.5 
7.7 
Pluronic .RTM. P105 
12-18 
Macol .RTM. TD3 8.0 
Triton X .RTM. 35 
7.8 
______________________________________ 
Additional studies were conducted to test Pluronics.RTM. as replacement 
surfactants for Brij.RTM. 35 as the nonionic surfactant in the peroxidase 
R1 reagent composition formulated in accordance with the invention. 
As described in Example 7 and shown in Tables 9 and 10, acceptable 
automated peroxidase method results using both Day 1 and Day 2 blood 
samples were obtained with surfactants having HLB values from about 9.3 to 
about 16.9. Surfactants having HLB values above about 17.3 as well as low 
HLB values, e.g., HLB=5.3, in the R1 reagent composition resulted in 
unacceptable data. 
Pluronics.RTM., including P105, possess structures which are distinctly 
different from the other polyethoxylates used in the experiments as 
described herein. In the structures of Pluronics.RTM., there are three 
domains: (POE).sub.n -(POP).sub.m -(POE).sub.n, in which POE and POP 
represent polyoxyethylene and polyoxypropylene, respectively. The POE 
domains are hydrophilic (i.e., "the hydrophiles") and the POP domain is 
hydrophobic (i.e., "the hydrophobe"). There also exists a Pluronic.RTM. 
"reverse" or "R" series, in which there are two POP domains flanking one 
POE domain, (see Pluronic.RTM. & Tetronic.RTM. Surfactants, BASF 
Corporation, 1987). Pluronics.RTM. have been assigned HLB values. For 
P105, the HLB value is 12-18. The wide HLB range assigned to P105 is in 
sharp contrast to that of the two domain polyethoxylates, which are 
assigned narrower HLB values. Because the Pluronic structure is 
significantly different from the other two domain polyethoxylates 
(polyethoxylated alcohols and phenols), Pluronics.RTM. represent a 
separate class of polyethoxylates. Accordingly, this distinction between 
two and three domain polyethoxylates allows the utilization of the HLB 
scale as a predictor of utility for the two domain polyethoxylates as 
potential nonionic surfactants capable of being employed successfully in 
the peroxidase reagent and method of the invention. 
The two domain class of polyethoxylates can be represented as 
hydrophobe-O-(POE).sub.n -OH. The hydrophobe can be either a long-chain, 
branched, or straight alcohol, such as Brij.RTM. 35. Alternatively, the 
other commonly used hydrophobe structures are the octaphenyl (e.g., the 
Triton X.RTM. series) or the nonaphenyl classes (e.g., Triton.RTM. series, 
Macol.RTM. NP series, Surfonic.RTM. NP series, and the like) in which a 
straight or branched chain hydrocarbon is bonded to a phenolic structure, 
which, in turn, is bonded to the POE domain. 
When peroxidase R1 reagent compositions were formulated to contain both SDS 
and polyethoxylated alcohols or phenols having HLB values from about 7.7 
to 8.9, and these compositions were used for the analysis of Day 2 blood 
samples, none of the cytograms exhibited a valley between the lymphocyte 
region and the noise region. Hence, the percent lymphocytes was high in 
all four cases and the percent neutrophils was low in three cases. The 
entire set of such reagents was judged to be unacceptable for use in the 
R1 reagent composition of the peroxidase method. The most likely cause of 
the observed unacceptable performance is that these polyethoxylate 
surfactants were too hydrophobic (i.e., the HLB was too low) for the 
peroxidase method application using an automated hematology analyzer. 
Therefore, it was concluded that for polyethoxylated alcohols or phenols, 
in the presence of an ionic surfactant such as SDS or TDAPS, the required 
HLB value should be between about 9.3 to about 17.3, more preferably about 
9.7 and 16.9. Because Pluronics.RTM. represent a separate class of 
nonionic surfactants and but one member of this class, i.e., Pluronic.RTM. 
P105, was determined not to be useful in the peroxidase reagent and 
method, it will be appreciated by those in the art, nonetheless, that 
other Pluronics.RTM. having different structures or properties might be 
useful due to their different HLB values which provide sufficient 
surfactant lyric properties that are conducive for use in the reagents of 
the invention. 
Accordingly, it was determined from these studies that for two domain 
polyethoxylate nonionic surfactants at 0.12 g/L, in the presence of 0.105 
g/L of SDS in the peroxidase R1 reagent composition, those with HLB values 
between about 5.3 and about 8.9, and greater than about 17.3, were 
unacceptable for use in the method. In contrast, surfactants (i.e., those 
surfactants possessing structures which include the three common types of 
hydrophobes) formulated into the R1 reagent and having HLB values in the 
range of about 9.7 to 16.9 provided acceptable results in the method. 
Pluronics.RTM. comprise a distinct class of polyethoxylate surfactants 
based on their three domain structures. As a consequence of its 
nonhemolytic characteristics, Pluronic.RTM. P105 was not acceptable when 
formulated into the peroxidase R1 reagent composition at 0.12 g/L (in the 
presence of 0.105 g/L of SDS), despite the fact that this Pluronic.RTM. 
has been assigned a wide HLB range of 12-18, which overlaps the useful HLB 
range for the two domain polyethoxylates. 
Example 9 
Analysis of other classes of ionic surfactants for suitability in the Px R1 
reagent composition and peroxidase method of leukocyte differential 
counting 
To determine whether or not other classes of ionic surfactants (e.g., 
cationic or zwitterionic surfactants) were suitable for use in the 
improved reagent composition and method of the invention, additional 
experiments were performed employing such ionic surfactants. These studies 
were designed to test if cationic and zwitterionic surfactants, rather 
than anionic surfactant such as SDS, were suitable for use in combination 
with 0.12 g/L of nonionic surfactant (e.g., Brij.RTM. 35) in the 
peroxidase R1 reagent composition. The experiments were performed within 
the context of an automated hematology analyzer and employed a rinse cycle 
with a Brij.RTM. 35-free rinse. An example of one cationic surfactant 
tested is tetradecyltrimethylammonium bromide or TTAB; and example of a 
zwitterionic surfactant tested is tetradecylammoniopropanesulfonate or 
TDAPS. 
Test R1 reagents which did not contain SDS (e.g., Px 1/No SDS) were 
prepared to contain Brij.RTM. 35 (4.0 mL of a 30 g/L solution in distilled 
water; sorbitol, 113.0 g; NaPhosphate, monobasic, 2.08 g (Mallinkrodt); 
NaPhosphate, dibasic, 11.89 g (Mallinkrodt); NaCl, 0.488 g (Mallinkrodt 
7581KMER); Na.sub.2 EDTA, 0.750 g (Mallinkrodt 4931KMHK); formaldehyde, 
37%, 150 ml (Mallinkrodt). The in-process pH of the assay was 7.23. The 
test reagents were filtered through a 0.2 micron polysulfone membrane 47 
mm disc (Gelman Supor). The ionic surfactants were added to Px 1/no SDS as 
follows: 200 .mu.L of a 30 g/L solution of either TDAPS or TTAB were added 
to 50.0 mL of Px 1/No SDS in a 60 ml polypropylene screw-top tube. A rinse 
reagent containing Pluronic.RTM. P105 was used in the rinse cycle of all 
test analyses. Five normal blood samples per set were collected from 
normal volunteers in Vacutainer.TM. tubes and anticoagulated with K.sub.3 
EDTA. Data were collected on an automated hematology analyzer of the 
TECHNICON H.cndot..TM. series with manual open-tube aspiration and the 
method was carded out as described in Example 2. 
In the standard or control method of leukocyte differential counting as 
described herein, the Px R1 reagent composition contained SDS as the sole 
surfactant and Brij.RTM. 35 was found to be supplied to the R1 phase as a 
result of rinse carryover (i.e., by the rinse which contains both 
Brij.RTM. 35 and SDS, see Example 5). In the test methods conducted in 
accordance with the findings of the present invention, the Px R1 reagent 
composition contained both the anionic surfactant SDS and the nonionic 
surfactant Brij.RTM. 35, and the rinse solution contained neither SDS nor 
Brij.RTM.. 
The effectiveness of the Px method employing a Px 1 reagent composition in 
which the anionic surfactant SDS was replaced either by the cationic, 
quarternary ammonium halide surfactant TrAB at a concentration of 0.12 
g/L, or by the zwitterionic surfactant TDAPS at a concentration of 0.12 
g/L, was assayed. The results obtained with the R1 reagent containing TTAB 
at 0.12 g/L and Brij.RTM. 35 also at 0.12 g/L ("Reagent 1") were 
surprising--no peroxidase data were obtained because of poor lysis of the 
red blood cells which led to a "streak" of red cells on the left side of 
the cytogram. By contrast, the R1 reagent containing TDAPS at a 
concentration of 0.12 g/L and Brij.RTM. 35 at 0.12 g/L provided acceptable 
data for both Day 1 and Day 2 blood samples assayed in the peroxidase 
method (see Table 11). These results demonstrated that zwitterionic 
surfactants such as TDAPS are suitable for use in conjunction with 
nonionic surfactant in the Px R1 reagent composition employed in the 
peroxidase method of the invention. 
TABLE 11 
__________________________________________________________________________ 
Performance of the peroxidase method using a dual-surfactant-containing 
R1 reagent composition 
formulated to contain Brij .RTM. 35 and either a cationic or a 
zwitterionic surfactant 
__________________________________________________________________________ 
Px R1 Surf 
Structure 
Structure 
Surfac- type 
Hydro- 
Hydro- 
Reagent 
tant WBCP 
% N % LYM 
% M % EOS 
% LUC 
% NOIS 
.dagger-dbl. 
phobe phile 
__________________________________________________________________________ 
Day 1 
Std SDS 6.09 
60.2 27.1 7.6 1.9 2.4 19.6 A C.sub.12 H.sub.25 --O 
SO3-- 
sd 0.11 
0.5 0.6 0.2 0.3 0.4 0.8 
1 TTAB and N.D. (non-lysis of red cells) 
N.D. N.D. C, N 
C.sub.14 H.sub.29 
N(CH3)3+ 
Brij .RTM. 35 
2 TDAPS.dagger. 
6.01 
61.4 27.2 *5.8 
2.4 2.6 20.9 Z, N 
C.sub.14 H.sub.19 
and 
Brij .RTM. 35 
sd 0.04 
0.8 0.7 0.5 0.2 0.2 0.7 
acc. spec 0.15 
1 0.5 0.5 0.2 0.5 none 
sd spec 0.16 
1.4 1.1 0.9 0.5 0.5 none 
__________________________________________________________________________ 
Day 2 
Reagent WBCP 
% NEU 
% LYM 
MON % EOS 
% LUC 
% NOIS 
__________________________________________________________________________ 
Std 5.96 
61.5 25.8 8.1 1.5 2.3 17.6 
1 TTAB N.D. 
N.D. N.D. N.D. N.D. 
and 
Brij .RTM. 35 
2 TDAPS.dagger. 
5.81 
62.0 26.6 6.6 *2.0 2.1 17.2 
and 
Brij .RTM. 35 
acc. spec. 
aged 0.32 
2.8 2.2 1.8 1.0 1.0 none 
__________________________________________________________________________ 
*exceeded automated hemotology analyzer specifications. 
.dagger. TDAPS = Ntetradecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate. 
.dagger-dbl. Surf type: surfactant type; A = anionic (e.g., SDS); C = 
cationic (e.g., TTAB); Z = switterionic (e.g., TDAPS); and N = nonionic 
(e.g., Brij .RTM. 35). 
N.D.: No data obtained due to nonlysis of cells. 
CH.sub.3 --(CH.sub.2).sub.13 --N(CH.sub.3).sub.2 --(CH.sub.2).sub.3 
--SO.sub.3 --, hydrophile = --N(CH.sub.3).sub.2 --(CH.sub.2).sub.3 
--SO.sub.3 
TDAPS is zwitterionic with a "+" charge on N, and a "-" charge on O 
TTAB: tetradecyltrimethylammonium bromide; the counterion is Br 
In summary, for the experiments conducted in Examples 7-9, the 
polyethoxlated alcohols and phenols with HLB values between about 7.7 and 
8.9 were unacceptable as substitutes for the nonionic surfactant Brij.RTM. 
35 in the Px R1 reagent composition. Plutonic.RTM. P105, a block copolymer 
(polyoxyethylene-polyoxypropylene-polyoxyethylene) was also unsatisfactory 
(it produced a high 5 Noise in the cytogram) in combination with the ionic 
surfactant SDS in the Px R1 reagent composition. It is noted that cationic 
surfactants, e.g., TTAB, were found to be unacceptable substitutes for SDS 
in the Px R1 reagent composition. However, zwitterionic surfactants, e.g., 
TDAPS, were found to be acceptable and useful substitutes for SDS. 
The contents of all patent applications, issued patents, texts, and 
published articles and references cited herein are hereby incorporated by 
reference in their entirety. 
As various changes can be made in the above compositions and methods 
without departing from the scope and spirit of the invention, it is 
intended that all subject matter contained in the above description, shown 
in the accompanying drawings, or defined in the appended claims will be 
interpreted as illustrative, and not in a limiting sense.