Patents

Hematology reagent and methods

Abstract
US7247484B2
A reagent system is provided for substantially lysing red blood cells in a whole blood sample prior to
United States
leukocyte analysis, which system includes a :rst reagent for substantially lysing the red blood cells
in the whole blood sample, and a second reagent for quenching the activity of the :rst reagent. The
:rst reagent is formulated to include an autoclaved saponin compound and an acid selected from Download PDF Find Prior Art Similar
the group consisting of a halogenated carboxylic acid, a phosphoric acid and a combination thereof.
The second reagent includes a base and has a pH value of about 8 to 12. Also provided is a method Inventor: Jiong Wu, Wayne L. Ryan
of lysing the red blood cells and stabilizing white blood cells present in a sample of whole blood.
Current Assignee : Streck Laboratories Inc

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Worldwide applications

2003 EP CA US

Application US10/699,502 events

2003-10-31 Application :led by Streck Laboratories Inc

2003-10-31 Priority to US10/699,502

2004-07-22 Publication of US20040142318A1

Classi9cations
2007-07-24 Application granted

G01N33/5094 Chemical analysis of biological material, e.g. blood, urine; Testing involving 2007-07-24 Publication of US7247484B2
biospeci:c ligand binding methods; Immunological testing involving human or animal cells for
Status Expired - Fee Related
blood cell populations
2025-01-09 Adjusted expiration
View 3 more classi.cations
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Info: Patent citations (29), Non-patent citations (12), Cited by

(20), Legal events, Similar documents, Priority and Related
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External links: USPTO, USPTO PatentCenter, USPTO

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Description Claims (7) Hide Dependent

This is a non-provisional of U.S. provisional application No. 60/423,060, :led Nov. 1. A reagent system for substantially lysing red blood cells in a whole blood
1, 2002 the contents of which are incorporated herein by reference. sample prior to leukocyte analysis, the reagent system comprising:

BACKGROUND
Analysis of leukocyte populations from whole blood samples is an important
diagnostic procedure. The ability to analyze the major subpopulations of
leukocytes in an automated manner has proved to be an effective tool for a rapid
diagnosis of one or more blood samples.
For a number of automated hematology analyzers, a blood sample is typically split
into a :rst portion that is subjected to techniques for analysis of red blood cells. A
second portion is also obtained from the sample, which is employed for white
blood cell analysis. It is important during analysis of the second portion that red
blood cells be substantially completely lysed so that they do not interfere with
white blood cell analysis. Thus far, several lysis reagents have been developed for
use in whole blood samples. The claimed subject matter constitutes an
improvement relative to existing reagents.
(a) a :rst reagent for substantially lysing the red blood cells in the whole
blood sample, wherein the :rst reagent includes a saponin compound, an
acid selected from the group consisting of a halogenated carboxylic acid, a
phosphoric acid and a combination thereof, and an additional surfactant
selected from the group consisting of a non-ionic surfactant, a cationic
surfactant and a combination thereof, wherein the :rst reagent is
autoclaved at about 121° C.; and
(b) a second reagent for quenching the activity of the :rst reagent, wherein
the second reagent includes a base and has a pH value of about 8 to 12.
2. The reagent system of claim 1, wherein the non-ionic surfactant is
selected from the group consisting of ethoxylated decylalcohols, an
ethoxylated and propoxylated linear (C8-C10) aliphatic alcohols, and a
combination thereof.

SUMMARY
The subject matter of the present disclosure is generally directed to a reagent
system for substantially lysing red blood cells in a whole blood sample prior to
leukocyte analysis. In one illustrative embodiment, the reagent system includes a
:rst reagent for substantially lysing the red blood cells in the whole blood sample,
and a second reagent for quenching the activity of the :rst reagent, wherein the
second reagent includes a base and has a pH value of about 8 to 12. A :nal acidic
medium, ranging from about pH 3 to about 6, preferably from about 4 to about 5, is
used to stabilize the white blood cells and continuously remove red blood cell
fragments. The :rst reagent is formulated to include: a saponin compound; an acid,
preferably selected from halogenated carboxylic acids, phosphoric acid or
combinations of these and similar compounds that should be known to one of skill
in the art. Optionally the :rst reagent may further include a surfactant preferably
selected from non-ionic surfactants, cationic surfactants and combinations of
these and similar compounds that should be known to one of skill in the art. In one
speci:c and illustrative embodiment, the surfactant is selected from ethoxylated
decylalcohols, ethoxylated and propoxylated linear (C8-C10) aliphatic alcohols, and
3. A method of lysing red blood cells and stabilizing white blood cells present in
a whole blood sample, the method comprising the steps of:
(a) combining a predetermined portion of the whole blood sample with a
predetermined portion of a :rst reagent to substantially lysing the red blood
cells in the whole blood sample, wherein the :rst reagent includes a saponin
compound, an acid and an additional surfactant selected from the group
consisting of a non-ionic surfactant, a cationic surfactant and a
combination thereof, wherein the :rst reagent is autoclaved at about 121°
C.; and
(b) quenching the lysing action of said :rst reagent by the addition of a
predetermined portion of a second reagent to result in a solution containing
leukocytes and substantially lysed red blood cells and having a pH value of
about 3 to about 6, wherein the second reagent includes a base and has a
pH value of about 8 to about 12.
4. The method of claim 3, wherein the acid is selected from the group

combinations of these and similar compounds that should be known to one of skill consisting of halogenated carboxylic acids, a phosphoric acid and a

in the art. It should be appreciated that the saponin compound is preferably combinations thereof.

selected from the group including saponin; heat-treated saponin, saponin modi:ed
5. The method of claim 3, wherein the non-ionic surfactant is selected
by heating in the presence of a halogenated carboxylic acid and combinations of
from the group consisting of an ethoxylated decylalcohols, an
these and similar compounds that should be known to one of skill in the art.
ethoxylated and propoxylated linear (C8-C10) aliphatic alcohols, and
Another illustrative embodiment of the claimed subject matter includes a reagent combinations thereof.
system formulated to include: a reagent for lysing red blood cells; and a quench;
6. A method of preparing a whole blood sample for leukacyte analysis,
such that the system is substantially free of compounds including: i. dye; ii. a
comprising the steps of:
combination of saponin and carboxylic acid; iii. an acid selected from formic acid,
acetic acid and mixtures thereof; iv. a combination of saponin and sulphonic acid; (a) substantially lysing red blood cells in at least a portion of the whole
v. a cross-linking agent such as an aldehyde; vi. an alkali metal salt of an alkyl blood sample by adding a saponin compound, an acid and an additional
sulfate anionic surfactant; vii. an ethoxylated long chain amine; and viii. surfactant selected from the group consisting of a non-ionic surfactant, a
combinations thereof. A :nal acidic medium, ranging from about pH 3 to about 6, cationic surfactant and a combination thereof, to the sample to form a
preferably from about 4 to about 5, is used to stabilize the white blood cells and mixture, wherein said saponin compound, acid and additional surfactant are
continuously remove red blood cell fragments. Preferably the illustrative reagent autoclaved together at about 121° C.; and
for lysing red blood cells includes a saponin compound and an acid. The saponin
compound can be selected from saponin; heat-treated saponin, saponin modi:ed (b) substantially quenching the mixture by bringing the pH value of the
by heating in the presence of a halogenated carboxylic acid and combinations of mixture to about 3 to about 6.
these and similar compounds that should be known to one of skill in the art. The
7. The method of claim 6, wherein the pH value of the mixture is from
acid portion of the reagent system is selected from halogenated carboxylic acids,
about 4 to about 5.
phosphoric acid or combinations of these and similar compounds that should be
known to one of skill in the art. The reagent for lysing red blood cells may further
include a surfactant. The surfactant can be selected from non-ionic surfactants,
cationic surfactants and combinations thereof and preferably the surfactant is
selected from ethoxylated decylalcohols, ethoxylated and propoxylated linear (C8-
C10) aliphatic alcohols, and combinations of these and similar such compounds.

It will also be appreciated by one or ordinary skill in the art that an illustrative
embodiment of the claimed subject matter includes a method of lysing the red
blood cells present in a sample of whole blood. In one such illustrative
embodiment, the method includes: combining a predetermined portion of the
sample of whole blood with a predetermined portion of a :rst reagent for
substantially lysing the red blood cells in the whole blood sample, wherein the :rst
reagent includes: a saponin compound; and an acid; and quenching the lysing
action of said :rst reagent by the addition of a predetermined portion of a second
reagent, wherein the second reagent includes a base and has a pH value of about 8
to about 12 to give a solution containing substantially lysed red blood cells,
leukocytes and a pH value of about 3 to about 6. It is preferred in one illustrative
embodiment that the saponin compound is selected from saponin; heat-treated
saponin, saponin modi:ed by heating in the presence of a halogenated carboxylic
acid and combinations of these and similar such compounds. Another preferred
and illustrative embodiment is formulated such that the acid is selected from
halogenated carboxylic acids, phosphoric acid or combinations of these and
similar such compounds. Further it should be noted that the reagent for lysing red
blood cells may further be formulated to include a surfactant, preferably selected
from non-ionic surfactants, cationic surfactants and combinations of these and
similar such compounds. More preferably, the surfactant is selected from
ethoxylated decylalcohols; ethoxylated and propoxylated linear (C8-C10) aliphatic
alcohols and combinations of these and similar such compounds.

These and other features of the claimed subject matter are more fully set forth in
the following description of preferred or illustrative embodiments.

BRIEF DESCRIPTION OF THE DRAWING

Features of the present invention as well as a preferred mode of use, further

objectives, and advantages thereof, will best be understood by reference to the
following detailed description of an illustrative embodiment when read in
conjunction with the accompanying drawing, wherein:

FIG. 1 illustrates comparison of lytic stability of the red blood cell lyse solutions at
40° C. in accordance with the present invention.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Red Blood Cell Lyse Agent

The red blood cell lyse agent of the claimed subject matter preferably includes :rst
component for lysing red blood cell components of a blood sample, a second
component that is an acid, and an optional third component that functions as a
surfactant. The combination of reagents is designed to achieve a :nal acidic
medium, ranging from about pH 3 to about 6, preferably from about 4 to about 5, is
used to stabilize the white blood cells and continuously remove red blood cell
fragments.

An example of a suitable :rst component is preferably saponin, although other art

disclosed agents including modi:ed saponin or saponin derivatives or other
saponin like compounds may also be employed. As the term is used herein, the
term “a saponin compound” or just “saponin” is de:ned as including such modi:ed
saponin or saponin derivatives that retain the functionality of saponin as well as
saponin itself. Modi:ed saponin derivatives are synthesized by heating at 121° C.
in solution containing chloroacetic acid and surfactant. Such saponin derivatives
are signi:cantly different from the original saponin. The modi:cation allows (1) a
much broader range of saponin derivative concentration, ranging from
0.020%-0.035%, which can be used in the red blood cell lyse; and (2) a signi:cantly
longer stability for the reagents.

The acid of the second component may be any suitable acid. For example, it may
be phosphoric acid, sulfuric acid, hydrochloric acid or other acid of like
characteristics for the present intended environment. The acid may be an organic
acid, an inorganic acid or a combination thereof. In one embodiment, it is a
halogenated organic acid. More preferably it is a halogenated carboxylic acid, such
as monochloroacetic acid. A preferred acid is one that is substantially free of
formic acid, acetic acid or respective mixtures thereof. In one embodiment, it is
contemplated that the third component is omitted altogether, with saponin or
saponin derivative acting instead also as a surfactant.

The surfactant may be one or more of a non-ionic, cationic, anionic, or amphoteric

surfactant. In a highly preferred embodiment it is a non-ionic surfactant or a
cationic surfactant. In one embodiment, it is preferable to employ a surfactant that
is substantially free of polyoxyethylene groups. For example, one preferred class of
surfactants includes halogen-capped (e.g., chlorine capped) surfactants, linear
alcohol based surfactants, alkoxylated (e.g., ethoxylated or propoxylated) alcohol
surfactants. Examples of suitable commercially available surfactants include,
without limitation, Rhodasurf DA630, Rhodasurf 10060, Rhodasurf 8 D 75, Antarox
BL 240 or the like. Preferably the surfactant is generally stable in either an acid or
an alkali medium. Rhodasurf DA 630 and other possible choices of surfactants are
used to effectively remove the RBC fragments during the procedure of lysis.

The amounts of the respective components are sujcient for yielding a red blood
cell lyse agent that is hypotonic. More preferably, the resulting lyse agent will have
an osmolality of less than about 50 mOsm, and more preferably less than about 35
mOsm. For example, a preferred range is about 10 to about 30 mOsm. The pH of
the red blood cell lyse agent preferably is about 2 to about 4, and more preferably
about 2.2 to about 3.2.

Thus, one preferred composition employs an acid in an amount of about 0.001%

weight per volume to about 1% weight per volume, and more preferably about 0.05
to about 0.5% weight per volume. Saponin or saponin derivative is employed in an
amount of about 0.005 to about 0.1% weight per volume, and more preferably
about 0.01 to about 0.04% weight per volume. Any surfactant employed is present
in an amount of about 0.005 to about 0.07% weight per volume, and more
preferably about 0.01 to about 0.05% weight per volume.

The red blood cell lyse is preferably contacted with a blood sample prior to analysis
by an automated instrument of one or more white blood cell populations (e.g., by
light scatter, impedance or other art-disclosed techniques). The sample is
contacted for a sujcient time so that red blood cells are lysed and will not
interfere with white blood cell analysis.

Quench

It is seen from the above that promptly after contacting the blood sample with the
red blood cell lyse, it is desirable to quench a substantial portion of the remaining
white blood cell fraction of the blood. This is accomplished by raising the
osmolality to isotonicity and bringing the pH to a value of approximately 4.5. The
quench is used to achieve a :nal acidic medium, ranging from about pH 3 to about
6, preferably from about 4 to about 5, which is designed to stabilize the white blood
cells and continuously remove red blood cell fragments.

A preferred quench composition will include a :rst component, which is generally

basic, and at least one other component that includes a salt (or another buffer
material, such as those disclosed elsewhere herein). For example, one preferred
embodiment contemplates the use of a metal sulfate, such as an alkali metal
sulfate (e.g., sodium sulfate) in an amount of about 0.5 to about 10% weight, more
preferably about 1 to about 7% weight, and still more preferably about 2 to about
4% weight. An alkali metal salt, halide salt, or a mixture thereof, is also preferred as
comprising the salt component, such as sodium chloride. The salt is present in an
amount of about 0.1 to about 10% weight, more preferably about 0.5 to about 7%
weight, and still more preferably about 1 to about 4% weight. A metal carbonate
may also be employed, such as sodium carbonate, e.g., in an amount ranging from
about 0.01 to about 1% weight.

The amounts of the respective components are sujcient for yielding a quench that
is generally hypertonic, though its use following a red blood cell lyse will generally
yield the blood sample subjected to a generally isotonic environment. More
preferably, the resulting quench will have an osmolality of greater than about 750
mOsm. For example, a preferred range is about 900 to about 1100 mOsm. The pH
of the quench preferably is about 8 to about 12, and more preferably about 9 to
about 11.

The amount of the quench utilized should result in a :nal solution having a pH
value of about 3.0 to about 6.0 and preferably from about 4 to about 5. That is to
say a slightly acid solution is produced to stabilize the leukocytes and preserve the
differentiation of leukocytes.

The quench is preferably :ltered in a suitable manner, such as by using a :lter

membrane (e.g., a cellulose nitrate membrane, such as a Nalgene 0.2 CN :lter) or
the like.

Diluent

The claimed subject matter also contemplates the employment of a diluent,

preferably after the sample has been quenched. The diluent thus functions as a
sheath agent for carrying the sample through a low cell of an instrument. A
preferred diluent is generally isotonic and has a pH of between about 6.5 and 7.
The diluent preferably is water or saline based composition and may include one or
a combination of a buffer, a chelating agent, a stabilizer or an anti-microbial.
Examples of suitable ingredients for the diluent include, without limitation, one or a
combination of ammonium sulfate, boric acid, EDTA, EDTA disodium, glycine,
potassium phosphate, sodium bicarbonate, sodium carbonate, sodium chloride,
sodium citrate, sodium phosphate, tris hydrochloride, omadine, procaine or the like.

The red blood cell lyse agent of the claimed subject matter is contacted with red
blood cells of a blood sample for lysing the same. The quench is thereafter (e.g.,
within about 30, and more preferably about 5-10 seconds of red blood cell lysing)
contacted with the blood sample. Following the quench, the white blood cells of
the sample are analyzed by an instrument (e.g., an instrument for differentiating
the subpopulations of white blood cells, a low cytometer, or other like instrument).

One illustrative composition includes a combination of two or more of the

following ingredients in a water base:

Sodium sulfate about 0.1 to about 3% weight

Sodium chloride about 0.1 to about 1% weight
Sodium phosphate dibasic about 0.1 to about 3% weight
Sodium phosphate monobasic about 0.01 to about 0.2% weight
Preservative (e.g., EDTA disodium) about 0.001 to about 0.5%
weight
Disinfectant (e.g. Sodium omadine) about 0.05 to about 1 mL/L.
Procaine about 0.001% to about 1% weight

Various other possible art-disclosed ingredients may be employed in the reagents

herein including for example those disclosed in U.S. Pat. Nos. 5,731,206; 5,928,949;
4,751,179; 5,155,044; 5,786,224; 5,817,518; 5,686,308, or the like, all of which are
hereby incorporated by reference.

It will be appreciated that the claimed subject matter also can be employed in the
analysis of blood controls, such as those found in one or more of U.S. Pat. Nos.
6,200,500; 6,221,668; 5,731,205; 5,529,933; 6,362,003; 5,994,139; 5,858,790;
6,444,471, or the like, all of which are hereby incorporated by reference. The
claimed subject matter thus also contemplates the packaging of the reagents of
the claimed subject matter along with the controls disclosed in the above patents
or as part of a kit as disclosed in the above patents. The claimed subject matter
additionally or alternatively may be packaged as part of a kit in combination with
other reagents, such as a lysing agent (e.g., a cyanide-free lysing agent). The
compositions of the claimed subject matter can also be employed as part of a
calibration procedure for an automated instrument.

In another preferred embodiment of the claimed subject matter the red blood cell
lyse, the quench, another reagent or a combination thereof can further include a
lipoprotein, and more preferably a high density lipoprotein (e.g., SUPERTRATE™,
from Bayer) that is brought into contact with the white blood cells of the blood
sample.

In the use of the claimed subject matter, it is contemplated in one aspect that a
blood sample is provided to a laboratory in a suitable container, and preferably one
that includes an anti-coagulant and optionally one or more agents for preserving
surface antigens on the surfaces of blood cells in the sample. An example of one
suitable system for such application is disclosed in commonly owned U.S. patent
application Ser. No. 60/418,978, :led Oct. 16, 2002, hereby incorporated by
reference (entitled: “METHOD AND DEVICE FOR COLLECTING AND PRESERVING
CELLS FOR ANALYSIS”).

In accordance with one highly preferred aspect of that technology, there is

contemplated a method of making a collection device for cells comprising:
providing a tube having an open end and a closed end; preloading compounds
including: i) an anticoagulant agent, and ii) a :xative agent consisting of
diazolidinyl urea into said tube, wherein said compounds are in a sujcient amount
to preserve said cells' original morphology and antigenic sites without signi:cant
dilution of said cells, and thereby allowing said cells to be directly analyzed by an
instrument such as a low cytometer without further treatment; inserting a closure
into said open end of said tube; and drawing a vacuum inside said tube to a
predetermined level to form said collection device.

It will be appreciated from the above that use of the reagents of the claimed
subject matter may be done in an environment that is substantially free of one or
more of the following: 1) dye (e.g., a luorochrome dye) 2) a combination of
saponin or saponin derivative and a nonhalogenated carboxylic acid; 3) an acid
selected from formic acid, acetic acid and mixtures thereof; 4) a combination of
saponin or saponin derivative and sulphonic acid; 5) a cross-linking agent such as
an aldehyde; 6) an alkali metal salt of an alkyl sulfate anionic surfactant (e.g., as
de:ned in U.S. Pat. No. 5,786,224); 7) an ethoxylated long chain amine (e.g., as
de:ned in U.S. Pat. No. 5,686,308); or 8) combinations of these compounds

The following examples are included as demonstrative preferred embodiments. It

should be appreciated by those of skill in the art that the techniques disclosed in
the examples which follow represent techniques discovered by the inventors to
function well in the practice of what is claimed, and thus can be considered to
constitute preferred modes of practice. However, those of skill in the art should, in
light of the present disclosure, appreciate that many changes can be made in the
speci:c embodiments which are disclosed and still obtain a like or similar result
without departing from the scope of what is claimed.

The following Examples 1-3, when employing an isotonic diluent, employ the
diluent substantially described in Example 4.

EXAMPLE 1

An illustrative red blood cell lyse solution was formulated as follows:

Chloroacetic acid   0.1% weight

Saponin 0.0225% weight
Rhodasurf DA 630  0.025% weight/volume
Water or isotonic diluent balance

The composition exhibits a pH of about 2.5 and an osmolality of about 15 mOsm.

It should be noted that Rhodasurf (formerly Emulphogene) DA 630: is an

ethoxylated decylalcohol, CAS 26183-52-8; non-ionic; liquid 100% conc.; HLB=12.5;
cloud point=42° C. (1% aq.); acid and alkali stable. It is described as being a low-
foaming rapid wetting agent; dispersant for industrial, institutional and household
cleaners, textile scouring; intermediate for manufacturing of esters for textile and
industrial applications; emulsi:er for deformers. Similar products include
Rhodasurf ID 060 (HLB=12) and Rhodasurf 8 D 75 (HLB=14).

EXAMPLE 2

An illustrative red blood cell lyse solution was formulated as follows:

Chloroacetic acid   0.1% weight

Saponin 0.0225% weight
Antarox BL 240  0.025% weight/volume
Water or isotonic diluent balance

The composition exhibits a pH of about 2.5 and an osmolality of about 15 mOsm.

It should be noted that Antarox BL 240: is described as an ethoxylated and

propoxylated linear (C8-C10) aliphatic alcohols, CAS 68603-25-8; non-ionic; liquid
100% act.; bland odor; water sol.; sp. gr.=0.99; visc.=35 cps; surf tension=28
dyns/cm (0.1%); cloud point=38-42° C. (1% aq.); lash point=124° C.; pour point
<21° C. biodegradable, most water-soluble member of BL-200 series. Recognized
uses include low-foaming detergent; wetting agent for metal cleaning, (high-
temperature) rinse aids, textiles, loor cleaners.

EXAMPLE 3

An illustrative quench solution was formulated as follows:

Sodium sulfate 3.13% weight

Sodium chloride 1.85% weight
Sodium carbonate 0.15% weight
Water or isotonic diluent balance

The composition is :ltered by a Nalgene 0.2 CN :lter. The composition exhibits a

pH of about 10.5 and an osmolality of about 1050 mOsm.

EXAMPLE 4

An illustrative isotonic diluent solution was formulated as follows:

Sodium sulfate  0.822% weight

Sodium chloride  0.484% weight
Sodium phosphate dibasic  0.103% weight
Sodium phosphate monobasic  0.059% weight
EDTA disodium  0.010% weight
Sodium omadine   0.25 mL/L
Procaine 0.0122% weight

The composition is :ltered a 0.45 μm capsule :lter. The composition exhibits a pH

of about 6.9 and an osmolality of about 315 mOsm.

The above reagents can be used in a STK-S, a GEN-S, a MAXM, and a LH 750
analyzer all commercially available and/or sold by BeckmanCoulter and yields
reliable and reproducible blood analysis data. The reagents of Examples 1 and 2
are contacted with red blood cells of a blood sample for lysing the same. The
reagent of Example 3 is thereafter (e.g., within about 30, and more preferably about
5-10 seconds of red blood cell lysing) contacted with the blood sample. Following
the quench, the instrument analyzes the white blood cells of the sample by passing
the cells through a low cell of the instrument (e.g., an instrument for differentiating
the subpopulations of white blood cells, a low cytometer, or other like instrument)
carried in a suitable diluent, such as that of Example 4. Successful results can also
be achieved when the chloroacetic acid of Examples 1 and 2 is substituted with a
phosphoric acid in an amount of about 0.05% weight.

EXAMPLE 5

Modi:ed Saponin can be used in the formulation of the lyse solutions substantially
described above. An early study showed that the red blood cell lyse using original
(0.0225%, un-heated) saponin failed to perform on Beckman Coulter hematology
analyzers after 8 months at room temperature. Another study showed that the red
blood cell lyse lost approximately 45% of the saponin activity after 8 months.

The purpose of this study is to explore the possibility to improve the saponin
stability by a heating procedure. It is known that such intensive heating procedure
reduces the saponin activity, possibly by removing the unstable lytic components
from saponin. The remaining lytic components in saponin may have a longer
stability in solution.

Stock solution (heated) contained:

Chloroacetic acid:  0.1% weight

Saponin 5.000% weight
DA-630 0.025% weight

Base solution (not heated) contained:

Chloroacetic acid:  0.1% weight

Saponin 5.000% weight
DA-630 0.025% weight

The stock solution is autoclaved (Cycle 1, 121° C. for 30 minutes) and double
:ltered using Nalgene 0.8 μm and 0.2 μm :lters. The red blood cell lyse is prepared
by mixing the stock solution and the base solution at a calculated ratio to achieve
the desired :nal formulation.

Modi:ed Saponin Derivatives: The procedure of heating results in a derivative of

saponin. The product, as well as the original saponin, were analyzed by HPLC.

A review of the results indicates that the most distinctive change due to the
modi:cation was the generation of an additional peak (eluted at 10 minute).
Further study showed that the additional peak did not have any lytic capability,
while the :rst three peaks retained the lytic function. This result suggests that the
heating procedure removed some unstable components of saponin. Without the
heating procedure, such unstable components may degrade slowly at room
temperature.

It has been found that a broader range of saponin derivative can be applied to the
red blood cell lyse solution formulation.

When the red blood cell lyse solution is outside of a very narrow concentration
range of saponin (i.e. 0.0200%-0.0225%), the red blood cell lyse does not perform
properly. This problem was especially true if too much or too little saponin was
used in the formulation. For example the following table is illustrative of the data
gathered.

Concentration of Saponin
0.0175% 0.0200% 0.0225% 0.0250%

STK-S Bad Good Good Bad

GEN•S Bad Marginal Good Marginal

The modi:cation of saponin by heating at 121° C. has been found to allow a much
broader concentration range (i.e. 0.0200%-0.0350%) of the saponin derivative in the
red blood cell lyse solution. Such a broad range for the concentration of the
saponin component brings a high lexibility to the red blood cell lyse, and
subsequently, a much longer stability. Data illustrative of this result is provided in
the following table:

Concentration of Saponin
0.020% 0.025% 0.030% 0.035% 0.040% 0.050%

STK-S Good N/A N/A Good N/A N/A

GEN•S Good Good Good Good Marginal Bad

The relative lytic strength of the red blood cell solution substantially disclosed
herein was compared to the prior art reagents commercially available from Coulter.
FIG. 1, provides an illustrative comparison of the 40-degree C. stability of the red
blood cell lyse solutions. The value of ΔNE (% NE using the reagents disclosed
herein—% NE using prior art reagents available from Coulter) for STaK Chex Low
was used to evaluate the possible loss of lytic strength.

Upon review of this data one of skill in the art should appreciate that no apparent
loss of lytic strength was revealed for the autoclaved saponin derivatives for 47
days at 40° C. In contrast a measurable amount of loss of lytic strength was
observed for the un-autoclaved saponin.

In view of the above disclosure, one of ordinary skill in the art should understand
and appreciate that one illustrative embodiment of the claimed subject matter
includes a reagent system for substantially lysing red blood cells in a whole blood
sample prior to leukocyte analysis. In one such illustrative embodiment, the
reagent system includes a :rst reagent for substantially lysing the red blood cells
in the whole blood sample, and a second reagent for quenching the activity of the
:rst reagent, wherein the second reagent includes a base and has a pH value of
about 8 to 12. A :nal acidic medium, ranging from about pH 3 to about 6, preferably
from about 4 to about 5, is used to stabilize the white blood cells and continuously
remove red blood cell fragments. The :rst reagent is formulated to include: a
saponin compound; an acid, preferably selected from halogenated carboxylic acids,
phosphoric acid or combinations of these and similar compounds that should be
known to one of skill in the art. Optionally the :rst reagent may further include a
surfactant preferably selected from non-ionic surfactants, cationic surfactants and
combinations of these and similar compounds that should be known to one of skill
in the art. In one speci:c and illustrative embodiment, the surfactant is selected
from ethoxylated decylalcohols, ethoxylated and propoxylated linear (C8-C10)
aliphatic alcohols, and combinations of these and similar compounds that should
be known to one of skill in the art. It should be appreciated that the saponin
compound is preferably selected from the group including saponin; heat-treated
saponin, saponin modi:ed by heating in the presence of a halogenated carboxylic
acid and combinations of these and similar compounds that should be known to
one of skill in the art.

Another illustrative embodiment of the claimed subject matter includes A reagent

system formulated to include: a reagent for lysing red blood cells; and a quench;
such that the system is substantially free of compounds including: i. dye; ii. a
combination of saponin and carboxylic acid; iii. an acid selected from formic acid,
acetic acid and mixtures thereof; iv. a combination of saponin and sulphonic acid;
v. a cross-linking agent such as an aldehyde; vi. an alkali metal salt of an alkyl
sulfate anionic surfactant; vii. an ethoxylated long chain amine; and combinations
thereof. Preferably the illustrative reagent for lysing red blood cells includes a
saponin compound and an acid. The saponin compound can be selected from
saponin; heat-treated saponin, saponin modi:ed by heating in the presence of a
halogenated carboxylic acid and combinations of these and similar compounds
that should be known to one of skill in the art. The acid portion of the reagent
system is selected from halogenated carboxylic acids, phosphoric acid or
combinations of these and similar compounds that should be known to one of skill
in the art. The reagent for lysing red blood cells may further includes a surfactant.
The surfactant can be selected from non-ionic surfactants, cationic surfactants
and combinations thereof and preferably the surfactant is selected from
ethoxylated decylalcohols, ethoxylated and propoxylated linear (C8-C10) aliphatic
alcohols, and combinations of these and similar such compounds.

It will also be appreciated by one or ordinary skill in the art that a present
illustrative embodiment of the claimed subject matter includes a method of lysing
the red blood cells and stabilizing white blood cells present in a sample of whole
blood. In one such illustrative embodiment, the method includes: combining a
predetermined portion of the sample of whole blood with a predetermined portion
of a :rst reagent for substantially lysing the red blood cells in the whole blood
sample, wherein the :rst reagent includes: a saponin compound; and an acid; and
quenching the lysing action of said :rst reagent by the addition of a predetermined
portion of a second reagent, wherein the second reagent includes a base and has a
pH value of about 8 to about 12 to give a solution containing substantially lysed red
blood cells, leukocytes and a pH value of about 3 to about 6. It is preferred in one
illustrative embodiment that the saponin compound is selected from saponin; heat-
treated saponin, saponin modi:ed by heating in the presence of a halogenated
carboxylic acid and combinations of these and similar such compounds. Another
preferred and illustrative embodiment is formulated such that the acid is selected
from halogenated carboxylic acids, phosphoric acid or combinations of these and
similar such compounds. Further it should be noted that the reagent for lysing red
blood cells may further be formulated to include a surfactant, preferably selected
from non-ionic surfactants, cationic surfactants and combinations of these and
similar such compounds. More preferably, the surfactant is selected from
ethoxylated decylalcohols, ethoxylated and propoxylated linear (C8-C10) aliphatic
alcohols, and combinations of these and similar such compounds.

While the apparatus, compositions and methods disclosed above have been
described in terms of preferred or illustrative embodiments, it will be apparent to
those of skill in the art that variations may be applied to the process described
herein without departing from the concept and scope of the claimed subject
matter. All such similar substitutes and modi:cations apparent to those skilled in
the art are deemed to be within the scope and concept of the subject matter as it is
set out in the following claims.

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Priority And Related Applications

Priority Applications (1)

Application Priority date Filing date Title

US10/699,502 2002-11-01 2003-10-31 Hematology reagent and methods

Applications Claiming Priority (2)

Application Filing date Title

US42306002P 2002-11-01

US10/699,502 2003-10-31 Hematology reagent and methods

Legal Events

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2004-04-01 AS Assignment Owner name: STRECK LABORATORIES INC., NEBRASKA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WU, JIONG;RYAN, WAYNE

L.;REEL/FRAME:015175/0720

Effective date: 20040329

2007-06-14 AS Assignment Owner name: STRECK, INC., NEBRASKA

Free format text: CHANGE OF NAME;ASSIGNOR:STRECK LABORATORIES,

INC.;REEL/FRAME:019453/0050

Effective date: 20040329

2007-10-02 CC Certi:cate of correction

2011-02-28 REMI Maintenance fee reminder mailed

2011-07-24 LAPS Lapse for failure to pay maintenance fees

2011-08-22 STCH Information on status: patent discontinuation Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR
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Concepts

machine-extracted Download Filter table

Name Image Sections Count Query match

chemical reaction reagent title,claims,abstract,description 150 0.000

quillaja saponaria molina bark claims,abstract,description 172 0.000

mixture claims,abstract,description 146 0.000

RBC claims,abstract,description 128 0.000

Erythrocytes claims,abstract,description 126 0.000

Blood claims,abstract,description 72 0.000

blood claims,abstract,description 72 0.000

lysing claims,abstract,description 72 0.000

saponin compound claims,abstract,description 68 0.000

Leukocytes claims,abstract,description 58 0.000

acid claims,abstract,description 58 0.000

quenching claims,abstract,description 48 0.000

phosphoric acid claims,abstract,description 44 0.000

immune cell claims,abstract,description 36 0.000

analytical method claims,abstract,description 28 0.000

carboxylic acid derivatives claims,abstract,description 24 0.000

aluminium phosphate claims,abstract,description 22 0.000

quenching claims,abstract,description 16 0.000

effects claims,abstract,description 12 0.000

stabilizing claims,abstract,description 6 0.000

surface-active agent claims,description 62 0.000

nonionic surfactant claims,description 26 0.000

cationic surfactant claims,description 22 0.000

1-Decanol claims,description 18 0.000

carboxylic acids claims,description 14 0.000

Show all concepts from the description section

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