U.S. patent application number 09/934463 was filed with the patent office on 2002-02-21 for hematology control and system for multi-parameter hematology measurements.
This patent application is currently assigned to Streck Laboratories, Inc.. Invention is credited to Ryan, Wayne L., Scholl, John W..
Application Number | 20020022271 09/934463 |
Document ID | / |
Family ID | 27008293 |
Filed Date | 2002-02-21 |
United States Patent
Application |
20020022271 |
Kind Code |
A1 |
Ryan, Wayne L. ; et
al. |
February 21, 2002 |
Hematology control and system for multi-parameter hematology
measurements
Abstract
Hematology control compositions and systems used to measure a
plurality of parameters in a blood sample are provided. The
hematology control compositions are particularly useful as a
control for multi-parameter, automated instrument systems. The
control compositions comprise a reticulocyte component, a white
blood cell component, a red blood cell component, a nucleated red
blood cell component, a platelet component and a reticulated
platelet component. Methods of making and using the control
compositions are also provided.
Inventors: |
Ryan, Wayne L.; (Omaha,
NE) ; Scholl, John W.; (Omaha, NE) |
Correspondence
Address: |
Eric M. Dobrusin
Dobrusin & Thennisch PC
Suite 311
401 South Old Woodward Avenue
Birmingham
MI
48009
US
|
Assignee: |
Streck Laboratories, Inc.
|
Family ID: |
27008293 |
Appl. No.: |
09/934463 |
Filed: |
August 21, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09934463 |
Aug 21, 2001 |
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09740509 |
Dec 19, 2000 |
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09740509 |
Dec 19, 2000 |
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09504816 |
Feb 16, 2000 |
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6221668 |
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09504816 |
Feb 16, 2000 |
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09378608 |
Aug 20, 1999 |
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6200500 |
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Current U.S.
Class: |
436/16 ;
422/50 |
Current CPC
Class: |
Y10T 436/107497
20150115; G01N 33/5094 20130101; Y10T 436/105831 20150115; Y10T
436/10 20150115; Y10T 436/2525 20150115; Y10S 436/807 20130101;
G01N 33/96 20130101; Y10T 436/108331 20150115; G01N 2015/1018
20130101; Y10T 436/101666 20150115; Y10T 436/106664 20150115; G01N
2001/2893 20130101 |
Class at
Publication: |
436/16 ;
422/50 |
International
Class: |
G01N 001/00 |
Claims
We claim:
1. A hematology control composition comprising: a) a stabilized
reticulocyte component; and b) a fixed and stabilized white blood
cell component capable of exhibiting a five-part differential.
2. The control composition of claim 1, wherein the reticulocyte
component comprises reticulocytes or an analog thereof
3. The control composition of claim 2, wherein the reticulocyte
component comprises reticulocytes prepared by human red blood cell
encapsulation
4. The control composition of claim 2, wherein the reticulocyte
component comprises reticulocytes prepared by isolation from whole
blood.
5. The control composition of claim 1, wherein the white blood cell
component comprises a member selected from the group consisting of
white blood cells for cellular types, white blood cells for all
phenotypes and mixtures thereof.
6. The control composition of claim 5, wherein the white blood cell
component comprises a member selected from the group consisting of
white blood cells that are stabilized with glutaraldehyde; with
glutaraldehyde and formaldehyde; or with 20% NuoSept 145.
7. The control composition of claim 5, wherein the white blood cell
component includes fixed cells exhibiting characteristics of each
of: a) lymphocytes in an amount of about 20 to 45% of said white
blood cell component; b) monocytes in an amount of about 2 to 10%
of said white blood cell component; c) neutrophils in an amount of
about 40 to about 75% of said white blood cell component; d)
eosinophils in an amount of about 1 to 6% of said white blood cell
component; and e) basophils in an amount up to about 1% of said
white blood cell component.
8. The control composition of claim 1, wherein the red blood cell
component comprises human red blood cells.
9. The control composition of claim 1, wherein the nucleated red
blood cell component comprises nucleated avian red blood cells.
10. The control composition of claim 9, wherein the nucleated avian
red blood cells comprise turkey red blood cells.
11. The control composition of claim 1, wherein the platelet
component comprises simulated platelets.
12. The control composition of claim 11, wherein the simulated
platelets comprise goat red blood cells.
13. The control composition of claim 1, wherein the platelet
component comprises human platelets.
14. The control composition of claim 1, wherein the reticulated
platelet component comprises goat red blood cells.
16. A method of analysis of whole blood comprising the steps of: a)
providing a blood control composition including a reticulocyte
component; and a white blood cell component capable of exhibiting
at least a three-part differential; b) providing a multi-parameter
automated hematology instrument; c) providing a sample of patient
blood; d) analyzing said blood control composition in said
instrument for white blood cell and reticulocyte readings; e)
analyzing said sample of patient blood in said instrument for white
blood cell and reticulocyte readings
17. A method of making a hematology control composition for use
with multi-parameter hematology measuring systems, comprising the
step of mixing a reticulocyte component, a white blood cell
component and a lipoprotein in an isotonic suspension medium.
18. The method of claim 17, wherein the reticulocyte component
comprises reticulocytes or an analog thereof
19. The method of claim 17, wherein the white blood cell component
comprises a member selected from the group consisting of white
blood cells for cellular types, white blood cells for all
phenotypes and mixtures thereof.
20. The method of claim 19, wherein the white blood cells for
cellular types comprise: a) lymphocytes; b) monocytes; c)
neutrophils; d) eosinophils; and e) basophils.
21. The method of claim 17, wherein the red blood cell component
comprises human red blood cells.
22. The method of claim 17, wherein the nucleated red blood cell
component comprises nucleated avian red blood cells.
23. The method of claim 22, wherein the nucleated avian red blood
cells comprise turkey red blood cells.
24. The method of claim 17, wherein the platelet component
comprises simulated platelets.
25. The method of claim 17, wherein the simulated platelets
comprise goat red blood cells.
26. The method of claim 25, wherein the reticulated platelet
component comprises goat red blood cells.
27. A system for measuring components of blood, comprising: (a) an
automated test instrument, and (b) a control for use in said test
instrument, said control comprising: 1. an isotonic suspension
medium having an agent therein for assuring a properly positioned
five-part scattergram is obtained of the white blood cell
subpopulations; 2. a component selected from the group consisting
of a reticulocyte component; a white blood cell component; a red
blood cell component; a nucleated red blood cell component; a
platelet component; a reticulated platelet component and mixtures
thereof; and 3. a reticulocyte component.
29. The system of claim 27, further comprising a device for
visually displaying the results of a test using said system.
30. The system of claim 27, further comprising of a bar code
scanner
31. The system of claim 27, wherein said instrument detects cells
using the Coulter principle.
32. The system of claim 27, wherein said instrument is a STKS
instrument.
33. The system of claim 27, wherein said instrument is a Gen-S
instrument.
Description
CLAIM OF BENEFIT OF EARLIER FILING DATE
[0001] The present invention is a continuation-in-part of
commonly-owned, co-pending U.S. patent application Ser. No.
09/378,608, filed Aug. 20, 1999, hereby expressly incorporated by
reference. Applicants hereby claim the benefit of the filing date
of such application for all purposes and to the extent permitted in
accordance with 35 U.S.C. Section 120.
FIELD OF THE INVENTION
[0002] The present invention relates generally to hematology
control compositions and systems and, more particularly, to a
hematology control composition and system used to measure a
plurality of parameters in a sample of blood with a multi-parameter
automated hematology instrument.
BACKGROUND OF THE INVENTION
[0003] Hematology controls for various automated instruments that
measure, for example, red and white blood cell counts and platelet
counts, are known in the art and are described in the following
U.S. Pat. Nos.: 3,558,522; 3,873,467; 4,179,398; 4,219,440;
4,299,726; 4,324,687; 4,358,394; and 4,436,821 (incorporated by
reference herein). Currently, blood analysis requires the use of
one or more of several different instruments and, subsequently,
different blood samples and blood sample preparations to analyze
the various components of blood. Several hematology instruments,
however, now have the capability of measuring various parameters of
blood without requiring separate sample preparation for each
parameter being tested. Such instruments include the Beckman
Coulter STKS or Gen-S Systems, the Abbott Cell-Dyn 4000 Hematology
System, Bayer ADVIA 120, and the Sysmex XE2100 System. These
improved automated instruments can measure one or more of: 1)
reticulocytes, 2) red blood cells, 3) nucleated red blood cells, 4)
platelets, 5) reticulated platelets, 6) white blood cells,
including lymphocytes, monocytes, neutrophils, eosinophils,
basophils, and 7) white blood cells with all phenotypes. It would
thus be desirable to provide a hematology control composition that
could be used as a control in connection with these instruments. 5
In the preferred embodiment, a hematology control composition for
use with a multi-parameter automated hematology instrument includes
a liquid suspension of particulates that has measureable
characteristics like whole blood. The control composition includes
one or more blood cells (i.e. cells handled or treated to simulate
such a component as found in whole blood) or their analogs
(collectively referred to as blood cell components), that may or
may not be fixed, stabilized, or prepared by other treatment prior
to final suspension. In different embodiments, the blood cell
components may be derived from a source that will exhibit the size,
shape or other measurable characteristics of human, animal, or
other whole blood. By way of examples, U.S. Pat. Nos. 4,198,206;
4,436,821; 5,008,021; 5,262,327; 5,270,208; 5,432,089; 5,460,797;
5,672,474; 5,677,145; 5,731,205; 5,811,099 and 5,981,282, which are
each hereby incorporated by reference, each contain examples of
these types of blood cell components. The control has one or more
blood component to resemble corresponding components in whole blood
when measured by the multi-parameter automated hematology
instrument. When so measured, the control composition would assist
in the calibration, operation, and accumulation of quality
assurance data for the multi-parameter automated hematology
instrument.
[0004] Also of potential interest may be U.S. Pat. No. 5,888,790
and "Improved Isolation of Normal Human Reticulocytes via
Exploitation of Chloride-Dependent Potassium Transport," Sorette et
al., Blood, Vol. 80, No. 1 (Jul. 1), 1992: pp. 249-254; hereby
incorporated by reference.
SUMMARY OF THE INVENTION
[0005] A hematology control and system for multi-parameter
hematology measurement is provided. The hematology control provides
values for the various constituents of blood that the
multi-parameter hematology instrument is capable of measuring. The
hematology control composition comprises components for simulating
reticulocyte, white blood cell, red blood cell, nucleated red blood
cell, platelet or reticulated platelet constituents of whole
blood.
[0006] Methods of making and using the hematology control
composition of the present invention are also provided herein.
[0007] The system of the present invention also includes a
hematology instrument, a control, and may further include output or
readout devices. In one embodiment the system includes other
peripheral devices, such as a device for tracking samples and
associating them with particular data, such as a bar-code scanner
system.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0008] The hematology control composition of the present invention
comprises components for simulating one or more of the following
constituents of whole blood: reticulocytes, white blood cells, red
blood cells, nucleated red blood cells, platelets, or reticulated
platelets. In one embodiment the components are suspended in an
isotonic medium, preferably including lipoprotein. The hematology
control composition of the present invention provides values for
various constituents of blood that a hematology instrument, such as
a multi-parameter hematology instrument, is capable of measuring.
Examples of multi-parameter hematology instruments include those
available commercially without limitation, under the designations
Beckman Coulter STKS or Gen-S Systems, the Abbott Cell-Dyn 4000
Hematology System, Bayer ADVIA 120 System, the Sysmex XE2100
System, or the like.
[0009] The present discussion includes multiple approaches to
making a control in accordance with the present invention, it being
recognized that one highly preferred embodiment contemplates a
control having components that simulate the characteristics of
whole blood for purposes of obtaining readouts on a Beckman Coulter
GEN-S instrument for red blood cells, the five populations of white
blood cells, reticulocytes and platelets. Thus, a suspension is
provided that includes a plurality of particles that exhibit
similar light scattering, conductivity, impedance, optical
(including fluorescence), photometric, or other property responses
detected by the instrument in operation, as would the corresponding
components of whole blood.
[0010] It will be appreciated that the term "control composition"
as used herein means one or more blood components (i.e., blood
constituents as well as analogs thereof), which when combined or
used alone, sufficiently simulate the relevant characteristics of
whole blood for which the instrument tests. The following addresses
the preparation of various of the constituent components. The
control of the present invention contemplates an admixture of two
or more blood components, and preferably a reticulocyte component
and a component simulating at least three, and preferably five
subpopulations of white blood cells. Percentages are by volume
unless otherwise indicated.
Reticulocyte Component
[0011] The reticulocyte component of the control composition
includes a component that exhibits the relevant characteristics for
detection of reticulocytes using a hematology instrument in
accordance with the present invention. Accordingly, the control may
suitably contain stabilized reticulocytes (that is, immature
anucleate red blood cells containing some ribonucleic acid) or an
analog thereof. For example, among possible embodiments, the
reticulocyte component may comprise true mammalian reticulocytes
prepared for instance by mammalian (e.g. human) red blood cell
encapsulation or by isolation from whole blood. The reticulocyte
component is prepared in any suitable manner. See, e.g., U.S. Pat.
No. 5,432,089, incorporated by reference. Alternatively, it is
possible to obtain suitable reticulocytes by obtaining blood from
an anemic animal (e.g., a pig, goat, rabbit or the like).
[0012] In one particularly preferred embodiment, the reticulocyte
component is prepared by an encapsulation method, using
non-reticulocyte blood cells, such as red blood cells from a human
or other source. The red blood cells are encapsulated and
stabilized. By way of illustration, in one embodiment, red blood
cells having a relatively high MCV (e.g., about 85 to about 95 fL,
and more preferably about 88 to about 92 fL) are provided.
[0013] The cells are washed in a suitable diluent (e.g., about 0.15
M NaCl) in one or more washing steps. The cells are then
concentrated to a desired hematocrit value, e.g., greater than
about 50%, and more preferably greater than about 70%.
[0014] The cells are then encapsulated with RNA. By way of example,
RNA is encapsulated into the red blood cells by a suitable lysis
step, e.g., by hypotonic lysis. This may be done in a number of
ways, including by mixing red blood cells with a solution of RNA
having an appropriate pH and osmolarity. For instance the solution
may contain a minor amount of RNA (more preferably about 1%), and
has a pH between about 7 and 8 (more preferably about 7.6). The
solution is adjusted (e.g. with NaCl) to obtain an osmolarity of
about 40 mOsm.
[0015] The red blood cells are mixed with the RNA solution in a
suitable proportion, which may vary as desired. In a present
preferred embodiment, the volume ratio of packed red blood cells to
RNA solution is about 0.8 to about 1.2:about 1 to about 2, and more
preferably is about 1:1.4. Before lysing, or at an early stage of
the lysing, the red blood cells and the RNA solution are
pre-incubated, such as by heating to above room temperature (e.g.,
about 37 degrees C.). The cells are lysed in the admixture, and
thereafter, the red blood cells are re-sealed. By way of example,
the red blood cells are re-sealed by introducing them to a salt
solution and then heating above room temperature (e.g., adding
about 0.15 volume of about 12% NaCl solution, and then heating or
annealing the cells to about 37 degrees C. for about one hour).
[0016] The resulting mixture is poured into a separatory funnel and
allowed to incubate for a suitable time, e.g., for at least about
18 hours at room temperature. Thereafter, cells from the bottom
portion of the funnel are collected (e.g., from about the bottom
70% or lower). The cells are washed with a suitable diluent, such
as the diluent from Table 1 or 4. Optionally, about 0.5 to about
5.0% and more preferably about 0.75% of Streck Laboratories product
number 233301, also known as STA-CELL (available from Streck
Laboratories (Omaha, Neb.)) is added. In the present illustrated
embodiment, the cells are thus treated in a manner that would make
them susceptible to a detection stain (e.g., methylene blue).
[0017] In another embodiment, the reticulocyte component is fixed
(such as with an aldehyde or other suitable fixative) in a lactose
or other suitable diluent. A like diluent absent the fixative may
also be employed for washing.
White Blood Cell Component
[0018] In the present embodiment, the component of the control that
is to simulate the characteristics of white blood cells in whole
blood is prepared from a biological material. More specifically,
the material is a cellular biological material, and preferably the
material includes human white blood cells (although blood cells
from any suitable animal may be employed). In a preferred
embodiment of the present invention, the white blood cell component
includes materials for replicating the relevant measurable
characteristics for some or all of each of the five white blood
cell types, namely, lymphocytes, monocytes, neutrophils,
eosinophils and basophils, and they in turn are provided at their
art-disclosed levels (see e.g., Table 6).
[0019] The white blood cell component of the hematology control
composition comprises a blood cell (e.g. white blood cell) or
analog thereof, selected from the group consisting of white blood
cells for various cellular types, white blood cells for all
phenotypes, and mixtures thereof. U.S. Pat. Nos. 5,270,208 and
5,262,327, incorporated herein by reference, provide examples of a
suitable white blood cell component (see also U.S. Pat. No.
5,529,933, hereby incorporated by reference). Of course, the
skilled artisan will appreciate that the subject invention is not
limited to white blood cell components prepared from only white
blood cells. Analogs prepared from any of a variety of other
sources are possible, including but not limited to red blood cells
from birds, reptiles, mammals, etc.
[0020] The cells are provided in packs (e.g., 2 to 3 packs per
bottle). The cells are treated by a series of steps for selectively
lysing any undesired blood cells present in the material as
provided; for washing the cells; and for fixing or otherwise
stabilizing the cells.
[0021] Selective lysing may be accomplished in any suitable manner,
for instance, by contacting the undesired blood cells with a lysing
agent. Any suitable lysing agent may be employed. Buffered halides,
such as ammonium chloride and Trizma Based (e.g., about 7.5 g
ammonium chloride and 2 g Tris per liter), illustrates one suitable
class of lysing agents, where the undesired cells include red blood
cells. Lysing is accomplished through a series of consecutive
washing steps with the lysing agent. Optionally, before the lysing,
the cells are subjected to a preliminary fixing step, such as by
contacting them with a suitable fixing agent, heating them or both.
For instance, the cells are contacted with a buffered antimicrobial
saline solution (optionally including a diluent as described in
Table 4) including a suitable amount of a fixative (e.g., about
0.11% formaldehyde).
[0022] The lysing may be done in a single step or a plurality of
steps (e.g., for about one hour, then for about 25 minutes), where
after each step, the lysing agent is removed and fresh agent is
introduced. After lysing, the cells are washed to remove the lysing
agent. Any suitable wash composition and technique may be employed.
For instance, the aforenoted buffered antimicrobial saline solution
(absent a fixing agent) may be employed. Using this solution the
cells are washed in at least one step and preferably two steps,
wherein the cells are centrifuged at a suitable rate (e.g. about
900 rpm for about 10 minutes) (e.g., about 200 xg).
[0023] Prior to fixing, it may be preferable to further pre-treat
the cells in an albumin-containing diluent, such as about 2% BSA in
a diluent such as that of Table 1. Preferably the diluent has a pH
of about 8 and an osmolarity of about 175 to about 300 (e.g. about
215). Any suitable period of pre-treatment may be employed, e.g.,
about one hour, when maintained at about 6 degrees C.
[0024] The cells are fixed in any suitable manner sufficient to
denature the protein on the cell surface. The cells may be heated,
contacted with a fixing agent or both. The skilled artisan will
recognize that, though the preferred fixing agent is an aldehyde,
any suitable agent (preferably in a hypotonic solution) may be
used, including for instance, those containing an aldehyde, an
alcohol, a heterocyclic urea (e.g., diazolidinyl urea (known as
DU), imidazolidinyl urea (known as IDU) or a mixture thereof) or a
mixture thereof. Among the suitable fixing agents, one particularly
effective alcohol-containing agent is 50% by
volume--[1-methyl-2-(5-methyl-3-oxazolidinyl)-ethoxy] methoxy]
methanol (e.g., NUOSEPT 145, from HULS America, Inc.), or a mixture
therof In one present preferred embodiment, the aldehyde is
selected from the group consisting of formaldehyde, glutaraldehyde,
and mixtures thereof.
[0025] By way of illustration, a fixing admixture is prepared to
include about 10 parts cells, about 20 parts distilled water, an
agent for enhancing osmolarity across the cell membrane, for aiding
in the formation of clusters in scattergram population readouts or
both (e.g., about 5 g/l of a sugar, such as sorbitol), an agent for
helping to stabilize the readout of monocytes (e.g., about 4% DU),
and a fixing agent, such as about about 4 parts formaldehyde, and
about 0.1 parts glutaraldehyde. Fixing is performed for a suitable
amount of time and at a suitable temperature. Using this fixing
agent, for example, fixing is performed for about 2 to about 3 days
at about 22 degrees C. (i.e., warmed prior to fixing after
refrigeration).
[0026] Fixed cells are washed with a suitable rinse material (e.g.,
a cell stabilizer in a diluent) to remove the fixing agent. By way
of illustration, two washes are made during centrifugation at about
900 rpm for about 10 minutes (e.g., about 200 xg), in a solution
including a metal halide (e.g., about 5% NaF) cell stabilizer in a
diluent (e.g. a diluent having the composition as outlined in Table
2).
[0027] In another illustrative embodiment, when preparing the white
blood cell component for the control composition of the present
invention, the cells are obtained by standard separation from whole
blood or from portion of previously fractionated whole blood
containing the desired cell population. The cells are resuspended,
for instance, in a phosphate buffered solution containing
polyethylene glycol 20,000 (PEG), ethylenediamine tetraacetic acid
(EDTA) and magnesium gluconate with 2% bovine serum albumin. The
osmolarity of this solution is preferably sufficient to swell the
white blood cells prior to fixation (e.g. about 215 mosm). The
cells may then be stored in this solution, e.g. at about 6.degree.
C. for 1 hour.
[0028] The cells are fixed in a suitable media in order preferably
to denature the surface or otherwise accomplish preserving the cell
morphology. To illustrate, in one embodiment, in a solution of
distilled water containing 5 g/l sorbitol, 7.4% formaldehyde and
0.125% glutaraldehyde. Of course, other suitable fixing agents may
be used in suitable amounts. In a highly preferred embodiment, the
white blood cells and the fix solution are maintained at a
temperature sufficient to provide a proper white blood cell
position (e.g. between about 4.degree. C. and 12.degree. C.). The
fixative is added to the cells at a suitable ratio. For example, in
one embodiment, a ratio of between 10 ml of cell suspension to 24
ml of fix solution is used. The distilled water in the fix solution
swells the white blood cells further, while the fixative stabilizes
the cell membrane. The cells are thus left in the fixative for 2
days at room temperature.
[0029] After fixation, the cells preferably are washed. In one
aspect, they are washed in a phosphate buffered solution. One such
solution contains polyethylene glycol, (PEG),
ethylenediaminetetraactic acid (EDTA), magnesium gluconate and
bovine serum albumin. Lipoprotein concentrate is added at 150 mg/dl
HDL to store the cells prior to use in order to improve the
stability of the scattergram position while the white blood cells
are waiting to be added to the other components of the control
composition.
[0030] It will be appreciated that white blood cells prepared as
described in U.S. Pat. No. 5,459,073 (incorporated herein by
reference) for flow cytometry may be employed for phenotyping. By
mixing the two types of white cells, both requirements can be met,
i.e., white blood cells for various cellular types and phenotypes,
as the cells prepared for phenotyping generally should not
interfere with the position of other white blood cells on the
histograms/scattergrams.
[0031] In controls for certain instruments, white blood cells may
need to be diluted or concentrated, for example, for the Cell-Dyn
instruments a dilution to a count of 10,000 is preferred.
Red Blood Cell Component
[0032] In the present embodiment, the component of the control that
is to simulate the characteristics of red blood cells in whole
blood is prepared from a bio-compatible material. More
specifically, the material is a cellular biological material, and
preferably the material includes human red blood cells (although
analog blood cells from any suitable animal may be employed).
[0033] The cells as provided are separated from the liquid medium
or supernatant in which it is supplied through any suitable
separation technique, including but not limited to centrifugation,
filtration, or the like. The cells are washed in a series of one or
more (e.g., 3) consecutive washing steps, pursuant to which excess
supernatant is removed. The cells preferably are washed in a (e.g.
such as the diluent of Table 2) and optionally one or more
additional components selected from the group consisting of a cell
stabilizer, an albumin, an agent for reducing the likelihood of
cell hemolysis in the presence of oxygen, and mixtures thereof In a
more preferred aspect, the additional components are selected from
the group consisting of a metal halide cell stabilizer, bovine
serum albumen (BSA), an antioxidant and mixtures thereof. In still
a more preferred embodiment, the diluent is one such as that of
Table 4, and it will include about 0.003 to about 0.010 (and more
preferably about 0.005%) NaF, about 2% BSA, about 0.005 to about
0.020 (and more preferably about 0.010%) sulfasalizine. The diluent
of Table 1 likewise may be used as desired.
[0034] Optionally, depending upon the end use and commercial
considerations, one or more agents for reducing the rate of
degradation is employed in a suitable amount; for example, about
0.25 to about 2.0%, and more preferably about 0.75% of a material
available from Streck Laboratories (Omaha, Neb.) under the product
number 233301 or the designation STA CELL (which material may also
be suitably added for one or more of the other components).
Further, optionally, the cells preferably are prepared in an
environment substantially free of glucose. In yet another optional
embodiment, the cells are fixed (e.g., by a suitable protein
denaturation step, such as by glutaraldehyde fixing) after washing
and then further washed.
[0035] The cells may be washed for any suitable period of time, and
resuspended (in the same wash or a different one, e.g., one having
a higher concentration) any suitable number of times.
[0036] The skilled artisan will appreciate that the red blood cells
may be washed free of all other cellular material, such as by using
a magnesium gluconate diluent.
[0037] By way of further illustration, in another embodiment,
concentrated red blood cells are provided, separated from
associated supernatant, and concentrated human red blood cell packs
are suspended in a solution (e.g. phosphate buffered solution
containing PEG (MW=20,000)) and allowed to settle overnight. The
supernatant is then removed and 0.5% NaCl with PEG is added in an
equal volume to the packed red blood cells and allowed to set at
room temperature for 4-5 hours. The supernatant is again removed
and the cells are resuspended in the NaCl solution and stored at
6.degree. C. overnight. The packs are further checked for excessive
hemolysis and removed from the inventory. The remaining packs are
pooled into batches based on the MCV's, wherein twelve to fourteen
packs are combined to make a batch. The batches are again
resuspended in the NaCl solution for about 4-5 hours at room
temperature. Of course, other times and temperatures may be
employed.
[0038] Each batch is resuspended into a phosphate buffered solution
containing PEG, EDTA and magnesium gluconate. The cells are allowed
to settle, the supernatant is removed and the cells are resuspended
in the above solution with lower PEG concentrations for storage up
to 90 days at 6.degree. C.
[0039] One diluent efficient in stabilizing the red blood cells on
the Coulter STKS includes a phosphate buffered solution containing
PEG, Na.sub.2EDTA, magnesium gluconate and an antioxidant (e.g.,
sulfasalazine or .alpha.-tocopherol), wherein the antioxidant is
added to prevent hemolysis when the lipoprotein is added to the
control composition of the present invention. The final diluent
also contains about 2% bovine serum albumin to improve the position
of the white blood cells. After the cells have been washed into
this diluent, STA-CELL from Streck Laboratories (Omaha, Neb.) is
added at 0.75% to the total volume of red blood cells to give added
stability to the MCV'S.
[0040] Optionally, in certain applications, it may be desirable to
fix the red blood cells, after removing excess red blood cells,
such as by a slow centrifugation. By way of example, for such an
embodiment, the diluent of Table 1 is employed. Cells are washed
and resuspended in the diluent to a suitable concentration (e.g.
about 4.times.10.sup.6/mm.sup.3). Preferably the pH is about 7 and
there is no glucose in the suspension. Approximately one to one
proportions of the cells are admixed with the diluent and a
suitable amount of a fixative (e.g., about 0.007% to about 0.01%
glutaraldehyde (by count)) for a suitable period and at a suitable
temperature (e.g., 22 degrees C. for about one or two days). The
resulting cells are then washed a plurality of times (e.g., about 3
to about 8) in a like diluent (preferably at a pH of between 7 and
8). The cells are centrifuged at about 1500 for about 15 minutes.
Decanting and sonication is performed as needed. Moreover, the
cells may be further treated as desired by the addition of a
suitable amount of STA-CELL (e.g., about 0.75%) from Streck
Laboratories, Inc. (Omaha, Neb.).
Nucleated Red Blood Cell Component
[0041] When employed, the nucleated red blood cell component of the
control composition of the present invention comprises nucleated
red blood cells or an analog thereof, such as avian red blood
cells, e.g., turkey or chicken red blood cells. For example, turkey
red blood cells are washed into a phosphate buffered solution and
set to a count of about 1.times.10.sup.6/mm3. The cells are fixed
with a phosphate solution (volume equal to the cell volume)+0.4%
v/v glutaraldehyde, at room temperature for one day and then washed
into a phosphate buffer.
[0042] The fixed turkey red blood cells are added to the control
composition of the present invention to yield a cell count equal to
at least 10% of the white blood cell count in order to produce NRBC
flags on the Coulter STKS or the Cell-Dyne 4000 manufactured by
Abbott Laboratories. Though the present example contemplates the
use of turkey cells, cells from other cell sources may be employed
as the skilled artisan will appreciate.
Platelet Component
[0043] In a preferred embodiment of the present invention, the
hematology control composition additionally provides a platelet
component, preferably a simulated platelet component. Among other
possible types, the platelet component may comprise stabilized
human platelets or platelets simulated from goat, bovine or porcine
blood cells. In one embodiment, they are prepared from red blood
cells. See, U.S. Pat. Nos. 4,160,644 and 4,198,206, incorporated
herein by reference, disclose an example of a suitable platelet
reference control and methods of preparation. The skilled artisan
will appreciate a number of other techniques for preparing
simulated platelets.
[0044] In general, how the platelets are prepared may depend upon
the source of the cells (i.e., whether they are animal blood cells
to be shrunken, swollen or otherwise sized or shaped to resemble
platelets, or whether they are platelets from blood). In general,
the cells are washed, optionally pre-fixed, sized and shaped, and
then fixed or otherwise stabilized in terms of size and shape, and
neutralized.
[0045] By way of example, the platelet component is prepared from
animal blood cells, such as goat red blood cells. The cells are
washed one or more times (e.g., about three times) in a suitable
buffered solution such as a buffered saline solution (e.g.,
phosphate buffered saline solution). The solution may also include
a suitable amount of a chelating agent (e.g., about 1% ethylene
diamine tetraacetic acid (EDTA)).
[0046] The cells optionally are prefixed in a suitable manner to
aid in the step of sizing and shaping them. By way of illustration,
the cells are contacted with a suitable pre-fix solution ((e.g.,
one to one in the initial wash solution having a fixative (e.g.,
about 0.0085% of glutaraldehyde)). The amount of such fixing agent
of course may be suitably adjusted as needed to control the sizing
rate (e.g., the shrinkage rate). Preferably the pre-fix solution is
warmed to an elevated temperature (e.g., about 30 degrees C.), and
prefixing is performed for a sufficient time at such temperature
(e.g., about 90 minutes). After the pre-fix step, the supernatant
is removed, such as by centrifuging, aspirating or both.
[0047] In instances where the sizing and shaping is performed to
shrink the cells and form a simulated platelet structure, the cells
preferably are lysed in a suitable manner (e.g., using a lytic
agent such as ammonium chloride tris). During lysing, the cell size
and shape may be monitored using a suitable instrument such as the
H3 by Bayer Corporation (a hematology analyzer with a laser optical
detection system). The cells are then washed one or more times with
a suitable diluent (e.g., the diluent of Table 3)
[0048] Like with the aforenoted blood cells, the simulated
platelets are fixed in any suitable manner, preferably one that
will denature the protein on the cell surface. In a particularly
preferred embodiment, the cells are fixed in a one to one solution
with a diluent such as that of Table 3 and about 0.1% formaldehyde.
Preferably the temperature of the fixing solution is elevated (e.g.
about 37 degrees C.) for a sutiable period of time (e.g., about 3
days).
[0049] The cells are then washed with a suitable wash to remove the
fixative, preferably also taking measures to neutralize any
unreacted fixative that has not bound to the cell surface. To
illustrate, preferably where the fixative is an aldehyde, a
suitable amount of a glycine solution is employed in the wash. One
or more additional washing steps may be performed, using one or
more additional washes, as desired. For instance, subsequent
washing steps may be employed using the wash composition of Table
3, Table 4 or a mixture thereof
[0050] It will be appreciated that the platelet component can be
made from human blood, using any suitable process. By way of
example, without limitation, human blood is provided (preferably in
a diluent having a fixative (e.g., a diluent such as that of Table
4 with about 0.10% formaldehyde). Optionally, red blood cells are
removed, and the resulting cells are admixed with fixed cells. For
instance, the red cells are centrifuged in quantities of about 400
ml per container for about 10 minutes at about 900 rpm).
[0051] As-provided cells are placed in a fixative (e.g., about one
to one proportions in a diluent such as that of Table 3 with a
suitable amount of a fixative (e.g., about 0.075% glutaraldehyde)).
Fixing is done at about 22 degrees C. for about 2 days. Following
fixation, centrifugation is performed at about 1800 rpm for about
20 minutes (for 400 ml container). The two separate collections of
cells are brought together and washed (such as in a 1x diluent like
that of Table 1), and then centrifuged (e.g. at about 1800 rpm for
about 20 minutes). Optionally, the platelets are decanted from
residual red blood cells. Sonication may be used as desired to
address platelet clumping. Decantation may also be used as desired
to assure debris and red blood cells are removed.
[0052] Resulting materials are then resuspended in a diluent. An
example of a suitable diluent would be that of Table I having no
glucose and a pH of about 7.
Reticulated Platelet Component
[0053] In another embodiment of the present invention, the control
composition comprises a reticulated platelet component. To
illustrate, without limitation, goat red blood cells with
encapsulated nucleic acids would constitute one example of a
reticulated platelet component useful for the present invention.
Other analogs may be used as well.
[0054] By way of further illustration, in another embodiment,
reticulated platelets for the control composition of the present
invention are prepared by inducing a porous blood cell membrane to
permit entry of RNA into a cell, hemoglobin to leave, or both. The
then cells are sized or shaped and stablizied. To illustrate, goat
red blood cells are placed in a solution of about 0.9% NaCl and
concentrated to 70-80% hematocrit (HCT). Equal volumes of
concentrated red blood cells and 4% RNA solution (20 mls of each)
adjusted to 300 mosm with a suitable salt (e.g., KCl) are mixed
together and dialyzed against 500 mls of a hypotonic solution, such
as one containing glycerol (osm=90-100), for 90 minutes at
6.degree. C. Dialysis is required to slowly change the osmolarity
without damaging the cells. The resulting osmolarity change in the
red blood cell solution is from about 300 mosm to about 150 mosm.
This process creates holes in the cell membrane to allow the RNA in
the red blood cell solution to enter the red blood cells.
[0055] The osmolarity is brought back to isotonicity by dialyzing
the red blood cells containing RNA against an isotonic solution.
This dialysis is at room temperature for 30 minutes and the final
osmolarity of the cells is about 260 mosm. This process reseals the
holes that were created by the hypotonic dialysis, thus trapping
the RNA inside the cells.
[0056] Eighty milliliters of the resealing diluent containing 0.1%
Nuosept 101 is added to the encapsulated red blood cells and the
mixture is heated at 37.degree. C. for 3 hours. This heating step
helps to lyse the weakened cells from the encapsulation process and
anneals the membranes of the encapsulated red blood cells.
[0057] To illustrate, goat red blood cells are separated from other
constituents of goat whole blood. For instance, cells are washed
into PBS three times to remove the plasma and white cells. The
concentration is adjusted to 8.times.10.sup.6/mm3 and fixed with a
volume of PBS equal to the cells that contain 0.224-0.320%
glutaraldehyde, providing the amount of protection needed to allow
proper Iysis during the shrinkage step. The cells are incubated at
30.degree. C. for one hour and centrifuged at 1200 RPM for 15
minutes. The supernatant is removed and the cell volume is adjusted
to one-fourth of fixed volume.
[0058] An ammonium chloride solution is added to the cells to equal
the original volume of fixed cells. Without intending to be bound
by theory, the ammonium chloride solution creates holes in the
membrane to allow hemoglobin to exit to the cells, while the
glutaraldehyde protects from total lysis. The cells are monitored
for hemoglobin loss based on (MPV) decreases on a Bayer H-1. When
the MPV is at 10 fl on the Bayer H-1, the cells are diluted with a
phosphate buffered solution and centrifuged at 1800 RPM for 20
minutes. The supernatant is removed and the cells are washed to
remove the free hemoglobin and shrink the membrane around the
hemoglobin to produce an MPV of approximately 10 fl on impedance
instruments, such as the S+IV, manufactured by Beckman Coulter.
[0059] When the MPV is at 10 fl on the H-1, the cells are diluted
with a phosphate buffered solution and centrifuged at 1800 RPM for
20 minutes. The supernatant is removed and the cells are washed to
remove the free hemoglobin and shrink the membrane around the
hemoglobin to produce an MPV of approximately 10 fl on impedance
instruments, such as the S+IV, manufactured by Beckman Coulter.
[0060] In a more preferred embodiment, to prevent further loss of
hemoglobin and shrinkage of the membrane, the cells are fixed more
than once, for instance, with 0.04% glutaraldehyde in a volume of
phosphate buffered solution equal to the cell volume at a count of
1.times.10.sup.6/mm3. The cells are left at room temperature
overnight and then washed. Though the present example contemplates
the use of goat cells, cells from other cell sources may be
employed (such as stabilized human platelets) as the skilled
artisan will appreciate.
Suspension Medium
[0061] The present invention relates to a method of making a
hematology control composition for use with multi-parameter
systems, comprising the step of mixing one or more of a
reticulocyte (retic) component, a white blood cell component, a red
blood cell component, a nucleated red blood cell component, a
platelet component, and a reticulated platelet component in an
isotonic suspension medium.
[0062] The components of the control preferably are suspended in
appropriate concentrations a suitable suspension medium that
permits the control to be processed through the automated
instrument. The suspension medium thus has a pH of about 6.5 to
about 8.5 and is isotonic. By way of example, among the possible
embodiments of the present invention, the isotonic suspension
medium may comprise a buffer, antioxidant, protein or a mixture
thereof, e.g., magnesium gluconate/ethylene diamine tetraacetic
acid (EDTA)/phospate buffer with nucleated red blood cells; the
same buffer with the additives HDL, sulfasalazine and alpha
tocopherol; or the same buffer with 3% albumin.
Lipoprotein
[0063] While BSA in any diluent present improves the white blood
cell position on the scattergram, lipoprotein is also preferably
used in an amount effective to provide a scattergram that
represents whole blood, including the proper positioning of the
five subpopulations of white blood cells. See U.S. Pat. Nos.
5,270,208 and 5,262,327 incorporated by reference. A lipoprotein
source, preferably one consisting essentially of high-density
lipoprotein (i.e., HDL) is added at about 0.5 to about 8.0% by
volume of the control, and more preferably at about 100-175 mg/dl
to the control composition and a-Tocopherol is further added to the
lipoprotein source to reduce peroxides produced by the oxidation of
the lipoproteins. An example of a suitable commercially available
form of lipoprotein is SUPERTRATE (available from Bayer).
Admixing Components
[0064] Stock volumes of the constituent components are prepared in
the following approximate concentrations:
1 RBC: 6.0 .times. 10.sup.6/mm.sup.3 WBC: 150,000/mm.sup.3
Platelets: 10 .times. 10.sup.6/mm.sup.3 Retics: 50% of 5.5 .times.
10/mm.sup.3 red count NRBC: 0.5 .times. 10.sup.6/mm.sup.3
[0065] To prepare the final control composition, for example, in a
5 liter volume, stock volumes of the constituent components are
combined as follows:
2 Approximate Approximate Target Count Volume Stock RBC: 4.5
.times. 10.sup.6/mm.sup.3 3,750 ml.sup. WBC: 8.0 .times.
10.sup.3/mm.sup.3 266 ml Plt (platelets) 225 .times.
10.sup.3/mm.sup.3.sup. 112 ml Retic(%) 3% 370 ml NRBC 0.01% 4.5
ml.sup.
[0066] The combined constituents are brought to a final total
volume of 5 liters by adding a suitable final diluent (e.g.,
prepared according to U.S. Pat. No. 5,262,327, incorporated herein
by reference (preferably including SUPERTRATE or a like
substance)). The skilled artisan will appreciate that there are
other means and procedures to prepare this and other embodiments of
the present invention. Moreover, concentrations can be varied to
provide controls having predetermined abnormal readings when
tested.
Using Control
[0067] The following discusses examples of methods of using the
control composition to determine the accuracy and reproducibility
of the operation of a multi-parameter automated hematology
instrument. By way of example, a multi-parameter automated
hematology instrument, such as a Beckman Coulter STKS or Gen-S
Systems, the Abbott Cell-Dyn 4000 Hematology System, Bayer ADVIA
120, and the Sysmex XE2100 System, is provided, optionally with a
slide preparation module. The claimed control composition is
obtained or prepared which includes, by way of example, a treated
stabilized human red blood cell component and a reticulocyte
component with quality control values in an appropriate range, for
example, 1.0%, 2.5%, and 9.0%, respectively. It is refrigerated
prior to use. At the beginning of testing, the control composition
is allowed to warm to room temperature for about fifteen minutes,
mixed manually, and checked for resuspension of contents.
[0068] The control composition is prepared and analyzed by the same
standard method as test samples which may be tested in batch
quantities by the use of a suitable cassette having apertures for
receiving test vials. After preparation, the control composition
and test samples are analyzed by counting the population number of
each subject component type with a multi-parameter automated
hematology instrument, which will yield a visual display of the
data.
[0069] For a Coulter System, the automated test instrument may
employ technology known generally as VCS Technology (as marketed by
Beckman Coulter). VCS generally analyzes cell samples in view of
simultaneous volume conductivity and Scatter measurements.
Ordinarily, a starting sample is employed in combination with
suitable reagents (which may comprise a component of a kit) and
physical agitation for lysing and cell measuring by way of flow
cytometry.
[0070] Accordingly, the sample may be tested by the Coulter
Principle of (DC) Impedance to measure the cell volume in an
isotonic suspension.
[0071] Conductivity may be employed, for instance, by applying
alternating current in the radio frequency range. Energy can
penetrate the cell by short circuiting the cell membrane's bipolar
lipid layer.
[0072] Information about the cells is also possible with light
scatter techniques, such as from the scatter characteristics
detected from cells in response to a coherent light source, e.g. a
laser beam.
[0073] Of course, by no means is the mode of sample testing limited
to the above. As mentioned other principles may be used.
[0074] The respective population counts obtained from the analysis
are compared either to known reference value for each component
type in the control composition, or by comparison of the population
counts for each component types in the test sample with the
corresponding values of components in the control composition. Data
relating to the measurement of components in control composition
and test samples is collected, monitored, stored, compared and
analyzed by electronic means, such as a computer programmed with
appropriate software and containing appropriate data file
structure.
3 TABLE 1 Most Preferred Reagents Concentration Distilled Water 0.9
liter Methyl Paraben 0.40 g/l PEG 20,000 3.00 g/l EDTA, Disodium
Salt 7.04 g/l Magnesium Gluconate 3.92 g/l Sodium Phosphate Dibasic
Anhydrous 2.68 g/l Glucose 6.0 g/l Sodium Hydroxide pellets 0.8 g/l
Adenosine 0.25 g/l Inosine 0.25 g/l Neomycin Sulfate 0.40 g/l
Chloramphenicol 0.15 g/l *q.s. to 1 Liter
[0075] Concentrated bovine serum albumin is added to the product at
the time of combining the various cell types to make the protein
concentration 3% of the liquid portion of the product.
4 TABLE 2 Most Preferred Reagents Concentration Distilled Water 0.9
liter Methyl Paraben 0.4 g/l PEG 20,000 3.00 g/l EDTA, Disodium
Salt 11.73 g/l Magnesium Gluconate 6.53 g/l Sodium Phosphate
Dibasic Anhydrous 4.47 g/l Glucose 10 g/l Sodium Hydroxide pellets
1.40 g/l Adenosine 0.25 g/l Inosine 0.25 g/l Neomycin Sulfate 0.40
g/l Sodium Fluoride 0.05 g/l Chloramphenicol 0.15 g/l *q.s. to 1
Liter
[0076]
5 TABLE 3 Most Preferred Reagents Concentration Distilled Water 0.9
liter Methyl Paraben 0.4 g/l PEG 20,000 3.00 g/l EDTA, Disodium
Salt 16.75 g/l Magnesium Gluconate 9.33 g/l Sodium Phosphate
Dibasic Anhydrous 6.39 g/l Sodium Hydroxide Pellets 2.04 g/l
Adenosine 0.25 g/1 Inosine 0.25 g/l Neomycin Sulfate 0.40 g/l
Chloramphenicol 0.15 g/l *q.s. to 1 Liter
[0077]
6 TABLE 4 Most Preferred Reagents Concentration Distilled Water 1
liter Methyl Paraben 0.40 g/l PEG 20,000 3.00 g/l EDTA, Disodium
Salt 11.73 g/l Magnesium Gluconate 6.53 g/l Sodium Phosphate
Dibasic Anhydrous 4.47 g/l Glucose 10 g/l Sodium Hydroxide pellets
1.40 g/l Adenosine 0.25 g/l Inosine 0.25 g/l Neomycin Sulfate 0.40
g/l Sodium Fluoride 0.05 g/l Bovine Serum Albumin 20 g/l
Sulfasalazine 0.10 g/l Chloramphenicol 0.15 g/l *Cholestrol
Supertrate containing .alpha. - Tocopherol is added to the product
at the time of combining the various cell types. The cholestrol
supertrate is added (2-3%) to make the .alpha. -Tocopherol
concentration about 5 mg % in the product.
[0078] The skilled artisan will appreciate that a number of the
ingredients have been disclosed by way of specific example, but
that any of a number of alternative ingredients at the suggested or
different concentration, may be suitably substituted for such
ingredients. The following Table 5 illustrates examples of various
alternatives, and also includes a brief discussion of the commonly
employed concentration ranges when employed in the diluents of
Tables 14. Though the ingredients are described by reference to a
particular function, it should be appreciated that such discussion
is presented without intending to be bound by theory. In some
instances, the ingredient will perform a different or an additional
function. Moreover, the skilled artisan will appreciate that
reference in Table 5 to the functions, in the context of the
present preferred embodiments, could be made to select additional
alternatives. Thus, there is no intention to be bound to the
breadth of any specific illustrative ingredient or concentration,
where it is apparent that others may be advantageously be employed
in addition to or as a substitute for such ingredient.
[0079] Likewise, as desired, ingredients may be deleted from the
diluent. Thus, a combination of some of the ingredients may be
suitably employed to achieve desired results.
[0080] Preferably, the diluent includes one or more agents that
function as a surfactant, hemolysis inhibitor, red blood cell
settler, MCV stabilizer, buffer, metabolite, osmolarity adjuster,
antimicrobial, antifungal, protein source, positioner of white
blood cell subpopulation, antioxidant, debris reducer or a mixture
thereof.
7TABLE 5 Preferred Range of Component Concentration concentration
Others Polyethylene Glycol 3.00 g/l 1.00-10.00 g/l PEG 8000, F68
(PEG MW 20,000) Disodium EDTA 11.75 g/l 7.00-17.00 g/l Tetrasodium
EDTA Magnesium Gluconate 6.50 g/l 3.5-9.5 g/l Lactose Sodium
Phosphate 4.50 g/l 2.5-6.5 g/1 Citrate, Borate, Trizma Base Sodium
Bicarbonate Glucose 10.00 g/l 0-10 g/l Other sugars Adenosine 0.25
g/l 0.1-1.0 g/l Inosine Neomycin sulfate 0.40 g/l 0.1-0.8 g/l
strepto-,kana-mycin Chloramphenicol 0.15 g/l 0.1-0.4 g/l
Piperacillin Methyl Paraben 0.40 g/l 0.20-1.20 g/l Other
antifungals Bovine Serum Albumin 30.0 g/l 0.0-60.0 g/l Other
protein sources Cholesterol Supertrate 30 ml/l 20-50 m//l
Triglyceride Supertrate, Cholesterol HDL Supertrate Cholesterol
(all available through Bayer) Sodium Fluoride 0.05 g/l 0.0-0.50 g/l
Other halides Sulfasalazine 0.10 g/l 0.0-0.50 g/l Other
antioxidants .alpha.-Tocopherol 0.05 g/l 0.0-0.30 g/l Ascorbic
acid, BHT, (Vitamin E) Deferoximine Mesylate, Probucol, Rutin
[0081]
8 TABLE 6 Cell Range (approximate % of white blood cells)
Lymphocytes 20 to 45% Monocytes 2 to 10% Neutrophils 40 to 75%
Eosinophil 1 to 6% Basophils Up to 1%
Further Alternative Exemplary Embodiment
[0082] For the red blood cell component, a diluent containing Mg
Gluconate and EDTA (e.g., about 3.92 g/l Mg Gluconate; 7.04 g/l
EDTA-Disodium; 2.68 g/l NA.sub.2HPO.sub.4; glucose 6 g/l; and
antimicrobials (pH of 7.1 and osmolarity using KCl of about 300))
stabilizes red cells so that red cell parameters are stable for 200
days.
[0083] The white blood cell component is prepared from fresh human
white blood cells. The cells are washed free of red blood cells and
platelets using the above diluent. The cells are suspended in
phosphate buffered saline with an osmolarity of 280 and pH of 7.2.
A solution of 20% Nuosept 145 (Huls America) is mixed with the
white blood cells 1:1 to give a final concentration of about 10%
(e.g., 9.11%). The mixture is placed at a temperature of about
37.degree. C. to about 50.degree. C. for six days or more.
Thereafter, the cells are washed one or more times (e.g., 3 times)
in a suitable diluent, e.g., a diluent such as Table 1, centrifuged
(e.g., about 900 rpm for 10 minutes) and resuspended in a diluent
such as in Table 1 (having no glucose and a pH of about 7).
[0084] The platelet component is prepared by washing human
platelets free from whole blood components by low speed
centrifugation (900 RPM for 10 min.). The platelets are then
stabilized by addition of a low level of glutaraldehyde. The
platelets are stabilized by mixing 1:1 with the magnesium gluconate
diluent containing 0.075% glutaraldehyde (final concentration of
0.037% glutaraldehyde). After a 22.degree. C. incubation, the cells
are again washed in the diluent and are ready for use. The same
procedure can be used for bovine or porcine.
[0085] The reticulocytes are prepared by encapsulation of yeast-RNA
as described in U.S. Pat. No. 5,432,089, incorporated by
reference.
[0086] The white blood cell component may be set up with red blood
cells for achieving differential stability, such as by maintaining
them at about 22.degree. C. for about 20 days.
[0087] The final control contains the following cellular
concentrations (Table 7), exhibits a histogram/scattergram profile
that includes a properly positioned five-part differential of white
blood cells and reticulocytes, substantially approximating whole
blood and is stable for 200 days or more. This control provides
such results for the Cell Dyn series, the Bayer H-3, and the Sysmex
SF instruments.
9TABLE 7 Values WBC % N % L % M % E % B RBC RBCo HCT MCV MCHC RDW %
R IRF PLTo PLTl MPV Low 3.65 43.1 41.7 11.0 1.94 2.32 3.82 3.80
30.8 80.6 34.9 13.1 5.38 .212 67.6 69.4 10.2 Normal 7.92 55.9 31.0
10.4 1.31 1.50 5.01 4.97 42.8 85.5 35.0 13.0 2.79 .184 179.0 196.0
9.90 High 19.2 59.9 23.3 14.8 1.86 .213 5.51 5.39 48.6 88.2 33.8
16.4 .736 .190 381.0 443.0 10.2
[0088] Accordingly, the foregoing discussion discloses and
describes merely exemplary embodiments of the present invention.
One skilled in the art will readily recognize from such discussion
and from the accompanying drawings and claims, that various
changes, modifications and variations can be made therein without
departing from the spirit and scope of the invention as defined in
the following claims. All patents and other publications cited
herein are expressly incorporated by reference.
* * * * *