USOO6653137B2

(12) United States Patent (10) Patent No.: US 6,653,137 B2

Ryan (45) Date of Patent: Nov. 25, 2003

(54) HEMATOLOGY REFERENCE CONTROL 5,731.205 A 3/1998 Ryan

5,736,402 A 4/1998 Francis et al.
(75) Inventor: Wayne L. Ryan, Omaha, NE (US) 5,811,099 A
5,811,303 A 9/1998 Ryan
9/1998 Ryan
(73) Assignee: Streck Laboratories Inc., La Vista, NE s A g FM is et al.
(US) 5858,790 A 1/1999 Kim et al.
5,874,310 A 2/1999 Li et al.
(*) Notice: Subject to any disclaimer, the term of this 5,874,311 A 2/1999 Li et al.
patent is extended or adjusted under 35 3. A
917,584
E. ith.
et al.
et al.
U.S.C. 154(b) by 38 days. 5,939,326 A 8/1999 Chupp et al.
5,945,340 A 8/1999 Francis et al.
(21) Appl. No.: 10/007,519 5.981282 A 11/1999 Ryan
5,994,139 A 11/1999 Jacobs et al.
(22) Filed: Dec. 3, 2001 6,060,322. A 5/2000 Horton et al.
O O 6,074,879 A 6/2000 Zelmanovic et al.
(65) Prior Publication Data 6,197.593 B1 * 3/2001 Deka et al. ................... 436/63
6,200,500 B1 3/2001 Ryan
US 2003/0104629 A1 Jun. 5, 2003 6,221,668 B1 4/2001 Ryan et al.
6.228,652 B1 5/2001 Rodriguez et al.
(51) Int. Cl. ................................................ G01N 31100 6,232,125 B1 5/2001 Deka et al.
(52) U.S. Cl. ............................... 436/10; 436/8; 436/63; 36. R : 383 A.fal 436/8
436/164; 422/73; 422/82.05; 435/2 6403377 B1 6/2002 Ryan et al. . ... 436/8
(58) Field of Search ................................ 436/8, 10, 17, 6,406,915 B2 * 6/2002 Ryan et al. .. ... 436/10
436/18, 63, 164, 174; 422/73, 82.05; 435/2 6,448,085 B1 * 9/2002 Wang et al. . ... 436/10
6,472.215 B1 * 10/2002 Huo et al. .................... 436/10
(56) References Cited
FOREIGN PATENT DOCUMENTS
U.S. PATENT DOCUMENTS
WO WO 93/17329 3/1993
3,558,522 A 1/1971 Louderback et al. WO WO 93/17330 3/1993
3,574,137 A 4/1971 DeCasperis et al. WO WO 01/14872 3/2001 .......... GO1N/31/00
3,607,783. A 9/1971 Tata
3,640,896 A 2/1972 DeCasperis OTHER PUBLICATIONS
3,873,467 A 3/1975 Hunt
4,099.917 A 7/1978 Kim International Search Report for PCT Application No. PCT/
4,160,644 A 13. 12 E. USO2/31718 dated Mar. 20, 2003.
3. A 33. SN Greenfield, et al., “Inhibition of Red Cell Membrane Lipid
4219440 A 8/1980 Runck et al. Peroxidation by Sulfasalazine and 5-Aminosalicylic Acid.”
4.264,470 A 4/1981 Chastain, Jr. et al. Gut, pp. 1156-1159, (1991).
4.299,726. A 11/1981 Crews et al. Lombarts, et al., “A Stable Human-Platelet-White Blood
:
2-1 :
A : 1: Myths, OceaCK e a
Cell Control for the Coulter Model S-Plus II,” Clinica.
4,358,394. A 11/1982 Crews et al. Chimica. Acta., pp. 95 102. (1982).
4,389,490 A 6/1983 Crews et al. Nergre-Salvayre, et al., “Protective Effect of a-Tocopherol,
4,390,632 A 6/1983 Carter, II Ascorbic Acid and Rutin against Peroxidative Stress
4.425,334 A 1/1984 Hunt Induced by Oxidized Lipoproteins on Lymphoid Cell
4,436.821 A 3/1984 Ryan Lines,” Biochem. Pharmacol., p. 450–453, (1991).
E. A E. th et al. Sorette, et al., “Improved Isolation of Normal Human
4704364 A 11/1987 E. et al. Reticulocytes via Exploitation of Chloride-Dependent
4,711852. A 12/1987 Jacobson et al. Potassium Transport,” Blood, vol. 80 (No. 1), P. 249-254,
4,745,071 A 5/1988 Lapicola et al. (Jul. 1, 1992).
E. A
2. . . .
8. SE al
ong et al.
Lombarts, et al. “A White Blood Cell Control for Long
5,008,021 A 4/1991 Conner et al. Term Stability,”22 Clinica.
YY k.u.
Chimica, Acta, 129:79–83 (1983).
5.008-201
2Y- - -
A 4/1991 Ryan sk -ted bw examiner
5,196,182 A 3/1993 Ryan cited by
5,262,327 A 11/1993 Ryan
5,270.208 A 12/1993 Ryan Primary Examiner Maureen M. Wallenhorst
5,320.964 A 6/1994 Young et al. (74) Attorney, Agent, or Firm-Howrey Simon Arnold &
5,380,664 A 1/1995 Carver et al. White LLP
5,389,664 A 2/1995 Baile et al.
5,432,089 A 7/1995 Ryan et al. (57) ABSTRACT
5,459,073. A 10/1995 Ryan
5,460,797 A 10/1995 Ryan An improved control for a hematology analyzer. In one
5.492.833 A 2/1996 Rodriguez et al. embodiment, blood cells are treated for permitting the cells
5,512.485 A 4/1996 Young et al. to Simulate nucleated red blood cells for detection or analy
A 3. g R.S.", al sis by the hematology analyzer.
5,672.474. A 9/1997 Ryan
5,677,145 A 10/1997 Ryan 14 Claims, No Drawings
 US 6,653,137 B2
1 2
HEMATOLOGY REFERENCE CONTROL prepared from red or white blood cells, adapted for Simu
lating at least three, and more preferably five Subpopulations
of white blood cells.
TECHNICAL FIELD In another embodiment, the white blood cell component
This invention relates generally to an improved method of is prepared from a Sample of human whole blood in which
making a hematology control composition, improved white blood cells are fixed prior to lysing of the red blood
compositions, and their use in an automated or Semi cells from the whole blood. After red blood cell lysis, the
automated hematology analyzer. white blood cells are again fixed for a period of at least about
1 hour, and more preferably about 4 to about 5 hours.
BACKGROUND In another embodiment, compositions in accordance with
the above are employed in a hematology analyzer as a
The proliferation of Semi-automated and automated reference control that detects blood cell characteristics on
hematology instruments in recent years and the increased the basis of light Scatter, electrical impedance, radiofre
regulation of clinical laboratories has placed an increasing quency or a mixture thereof.
demand for high performance, long-term Stable reference 15
controls. Certain instruments characterize a Sample of blood DETAILED DESCRIPTION OF PREFERRED
by detecting the impedance or light Scatter or radiofrequency EMBODIMENT
characteristics of cells in a Sample. It has become especially Blood Cell Source
popular to use these instruments for differentiating cells One object of the present invention, as gathered from the
relative to each other, as well as for other flow cytometry foregoing, is to provide a component for a hematology
techniques. reference control that Simulates nucleated red blood cells of
In both U.S. Pat. Nos. 6,187.590 and 5,858,790 (both human blood. In this regard, the Simulation does not nec
incorporated by reference) the patentee identifies examples essarily mean that cells be chemically similar to the intended
of certain of these instruments, including the ABBOTT corresponding human cells. Rather, the desired Simulation is
CELL-DYNCR 4000 Analyzer and the STKS(R) Analyzer 25 in a characteristic that would be detected by an instrument
from Beckman Coulter. In the latter patents, differences in Suitable for analysis of Such cells. For example, by way of
the modes of detection of the respective instruments are Summary, an instrument might detect the presence or amount
emphasized as determinative of whether a particular control of a particular cell by measuring one or more of light Scatter,
may properly function in the instrument. The differences are impedence, radiofrequency response or Some other physical
emphasized with particular reference to a control including response or lack thereof to an applied Stimulus.
a nucleated red blood cell component prepared from mam Thus, as is well known in the art pertaining to blood cell
malian nucleated blood cells or avian or fish erythrocytes. analogs, as exemplified in a number of teachings Spanning
The control disclosed is intended specifically for use in a the past few decades, such as (without limitation) U.S. Pat.
multi-angle light Scatter hematology analyzer, Such as a Nos. 3,574,137; 3,640,896; 3,873,467; 4,219,440; 4,704,
CELL DYNCE Analyzers. The nucleated red blood cell 35 364; 5,207,208; 5,262,327; 5,320,964; 5,389,664; 5,994,
component is made by Stripping a membrane from a nucle 139; 6,187.590; 6,187.590 or the like (all of which are
ated cell. expressly incorporated by reference), the Simulated blood
It would be particularly attractive to provide a control cell component need not originate from a human blood cell.
including a simulated nucleated red blood cell component, In fact, though human blood cells may be Suitably employed
wherein after preparation the nucleus remains at least 40 in the present invention for any of the particular
partially, if not Substantially wholly encapsulated with a components, a preferred component for Simulating a blood
cell will be one that is derived from a blood cell other than
membrane, and especially the natural membrane of the cell; a human blood cell.
thus also rendering the control Sensitive to components of
the instrument System (e.g., lysing agent, stain or dye, or the In the context of one aspect of the present invention, by
like). It would also be attractive to have a simulated nucle 45 way of illustration, and though not intended as limiting,
ated red blood cell component that is useful in a control for examples of Sources of a cell component for Simulating a
any or all of instruments that measure hematological param human nucleated red blood cell include reptile nucleated
eters by (for instance) light Scatter, radiofrequency or elec blood cells, avian nucleated blood cells, fish nucleated blood
trical impedance. cells or the like. Other mammalian blood cells may be
50 employed as well, Such as bovine cells, porcine cells, goat
SUMMARY OF THE INVENTION cells or otherwise. In a particularly preferred embodiment,
the reptilian Source of a nucleated blood cell is an alligator,
The present invention meets the above needs by providing the fish Source is a Salmon and the avian Source is a turkey
an improved control composition, and method of making or chicken. Most preferably, the source of blood cell is an
and using the same. 55 alligator, a Salmon or a mixture thereof. It should be appre
In a particularly preferred embodiment a blood cell is ciated from the above that the Source of the cell component
provided for simulating a nucleated red blood cell. When for Simulating a human nucleated red blood cell need not be
provided, the blood cell has a nucleus and cytoplasm a red blood cell, but may indeed be a nucleated white blood
enclosed by a membrane. Cytoplasm is optionally removed cell or other cell.
from within the membrane and the membrane is handled for 60 Starting materials for providing these components are
preserving it Substantially intact at least partially encapsu widely available and preferably they are provided in a
lating the nucleus. The resulting cell form is Suspended in a Suitable Suspension. For example, the Starting material for a
Suitable Suspension medium and is capable of functioning as cell for Simulating a human nucleated red blood cell may be
a simulated nucleated red blood cell. Suitably Supplied in whole blood form or in a Suspension
In another embodiment, the resulting cell form is incor 65 form or otherwise in a Suitable handling medium, (e.g.,
porated into a multi-parameter hematology control, and without limitation, a liquid or gel medium). Such medium
particularly one that includes a white blood cell component, preferably includes a Suitable amount of an anti-coagulant,
 US 6,653,137 B2
3 4
and may include other ingredients for Stabilizing the cells The fixative agent may be any Suitable art disclosed
during Storage, transport and handling. fixative, including but not limited to those including an
Early Stage Cell Preparation aldehyde, oxazolidine, alcohol, cyclic urea, or the like.
For preparing nucleated red blood cells in accordance Examples of particularly preferred fixatives include, without
with the present invention, upon providing cells from a 5 limitation, formaldehyde, glutaraldehyde, diazolidinyl urea
Suitable Source, the cells are treated as desired for removing (DU), imidazolidinyl urea (IDU), dimethylol urea,
any of the handling medium and for Subsequent processing dimethylol-5,5-dimethylhydantoin, 2-bromo-2-
for forming a simulated human nucleated red blood cell. In nitro propane-1,3-diol, quaternary a damantine;
one preferred embodiment, this can be accomplished by -hydroxymethyl-1-aza3,7-dioxabicyclo (3.3.0)octane and
Simply washing the cells with a Suitable Solution, preferably 5-hydroxymethyl-1-aza-3,7-dioxabicyclo (3.3.0)octane and
a buffered solution and still more preferably an isotonic 5-hydroxy poly-methylene oxy-methyl-1-aza-3,7-
wash Solution, Such as that described (without limitation) in dioxabicyclo (3.3.0)octane, sodium hydroxymethyl
further detail herein, under the heading “Isotonic Wash glycinate, and mixtures thereof, and or the like.
Solution. Other fixatives may be used, Such as those disclosed in
This treatment step may be done below, at or above room 15 U.S. Pat. Nos. 5,196, 182; 5,262,327; 5,460,797; 5,811,099;
temperature, but preferably under conditions for Substan 5,849,517; 6,221,668, and 5,529,933; 6,187,590; 6,187,590,
tially maintaining the integrity of the nucleus of the cell and all of which are hereby incorporated by reference. As will be
a major portion of the cell membrane. appreciated from the above, if desired, the fixative may be
Lysis of Nucleated Blood Cell Selected for preserving one or more of the Surface antigens
In one particularly preferred embodiment of the present present on the blood cells, for permitting Stain or dyeing of
invention, though not in every embodiment, lysis is per the blood cells, or for enabling some other treatment of the
formed upon the nucleated blood cell for permitting cyto cells before or during analysis by a hematology instrument.
plasm from within the cell membrane to escape through the Preferably the volume concentration ratios of fixative to
membrane. It is preferred, in these instances, that the mem cell Suspension ranges from about 1:100 to about 100:1,
brane remain at least partially Surrounding the remaining 25 more preferably about 1:20 to about 20:1, and still more
nucleus. Lysis therefore preferably is a controlled lysis by preferably it is about 1:10 to about 10:1. One highly pre
which the membrane is permeated or otherwise penetrated ferred example of a ratio is about 1:10.
for permitting the cytoplasm to escape, but Still maintaining Isotonic Wash Solution
a Sufficiently rigid structure for at least partially, if not AS indicated in the above, the present invention makes use
Substantially entirely, Surrounding the remaining nucleus. of a Suitable isotonic wash Solution in various Steps of the
Further, the lysis is Sufficient for permitting a stain or dye to Washing, fixing or other handling of the blood cells for
enter in later processing Steps. making a simulated nucleated red blood cell component or
Any Suitable lysing agent may be employed in its art a control. Any Suitable Solution may be employed.
disclosed amount. By way of illustration a Solution includ Preferably, the solution will have a pH ranging from about
ing about 3 to about 20, and more preferably about 5 to about 35 7.0 to about 7.8, and will have an osmolarity ranging from
10 mg percent by weight Saponin may be employed about 270 to about 340. Preferably the solution will include
(wherein mg% refers to mg per 100 ml). Other lysing agents at least one or more, more preferably two or more, Still more
may be employed Such as alcohols (e.g. methanol), a Sur preferably three or more, and still more preferably four or
factant (e.g., Sodium dodecyl Sulfate or a cationic Surfactant more and still even more preferably all of the following
such as Triton X-100TM), or those disclosed in U.S. Pat. Nos. 40 ingredients:
5,858,790 and 6,187,590. 1) a fungicide;
In another embodiment, as illustrated in the Examples 2) an antimicrobial;
Section herein, the present Step of lysing is omitted entirely. 3) a Surfactant;
Later Stage Fixing of Cells
The cells for simulating nucleated red blood cells of the 45 4) a buffer;
present invention may optionally be fixed in a later Stage 5) a metal chelating agent;
fixing Step (e.g., after any optional step of lysing, or prior to 6) a cell nutrient; or
admixing the cells into the medium for forming the resulting 7) an agent for maintaining tonicity.
control) for Substantially preserving the morphology and Though other quantitative ranges may be employed, in
Size of the cells. Any Suitable fixative technique may be 50 one preferred embodiment, at least a buffer, a Surfactant, a
employed, Such as by contact with a fixative agent, heating metal chelating agent and an agent for maintaining tonicity
or a combination thereof. is employed. By way of example, for Such an embodiment
The fixative is contacted with the cells for a time Sufficient the relative amounts of the above respective ingredients may
for Substantially preserving cell morphology and size of the fall within the following (expressed in parts by weight):
cells for at least about 2 weeks and more preferably at least 55 1) a fungicide up to about 5 parts;
about 45 days, and still more preferably at least about 75 2) an antimicrobial up to about 5 parts;
days. The time may vary with factorS Such as the concen 3) a surfactant in about 5 to about 20 parts;
tration of cells to fixative agent, the Strength of the fixative
or the like. However, it is preferred that fixing is accom 4) a buffer in about 5 to about 30 parts;
plished over a duration of at least about 2 hours, more 60 5) a metal chelating agent in about 25 to about 50 parts;
preferably at least about 12 hours; still more preferably at 6) a cell nutrient up to about 5 parts; and
least bout 24 hours; even still more preferably at least about 7) an agent for maintaining tonicity in about 15 to about
48 hours; and even still more preferably at least about 96 35 parts.
hours. At higher temperatures, these times may be shorter. Of course, other ingredients may also be employed in
Fixing may occur with the fixative agent at, below or 65 their art-disclosed quantities, such as (without limitation)
above room temperature. In a highly preferred embodiment that described in U.S. Pat. Nos. 5,858,790 or 6,187,590,
it occurs at about room temperature. hereby incorporated by reference.
 US 6,653,137 B2
S 6
Optionally, though certain of the above may already for Simulating a nucleated red blood cell component is
perform Such functions, the Solution may also include one or provided in a control or kit that also includes an absorbance
more agents that function as a hemolysis inhibitor, an agent (e.g., a dye or stain with a red color Such as, without
aggregating agent, cell size, shape or Volume Stabilizer, limitation, monoaZO monochlorotriazinyl dyes, Such as
metabolite, protein Source, an agent for properly positioning Cibacron Brilliant Red 3B-A, pyrimidinyl dyes, such as
the white blood cell Subpopulation, an antioxidant, a debris Procion Brilliant Red HE-7B; Poncau 3R Red Dye (Cl
reducer or a mixture thereof. 16155); and FD&C Red #40) for simulating a predetermined
Other Control Ingredients hemoglobin concentration, as discussed in U.S. Pat. No.
The Simulated nucleated red blood cells prepared in 5,994,139, hereby incorporated by reference.
accordance with the method disclosed herein may be used In another embodiment, a Stabilizing agent may be
alone or with one or more other control ingredients. The employed for Stabilizing the size of a blood cell component
control ingredients may be part of a kit in which one or more (i.e., analog) when the control product is Subjected to
of the components for Simulating a component of whole temperature ranging from -15 C. to 45 C., as discussed in
blood is provided separately (by itself or with another U.S. Pat. No. 5,994,139, hereby incorporated by reference.
component) from the nucleated red blood cell component, 15 Examples of Suitable Stabilizing agents include Salts,
Such as a component for Simulating a reticulocyte, being polyols, and dimethyl Sulfoxide, e.g., an agent Selected from
provided Separately from the Simulated nucleated red blood glycerol, ethylene glycol and propylene glycol and mixtures
cell component. thereof. The Stabilizing agent may be used at any Suitable
Thus, the present simulated nucleated red blood cell concentration (e.g.,in the Volume percent range of about 5%
components may be used to make a control composition or to about 30%).
kit including one or more additional ingredients Selected AS discussed previously, in another embodiment, the
from a reticulocyte component; b) a white blood cell com nucleated red blood cell component of the present invention
ponent; c) a red blood cell component; d) a nucleated red is combined with a white blood cell component (analogs
blood cell component; e) a platelet component; or f) a prepared from red or white blood cells).
reticulated platelet component, preferably being mixed in a 25 By way of example (without limitation), the white blood
Suitable isotonic Suspension medium. Such a control or cell component may be prepared from a Sample of whole
components thereof may be prepared in accordance with the blood in which white blood cells are fixed prior to lysing of
teachings of U.S. Pat. Nos. 4,436,821; 5,008,201; 5,270, the red blood cells (e.g., with a Suitable lysing agent Such as
208; 5,262,327; 5,432,089; 6,200,500; 6,221,668, all of Ammonium Chloride Trissolution) to remove them from the
which are hereby incorporated by reference. Additional whole blood. After red blood cell lysis, the white blood cells
controls may be prepared in accordance with the Subject are again fixed for a period of at least about 1 hour, and more
matter set forth in U.S. Pat. Nos. 5,529,933; 5,994,139; preferably about 4 to about 5 hours.
5,858,790; or 6,187,590, all of which are hereby incorpo The fixative agent may be any Suitable art disclosed
rated by reference. fixative, including but not limited to those including an
Optionally, for use in a control for attaining a 3 or 5 part 35 aldehyde, oxazolidine, alcohol, cyclic urea, or the like.
differential of white blood cells, the control may contain a Examples of particularly preferred fixatives include, without
lipoprotein. While BSA in any diluent present improves the limitation, formaldehyde, glutaraldehyde, diazolidinyl urea
white blood cell position on the Scattergram, lipoprotein may (DU), imidazolidinyl urea (IDU), dimethylol urea,
be used in an amount effective to provide a Scattergram that dimethylol-5,5-dimethylhydantoin, 2-bromo-2-
represents whole blood, including the proper positioning of 40 nitro propane-1,3-diol, quaternary a damantine;
the five subpopulations of white blood cells. See, e.g., U.S. -hydroxymethyl-1-aza3,7-dioxabicyclo (3.3.0)octane and
Pat. Nos. 5,270,208 and 5,262,327 incorporated by refer 5-hydroxymethyl-1-aza-3,7-dioxabicyclo (3.3.0)octane and
ence. By way of illustration, a lipoprotein Source, preferably 5-hydroxy poly-methylene oxy-methyl-1-aza-3,7-
one consisting essentially of high-density lipoprotein (i.e., dioxabicyclo (3.3.0)octane, sodium hydroxymethyl
HDL) is added at about 0.5 to about 8.0% by volume of the 45 glycinate, and mixtures thereof, and or the like.
control, and more preferably at about 100-175 mg/dl to the Other fixatives may be used, Such as those disclosed in
control composition and a-Tocopherol is further added to the U.S. Pat. Nos. 5,196, 182; 5,262,327; 5,460,797; 5,811,099;
lipoprotein Source to reduce peroxides produced by the 5,849,517; 6,221,668, and 5,529,933; 6,187,590; 6,187,590,
oxidation of the lipoproteins. An example of a Suitable all of which are hereby incorporated by reference. As will be
commercially available form of lipoprotein is SUPER 50 appreciated from the above, if desired, the fixative may be
TRATE (available from Bayer). It will be appreciated that Selected for preserving one or more of the Surface antigens
treatment with the lipoprotein may take place by adding the present on the blood cells, for permitting Stain or dyeing of
lipoprotein to a Suspension medium for the control, it may the blood cells, or for enabling some other treatment of the
take place by a pretreatment outside of the Suspension cells before or during analysis by a hematology instrument.
medium, or a combination thereof. 55 Preferably the volume concentration ratios of fixative to
Examples of commercially available media into which the cell ranges from about 1:100 to about 100:1, more preferably
nucleated red blood cell component of the present invention about 1:20 to about 20:1, and still more preferably it is about
may be added include the STAKCHEXOR) product available 1:10 to about 10:1. A highly preferred example is about 1:10.
commercially from Streck Laboratories. In another example, The fixing preferably takes place for a Suitable period of
5CTM control, from BeckmanCoulter is employed in com 60 time, Such as ranging from about 1 to about 10 hours, more
bination with the nucleated red blood cell component of the preferably about 3 to about 7 hours and still more preferably
present invention. about 4 to about 5 hours. Preferably the fixative agent is
In another embodiment, the cells are Suspended in a maintained at about room temperature, though higher or
Suitable isotonic wash Solution, Such as described herein. lower temperatures may be Suitably employed.
One or a combination of more than one other agents may 65 Lysing or Fixing Omitted
also be included in the control or provided along with the In another embodiment of the present invention, a control
control. For example, in one embodiment, the component is prepared in which one and, more preferably, all of the
 US 6,653,137 B2
7 8
above discussed fixing and lysing Steps are omitted alto Suspension of cells, 6.232,125 (Stating that "Light Scattering
gether in the preparation of the nucleated red blood cell characteristics of the leukocytes are determined within five
component. This is the approach that is particularly pre different angular ranges, all being lower than 40 degrees”);
ferred if the simulated nucleated red blood cell component 6,228,652 (discussing use of “single transducer for simul
is prepared from an alligator red blood cell. In this approach, taneously measuring the DC volume, RF conductivity, light
the cells are provided from their respective Source (e.g., an Scattering and fluorescence characteristics of blood cells
alligator). They are washed with an isotonic wash Solution in passing through a cell-interrogation Zone'); 5,917,584
one or more Washing Steps. In one preferred embodiment, (discussing “differentiation and enumeration of nucleated
they are kept in Such a Solution while being maintained at a red blood cells without using fluorescence”); 5,874,311
temperature of about 10 to about 40°C., and more preferably (discussing “measuring low angle light Scatter signal
about 18 to about 30° C. for longer than about one hour, detected in less than 10 to differentiate reticulocytes from
more preferably longer than about 3 hours and Still more other cell types”) 5,874,310(discusses “exposing a blood
preferably longer than about 12 hours (e.g., about 22 to cell Sample to a reagent System to lyse mature red blood cells
about 30 hours). The resulting cells are then Suspended in a and Subsequently analyzing nucleated red blood cells in a
Suitable medium (as discussed for example in the Section 15 flow cell by optical analysis” and the use of “two angles of
herein entitled “Other Control Ingredients”, optionally with light Scatter Signals' Such as “low angle light Scatter Signals
one or a plurality of other simulated blood components, for detected in less than 10'). Other techniques are also dis
use as a control. cussed in U.S. Pat. Nos. 5,858,790 and 6,187,590. Of
Using Control course, by no means is the mode of Sample testing limited
The following discusses examples of methods of using the to the above. AS mentioned other principles may be used.
control composition to determine the accuracy and repro Thus, in one embodiment, the present invention contem
ducibility of the operation of a multi-parameter automated plates a method of using a control including a nucleated red
hematology instrument. By way of example, a multi blood cell component including the Steps of providing a
parameter automated hematology instrument, Such as a control including a stabilized blood cell suitable for simu
Beckman Coulter Gen-S or LH-700 System (optionally 25 lating a nucleated red blood cell. The control (which may be
employing ACCUCOUNT technology offered by provided in a kit) may also have other components Such as
BeckmanCoulter), the Abbott Cell-Dyn 4000 Hematology those described herein, Such as a white blood cell component
System, Bayer ADVIA 120, and the Sysmex XE2100 for Simulating at least five Subpopulations of white blood
System, is provided, optionally with a slide preparation cells. In one preferred embodiment, though not required, the
module. The claimed control composition is obtained or white blood cell component has been prepared from a red
prepared, optionally to illustrate low, normal or high values blood cell, a white blood cell, or a mixture thereof, at least
of a blood cell component. The control optionally is refrig one of which has been contacted with a lipoprotein.
erated prior to use. If So, at the beginning of testing, the A hematology analyzer is provided. Preferably the ana
control composition is allowed to warm to room temperature lyzer is also adapted for differentiating white blood cells and
for about fifteen minutes, mixed manually, and checked for 35 for analyzing a blood cell Sample by light Scatter, and more
resuspension of contents. preferably by two angles of light Scatter measurement,
The control composition is prepared and analyzed by the which may include a medium angle light Scatter Signal and
Same Standard method as test Samples which may be tested a right-angle light Scatter. Preferably both Signals are leSS
in batch quantities by the use of a Suitable cassette having than about 10 (e.g., one light Scatter angle is in the range of
apertures for receiving test Vials. After preparation, the 40 about 0 to about 4 and the other light scatter angle is in the
control composition and test Samples are analyzed by detect range of about 3 to about 7) to differentiate nucleated red
ing the presence of or counting the population number of blood cells from other cell types. The control is passed
each Subject component type with a multi-parameter auto through the hematology analyzer at a Suitable temperature
mated hematology instrument, which will preferably yield a (e.g., at a temperature in the range of about 18 to about 28
visual display of the data. In one embodiment the control of 45 C., though higher or lower temperatures are also possible).
the present invention is provided in combination with a Optionally, the detection of the Simulated nucleated red
peripheral devices, Such as a device for tracking Samples and blood cell is performed in the absence of a fluorescent Stain
asSociating them with particular data, Such as a bar-code or dye. In one embodiment, the control is passed though the
Scanner System. The control may also be provided in com instrument only a Single time, in order to obtain a Satisfac
bination with a slide preparation kit, Stain or dye-resistant 50 tory result. In another embodiment, the control is repeatedly
labels, lytic reagents (e.g., containing a quaternary ammo passed though the instrument to assure test integrity.
nium salt), blood diluents, or other like components used in The results of the analysis, which will resemble that of
a clinical laboratory Setting. whole blood, may then be analyzed and reported. For
The automated test instrument may employ technology example, the respective population counts obtained from the
that analyzes cell Samples in View of Simultaneous Volume 55 analysis are compared either to known reference value for
conductivity and light Scatter measurements, or Solely by each component type in the control composition, or by
light Scatter. Ordinarily, a starting Sample is employed in comparison of the population counts for each component
combination with Suitable reagents (which may comprise a types in the test Sample with the corresponding values of
component of a kit) and physical agitation for lysing and cell components in the control composition. Data relating to the
measuring by way of flow cytometry. 60 measurement of components in control composition and test
Examples of the various analysis techniques that might be Samples is collected, monitored, Stored, compared and ana
employed will be apparent by familiarity with the above lyzed by electronic means, Such as part of a System including
identified commercially available instruments, as well as by a computer programmed with appropriate Software and
reference to art-disclosed techniques discussed in U.S. Pat. containing appropriate data file Structure, and preferably
Nos. 6,060,322 (discussing “mixing a blood cell sample 65 coupled with one or more devices for outputting or Storing
containing reticulated cells with a reagent composition com the data (e.g., a monitor, a printer, an electronic data Storage
prising a metachromatic dye and a sphering agent to form a medium or the like).
 US 6,653,137 B2
10
The skilled artisan will appreciate that a number of the to a diluent, Such as a multi-parameter hematology control
StepS and ingredients have been disclosed by way of Specific (e.g., STAK-CHEX(R), from Streck Laboratories or 5C, from
example, but that any of a number of alternative Steps or BeckmanCoulter), in a count of approximately 1.5–2.9x10
ingredients at the Suggested or different parameter or cells per mm, to thus form 3 sets controls (fixed, unfixed
concentration, may be Suitably Substituted. Though the and mixture of fixed and unfixed). The resulting 3 sets of
ingredients or Steps have been, in certain instances, controls each indicate the presence of nucleated red blood
described by reference to a particular function or result, it cells when run through an automated hematology analyzer,
should be appreciated that Such discussion is presented Such as the STK-S or GEN-S instruments available from
without intending to be bound by theory. In Some instances, BeckmanCoulter.
the ingredient or Step will perform a different or an addi
tional function or achieve a different result, or multiple other EXAMPLE 2
ingredients or StepS may be Substituted to perform Such In this Example, a nucleated red blood cell component is
function or achieve Such result. Thus, there is no intention to prepared for a control for an automated instrument, Such as
be bound to the breadth of any specific illustrative step, those employing the detection or characterization technol
parameter, ingredient or concentration, where it is apparent 15
ogy of the CELL-DYN 4000 Instrument offered commer
that others may be advantageously be employed in addition cially by Abbott Laboratories. The present Example makes
to or as a Substitute. use of blood obtained from a reptile, Specifically an alligator.
The present invention is further illustrated by particular The blood is obtained from any suitable supplier, and is
reference to the following Examples, it being understood preferably provided in a Suitable medium containing an
that variations of the same may be made while Still remain anticoagulant, Such as in an Alsevers anticoagulant.
ing within the Scope of the invention. The alligator cells are then washed in an isotonic wash
EXAMPLE 1. solution having a pH of about 7.1 and an osmolarity of about
305-325 mC)sm. The wash solution preferably includes one
In this Example, a nucleated red blood cell component is 25 or more of a Surfactant, an antibiotic, a preservative or other
prepared for a control for an automated instrument, Such as ingredients, Such as illustrated by the following (expressed
those employing the detection or characterization technol in approximate amounts) “Table of Illustrative Wash Solu
ogy of the STK-S or GEN-S Instruments offered commer tion Ingredients”:
cially by Beckman Coulter. The present Example makes use
of blood obtained from a reptile, Specifically an alligator.
The blood is obtained from any suitable supplier, and is
preferably provided in a Suitable medium containing an Table of Illustrative Wash Solution Ingredients
anticoagulant, Such as in an Alsevers anticoagulant. 40 mg% Methyl Paraben
The alligator cells are then washed in an isotonic wash 300 mg% Polyethylene Glycol-molecular weight 20,000
solution having a pH of about 7.1 and an osmolarity of about 1675 mg% Ethylenediaminetetraacetic Acid
35 933 mg% Magnesium Gluconate
305-325 mC)sm. The wash solution preferably includes one 639 mg% Sodium Phosphate Dibasic anhydrous
or more of a Surfactant, an antibiotic, a preservative or other 25 mg% Adenosine
ingredients, Such as illustrated by the following (expressed 25 mg% Inosine
40 mg% Neomycin Sulfate
in approximate amounts) “Table of Illustrative Wash Solu 15 mg% Chloramphenicol
tion Ingredients: 40

One part by Volume concentrated Alligator cells is treated

with ten parts by Volume of a lysing agent, Such as approxi
Table of Illustrative Wash Solution Ingredients mately 7-9mg % Saponin, introduced into a suitable iso
40 mg% Methyl Paraben 45 tonic wash Solution, Such as describe previously. The cells
300 mg% Polyethylene Glycol-molecular weight 20,000 are left in this solution for about 22–30 hours at 18-30° C.
1675 mg% Ethylenediaminetetraacetic Acid The resulting Supernatant containing the released hemoglo
933 mg% Magnesium Gluconate bin is removed and the lysed alligator cells are resuspended
639 mg% Sodium Phosphate Dibasic anhydrous
25 mg% Adenosine in an isotonic wash Solution, Such as that described previ
25 mg% Inosine 50 ously. Though hemoglobin has been Substantially removed
40 mg% Neomycin Sulfate from the cells, the cytoplasmic membrane is still Substan
15 mg% Chloramphenicol
tially intact, Surrounding the nucleus.
About 1 part by Volume of concentrated, lysed alligator
A portion of the alligator cells are left unfixed and will be cells is fixed in an aldehyde fixative (e.g., in about ten parts
employed in at least two of the controls prepared according 55 of about 1 to about 5% formaldehyde) in the above isotonic
to the present example. The remaining portion is fixed with wash Solution. The cells are fixed in this solution for about
an aldehyde fixative (e.g., formaldehyde, glutaraldehyde or 22–30 hours at about 18-30° C.
a mixture thereof) in a Suitable proportion (e.g., about 1:1). The fixative is then removed from the cells by resuspend
(e.g., about 0.01%–0.05% glutaraldehyde, 0.05% -0.1% ing the cells in an isotonic wash Solution, Such as described
formaldehyde, or mixtures thereof). The glutaraldehyde 60 in the above discussion. The resulting fixed and lysed
Samples are fixed according to cell concentration, while the alligator cells are added to a Suitable medium, Such as a
formaldehyde Samples are fixed by Volume. After contact multi-parameter hematology control (e.g., Para 12 Plus
with any aldehyde, the cells are placed in an isotonic wash ReticsTM, from Streck Laboratories, Inc. in which the white
Solution (e.g., as described above) for 22–30 hours at a blood cell component is prepared with a step of fixing prior
temperature ranging from about 18-30° C. 65 to lysis of red blood cell components, or the control
Each of the fixed cells (for each fixative type), the unfixed described generally in the following Table of Illustrative
cells and a mixture of fixed and unfixed cells are then added Control Ingredients, also optionally including a reticulocyte
 US 6,653,137 B2
11 12
analog) at a count approximating about 1.5–2.9x10 (or
approximately 10% of the white blood cell component
population) of nucleated red blood cells per mm in the Table of Illustrative Diluent
resulting control.
1173 mg% Ethylenediaminetetraacetic Acid
653 mg% Magnesium Gluconate
447 mg% Sodium Phosphate Dibasic Anhydrous
300 mg% Polyethylene Glycol-molecular weight 20,000
Table of Illustrative Control Ingredients 25 mg% Adenosine
-Unfixed red blood cells 25 mg% Inosine
-Human white blood cells that are suspended about 1 part phosphate 1O 140 mg% Sodium Hydroxide
buffered saline having an osm of about 280 and including about 0.2% 40 mg% Methyl Paraben
EDTA and about 1 part phosphate buffered saline having an osm of 40 mg% Neomycin Sulfate
about 280 and including about 0.2% EDTA and about 15% Nuosept 145, 5 mg% Sodium Fluoride
held at about 50° C. for about 6 days 1000 mg% Glucose
-Human platelets that are treated in a solution of about 1:1 isotonic 15 mg% Chloramphenicol
wash solution (described above) plus about .025% glutaraldehyde at about 15
22 C. for about 2 days
-About 0.03% Soybean protease inhibitor The resulting fixed and lysed Salmon cells are added to a
-Diluent (as set forth in the “Table of Illustrative Diluent of Suitable medium, Such as a multi-parameter hematology
Example 3, plus about 50 mg% NaF and about 10 mg % Sulfasalazine) control (e.g., e-CHEXTM Control from Sysmex) at a count
approximating about 1.5-2.9x10 cells per mm in the
resulting control. The resulting control indicates the pres
The resulting control indicates the presence of nucleated ence of nucleated red blood cells when run through an
red blood cells when run through an automated hematology automated hematology analyzer, such as the XE2100 from
analyzer, such as the CELL-DYN 4000 from Abbott Labo Sysmex.
ratories. 25 Like results are also obtained when the concentrations of
the nucleated red blood cell component are varied to Simu
EXAMPLE 3
late normal, low or high values of the components.
Quantitative amounts of the nucleated red blood cell
component may also be obtained with the above controls, on
In this Example, a nucleated red blood cell component is an instrument employing a detector Suitable for counting.
prepared for a control for an automated instrument, Such as While the above has been described in connection with
those employing the detection or characterization technol simulation of human nucleated red blood cells, it should be
ogy of the XE 2100 Hematology Instrument offered com appreciated that the invention may be modified within the
mercially by SySmeX. The present Example makes use of Scope of the present invention for achieving a simulation of
35 nucleated red blood cells in mammals other than humans, or
blood obtained from a fish, specifically a salmon. The blood
is obtained from any Suitable Supplier, and is preferably other animals. Thus, the present invention is not intended as
provided in a Suitable medium containing an anticoagulant, limited to the use of the subject matter herein for clinical
Such as in an Alsevers anticoagulant. analysis of human blood, but may be extended as desired to
a variety of Veterinary or other applications where it is
40 desired to simulate a nucleated cell.
The blood cells are washed into a Suitable medium (e.g.,
having a pH of about 7.4 and an osmolarity of about The foregoing discussion discloses and describes merely
320-340 mOsm, Such as Hanks’ Balanced Salt Solution exemplary embodiments of the present invention. One
with Urea, having the following approximate composition: skilled in the art will readily recognize from Such discussion
45
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
18.5 mg% Calcium Chloride Dihydrate invention as defined in the following claims. All patents and
9.8 mg% Magnesium Sulfate Anhydrous
40 mg% Potassium Chloride other publications cited herein are expressly incorporated by
6 mg% Potassium Phosphate Monobasic Anhydrous reference.
4.9 mg% Sodium Phosphate Dibasic Anhydrous 50
What is claimed is:
100 mg% Glucose
1. A method of using a control including a nucleated red
35 mg% Sodium Bicarbonate
2 mg% Antimicrobial blood cell component comprising the Steps of
250 mg% Urea a) providing a control including a stabilized blood cell
700 mg% NaCl
Suitable for Simulating a nucleated red blood cell, the
55
Stabilized blood cell including a membrane enclosing a
nucleus and wherein the cytoplasm of the Stabilized
Salmon cells are then fixed 1:1 (by volume) with a blood cell has been substantially removed from within
suitable aldehyde fixative (e.g., about 0.3-1.5% Said membrane,
formaldehyde) in a liquid medium as described above, Such 60 b) providing a hematology analyzer for analyzing a blood
as the Hank's Solution. The cells are fixed in this solution for cell Sample by at least two angles of light Scatter
about 22–30 hours at about 18-30° C. The fixative is then measurement to differentiate nucleated red blood cells
removed from the Salmon cells by resuspending the cells in from other cell types;
a Suitable diluent, Such as that having a pH of about 7.1 and c) passing the control through the hematology analyzer
an osmolarity of about 285-305 mC)sm and including one or 65 for detection of the simulated nucleated red blood cells;
more of a Surfactant, an antibiotic, a preservative or other
ingredients, Such as approximated in the following: d) reporting nucleated red blood cells in Said control.
 US 6,653,137 B2
13 14
2. The method of claim 1, wherein a first angle of light ii) a white blood cell component for simulating at least
Scatter measurement is in the range of about 0 to about 4 five subpopulations of white blood cells, the white
and a Second angle of light Scatter measurement is in the blood cell component having been prepared from a
range of about 4 to about 7. red blood cell, a white blood cell, or a mixture
3. The method of claim 1, wherein the detection is thereof, at least one of which has been contacted with
performed in the absence of a fluorescent Stain or dye. a lipoprotein;
4. The method of claim 1, wherein the passing Step takes
place at a temperature in the range of about 18 to about 28 b) providing a hematology analyzer for differentiating
C. white blood cells and for analyzing a blood cell Sample
5. A method of using a control including a nucleated red by at least two angles of light Scatter measurement both
blood cell component comprising the Steps of being less than about 10 to differentiate nucleated red
a) providing a control that is Suitable for use for at least blood cells from other cell types;
3 days, including: c) passing the control through the hematology analyzer at
i) a stabilized blood cell suitable for simulating a a temperature in the range of about 18 to about 28 C.
nucleated red blood cell, the stabilized blood cell 15 for detection of the simulated nucleated red blood cells
including a membrane enclosing a nucleus and
wherein the cytoplasm of the stabilized blood cell and subpopulations of white blood cells;
has been substantially removed from within said d) reporting simulated white blood cells in the control;
membrane, and
ii) a white blood cell component for simulating at least e) reporting simulated nucleated red blood cells in the
three subpopulations of white blood cells; control.
b) providing a hematology analyzer differentiating white 10. The method of claim 9, wherein a first light scatter
blood cells and for analyzing a blood cell Sample by at angle is in the range of about 0 to about 4 and a second light
least one angle of light Scatter measurement to differ Scatter angle is in the range of about 3 to 7.
entiate nucleated red blood cells from other cell types; 11. The method of claim 9, wherein the control is passed
c) passing the control through the hematology analyzer 25
only a single time through the hematology analyzer for
for detection of the simulated nucleated red blood cells
and subpopulations of white blood cells; assuring a reliable result.
12. The method of claim 9, wherein the stabilized blood
d) reporting simulated white blood cells in the control; cell Suitable for Simulating a nucleated red blood cell is
and unfixed.
e) reporting simulated nucleated red blood cells in the 13. A method of using a control including a nucleated red
control. blood cell component comprising the Steps of
6. The method of claim 5, wherein a first angle of light a) providing a control including a stabilized blood cell
Scatter measurement is in the range of about 0 to about 4 Suitable for Simulating a nucleated red blood cell, the
and a Second angle of light Scatter measurement is in the blood cell including a membrane enclosing a nucleus
range of about 4 to about 7. 35
and wherein the stabilized blood cell is selected from
7. The method of claim 5, wherein the detection is the group consisting of reptile blood, fish blood and
performed in the absence of a fluorescent Stain or dye. mixtures thereof;
8. The method of claim 5, wherein the passing step takes b) providing a hematology analyzer or analyzing a blood
place at a temperature in the range of about 18 to about 28
C. 40 cell Sample by at least two angles of light Scatter
9. A method of using a control including a nucleated red measurement to differentiate nucleated red blood cells
blood cell component comprising the Steps of from other cell types;
a) providing a control that is Suitable for use for at least c) passing the control through the hematology analyzer
45 days, including: for detection of the simulated nucleated red blood cells;
i) a stabilized blood cell suitable for simulating a 45 d) reporting nucleated red blood cells in Said control.
nucleated red blood cell the stabilized blood cell 14. The method of claim 13, wherein the stabilized blood
including a membrane enclosing a nucleus and cell Suitable for Simulating a nucleated red blood cell is
wherein the cytoplasm of the stabilized blood cell unfixed.
has been substantially removed from within said
membrane,
 UNITED STATES PATENT AND TRADEMARK OFFICE
CERTIFICATE OF CORRECTION

PATENT NO. : 6,653,137 B2 Page 1 of 1

DATED : November 25, 2003
INVENTOR(S) : Wayne L. Ryan

It is certified that error appears in the above-identified patent and that said Letters Patent is
hereby corrected as shown below:

Column 12
Line 66, delete “a” and insert -- and --.
Column 13
Line 46, after “red blood cell insert --, --.
Column 14
Line 9, after “measurement' insert --, --.
Line 10, after “10 insert --, --.
Line 15, delete “C.” and insert -- C --.
Line 34, before “blood' insert -- Stabilized --.
Line 39, delete 'or' and insert -- for --.
Line 44, after “cells; insert -- and --.

Signed and Sealed this

Fourteenth Day of September, 2004

WDJ
JON W. DUDAS
Director of the United States Patent and Trademark Office