U.S. patent application number 11/136889 was filed with the patent office on 2005-10-06 for quality control method.
Invention is credited to Elliott, Michael N., Fischer, Timothy J., Naylor-Schlipp, Nancy R., Young, Carole J..
Application Number | 20050221496 11/136889 |
Document ID | / |
Family ID | 27491629 |
Filed Date | 2005-10-06 |
United States Patent
Application |
20050221496 |
Kind Code |
A1 |
Young, Carole J. ; et
al. |
October 6, 2005 |
Quality control method
Abstract
A method of quality control to diagnose the cause of a
malfunction of an instrument. The method uses measurements of the
physical property of a sample to diagnose the cause of a
malfunction of an instrument. The spatial position of a control
product sample is analyzed. Alternatively, the spatial position of
a statistically significant number of patient blood samples can be
used. The method enables the monitoring of an instrument for
problems associated with debris and noise caused by red cell lysis
inefficiency; instrument reagents pup volume settings; instrument
laser alignments; instrument gain settings; and flow noise caused
by partial plugs, residual plugs or other flow problems. The method
provides a more specific indication of the type and cause of an
instrument malfunctioning than non specific flagging provided by
prior art methods.
Inventors: |
Young, Carole J.; (Raleigh,
NC) ; Elliott, Michael N.; (Fort Lauderdale, FL)
; Naylor-Schlipp, Nancy R.; (Waverly, PA) ;
Fischer, Timothy J.; (Raleigh, NC) |
Correspondence
Address: |
BECKMAN COULTER, INC
PO BOX 169015
MAIL CODE 32-A02
MIAMI
FL
33116-9015
US
|
Family ID: |
27491629 |
Appl. No.: |
11/136889 |
Filed: |
May 25, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11136889 |
May 25, 2005 |
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10996863 |
Nov 24, 2004 |
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10996863 |
Nov 24, 2004 |
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10909594 |
Aug 2, 2004 |
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10909594 |
Aug 2, 2004 |
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10214717 |
Aug 9, 2002 |
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10214717 |
Aug 9, 2002 |
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08787408 |
Jan 22, 1997 |
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6509192 |
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08787408 |
Jan 22, 1997 |
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08432435 |
Apr 28, 1995 |
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08432435 |
Apr 28, 1995 |
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08386711 |
Feb 8, 1995 |
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5529933 |
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08386711 |
Feb 8, 1995 |
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08081529 |
Jun 23, 1993 |
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08081529 |
Jun 23, 1993 |
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07840438 |
Feb 24, 1992 |
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Current U.S.
Class: |
436/63 ;
436/16 |
Current CPC
Class: |
Y10T 436/101666
20150115; G01N 2015/1493 20130101; G01N 2015/1062 20130101; G01N
33/96 20130101; G01N 33/92 20130101; G01N 15/1012 20130101; G01N
15/12 20130101; Y10T 436/105831 20150115; G01N 2015/1486 20130101;
Y10T 436/108331 20150115; Y10T 436/102499 20150115; G01N 2015/1087
20130101; Y10T 436/106664 20150115; G01N 2496/05 20130101; Y10T
436/107497 20150115; G01N 2496/10 20130101; G01N 33/5094
20130101 |
Class at
Publication: |
436/063 ;
436/016 |
International
Class: |
G01N 033/48 |
Claims
1-39. (canceled)
40. A method comprising the steps of: (a) providing a composition
comprising white blood cells and red blood cells; (b) treating the
composition resulting from step (a) with a lytic reagent to lyse
the red blood cells from the white cells; (c) collecting the white
blood cells resulting from step (b); (d) fixing the white blood
cells collected in step (c); (e) washing the fixed white blood
cells; and, (f) admixing the product of step (e) in a suspension
medium suitable for delivering said product to a hematology
analyzer for analysis.
41. A method comprising the steps of: (a) providing a quantity of
avian nucleated red blood cells; (b) treating said nucleated red
blood cells with a lytic reagent; (c) collecting the blood cells
resulting from step (b); (d) fixing the blood cells collected in
step (c); (e) washing the fixed blood cells; and, (f) admixing the
product of step (e) in a suspension medium suitable for delivering
said product to a hematology analyzer for analysis.
42. A method comprising the steps of: (a) providing a quantity of
mammalian nucleated red blood cells; (b) treating said nucleated
red blood cells with a lytic reagent; (c) collecting the blood
cells resulting from step (b); (d) fixing the blood cells collected
in step (c); (e) washing the fixed blood cells; and, (f) admixing
the product of step (e) in a suspension medium suitable for
delivering said product to a hematology analyzer for analysis.
43. A method comprising the steps of: (a) providing a hematology
control product including a nucleated blood cell analog, wherein
the analog is a blood cell that has been treated with a lytic
reagent and a fixative; (b) providing an instrument for analyzing a
blood cell sample by light scatter; (c) passing the control product
through the instrument for detection of said nucleated blood cell
analog; and (d) reporting said nucleated blood cell analog in the
control product.
44. A method according to claim 43, said instrument having the
capability to analyze a blood cell sample by light scatter at
various angular ranges.
45. A method according to claim 44, said instrument having the
capability to analyze a blood cell sample by at least one of low
angle light scatter, median angle light scatter, and high angle
light scatter.
46. A method comprising the steps of: (a) providing a hematology
control product including a white blood cell analog, wherein the
analog is a blood cell that has been treated with a lytic reagent
and a fixative; (b) providing an instrument for analyzing a blood
cell sample by light scatter; (c) passing the control product
through the instrument for detection of said nucleated blood cell
analog; and (d) reporting said nucleated blood cell analog in the
control product.
47. A method according to claim 46, said instrument having the
capability to analyze a blood cell sample by light scatter at
various angular ranges.
48. A method according to claim 47, said instrument having the
capability to analyze a blood cell sample by at least one of low
angle light scatter, median angle light scatter, and high angle
light scatter.
49. A blood cell analog produced by (a) providing a composition
comprising white blood cells and red blood cells; (b) treating the
composition resulting from step (a) with a lytic reagent to lyse
the red blood cells from the white cells; (c) collecting the white
blood cells resulting from step (b); (d) fixing the white blood
cells collected in step (c); (e) washing the fixed white blood
cells; and, (f) admixing the product of step (e) in a suspension
medium suitable for delivering said product to a hematology
analyzer for analysis, said analog having the light scatter
characteristics of a white blood cell of interest.
50. A blood cell analog produced by: (a) providing a quantity of
avian nucleated red blood cells; (b) treating said nucleated red
blood cells with a lytic reagent; (c) collecting the blood cells
resulting from step (b); (d) fixing the blood cells collected in
step (c); (e) washing the fixed blood cells; and, (f) admixing the
product of step (e) in a suspension medium suitable for delivering
said product to a hematology analyzer for analysis said analog
having the light scatter characteristics of a nucleated blood cell
of interest.
51. A blood cell analog produced by: (a) providing a quantity of
mammalian nucleated red blood cells; (b) treating said nucleated
red blood cells with a lytic reagent; (c) collecting the blood
cells resulting from step (b); (d) fixing the blood cells collected
in step (c); (e) washing the fixed blood cells; and, (f) admixing
the product of step (e) in a suspension medium suitable for
delivering said product to a hematology analyzer for analysis said
analog having the light scatter characteristics of a nucleated
blood cell of interest.
52. A blood cell analog produced by: (a) providing a quantity of
nucleated blood cells selected from avian, mammalian, and reptile
nucleated blood cells; (b) treating said nucleated red blood cells
with a lytic reagent; (c) collecting the blood cells resulting from
step (b); (d) fixing the blood cells collected in step (c); (e)
washing the fixed blood cells; and, (f) admixing the product of
step (e) in a suspension medium suitable for delivering said
product to a hematology analyzer for analysis said analog having
the light scatter characteristics of a nucleated blood cell of
interest.
53. A method comprising the steps of: (a) providing a quantity of
nucleated red blood cells selected from avian, mammalian, and
reptile nucleated red blood cells; (b) treating said nucleated red
blood cells with a lytic reagent; (c) collecting the blood cells
resulting from step (b); (d) fixing the blood cells collected in
step (c); (e) washing the fixed blood cells; and, (f) admixing the
product of step (e) in a suspension medium suitable for delivering
said product to a hematology analyzer for analysis.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of copending
application U.S. Ser. No. 08/386,711 filed Feb. 8, 1995, which is a
continuation or U.S. Ser. No. 08/081,529 filed Jun. 23, 1993 which
is now abandoned, which was a continuation of U.S. Ser. No.
07/840,438 filed Feb. 24, 1992 which is now abandoned.
FIELD OF INVENTION
[0002] This invention relates to a novel method to increase the
system quality control capabilities of hematology instruments. The
method has particular utility for instruments which measure (1)
volume measured by D.C. current, (2) high frequency (RF) size, (3)
opacity, and (4) light scatter to discriminate cell populations of
blood. The method has found additional utility for instruments
which measure (1) volume measured by D.C. current and (2) high
frequency (RF) size. The method has also found utility for
instruments that measure cell volume only by D.C. current.
BACKGROUND OF THE INVENTION
[0003] Quality control long has been a necessary and routine
procedure in clinical hematology. Accuracy in the counting of red
blood cells and white blood cells, including differentiating among
the subpopulations of white blood cells is dependent, in part, upon
the use of adequate control products and methods of using the
control products. With the numerous types of equipment for particle
counting now available, quality control by the use of control
products is necessary, since the possibility of malfunctioning of
the instrument is ever present. The traditional method of
maintaining a quality control program for automatic particle
counting equipment has consisted of providing fresh human blood as
a whole blood standard. However, this fresh blood is usable for
only one day, therefore, durable blood products were developed.
[0004] Hematology control products, which contain reference blood
cell analogs, which monitor the accuracy and precision of blood
cell counting devices are important. It is recognized that there is
a present need for new methods of using blood cell analogs for
maintaining the accuracy of white cell differentiation and other
parameters when employing such blood cell counting devices.
[0005] The control products should approximate that of fresh whole
blood as closely as possible. Attempts have been made to provide
suitably sized particles in stable suspensions by the use of
ragweed pollen, polystyrene, latex, various organic materials and
fixed human red cells. None of these suspensions have proved
suitable for use as a control product for white cell
differentiation of at least four subpopulations of leukocytes.
[0006] The material used for maintaining quality control,
hereinafter called a hematology control product or control product,
can under specific circumstances be used also to calibrate
hematology instruments. For the purposes of this invention, the
control product used in the method to determine whether an
instrument is properly functioning will contain one or more analogs
suspended in a liquid media, which when analyzed simulates at least
one physical or biological property of blood which the instrument
is capable of analyzing. As used herein, an analog is defined as a
particle which simulates at least one physical or biological
property of a target population. As such, some automatic machines
are able to analyze only certain components of a control product,
despite the control product having additional parameter components
susceptible to analysis by other machines. Heretofore, there has
been an absence of methods developed for using a control product to
provide quality control of the instrument's performance. Control
products typically provide checks for at least four subgroups of
leukocytes namely, lymphocytes, monocytes, neutrophils and
eosinophils. Prior art use of control products have focused upon
checking whether the instrument provides the proper count and
percentage of the analogs. However, the method of this invention
provides additional information to evaluate and diagnose instrument
performance.
[0007] It is evident that a control product must accurately
indicate, on a comparative basis, what a test sample of fresh blood
constitutes with regard to the determinations in question. It is
further evident how important it is for the control product to
simulate fresh blood, since blood components, such as red blood
cells, can hemolyze slowly and undergo changes in size and shape
within hours after removal from a blood donor. Similarly, non
stabilized white blood cells suffer degenerative changes.
[0008] In general, the process of the prior art for making analogs
focused on using red blood cells which had maintained or reduced
their original volume prior to fixation. Shrinking or expansion of
the cells by manipulating their osmotic environment prior to
fixation has had its limitations. Previously, shrinking or swelling
non-human erythrocytes more than about 30% to 50% caused excessive
cell association or lysis of the cell.
[0009] U.S. Pat. No. 3,873,467 to Hunt teaches a hematologic
reference control comprising a suspension of washed, stabilized
human red blood cells in a nonproteinaceous aqueous suspension
fluid that replaces the plasma in human blood. Stability in the
reference control is attained by conditioning the cells by the
inclusion in the aqueous suspension fluid of materials tending to
make the cells assume a spherical shape, without substantial change
in the mean cell volume of the cells, as well as imparting to the
cells a resistance to the normal tendency of degrading with time.
The aqueous suspension fluid furthermore produces an environment
for the cells inhibiting biological activity. In a preferred
embodiment there is further included in the reference control a
minor amount of fixed human red blood cells, processed to have a
substantially increased mean cell volume. The fixed cells are
resistant to a change in cell volume, and to dissolution under the
action of lysing reagents producing lysing of the stabilized cells.
The fixed red blood cells in the reference control substitute for
the white cell population in human blood.
[0010] In U.S. Pat. No. 4,704,364, to Carver, et al., there are
disclosed controls for thresholds and additional operational
performances for electronic particle counters typified by the
COULTER COUNTER.RTM. Model S-Plus type analyzers. However, there is
now a need for new methods of using a whole blood cell control
product for electronic optical particle counters typified by the
COULTER.RTM. VCS analyzer. The VCS analyzer permits the
differentiation of at least four populations of leukocytes.
[0011] Any system for automated differential counting of human
leukocytes, which distinguishes at least four populations of
leukocytes from other cells in the blood on the basis of size
range, volume distribution, light scatter range, and electrical
opacity and conductivity sensitivities requires that the control
product closely simulate the range, distribution and sensitivities
characteristics of the respective cells in normal human blood.
[0012] Human lymphocytes, monocytes, neutrophils, basophils and
eosinophils have a specific size distribution range and optical
characteristics. Both the upper and lower size limits for each
subpopulation of leukocytes should be represented in a reference
control product. In addition, the mean cell volume of each
leukocyte subpopulation in the control product should approximate
that oaf normal human blood. Moreover, it is necessary that the
liquid suspension media used for the control product does not cause
significant shrinking or swelling of the cells. Still further, the
aging of the control product should not result in deterioration of
the volume distribution histogram characteristics or other
parameters. A further requirement for the leukocyte analogs in the
control product for multi-parameter instruments is that in order to
be counted and differentiated, the analog cells in a whole blood
control product must not be completely lysed by the lytic
reagent.
[0013] A variety of media have been used in conjunction with blood
cell analogs. In U.S. Pat. No. 4,299,726, a multi-purpose diluent
and a media is disclosed. The diluent is used to precondition red
blood cells and consists essentially of lactose, sodium azide and a
non-ionic surfactant; is pH adjusted and osmolality adjusted. The
media is used for a carrier of the whole blood control product and
includes lactose, fungicides and antibiotics. It also-includes
additional components which alter red blood cell membranes,
including bile salts and cholic acid derivatives, phenothiazine
compounds and the salts thereof having antihistamine properties,
and 4-amino-benzoic acid ester derivatives and their salts having
local anesthetic properties.
[0014] One disadvantage of the prior art medias is that, when used
in conjunction with red blood cells and fixed human white blood
cells or white blood cell analogs, the control product does not
simulate a whole blood sample in instruments which differentiate at
least four subpopulations of leukocytes. The specific parameters of
the red and white blood cells which it is desirable to measure
dictate some of the necessary characteristics of a suitable media
for a whole blood reference control product. It is desirable to
know the volume of the red cell. Once this measurement is
ascertained and the red cells have been counted, the packed cell
volume or hematocrit can be computed. Therefore, the suspension
media of the control product should be capable of equilibrating and
stabilizing the volume of red blood cells in the sample so that its
mean cell volume can be measured (MCV).
[0015] A control product should also be rendered free of any
particulate matter that would perhaps demonstrate interference in
lower size thresholds corresponding to that of human platelet size
and distribution. Concomitantly, the suspension media would
optionally include bacteriostatic agents to prevent the growth of
microorganisms after packaging the control product.
[0016] Although red blood cells (erythrocytes) and white blood
cells (leukocytes) nominally have different sizes, their size
ranges tend to overlap, or at least under certain conditions of
health could overlap. Moreover, the opacity of these two types of
blood cells also may overlap. Erythrocytes and the lymphoid
leukocytes unfortunately overlap considerably in cell sizes, and it
is not practical to count one in the presence of the other by size
discrimination alone. Traditional practice involved the use of a
strong lytic reagent that stromatolyses the erythrocytes, reducing
them to very small particles or causing membrane solubilization, to
eliminate them from being counted; and strips most, if not all, of
the cytoplasm from the leukocytes, leaving only their
lyse-resistant nuclei to be counted. Since original leukocyte cell
volume is drastically affected and reduced to a minimum, only a
single leukocyte population is discernible by this older form of
blood cell size analysis.
[0017] U.S. Pat. No. 3,741,875, Ansley et al., describes a process
for obtaining a differential white blood cell count. A cytological
fixing agent, which is a monoaldehyde, such as formaldehyde, is
added to a blood sample. A hemolyzing agent is added after the
fixation step to cause the red blood cells to release their
hemoglobin content into solution. Addition of a specific
cytochemical substrate, chromogenic precipitating coupling reagent,
and pH buffer causes deposition of an insoluble dye in a specific
type of cell containing an immobilized enzyme. The solution
containing the dyed blood cells then is passed through a
photometric counter. Using different specific substrates for
different enzymes contained in specific kinds of cells, absolute
and relative counts of the different kinds of cells are obtained.
The cytological fixing solution utilized only a monoaldehyde.
Dialdehydes are stated to be unsuitable, since they cross-link and
produce extracellular precipitates.
[0018] U.S. Pat. No. 4,485,175, to Ledis, et al., concerns a method
and reagent system for three-volume differential determination of
lymphocyte, mononuclear, and granulocyte populations of leukocytes,
using quaternary ammonium salts as lysing agents and the COULTER
COUNTER.RTM. Model-S-Plus automated blood counter, which instrument
employs only direct current field excitation.
[0019] U.S. Pat. No. 4,751,179 to Ledis, et al. describes a reagent
system, including saponin in a lysing reagent and a rapidly active
cross-linking agent such as glutaraldehyde as a fixing reagent,
which reproducibly affects whole blood to cause the red blood cells
to stromatolyze and modifies the leukocytes to generate data to
define four distinct clusters for detection and classification by
flow analysis instrumentation. The clusters represent the four
major leukocyte types found in blood: lymphocytes, monocytes,
neutrophils and eosinophils, thus providing a method of leukocyte
differential analysis. According to Ledis, et al., previous methods
of flow analysis of leukocytes using D.C. volume, or light scatter
at various angles have shown only three clusters of leukocytes,
corresponding to lymphocytes, granulocytes and monocytes. The
parameters used by Ledis, et al. for the leukocyte classification
include combinations of two or more of DC (Coulter) volume, high
frequency (RF) size, Coulter opacity (RF size/DC volume), light
scatter at various angular ranges, and fluorescence at various
wavelengths of illumination.
[0020] Electronic counters which employ the Coulter Principle,
first described in U.S. Pat. No. 2,656,508, express a true
reflection of particle counts. According to the Coulter Principle,
when a particle of microscopic size is suspended in an electrolyte
liquid, is passed throgh an electrical field of small dimensions of
an order approaching those of a particle, there will be a momentary
change in the field's electric impedance. If the electrical field
is excited by a direct (DC) or low frequency current, the
electrical change is closely proportional to the volume of the
particle. In commercial apparatus, the changes are detected by some
suitable means and used to operate counters and analyzers. The
analyzers associated with such apparatus classify and size
particles into populations based upon particle volume and record
the data obtained.
[0021] The Coulter Principle invention was expanded materially in
U.S. Pat. No. 3,502,974, Coulter, et al., using radio frequency
(RF) current in addition to DC current field excitation, to provide
not only DC volume information concerning the particle studied, but
also information due to the composition and nature of the material
constituting the particle. This patent discloses apparatus capable
of distinguishing between particles of identical size, but of
different material. By generating the particle sensing field by
means of both a low frequency or direct current (DC) and radio
frequency (RF) current excitation, two or more interrelated output
signals can be derived from the passage of a single particle
through the electrical field. This is due to the fact that,
although the particles, such as blood cells, are nearly always
insulators with respect to low frequency or direct current fields,
they are capable of carrying or impeding radio frequency current
differently from the surrounding electrolyte. This may be due to
differences in the dielectric constant in the case of homogeneous
particles, or to the sac-like structure in the case of blood cells
which have, enclosed in an extremely thin membrane, contents having
conductivities different from the electrolyte. Thus, while all the
DC current goes around a blood cell, some of the RF current will go
through it. The ease with which RF current will go through a
particle is a measure of what is termed its "electrical
transparency", or simply "transparency", in analogy with light
transmission; whereas, a particle's ability to impede RF current is
termed its "opacity". In later publications, "opacity" is defined
as the RF impedance divided by the DC impedance.
[0022] The relative electrical opacity of a particle becomes an
identifying feature of the particle contents and hence its particle
type for classification purposes. To the extent that dirrerent
types of particles each possess a different opacity, the difference
between them is detectable. However, significantly different
particles can possess substantially the same opacity and such
particles cannot be classified effectively in this manner. In U.S.
Pat. No. 3,836,849, Coulter, et al. taught that it is possible to
change selectively the opacity of particle types by treatment of
the particles, so that detectable differences result.
[0023] The COULTER COUNTER.RTM. Model S-Plus automated blood cell
counter is designed to dilute a sample of whole blood in an
isotonic diluent, add a lysing agent, and shortly thereafter begin
counting. Thus, a diluent-lysing system must provide erythrocyte
lysing kinetics sufficiently rapid to effect complete
stromatolysation of the red blood cells (erythrocytes) during the
lysing period. In addition, changes in leukocyte volume must be
minimal during the data collection step, and ideally should be
stable for several minutes.
[0024] COULTER Model VCS is a semi-automated analytical instrument
that analyzes blood by using DC (Coulter) volume, Coulter opacity
and light scatter at various angular ranges. The COULTER Model VCS
uses a reagent system to obtain a five part differentiation in the
total leukocyte count which provide quantitative analysis of the
lymphocyte, monocyte, neutrophil, eosinophil and basophil
population. The reagent system includes a quench, added after the
weak "acid" lyse, the operation of which is to greatly reduce lytic
action on the white cells. Shortly after the quench, the instrument
begins measuring the volume, opacity and light scattering
characteristics of the remaining white blood cells. The Model VCS
must provide erythrocyte lysing kinetics sufficiently rapid to
effect complete stromatolysation of the red blood cells during the
lysing period while not affecting the leukocyte cells as to their
volume, Coulter opacity and light scattering properties. The
COULTER COUNTERS.RTM. instruments, with which this invention can be
used, are the VCS, STKS and MAXM. However, the Model S and S-Plus
types are not able to differentiate all of the subpopulations of
leukocyte analogs which are in a whole blood control product, but
rather can provide a total count of the leukocyte analogs. Certain
of the S-Plus types are further able to differentiate two leukocyte
subpopulations.
[0025] New electronic optical particle counting devices have made
it necessary to develop new methods to determine whether an
instrument is properly functioning within manufacturer's
specification and diagnosing the cause of an instrument
malfunction. Although this Specification will be directed primarily
to method of using hematology control product useful with particle
counters of the COULTER.RTM. type, it should be understood that the
suspension media, analogs and control products disclosed herein,
and their methods of use described herein, find wide application
with particle counters generally. Accordingly, the term "electronic
optical particle counter" should be understood to include, in
addition to COULTER COUNTER.RTM. instruments, any other type of
particle counter which discriminates between particles of various
sizes by the use of electronic discriminator circuits
("thresholds") which respond electronically to signals indicative
of particle size, mass, volume, opacity or light scatter. COULTER
and COULTER COUNTER are Registered Trademarks of Coulter
Corporation.
SUMMARY OF INVENTION
[0026] This invention relates to a method for using a hematology
control product comprising placing a hematology control product in
an instrument, said control product containing at least one
leukocyte analog which has been derived from a blood cell which has
been treated so that it is resistant to degradation by the lytic
reagents used in the hematological test procedures, and the analog
remains responsive to the reagents used in the performance of the
instrument. The spatial position of the control product is analyzed
from at least one member selected form the group comprising D.C.
volume, RF size, opacity, and light scatter using new control
parameters. More preferably, at least two different physical
properties are measured. The results of such measurement are then
reported to diagnose the cause of a malfunction of an
instrument.
[0027] The invention further relates to a method of using a control
product which contains at least one leukocyte analog population to
determine if the instrument is functioning within manufacturer's
analytical specifications. The method comprises placing a
hematology control product in an instrument, said control product
containing at least one leukocyte analog which has been derived
from a blood cell which has been treated so that it is resistant to
degradation by the lytic reagents used in the hematological test
procedures, and the analog remains responsive to the performance of
the instrument. The control product simulates at least one physical
property of a human leukocyte, said property selected from the
group comprising volume measured by D.C. current, high frequency
(RF) size, opacity, and light scatter. More preferably, the control
product simulates at least two physical properties of a human
leukocyte. Then at least one, preferably two of the spatial
positions from physical properties of the control product are
measured. The results or such measurement are reported to diagnose
the cause of a malfunction of an instrument.
[0028] Still further, the invention relates to a method comprising
analyzing the spatial position of a leukocyte subpopulation in a
quantity of patient blood samples to obtain a statistically
significant value of a measured parameter, said analysis selected
from at least one member of the group comprising D.C. volume, RF
size, opacity, and light scatter; and reporting the results of such
measurement in an instrument to diagnose the cause of a malfunction
of an instrument.
[0029] Moreover, this invention also relates to combining a
hematological sample with a cell suspension media comprising an
aqueous solution of a plasma substance; and analyzing the resulting
mixture in an instrument to diagnose the cause of an instrument
malfunction, said analysis selected from at least one member and
more preferably at least two members of the group comprising D.C.
volume, RF size, opacity, and light scatter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 is a comparison of the COUNT RATIO of the white blood
cells compared to the conductivity of the diluted blood sample in
the core that is to be tested.
[0031] FIG. 2 is a comparison of the NEUTROPHIL DC MEAN compared to
the osmolality of the blood sample being analyzed after it has been
diluted with the lytic and quench reagents.
DETAILED DESCRIPTION OF THE INVENTION
[0032] For the purposes of understanding and explaining this
invention, the following terms are defined.
[0033] Beads: Beads are particles (made, usually, of latex or some
form of polystyrene) that can be used as stable and inert standards
for flow cytometric analysis. They can be obtained in different
narrowly defined sizes in order to standardize the FSC (standard
format for flow cytometric data storage) settings. They can also be
obtained conjugated to various fluorochromes in order to
standardize the fluorescence detection settings.
[0034] Channel: Channel is the term by which a flow cytometer
characterizes the intensity of the signal emitting by a particle.
Most cytometers divide intensity of light signals into 256 for 1024
channels. Signals with high channel numbers are brighter than
signals with low channel numbers; however, the quantitative
relationship between signals defined by one channel number and
those defined by another will depend on the amplifier and
photodetector voltage characteristic of a given protocol.
[0035] Coaxial flow: The flow of a narrow core of liquid within the
center of a wider stream. Flow of this type is important in flow
cytometry because it provides a means by which particles flowing
through a relatively wide nozzle can be tightly confined in space,
allowing accurate and stable illumination as they pass one by one
through a light beam.
[0036] Compensation: Compensation is the ability of a flow
cytometer to correct for the overlap between the fluorescence
spectra of different fluorochromes. Without compensation,
fluorescence from a given fluorochrome may register to some extent
on a photodetector assigned to the detection of a different
fluorochrome.
[0037] Coulter volume: The increase in electrical resistance that
occurs as a particle displaces electrolyte when it flows through a
narrow nozzle is the particle's Coulter volume. This increase in
resistance is only roughly related to the volume of the
particle.
[0038] Core: The core is the stream-within-a-stream that has been
injected into the center of the sheath stream and is maintained
there by the hydrodynamic considerations of laminar flow. The core
contains the sample particles that are to be analyzed in the flow
cytometer.
[0039] Cross-talk: Cross-talk is the signal from the "wrong"
photodetector that results because the fluorescent light emitted by
one fluorochrome contains some light of a wavelength that gets
through the filters on the photodetector that is normally specific
for the fluorescence from a different fluorochrome. See
"Compensation."
[0040] CV: The coefficient of variation is defined as the standard
deviation of a series of values divided by the mean of those
values. It is used in flow cytometry to describe the width of a
histogram peak. Where in some protocols it can be used to assess
the variation in particle characteristics within a population. In
DNA analysis (where all normal particles are assumed to have
identical characteristics), it is frequently used to assess the
alignment of a flow cytometer (and the skill of the operator).
[0041] Fixation: A process by which the protein of cells is
denatured. Fixation in flow cytometry is used to inactivate
hazardous biological material and also to preserve stained cells
where there is not immediate access to a flow cytometer.
Paraformaldehyde is the fixative of choice for flow cytometry
because it preserves the forward and side scatter characteristics
of cells (but causes some increase in their autofluorescence).
[0042] Flow Cell: The flow cell is the device in the flow cytometer
that delivers the sample stream to the center of the sheath stream.
In some cytometric configurations, the illumination occurs "in air"
after the stream has left the flow cell.
[0043] Fluorochrome: A flurorochrome is a dye that absorbs light
and then emits light of a different color (always of a longer
wavelength).
[0044] Forward scatter: Forward scatter is light from the
illuminating beam that has been bent (refracted or otherwise
deflected) as it passes through a particle so as to diverge from
the original-direction of the beam. The intensity of the light bent
to a small angle from the illuminating beam is related to the
refractive index of the particle as well as to its cross-sectional
area. The forward scatter signal is not correlated with a cell's
volume.
[0045] Gain: Gain is the electronic control on an amplifier that
determines the current intensity that results when a given signal
is received by a photomultiplier tube. Variation in the gain on
photomultiplier tube amplifiers may vary the appearance of the
output signals as they are converted into flow cytometric data.
[0046] Gate: Gate is a restriction placed on the flow cytometric
data that will be included in subsequent analysis. A live gate
restricts the data that will be accepted by a computer for storage;
an analysis gate simply excludes certain stored data from a
particular analysis procedure. A gate is used to restrict analysis
of a mixed population to certain cells within that mixed
population.
[0047] Granularity: Granularity is a term used synonymously with
side scatter to describe the light that is deflected to a right
angle from the illuminating beam in a flow cytometer. The intensity
of this light is related, in an imprecise way, to internal or
surface irregularities of the particles' flow through the beam.
[0048] Light scatter: Median angle light scatter (MALS) is defined
as that light scatter information obtained at angles between
10.degree. and 70.degree.. Low angle light scatter (LALS) is light
scatter information obtained at angles below between 10.degree.
relative to the beam axis, excluding 0.degree.. High angle light
scatter (HALS) is light scatter information centered at 90.degree.
to the laser axis.
[0049] Linear amplifier: A linear amplifier is one means of
increasing the signal from a photomultiplier tube to make it
measurable. A linear amplifier increases the signal in such a way
that the output current from the amplifier is directly proportional
to the input current derived from the photodetector.
[0050] Logarithmic amplifier: Logarithmic amplification is one
means of modifying the signal from a photomultiplier tube to make
it measurable. A logarithmic amplifier modifies the signal is such
a way that the output current from the amplifier is in proportion
to the logarithm of the input current derived from the
photodetector.
[0051] Photodetector: A photodetector is a device that senses light
and converts the energy from that light into an electrical signal.
Within the operating range of the detector, the intensity of the
electrical signal is proportional to the intensity of the light.
Photomultiplier tubes and photodiodes are two types of
photodetectors.
[0052] Photodiode: A type of photodetector used to detect
relatively intense light signals. It does not have a high voltage
applied to increase the current flow at its anode (output) end.
[0053] Photomultiplier tube: A photomultiplier tube is a type or
photodetector used to detect a relatively weak signal. Its output
current is increased by means of high voltage applied.
[0054] Rotated light scatter: Rotated light scatter (RLS) is a
transformation of the data derived from a ratio of the MALS/DC
pulse peak information. The RLS function has the effect of removing
the size component of the cell, yielding a measurement which is
more related to the internal structure of the cell.
[0055] Sheath: Sheath is the fluid within which the central sample
core is contained during coaxial flow from or within the flow cell
of a flow cytometer.
[0056] Side scatter: Side scatter is light of the same color as the
illuminating beam that bounces off particles in that beam and is
deflected to the side. The "side" is usually defined by a lens at
right angle (orthogonal orientation) to the line of the laser beam.
It may also be alternatively called right angle light scatter or
90.degree. LS. The intensity of this light scattered to the side is
related in a general way to the roughness or irregularity of the
surface or internal constituents of a particle.
[0057] Spatial position: Spatial position is the position of the
analyzed population in the domain of analysis. In the example of a
VCS instrument, the domain of analysis is volume measured by D.C.
current, high frequency (RF) size and light scatter. Another
example is that if the analyzed population is only by volume
measured by D.C. current, the spatial position would be the mean
position of the analyzed population within the D.C. domain.
[0058] Threshold: The threshold is an electronic device by which an
ADC can be made to ignore signals below a certain intensity. A
forward scatter threshold is most commonly used in flow cytometry
to exclude very small partices, debris and electronic or optical
noise from acquisition.
[0059] VCS technology: An instrument that analyzes blood by using
DC (Coulter) volume, Coulter opacity or RF size, and light scatter
at various angular ranges.
[0060] Wavelength: A wavelength is a characteristic of light that
is related exactly to its energy content and also (with light to
which our eyes are sensitive) to its color. Light of short
wavelength has more energy than light of longer wavelength.
[0061] Current multiple white blood cell population analysis
requires analogs of specific size and volume increments and
specific light scatter characteristics for use as a quality
control. In the method of this invention, it is. necessary to
prepare at least one analog of the major leukocyte components which
are the lymphocytes, monocytes, neutrophils, and eosinophils in
order to check the threshold settings of electronic optical
particle counters. Preferably, for the method of this invention, at
least a neutrophil analog needs to be prepared. More preferably, a
neutrophil and a lymphocyte analog needs to be prepared. Prior
hereto, an increased volume was correlated with an increased light
scatter which impeded the making of at least four different
populations of leukocyte analogs from other than human white blood
cells. As the analogs and instruments used to analyze the analogs
have become more complex, the suspension media for the analog has
to compliment their complexity. More specifically, the suspension
media must be compatible with these analogs and instruments, and
compliment the physical and biological properties of the
analogs.
[0062] The suspension media is primarily used with leukocyte
analogs produced from blood cells. One process to produce leukocyte
analogs provides treated blood cells from dirrerent sources to
match a plurality of threshold settings for many types of blood
counting instruments. In the selection of the blood cells, the main
limitation is the mean cell volume of the original cells as it
relates to the mean cell volume of the desired analog. Without
limiting the scope of this method, specific reference will be made
to blood cells from particular animals, with the understanding that
red and white blood cells from other animals may be employed in the
method of this invention.
[0063] One process for the manufacture of the leukocyte analogs
that are useful in the method of this invention comprises mixing a
red blood cell with a hypoosmotic solution to expand the volume of
the cell; changing the hemoglobin content of the cell to simulate
the light scatter and opacity properties of human leukocyte cells;
and, fixing the cell so that it is resistant to degradation by
lytic reagents used in the hematological test procedure and said
fixed cell having at least two properties selected from the group
comprising volume measured by D.C. current, high frequency (RF)
size, opacity and light scatter similar to human leukocytes
properties. The process for making the eosinophil blood cell analog
is similar, but the changing of the hemoglobin content is
accomplished by denaturing it in the cell rather than leaking it
from the cell. This additional embodiment results in an analog
having volume and light scattering characteristics of a human
leukocyte.
[0064] This process also enables the swelling of red blood cells
greater than 50% of their original volume, which provides a wider
latitude in the selection of animal cells for producing the desired
analogs. In a preferred process, the red blood cells are swollen
greater than 75% of their original volume.
[0065] For the purpose of making analogs suitable for use with the
method of this invention, it has been found that fowl red blood
cells such as turkey, chicken, duck, and preferably goose red blood
cells, land themselves to an aldehyde stabilization process to
produce the smaller lymphocyte analogs. It has also been found that
other non-human vertebrates including "fishes", particularly
members of the shark family, and reptiles, preferably alligators,
have red blood cells in the desired size range which when properly
treated yield in an analog similar to the larger sizes of the human
monocytes, neutrophils and eosinophils. These erythrocytes
generally show excellent suspension stability and highly
reproducible volume distribution characteristics. However,
considerations, such as availability in quantity at reasonable
expense, must be considered.
[0066] Moreover, the red blood cells are fixed so that they are
resistant to degradation by the lytic reagent used in the
hematological test procedures when determining the white blood cell
parameters in the whole blood control product.
[0067] The cells of avian, alligators and nurse sharks, are
nucleated, but the presence of a nucleus is neither essential nor
detrimental for their use as a substitute for human white blood
cells, given the process described herein which permits a regulated
hemolysis of the red blood cell. Preferably between 20% to 80% by
weight and most preferably 30% to 70% by weight of the hemoglobin
in the cell is released. The cells are further stabilized with a
fixing agent, such as an organic aldehyde which prevents disruption
of the cell membrane and further loss of hemoglobin.
[0068] Another process for the manufacture of the leukocyte analogs
that are useful in the method of this invention includes the
stabilizing of human white blood cells to simulate at least one of
the five subpopulations of leukocytes.
[0069] In addition, the method of this invention is useful with
leukocyte analogs prepared by other processes known in the art.
These stabilized leukocyte analog cells provide a satisfactory
substitute for human leukocyte cells in a control product.
[0070] A preferred process to produce a control product suitable
for use in the method of this invention, embodies a composition
prepared by mixing a suspension of fixed goose red blood calls to
simulate human lymphocytes, fixed alligator red blood cells to
simulate human monocytes, neutrophils, and eosinophils, all
assembled in the suspension media and in such proportions as to
provide a single composition to simulate human white cells. This
control product then is commingled with lysable human red blood
cells, and stabilized platelets or platelet analogs, to provide a
single multiple-analysis control product.
[0071] The following description describes a preferred process of
making a control product using red blood cells for use in the
method of this invention.
[0072] In the collecting step, the red blood cells are suspended in
an anticoagulant, such as an alkali metal salt of
ethylenediaminetetraacetic acid (EDTA) dissolved in a physiological
saline solution (sodium chloride). It is envisioned that other
anticoagulants and salts will do, as long as they do not cause
undue hemolysis or cell association.
[0073] Fresh red blood cells must be washed to remove donor
specific plasma proteins. This will reduce the probability of cell
agglutination when mixing red cells from multiple blood cell
donors. The cells are pooled together to obtain a homogeneous
composite.
[0074] The cell pool may be pretreated with a serum substance as a
processing aid. The pretreatment with the serum substance permits
swelling of the cell without causing the cell to rupture. Exposure
of the erythrocytes to a hypoosmotic environment has the principal
effect of increasing the mean corpuscular volume, and decreasing
the widths of the light scatter histogram. The blood cells are
increased in size as a result of the hypoosmotic environment having
a solute concentration which is reduced from the solute
concentration of the cells. When the concentration of solute inside
the cell is greater than the concentration outside the cell, there
is a tendency for the water to move into the cell to equilibrate
concentration. As such, the moving of water inside the cell causes
swelling. The hypoosmotic environment can include a solution of
sodium compounds, potassium compounds, or both sodium and potassium
or other compositions known to those skilled in the art to provide
the desired solute concentration.
[0075] As defined herein, serum comprises cholesterol, cholesterol
esters, and cholesterol which has been combined with one or more
other compounds found in serum plasma, and mixtures thereof.
Preferably, such other compounds further comprise lipoproteins and
phospholipids, and mixtures thereof. As appreciated by those
skilled in the art, typically cholesterol will contain
approximately 30% esters. As further appreciated by those skilled
in the art, the lipoprotein will maintain the cholesterol in an
aqueous solution. Preferably, the serum substance in the
pretreatment is selected from the group comprising cholesterol,
cholesterol esters, lipoprotein cholesterol, lipoprotein
cholesterol esters, cholesterol combined with phospholipids and
mixtures thereof. Most preferably, the serum substance comprises
cholesterol in combination with phospholipids. A suitable
commercially available example of such preferred embodiment is
Pentex.RTM. Cholesterol Super-Trate by Miles, Inc., which is a high
density lipoprotein cholesterol and lipoprotein cholesterol esters
in combination with phospholipids. Thus, when smaller cells are
expanded greater than 30% to 50% of their original volume, the
pretreatment is necessary. It is further believed that the
concentration of the serum substance used is both a function of the
amount of cell expansion, caused by the hypoosmotic solution, as
well as, the process conditions of the fixation reaction which
permits the cell's hemoglobin to leak from the cell. In processes
which fix the cell in less than approximately 2 hours due mainly to
the aldehyde concentration at room temperature, and wherein the
hypoosmotic pressure is greater than approximately 150
milliosmoles, no pretreatment appears necessary. When the
pretreatment is used, preferably the concentration of the
cholesterol is from 0.1 to 5.0 milligrams to a cell count of
1.times.10.sup.6 cells per microliter. If too high of a cholesterol
concentration used, then the cells will tend to lyse. If too low of
cholesterol concentration is used, the cell will rupture when
swelled.
[0076] Prior art attempts at-swelling cells without bursting them
have focused on the use of a processing aid, such as potassium
sodium tartrate, which functions to strengthen the cell membrane.
However, this approach does not permit expansion greater than the
expected 30 to 50%, nor provide the cell with regulated
hemolysis.
[0077] Although the present process is disclosed in terms of
simultaneously swelling and fixing of the cell in a one step
process, it is within the contemplation of this process that more
than one step could be used to pretreat the cell with the serum
substance, swell the cell to permit a controlled release of
hemoglobin and thereafter fix the cell. However, such procedure
would be expected to have the problems of controlling the process
conditions for each step, and more specifically, the timing of the
fixation of the blood cell.
[0078] In a preferred process to produce the control product for
use in the method of this invention, the hypoosmotic solution is
formed by combining an aqueous solution of sodium phosphate with
the fixative reagent to the desired osmotic pressure. The lower the
osmotic pressure relative to the normal tonicity of the native
blood, the more that the cell will swell due in part because of the
water moving from outside the cell to inside the cell. The osmotic
pressure will preferably range from 0 to 150 milliosmoles,
depending upon the initial cell size, cell count, and the desired
final cell size; even more preferably from 65 to 95 milliosmoles
for the eosinophil analog; 0 to 20 milliosmoles for the monocyte
analog; 5 to 35 milliosmoles for the lymphocyte analog; and from 45
to 65 milliosmoles for the neutrophil analog. The above preferred
ranges are based upon blood cells that have been washed with an
isotonic saline solution and are further based upon a cell count in
the fixative reaction of approximately 20,000 to 50,000 cells per
microliter.
[0079] Concomitantly, temperature does not appear to independently
affect the swelling rate of the cell, but does affect the rate of
the fixation reaction. As the cell expands, the hemoglobin leaks
out of the cell at a controlled rate until the fixation reaction
prevents further release of hemoglobin. The majority of the
hemoglobin will be released within the first five minutes of the
hypoosmotic treatment. Thus, in the simultaneous swelling and
fixing of the cells, reducing the temperature of the fixation in
solution enables the control of the fixation process and hemoglobin
release rates during which time the cell is swelling. Upon
completion of the fixation reaction, the cell is resistant to
dissolution or degradation under the influence of the usual lysing
reagents used in hematological test procedures.
[0080] In a further process to produce control products for use in
the method of this invention, the blood cells are added to a
chilled hypotonic solution containing glutaraldehyde. The chilled
fixing solution is at a temperature or 0.degree. to 15.degree. C.,
and more preferably, from 1.degree. to 10.degree. C., most
preferably, the fixation treatment is at 2.degree. to 8.degree. C.
for the lymphocyte and monocyte analogs and at room temperature for
the neutrophil and eosinophil analogs. The reduced temperature has
been shown to provide a qualitatively different cell as measured on
a sizing apparatus such as a COULTER COUNTER.RTM. Model VCS
analyzer. A qualitative difference includes a higher mean cell
volume compared to fixing at room temperature.
[0081] Fixing of the swollen cells is important to toughen the cell
membranes and to prevent degradation of the membranes. This is
accomplished by contacting the cells with a solution of an organic
aldehyde, including monoaldehydes such as formaldehyde, or
dialdehydes such as glutaraldehyde. Glutaraldehyde is the preferred
aldehyde, since it reacts more speedily than formaldehyde.
Glutaraldehyde can be added in higher concentrations than the final
concentration, so long as the final concentration is in the range
of about 0.05% to 0.8% and more preferably 0.1% to 0.6%, based upon
a cell count of approximately 20,000 to 50,000 cells per
microliter. The practical limitations on selection of an
appropriate aldehyde and concentration thereof are the functional
limitations of the number of cells, elimination of undue cell
association, and as a parameter in controlling the fixation
reaction. The fixation reaction conditions will vary for the
specific animal cell used and the leukocyte analog being
manufactured.
[0082] Although most room temperature fixation with glutaraldehyde
occurs within two hours, more time is required for the red blood
cells to be totally resistant to the usual red blood cell lytic
agents employed in COULTER COUNTER.RTM. hematology instruments.
With careful selection of the red blood cells, the length of time
for fixation with glutaraldehyde will range between 2 and 72 hours,
preferably between 3 to 30 hours, depending upon temperature,
concentration of glutaraldehyde, number of cells and desired amount
of hemoglobin released. In a most preferred embodiment, the
fixation time for a cell count of approximately 20,000 to 50,000
cells per microliter is between 10 to 24 hours for the monocyte and
lymphocyte analogs and 3 to 18 hours for the eosinophil and
neutrophil analogs. Under-fixation may result in a partially fixed
red blood cell with a mean cell volume less than that for the
targeted human leukocyte population. Generally, the upper time
limit of fixation is based upon manufacturing convenience. After
fixation, the cells are separated from the liquid phase by a
centrifugation or gravitation means and then are washed with a
phosphate buffered saline solution.
[0083] The pH of the fixing solution ranges from 7.0 to 9.0. If the
pH of the fixing solution is too low, agglutination may occur; and
if too high, the cell may rupture. In addition, the pH affects the
release of hemoglobin. If the fixation reaction occurs too quickly,
the cell will not be able to leak the hemoglobin. Thus, the pH
range is approximately 7.0 to 9.0, and preferably 7.5 to 8.5. In a
most preferred embodiment, the pH of the fixation solution is
8.0.+-.0.2 for the neutrophil and eosinophil analogs, and
7.8.+-.0.1 for the monocyte and lymphocyte analogs.
[0084] The eosinophil analog is prepared in a similar process
except, the hypotonic glutaraldehyde solution is preferably at room
temperature and the hypotonic glutaraldehyde solution is primarily
used to lightly cross link the hemoglobin in the blood cells,
rather than to completely fix the cell. As such, the glutaraldehyde
concentration for a cell count of approximately 20,000 to 50,000
cells per microliter is between approximately 0.1 and 0.4%, and
more preferably from 0.2 to 0.3%. After lightly cross linking the
hemoglobin and washing with a phosphate buffered saline solution,
the cells are further treated with a protein denaturing reagent,
such as a guaternary ammonium compound, or other denaturing agent
known to those skilled in the art to precipitate the hemoglobin
within the cell. The pH of the denaturing solution should be
between 9.0 and 12.0, and preferably between 10.0 and 11.0. This
treatment does not reduce the volume of the cell. The treatment
with the protein denaturing reagent increases the light scatter
characteristics of the swollen cell to provide the swollen cell
with the requisite light scattering characteristics similar to the
human eosinophil. Both the denaturation of the hemoglobin and the
controlled release of the hemoglobin have the effect of changing
the hemoglobin composition in the cell. However, the light scatter
properties are distinctly different between the controlled release
of the hemoglobin in the monocyte and lymphocyte analogs and the
denaturation of hemoglobin in the eosinophil analog. Generally, the
leaking of hemoglobin from the cell will reduce the light scatter
and opacity of the cell. Denaturing the hemoglobin in the cell will
increase the light scatter of the cell.
[0085] The preferred process of preparing the eosinophil analog
comprises pretreating the red cell pool with an aqueous serum
substance, swelling the cell, denaturing the hemoglobin in the cell
and fixing the cell. As appreciated by one skilled in the art, it
is within the contemplation of this process to produce a control
product that one could choose an appropriate sized red blood cell
which did not require the amount of swelling which would
necessitate the pretreatment with the serum substance. In such
case, the process would comprise denaturing the hemoglobin in the
cell to simulate the light scatter properties of a human leukocyte
cell and fixing the cell so that it is resistant to degradation by
lytic reagents used in hematological test procedures. As such, the
treated red cell would have light scatter and volume properties
similar to human leukocytes. However, if the cell is not swelled to
some extent, it would be expected that since the red blood cell is
not by nature spherical, the standard deviation of the light
scatter would not be within boundary of the targeted cell
population. The addition of a sphering agent may obviate this
problem.
[0086] By using a combination of the above disclosed processing
steps, of swelling the cell, leaking of hemoglobin from the cell,
denaturing the hemoglobin in the cell, as well as, shrinking the
cell by processes known to those skilled in the art, one is
effectively provided with processes to design an analog having a
plurality of different physical parameters of D.C. volume, RF size,
opacity and light scatter which can be used in new methods to
diagnose the cause of a malfunction of an instrument. More
specifically, shrinking and swelling of the cell can affect all of
the above listed parameters, while changing the hemoglobin in the
cell can affect the opacity and light scatter characteristics.
[0087] The suspension media disclosed herein, is also used with
leukocyte analogs prepared by other processes known in the art. One
such other process includes the fixing of human white blood cells
to simulate five subpopulations of leukocytes as described in
Example 5 herein.
[0088] The reference blood cell control product can include one or
more of the leukocyte analogs prepared by any process known to
those skilled in the art or any of the above described processes.
The leukocyte analog can be stored in any suitable media such as
phosphate buffered saline solution and those fully described in
U.S. Pat. Nos. 4,213,876; 4,299,726, 4,358,394 and 3,873,467.
[0089] The following specific example is disclosed in U.S. Pat. No.
4,299,726:
1 Stabilizlng Media for Conferring Long Term Stability on Red Blood
Cells-preferred Formulation Approximate Amounts Liter Formulation
1. Distilled water 500 ml 2. Propyl paraben 0.3 to 1.0 gm 3. Methyl
paraben 0.5 to 1.0 gm 4. Procaine hydrochloride 0.1 to 0.5 gm 5.
Deoxycholic acid 0.1 to 0.9 gm 6. Lactose 10.0 to 50.0 gm 7.
Actidione 0.1 to 0.6 gm 8. Trisodium citrate dihydrate 3.0 to 8.0
gm 9. Citric acid monohydrate 0.3 to 0.9 gm 10. Sodium dihydrogen
phosphate 0.8 to 2.5 gm monohydrate 11. Phenergan hydrochloride 0.1
to 1.0 gm 12. Colistimethate, sodium 0.2 to 0.9 gm 13. Penicillin
G., sodium 0.5 .times. 10.sup.6 to 3 .times. 10.sup.6 units 14.
Kanamycin sulfate 0.2 to 0.8 gm 15. Neomycin sulfate 0.2 to 1.0 gm
16. 5'-AMP 0.4 to 1.0 gm 17. Adenine 0.2 to 0.8 gm 18. Inosine 0.4
to 1.0 gm 19. Dihydrostreptomycin sulfate 0.2 to 1.0 gm 20.
Tetracycline hydrochloride 0.2 to 1.0 gm 21. 30% Bovine albumin 100
to 350 ml 22. q.s. to 1 liter with in distilled water
[0090] Since many of the chemicals listed above are known
commercially by several names, the name given is a common name
listed in the Merck Index, Eleventh Edition (1989), published by
Merck and Co., Inc., Rahway, N.J.
[0091] When making the control product, the supernatant fluid is
removed from the leukocyte analogs and they are then resuspended in
the suspension media. The preferred suspension media comprises an
aqueous solution of a plasma substance. As defined herein, an
aqueous solution of a plasma substance comprises an aqueous
solution of a serum substance (as previously defined), serum
substance in combination with a plasma protein and mixtures therof.
As further defined herein, plasma protein comprises one or more of
the proteins contained in plasma. Preferably, such plasma proteins
comprise albumin, lipoproteins, globulins, fibrinogens, and
mixtures thereof. More preferably, the plasma substance is selected
from the group comprising cholesterol, cholesterol esters,
lipoprotein cholesterol, lipoprotein cholesterol esters,
cholesterol combined with phospholipids, cholesterol combined with
albumin, cholesterol esters combined with albumin, lipoprotein
cholesterol combined with phospholipids, lipoprotein cholesterol
combined with albumin, and mixtures thereof.
[0092] To confirm the utility of the plasma substance for red blood
cell lysis in a saponin based lytic system, an aqueous plasma
substance was added to washed red blood cells. The aqueous solution
comprised 3% plasma substance in a phosphate buffered saline
solution. The results are as follows:
2 Sample Plasma Substance Lysis 1. Human albumin Yes 2. Albumin
with fatty acid removed No 3. Sample 2 with lipoprotein cholesterol
Yes 4. Bovine serum albumin No 5. Sample 4 with lipoprotein
cholesterol Yes 6. Albumin bound cholesterol Yes 7. Monomer albumin
Yes 8. Bovine serum albumin capalate No stabilized 9. Sample 8 with
lipoprotein cholesterol Yes 10. Polymer enhanced bovine serum Yes
albumin 11. Human albumin Yes 12. Swine albumin Yes 13. Media of
U.S. Pat. 4,299,726 No 14. Sample 13 with lipoprotein Yes
cholesterol 15. Cholesterol bound with a surfactant No 16.
Phosphate buffered saline (PBS) No solution 18. Lecithin No 19. PBS
with lipoprotein cholesterol Yes
[0093] From the test results given above, it has been determined
that the addition of a plasma substance to washed red blood
cells-enables lysis in a saponin lytic system. It is believed that
the albumin is interacting with the red blood cell and saponin to
effect the lytic action. However, when the washed red blood cells
and bovine serum albumin are further combined with other
ingredients such as those disclosed in U.S. Pat. No. 4,299,726, the
albumin does not cause the lysis. However, when a lipoprotein
cholesterol is added, lysis is effected. Moreover, when a
lipoprotein cholesterol is added to the PBS, lysis is effected.
[0094] When using the leukocyte analogs prepared from red blood
cells as described above, the aqueous solution of a plasma
substance is preferably added to the hematological composition at
least 12 hours before being used in an instrument. When one or more
leukocyte analogs are combined with lysable human red blood cells
to provide a single multiple analysis reference blood cell control
product for instruments which use lytic reagents, it is most
preferred that the aqueous solution of the plasma substance
comprises bound cholesterol. A suitable example of the most
preferred plasma substance is Moducyte.RTM., as described in U.S.
Pat. No. 4,290,774, assigned to Miles, Inc., which is a high
density lipoprotein cholesterol bound with albumen. The final
concentration of cholesterol in the suspension media ranges from
400 to 1,200, and preferably 600 to 1,000 milligrams per liter
depending upon the cell count in the final control product.
[0095] For a control product using the leukocyte analogs prepared
by the preferred processes disclosed herein, if an insufficient
concentration of the cholesterol is used in the preferred media,
the red blood cells in the reference blood cell control product
will not efficiently lyse to dissolve the cell membrane so that
there is an absence of noise and debris when using a saponin lytic
reagent system and the leukocyte analogs will have a mean cell
volume below the required size due to the lytic reaction. If the
media contains too high of a concentration of cholesterol, the red
blood cells in the reference blood cell control will not
efficiently lyse to dissolve the cell membrane so that there is an
absence of noise and debris.
[0096] More specifically, when the control product is used in
instruments, such as those that employ the Coulter Model VCS
technology, which uses a reagent system such as described in U.S.
Pat. No. 4,751,179, in order to distinguish at least two
populations of leukocytes, (1) lymphoids (lymphocytes) and (2)
myeloids (neutrophils, monocytas, eosinophils and basophils), the
preferred suspension media enables the reaction between the weaker
lytic reagent and the non fixed red blood cells to occur so that
the red blood cells lyse while the leukocyte analogs remain
substantially unaffected, enabling each type of leukocyte analog to
be counted. As taught by U.S. Pat. No. 4,751,179, the lysing
reagent has two forms: (1) a lytic diluent containing saponin,
which simultaneously functions to dilute the whole blood sample and
stromatolyse its red blood cells; or (2) a two part system
comprised of non-lytic blood diluent followed by a lytic reagent
containing saponin.
[0097] When prior art medias, such as those described in U.S. Pat.
Nos. 4,213,876; 4,299,726; or 4,358,395, are used, the leukocyte
analogs prepared by the preferred processes disclosed herein are
lower in volume than desired for the targeted leukocyte population.
More specifically, when the preferred suspension media is used with
leukocyte analogs, which have been prepared from either red or
white blood cells, the D.C. volume of the analog is within the
desired range for the targeted leukocyte population.
[0098] In a more preferred embodiment, the suspension media would
further comprise the addition of a non-ionic surfactant. The
surfactant will have a high hydrophile-lipophile balance (HLB). The
HLB typically has a value greater than 15 and more preferably
greater than 17. Typically, the surfactant is in an amount
effective to make the lytic action more specific to the red blood
cells without detrimentally affecting the leukocyte analogs. In
addition, the surfactant will stabilize any free cholesterol in the
control product so that it does not separate out in solution. As
appreciated by those skilled in the art, the effective amount of
surfactant may be empirically determined, but is typically less
than 0.5% by weight of the control product.
[0099] Suitable non-ionic surfactants include alkyl polyether
alcohols of the general formula: R--X--(y).sub.n--H, where R is a
lipophilic chain C.sub.8-C.sub.18 carbon atoms; where X is --O--,
1
[0100] --COO--; and Y is CH.sub.2 CH.sub.2O-- or C CH.sub.2
CH.sub.2O; n is an integer of 15-50. Suitable commercial examples
of these surfactants include Diazopan.RTM. SS-837 by GAF chemical
Corp., Triton.RTM. X405 by Rohm and Haas, and Pluronic F.RTM.-127
PRILL by BASF Wyandotte Corp.
[0101] While not desiring to be bound by any theory, it is
presently believed that there is an interaction among the red blood
cells, weak lytic agent (e.g., saponin), and the plasma substance
in the preferred suspension media causes the red blood cells to
lyse. More specifically, it is presently believed that the plasma
substance may be affecting the cell membrane cholesterol which
further affects the leukocyte analog's response to the lytic
reagent. Moreover, it is further believed that the surfactant makes
the lytic reaction more specific to the red blood cells and yet
does not detrimentally affect the leukocyte analogs as to measured
control parameters. In addition, it is further believed that the
surfactant may also be affecting the cholesterol found in the cell
membrane or in the plasma substance.
[0102] The preferred suspension media for the hematological
reference control product having stability up to six months
includes an aqueous solution of the plasma substance and optional
compatible fungicidal and bactericidal agents, and optional
supplementary agents such as purine nucleoside, bile salt, and
cholic acid derivatives, phenothiazine compounds and the salts
thereof having antihistamine properties, and 4-aminobenzoic acid
esters and derivatives and their salts having aesthetic properties,
as well as, sphering agents for the red blood cells, or
combinations thereof. Since one or more of the leukocyte analogs
may be combined into a single reference blood cell control product
for use with the known lysing agent for the red blood cells, the
formulation for the preferred suspension media is the same for all
of the leukocyte analogs.
[0103] As appreciated by one skilled in the art, the suspension
media should have sufficient tonicity to avoid cell lysis. The
preferred formula for the suspension media is:
3 Suspension Media Approximate Amounts Liter Formulation 1.
Distilled water 500 ml *2. Propyl paraben 0.3 to 1.0 gm *3. Methyl
paraben 0.5 to 1.0 gm *4. Procaine hydrochloride 0.1 to 0.5 gm *5.
Deoxycholic acid 0.1 to 0.9 gm *6. Lactose 10.0 to 50.0 gm *7.
Actidione 0.1 to 0.6 gm *8. Trisodium citrate dihydrate 3.0 to 8.0
gm *9. Citric acid monohydrate 03 to 0.9 gm *10. Sodium dihydrogen
phosphate 0.8 to 2.5 gm monohydrate *11. Phenergan hydrochloride
0.1 to 1.0 gm *12. Colistimethate, sodium 0.2 to to -0.9 gm *13.
Penicillin C., sodium 0.5 .times. 10.sup.6 to 3 .times. 10.sup.6
units *14. Kanamycin sulfate 0.2 to 0.8 gm *15. Neomycin sulfate
0.2 to 1.0 gm *16. 5'-AMP 0.4 to 1.0 gm *17. Adenine 0.2 to 0.8 gm
*18. Inosine 0.4 to 1.0 gm *19. Dihydrostreptomycin sulfate 0.2 to
1.0 gm *20. Tetracycline hydrochloride 0.2 to 1.0 gm *21. 30%
Bovine albumin 100 to 350 ml 22. Lipoprotein Cholesterol 400 to
1,200 mg 23. q.s. to 1 liter with distilled water *Optional
ingredient preferred for conferring long term stability for red
blood cells and analogs.
[0104] The manufactured control product can be used to monitor and
diagnose the cause of a malfunction of an instrument. In this
invention, a method for using a hematol control product has been
developed that enables the monitoring and diagnosing of an
instrument for problems associated with:
[0105] 1. lysis debris and noise
[0106] 2. instrument reagents pump volume settings
[0107] 3. instrument laser alignments
[0108] 4. instrument gain settings
[0109] 5. inconsistency of flow rate in the flow cell
[0110] The method provides a more specific indication of the type
and cause of an instrument malfunction than non specific flagging
that is diagnostically non specific or inspection of the test
results by the instrument operator which are provided by prior art
methods. The following description describes a new method of using
the previously described control product. As appreciated by one
skilled in the art, the control product must contain lyseable
erthryocytes to monitor any cellular debris and noise caused by
ineffective red cell lysis. The method uses new control parameters
of the physical properties of the control product to determine the
cause of an instrument malfunction. These physical properties are
selected from the group comprising:
[0111] (1) volume measured by D.C. current,
[0112] (2) high frequency (RF) size,
[0113] (3) opacity, and
[0114] (4) light scatter.
[0115] The leukocyte populations are used as the indicator of
system performance. Preferably the neutrophil and lymphocyte
subpopulations are used as control parameters and are measured to
determine instrument performance. More preferably the neutrophil
population is used as a control parameter and is measured as an
indication of system performance.
[0116] In order to determine whether there is a noise problem due
to cellular debris the use of two control parameters are employed
The control parameters are COUNT RATIO and ELAPSED TIME. The COUNT
RADTO is a measure of the number of white blood cells in an
analysis compared to the total count of events that are recorded in
the analysis during a specific ELAPSED TIME. ELAPSED TIME is a
measured period of time, usually in seconds. As the . ratio of
white blood cells counted and total event counted approaches 100
percent, the cellular debris or noise problem associated with an
instrument malfunction is eliminated.
[0117] An example of the use of COUNT RATIO in an instrument
employing VCS technology would be a comparison of the number of
white blood cells analogs compared to a total count of 8192
particles obtained for a specific ELAPSED TIME. If the ratio is
less than about 95%, there is an indication that noise or
interference is a problem. The specific problem is further
confirmed by additional tests that are further explained in this
disclosure.
[0118] Another approach to monitor and diagnose an instrument for
problems is to use fresh blood monitoring of the running average of
the same set of parameters of a statistically significant number of
patient normal bloods. When the average is outside the expected
ranges of the control parameter there is an early indication of the
instrument malfunction. The instrument malfunction can be further
verified using the control product described herein.
[0119] One indication of whether an instrument is properly
functioning is to determine the cellular debris or noise that is
measured by the instrument. Excess cell debris can be caused by
incomplete lysis, changes in the reaction kindetics due to
temperature, conditions or interference with the lytic reaction.
since various instrument systems have cell count and data
accumulation time limitations, excess debris or noise from any
source interferes with the proper acquisition and analysis of the
white cell populations. The cause of the cellular debris or noise
problem can be attributed to several problems including:
[0120] A. conductivity noise due to sheath fluid and the blood
sample stream imbalance.
[0121] B. Lysing noise caused by improper red cell lysing.
[0122] C. Flow noise caused by partial plugs, residual plugs or
other flow problems
[0123] In order to determine whether the malfunction is due to
conductivity noise caused by mismatches between the sample stream
and the sheath fluid stream conductivity, one first measures the
reagent pump volumes. A calibrated container is employed to measure
each of the reagents that are added to the blood sample. The volume
of reagent that is transferred should be within a predetermined
value. If the volume of a reagent is not within the prescribed
limits, then the reagent pump is adjusted to increase or decrease
the reagent volume that will be dispensed.
[0124] In the example of an instrument that employs Vcs technology,
one measures the pump volumes of the lytic reagent and quench
reagent streams. A calibrated container is employed to measure each
of these volumes. The volume of the reagent should be within a
predetermined value. If the volume of the reagent is not within the
prescribed limits, then the pump is adjusted to increase or
decrease the volume that will be provided.
[0125] The prepared control product containing fixed cell analog
populations that are osmotically sensitive are further used, to
monitor the reagent pump volumes. Changes in volume of lytic
reagents will affect the final osmolality of the diluted blood
sample and affect the measured volume of the analog population.
Lower osmolality increases the measured volume of the control cell
analog and higher osmolality decreases the measured volume of the
control cell analog. Variations of reagent pump volumes beyond the
limits of conductivity required to electrically balance the sheath
fluid will produce noise in addition to the changes in the volume
of the control cell.
[0126] Therefore, after the conductivity noise has been minimized
by the preceding process, lysing noise resulting from improper red
cell lysing is checked with a control product containing a known
number of stabilized red blood cells and at least one leukocyte
analog cell subpopulation. If lysing is a problem, then the control
cells will show a COUNT RATIO less than about 95% of the maximum
COUNT RATIO. In addition, although the reagent volumes can be
adjusted by the preceding process, the lysing kinetics relating to
the temperature of the lysing reaction will be affected. More
specifically, it has been found that if the room temperature of a
laboratory instrument is at 50.degree. F., the lysing reaction will
require a different lytic and quench reagent volume than if the
temperature is at 90.degree. F., to obtain a minimum of lysing
noise.
[0127] In the example of an instrument which employs the VCS type
technology, the lytic reagent and quench reagent are reacted with a
blood sample to provide a lysed and diluted suspension of white
blood cells suitable for measurement. In the VCS type instrument,
blood cells are reacted with the lytic reagent causing hypotonic
cell swelling, red call and platelet lysis and dissolution of red
cell and platelet membranes. Reaction with the quench reagent stops
the swelling and lysis and begins a process of re-equilibration of
the white cells to their native size. Because of the nature of the
reactions, the lytic reagent and quench reagent are very different
in both osmolality and conductivity. The final osmolality and
conductivity of the sample stream is proportional to the volume,
osmolality and conductivity of the lytic reagent, quench reagent
and blood sample.
[0128] The use of two control parameters are employed to determine
if the reagent pump volumes are within specification or determining
the cause of the malfunction. The monitoring the COUNT RATIO and
NEUTROPHIL DC MEAN assists one in differentiating pump volume
changes within conductivity tolerances and beyond conductivity
tolerances. The NEUTROPHIL DC MEAN is the neutrophil mean value in
the DC channel.
[0129] Another approach to monitor and diagnose an instrument for
problems is to use fresh blood monitoring of the running average of
the same set of parameters of a statistically significant number of
patient normal bloods. When the average is outside the expected
ranges of the control parameter there is an early indication of the
instrument malfunction. The instrument malfunction can be further
verified using the control product described herein.
[0130] It has been found that the COUNT RATIO of the white blood
cells is related to the reagent pump-volumes calculated from known
reagent concentrations and measured pump volumes, expressed as
either dilution conductivity or osmolality. FIG. 1 represents a
comparison of the COUNT RATIO of the white blood cells compared to
the conductivity of the diluted blood sample in the diluted blood
sample stream that is to be tested. As shown in FIG. 1, as the
quench reagent volume is increased, the COUNT RATIO increases to a
plateau. When the quench volume is increased beyond the optimum
range the COUNT RATIO is substantially reduced.
[0131] To obtain a comparable FIG. 1 using a control product, the
lytic reagent volume is provided at a first volume that would be
expected to provide proper lysis. The quench volume is adjusted
from providing insufficient quench reagent to providing an excess
amount of quench reagent. When the quench reagent volume is
insufficient, the COUNT RATIO is significantly reduced. As the
quench volume is increased the COUNT RATIO rises and plateaus over
the optimum performance range. Increasing the quench volume above
the optimum range causes the COUNT RATIO to be substantially
reduced. Upon obtaining this information, the pump for the quench
reagent can be adjusted to provide a quench reagent volume that is
in the middle or the plateau.
[0132] After the quench volume has been adjusted, the lytic reagent
pump can be re-adjusted. It has been found that the NEUTROPHIL DC
MEAN of the control product is related to the lytic reagent pump
volume. The monocyte population shows sensitivity similar to the
neutrophil population. The pump for the lyse reagent is adjusted to
provide an osmolality which corresponds to a previously measured
NEUTROPHIL DC MEAN of the control product. The previously measured
NEUTROPHIL DC MEAN is provided as an assay value from a reference
instrument.
[0133] FIG. 2 represents a comparison of the NEUTROPHIL DC MEAN
compared to the osmolality of the blood sample being analyzed after
it has been diluted with the lytic and quench reagents. As shown in
FIG. 2, as the osmolality is increased, the NEUTROPHIL DC MEAN of
the control product decreases.
[0134] In order to determine whether there is a problem because of
left over plugs or partial plugs, two control parameters of WHITE
TIME and DATE TIME are used. The monitoring of these two control
parameters assists one in determining these type of problems. WHITE
TIME is the product of the number of white blood cell analogs
multiplied by the time required for analysis. DATE TIME is the day
that a control sample has been analyzed. It has been found that
using these two control parameters on a routine basis with a
control product that contains a known number of leukocyte analogs
provides information as to instrument performance problems.
[0135] In the method of this invention, the control product is used
at least daily. The WHITE TIME and DATE TIME are recorded to
provide meaningful information about the existence of flow
problems. More specifically, when the WHITE TIME is graphed versus
the DATE TIME, one is able to obtain a two dimension graph of the
performance of the instrument to determine trends of the flow
performance of the instrument.
[0136] In a VCS type instrument, an alternate method is provided to
check the sample flow rate. The VCS instrument records the WHITE
TIME and DATE TIME on each patient blood sample that has been
analyzed by the instrument. By comparing these control parameters
on a statistically significant number of patient blood samples, an
expanded data base is available to monitor the history of the
instrument's flow performance. More specifically, by recording the
WHITE TIME and DATE TIME on each patient blood sample, one is able
to detect whether left over plugs or partial plugs have occurred.
Left over plugs can restrict the flow of subsequent samples, but
may produce no detectable change in the number of cells per second.
Partial plugs can increase the time required to obtain the white
blood cell count because of a reduced flow of cells through the
flow cell.
[0137] In addition, when these control parameters are monitored for
the patient samples, a two dimension analysis can be obtained to
discover if the flow rate is deteriorating and whether noise and
debris levels are interfering with the expected time to obtain the
white blood cell count. Excessive debris will decrease the amount
of time necessary to obtain a specified number of white blood
cells. This two dimensional analysis has provided an improvement
over complex multidimensional analysis of the prior art.
[0138] Prior art methods to determine problems due to the
consistency of the flow cell has been to measure the number of
leukocytes that are detected during a predetermined time. More
specifically, the instrument operator would use a specially
selected fresh blood that had a known number of white blood cells
and measure the time the instrument required to detect the white
blood cells. If the instrument detected the white blood cells
within a specified time, it was presumed that there was an absence
of flow cell problems. However, this measurement only provided
information about the instrument for that particular analysis. This
measurement did not provide meaningful information concerning the
performance of the instrument over an extended time period. More
specifically, graphing the count of white blood cells per unit time
only provides an instantaneous view of the instrument's performance
on that one day. To obtain a view of the performance of an
instrument over several days, a three dimensional charting of the
results was necessary. Moreover, the method of this invention uses
a control product with a known number of white blood cells which
eliminates the necessity of obtaining a specially selected fresh
blood and determining the number of white blood cells it
contains.
[0139] In addition, a control product can be used to monitor the
instrument to determine if there is a problem with the instrument's
laser, the laser optics or the laser alignment. Laser alignment has
three dimensions which corresponds to X, Y and Z axes.
[0140] Currently, the material used to set and then monitor laser
alignment and gain adjustments is latex microparticles. The method
of using the latex particle is to monitor the population mean and
coefficient of variation of the latex population. However, if the
laser lens accumulates dirt or other debris which obscures the
laser beam signal, the prior art practice has permitted obscuring
the alignment problem by increasing the gain of the laser to
overcome the interference to the laser beam. In addition, the latex
particles are not responsive to the reagents that are used in the
instrument. Moreover, the latex particles are a separate control
product that is required to be run in a calibration mode in the
instrument. The latex particles require additional work to be
undertaken to monitor instrument's performance. Therefore, it is
advantageous to have a single hematological control product that is
both responsive to the reagents used in the instrument and provides
and indication of the performance of the laser in the
instrument.
[0141] It has been determined that X-axis alignment is the most
sensitive of the-axes and produces the greatest affects on a
control product's light scatter position and distribution. The
rotated light scatter position of the control product is at a
maximum when the X axis is properly aligned and the distribution
width is at its minimum. As the alignment degrades to slight
misalignment and then to gross misalignment, the control product's
position in the histogram shifts to the left, the mean value
decreases and the rotated light scatter channel distribution
widens.
[0142] When analyzing fresh blood and having X-axis misalignment,
the white cell populations are shifted left (low light scatter)
producing a loss of white cells counted and an increase in noise.
As misalignment becomes more extreme, the effect on fresh blood
analysis is that the subpopulations of leukocytes merge and
differentiation is lost.
[0143] Y-axis and Z-axis misalignment do not show the same kind of
change of position of the white cell population as exhibited by the
white blood cells when caused by X-axis misalignment. It has been
determined-that Z-axis alignment is related to whether the,lens is
in a proper focal length which will provide the sharpest image. If
the Z-axis is misaligned, then the resulting scatterplot will not
have clear distinct clusters of leukocytes. More specifically, in a
histogram of light scatter versus DC volume, the separation of the
populations of lymphocytes and monocytes, from the population of
neutrophils will not be distinct. There will be a degradation of
the separation of the light scatter peaks of the volume
populations. More specifically, there will be a reduction in the
individual populations in amplitudes and an increase in the
amplitude of the valleys between the populations.
[0144] It has been found that the lymphocyte population of the
control product provides information to determine low light scatter
while the neutrophil population provides information to determine
high light scatter. The relationship between the position of these
two populations monitors system variables that produce both bias
and proportional effects on populations position such as laser
alignment (bias) and DC, RF and LS gain (proportional)
adjustments.
[0145] The use of two control parameters can be employed to
determine the problem with the laser's operation. The monitoring of
the NEUTROPHIL RLS MEAN and the PEAK TO VALLEY RATIO will provide
an indication of the performance of the laser and X axis and Z axis
alignment. The LYMPHOCYTE RLS MEAN shows sensitivity similar to the
NEUTROPHIL RLS MEAN. The NEUTROPHIL RLS MEAN is the neutrophil mean
value in the light scatter channel. The LYMPHOCYTE RLS MEAN is the
lymphocyte mean value in the light scatter channel. The PEAK TO
VALLEY RATIO (PVR) is the ratio of the amplitude of the rotated
light scatter signal of the neutrophil population compared to the
rotated light scatter signal of the valley formed between the
lymphocyte and monocyte populations, and the neutrophil
population.
[0146] The PVR provides a reliable quality control parameter to be
used in a method to determine whether a problem exists with the
laser. When using a control product the PEAK TO VALLEY RATIO should
meet a threshold number which has been predetermined. If the PVR
meets or exceeds this number, the laser is operating optimally.
More specifically, a PVR that meets the predetermined number
indicates that there is a clear separation of the neutrophil
population and lymphocyte and monocyte populations. If the PVR is
less than the predetermined number, then there is an indication
that a problem with the laser exists.
[0147] As previously noted, the prior art practice has permitted
increasing the laser gain to compensate for a dirty lens. However,
a latex particle control does not provide specific information
concerning the misadjustment of the light scatter signal gain when
optical interference exists. It has been determined that when the
gain adjustment has been improperly increased that the entire
neutrophil population position in the rotated light scatter
histogram, including the NEUTROPHIL RLS MEAN, is shifted to the
right.
[0148] In accordance with the method of this invention, the
determination of a PVR that is less than the predetermined value,
and the NEUTROPHIL RLS MEAN is above the predetermined value, it
indicates that the instrument has a problem with either optical
interference or X-axis or Z-axis alignment. Therefore, the
technician will know to clean the lens and calibrate the laser
alignment. In addition, it has also been determined that if the
determination of a PVR that is less than the predetermined value,
and the NEUTROPHIL RLS MEAN is below the predetermined value, it
indicates that the instrument has a problem with either optical
interference which has not been compensated by increasing the gain
or X-axis or Z-axis alignment. Moreover, if the PVR is within the
predetermined value and the NEUTROPHIL RLS MEAN is either greater
than or less than the predetermined value, it indicates that the
laser gain is set improperly. Still further, if the PVR is below
the predetermined value and the NEUTROPHIL RLS MEAN is within the
predetermined value, it indicates that the laser needs replacing
because it is losing power.
[0149] While the foregoing specification explains the use of some
of the control parameters to determine whether an instrument is
performing according to manufacturer's specification and the use of
the control parameter to diagnose the cause of the malfunction,
Table 1 provides a detailed analysis of which control parameters
can be used for a control product or for a statistically
significant value of patient blood samples to detect and diagnose
problems with an instrument's system. In this Table, the following
abbreviations are used:
[0150] s=sensitivity for the applicable instrument function
[0151] SD=standard deviation
[0152] OP=opacity
[0153] x=measurement of Control Product or Sample
4TABLE 1 Patient Control Control Blood Cell/Type Parameter Product
Sample Lysis Flow Alignment Gain Chemistry Neutrophil DC Mean x x s
s DC SD x x s RLS Mean x x s s RLS SD x x s s OP Mean x x s OP SD x
x s PVR x s Lymphocyte DC Mean x x s DC SD x x s RLS Mean x x s s
RLS SD x x s s OP Mean x x s OP SD x x s PVR x s Monocyte DC Mean x
x s DC SD x x s RLS Mean x x s s RLS SD x x s s OP Mean x x s OP SD
x x s Latex Particle DC Mean x x s Sample DC SD x x s Analysis RLS
Mean x x s Mode RLS SD x x s s OP Mean x x s OP SD x x s Noise
COUNT RATIO x x s s s Flow WHITE TIME x x s
[0154] In addition, a latex, polystyrene or other plastic bead can
be added to the control product to provide a single reference
control product that can further indicate instrument performance.
More specifically, the use of this type of control product with the
control parameters can provide information about the instruments
performance. For example, if the latex particle DC gain is within
specification, but the control product has a DC mean not within
specification, then there is an indication that there is a problem
with the reagent pump volumes.
[0155] When this type of control product is used in an instrument
that uses VCS technology, the above described example would
indicate that there is a problem with the lytic or quench reagent
volumes. More specifically, in the VCS type instrument, if the
latex particle DC gain is within specification, but the control
product has a DC mean that is above the manufacturer's
specification, then there is an indication that the lytic reagent
volume can be above the optimum level or the quench reagent volume
can be below the optimum level. Still further in the VCS example,
if the latex particle DC gain is within specification, but the
control product has a DC mean that is below the manufacturer's
specification, then there is an indication that the lytic reagent
volume can be below the optimum level or the quench reagent volume
can be above the optimum level.
[0156] The process for preparing leukocyte analogs for use in the
method of this invention is hereinafter provided in the Examples.
Example 1 is a specific example of preferred reagents and
techniques for treating goose cells, it being understood that the
formulations are only illustrative of those that can be used in the
method of this invention. Examples 2, 3 and 4 are specific examples
of preferred reagents and techniques for treating the alligator
cells, it being understood that the formulations are only
illustrative of those that can be used in the method of this
invention. Example 5 shows an example of using human white blood
cells to produce five subpopulations of leukocyte analogs, it being
understood that the formulations are only illustrative of those
that can be used in the method of this invention. Example 6 shows
an assembly of the four leukocyte populations. It should be
appreciated that these Examples provide formulations for the
leukocyte analogs which are only illustrative. The reagents and/or
techniques described can also be applicable to blood cells from
animals other than geese and alligators. Other ingredients and
proportions can be employed, in accordance with this
disclosure.
EXAMPLE 1
Lymphocyte Analog from Goose Red Blood Cells
[0157] The following is a specific example of preferred reagents
and recommended specific procedural steps for treating goose red
blood cells to obtain a normal sized lymphocyte analog. It will be
understood that the formulations and the procedures only are
illustrative and that other ingredients, proportions and procedures
can be employed, in accordance with the disclosures in this
invention.
Phosphate Buffered Saline Solution (PBS) Liter Formulation
[0158] 1. Sodium phosphate monobasic: 0.2 g
[0159] 2. Sodium phosphate dibasic.7H.sub.2O: 2.0 g
[0160] 3. Sodium azide: 0.1 g
[0161] 4. Sodium chloride: 9.4 g
[0162] 5. q.s. to 1 liter with distilled water:pH approximately
7.4; osmolality 315 to 345 mOsm/kg.
Lymphocyte Hypotonic Solution
[0163] 1. Sodium phosphate monobasic: 0.2 q
[0164] 2. Sodium phosphate dibasic.7H.sub.2O: 2.0 g
[0165] 3. q.s. to 1 liter with distilled water:pH approximately
7.8; osmolality 15 to 25 mOsm/kg.
Procedure
[0166] 1. Select avian red blood cells having a mean cell volume
range of about 140 to 170 fL. Wash the packed avian red blood cells
with the phosphate buffered saline solution (PBS).
[0167] 2. Add 1.0 to 5.0 milligrams of cholesterol to a cell count
of 2.times.10.sup.6 per microliter and incubate for 2 to 6 hours,
at room temperature.
[0168] 3. Prepare a glutaraldehyde fixative reagent having a
glutaraldehyde content of about 0.1 to 0.8% by adding a commercial
25% glutaraldehyde product to the chilled Lymphocyte Hypotonic
Solution. Preferably, the temperature is from 2.degree. to
8.degree. C. The preferred concentration of glutaraldehyde is
approximately 0.35%.
[0169] 4. Add the washed red blood cells to a measured amount of
the fixative of step 3 at a 1:35 dilution. Transfer to sealed
containers which are rolled slowly for 18 to 24 hours at 2.degree.
to 8.degree. C. The reduction in hemoglobin content is calculated
to be approximately 60% by weight.
[0170] 5. Remove the supernatant fluid, wash cells several times
with the PBS, then resuspend in a suitable storing solution.
[0171] 6. For a stand alone lymphocyte analog, resuspend the washed
fixed cells in the suspension media of this invention and adjust
the concentration to simulate that of human lymphocyte cells in
normal human blood.
[0172] 7. For multiple hematological parameters for a control
product, add the washed fixed cells in the suspension media of this
invention with other hematological compositions and analogs desired
for the multiple parameter hematology control product, the cell
count being appropriate to measure lymphocyte proportions.
[0173] 8. With suitable stabilizers, the fixed cells can be stored
for a time period in excess of six months.
[0174] In accordance with the above example, but starting with
other types of mammalian red blood cells, comparable results are
obtained.
EXAMPLE 2
Monocyte Cell Analog from Alligator Red Blood Cells
[0175] The following is a specific example of preferred reagents
and recommended specific procedural steps for treating alligator
red blood cells to obtain the monocyte cell analog. It will be
understood that the formulations and the procedures are only
illustrative and that other ingredients, proportions and procedures
may be employed, in accordance with the disclosures in this
invention.
Monocyte Hypotonic Solution
[0176] 1. Sodium phosphate monobasic: 0.1 g
[0177] 2. Sodium phosphate dibasic 1.0 g
[0178] 3. q.s. to 1 liter with distilled water; pH approximately
7.8; osmolality 5 to 15 mOsm/kg.
[0179] Washing solution for cells (PBS), as set forth in Example
1.
Procedure
[0180] 1. Select alligator red blood cells having a mean cell
volume range of about 350 to 450 fL. Wash the packed alligator red
blood cells with PBS.
[0181] 2. Add 1.0 to 5.0 milligrams of cholesterol to a cell count
of 1.times.10.sup.6 per microliter and incubate 3 to 5 hours at
room temperature.
[0182] 3. Prepare a glutaraldehyde fixing reagent having a
glutaraldehyde content of about 0.1 to 0.8% by adding a commercial
25% glutaraldehyde product to the chilled Monocyte Hypotonic
Solution. Preferably the temperature is from 2.degree. to 8.degree.
C. The preferred concentration of glutaraldehyde is approximately
0.15%.
[0183] 4. Add the washed red blood cells to a measured amount of
the fixative of step 3 at a 1:50 dilution. Transfer to sealed
containers which are rolled slowly for 18 to 24 hours at room
temperature. The reduction in hemoglobin content is calculated to
be approximately 40% by weight.
[0184] 5. Remove the supernatant fluid, wash cells several times
with the PBS, then resuspend in a suitable storing solution.
[0185] 6. For a stand alone monocyte analog, resuspend the washed
fixed cells in the suspension media of this invention and adjust
the concentration to simulate that of human monocyte cells in
normal human blood.
[0186] 7. For multiple hematological control product, add the
washed fixed cells in the suspension media of this invention with
other hematological compositions and analogs desired for the
multiple parameter control product in the appropriate concentration
to measure monocyte cells.
[0187] 8. With suitable stabilizers, the fixed cells can be stored
for a time period in excess of six months.
EXAMPLE 3
Eosinophil Analog from Red Blood Cells of the Alligator
[0188] The following is a specific example of preferred reagents
and recommended specific procedural steps for treating red blood
cells of the alligator to obtain the eosinophil analog. It will be
understood that the formulations and the procedures are only
illustrative, and that other ingredients, proportions and
procedures may be employed, in accordance with the disclosures in
this invention.
Eosinophil Hypotonic Solution
[0189] 1. Sodium phosphate monobasic: 0.32 grams
[0190] 2. Sodium phosphate dibasic 8.08 grams
[0191] 3. q.s. to 1 liter with distilled water; pH approximately
8.0; osmolality 75 to 85 mOsm/kg.
Eosinophil Hemoglobin Denaturing Treatment Solution
[0192] 1. dimethyldicocoammonium chloride 2.5 grams
[0193] 2. tris(hydroxymethyl)amino methane 6.06 grams (organic
buffer)
[0194] 3. q.s. to 1 liter with distilled water: pH approximately
10.5.
Eosinophil Post-Treatment Wash Solution
[0195] 1. polyoxethylated alkylphenol 5 grams (Diazopan.RTM. SS-837
by GAF Chemical Corp.)
[0196] 2. q.s. to 1 liter with distilled water
[0197] Washing solution for cells (PBS), as set forth in Example
1.
Procedure
[0198] 1. Select alligator red blood cells having a mean cell
volume range of about 400 to 500 fL. Wash the packed alligator red
blood cells with PBS.
[0199] 2. Add 0.25 to 1.25 milligrams of cholesterol to a cell
count of 1.times.10.sup.6 per microliter and incubate 2 to 5 hours,
at room temperature.
[0200] 3. Prepare a glutaraldehyde cross linking reagent having a
glutaraldehyde content of about 0.1 to 0.8% by adding a commercial
25% glutaraldehyde product to the Eosinophil Hypotonic Solution.
The preferred concentration of glutaraldehyde is approximately
0.2%.
[0201] 4. Add the washed red blood cells to a measured amount of
the cross linking of step 3 at a 1:50 dilution. Transfer to sealed
containers which are rolled slowly for 18 to 24 hours at room
temperature.
[0202] 5. Remove the supernatant fluid, wash cells several times
with the PBS.
[0203] 6. Add the washed red blood cells to the Eosinophil
Hemoglobin Denaturing Treatment Solution at a 1:10 dilution.
Transfer to sealed containers which are rolled slowly for 2-4 hours
at room temperature.
[0204] 7. Remove the supernatant fluid, wash cells several times
with the Eosinophil Post-Treatment Wash Solution to remove the
Eosinophil Hemoglobin Denaturing Treatment Solution. Then resuspend
in a suitable storage solution.
[0205] 8. For a stand alone eosinophil analog, resuspend the washed
fixed cells in the suspension media of this invention and adjust
the concentration to simulate that of human eosinophil cells in
normal human blood.
[0206] 9. For multiple hematological control products, add the
washed fixed cells in the suspension media of this invention with
other hematological compositions and analogs desired for the
multiple parameter control product in the appropriate concentration
to measure eosinophil cells.
[0207] 10. With suitable stabilizers, the fixed cells can be stored
for a time in excess of six months.
EXAMPLE 4
Neutrophil Cell Analog from Alligator Red Blood Cells
[0208] The following is a specific example of preferred reagents
and recommended specific procedural steps for treating alligator
red blood cells to obtain the monocyte cell analog. It will be
understood that the formulations and the procedures are only
illustrative and that other ingredients, proportions and procedures
may be employed, in accordance with the disclosures in this
invention.
Neutrophil Hypotonic Solution
[0209] 1. Sodium phosphate monobasic: 0.23 g
[0210] 2;Sodium phosphate dibasic 5.32 g
[0211] 3. q.s. to 1 liter with distilled water; pH approximately
8.0; osmolality 45 to 65 mOsm/kg.
[0212] Washing solution for cells (PBS), as set forth in Example
1.
Procedure
[0213] 1. Select alligator red blood cells having a mean cell
volume range or about 400 to 500 fL. Wash the packed alligator red
blood cells with PBS.
[0214] 2. Prepare a glutaraldehyde fixing reagent having a
glutaraldehyde content of about 0.1 to 0.8% by adding a commercial
25% glutaraldehyde product to the Neutrophil Hypotonic Solution.
The preferred concentration of glutaraldehyde is approximately
0.4%.
[0215] 3. Add the washed red blood cells at a count of
1.times.10.sup.6 to a measured amount of the fixative of step 3 at
a 1:50 dilution. Transfer to sealed containers which are rolled
slowly for 18 to 24 hours at room temperature.
[0216] 4. Remove the supernatant fluid, wash cells several times
with the PBS, then resuspend in a suitable storing solution.
[0217] 5. Add packed cells to a nonionic surfactant solution. Said
solution tends to standardize the volume of donor cells. The
solution comprises 0.5 grams of octylphenoxy polyethoxy ethanol
having an HLB of approximately 13.5 (Triton.RTM. X-100 by Rohm and
Haas Co.,) in 1 liter of distilled water.
[0218] 6. Remove the supernatant fluid, wash cells several times
with the PBS, then resuspend in a suitable storing solution.
[0219] 7. For a stand alone neutrophil analog, resuspend the washed
fixed cells in the suspension media of this invention and adjust
the concentration to simulate that of human neutrophil cells in
normal human blood.
[0220] 8. For multiple hematological control product, add the
washed fixed cells in the suspension media of this invention with
other hematological compositions And analogs desired for the
multiple-parameter control product in the appropriate concentration
to measure neutrophil cells.
[0221] 9. with suitable stabilizers, the fixed cells can be stored
for a time period in excess of six months.
EXAMPLE 5
Five Subpopulations of Leukocyte Analogs from Human White Blood
Cells
[0222] 1. Add whole blood to a Dextran (molecular weight 100,000 to
500,000) solution at a dilution of 1:10 and allow to settle by
gravity means for 1 to 3 hours.
[0223] 2. Remove the supernatant, which includes the white blood
cells, platelets and some residual red blood cells.
[0224] 3;Centrifuge the product of step 2 at less than 300 RCF for
about 10 minutes. Aspirate the platelets leaving the button of
white blood cells, residual red blood cells, and a small amount of
plasma with which to resuspend the cells.
[0225] 4. Add a suitable lytic agent, such as water, to lyse the
red blood cells from the white blood cells.
[0226] 5. Centrifuge the product of step 4 and remove supernatant,
leaving packed white blood cells. Resuspend the packed white blood
cells in an approximately equal volume of saline solution.
[0227] 6. Add white blood cells to a suitable isoosmotic fixative
solution, such as 5% formaldehyde and 95% PBS (volume percent), in
a 1:10 dilution and transfer to sealed containers which are rolled
slowly for 18-30 hours at room temperature.
[0228] 7. Add a glutaraldehyde fixative solution having
approximately a 0.1% concentration of glutaraldehyde at a 1:1
dilution to the pool, and continue fixation for an additional 8-12
hours.
[0229] 8. Remove the supernatant fluid, wash cells several times
with the PBS, then resuspend in a suitable storing solution.
[0230] 9. For stand alone five subpopulation leukocyte analogs
assembly, resuspend the washed fixed cells in the suspension media
of this invention and adjust the concentration to simulate that of
human leukocyte cells in normal human blood.
[0231] 10. For multiple hematological control product, add the
washed fixed cells in the suspension media of this invention with
other hematological compositions and analogs desired for the
multiple parameter hematology control product, the cell count being
appropriate to measure leukocytes proportions.
[0232] 11. With suitable stabilizers, the fixed cells can be stored
for a time period in excess of six months.
EXAMPLE 6
[0233] In a sub-assembly for simulating the targeted composition of
white blood cells in a normal human blood sample, the following
quantities of the individual components are employed:
5 STOCK SOLUTION 0.150 L Example 1 lymphocytes 500 .times.
10.sup.3/uL 0.040 L Example 2 monocytes 500 .times. 10.sup.3/uL
0.030 L Example 3 eosinophils 500 .times. 10.sup.3/uL 0.280 L
Example 4 neutrophils 500 .times. 10.sup.3/uL 0.500 L diluent
phosphate buffered saline
[0234] In the final assembly of the four leukocyte populations,
remove the supernatant fluid, then resuspend the cells in 1.0 liter
of an aqueous solution of Moducyte.RTM. having a final
concentration of 800 milligrams or cholesterol.
[0235] This assembly can be stored for up to about six months with
the addition of known suitable stabilizers.
[0236] The ratio and total cell count for the leukocyte populations
can be adjusted to represent pathological, as well as normal
conditions in human blood. These compositions are useful likewise
in control and calibrator products particularly for automated
particle analysis instruments employing the Coulter Principle. In
addition, latex particles can be added to the control product to
provide a single control product that can further indicate
instrument performance.
[0237] Suspensions of untreated human red blood cells, simulated
white blood cells, and stabilized or simulated platelets can be
thereafter added in such proportion that the final red blood cell,
white blood cell and platelet counts, as well as hemoglobin content
and hematocrit fall in the desired range.
[0238] Stabilized platelets are furnished by processes known in the
art. Useful processes include:
[0239] 1. A combination of iodoacetamide and an iminodiacetic acid
or salt thereof, together with a compatible bacteriostatic agent in
an aqueous solution which is maintained at a preselected range of
pH and osmolality as is described in U.S. Pat. No. 4,405,719.
[0240] 2. A fixative-stabilizing composition containing a
glutaraldehyde concentration of 0.1% to 5% and a non-ionic
surfactant which is a mixture of ethoxylates of certain isomeric
linear alcohols, as is more fully described in U.S. Pat. No.
4,389,490.
[0241] 3. A human platelet analog comprising goat erythrocytes
stabilized, combined and blended an necessary to have a size range
and volume distribution close to that of human platelets, as is
described in U.S. Pat. No. 4,264,470.
[0242] The values for each of the hematological parameters can be
varied to represent abnormal low and abnormal high conditions. The
white blood cell count in normal blood is 5,000 to 11,000 per
microliter (uL) with a lymphocyte value of 20 to 40%, mononuclear
cell value of less than 10%, a granulocyte value of 60 to 80%,
eosinophil value less than approximately 5%, and basophil value
less than approximately 2%. The normal range in human blood for red
blood cells is 4,000,000 to 5,000,000 cells per microliter. The
normal hemoglobin value is 12 to 16 grams/100 ml. The term
"hematocrit" is defined as the ratio of volume of packed red blood
cells to the volume of whole blood. The normal ratio in humans is
about 45%. The mean corpuscular volume is the ratio of the volume
of packed red blood cells in ml per liter of blood to red blood
cells in millions per microliter. The mean corpuscular hemoglobin
concentration is an index indicating the mean or average weight of
hemoglobin per 100 ml of packed red blood cells in terms of
percent. The mean corpuscular hemoglobin is the ratio of hemoglobin
content, in grams per liter, to red blood cells, in millions per
microliter.
[0243] While in the foregoing specification, a detailed description
of the invention has been set down for the purpose of illustration,
many variations in the details herein give may be made by those
skilled in the art without departing from the spirit and scope of
the invention.
* * * * *