Chemistry: analytical and immunological testing – Composition for standardization – calibration – simulation,... – Particle count or volume standard or control
Reexamination Certificate
1998-10-16
2001-02-13
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Composition for standardization, calibration, simulation,...
Particle count or volume standard or control
C436S008000, C436S016000, C436S017000, C436S018000, C436S063000, C436S164000, C436S166000, C436S175000
Reexamination Certificate
active
06187590
ABSTRACT:
BACKGROUND
This invention relates to hematology control compositions and their methods of preparation and use in a reference standard. More particularly, this invention relates to a hematology reference control suspension containing stabilized white blood cells (“WBC”) and the nuclei of mammalian WBC or avian or fish erythrocytes. Even more particularly this invention relates to a hematology reference control suspension containing stabilized WBC that have been subjected to a whole blood lysing process during their preparation such that in their stabilized state they retain multi-angle light scattering properties. This enables the stablized cells, when utilized in a hematology analyzer that differentiates WBC based solely on multi-angle light scatter signals to produce multi-angle light scatter signal that mimic whole blood WBC signals. Currently there are several different brands of automated hematology instruments in the marketplace. These different analyzers utilize varying detection techniques to quantify neutrophils, lymphocytes, monocytes, eosinophils and basophils. Among the detection techniques utilized are: electronic impedance, forward light scatter, polarized 90° angle light scatter, depolarized 90° angle light scatter, light absorption, radio-frequency and combinations thereof. The different optical bench designs significantly affect the characteristics of the optical signals obtained from stabilized control cells. Since these instruments utilize different detection methods for white blood cell differential analysis (“WBC/Diff”), it has become necessary to utilize different types of WBC/Diff control solutions in order to obtain control cell signatures that are similar to those of whole blood cells when run on that particular type of instrument.
The current class of instruments must utilize either impedance, impedance and light scatter (but not necessarily multi-angle light scatter), or light scatter, impedance and radio frequency signals to differentiate and determine cells from one another. Further, these currently available hematology instruments are not able to quantify nucleated red blood cells (“NRBC”). NRBC interfere with an accurate WBC/Diff analysis. The currently available hematology instruments only “flag” for the existence of NRBC in a sample. However, the soon to be released Abbott Cell-Dyn® 4000 hematology analyzer system will be capable of performing a simultaneous whole blood analysis of WBC/Diff and NRBC. The Cell-Dyn® 4000 instrument will perform a simultaneous, whole blood analysis by utilizing only multi-angle light scatter signals, including on occasion fluorescence to differentiate among WBC and NRBC. Consequently, it has become necessary to develop a new hematology control for WBC/Diff and NRBC analysis. The control cells of this new reference control must possess all of the multi-angle light scattering capabilities of the cells of the whole blood sample that they are suppose to mimic.
There are in the current realm of art, several patents which describe methods and reagent systems for preparing hematology reference control materials for the current class of analyzers, i.e. those that do not perform an exclusive multi-angle light scatter WBC/Diff analysis. The applicants are not aware of any art describing a method or reagent system for the preparation or utilization of a hematology reference control for the multi-angle light scatter analysis of WBC or NRBC.
U.S. Pat. No. 4,704,364 to Carver et al. and assigned to Coulter Instrument Corp., discloses a method for preparing a three component system which simulates the three major components of human leukocytes. However these simulated cells are detected by an impedance based detection system, not a multi-angle light scatter detection system. Carver et al. use fixed, red blood cells (“RBC”) from the nurse shark to simulate human granulocytes; fixed RBC from turkeys to simulate human mononuclear cells; and fixed human RBC to simulate human lymphocytes. The hematology control produce by the teachings of Carver et al. is useful only for electronic impedance measurement of a 3 part WBC/Diff since the three components are only distinguishable by size (impedance), not by optical properties.
The three components produced by Carver et al.'s method do not have similar cell surface structure or cytoplasmic granularity substantially the same as that of human WBC. Therefore, they are not usable as a reference control on a multi-angle light scatter based system. The applicants tested these simulated cells on the soon to be commercially available Cell-Dyn® 4000 analyzer, and found that the light scattering characteristics of the simulated WBC in the Carver et al. control produced very different multi-angle light scatter signals than that of a normal blood sample.
FIGS. 1
a
-
1
c
are reproductions of the dot plots obtained on a Cell-Dyn® 4000 hematology analyzer for normal blood.
[Abbreviations used to label the axis in the following figures:
ALL or WBC ALL:
Axial Light Loss (0°)
IAS or WBC IAS:
Intermediate Angle Scatter
(3°-10°)
PSS or WBC PSS:
Polarized Light Scatter (90°)
DSS or WBC DSS:
Depolarized Light Scatter (90°)
FL3 or WBC FL3:
Red Fluorescence (515-545 nm)
G:
Granulocyte Cluster
M:
Monocyte Cluster
B:
Basophil Cluster
L:
Lymphocyte Cluster
E or Eos:
Eosinophil Cluster
N:
Neutrophils
S:
Noise signals from RBC Stroma
& PLT
NRBC:
Nucleated Red Blood Cells
FIGS. 4
a
-
4
c
show the results obtained on a Cell Dyn® 4000 analyzer for the simulated WBC control of Carver et al. and embodied in the Coulter Corp. Product 4C® Plus Cell control. As can be seen in
FIGS. 4
a
-
4
c
the clusters are not identifiable.
U.S. Pat. No. 5,270,208 to Ryan, discloses a different method of preparing a hematology reference control for WBC/Diff analysis. In the Ryan method, aldehyde-fixed human WBC's are suspended in an isotonic aqueous medium comprising lipoprotein in an amount sufficient to provide a mixture that gives a WBC signature profile that is substantially similar to that obtained from whole blood. To support his claim, Ryan exhibited a WBC distribution of a Coulter Corp. STK-S® analyzer dot plot, DF1 (abscissa) vs. Volume (ordinate). It is believed that Ryan's WBC preparation is commercially available under the name of PARA 12® (Low, Normal & High) Tri-level hematology control from Streck Laboratories. The applicants tested this commercial material on a Cell-Dyn® 4000 analyzer which performs a simultaneous analysis of WBC/Diff and NRBC by multi-angle light scatter (axial light loss, multi-dimensional light scatter and fluorescence.) The results (see
FIGS. 5
a
-
5
c
) reveal that the WBC component of the Ryan preparation generates a significantly different light scatter signature than that obtained from whole blood. As can be seen in
FIGS. 5
a
-
5
c
the neutrophil components of the product generated much smaller axial light loss and polarized side scatter signals than that of whole blood; the depolarized side scatter signals from neutrophils are much too large to be separated from that of eosinophils; monocyte cluster does not separate from neutrophil cluster at all; the lymphocyte component generate much higher intermediate angle scatter (7°) signals than that of whole blood and thus wiping out the region reserved for basophils by overlapping.
U.S. Pat. No. 5,320,964 to Young et al. discloses methods and reagent compositions for preparing leukocyte analogs. The Young et al. lymphocyte analogs are prepared from fixed goose RBC in a hypotonic phosphate buffered solution (15-25 mOsm/kg); the Monocyte analogs are prepared from fixed alligator RBC in a hypotonic buffered solution (5-15 mOsm/kg); the Eosinophil analogs are also prepared from fixed alligator RBC in a hypotonic solution (75-85 mOsm/kg); and the Neutrophil analogs are prepared from alligator RBC in hypotonic buffered solution (45-65 mOsm/kg). Both goose RBC and alligator RBC are elliptical, nucleated and have a smooth cell surface. Young et al. claim that the fixed cells prepared according to their procedures simulate at least two
Kihara Jean Emiko
Kim Young Ran
Abbott Laboratories
Goller Mimi C.
Wallenhorst Maureen M.
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