Stable reference materials for automated hematology testing platforms
This invention describes a method for preparation of stable hematology reference materials by producing synthetic hydrogel blood cell surrogates, which mimic human blood components in size, morphology, performance and functionality when analyzed using an automated hematology analyzer employing multiple detection technologies. Different hydrogel particles can be combined and mixed to prepare multi-parameter and multi-level hematology reference materials, which could be used for calibration, linearity verification, proficiency evaluation, and routine performance monitoring of modern automated hematology analyzers. These hydrogel particles can also be combined with processed and stabilized human blood components to prepare the reference materials of this invention.
1 . A hematology reference material (HRM) comprising a suspension fluid and:
a red blood cell (RBC) component that comprises human erythrocytes fixed with a crosslinking agent;
a platelet component that comprises goat erythrocytes fixed with a crosslinking agent;
a reticulocyte component that comprises fixed human erythrocytes encapsulated with RNA;
a lymphocyte component that comprises chicken erythrocytes fixed with a crosslinking agent;
a monocyte component that comprises turkey erythrocytes fixed with a crosslinking agent;
a basophil component that comprises alligator erythrocytes fixed with a crosslinking agent;
a neutrophil component that comprises alligator erythrocytes fixed with a crosslinking agent;
an eosinophil component that comprises alligator erythrocytes fixed with a crosslinking agent;
an immature reticulocyte component that comprises fixed human erythrocytes encapsulated with RNA;
an immature granulocyte component that comprises alligator erythrocytes fixed with a crosslinking agent;
an immature thrombocyte component that comprises goat erythrocytes fixed with a crosslinking agent; and
a nucleated red blood cell component that comprises chicken erythrocytes fixed a crosslinking agent,
wherein each of said components exhibit a forward scatter profile or a side scatter profile that is at least 95% similar to its corresponding component in a human blood sample.
2 . The HRM according to claim 1 , wherein the suspension fluid is an aqueous isotonic solution with a pH of 7.0 to about 7.6 and containing additives selected from anti-microbial agents, surfactants, stabilizers, buffers, salts, enzyme inhibitors, albumin, and lipoproteins.
3 . The HRM according claim 1 , said HRM further comprising hydrogel particles comprising blood group antigens.
4 . The HRM according to claim 3 , wherein said HRM comprises a population of hydrogel particles comprising blood group antigen A, a population of hydrogel particles comprising blood group antigen B, a population of hydrogel particles comprising blood group antigen RhD, or a combination of said populations of hydrogel particles.
5 . The HRM according to claim 4 , wherein the population of hydrogel particles comprises blood group antigen A and blood group antigen RhD attached to the hydrogel particles.
6 . The HRM according to claim 4 , wherein the population of hydrogel particles comprises blood group antigen B and blood group antigen RhD attached to the hydrogel particles.
7 . The HRM according to claim 4 , wherein the population of hydrogel particles comprising blood group antigen A does not comprise blood group antigen RhD attached to the hydrogel particles.
8 . The HRM according to claim 4 , wherein the population of hydrogel particles comprising blood group antigen B does not comprise blood group antigen RhD attached to the hydrogel particles.
9 . A method of agglutinating a HRM containing hydrogel particles comprising blood group antigens comprising contacting an HRM according to claim 3 with antibodies specific to blood group antigens and agglutinating the hydrogel particles in said HRM.
10 . The method according to claim 9 , wherein said HRM comprises a population of hydrogel particles comprising blood group antigen A, a population of hydrogel particles comprising blood group antigen B, a population of hydrogel particles comprising blood group antigen RhD, or a combination of said populations of hydrogel particles.
11 . The method according to claim 10 , wherein the population of hydrogel particles comprises blood group antigen A and blood group antigen RhD attached to the hydrogel particles.
12 . The method according to claim 10 , wherein the population of hydrogel particles comprises blood group antigen B and blood group antigen RhD attached to the hydrogel particles.
13 . The method according to claim 10 , wherein the population of hydrogel particles comprising blood group antigen A does not comprise blood group antigen RhD attached to the hydrogel particles.
14 . The method according to claim 10 , wherein the population of hydrogel particles comprising blood group antigen B does not comprise blood group antigen RhD attached to the hydrogel particles.
15 . The HRM according to claim 1 , wherein the HRM comprises a white blood cell count (WBC), a red blood cell count (RBC), a platelet count (PLT), a hemoglobin concentration (HGB), a red cell distribution width (RDW), a platelet distribution width (PDW), a hematocrit (HCT), a mean cell volume (MCV), a mean platelet volume (MPV), a eosinophil count (EOS), a eosinophil percent (EOS %), a basophil count (BASO), a basophil percent (BASO %), a monocyte count (MONO), a monocyte percent (MONO %), a lymphocyte count (LYM), a lymphocyte percent (LYM %), a reticulocyte count (RETIC), a reticulocyte percent (RETIC %), and nucleated red blood cells (NRBC), the components having a lower limit (LL) and upper limit (UL) as follows:
Range of HRMs Blood Variables
Variable
Units
LL
UL
WBC
K/μL
2.8
26.1
NEUT
K/μL
1
20.1
NEUT
%
40.5
84.6
LYMPH
K/μL
0.6
6.2
LYMPH
%
24.5
26
MONO
K/μL
0
e1.2
MONO
%
1
6
EOS
K/μL
0
1.8
EOS
%
0
8.4
BASO
K/μL
0
0.6
BASO
%
0
2.4
RBC
M/μL
1.99
5.49
HGB
g/dL
5.1
17.2
HCT
%
13.6
46.2
MCV
fL
66
87
MCH
pg
22.5
33.9
MCHC
g/dL
32.1
41.4
RDW
%
10.8
13
PLT
K/μL
53
555
MPV
fL
2.5
8
NRBC
K/μL
0
5.5.
16 . A method for calibrating a cytometric device for analysis of a biological sample comprising:
inserting, into a cytometric device, a HRM according to claim 1 ;
measuring the side scatter, forward scatter, and hydrodynamic properties of the components of said HRM using the cytometric device; and
calibrating the cytometric device for analysis of cells in a biological sample based on the measured side scatter, forward scatter, and hydrodynamic properties of the HRM.
17 . A method for determining if a sample includes one or more cell types comprising:
inserting, into a cytometric device, a HRM according to claim 1 ;
measuring the forward scatter, side scatter, and hydrodynamic properties of the HRM using the cytometric device;
calibrating the cytometric device based on the measured forward scatter, side scatter, and hydrodynamic properties of the HRM;
adding a sample comprising a plurality of cells to the calibrated cytometric device; and
measuring the forward scatter, side scatter, and hydrodynamic properties of each cell of the plurality of cells using the calibrated cytometric device to determine if the sample includes one or more of a specific cell type.
18 . A hematology reference material (HRM) comprising a suspension fluid and:
a red blood cell (RBC) component that comprises human erythrocytes fixed with a crosslinking agent and hydrogel particles substantially similar to human red blood cells containing hemoglobin;
a platelet component that comprises goat erythrocytes fixed with a crosslinking agent and hydrogel particles substantially similar to human platelets;
a reticulocyte component that comprises fixed human erythrocytes encapsulated with RNA and hydrogel particles substantially similar to human reticulocytes;
a lymphocyte component that comprises chicken erythrocytes fixed with a crosslinking agent and hydrogel particles substantially similar to human lymphocytes;
a monocyte component that comprises turkey erythrocytes fixed with a crosslinking agent and hydrogel particles substantially similar to human monocytes;
a basophil component that comprises alligator erythrocytes fixed with a crosslinking agent and hydrogel particles substantially similar to human basophils;
a neutrophil component that comprises alligator erythrocytes fixed with a crosslinking agent and hydrogel particles substantially similar to human neutrophils;
an eosinophil component that comprises alligator erythrocytes fixed with a crosslinking agent and hydrogel particles substantially similar to human eosinophils;
an immature reticulocyte component that comprises fixed human erythrocytes encapsulated with RNA and hydrogel particles substantially similar to immature human reticulocytes;
an immature granulocyte component that comprises alligator erythrocytes fixed with a crosslinking agent and hydrogel particles substantially similar to immature human granulocytes;
an immature thrombocyte component that comprises goat erythrocytes fixed with a crosslinking agent and hydrogel particles substantially similar immature human thrombocytes; and
a nucleated red blood cell component that comprises chicken erythrocytes fixed a crosslinking agent and hydrogel particles substantially similar to nucleated human red blood cells,
wherein each of said components exhibit a forward scatter profile or a side scatter profile that is at least 95% similar to its corresponding component in a human blood sample.
19 . The HRM according to claim 18 , wherein each component comprises about 0.1% to about 99.9% biological component and about 0.1% to about 99.9% hydrogel component, the percentages of which total 100% for said components.
20 . The HRM according to claim 18 , wherein the suspension fluid is an aqueous isotonic solution with a pH of 7.0 to about 7.6 and containing additives selected from anti-microbial agents, surfactants, stabilizers, buffers, salts, enzyme inhibitors, albumin, and lipoproteins.
21 . A method for calibrating a cytometric device for analysis of a biological sample comprising:
inserting, into a cytometric device, a HRM according to claim 18 ;
measuring the side scatter, forward scatter, and hydrodynamic properties of the components of said HRM using the cytometric device; and
calibrating the cytometric device for analysis of cells in a biological sample based on the measured side scatter, forward scatter, and hydrodynamic properties of the HRM.
22 . A method for determining if a sample includes one or more cell types comprising:
inserting, into a cytometric device, a HRM according to claim 18 ;
measuring the forward scatter, side scatter, and hydrodynamic properties of the HRM using the cytometric device;
calibrating the cytometric device based on the measured forward scatter, side scatter, and hydrodynamic properties of the HRM;
adding a sample comprising a plurality of cells to the calibrated cytometric device; and
measuring the forward scatter, side scatter, and hydrodynamic properties of each cell of the plurality of cells using the calibrated cytometric device to determine if the sample includes one or more of a specific cell type.
23 . The HRM according to claim 18 , wherein the HRM comprises a white blood cell count (WBC), a red blood cell count (RBC), a platelet count (PLT), a hemoglobin concentration (HGB), a red cell distribution width (RDW), a platelet distribution width (PDW), a hematocrit (HCT), a mean cell volume (MCV), a mean platelet volume (MPV), a eosinophil count (EOS), a eosinophil percent (EOS %), a basophil count (BASO), a basophil percent (BASO %), a monocyte count (MONO), a monocyte percent (MONO %), a lymphocyte count (LYM), a lymphocyte percent (LYM %), a reticulocyte count (RETIC), a reticulocyte percent (RETIC %), and nucleated red blood cells (NRBC), the components having a lower limit (LL) and upper limit (UL) as follows:
Range of HRMs Blood Variables
Variable
Units
LL
UL
WBC
K/μL
2.8
26.1
NEUT
K/μL
1
20.1
NEUT
%
40.5
84.6
LYMPH
K/μL
0.6
6.2
LYMPH
%
24.5
26
MONO
K/μL
0
e1.2
MONO
%
1
6
EOS
K/μL
0
1.8
EOS
%
0
8.4
BASO
K/μL
0
0.6
BASO
%
0
2.4
RBC
M/μL
1.99
5.49
HGB
g/dL
5.1
17.2
HCT
%
13.6
46.2
MCV
fL
66
87
MCH
pg
22.5
33.9
MCHC
g/dL
32.1
41.4
RDW
%
10.8
13
PLT
K/μL
53
555
MPV
fL
2.5
8
NRBC
K/μL
0
5.5.