Preparation of nucleated RBC (NRBC) analogs for use as reference hematology controls in automated hematology analyzers
The subject invention pertains to compositions of novel analogs of red blood cells that are distinguishable from white blood cells in a hematological instrument and processes for manufacturing such analogs. The processes for creating the compositions include washing, shrinking, and fixing cells at temperatures at or below room temperature.
1. A process for manufacture of red blood cells which are distinguishable from white blood cells for use as a reference control in a blood cell analyzer, the process comprising the steps of:
a) shrinking and washing red blood cells of a non-human vertebrate by application of a salt solution comprising a denaturing agent, EDTA, potassium cyanide, potassium ferricyanide, sodium nitrate, potassium hydroxide, sodium hydroxide, MOPS, sodium fluoride, mecaptosuccinic acid, sodium omadine, amikacin, tetracycline, gentamycin sulfate, and reagent alcohol to extract cellular fluid from the red blood cells; and
b) applying a quantity of a fixing agent to the red blood cells from step a).
2. The process of claim 1 , wherein the non-human vertebrate is a chicken.
3. The process of claim 1 , wherein the red blood cells are nucleated red blood cells.
4. The process of claim 1 , wherein the salt solution comprises a salt and a naphthol or a derivative thereof as the denaturing agent.
5. The process of claim 4 , wherein the denaturing agent is α-naphthol.
6. The process of claim 4 , wherein the denaturing agent has a concentration of about 0.1 g/L to about 100 g/L.
7. The process of claim 4 , wherein the salt is present at a concentration of about 0.1 g/L to about 100 g/L.
8. The process of claim 1 , wherein the fixing agent is glutaraldehyde.
9. The process of claim 1 , wherein the fixing agent has a concentration of about 0.01% to about 10%.
10. The process of claim 1 , wherein the salt solution has an osmolality of about 600 mOsm to about 1170 mOsm.
11. Modified red blood cells for use as reference controls in blood cell analyzers prepared by the process of claim 1 .
12. A method of using a reference control containing a modified red blood cell component comprising the steps of:
a) providing a reference control containing the modified red blood cells of claim 11 ;
b) providing a blood analyzer adapted for analyzing said reference control and differentiating the modified red blood cells from other cell types;
c) passing said reference control through said blood analyzer for detection and differentiation of said modified red blood cells from other cell types; and
d) reporting modified red blood cells in said reference control.
13. The method of claim 12 , wherein said differentiation of the modified red blood cells is performed using an impedance measurement.
14. The method of claim 12 , wherein said differentiation of the modified red blood cells is performed using an optical measurement.
15. The method of claim 14 , wherein said optical measurement is one or more measurements selected from the group consisting of fluorescence, light scatter and axial light loss measurements.
16. The method of claim 12 , wherein said differentiation of the modified red blood cells is performed using a combination of impedance and optical measurements.
17. The process of claim 1 , wherein the salt solution comprises:
Concentration
concentration
Material
(g/L)
(mL/L)
EDTA
6.3
1-Naphthol, 3-6-Disulfonic Acid
4.5
Sodium nitrate
4.0
Potassium Hydroxide
2.6
Sodium hydroxide
1.2
MOPS
2
Sodium Fluoride
0.5
Mercaptosuccinic Acid
0.1
Sodium Omadine
0.25
Potassium Ferric Cyanide
0.4
Potassium Cyanide
0.2
Amikacin
0.2
Tetracycline
0.2
Gentamycin Sulfate
0.1
Reagent Alcohol (90% 200 proof
35
ethyl alcohol, 5% methyl alcohol,
and 5% isopropyl alcohol)
18. The process of claim 1 , wherein the denaturing agent is naphthol.
19. The process of claim 1 , wherein the denaturing agent is α-naphthol or β-naphthol.
20. The process of claim 1 , wherein the denaturing agent is 4-chloro-1-naphthol, 1,5-dihydroxynaphthalene, 1,7-dihydroxynaphthalene, 1-methoxy-4-nitronaphtalene, 4-fluorosulfonyl-1-hydroxy-2-naphthoic acid, 1,3-dihydroxynaphthalene, 4-methoxy-1-naphthonitrile, 2-nitro-1-naphthol, 4-methoxy-1-naphthol, 2-acetyl-1-naphthol, 9-phenanthrol, 1-naphthol-3,6-disulfonic acid disodium salt hydrate, alpha-hydroxy-heptafluoronaphthalene, 5-amino-1-naphthol, N-(2-acetamidophenethyl)-1-hydroxy-2-naphthamide, 8-amino-1-naphthol-3,6-disulfonic acid or a monosodium salt monohydrate thereof, 8-amino-1-naphthol-5-sulfonic acid, chromotropic acid or a salt thereof, 2,4-dichloro-1-naphthol, 1-naphthol-2-sulfonic acid or a potassium salt thereof, 1-naphthol-4-sulfonic acid or a salt thereof, 1,7-dihydroxynaphthalene, 4-aminosulfonyl-1-hydroxy-2-naphthoic acid, 5-hydroxy-1-naphthalenesulfonamide, 6-amino-1-naphthol, 2-(2-hydroxy-1,1,1,3,3,3-hexafluoropropyl)-1-naphthol, 1,4-dihydroxy-2-naphthoic acid, 1,4-Dihydroxy-2-naphthoic acid phenyl ester, 1,6-dihydroxynaphthalene, 4-hydroxy-1-naphthaldehyde, 7-anilino-1-naphthol-3-sulfonic acid, 6-amino-1-naphthol-3-sulfonic acid, 4-nitro-1-naphthol, 4-hydroxy-6,7-di(methoxycarbonyl)-1-naphthol, 2,4,6,8-tetranitro-5-hydroxy-1-naphthol, 4,6-dinitroso-5-hydroxy-1-naphthol, 4,6-diamino-5-hydroxy-1-naphthol, 2,2′-binaphthyl-1,1′-diol, 2,3-di(methoxycarbonyl)-1-naphthol, 2-acetyl-3-butyl 1-naphthol, 3-phenyl-1-naphthol, 1-naphthol-8-sulfonic acid or a salt thereof, 3-chloro-1,4-dihydroxynaphthalene, 1-amino-5-naphthol-7-sulfonic acid, 2-fluoro-1-naphthol, 3,5-dihydroxy-2-naphthoic acid, 1-naphthol-3-sulfonic acid or a salt thereof, 2,4-dibromo-1-naphthol, 6-amino-1-naphthol, 2-amino-1-naphtholhydrochloride, 2,3-dicyano-1,4-dihydroxy-5-nitronaphthalene, 1,2-dihydroxynaphthalene, 1-hydroxy-2-naphthaldehyde, 4-phenylsulfonamido-1-naphthol, 2-(4-phenylsulfonyl)-4-(4-chlorophenylsulfonamido) 1-naphthol or 4-acetamido-1-naphthol.
21. The process of claim 1 , wherein the denaturing agent is a di-alkali metal salt of a naphthol-disulfonic acid.