Automated method and reagent therefor assaying body fluid samples such as cerebrospinal fluid (CSF)
View Patent ↗The present invention describes semi- and fully-automated methods and reagents therefor for the assay and analysis of body fluid samples, particularly non-blood samples. The methods and reagents are especially useful for the assay and analysis of cerebrospinal fluid (CSF) samples. The reagent compositions sphere and fix all cells in the sample in suspension. Reported results can include red blood cell (RBC) and white blood cell (WBC) counts, WBC differential values, cell-by-cell volumes and dry-mass concentrations.
1. An automated method for the analysis of a non-blood body fluid, comprising: (a) mixing an aliquot of the body fluid with a reagent composition comprising, in aqueous solution, at least one aldehyde, at least one surface-active agent and cyclodextrin to form a reagent mixture to prevent leakage from cells; (b) aspirating the reagent mixture into an automated analyzer; (c) analyzing the components in the reagent mixture virtually one cell at a time by direct cytometry; and (d) obtaining counts of cell components and cell-by-cell volumes and dry-mass concentrations of the cell components in the body fluid using said direct cytometry.
2. The method according to claim 1 , further comprising: obtaining a cytogram of the cell components in the body fluid.
3. The method according to claim 1 , wherein the method is semi-automated or fully-automated.
4. The method according to claim 1 , wherein the method is semi-automated.
5. The method according to claim 1 , wherein the body fluid is selected from the group consisting of cerebrospinal fluid (CSF), pleural fluid, pulmonary lavage fluid, bronchial lavage fluid, synovial fluid, peritoneal fluid, bone marrow aspirate fluid, ascites fluid, sputum, saliva, lymph, tears, serum, plasma, semen, urine, and bladder wash.
6. The method according to claim 5 , wherein the body fluid is cerebrospinal fluid (CSF).
7. The method according to claim 1 , wherein the at least one aldehyde in the reagent composition is glutaraldehyde, formaldehyde, or a combination of glutaraldehyde and formaldehyde.
8. The method according to claim 7 , wherein the surface active agent is a zwitterionic surfactant.
9. The method according to claim 1 , wherein the surface active agent is a zwitterionic surfactant.
10. The method according to claim 9 , wherein the zwitterionic surfactant is selected from the group consisting of betaines, alkylbetaines, carboxybetaines, sulfobetaines, amidobetaines and sulfoamidobetaines.
11. The method according to claim 10 , wherein the sulfobetaine is selected from the group consisting of n-alkyl dimethylammonio methane sulfonate (DAMS), n-alkyl dimethylammonio ethane sulfonate (DABS), n-alkyl dimethylammonio propane sulfonate (DAPS), n-alkyl dimethylammonio butane sulfonate (DABS), n-tetradecyl dimethylammonio propane sulfonate (TDAPS), and dodecyl dimethylammonio propane sulfonate (DDAPS).
12. The method according to claim 9 , wherein the zwitterionic surfactant is n-tetradecyl dimethylammonio propane sulfonate (TDAPS).
13. The method according to claim 9 , wherein the zwitterionic surfactant is 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO).
14. The method according to claim 1 , wherein the surface active agent is a nonionic surfactant.
15. The method according to claim 7 , wherein the surface active agent is a nonionic surfactant.
16. The method according to claim 14 , wherein the nonionic surfactant is an alkylglycoside.
17. The method according to claim 16 , wherein the alkylglycoside nonionic surfactant is selected from the group consisting of n-dodecyl-β-D-maltoside, n-tetradecyl-β-D-maltoside and n-tetradecyl-β-D-glucoside.
18. The method according claim 1 , wherein the surface active agent is present in the composition in an amount of from about 1.5 g/L to about 3 g/L.
19. The method according to claim 1 , wherein the surface active agent is present in the composition in an amount of from about 1.8 g/L to about 2.5 g/L.
20. The method according to claim 1 , wherein the cyclodextrin is selected from alpha (α) cyclodextrin, beta (β) cyclodextrin, or gamma (γ) cyclodextrin.
21. The method according claim 1 , wherein the cyclodextrin is hydroxypropyl-β-cyclodextrin.
22. The method according to claim 1 , wherein the cyclodextrin is present in the composition in an amount of from about 10 g/L to about 35 g/L.
23. The method according to claim 1 , wherein the cyclodextrin is present in the composition in an amount of from about 15 g/L to about 21 g/L.
24. The method according to claim 1 , wherein the reagent composition further comprises a buffering agent to maintain reagent pH in the range of about pH 4.0 to pH 7.0.
25. The method according to claim 24 , wherein the pH of the reagent composition is in the range of about pH 4.5 to pH 6.0.
26. The method according to claim 24 , wherein the buffering agent comprises one or more of Na 2 HPO 4 and/or NaH 2 PO 4 , citric acid and its salts, succinic acid and its salts, and ethylene diamene tetra acetic acid (EDTA) and its salts.
27. The method according to claim 7 , wherein formaldehyde is present in the reagent composition in an amount of from about 10 g/L to about 25 g/L, and glutaraldehyde is present in the reagent composition in an amount of from about 1 g/L to about 5 g/L.
28. The method according to claim 7 , wherein formaldehyde is present in the reagent composition in an amount of from about 15 g/L to about 23 g/L, and glutaraldehyde is present in the reagent composition in an amount of from about 2 g/L to about 3 g/L.
29. The method according to claim 7 , wherein formaldehyde is present in the reagent composition in an amount of from about 18 g/L to about 21 g/L, and glutaraldehyde is present in the reagent composition in an amount of from about 2.4 g/L to about 2.6 g/L.