Screening test for beta-thalassemia trait and a diagnostic kit for its individualization

Claim 1

A method for detecting quantitatively beta-thalassemia in a patient which consists of: (a) preparing an aqueous solution A containing 148.5 mmoles of glycerol, 58 mmoles of sodium chloride, 7 mmoles of sodium dihydrogen phosphate, 3 mmoles of disodium monohydrogen phoshate, 1 mmole of sodium azide, 0.04 mmoles of Acid Green 5 (C 37 H 34 N 2 Na 2 O 3 S 9 ) of pH 6.5 per liter of solution; (b) placing 3 cc of said solution A from step (a) in a test tube; (c) adding to said solution A 5 microliters of capillary freshly drawn blood or venous blood collected in the presence of an anticoagulant, stirring therewith to obtain a suspension and incubating said suspension for 30-45 minutes at room temperature to obtain the test suspension; (d) preparing solution B from 150 mmoles of sodium chloride and 0.04 mmoles of Acid Green 5, said solution B being the 100% turbidity solution; (e) placing 3 ml of said solution B from step (d) in a second test tube; (f) preparing a blood suspension having 100% turbidity by shaking said solution B with 5 microliters of the blood from the same patient to obtain the comparison suspension; (g) separately measuring at 495 nm wavelength the optical densities of said carefully stirred blood suspensions from steps (c) and (f) against said solution A from step (a) without blood added, and using said solution A as a blank; (h) comparing the degree of turbidity based on values calculated as percent turbidity according to the equation ##EQU2## and when the turbidity value is lower than 30%, said patient is normal, when the turbidity is between 30 and 50%, said patient is suspect and when the turbidity value is greater than 50%, said patient is a carrier of beta-thalassemia trait.

Claim 2

A method for detecting quantitatively beta-thalassemia in a patient which consists of: (a) preparing an aqueous solution A containing 148.5 mmoles of glycerol, 58 mmoles of sodium chloride, 7 mmoles of sodium dihydrogen phosphate, 3 mmoles of disodium monohydrogen phoshate, 1 mmole of sodium azide, 0.04 mmoles of Acid Green 5 (C 37 H 34 N 2 Na 2 O 3 S 9 ) of pH 6.5 per liter of solution; (b) placing 3 cc of said solution A from step (a) in a test tube; (c) adding to said solution A 5 microliters of capillary freshly drawn blood or venous blood collected in the presence of an anticoagulant, stirring therewith to obtain a suspension and incubating said suspension for 30-45 minutes at room temperature to obtain the test suspension; (d) preparing solution B from 150 mmoles of sodium chloride and 0.04 mmoles of Acid Green 5, said solution B being the 100% turbidity solution; (e) placing 3 ml of said solution B from step (d) in a second test tube; (f) preparing a blood suspension having 100% turbidity by shaking said solution B with 5 microliters of the blood from the same patient to obtain the comparison suspension; (g) separately measuring at 495 nm wavelength the optical densities of said carefully stirred blood suspensions from steps (c) and (f) against said solution A from step (a) without blood added, and using said solution A as a blank; (h) comparing the degree of turbidity based on values calculated as percent turbidity according to the equation ##EQU2## and when the turbidity value is lower than 30%, said patient is normal, when the turbidity is between 30 and 50%, said patient is suspect and when the turbidity value is greater than 50%, said patient is a carrier of beta-thalassemia trait.

Claim 3

A method for carrying out a test to detect beta-thalassemia in a patient, which consists of: (a) preparing an aqueous solution A containing 148.5 mmoles of glycerol, 58 mmoles of sodium chloride, 7 mmoles of sodium dihydrogen phosphate, 3 mmoles of disodium monohydrogen phosphate, 1.5 mmole of sodium azide, 0.04 mmole of Acid Green 5 (C 37 H 34 N 2 Na 2 O 3 S 9 ) of pH 6.5 per liter of solution; (b) placing 2 cc of the solution A from step (a) in one well of a well cluster of a plurality of cylindrical wells, having transparent bottom; (c) adding to said solution A in said well 10 microliters of capillary freshly drawn blood or venous blood collected in the presence of an anticoagulant, stirring therewith to obtain a suspension and incubating said suspension for 30-45 minutes at room temperature; (d) juxtaposing the wells cluster to a grid consisting of a thin card having groups of three parallel lines of different thickness so that each group of three lines cross the median bottom zone of said well; and (e) measuring after gentle stirring the degree of turbidity of said suspension by the degree of visibility of said grid lines, so that said test is considered negative when all three grid lines are clearly visible, said test is slightly positive indicative of a suspect patient when only the larger grid lines are faintly visible, and said test is positive when all three grid lines are not visible.

Claim 4

A method for carrying out a test to detect beta-thalassemia in a patient, which consists of: (a) preparing an aqueous solution A containing 148.5 mmoles of glycerol, 58 mmoles of sodium chloride, 7 mmoles of sodium dihydrogen phosphate, 3 mmoles of disodium monohydrogen phosphate, 1.5 mmole of sodium azide, 0.04 mmole of Acid Green 5 (C 37 H 34 N 2 Na 2 O 3 S 9 ) of pH 6.5 per liter of solution; (b) placing 2 cc of the solution A from step (a) in one well of a well cluster of a plurality of cylindrical wells, having transparent bottom; (c) adding to said solution A in said well 10 microliters of capillary freshly drawn blood or venous blood collected in the presence of an anticoagulant, stirring therewith to obtain a suspension and incubating said suspension for 30-45 minutes at room temperature; (d) juxtaposing the wells cluster to a grid consisting of a thin card having groups of three parallel lines of different thickness so that each group of three lines cross the median bottom zone of said well; and (e) measuring after gentle stirring the degree of turbidity of said suspension by the degree of visibility of said grid lines, so that said test is considered negative when all three grid lines are clearly visible, said test is slightly positive indicative of a suspect patient when only the larger grid lines are faintly visible, and said test is positive when all three grid lines are not visible.