Normalizing the response of a fluorescence instrument using spectral response
A method to normalize at least one of a population of subordinate clinical diagnostic analyzer to a master clinical diagnostic analyzer such that an assay result from a subordinate clinical diagnostic analyzer can be converted to the equivalent result of the master clinical diagnostic analyzer by using a simple multiplicative factor when the assay executed on each analyzer uses a common fluorescently labeled dye. Also a method to re-normalize a subordinate clinical diagnostic analyzer assay result to a master clinical diagnostic analyzer assay result by using a simple multiplicative factor when the assay executed on the subordinate clinical diagnostic analyzer uses a different fluorescently labeled dye than the assay executed on the master clinical diagnostic analyzer.
1. A method of normalizing a first diagnostic result of a subordinate clinical diagnostic analyzer to a second diagnostic result of a master clinical diagnostic analyzer, said method comprising the steps of:
(i) obtaining a normalized excitation intensity spectrum of the master clinical diagnostic analyzer,
(ii) obtaining a normalized excitation intensity spectrum of the subordinate clinical diagnostic analyzer,
(iii) obtaining a normalized responsivity intensity spectrum of the master clinical diagnostic analyzer,
(iv) obtaining a normalized responsivity intensity spectrum of the subordinate clinical diagnostic analyzer,
(v) obtaining a normalized excitation/emission spectrum of a solid inorganic photostable fluorophore calibration target,
determining a ratio of spectral differences between the master clinical diagnostic analyzer and the subordinate clinical diagnostic analyzer using at least (i) to (v),
reading the solid inorganic photostable fluorophore calibration target in the master clinical diagnostic analyzer thereby obtaining a first response value (E A ),
reading the solid inorganic photostable fluorophore calibration target in the subordinate clinical diagnostic analyzer thereby obtaining a second response value (E B ),
determining a gain ratio (G R ) of the master clinical diagnostic analyzer to the subordinate clinical diagnostic analyzer based upon (a) a ratio of the first response value and the second response value (E A /E B ), and (b) the ratio of spectral differences,
using the determined gain ratio (G R ) to determine a multiplicative normalization factor between a normalized subordinate clinical diagnostic analyzer and the master clinical diagnostic analyzer,
determining a relative absorption/emission spectrum of a first fluorescently labeled dye whereas the first fluorescently labeled dye is a diagnostic assay component,
obtaining a first diagnostic result from a specific patient specimen or sample incorporating the first fluorescently labeled dye using the normalized subordinate clinical diagnostic analyzer, and
modifying the first diagnostic result by the multiplicative normalization factor to obtain a second diagnostic result, wherein the second diagnostic result is a normalized approximation to a diagnostic result which would be obtained by analyzing the specific patient specimen or sample on the master clinical diagnostic analyzer, and wherein the gain ratio (G R ) is not dependent upon the first fluorescently labeled dye to enable subsequent re-normalization using only absorption/emissivity spectra of a second or subsequent fluorescently labeled dyes substituted for the first fluorescently labeled dye, and
wherein the multiplicative normalization factor is based on the relative absorption/emission spectrum of the first fluorescently labeled dye.
2. A method as claimed in claim 1 , wherein the solid inorganic photostable fluorophore is a phosphate matrix glass.
3. A method as claimed in claim 2 , wherein the phosphate matrix glass is a bismuth-doped phosphate glass.
4. A method as claimed in claim 2 , wherein the phosphate matrix glass is a copper-doped phosphate glass.
5. A method as claimed in claim 1 , wherein the modification of the first diagnostic result by the multiplicative normalization factor to obtain the second diagnostic result is accomplished by using a linear calibration curve.
6. A method as claimed in claim 1 , in which the gain ratio is determined by the relation:
G
R
=
(
G
A
·
φ
A
G
B
·
φ
B
)
=
(
E
A
E
B
)
·
{
∑
χ
S
RB
(
χ
)
·
[
∑
ξ
S
B
(
ξ
)
·
ECglass
(
ξ
,
χ
)
]
∑
χ
S
RA
(
χ
)
·
[
∑
ξ
S
A
(
ξ
)
·
ECglass
(
ξ
,
χ
)
]
}
wherein S RA (χ) corresponds to (i), S RB (χ) corresponds to (ii), S A (ξ) corresponds to (iii), S B (ξ) corresponds to (iv), and ECglass(ξ, χ) corresponds to (v).
7. A method to re-normalize a subordinate clinical diagnostic analyzer assay result as compared to a master clinical diagnostic analyzer assay result comprising the steps of:
normalizing the subordinate clinical diagnostic analyzer as claimed in claim 1 ,
determining a relative absorption/intensity spectrum of a second fluorescently labeled dye whereas the second fluorescently labeled dye is a diagnostic assay component,
determining a re-normalization multiplicative factor between a subordinate clinical diagnostic analyzer and a master clinical diagnostic analyzer,
obtaining a first diagnostic result from a specific patient specimen or sample incorporating the second fluorescently labeled dye using the normalized subordinate clinical diagnostic analyzer, and
modifying the first diagnostic result by the re-normalization factor to obtain a second diagnostic result wherein the second diagnostic result is a normalized approximation to a diagnostic result which would be obtained by analyzing the specific patient specimen or sample on the master clinical diagnostic analyzer.
8. A method as claimed in claim 7 , wherein the modification of the first diagnostic result by the multiplicative re-normalization factor to obtain the second diagnostic result is accomplished by using a linear calibration curve.
9. A method of normalizing a first diagnostic result of a subordinate clinical diagnostic analyzer to a second diagnostic result of a master clinical diagnostic analyzer, said method comprising the steps of:
determining a ratio of spectral differences between the master clinical diagnostic analyzer and the subordinate clinical diagnostic analyzer using at least (i) a normalized excitation intensity spectrum of the master clinical diagnostic analyzer, (ii) a normalized excitation intensity spectrum of the subordinate clinical diagnostic analyzer, (iii) a normalized responsivity intensity spectrum of the master clinical diagnostic analyzer, (iv) a normalized responsivity intensity spectrum of the subordinate clinical diagnostic analyzer, and (v) a normalized excitation/emission spectrum of solid inorganic photostable fluorophore calibration target;
obtaining a first response value (E A ) as the solid inorganic photostable fluorophore calibration target in the master clinical diagnostic analyzer;
obtaining a second response value (E B ) as the solid inorganic photostable fluorophore calibration target in the subordinate clinical diagnostic analyzer;
determining a gain ratio (G R ) of the master clinical diagnostic analyzer to the subordinate clinical diagnostic analyzer based upon (a) a ratio of the first response value and the second response value (E A /E B ), and (b) the ratio of spectral differences;
using the determined gain ratio (G R ) to determine a multiplicative normalization factor between a normalized subordinate clinical diagnostic analyzer and the master clinical diagnostic analyzer;
determining a relative absorption/emission spectrum of a first fluorescently labeled dye whereas the first fluorescently labeled dye is a diagnostic assay component;
obtaining a first diagnostic result from a specific patient specimen or sample incorporating the first fluorescently labeled dye using the normalized subordinate clinical diagnostic analyzer; and
modifying the first diagnostic result by the multiplicative normalization factor to obtain a second diagnostic result,
wherein the second diagnostic result is a normalized approximation to a diagnostic result which would be obtained by analyzing the specific patient specimen or sample on the master clinical diagnostic analyzer, and
wherein the multiplicative normalization factor is based on the relative absorption/emission spectrum of the first fluorescently labeled dye.
10. A method as claimed in claim 9 , in which the gain ratio is determined by the relation:
G
R
=
(
G
A
·
φ
A
G
B
·
φ
B
)
=
(
E
A
E
B
)
·
{
Σ
χ
S
RB
(
χ
)
·
[
Σ
ξ
S
B
(
ξ
)
·
EC
glass
(
ξ
,
χ
)
]
Σ
χ
S
RA
(
χ
)
·
[
Σ
ξ
S
A
(
ξ
)
·
EC
glass
(
ξ
,
χ
)
]
}
wherein S RA (χ) corresponds to (i), S RB (χ) corresponds to (ii), S A (ξ) corresponds to (iii), S B (ξ) corresponds to (iv), and ECglass(ξ, χ) corresponds to (v).
11. A method as claimed in claim 9 , wherein the solid inorganic photostable fluorophore is a phosphate matrix glass.
12. A method as claimed in claim 11 , wherein the phosphate matrix glass is a bismuth-doped phosphate glass, or a copper-doped phosphate glass.
13. A method as claimed in claim 9 , wherein the modification of the first diagnostic result by the multiplicative normalization factor to obtain the second diagnostic result is accomplished by using a linear calibration curve.
14. A method as claimed in claim 9 , wherein a plurality of subordinate clinical diagnostic analyzers are associated with the master clinical diagnostic analyzer, and the method of claim 9 is carried out for the plurality of subordinate clinical diagnostic analyzers.
15. A method to re-normalize a subordinate clinical diagnostic analyzer assay result as compared to a master clinical diagnostic analyzer assay result comprising the steps of:
normalizing the subordinate clinical diagnostic analyzer as claimed in claim 9 ,
determining a relative absorption/intensity spectrum of a second fluorescently labeled dye whereas the second fluorescently labeled dye is a diagnostic assay component,
determining a re-normalization multiplicative factor between a subordinate clinical diagnostic analyzer and a master clinical diagnostic analyzer,
obtaining a first diagnostic result from a specific patient specimen or sample incorporating the second fluorescently labeled dye using the normalized subordinate clinical diagnostic analyzer, and
modifying the first diagnostic result by the re-normalization factor to obtain a second diagnostic result wherein the second diagnostic result is a normalized approximation to a diagnostic result which would be obtained by analyzing the specific patient specimen or sample on the master clinical diagnostic analyzer.