FIELD

The disclosed subject matter relates to a system for fluorescence and reflectance measurements. Particularly, the present disclosed subject matter is directed to the quantitative determination of blood and/or blood bound fluorophores.

BACKGROUND OF THE DISCLOSED SUBJECT MATTER

When measuring the fluorescence of a blood or blood-bound fluorophore, e.g., the red blood cell bound fluorophore zinc protoporphyrin, from tissue, e.g., the oral mucosa, the fluorescence intensity depends on the tissue structure and tissue optical properties. This may be problematic for achieving accurate measurements. Generally, the parameter with the largest influence is the amount of blood found in the tissue. There have been efforts to detect the amount of influence imposed by relevant parameters (tissue structure, tissue optical properties). These efforts include: extraction of a parameter quantifying the influence of tissue structure and tissue optical properties from the autofluorescence spectrum measured upon blue light excitation, and extraction of this parameter by measuring the amount of remitted light at the excitation wavelength and a second wavelength. There is a need to reliably measure the intensity of fluorescent light emitted from blood and/or blood bound fluorophores that takes account of the influences on measurements caused by the tissue structure and tissue optical properties.

Measuring the intensity of fluorescent light emitted from one or more blood and/or blood bound fluorophores is necessary for various diagnostic methods. A determination of the concentration of the fluorophores or the concentration relative to the absorbing heme or hemoglobin can be made from the intensity measurement. For example, measuring the concentration-ratio of erythrocyte-bound zinc protoporphyrin (ZnPP), and/or protoporphyrin IX (PP) to heme can be used for the diagnosis of iron deficiency, lead poisoning, and different porphyrias.

To determine the ZnPP and PP concentrations, blood is typically taken from the subject and fluorescence spectroscopic measurements are conducted, either directly on the blood sample or after separation of the fluorophores from the sample. In this way, ZnPP and PP can be separated and quantitatively determined by HPLC (high performance liquid chromatography). For direct measurements on a blood sample, the “hematofluorometer” (e.g., model 206D of the company AVIV Biomedical, Inc., Lakewood, N.J., USA) illuminates a drop of blood in vitro and estimates the ratio ZnPP/heme from the measured fluorescence intensity.

Alternatively, fluorescence spectroscopic measurements can be made on intact tissue in vivo. In a manner similar to the hematofluorometer, intact tissue (e.g., the oral mucosa) is illuminated with excitation light. The fluorescence intensity from the erythrocyte-bound ZnPP and PP is measured, and the ratio of ZnPP/heme and PP/heme can be derived from this measurement. In contrast to measurement from a blood sample, however, the precise structure of the tissue, e.g., thickness of the epithelium which does not contain blood and does not contribute to the ZnPP fluorescence signal, and other tissue parameters are unknown. These tissue parameters include tissue absorption, scattering, blood vessel diameter and the blood volume fraction (BVF), which is the relative proportion of the blood volume to the tissue volume. These tissue parameters can substantially influence the fluorescence intensity value, especially at low BVF. As a consequence, the measured intensity of the fluorescent light varies in an unknown, intra- and inter-individual way. Consequently, if the desired values (the ratios ZnPP/heme and PP/heme) are derived from the measured fluorescence signal, these values will also be influenced by this unknown variation. In principle, determination of the tissue parameters is possible, for example, by white light reflectance measurements but such measurements are complicated, expensive, and have uncertain effectiveness.

SUMMARY

As described and embodied herein, an apparatus and method is provided to overcome the problems of inaccurate measurement of a blood analyte in tissue. The apparatus and method reliably measures the blood analyte. The invention is directed to an apparatus for reliable quantitative measurement of a fluorescent blood analyte in tissue comprising the features of claim1and a method for a quantitative measurement of a fluorescent blood analyte in tissue with the features of claim19. Advantageous configurations of the apparatus and the method are also apparent from the respectively dependent claims.

In accordance with one aspect of the described subject matter, an apparatus is provided for measurement of a blood analyte in tissue comprising: At least one light source, the light source emitting excitation light at least at a first wavelength range between 350 nm and 450 nm to the tissue, a detection unit, the detection unit measuring: a) a portion of the fluorescent light emitted by the fluorescent blood analyte excited at the first wavelength range; and b) a portion of the autofluorescence emitted by the tissue; and/or c1) a portion of the remitted excitation light at the first wavelength range, and c2) a portion of the remitted light at a second wavelength range. The apparatus further includes a control unit, the control unit operating the light source and detection unit. Since the tissue autofluorescence is partially absorbed by the blood before it reaches the detector, the autofluorescence can serve as a measure of the amount of blood in the tissue volume, the blood volume fraction (BVF), as well as other tissue parameters. Therefore, this measure can serve for excluding measurements unsuitable for reliable quantitative determination of the fluorescent blood analyte. Further, remission, the propagation of light back from tissue toward the light source, includes both the light reflected from the tissue surface and the light scattered within the tissue that then passes through the tissue surface back to the source. Light at the excitation wavelength is strongly absorbed by blood so that the amount of remitted light strongly depends on the BVF, while light in another wavelength range is less influenced by blood and the amount of remitted light therefore mainly depends on light scattering and epithelium layer thickness. Therefore, both measurements can be combined to derive a parameter identifying measurements and/or tissue sites suitable for quantitative fluorescence measurements of the blood analyte. Further, it is possible to use only the detected intensity of the remitted excitation light at the first wavelength range or the detected intensity of the remitted excitation light at the second wavelength range to derive a parameter identifying measurements and/or tissue sites suitable for quantitative fluorescence measurements of the blood analyte. With the described apparatus, locations at a tissue site can be identified where the thickness of the epithelial layer, the blood volume fraction, and other parameters of the tissue, are suitable to allow for quantitative evaluation of the measured analyte fluorescence.

In some embodiments, the apparatus comprises a processor configured to configured to determine a parameter from at least one intensity of said portion of the autofluorescence emitted by the tissue at a wavelength range between about 530 and 600 nm, especially about 580 nm, to identify measurements and/or tissue sites, where the detected amount of fluorescence light is a quantitative measure of the fluorescent blood analyte. Further, it is possible that the processor is configured or is additionally configured to derive a correction factor based on at least one intensity of said portion of the autofluorescence emitted by the tissue at a wavelength range between about 530 and 600 nm, especially about 580 nm, which allows scaling the measured fluorescence intensity, so that it is a quantitative measure of the fluorescent blood analyte. A wavelength range between about 530 and 600 nm means that the wavelength could be at 530 nm, 531 nm, 532 nm, 533 nm, 534 nm, 535 nm, 536 nm, 537 nm, 538 nm, 539 nm, 540 nm, 541 nm, 542 nm, 543 nm, 544 nm, 545 nm, 546 nm, 547 nm, 548 nm, 549 nm, 550 nm, 551 nm, 552 nm, 553 nm, 554 nm, 555 nm, 556 nm, 557 nm, 558 nm, 559 nm, 560 nm, 561 nm, 562 nm, 563 nm, 564 nm, 565 nm, 566 nm, 567 nm, 568 nm, 569 nm, 570 nm, 571 nm, 572 nm, 573 nm, 574 nm, 575 nm, 576 nm, 577 nm, 578 nm, 579 nm, 580 nm, 581 nm, 582 nm, 583 nm, 584 nm, 585 nm, 586 nm, 587 nm, 588 nm, 589 nm, 590 nm, 591 nm, 592 nm, 593 nm, 594 nm, 595 nm, 596 nm, 597 nm, 598 nm, 599 nm, 600 nm. As an example, the autofluorescence emitted by the tissue at a wavelength of 545 nm and/or 576 nm could be measured.

Further, it is possible that the processor is configured or is additionally configured to determine a parameter from the remitted light at the first wavelength range and the second wavelength range to identify measurements and/or tissue sites, where the detected amount of fluorescence light is a quantitative measure of the blood analyte. Further, it is possible that the processor is configured or is additionally configured to derive a correction factor from the remitted light at the first wavelength range and the second wavelength range, which allows for scaling the measured fluorescence intensity, so that it is a quantitative measure of the blood analyte. The ratio of a portion of the remitted excitation light at the first wavelength range (R1) and a portion of the remitted light at a second wavelength range (R2) could be corrected by a factor which is specific for the construction of the apparatus used. For example, the ratio R1/R2could be corrected as R10.8/R2with 0.8 as the correction factor specified for the apparatus. Another function of R1and R2, containing correction factors specific for the apparatus could be applied as well.

In some embodiments, the second wavelength range is between 450 nm and 750 nm. This means that the second wavelength could be at 450 nm, 455 nm, 460 nm, 465 nm, 470 nm, 475 nm, 480 nm, 485 nm, 490 nm, 495 nm, 500 nm, 505 nm, 510 nm, 515 nm, 520 nm, 525 nm, 530 nm, 535 nm, 540 nm, 545 nm, 550 nm, 555 nm, 560 nm, 565 nm, 570 nm, 575 nm, 580 nm, 585 nm, 590 nm, 595 nm, 600 nm, 605 nm, 610 nm, 615 nm, 620 nm, 625 nm, 630 nm, 635 nm, 640 nm, 645 nm, 650 nm, 655 nm, 660 nm, 665 nm, 670 nm, 675 nm, 680 nm, 685 nm, 690 nm, 695 nm, 700 nm, 705 nm, 710 nm, 715 nm, 720 nm, 725 nm, 730 nm, 735 nm, 740 nm, 745 nm, 750 nm. Other wavelengths within the mention range are possible. Further, the first wavelength could be at 350 nm, 351 nm, 352 nm, 353 nm, 354 nm, 355 nm, 356 nm, 357 nm, 358 nm, 359 nm, 360 nm, 361 nm, 362 nm, 363 nm, 364 nm, 365 nm, 366 nm, 367 nm, 368 nm, 369 nm, 370 nm, 371 nm, 372 nm, 373 nm, 374 nm, 375 nm, 376 nm, 377 nm, 378 nm, 379 nm, 380 nm, 381 nm, 382 nm, 383 nm, 384 nm, 385 nm, 386 nm, 387 nm, 388 nm, 389 nm, 390 nm, 391 nm, 392 nm, 393 nm, 394 nm, 395 nm, 396 nm, 397 nm, 398 nm, 399 nm, 400 nm, 401 nm, 402 nm, 403 nm, 404 nm, 405 nm, 406 nm, 407 nm, 408 nm, 409 nm, 410 nm, 411 nm, 412 nm, 413 nm, 414 nm, 415 nm, 416 nm, 417 nm, 418 nm, 419 nm, 420 nm, 421 nm, 422 nm, 423 nm, 424 nm, 425 nm, 426 nm, 427 nm, 428 nm, 429 nm, 430 nm, 431 nm, 432 nm, 433 nm, 434 nm, 435 nm, 436 nm, 437 nm, 438 nm, 439 nm, 440 nm, 441 nm, 442 nm, 443 nm, 444 nm, 445 nm, 446 nm, 447 nm, 448 nm, 449 nm or 450 nm. Additionally, the light source and detection unit can be configured as a single integral component. Other components of the inventive apparatus can also be part of the single integral component.

In some embodiments, the detection unit comprises optical means for collecting emitted fluorescent light and/or remitted light at the first and/or second wavelength ranges. Further, it is possible that the detection unit comprises a probe having a proximal end and a distal end, wherein the distal end is configured to be applied directly or indirectly to the tissue. In another embodiment the detection unit is configured as a probe having a proximal end and a distal end, wherein the distal end is configured to be applied directly or indirectly to the tissue. Furthermore, the probe could be designed to comprise at least one suction means formed at the distal end of the probe. Said suction means could comprise at least one suction aperture formed at the distal end of the probe. In yet another embodiment of the invention said probe comprises a cover attached to the distal end wherein the cover is configured to be applied directly or indirectly to the tissue. The at least one suction means could be formed within the cover. In a further embodiment, at least the detection unit is configured as a handheld device.

In some embodiments, the apparatus comprises a light guide guiding excitation light to the tissue and/or collecting emitted fluorescent light and/or remitted light at the first and/or second wavelength ranges. According to one embodiment of the apparatus, the light guide is an optical fiber or fiber bundle.

In certain embodiments, the measured fluorescent blood analyte includes the zinc protoporphyrin (ZnPP)/heme and/or protoporphyrin IX (PP)/heme ratios and/or ZnPP and/or PP concentrations. In some embodiments the apparatus is adapted to estimate the BVF.

In accordance with another aspect of the invention, a method is provided for measuring a blood analyte in tissue comprising the steps of: emitting excitation light at least at a first wavelength range between 350 nm and 450 nm to the tissue; and further a) detecting a portion of the fluorescent light emitted by the fluorescent blood analyte excited at the first wavelength range, and b) detecting a portion of the autofluorescence emitted by the tissue; and/or c1) detecting a portion of the remitted excitation light at the first wavelength range, and c2) detecting a portion of the remitted light at a second wavelength range; and determining an information for excluding measurements and/or tissue sites unsuitable for reliable quantitative determination of the fluorescent blood analyte based on at least one intensity of said autofluorescence portion emitted by the tissue at a wavelength range between about 530 and 600 nm, especially about 580 nm, and/or based on a parameter such as the ratio of the detected intensity of the remitted excitation light at the first wavelength range to the detected intensity of the remitted light at the second wavelength range or vice versa. Since the tissue autofluorescence is partially absorbed by the blood before it reaches the detector, the autofluorescence can serve as a measure of the amount of blood in the tissue volume, the blood volume fraction (BVF), as well as other tissue properties. Therefore, this measure serves for excluding measurements and/or tissue sites unsuitable for reliable determination of the fluorescent blood analyte concentration. A wavelength range between about 530 and 600 nm means that the wavelength could be at 530 nm, 531 nm, 532 nm, 533 nm, 534 nm, 535 nm, 536 nm, 537 nm, 538 nm, 539 nm, 540 nm, 541 nm, 542 nm, 543 nm, 544 nm, 545 nm, 546 nm, 547 nm, 548 nm, 549 nm, 550 nm, 551 nm, 552 nm, 553 nm, 554 nm, 555 nm, 556 nm, 557 nm, 558 nm, 559 nm, 560 nm, 561 nm, 562 nm, 563 nm, 564 nm, 565 nm, 566 nm, 567 nm, 568 nm, 569 nm, 570 nm, 571 nm, 572 nm, 573 nm, 574 nm, 575 nm, 576 nm, 577 nm, 578 nm, 579 nm, 580 nm, 581 nm, 582 nm, 583 nm, 584 nm, 585 nm, 586 nm, 587 nm, 588 nm, 589 nm, 590 nm, 591 nm, 592 nm, 593 nm, 594 nm, 595 nm, 596 nm, 597 nm, 598 nm, 599 nm, 600 nm. As an example, the autofluorescence emitted by the tissue at a wavelength of 545 nm and/or 576 nm could be measured.

Further, it is possible to use only the detected intensity of the remitted excitation light at the first wavelength range or the detected intensity of the remitted light at the second wavelength range to derive a parameter identifying measurements and/or tissue sites suitable for quantitative fluorescence measurements of the blood analyte. Further, remission, the propagation of light back from tissue toward the detector, includes the light reflected from the tissue surface and/or the light scattered within the tissue that then passes through the tissue surface back to the source. The ratio of a portion of the remitted excitation light at the first wavelength range (R1) and a portion of the remitted light at a second wavelength range (R2) could be corrected by a factor which is specific for the construction of the apparatus used. For example, the ratio R1/R2could corrected as R10.8/R2with 0.8 as the correction factor specified for the apparatus. Another function of R1and R2, containing correction factors specific for the apparatus could be applied as well.

In some embodiments, the second wavelength range is between 450 nm and 750 nm. This means that the second wavelength could be at 450 nm, 455 nm, 460 nm, 465 nm, 470 nm, 475 nm, 480 nm, 485 nm, 490 nm, 495 nm, 500 nm, 505 nm, 510 nm, 515 nm, 520 nm, 525 nm, 530 nm, 535 nm, 540 nm, 545 nm, 550 nm, 555 nm, 560 nm, 565 nm, 570 nm, 575 nm, 580 nm, 585 nm, 590 nm, 595 nm, 600 nm, 605 nm, 610 nm, 615 nm, 620 nm, 625 nm, 630 nm, 635 nm, 640 nm, 645 nm, 650 nm, 655 nm, 660 nm, 665 nm, 670 nm, 675 nm, 680 nm, 685 nm, 690 nm, 695 nm, 700 nm, 705 nm, 710 nm, 715 nm, 720 nm, 725 nm, 730 nm, 735 nm, 740 nm, 745 nm, 750 nm. Other wavelengths within the mention range are possible. Further, the first wavelength could be at 350 nm, 351 nm, 352 nm, 353 nm, 354 nm, 355 nm, 356 nm, 357 nm, 358 nm, 359 nm, 360 nm, 361 nm, 362 nm, 363 nm, 364 nm, 365 nm, 366 nm, 367 nm, 368 nm, 369 nm, 370 nm, 371 nm, 372 nm, 373 nm, 374 nm, 375 nm, 376 nm, 377 nm, 378 nm, 379 nm, 380 nm, 381 nm, 382 nm, 383 nm, 384 nm, 385 nm, 386 nm, 387 nm, 388 nm, 389 nm, 390 nm, 391 nm, 392 nm, 393 nm, 394 nm, 395 nm, 396 nm, 397 nm, 398 nm, 399 nm, 400 nm, 401 nm, 402 nm, 403 nm, 404 nm, 405 nm, 406 nm, 407 nm, 408 nm, 409 nm, 410 nm, 411 nm, 412 nm, 413 nm, 414 nm, 415 nm, 416 nm, 417 nm, 418 nm, 419 nm, 420 nm, 421 nm, 422 nm, 423 nm, 424 nm, 425 nm, 426 nm, 427 nm, 428 nm, 429 nm, 430 nm, 431 nm, 432 nm, 433 nm, 434 nm, 435 nm, 436 nm, 437 nm, 438 nm, 439 nm, 440 nm, 441 nm, 442 nm, 443 nm, 444 nm, 445 nm, 446 nm, 447 nm, 448 nm, 449 nm or 450 nm.

With the described method, tissue sites can be identified where the thickness of the epithelial layer, the blood volume fraction, and the optical parameters of the tissue, are suitable to allow for quantitative evaluation of the measured analyte fluorescence.

In one embodiment, the quantitatively determined blood analyte is the ZnPP/heme ratio, and the first wavelength is in the range of 395 to 435 nm and the second wavelength is about 520 nm. The remitted light is used to identify tissue sites suitable for reliable fluorescence measurements and/or to correct the measured fluorescence intensity so that it is reliable measure of the ZnPP/heme ratio.

In another embodiment, the quantitatively determined blood analyte is the ZnPP/heme ratio, and the first wavelength is in the range of 395 to 435 nm. The tissue autofluorescence in the wavelength range of 560 to 600 nm is used to identify tissue sites suitable for reliable fluorescence measurements and/or to correct the measured fluorescence intensity so that it is reliable measure of the ZnPP/heme ratio.

In another embodiment, the quantitatively determined blood analyte is a vitamin, e.g. vitamin A (retinol), and the first wavelength is in the range of 350 to 400 nm. Remitted light and/or tissue autofluorescence is used to identify tissue sites suitable for reliable fluorescence measurements and/or to correct the measured fluorescence intensity so that it is reliable measure of said vitamin.

