CA2460898A1

CA2460898A1 - Apparatus and method for combining in vivo and in vitro testing

Info

Publication number: CA2460898A1
Application number: CA002460898A
Authority: CA
Inventors: James Samsoondar
Original assignee: Individual
Current assignee: Individual
Priority date: 2004-03-09
Filing date: 2004-03-09
Publication date: 2005-09-09
Also published as: WO2005084527A1; US20050203356A1

Abstract

In vivo testing is painless with minimal risks of infection. The location of analytes in different fluid compartments in the body, and accumulation of the analytes in different concentrations in the fluid compartments, make spectroscopic in vivo testing very inaccurate.
The present invention provides a single apparatus and method for combining in vivo testing and in vitro testing. The apparatus comprises one or more source of electromagnetic radiation (EMR) within the wavelength range of 300nm to 2500nm, one or more slots for a biological sample, one or more receptor for a body part, a spectrometer, and electronics. The apparatus optionally contains a computer processor and appropriate software. Either the biological sample or the body part can be irradiated with EMR, and calibration algorithms can be applied to the emerging EMR, to predict analyte concentrations or other parameters related to analytes in biological samples.
Measurement of the analytes and other parameters will compensate for some of the limitations of in vivo testing, while capturing the benefits of in vivo testing.

Description

Apparatus and Method for Combining In vivo and In vitro Testing Field of invention This invention relates to the field of spectroscopic measurement of analytes in samples.
More specifically, the invention relates to a combination of in vitro and in vivo testing.
Background of the Invention Great efforts have been made over the last decade to develop a method for measuring glucose non-invasively (i.e., in vivo testing), to help diabetic patients monitor their blood glucose frequently and in a painless manner. It should be understood that the terms "in vivo testing" and "non-invasive testing" are used interchangeably in the following text to refer to testing without breaking the skin, in contrast to "in vitro testing," which requires at least breaking the skin to obtain a sample, for example blood. Near Infrared (NIR) spectroscopic methods are the most common non-invasive methods under investigation, but currently there is no spectroscopic apparatus available, and very little progress has been made in the development of a spectroscopic apparatus for in vivo measurement of blood glucose. In order to understand in vivo spectroscopic measurement, glucose will be discussed. In order to understand in vivo measurement of glucose, one must understand how the body processes glucose, and how spectroscopic methods work.
Dietary glucose is absorbed in the rich blood supply to the gut, where the glucose is then circulated around the other parts of the body. All the blood in the circulation traverses the entire circuit of the circulation an average of once each minute when the body is at rest, and as many as six times when a person becomes extremely active. The capillaries are the smallest blood vessels with walls of a single layer of cells and of diameter barely large enough for red blood cells to squeeze through. The capillary walls are permeable to small molecules like water and glucose.
As the blood passes through the capillaries, the glucose and water rapidly diffuses from the vascular compartment into the interstitial compartments, where the glucose concentration in both vascular and interstitial compartments equilibrate. Most body cells (e.g.
muscle cells) require insulin for glucose uptake. The internalized glucose is rapidly metabolized to provide energy, leaving a very low glucose concentration in the intracellular compartment, resulting in the compartment of highest fluid volume (the intracellular compartment) having the lowest glucose concentration.
In an average young male, 18% of the body weight is protein and related substances, 7% is mineral, and 15% is fat; the remaining 60% is water. The intracellular compartment of the body water accounts for about 55% of the body water and the extracellular compartment accounts for about 45%. In terms of volume, the average body water is about 42L (23L
intracellular, and 19L
extracellular of which 8L are interstitial and 3L plasma). Many factors affect the volumes of the fluid compartments, e.g., height, weight, gender, diseases, and age. Within an individual, these volumes can also be affected by activity level, diet, hormone fluctuations, pharmaceuticals, and body part.
In vivo NIR spectroscopic measurement of an analyte depends omthe correlation between the wavelength-specific absorbances of electromagnetic radiation (EMR) by the body part, and the blood concentrations of the analyte. Sophisticated statistical techniques are used to develop a calibration equation or algorithm (sometimes also referred to as a calibration model), using the blood analyte concentration (measured in, for example, millimoles per liter or milligrams per deciliter glucose) as the independent variable, and the absorbances at many wavelengths as the dependent variables. However, the NIR interacts or "sees"
all tissue in its path, i.e., both blood and non-blood tissue; and all the tissue contribute to the absorbance spectrum. Therefore, what the NIR "sees" is the tissue analyte concentration, which is much lower than the blood glucose concentration. In order to understand the concept of tissue glucose, one can think of a dilution of the blood glucose by the intracellular fluid (which has very little glucose and accounts for about 55% volume of body water), and hence one can consider the concept of "average tissue glucose." If there is a good correlation between blood analyte concentration and average tissue analyte concentration, there should be no significant difficulties in developing a calibration algorithm for that analyte, assuming that the analyte possesses sufficient absorbance signal. Because the correlation between blood glucose concentration and the average tissue glucose concentration is unreliable, the errors in the NIR
in vivo method would be random (referred to as random inaccuracies). By performing replicates, the magnitude of "random inaccuracies" cannot be diminished, as would be when other forms of random error occur. The correlation between blood glucose concentration and average tissue glucose concentration is based on the percent volume of fluid that is interstitial, intracellular and blood, and also the glucose concentration in each fluid.
Therefore, assuming that the glucose absorbance signal is easily detectable, it appears that the tissue compartmentalization of body fluids is the main reason for the difficulties encountered in the development of an in vivo apparatus for measuring blood glucose. If we assume that the main reason for the difficulties encountered in the development of an in vivo apparatus for measuring blood glucose is the lack of sufficient absorbance by glucose (i.e., the glucose signal), then it should be a simple task to develop an in vivo calibration algorithm fox Hemoglobin (Hb) in blood; Hb is a substance in abundance in the blood, and the absorbance signals are very large. The hemoglobin absorbance spectrum is well known and displayed in Figures 1 & 2. The fact that Hb is only contained in the vascular compartment should make the task even simpler. Accurate spectroscopic reagentless in vitro measurement of hemoglobin using NIR radiation is a very simple task, and is described by Samsoondar et al in US Pat. Nos. 6,353,471, and 6,268,910. Surprisingly, a non-invasive method for measuring blood hemoglobin is not a simple task, as discovered by M. Rendell et al (Determination of hemoglobin levels in the finger using NIR spectroscopy, Clin. Lab. Haem., 2003, 25, p93-97). Rendell et al concluded that they could only discriminate between patients who had normal hemoglobin, lov hemoglobin, and very low hemoglobin.
Therefore, the inventor concludes that compartmentalization of body fluids as explained above, is the major challenge facing the development of in vivo apparatus for measuring the concentration of blood analytes.
Pulse Oximetry is one area of in vivo testing or measurement, which is performed with relative success. Pulse Oximetry is described in details by Y. Mendelson in "Pulse Oximetry:
Theory and application for noninvasive monitoring " (Clinical Chemistry, 38/9:
1601-1607, 1992), and by K. K. Tremper in "Pulse Oximetry" (Anesthesiology, 70: 98-108, 1989). Pulse Oximetry monitors a patient's hemoglobin oxygen saturation or blood oxygen saturation (also referred to as Sa0?) by measuring the light attenuated by a finger, at two wavelengths (about 660nm and about 940nrn), at the peak and trough of a pulse. The measurement at the trough of a pulse represents the background absorbance by all the tissue (including the venous blood), which is subtracted from the absorbance at the peak of the pulse. The reason for the

2 relative success of Pulse Oximetry lies in the fact that hemoglobin oxygen saturation is the ratio of the concentration of Oxy-hemoglobin (Oxy-Hb} to Total-Hemoglobin (Tot-Hb), assuming that Tot-Hb comprises mainly Oxy-Hb, and Deoxy-hemoglobin (Deoxy-Hb).
The absorbance spectra of same amounts of Oxy-Hb and Deoxy-Hb, cross at about 800nm, as displayed in Figure 1. Figure 1 is reproduced from the two references cited above (Mendelson and Tremper). Therefore, a good correlation between hemoglobin oxygen saturation and the ratio of absorbances at 940nm and 660nm exists. Since the hemoglobin-saturation of arterial blood is more clinically relevant; the measurements must be synchronized with the pulse. As expected, Pulse Oximetry errors increase when other hemoglobin derivatives or species are present in elevated concentrations, e.g., elevated levels of Methemoglobin (Met-Hb) or Carboxy-Hemoglobin (Carboxy-Hb). Errors in Hb oxygen saturation caused by Met-Hb and Carboxy-Hb could be appreciated by examining the absorbance spectra in Figures 1 & 2. The National Committee for Clinical Laboratory Standards (NCCLS) (Document C25-P, page 22, published April 1990) recommended that the presence of dyshemoglobins (i.e:, Met-Hb and Carboxy-Hb in particular) must be assessed before using in vivo oximeters (e.g., Pulse Oximeters). However, there is no suggestion of use of a single apparatus that can be used for both in vivo and in vitro testing.
Another area of relative success in non-invasive measurement of blood analytes as described in Yamanishi US Pat. No. 4,267,844 (Medical instrument for determining jaundice).
Jaundice is a yellowing of skin due to the presence of high levels of bilirubin in blood and non-blood tissue. Monitoring blood bilirubin in newborn babies (neonates) is very important because when blood albumin (about 60% otal plasma protein) becomes saturated with bilirubin, the excess bilirubin can enter the interstitial space and hence cross the blood brain barrier. When the blood brain baxrier is crossed, the bilirubin can cause permanent brain damage. Therefore measurement of blood bilirubin in neonates is very important, and a non-invasive method is preferred to drawing blood from the neonates by heel prick.
Bilirubin, like hemoglobin, absorbs an abundance of visible light, and it should be a simple task to measure bilirubin non-invasively. Again; the situation is complicated by the location of bilirubin in more than one fluid compartment, in an unpredictable manner.
Yamanishi described the measurement of bilirubin in skin tissue in US Pat. No.
4,267,844, by squeezing out some of the blood from the tissue measurement site. The difficulties encountered in measuring blood bilirubin non-invasively is partly overcome by relying on the correlation between blood bilirubin and non-blood bilirubin (or skin bilirubin). For those skilled in the art of bilirubin measurement and its clinical significance; it is well known that the clinically relevant bilirubin is blood bilirubiy, and that the correlation between blood bilirubin and non-blood bilirubin is very unreliable. In vivo measurement of bilirubin is discussed more in "Detailed Description of the T.nvention." There is no suggestion in the prior art regarding bilirubin measurement, of an apparatus that combines both in vivo measurement with in vitro measurement of bilirubin.
Rosenthal US Pat. No. 6,066,847 describes the use of an in vitro glucose meter in verifying the accuracy of a non-invasive (in vivo) blood glucose measurement instrument.
There was no suggestion in US. Pat. No. 6,066,847, of a single instrument that can perform the two measurements, and there was no suggestion that the in vitro results could be used in any way to improve the accuracy of the in vivo results. Also not suggested in US. Pat.
No. 6,066,847 was the use of an iri vivo parameter or another analyte measured non-invasively, which can be used adjunctively with the in vitro glucose measurement.

3 In US patent application 10/136,329 (Publication Number 2003-0138960 A1), the present inventor described the use of Met-Hb as an indicator of degradation of Hb-based blood substitutes, and mentioned that measurements can be either in vivo, in vitYO, or both in vivo and in vitro. US Patent application 10/136329 did not describe any apparatus that can be used for a combination of in vivo and in vitro testing, and US Patent application 10/136329 did not suggest any method that would combine the in vivo and in vitro testing, using a single apparatus.
In vivo testing or measurement of blood analytes is in great demand because no blood sample has to be drawn. Elimination of blood samples eliminates the pain encountered, and the risk of infection during the handling of blood. The inventor has described three different in vivo systems, and their severe limitations, and to date, there is no in vivo testing method known to the inventor that overcomes these limitations. Unless an analyte is distributed across all fluid compartments in equal concentrations, the inventor believes that it is impossible to develop a calibration algorithm for the analyte that will predict blood analyte concentrations with accuracy comparable to the in vitro systems. Even a patient-specific in vivo calibration algorithm for a blood analyte will fall short of predicting accurate results.
It is the intent of the inventor to describe an apparatus and method that can combine both in vivo and in vitro testing, in a manner that .will compensate for some of the limitations of in vivo testing, and capture some of the benefits of in vivo testing.
Summary of the Invention In vivo testing is painless with minimal risks of infection. The location of analytes in different fluid compartments in the body, :and accumulation of the analytes in different concentrations in the fluid compartments, make spectroscopic in vivo testing very inaccurate.
The present invention provides a single apparatus and method for combining in vivo testing and in vitro testing. An apparatus for combining in vivo testing and in vitro testing is described, comprising:
a. one or more sources of electromagnetic radiation (EMR);
b. one or more photodetectors;
c. one or more slots in the host system of the apparatus far a sample vessel for in vitro testing of a biological sample taken from a patient;
d. one or more receptors for a body part of the patient for in vivo testing, wherein one or more receptor is located in the host system of the apparatus, or one or more remote receptors are connected to the host system of the apparatus, or a combination thereof;
and e. electronics.
The apparatus optionally comprises a computer processor and software.
In another embodiment, the apparatus further contains one or more remote receptors, wherein the one or more remote receptors are connected to the host system by a method selected from the group consisting of, a wireless method, one or more electrical wires, one or more fiber optic cables, and any combination thereof.
In yet another embodiment, one of the one of the one or more remote receptors comprises, one or more light emitting diodes (LED's), one ox more photodetectors, electronics, a

4 transmitter, and the host system further comprises a receiver that is compatible with the transmitter.
In yet another embodiment, the the one or more receptors are shaped to accept the body part, and the one or more receptors is adapted to allow EMR to enter the body part at a first surface of the body part, and the one or more receptors are also adapted to allow passage of at least some of the EMR, wherein the emerging EMR emerges from a second surface of the body part, and wherein the second surface is the same as or different from the first surface.
In yet another embodiment, the ane or more receptors are shaped to accept the body part, and the one or more receptors are adapted to allow EMR to enter the body part at the front surface of the body part, and the one or more receptors are also adapted to allow passage of at least some of the EMR, wherein the emerging EMR emerges from the back side of the body part, to be reflected off areflective surface in the one or more receptors adjacent to the back side of the body part, and the reflected EMR is collected either at the front surface or a different surface of the body part.
In yet another embodiment, the slot is adapted to allow EMR to enter a front side of the slot housing the sample vessel, and the transmitted EMR is collected at the back side of the slot.
In yet another embodiment, the slot, is adapted to allow EMR to enter a front side of the slot housing the sample :vessel, and the transmitted EMR is reflected off a reflective surface located at either the back side of the slot, or the side of the sample vessel adjacent to the back side of the slot, andthe reflected EMR is collected at the front side of the slot.
In yet another embodiment, the sample vessel is selected from the group consisting of, a cuvette, a sample tab, a pipette tip, tubing, labeled test tubes, unlabeled test tubes, blood bag tubing, any transparent sample container, any translucent sample container, and a flow-through cuvette.
In yet another embodiment, the sample vessel contains one or more reagents In yet another embodiment, the sample vessel is either a cuvette or a sample tab, and the cuvette or the sample tab contains one or more reagents.
In yet another embodiment, the sample vessel is a sample tab, and the slot is designed to accept the sample tab in a horizontal direction.
In yet another embodiment, the one or more sources of EMR is selected from the group consisting of, a tungsten lamp, one or more light emitting diodes (LED's), one or more lasers, and any combination thereof.
In yet another embodiment, the one or more photodetectors is selected from the group consisting of, a single photo diode, an array of photo diodes, an array of charged coupled detectors, and any combination thereof.
In yet another embodiment, the vessel is a sample tab comprising of a base plate with a sample well and a cover, wherein at least a portion of the base plate and at least a portion of the cover, is adapted to permit transmission of EMR therethrough.

In yet another embodiment, the vessel is a sample tab comprising of a base plate with a sample well and a cover, wherein at least a portion of the base plate is adapted to permit transmission of EMR through the sample, :and at least a portion of the cover is adapted to reflect EMR emerging from the sample, and wherein the reflected EMR is allowed to travexse the sample before leaving the sample tab at the base plate, or wherein at least a portion of the cover is adapted to permit transmission of EMR through the sample, and at least a portion of the base plate is adapted to reflect EMR emerging from the sample, and wherein he reflected EMR is allowed to traverse the sample before leaving the sample tab at the cover.
In yet another embodiment, the biological sample is selected from the group consisting of whole blood, a pinprick capillary blood sample, serum, plasma, urine, cerebrospinal fluid, sputum, synovial fluid, lymphatic fluid, feces.
In yet another embodiment, the body part is selected from the group consisting of a finger, an ear lobe, a forearm, a web between two fingers, a fold of skin, or the surface of any body part.
In yet another embodiment, the one or more sources of EMR provides EMR at one or more wavelengths selected from the wavelength range of 300nm to 2500 nm.
In still another aspect of the invention, a method is described, that combines in vivo testing (step a) and in vitro testing (step b) using the apparatus of the present invention, wherein the in vitro testing is performed at least once, and the in vivo testing is performed as frequently as necessary for monitoring a patient depending on the clinical usefulness of such testing, comprising:
a. obtaining a value of one or more analytes in a biological sample obtained from the patient, by applying one or more calibration algorithm to the order derivative of absorbance obtained from the biological sample in a vessel, at one or more wavelengths of a standard set of wavelengths;
b. calculating one or more parameters from one or more sets of order derivative of absorbances obtained from the body part of the patient, wherein the one or more sets of order derivative of absorbances are obtained at one or more wavelengths of a standard set of wavelengths, and wherein the one or more parameters are the same as or different from the one or more: analytes.
In yet another aspect of the invention, the one or more parameters are the same as or different from the one or more analytes, and the orie or more in vivo parameters are used adjunctly with the one or moi:e in vitro analytes.
In yet another aspect of the invention, the method further comprises, calculating the one or more parameters from the values obtained from the one or more analytes measured in the biological sample, for one or more purposes selected from the group consisting of, confirming the results of the in vivo testing, assessing the integrity of the results of the in vivo testing, correcting the results of the in vivo testing, and any combination thereof.
In yet another aspect of the invention, the value of one or more analytes measured in the biological sample, is used for one or more purposes selected from the group consisting of confirming the results of the in vivo testing, assessing the integrity of the results of the in vivo testing, correcting the results of the in vivo testing, and any combination thereof.

In yet another aspect of the invention, part of the in vivo testing is performed by applying a calibration algorithm to the absorbance for the body part at two or more wavelengths, wherein the calibration algorithm is a linear equation containing a constant plus one or more terms, wherein each of the one or more terms is an independent variable multiplied by a constant, and wherein each of the independent variable is the ratio of absorbances at two different wavelengths.
In yet another aspect of the invention; part of the in vitro testing is performed by applying a calibration algorithm to an order derivative of the absorbance at one or more wavelengths of the biological sample in the sample vessel; and wherein part of the in vivo testing is performed by applying a calibration algorithm to an order derivative of the absorbance for the body part, at one or more wavelengths.
In yet another aspect of the invention, the body part is selected from the group consisting of, a finger, an ear Lobe; a forearm, a web between two fingers, a fold of skin, mucous membrane, the surface of any body part.
In yet another aspect of the invention, the biological sample is selected from the group consisting of, whole blood, serum, plasma, urine, cerebrospinal fluid, sputum, synovial fluid, lymphatic fluid, feces.
In yet another aspect of the invention, the whole blood is a pinprick capillary blood sample.
In yet another aspect of the invention, the 'sample vessel is a cuvette or a sample tab.
In yet another aspect of the invention, the vessel contains one or more reagents, and an altered absorbance zs obtained, after reaction between the biological sample and the one or more reagents.
In yet another aspect of the invention, the one or more parameters is selected from the group consisting of, the proportion of Hemoglobin-based blood substitute in its Methemoglobin form, proportion of hemoglobin in its Carboxy-Hemoglobin form, proportion of hemoglobin in its Methemoglobin form, hemoglobin oxygen saturation, a ratio of bilirubin concentration to biliverdin concentration, and any combination thereof.
In yet another aspect of the invention, the one or more wavelengths in step (a) and step (b) of the method described above, are selected from the wavelength i:a.nge of 300nm to 2500 nm.
In yet another aspect of the invention, the order derivative of absorbance is selected from the group consisting of, zero order, first order, second order, and third order.
Brief Description of the Drawings These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
Figure 1 is a graphic representation of the absorbance spectra of four different hemoglobin species, as shown, in the wavelength range of 600 - 1 OOOnm plotted on the x-axis, and log of extinction coefficient plotted on the y-axis.

Figure 2 is a, graphic representation of the absorbance spectra of four different hemoglobin species, as shown, in the wavelength range of S00 - 700nm plotted on the x-axis, and absorbance of the same concentration of each specie (equivalent to extinction coefficient) on the y-axis.
Figure 3 is a graphic representation of the absorbance spectra of three different concentrations of total Hb, from the same pool, which was allowed to become partly oxidized to produce Met-Hb, which is also shown.
Figure 4a is a schematic view of a preferred embodiment of the present invention, with one host slot, one host receptor, and no remote receptor.
Figure 4b is a schematic view of a preferred embodiment of the present invention, with one host slot, no host receptor, and one remote receptor.
Figure 4c is'a schematic view of a preferred embodiment o.f the present invention, with one host slot, no host receptor, and one remote receptor, and a separate reference beam.
Figure 4d is a schematic view of a preferred embodiment of the present invention, with one host slot, one host receptor, one remote receptor, and communication channel between host system and other instruments.
Figure 4e is a schematic view of a preferred embodiment of the present invention, with one host slot and one remote receptor, which is place in contact with the surface of a body part.
Figure 5 is a graphic representation of a finger inserted into a receptor.
Figure 6 depicts various aspects of an embodiment of a sample tab used in the present invention. Figure 6a illustrates oblique views of a sample tab and a slot.
Figure 6b -exhibits a side view of the sample tab inserted into the slot:
Figure 7 depicts various aspects of an alternate embodiment of a sample tab used in the present invention. Figure 7a illustrates an oblique view of the sample tab. Figure 7b exhibits a side view of the sample tab Detailed Description of the Invention The current approaches to non-invasive testing (in vivo testing) of blood analytes have not been successful except, to the knowledge of the inventor, in pulse oximetry and bilirubin measurement. Yet, both pulse oximetry and the bilirubin measurement systems contain significant limitations. The present invention describes an apparatus and method used for combining in vivo and in vitro measurements. The combination of in vivo and in vitro testing is useful for improving patient care and the patient care process as follows:
1 ) Minimize the risks of infectious diseases.
The present invention could minimize the risks of infectious diseases during the handling of blood, by reducing the number of blood samples drawn. It should be understood that the risk of infection is not limited to blood, but applies to all biological samples, and in vitro testing of any biological sample is within the scope of the present invention.
2) Minimize the pain experienced by patients.
The present invention will minimize the pain experienced by the patient, by reducing the number of blood samples drawn. It should be understood that pain is not limited to obtaining blood samples, but also applies to obtaining other biological samples, e.g., cerebrospinal fluid and synovial fluid.
3) Minimize errors in diagnosis and treatment of patients.

The present invention could minimize the errors in diagnosis and treatment of patients, if in vivo testing alone is relied upon for diagnosis and treatment, and could compensate for some of the limitations regarding in vivo testing: Blood bilirubin measurement by the in vivo jaundice meter described in "Background of the Invention" (Yamanishi-US
Pat. No.
4;267,844) could be used for illustration as follows: Bilirubin could be underestimated when the blood bilirubin is increasing, since significant amounts of bilirubin can enter the interstitial fluids only after the blood albumin becomes saturated with bilirubin.
Therefore, if the in vivo jaundice meter is relied upon to administer phototherapy to a neonate, treatment could become started later than the appropriate time.
Similarly, phototherapy can become halted later than the appropriate time because the blood is cleared of bilirubin much faster than the non-blood tissue, causing an overestimation of the blood bilirubin by the in vivo jaundice meter.
4) Minimize transportation of biological samples.
By using one apparatus that combines in viva and in vitro testing, the present invention will eliminate the transportation of biological samples from the in vivo testing site to a remote in vitro apparatus.
For clarity, the inventor would like to differentiate a "slot" from a "receptor." In brief, by "slot" it is meant an opening through which the sample vessel is to be placed, or a groove or channel or slit into which the sample vessel fits; an example of a slot 60, is shown in Figure 6a and Figure 6b, and an example of a receptor S4 is shown in Figure 5. A slot is required for in vitro testing, and a receptor is required for in vivo testing.
The present invention is preferably used for measurement of blood analytes, measurement of blood parameters, or calculation of blood 'parameters. More preferably, the analytes are restricted to the vascular compartment (see "background of the invention" for more details on fluid compartments), and a useful parameter can be measured, or calculated as a ratio of the concentrations of two analytes. However, analytes that are not restricted to the vascular compartment are also considered to be within the scope of the present invention. It should be understood that, unless specified, when "an" analyte or "a" parameter is mentioned, "one or more" analyzes and '"one or more" parameters respectively are implied. By "analyte" it is meant a substance being measured in a sample. By "parameter" it is meant an analyte or some other value measured directly (for example, the Pulse Oximeter uses a calibration algorithm for measuring the parameter "blood oxygen saturation"), or a value calculated from other measurements (for example, "blood oxygen saturation" can be calculated as the percent of Tot-Hb that is in the form of Oxy-Hb, after measuring total-Hb and Oxy-Hb concentration). The in vivo parameter could be calculated from an order derivative of absorbance of the body part, at one or more wavelengths, or the in vivo parameter could be the same as the analyte measured by in vitro testing of a biological sample obtained from the same patient subjected to in vivo testing. Furthermore, the parameter could be calculated from sets of order derivative of absorbance measurement. The in vitro measurement of one or more analytes, of the present invention; could be used for diagnosis, treatment, confirmation of an in vivo result; assistance in interpreting the in vivo measurement, assistance in providing a useful in vivo parameter, and can be used as a form of quality assurance for the in vivo testing. The in vivo measurement of the present invention could be a parameter that is monitored more frequently or continuously, optionally as an adjunct to the in vitro analyte measurements. Conversely, the in vitro test could also be an adjunct to the in vivo test.

To the inventor's knowledge, there is no prior description of an apparatus capable of performing both in vivo and in vitro testing. Examples of specific measured analytes and specific measured or calculated parameters, which are used to illustrate usefulness of the present invention are discussed below. The examples given are for illustration only, and should not be considered limiting in any way, to the use of the apparatus.
MONITORING HEMOGLOBIN OXYGEN SATURATION
The ratio of Oxy-Hb to Total-Hb provides a parameter called hemoglobin oxygen saturation or blood oxygen saturation (also referred to as Sa02) by measuring the light attenuated by a anger, at two wavelengths. These measurements can be synchronized with the pulse, to monitor oxygen saturation of arterial blood. Mendelson in "Pulse Oximetry: Theory and application for noninvasive monitoring " (Clinical Chemistry, 38/9: 1601-1607, 1992), describes the process in brief as follows: Two wavelengths of light (660nm or manometers and 940nrn) are used. The pulse oximeter first determines the AC component of absorbance at each wavelength and devides this by the corresponding DC component to obtain a "pulse-added" absorbance that is independent of the incident light intensity. The ratio (R) of these pulse-added absorbances, which is empirically related to SaO2, is then calculated as follows:
R = (AC660/DC660)/(AC940/DC940) The DC component is the absorbance at the trough of a pulse, and the AC
component is the difference between the absorbance at the peak of a pulse and the DC
component.
The absorbance spectra of same amounts of Oxy-Hb and Deoxy-Hb, cross at about 805nm, i.e., an i'sosbestic wavelength; where the absorbance of EMR is independent of blood oxygenation. At 660nm, the absorbance of Oxy-Hb is less than the absorbanee of Deoxy-Hb; and the reverse is true at 940nm (See Figure 1, reproduced from the two references cited above by Mendelson and Tremper).
As expected, errors in Pulse Oximetry increase when other hemoglobin derivatives (or species) are present in elevated concentrations, e.g., elevated levels of Met-Hb or Carboxy-Hb (referred to as dyshemoglobins). The National Committee for Clinical Laboratory Standards (NCCLS) (Document C25-P, page 22, published April 1990) recommended that the presence of dyshemoglobins must be assessed before using in vivo oximeters (e.g., Pulse Oximeters). Although an arterial blood sample, which has to be obtained by a physician and cannot be exposed to air, is essential for in vitro measurement of oxygen saturation, an in vitro measurement for ruling out the presence of significant amounts of dyshemoglobins does not require an arterial blood sample. In the present invention, the in vivo parameter could be oxygen saturation, which is monitored frequently, and the in vitro measurement, which is preferably performed once, is used to assess the presence of dyshemoglobins. The in vitro measurement preferably uses a pinprick blood sample.
2. MONITORING OXIDATION OF HEMOGLOBIN
Oxidation of the iron in the heme moiety of Hb molecules is a normal process that occurs in vivo. Enzymes are continually at work reversing the oxidation process and thus preventing the accumulation of Met-Hb. Methemoglobinemia is a condition of people that lack enzymes, e.g., NADH methemoglobim reductase, required to reverse the oxidation process.
The Met-Hb reductase system maybe underdeveloped in infants. making methemoglobinemia more prevalent among infants. Another reason for the higher incidence of methemoglobinemia among infants and neonates is an underdeveloped gastrointestinal system in some infants. In an underdeveloped gastrointestinal system, bacteria level could rise due to a decrease secretion of gastric acid. Nitrates are usually converted into nitrites by bacteria of the gastrointestinal system, and the nitrites in turn react with the Hb to produce Met-Hb. Blood loss is critical in neonates, and even a heel-prick blood sample from a pre-matured neonate: is considered critical blood loss. In these neonates, the decrease frequency of blood sampling due to the use of an in vivo apparatus that monitors %Met-Hb, would be especially useful.
Lack of Met-Hb reductase enzymes in hemolyzed serum causes spontaneous oxidation of Hb to Met-Hb over time, causing the sample to darken in the color. Figure 3 illustrates how the absorbance spectra of a hemolyzed sample changes as it ages. The absorbance peak at about 632nm that accompanies the darkening of color indicates a conversion of Hb to Met-Hb.
Accumulation of Met-Hb could also occur in patients who are not lacking the Met-Hb reductase enzymes. In these patients, the accumulation of Met-Hb could be induced by the intake of certain therapeutic drugs and other chemicals, for examples, which should not be considered limiting in any way: dapsone, chloroquine, phenazopyridine, phenacetin, nitrates, nitrites, phenols; and aniline. Patients with high levels of Met-Hb, whatever the cause, should be monitored for the increase of Met-Hb, or the decrease of Met-Hb after treatment, or both the increase and decrease.
In a normal person, the composition of Hb (% of Tot-Hb) in the arterial blood is about 95 Oxy-Hb, about 2% Deoxy-Hb, about 2% Carboxy-Hb and about 1 % Met-Hb, as measured by CO-Oximetry. In a heavy smoker; the %Carboxy-Hb can be about 10%. It should be understood that the Hb composition depends on the CO-Oximeters used to measure the % of the Hb species. Newer CO-Oximeters tend to give different numbers, which are supposedly more reliable, since the measurements in the newer CO-Oximeters are performed at more wavelengths. More wavelengths could help compensate for interfering substances like, for example, biliruliin, turbidity, Sulfhemoglobin, and fetal hemoglobin. It should also be understood that although CO-Oximeters are considered by some as reference instruments for measuring the °~o Hb species, the methods using CO-Oximeters are not true reference methods for measuring the % of the Hb species in a blood sample.
The Tot-Hb and Met-Hb could be measured once in a pinprick blood sample (in vitro) and the %Met-Hb calculated. The in vitro measurement of %Met-Hb can be used to verify the in vivo measurement of the %Met-Hb, which can be monitored more frequently or continuously. The calibration algorithm for in vivo measurement of %Met-Hb could be developed empirically by taking the ratio of absorbances of a body part at two different wavelengths, for example about 630nm and about 560nm. In Figure 2, it is clear that the absorbance at about 630nm is greater for Met-Hb than for the same amount of each of the other species shown; the reverse is true at about 560nm. A calibration algorithm for in vivo %Met-Hb can also be develop empirically using the ratio of absorbances at ~60nm and 940 nm (see Figure 1), using the same logic. These wavelengths are just examples that can be used, and should not be considered limiting in any way. Furthermore, the ratio of absorbances at 560nm and 940 nm, could be one of more than one ratio term in a calibration algorithm for %Met-Hb. It should be understood, that the use of ratio of absorbances as a single term, or the use of the sum of more than one similar term in a calibration algorithn is preferred. However, any statistical technique used to develop a calibration algorithm is considered to be within the scope of the present invention. It should be obvious to those skilled in the art that this approach is very similar to the approach used to develop calibration algorithms for blood oxygen saturation obtained by pulse oximetry, described in the previous section. Since the %Met-Hb should be the same in arterial and venous blood, the in vivo measurement of %Met-Hb would not have to be synchronized with the pulse, making development of the calibration algorithm easier. The more difficult task is to obtain patients with varying amounts of Met-Hb. This task can be accomplished by selecting patients with an enzyme deficiency that causes methemogiobinemia, who will have different %Met-Hb at different times. Alternatively, patients infused with Hb-based blood substitutes, as discussed later, can be used. Samsoondar in US Pat. No. 6,689,612, the contents of which are incorporated herein by reference, describes a method for correcting the measurement of Tot-Hb (used as an indicator of hemolysis in serum and plasma), for the presence of Met-Hb.
The methods described for in vitro calibration of Met-Hb can also be used for whole blood samples.
The in vivo measurement of %Met-Hb is expected to produce higher accuracy that the in vivo measurement of the concentration of Met-Hb (e.g. Met-Hb, measured in grams /L); as mentioned before, Rendell et al demonstrated the difficulty in developing an in vivo calibration algorithm for Tot-Hb. Therefore, the concentration of a Hb specie (in this case, g/L Met-Hb) could be obtained by multiplying the in vivo %Met-Hb (i.e., obtained non-invasively), to the in vitro Tot-Hb (i.e., obtained from a blood sa.mple). For example, if the in vivo parameter is 10 % Met-Hb (i.e.; 10% of Tot-Hb), and the in vitro measurement is 12C
grams/L Tot-Hb, then the calculated grams/L of Met-Hb would be 12 grams/L. In this example, the in vitro Tot-Hb could be measured once over several days, and the in vivo %Met-Hb could be measured more frequently, for example, every few minutes.
Assuming there is no significant blood loss or dehydration, the change in Tot-Hb over the few days will be small, and the calculated g/L Met-Hb will be more accurate than a result obtained through an in vivo calibration for grams/L Met-Hb.
Uncontrollable spontaneous oxidation of Hb-based blood substitutes is another source of Met-Hb, and Hb-based blood substitutes are discussed in details later.
3. MONITORING METHYLENE BLUE TREATMENT FOR METHEMOGLOBINEMIA
One method used to treat methemoglobinemia is intravenous administration of methylene blue. Since high doses of methylene blue can also produce Met-Hb, monitoring the treatment is very important. In vitro measurement of methylene blue is a useful method for monitoring the treatment of methemoglobinemia, and is a useful adjunct to the in vivo monitoring of the %Met-Hb. Samsoondar et al in US Pat. No. 6,268,910, the contents of which are incorporated herein by reference, discloses spectroscopic in vitro measurement of methylene blue. Therefore, it should be understood that the present invention is not limited to the manner in which the in vitro aald in vivo measurements are used. Furthermore, the in vitro analyte could be different from the analyte or parameter measured in vivo, and also, the analyte measurement in vitro does not necessarily have to be the one that is used to calculate the in vivo parameter that is monitored. In the example of administration of methylene blue, the patient can be cared for in a better way if both the methylene blue (the in vitro analyte) and the %Met-Hb (the in vivo parameter) are measured.
4. MONITORING OXIDATION OF HEMOGLOBIN-BASED BLOOD SUBSTITUTES
Blood transfusion is a life-saving process that is performed after severe blood loss after trauma or during surgery. Some advantages of using a blood substitute instead of whole blood (by "whole blood" it is meant the combination the cellular and non-cellular components of blood) or red blood cells are as follows:
a) Blood substitutes are expected to be universally compatible with all blood types, therefore cross matching will not be necessary. b) Maximum storage time of blood is 42 days, whereas the blood substitutes could have a much longer shelf life. c) The purification process of the blood substitute may include heat treatment, which can minimize the threat of hazardous viruses.
Most blood substitutes under development are made from human Hb, bovine Hb, or recombinant DNA technology (recombinant Hb). Hemoglobin comprises four protein subunits, which are two pairs of identical polypeptide chains. Each subunit has a molecular weight of about 16,000, with a cleft that contains a heme (iron-porphyrin) group, the site of oxygen uptake. The subunits are not covalently linked, and require the red cell membrane to keep the subunits together. A hemoglobin molecule is too large to penetrate the kidney, but the subunits are small enough to become lodged in the kidney and cause kidney failure. In Hb-based blood substitutes, the subunits of the Hb could be chemically cross-linked with each other or to :large polymers, or the Hb molecules could be linked to other Hb molecules to form poly-Hb, for stability. The Hb subunits may be inter- or infra-molecularly cross-linked. Regardless of the protein or pol3~rner surrounding the heme groups, the absorbance spectrum of Hb=based blood substitutes is almost identical to normal Hb, but subtle differences at certain wavelengths may be present. The Hb-based blood substitutes are not protected from uncontrollable spontaneous oxidation into Met-Hb since they are no longer housed within the red cell membrane, where the Hb is usually in contact with Met-Hb reductase enzymes. T.M.S. Chang provides a detailed review of blood substitutes in vohunes I and II of "Blood Substitutes: Principles, Methods, Products and Clinical Trials" 1998, published by Karger Landes Systems. It should be understood that any form of Hb-based blood substitutes is considered to be within the scope of the present invention.
Due to the absence of the Met-Hb reduetase enzymes, accumulation of Met-Hb could occur in the plasma of patients transfused with Hb-based blood substitutes:
Measurement or calculation of the ratio of Met-Hb to Total-Hb is useful for monitoring the degradation of Hb-based blood substitutes to its Met-Hb form, or for monitoring the reversal of the oxidation process after for example, administration of one or more therapeutic agents, or monitoring a retardation in the spontaneous oxidation process by encapsulating the Hb-based blood substitutes with enzymes like NADH methemoglobin reductase or other reducing agents. In this example, the two blood analytes are the Hb-based blood substitute, and the Met-Hb form of the Hb-based blood substitute. In a patient transfused with one or more types of Hb-based blood substitutes, it should be understood that the Total-Hb could include both the one or more Hb-based blood substitutes and endogenous Hb, and the Met-Hb could include both the Met-Hb forms of the Hb-based blood substitutes and endogenous Met-Hb.
A method for monitoring degradation (or oxidation to be more specific) of Hb-based blood substitutes i~ vivo and in vitro, requires development of calibration algorithms for Met-Hb and the Hb-based blood substitute. The calibration algorithms can be developed by optionally using any statistical technique to process EMR absorbed by a sample at one or more wavelengths. The concentration of the one or more Hb-based blood substitutes and the Met-Hb can then be determined by applying the respective calibration algorithm to the absorbance of the sample at one or more wavelengths. Using a calibration algorithm for Met-Hb and another calibration algorithm for the Hb-based blood substitute, will allow the Met-Hb to be reported as a fraction of the total Hb-based blood substitute.
Alternatively, a calibration algorithm could be developed for the fraction of the total Hb-based blood substitute that is in the form of Met-Hb.
Samsoondar in US Patent Application No. 10/136,329 (Publication Number 2003-Al), the contents of which are incorporated herein by reference, describes a method of monitoring the degradation of Hb-based blood substitutes by monitoring the production of the Met-Hb derivative of the Hb-based blood substitutes. The application teaches that the sample can be whole blood, serum, plasma, or a body part from the patient infused with the blood substitute. For the convenience and comfort of the patients, it is preferred that the sample is a body part, where the measurement is performed non-invasively. Due to the limitations described in the background of the invention regarding non-invasive measurement of blood analytes, an aspect of the present invention is to provide an apparatus that can confirm the analyte measurement on a blood sample, i.e.; in vitro measurement.
In order to monitor the production of Met-Hb as an indicator of degradation of blood substitutes, the present invention could permit the in vivo measurement of % Met-Hb frequently or continuously, and the absolute concentration of Met-Hb and total Hb could be measured in a blood sample (in vitro).
Samsoondar in US Pat. No. 6,689,612, the contents of which are incorporated herein by reference, describes a method for correcting the measurement of Tot Hb (used as an indicator of hemolysis in serum and plasma), for the presence of Met-Hb. Methods of in vitro calibration for 'fot-Hb and Met-Hb are described. Similar methods can be used for in vivo calibration for °XoMet-Hb (of Tot-Hb), except the sample is a body part of one or more patients, with varying amounts of °f°Met-Hb. Preferably, several patients should be used, with variation in Tot-Hb and interfering substances like, for example, bilirubin, turbidity, Sulfhemoglobin, fetal hemoglobin, and other Hb species. More variation included in the calibration set; help to develop a more robust calibration algorithm. The calibration algorithm can be as simple as: %Met-Hb is equal to the ratio of absorbances at two wavelengths multiplied by a constant; the algorithm can be developed empirically as done for Pulse-Oximetry, except the pulse can be ignored since the %Met-Hb would be the same in both arterial and venous blood. Wavelengths can betaken from Figures 1& 2, for example, about 630nm or about 1000nm (where the extinction coefficient is largest for Met-Hb), and about S60nm (where the extinction coefficient is lowest for Met-Hb). It should be understood that these are just examples, which should not be considered limiting in any way.
Furthermore, the sum of more than one ratio terms can be used to compensate for interfering substances.
In another aspect of the invention, the in vitro results can be used to adjust the in vivo measurement of absolute concentrations of analytes in blood, by calculating the ratio of the in vitro concentration to the in vivo concentration of the same analyte; the calibration algorithm for the in vivo measurement of the absolute concentration could be modified by the ratio calculated previously.
S. MONITORING ACCUMULATION AND DISAPPEARANCE OF CARBOXY-HEMOGLOBIN
Measurement of the ratio of Carboxy-Hb to Total-Hb could be used for monitoring the accumulation of Carboxy-Hb after exposure to carbon monoxide, and could also be used for monitoring the re-conversion of Carboxy-Hb into Oxy-Hb, after optional treatment with oxygen. The two blood analyzes are total Hb, and Carboxy-Hb. As another example, the Carboxy-Hb can' be monitored frequently or continuously using the in vivo measurement and the absolute concentrations of Carboxy-Hb and Total-Hb can be measured in a blood sample (in vitro). Again, as in the case of Met-Hb, a correction factor can be used to correct the in vivo measurements of absolute concentrations of Carboxy-Hb and Total-Hb, if these are also measured. This 'invention is useful for monitoring patients (e.g., fire fighters) exposed to smoke, and subsequently treated with oxygen. Increased oxygen level in the gas the patients are allowed to breathe (or administered by a ventilator), would speed up the conversion of Carboxy-Hb to oxy-Hb. This process could be monitored frequently in vivo, and having to draw as little as one blood sample for in vitro measurement. An arterial blood sample is not required therefore a doctor is not required for drawing the sample. Either a venous or a capillary blood sample could be used, and the sample can be exposed to air without compromising the quality of the results. Preferably, the sample is a capillary blood sample, and can be obtained by any person, including the patient. To the best knowledge of the inventor, there is no prior art that describes a single apparatus that can perform both in vivo and in vitro measurement as described.
The in vivo calibration algorithm can be as simple as: %Carboxy-Hb is equal to the ratio of absorbances at two wavelengths multiplied by a constant; the algorithm can be developed empirically as done for Pulse-Oximetry, except the pulse can be ignored since the %Carboxy-Hb would be the same in both arterial and venous blood. In vivo calibration data has to be obtained from patients presenting to the emergency department for smoke inhalation and other forms of carbon monoxide poisoning, since it is not ethical to administer carbon monoxide to a person, as one would administer oxygen, for pulse-oximetry calibration algorithm development. The ratio of absorbance at wavelengths can be taken from Figures 1 & 2, for example, which should not be considered limiting in any way, 568nm and 805nm. At 568nm, Carboxy-Hb has the highest extinction coefficient (Figure 2), and at 805nm, Carboxy-Hb has the lowest extinction coefficient (Figure 1). At 805nm, both Oxy-Hb and Deoxy-Hb have the same extinction coefficient. It should be understood that these are just examples, which should not be considered limiting to the scope of the present invention in any way. Furthermore, the sum of more than one ratio terms can be used to develop more robust calibration algorithms.
Although the examples show so far, for use of the present invention seem to focus on Hb, it should be understood that any other analytes are considered to be within the scope of the present invention, as will be illustrated in the next example.
6. MONITORING THE RATIO OF BILIVERDIN TO BILIRUB1N.
Although not proven clinically, the ration of biliverdin to bilirubin could be used to monitor various conditions of the liver, including liver diseases and liver transplant. The two blood analytes are biliverdin and bilirubin. Samsoondar discloses in US Pat. No.

5,939,327, the contents of which are incorporated herein by reference, clinical relevance of the ratio of biliverdin to bilirubin in liver transplant patients compared to patients with liver cancer. The in vivo parameter could be the ratio of biliverdin to bilirubin, and the in vitro analytes would be biliverdin and bilirubin. Samsoondar et al discloses in US Pat. No.

6,268,910, the contents of which are incorporated herein by reference, methods of development of in vitro calibration algorithms for biiiverdin and bilirubin. Bilirubin and biliverdin are examples of analytes that are not restricted to the vascular compartment.

It should be understood that a ratio of two analytes could be optionally reported as a fraction, a proportion, or as a percent (%). Although the examples used for in vivo testing describe the ratio of two analytes, it should be understood that any in vivo testing is considered to be within the scope of the present invention, and the examples should not limit the scope of the present invention to in vivo measurement of the ratio of two analytes.
It should also be understood that any form of statistical analysis and data pre-processing is within the scope of the present invention. A primary calibration algorithm can also be obtained as follows: Absorbance spectra are obtained for several samples that cover a concentration range of a given analyte for which the primary calibration algorithm is being developed. It is preferred that the samples include all the absorbance variability expected in a sample, whereby the sample variability becomes 'built into the primary calibration algorithm. A multiple linear regression is then performed to develop a linear combination having the order derivative of absorbance at specific wavelengths as the independent variable, and the concentration of the analyte as the dependent variable. Other statistical methods, for example simple linear regression that uses only one wavelength, paitial least squares (PL,S), principal component analysis (PCA), neural network, and genetic algorithm may also be used. The equation thus obtained is a primary calibration algorithm.
By "Primary Calibration" it is meant a process used to develop a primary calibration algorithm for a first apparatus for an analyte or optionally for more that one first apparatus. The sample set used for calibration is relatively large, and the samples are natural or very close to natural samples. The primary calibration set should include all the variability expected in a sample, in order to develop robust calibration algorithm(s). Furthermore, one, or more than one sample of the primary calibration set could be measured on one or more than one first apparatus and combined, in order to develop a more robust calibration algorithms) that also includes inter-apparatus variability. Such a calibration algorithm would be developed using a combination of measurements obtained from one, or more than one, similar apparatus. Any form of statistical data analysis and optionally any form of data pre-processing, for example but not limited to, smoothing, calculation of first and higher order derivative of absorbance, photometric correction, data transformation, interpolation of absorbanee, or multiplicative scatter correction, may be used, depending on; the required accuracy of the analyte prediction. For example, by including data from more than one first apparatus, a lower level of precision and hence a lower Level of accuracy (poor precision translates into poor accuracy) may be obtained across many second apparatus. Such a type of primary calibration would be suitable if a simple yes/no answer to the presence of an analyte in a sample is all that is required, and is within the scope of this invention.
In another embodiment, a smaller set of samples like those of the primary calibration set, or a subset of the primary calibration set, or both; can be measured on a second apparatus, and the data combined with some or all of the original data from the primary calibration set, to develop one, or more than one, "upgraded primary calibration algorithm." Zero order derivative of absorbance (also referred to as raw absorbance) or any order derivative of absorbance may be used in the calibration process with second order derivative of absorbance being preferred, and first order derivative of absorbance being more preferred.
By "Data Pre-processing" it is meant any mathematical manipulation of spectroscopic data, which can be used to facilitate measurement of an analyte on an apparatus, including a first, second, or both, apparatus. Examples of data pre-processing, which should not be considered limiting in any way are: calculation of absorbance of EMR transmitted through or reflected from a sample; calculation of interpolated absorbances; smoothing of absorbances;
calculation of a first and higher order derivative of absorbance; multiplicative scatter correction; data transformation; photometric correction. It should be understood that any one or more forms of data pre-processing can be used prior to development of a calibration algorithm, and any one or more forms of data pre-processing can be used on the data from a second apparatus, prior to applying the calibration algorithm for calculating the concentration of an analyte. A non-limiting example of smoothing includes averaging of data:
By "smoothing" a curve, for example an absorbance spectrum, it is meant applying a mathematical function to the digital data to produce a "continuous spectrum"
and thereby reduce the "noise" in the spectrum. Various degrees of smoothing may be applied to a curve. The loss of analyte signal may be a price paid for smoothing.
For the convenience of transferring a calibration algorithm form a first apparatus to a second apparatus, it is preferred that a standard set of wavelengths are used.
Calibration algorithm transfer is covered in details by Samsoondar in US Pat. No. 6,65I,015 and related patents, the contents of which are incorporated herein by reference.
Software tools used for developing primary calibration algorithms comprises of the following:
MatlabTM used to create~rograms for smoothing absorbances and obtaining derivative of absorbances. MS Excel M may be used to develop macros for calculating derivative of absorbances; StatViewTM used to create algorithms by a process called "step-wise multiple linear regression." In the step-wise linear regression; the order derivative of absorbance measurements for all the wavelengths is presented to the StatViewTM program; only the wavelengths at which the order derivative of absorbance contribute to the calibration fit at a predetermined level of significance are selected for the algorithms. PirouetteTM may be used to create calibration algorithms by PLS or PCA, using the measurements for all the wavelengths; or selected sections of the absorbance spectra. Calibration algorithms may also include the techniques of neural network and genetic algorithms, although any statistical technique is considered to be within the scope of the present invention. It will be appreciated however that other software tools may also be used. Many examples of the primary calibration procedure, in respect of blood aiialytes, are shown in the references incorporated within this application. It will be appreciated that a primary calibration'algorithm may contain from a single wavelength term, in the simplest case, to multiple terms that use many wavelengths. The Primary Calibration Algorithms could be obtained by a process of simple linear regression, multiple linear regression and multivariate analysis. Some examples of multivariate analysis are PLS, PCA, Genetic Algorithm, and Neural Network. It should be understood that any order derivative of absorbance can be used, and it should also be understood, that the robustness of a primary calibration algorithm depends on the inclusion of interfering substances in the primary calibration sets, one expect to encounter in real samples. The chemometrics methods referred to should not be considered limiting in any way, and an.y form of chemometrics and data processing are within the scope of the present invention.
By "Data Transformation" it is meant any mathematical technique that can be applied to either the spectroscopic data or the analyte concentration data. Examples, which should not be considered limiting in any way, are Fourier Transformation of spectroscopic data, and calculation of the log or anti-log of an analyte concentration. It should be understood that smoothing can also be considered as data transformation, for example when the Savitzky-Golay method (Savitzky and Golay 1964, Anal Chern., 36:1627-1638) is used. By zero order derivative of absorbance it is meant the measured absorbance. The first order derivative of __ absorbance at a particular wavelength is the slope of the absorbance spectrum at that wavelength;
the second order derivative of absorbance at a particular wavelength is the slope of the first derivative absorbance spectrum at the wavelength. Higher order derivative (third; fourth etc.) of absorbance can similarly be obtained by taking the slope of the derivative absorbance spectrum of the order immediately below (second, third ete.) Methods of calculating a derivative of absorbance at a particular wavelength are well known by those skilled in the art. The calculation of the first derivative of absorbance at a particular wavelength may consist in taking the difference in absorbances at the two wavelengths that encompass the wavelength of interest.
Other methods of calculating derivative of absorbance may use the absorbances at several different wavelengths, where smoothing is an integral part of the derivative process. It should be understood that with a greater degree of smoothing, there is also a greater Loss of signal details in the absorbance spectrum or derivative of absorbance spectrum. The minimum number of wavelengths that may be used to calculate a derivative of absorbance is two wavelengths.
Smoothing, data transformation, and calculation of order derivatives of absorbances are non-limiting examples of data pre-processing. Other forms of data pre-processing may be performed either before or after calculation of an order derivative of absorbance, and include but are not limited to multiplicative scatter correction.
"Multiplicative Scatter Correction" (also known as multiplicative signal correction) is a mathematical technique that may be used to remove at least some of the light scattering effect in the spectroscopic data obtained from a sample set. The technique rotates each spectrum so that it fits as closely as possible to the mean spectrum. The technique is described in more details in:
Martens, H and Naes, T (Multivariate Calibration; 1993, Published by John Wiley & Sons); and Osborne, B.G., Fearn, T & Hindle, P.H. (Practical NIR Spectroscopy with Applications in Food and Beverage Analysis, 1993, Published by Longman Scientif c & Technical), both of which are incorporated herein by reference. It should be understood that the mean spectrum for a sample set can be obtained after combining one or mare sample measurements obtained from one or more than one apparatus.
By "Derivative of Absorbance" it is meant an order derivative of the absorbance spectrum. Zero order derivative of absorbance is the measured absorbance. The first order derivative of absorbance at a particular wavelength is the slope of the absorbance spectrum at that wavelength;
the second order derivative of absorbarice at a particular wavelength is the slope of the first derivative absorbarice spectrum at the wavelength. Higher order derivative (third, fourth etc.) of absorbance can similarly be obtained by taking the slope of the derivative absorbance spectrum of the order immediately below (second, third ete.) Methods of calculating a derivative of absorbance at a particular wavelength are well known by those skilled in the art. The calculation of the first derivative of absorbance at a particular wavelength may consist in taking the difference in absorbances at the two wavelengths that encompass the wavelength of interest.
Other methods of calculating derivative of absorbance may use the absorbances at several different wavelengths, where smoothing is an integral part of the derivative process. It should be understood that with a greater degree of smoothing, there is also a greater loss of signal details in the absorbance spectrum or derivative of absorbance spectrum. The minimum number of wavelengths that inay be used to calculate a derivative of absorbance is two wavelengths.
Smoothing, data transformation, and calculation of order derivatives of absorbances are non-limiting examples of data pre-processing. Other forms of data pre-processing may be performed either before or after calculation of an order derivative of absorbance, and include but are not limited to multiplicative scatter correction.

__ __ _. __ ~._ __ ~ _ ..

By "First Apparatus" it is meant an apparatus used to develop the at least one primary calibration algorithm.
By "Second Apparatus" it is meant an apparatus that is allowed to function like a first apparatus, whereby the second apparatus need not be calibrated in the same way in which the first apparatus was calibrated, i.e., by conducting a primary calibration. Samples similar to those of the primary calibration set, or a subset of the primary calibration set, may be measured on a second apparatus to develop an upgraded primary calibration algorithm, if desired.
The main aspect of the present invention is a single apparatus that provides a combination of in vivo testing and in vitro testing. In addition to using a patient's body part for measurement of EMR transmitted through or reflected from the body part (in vivo testing), the patient may be required to donate a biological sample. Examples of biological samples, which should not be considered limiting in any way are: whole blood, a pinprick capillary blood sample, serum, plasma, urine, cerebrospinal fluid, sputum, synovial fluid, lymphatic fluid, or feces. Non-limiting examples of body parts that may be used in this manner include, but are not limited to a finger, an ear lobe, a fold of skin, tlae web of the finger, skin, or mucous membrane. Hall et al in US 5,361,758, the contents of which are incorporated herein by reference, describes an apparatus used for in vivo testing; Cadell et al in US 5,429,128, the contents of which are incorporated herein by reference, describes a finger receptor that could be used with the apparatus described in US 5,361,758. It should be understood that these are just examples of hardware that can be incorporated in the present invention, for the in vivo testing; and should not be considered Limiting in any way: Figure 5 shows a finger 56 inserted into a receptor 54 for in vivo testing, and Figures 6a & 6b shows a slot 60 with a sample tab 62 for in vitro testing.
The following description of components of the apparatus is of preferred embodiments by way of example only and without limitation to the combination of features and parts necessary for carrying the invention into effect:
In a broad view, the apparatus for combining in vivo and in vitro testing comprises:
a. one or more sources of electromagnetic radiation (EMR);
b. one or more; photodetectors;
c. one or more slots in the host system of the apparatus for a sample vessel for in vitro testing of a biological sample taken from a patient;
d. one or more receptors for a body part of the patient for in vivo testing, wherein one or more receptor is located in the host system of the apparatus, or one or more remote receptors are connected to the host system of the apparatus, or a combination thereof;
e. electronics; and f. software.
In a preferred embodiment, the apparatus also contains a computer processor, but it should be understood that an apparatus without a computer processor, could be operated with a personal computer, and as such, is considered to be within the scope of the present invention.
In an embodiment with one or more receptors for a body part of the patient, one or more receptors axe located in the host system (referred to as the host receptor), and optionally one or more receptors axe remotely connected to the host system (referred to as the remote receptor). By "host system" of the apparatus it is meant the apparatus without the optionally one or more remote receptors, with the exception of the embodiment illustrated by Figure 4e, and without any communication with other instruments or equipment. It should be understood that the apparatus of the embodiment illustrated by Figure 4e, is one that has a remote receptor that is integrated with the host system, and may be regarded as a part of the host system.
It should be obvious from Figure 4a that the host system 434a is the entire apparatus, since there is no remote receptor, and there is no communication with other instruments.
Communication with other instruments, for example, which should not be considered limiting in any way, a monitor 436d and a relay system 438d are shown in the embodiment illustrated by Figure 4d. Remote receptors 424b, 424c and 424d are shown in Figure 4b, Figure 4c and Figure 4d respectivelf. In the embodiment illustrated by Figure 4e, the interface between the body part 40'7e and the bi-directional bundle of optical fibers 405e is considered to be the receptor, and this receptor is also considered to be a remote receptor.
HOST RECEPTOR
The host receptor is adapted to allow EMR to enter the body part at a first surface of the body part, and the receptor is also adapted to allow passage of at least some of the EMR, wherein the emerging EMR emerges from a second surface of the body part, and wherein the second surface is the wine as or different from the frst surface. Alternatively, the host receptor that is shaped to accept the body part, is adapted to allow EMR to enter the body part at the front surface of the body part, and the receptor is also adapted to allow passage of at least some of the EMR, wherein the emerging EMR emerges from the back side of the body part, to be reflected off a reflective surface in the receptor adjacent to the back side of the body part, and the reflected EIVIR is collected either at the front surface or a different surface of the body part. The body part may be, for example, which should not be considered limiting in any way, a finger, a toe, an ear lobe, a forearm, a web between two fingers, a fold of skin, or the surface of any body part. It should be understood that the one or more host receptors could be designed to accommodate, for example, which should not be considered limiting in any way, different body parts, or different sizes of body parts.
REMOTE RECEPTOR
The remote receptor is adapted to allow EMR to enter the body part at a first surface of the body part, and the receptor is also adapted to allow passage of at least some of the EMR, wherein the emerging EMR emerges from a second surface of the body part, and wherein the second surface is the same as or different from the first urface.
Alternatively, the remote receptor that is shaped to accept the body part, is adapted to allow EMR to enter the body part at the front surface of the body part, and the receptor is also adapted to allow passage of at least some of the EMR, wherein the emerging EMR emerges from the back side of the body part, to be reflected off a reflective surface in the receptor adjacent to the back side of the body part, and the reflected EMR is collected either at the front surface or a different surface of the body part. The body part may be, for example, which should not be considered limiting in any way, a finger; an ear lobe, a forearm, a web between two fingers, a fold of skin, or the surface of any body part: In another embodiment of the present invention, more than one remote receptor could be linked to the host system, allowing in vivo measurement on more than one patient to be conducted. The more than one remote receptors could also be attached to the same patient, if the receptors monitor different information, for example, which should not be considered limiting in any way, one receptor may monitor blood oxygen saturation, and another could monitor %Met-Hb. It should also be understood that the one or more remote receptors could be designed to accommodate, for example, which should not be considered limiting in any way, different body parts, or different sizes of body parts.
From the description of the host receptors and the remote receptors, it should be obvious that the EMR that is measured by the detector is one of transmitted EMR, reflected EMR, or a combination (sometimes referred to as transflectance), is within the scope of the invention. The EMR is then converted to a value called absorbance. By "absorbance" it is meant a measurement of the reduction of EMR intensity caused by a sample.
According to Beer's law, Absorbance = Log(1/Transmitted light), which applies to non-light-scattering samples. The measured parameter is the amount of EMR
transmitted through a sample; and the transmitted EMR (or transmittance) is then converted to absorbance units. When a sample is light-scattering and Beer's law is applied, an apparatus cannot distinguish "true absorbance" from loss of EMR due to scattering, hence the term "apparent absorbance" should be used. It should be understood that when the term "absorbance" is used, it could mean either "true absorbance" or "apparent absorbance," or both, since it is not always obvious whether the sample is EMR
-scattering or non- EMR -scattering. It should be understood that absorbance can be , replaced with Log(1/Reflectance), when reflectance is measured instead of transmittance, and reflectance (as well as transflectance) measurement is within the scope of the present invention. It should also be understood that the EMR reflected from a sample could first undergo any level of penetration of the sample, before the emerging EMR
emerges from the incident surface of the sample. Furthermore, the EMR can penetrate the sample once, and then a second time after the penetrated or transmitted EMR is reflected off a reflective surface situated on the opposite side of the sample; the reflective surface can be located on the sample vessel, slot, or receptor for a body part. It should be understood that the terms transmission and transmittance are used interchangeably to convey the same meaning. It should also be understood that the terms reflection and reflectance are used interchangeably to convey the same meaning The source of EMR for the remote receptor could be the same source of EMR in the host system, where some of the EMR is channeled to the remote receptor by one or more fiber optic cables. Preferably, the remote receptor is equipped with its own source of EMR, and the preferred source of EMR, which should not be considered limiting in any way, is one or more light emitting diodes (LED's). The EMR emerging from the body part could be channeled to one or more detectors located in the host system. Preferably, the remote receptor is equipped with its own one or more detectors, and one or more wires could transmit the electrical signal to the host system. In another embodiment of the present invention, one or more than one remote receptors could be linked to the host system, allowing in vivo measurement on more than one patient to be conducted, or the more than one remote receptors could also be attached to the same patient; if the receptors monitor different information, for example, which should not be considered limiting in any way, one receptor may monitor oxygen saturation, and another could monitor %Met-Hb.
In a preferred embodiment where more than one remote receptors are used, the receptors are wireless receptors, but wired multiple remote receptors should be considered to be within the scope of the present invention.
In a preferred embodiment, the wireless remote receptor connected to the host system of the apparatus comprises, one or more light emitting diodes (LED's), one or more photodetectors, electronics, a transmitter, and the host system further comprises a receiver ~- ~-__._._._ that is compatible with the transmitter. It should be understood that the use of a relay is considered to bean aspect of another embodiment of the present invention, when the transmitter is not powerful enough to reach the host system. In another embodiment of the present invention, one of the one or more wireless remote receptors is equipped with one or more LED's, one or more LED drivers, one or more photodetectors, one or more amplifiers, one or more filtering mechanisms, one or more analog-to-digital converter, and one or more transmitters for transmitting the signal to the host system;
the host system is then required to carry a receiver for receiving the digital signals from the remote receptor. It should be understood that any transmitting and receiving frequency is considered to be within the scope of the present invention, but in the preferred embodiment, the transmitting frequency used should one that does not interfere with other electronic equipment, and should be one that expose the receivers to emission by other electronic equipment. It should also be understood that any level of power is considered to be within the scope of the present invention; low power could be sufficient since the transmitting range is expected to be small, for example, within a room, or from emergency equipment (e.g., a fire engine) to an adjacent site.
Remote receptors are shown in the embodiments illustrated by Figure 4b, Figure 4c and Figure 4d, as 424b, 424c and 424d respectively. The components in each embodiment are the sources of EMR 426b:, 426c and 426d; the detectors are 428b, 428c and 428d; the analog-to-digital converters are 430b, 430c and 430d; the transmitters are 432b, 432c and 432d, in the embodiments illustrated by Figure 4b, Figure 4c and Figure 4d respectively.
In another embodiment, illustrated by Figure 4e, a bi-directional bundle of optical fibers 405e is used such that some of the fibers transmit EMR (shown as 401e) to a body part 407e, and some'of the fibers within the same bundle receive some of the EMR
returning from the body part (shown as 403e). Since the cross sectional surface of the fiber bundle is in contact with the body part, and as such "accepts" the body part, the tip of the optical fiber bundle, which is like a cross section of the fiber optic bundle, is considered to be a receptor for a body part. In this embodiment, the interface between the body part 407e and the bi-directional bundle of optical fibers 405e is considered to be the receptor, and this receptor is also considered to be a remote receptor. Therefore, it should be understood that a receptor does not have to be a part that accepts a body part as depicted in Figure 5. In another aspect of this embodiment, there is also a host receptor like 408a in the embodiment illustrated by Figure 4a. In this aspect of the embodiment, the shutter 404a, will direct EMR either through arm 401e of the bi-directional bundle of optical fibers 504e, or to the host receptor like 408a in the embodiment illustrated by Figure 4a.
In the embodiment illustrated by Figure 4e, reflectance is preferred to transmittance of EMR.
The apparatus comprises one or more sources of EMR, for example, which should not be considered limiting in any way, a tungsten lamp, one or more pulsed or continuous LED's, one or more pulsed or continuous lasers, or any combination thereof. As an example, a blue diode, or an ultraviolet diode or both, could be combined with a tungsten lamp, where the diodes could enrich the EMR from the tungsten lamp, in the blue and/or ultraviolet wavelengths. As another example, a tungsten lamp could be the source of EMR in the host system, and the source of EMR in the optional remote receptor.
The wavelengths of the EMR could be one or more wavelengths selected from the range of about 300nm to about 2500 nm.
The apparatus comprises one or more photodetectors, for example, which should not be considered limiting in any way, a single photo diode, an array of photo diodes, one or more chaxged coupled detectors (CCD), or any combination thereof.
The apparatus comprises one or more power supplies. Preferably, the host system is powered by alternating current, and the optional remote detector is battery powered.
More preferably; both the host system and the optional remote receptor are battery powered. It should be understood that the distance of signal transmission could determine the size of the battery, and any size or type of battery is considered to be within the scope of the present invention.
SLOT
The host system comprises a slot for a sample vessel, fox in vitro testing of a biological sample taken from the patient who donated the biological sample. By "slot" it is meant an opening through which the sample vessel is to be put, or a groove or channel ox slit into which the sample vessel fits. It should be understood that the slot could be oriented in any direction, but in the preferred embodiment, it is a horizontal slot, such that the EMR
travels in the vertical direction. The slot is adapted to allow EMR to enter a front side of the slot housing the sample vessel, and the transmitted EMR is collected at the back side of the slot. The slot may also be adapted to allow EMR to enter a .front side of the slot housing the sample vessel, where the transmitted EMR is reflected off a reflective surface located at either the back side of the slot, or the side of the sample vessel adjacent to the back side of the slot, and the reflected EMR is collected at the front side of the slot. The sample vessel may optionally contain one or more reagents; and the sample vessel may also be either a cuvette or a sample tab, and the cuvette or the sample tab may optionally contain one or more reagents. In the preferred embodiment; the sample vessel is a sample tab, and the slot is designed to accept the sample tab in a horizontal direction.
This aspect of the invention is particularly important when the sample is whole blood.
When whole blood is allowed to settle, the RBC's tend to precipitate.
Therefore, in order for the RBC's to remain in the path of the EMR, the EMR should travel in the vertical direction. However, any configuration of the sample slot is considered to be within the scope of the present invention. The sample vessel may also be a cuvette designed to draw in a sample by capillary action, and may optionally contain one or more reagents. In the preferred embodiment, the sample tab comprises a base plate with a sample well and a cover, wherein at least a portion of the base plate and at least a portion of the cover, is adapted to permit transmission of EMR therethrough. Alternatively, the sample tab comprises a base plate with a sample well and a cover, wherein at least a poxtion of the base plate is adapted to permit transmission of EMR through the sample, and at least a portion of the cover is adapted to reflect EMR emerging from the sample, and wherein the reflected EMR is allowed to traverse the sample before leaving the sample tab at the base plate, or wherein at least a portion of the cover is adapted to permit transmission o.f EMR
through the sample, and at least a portion of the base plate is adapted to reflect EMR
emerging from he sample, and wherein the reflected EMR is allowed to traverse the sample before leaving the sample tab at the cover. The biological sample may be, for example, which should not be considered limiting in any way; whole blood, a pinprick capillary blood sample, serum, plasma, urine, cerebrospinal fluid, sputum, synovial fluid, lymphatic fluid, or feces.
By "sample vessel" it is meant any transparent or translucent container capable of holding a sample, preferably fluid sample, to enable measurement of absorbance, reflectance, or both absorbance and reflectance of EMR from the sample. Examples of vessels include, but are not limited to, sample tab, pipette tips, tubing, cuvettes, labeled test tubes, unlabeled test tubes, blood bag tubing, any transparent sample container, and any translucent sample container. In the case of a cuvette, it should be understood that the cuvette could be designed as a flow-through cuvette, which requires that the sample be injected into the reuseable cuvette. However, a flow-through cuvette is not preferred due~to the requirement of a wash system, but a flow-through cuvettte is still considered to be within the scope of the present invention. The sample vessel may optionally contain one or more reagents. In the case of a body part, a receptor is required instead of a sample vessel. In vivo testing is usually reagentless, and since spectroscopic apparatus for in vitro testing can also be reagentless, spectroscopic methods were chosen for the preferred embodiment. However, because the apparatus uses spectroscopy, the present invention should not be limited to a reagentless system, and the use of one or more reagents in the in vitro sample vessel is regarded as an enhancement of a reagentless in vitro system. Lilja et al in US Pat. No.
4,088,448 describe a cuvette for sampling, with a cavity that is defined by two planar surfaces, which are placed at a predetermined distance from one another; wherein the cavity contains a reagent, and the sample is optionally drawn into the cavity by capillary force. It should be understood that the use of such cuvette or any similar cuvette is considered to be within the scope of the present invention.
By "blood bag tubing" it is meant the tubing connecting a first plastic bag that contains whole blood and a second plastic bag that may contain plasma obtained from the first bag. The tubing and bags'may be made from transparent or translucent flexible plastic.
By "sample tab" (Figure 7a, & Figure 7b) it is meant a sayple vessel comprising, a base plate having atop surface and a bottom surface, at least a portion of the base plate adapted to permit transmission of EMR therethrough, a well disposed on the top surface of the base plate for retaining a sample, the well defined by a closed wall extending above the top surface of the base plate, and a cover plate, preferably attached to the base plate, hingedly, and moveable between an open and a closed position, wherein at least a portion of the cover plate permits transmission of EMR therethrough, so that when the cover plate is in the closed position an optical path is formed through the portion of the base plate that permits transmission of EMR, the well, and the portion of the cover plate that permits transmission of EMR. Another embodiment of the sample tab permits the EMR to be reflected off the opposite side of the sample tab, thereby doubling the direct pathlength. The sample tab as described above wherein the closed wall comprises one or more overflow openings, the closed wall surrounded by a containment wall defining an overflow ring therebetween. The cover plate may be attached to the base plate or may be separate. Further, the sample tab may comprise a locking member that associates with to a corresponding mating member, thereby permitting the cover plate to be attached to the base plate. The locking member may comprise, but is not limited to, a circular ring capable of frictionally engaging an outer portion of a containment wall or one or more clips capable of frictionally engaging and attaching the cover plate to the base plate. The locking members may be located on the base plate, cover plate or both the base plate, and the cover plate. Similarly, the associated mating member that receives the locking member may be located on the base plate, cover plate or both the base plate, and the cover plate. Also provided by the sample tab as defined above is a wall surrounded by a containment wall defining an overflow ring therebetween. The containment wall may comprise a sealing member on its upper surface. The sealing member may be an O-ring, or a pliable material integral with the containment wall. In a preferred embodiment of the present invention, the sample well contains one or more openings or grooves and an overflow ring for collecting excess sample., as closing the cover plate squeezes out excess sample. Preferably, the cover plate is attached to the tab so that the sample proximate the cover plate hinge makes contact with the cover plate first, and as the cover plate closes, excess sample is squeezed out through the two grooves and into the overflow ring. Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the sample tab are given by way of illustration only. Various designs of sample tabs are described by Samsoondar in US patent application 10/042,258; (Publication Number 2002-0110496 Al), the contents of which are incorporated herein by reference.
The host system of the apparatus comprises: electronics, which could include one or more amplification systems, one or more filtering systems, one or more analog to digital converters, one or more interface between the one or more detectors; a microprocessor or computer processor; and software. The optional wireless remote receptor comprises:
electronics, which could include one or more amplification systems, one or more filtering systems; and one or more analog-to-digital converters.
The software of the apparatus optionally comprises: features for utilizing the calibration algorithm(s); features for calibrating the apparatus for respective analytes or parameters;
features for interpolating absorbances; features for mapping absorbances to a standard set of wavelengths; features for smoothing; features for creating derivatives of absorbances;
features for calculating one or more parameters; and features for calculating analyte concentrations to produce one or more predicted values.
By "predicted value," it is meant a value of an analyte obtained when the primary calibration algorithm for the analyte is applied to spectrophotometric data, with optional pre-processing, of a sample. A primary calibration algorithm is an equation comprising a predicted value of the analyte as the dependant variable and a constant, and one or more terms, preferably a linear summation of the constant and the terms. Preferably each term is the product of a constant and an independent variable as shown in the examples shown by Samsoondar in US Pat. No. 6,651,015. It should be understood that the use of non-linear primary calibration algorithms is within the scope of this invention.
The independent variable is the optionally pre-processed absorbance of the sample at a standard wavelength.
In another embodiment of the present invention, the host system is equipped with a monitor or screen, and contains a communication link with a, relay system, which enables the monitor of the host system to display other patient data, for example, which should not be considered limiting in any way, temperature, heart rate, blood pressure, and electrocardiograms. In yet another embodiment of the present invention, the host system is not equipped with a monitor or screen, and contains a communication link with another display system for displaying results from the present invention, and the display system is optionally used to display other patient data, for example, which should not be considered limiting in any way, temperature, heart rate, blood pressure, and electrocardiograms. As another aspect of the present invention, the communication devices shown in Figure 4d between the computer processor 415d and a remote monitor 436d, or between the computer processor 415d and a relay system 438d, or both, are replaced with one or more wireless communication devices.
The apparatus should contain one or more receivers, or one or more receivers with one or more channels, for receiving information transmitted from the optionally one or more remote receptors.
The apparatus should optionally contain means for synchronizing body part measurement with the patient's pulse. Examples of means for synchronizing body part measurement with the patient'.s pulse when synchronization with the pulse is necessary, which should not be considered limiting in any way, are the use of electrocardiogram or electrical output of the heart, and the measurement of Hb absorbance at one or more wavelengths.
It should be understood that the preferred embodiment of the apparatus does not necessarily contain all the features included in the above. A biological sample from the patient is required for the in vitro testing; and a body part is required for the in vivo testing is performed as frequently as necessary for monitoring the patient, which depends on the clinical usefulness of such measurements.
Referring now to Figure 4a, Figure 4b, Figure 4c, Figure 4d and Figure 4e,which are schematic views of the apparatus that combines both in vivo and in vitro testing. In order to compare the components in the different embodiments of the present invention, the representation of each component in different embodiments is labeled with the same number, with the appropriate letters a, b, c, d and a added to correspond with Figure 4a, Figure 4b, Figure 4c, Figure 4d, and Figure 4e respectively. Each of the four examples of preferred embodiments shows a host system 434a, 434b, 434c and 434d and all but one (Figure 4a) show a remote receptor. Figure 4a is a schematic view of the invention with no remote receptor; and Figure 4b, Figure 4c and Figure 4d are schematic views of the invention with remote receptors 424b, 424c and 424d. Figure 4e is a schematic view of the invention with a remote receptor, wherein the interface between the body part 407e and the bi-directional bundle of optical fibers 405e is considered to be the remote receptor.
Referring now to Figure 4a, there is shown a source of EMR 400a, which is preferably a tungsten lamp, is split into two paths by a bifurcated optical fiber, so that EMR can be supplied to slot 406a for in vitro testing, and a receptor 408a for in vivo testing. In a preferred embodiment, the amount of EMR directed to the slot and receptor is about the same, but it should be understood that the ratio EMR to slot and receptor can vary depending on the EMR attenuation caused by the in vitro sample and the EMR attenuation caused by the body part. Before reaching the slot and receptor, the EMR travels through two shutters, shown as the slot shutter 402a and the receptor shutter 404a. Either shutter or both shutters can be closed at any time, cutting off the source of EMR to the slot, the receptor, or both.
Although the embodiment illustrated in Figure 4a appears to be a dual beam system., it is not because both beams are for samples; an in vitro sample and an in vivo sample.
In a dual beam system, for example as illustrated in Figure 4c, one of the two beams is used as a reference beam. In the embodiment described by Fibure 4a, the EMR traveling through the slot, with the slot shutter open and the receptor shutter closed, can be attenuated by a member that fits in the slot like a sample tab, and the attenuated EMR is used as the reference beam.
Therefore, the embodiment illustrated in Figure 4a, contains a single beam spectrometer. It should be understood by those skilled in the art that attenuation is required to prevent saturation of the detector. Also, it should be understood that reference measurement can be taken before or after a sample measurement, for both the in vitro measurement and the in vivo measurement, or the reference measurement can be stored and reused any number of times. By closing both shutters, a dark current measurement can be made. By "dark current"
it is meant the detector response when the detector is not exposed to EMR. It should be understood that subtraction of dark current measurement is optional. Due to the location of the shutters 402a and 404a, any room light entering the slot or receptor would be included in the dark current measurement, and could be subtracted out from both the sample measurement and the reference measurement: During the in vitro measurement, the slot shutter must be open and the receptor shutter must be closed. During the in vivo measurement, the receptor shutter must be open and the slot shutter must be closed. The EMR emerging from either the body part in the receptor or the biological sample in the slot can enter the spectrometer, which comprises the diffraction grating 410a and the detector 412a. It should be understood that either a transmission or reflection grating is within the scope of the present invention. A grating is a dispersing element, which separates out the EMR component by wavelengths. It should be understood that the use of LED's is considered to be within the scope of the present invention, and. with the use of LED's, a grating may not be required. In the preferred embodiment, the detector 412a is an array of photodectors, but the use of a single detector instead of an array of detectors is considered to be within the scope of the present invention. As an example, which should not be considered limiting in anyway, a single detector could be used when the source of EMR
400a is one or more LED's. The electronic signal is proportional to the time that the detector integrates the optical signal. The electronic signal is amplified by analog electronic amplifiers (not shown) and converted to a digital signal by an analog-to-digital converter or ADC
414a. Absorbance is calculated in the computer processor 415a as:
Absorbance; = log{(Reference Light; - Reference Dark; ) / (Sample Light;-Sample Dark;)~+ log (ITS / ITR) where:
Absorbance; = Absorbance at pixel i;

Reference Light;= Reference pixel i readings;

Reference Dark;= Reference pixel i readings;

Sample Light; = Sample pixel i readings;

Sample Dark; = Sample pixel i readings;

ITS = Integration time for sample measurement;

ITR = Integration time for reference measurement;
and i = the particular pixel (wavelength) in the array of detectors The sample can be either an in vitro sample (a biological sample) or an in vivo sample (a body part).

Still referring to Figure 4a, commands can be executed from a keyboard or keypad 418a, and data, for example results, which should not be considered limiting in any way, are displayed on a monitor or screen 420a. A printer 416a is shown for producing reports, but it should be understood that a printer is not essential for the present invention.
Communication ports, which are not shown, are optional. It should be understood that appropriate shielding of the slot and receptors from room light is within the scope of the present invention, but the extent of shielding depends on the analyte or parameter measured, and the use of dark current measurement.
Figure 4b, Figure 4e and Figure 4d are schematic views of the invention with remote receptors 424b, 424e and 424d, and receivers 422b, 422c and 4224 in the host systems 434b; 434c and 434d respectively.
Referring now to Figure 4b, the embodiment described operates in a single beam mode (i.e., the spectrometer is a single beam spectrometer); similar to the embodiment described by Figure 4a.
No receptor and no shutters are shown in the host system 434b. In another embodiment, a shutter like 402a as' illustrated in Figure 4a is installed. Dark current measurement is optional with or without a shutter. By inserting an opaque member into the slot 406b of the embodiment illustrated by Figure 4b, a dark current measurement could be made.
Referring now to Figure 4c, there is shown a system that is similar to the embodiment illustrated by Figure 4b, except a bifurcated optical fiber is used to split the EMR
source 404c, and a slot shutter 402c and a reference beam shutter 404c are added: The reference beam shutter 404c is like the receptor shutter 404a in the embodiment illustrated by Figure 4a. The embodiment described by Figure 4c operates in a dual beam mode (i.e., the spectrometer is a dual beam spectrometer), unlike the embodiment described by Figure 4a. Still referring to Figure 4c, the EMR source 400c is split so that about 99% of the EMR is directed to the slot shutter 402c, and about 1% of the EMR is directed to shutter 402c, and used as the reference beam. It should be understood that the ratio EMR directed to slot and EMR directed to shutter 402c, can vary depending on the attenuation of the EMR in the two optical paths, and the sensitivity of the detector.
Referring now to Figure 4d, there is shown a system similar to the embodiment described by Figure 4a, except for the inclusion of a remote receptor 424d, a receiver 422d and a remote monitor 436d for displaying results of the present invention, along with other results, for example, which should not be considered limiting in any way, temperature, heart rate, blood pressure, and electrocardiogram. Also shown in Figure 4d, is a relay system 438d connected to the host system 434d, wherein other results, for example, which should not be considered limiting in any way; temperature, heart rate, blood pressure, and electrocardiogram, could be displayed on the monitor 420d of the host system 434d.
Referring now to Figure 4e, there is shown a system similar to the embodiment described by Figure 4a, except for bi-directional bundle of optical fibers 405e, which interfaces with a body part 407e, and the interface is considered to be a remote receptor. In another embodiment, illustrated by Figure 4e, reflectance is preferred over transmittance.
Turning now to the remote receptor, as may be seen in Figure 5. Although Figure 5 depicts a finger receptor, it should be understood that the body part does not necessarily have to be a finger, and that any other body part is considered to be within the scope of the present invention.
It should also be understood that the receptor could be the end of a bi-directional optical fiber bundle that makes contact with any body part.

Still referring to Figure 5, there is shown the finger 56 inserted into the receptor 54. Also shown is a wire 52, which is optionally the wire that connects the receptor 54 to the host systems 434b 434c and 4344. It should be understood that the remote receptor could be wired but a wireless system is preferred. The wire 54 could also be a connection to a power supply (not shown) that is strapped to the wrist. Also not shown is the one or more LED's, one or more photoreceptor, an analog-to-digital converter, and a transmitter. As an example of a wireless remote receptor, which should not be considered limiting in any way, is the 4100 Digital Pulse Oximeter from Nonin Medical, Inc. The receptor 54 could also be a finger receptor in the host system, illustrated by Figure 4a and Figure 4d as 408a and 408d respectively.
Turning now to Figure 6a & Figure 6b; there is shown the slot and sample tab, with EMR
delivered to the sample in the sample tab 62 through a source or incident optical fiber 68 while the sample in sample tab rests in slot 60 within a slot housing 64. By "slot housing" it is meant the section of the apparatus showing the location of the slot relative to the optical fibers. The electromagnetic radiation passing through the sample tab and specimen is received by a receiving optical fiber 66, and processed further to determine, the concentrations of one or more analytes, or one or more parameters or both. It should be understood that the incident fiber could be 66 and the receiving fiber could be 68; and is within the scope of the present invention.
SAMPLE TAB
According to an aspect of the present invention; there is provided a sample tab for retaining a sample for in vitro analysis. It should be understood that the sample tab is used as an example of a sample vessel, and should not be considered limiting in any way.
The sample tab 62 in Figure 6a & Figure 6b comprises, a) a base plate with a top surface and bottom surface, the base plate characterized as having at least a portion that permits transmission or reflectance of electromagnetic radiation;
b) a well or sample cavity disposed on the top surface of the base plate for retaining the sample, the well is defined by a closed wall extending above the top surface of the base plate. The well maybe of any desired volume and may be of any shape;
c) at least one overflow groove or opening in the wall of the well permitting drainage of excess sample from within the well;
d) a cover plate having at least a portion that permits transmission or reflection of EMR.
In use, a sample is retained in the well between the base plate and the cover plate so that electromagnetic radiation may pass through the base plate, through a sample in the well, and the cover plate. However, it is within the scope of the present invention that the radiation beam may travel though the sample, and be reflected off either the base plate or cover plate thereby doubling the path length of the radiation beam. By doubling the path length, a reduced volume of sample may be used during analysis. Either the base plate or the cover plate may have a reflective surface, or may be made of, reflective material.

The sample well def ned by a closed wall contains one or more openings or grooves and an overflow ring for collecting excess sample as it is squeezed out by the closing cover plate.
Preferably, the cover plate is attached to the tab so that the sample proximate the cover plate hinge makes contact with the cover plate first, and as the cover plate closes, excess sample is squeezed out through the grooves, which are preferably situated at the side where the cover plate makes final contact with the rest of the tab, and into the overflow ring. The hinged design helps the sample tab slide into the receptor of an instrument, such as a spectrophotometer.
Referring now to Figure 7a & Figure 7b, there is shown an aspect of an embodiment of the sample tab, which should not be considered limiting in any way. Shown in Figure 7a & Figure 7b, is sample tab 720 comprising base plate 718, cover plate 702 and sample well 714 defined by closed wall 706. Sample well 714 may be of any volume required, for example, but not limited to, a size sufficient to allow a drop of blood to fill the well, preferably with some excess. In an embodiment, which is not meant to be considered limiting in any manner, the well is circular and comprises dimensions of about 4 mm in diameter and about 2 znm in depth.
Overflow openings or grooves 716 in closed wall 706 allow excess sample to flow out of sample well 714 when cover plate 702 is closed over sample well 714 and base plate 718. A second wall, such as, but not limited to, a containment wall 712 may be employed to retain the sample that overflows sample well 714, into an overflow ring (circular groove between wall 706 and wall 712) to prevent leakage of fluid from the sample tab, while permitting a sample of sufficient volume to f 11 the well. In this regard, the vertical height of containment wall 712 is less than or equal to the height of closed wall 706 defining sample well 714. More preferably it is equal to the height of closed wall 706 defining sample well 7I4. Cover plate 702 is preferably attached to base plate 718 by hinge 710 or other suitable attachment means known in the art. However, a non-hinged cover plate may also be used, where the cover plate may be snapped on to the base plate.
The sample tab may be manufactured from any suitable material known in the art, for example, but not limited to, a transparent, translucent material, such as glass, plastic or a combination thereof, or a reflective material. If the base plate and cover plate are transparent or translucent, then it is preferred that the base plate, and cover plate comprise a transparent or translucent plastic, such as but not limited to polypropylene, polycarbonate, polyethylene, or polystyrene, however, a glass plate may also be used. If either of the base plate or cover plate is reflective, then a reflective material, for example but not limited to a ceramic coating, barium sulfate, Spectralon TM, SpectraflectTM, or DuraflectTM may be used for one of the base or cover plates.
Optionally, the sample tab of the pxesent invention may comprise a locking member to lock cover plate 702 to the base plate 718. The locking member may comprise a portion of the cover plate, base plate or both. Further, the locking member may reversibly or irreversibly lock the cover to the base plate. Any locking member known in the art may be employed with the sample tab of the present invention, for example, but not limited to those as shown by $amsoondar in US
Patent Application No.10/042,258 (Publication Number 2002-0110496 A1), the contents of which are incorporated by reference. The use of a containment wall ensures that the sample is retained within the sample tab and reduces contamination between samples:
Furthermore, by locking the cover prate of the sample tab in a closed position, the sample tab may be readily disposed of after use without sample leakage, or the sample tab may be used in a vertical position, for example within a cuvette holder adapted for use within spectrophotometers.

Also shown is a locking member 704 which permits cover plate 702 to be fastened to base plate 718. The locking member 704 comprises a circular ring, capable of frictionally engaging containment wall 712, thereby reversibly attaching cover plate 702 to base plate 718, preventing the escape of a sample from the sample tab.
When the cover plate is closed over the well, and attached to the base plate, it is preferred that the top surface 708 of the containment wall 712 seals against the lower surface of the cover slip.
However, the locking member may also be used to help seal the sample within the sample tab should any leakage occur past the containment wall.
According to another aspect of the sample tab, the absorbance can be calculated from reflectance instead of transmittance. In the case of reflectance, either the base plate or the cover plate may have a reflective surface or may be made of reflective material. Such a reflective surface or material could include any suitable reflective coatinfor example, but not limited to, a ceramic coating, barium sulfate, Spectralon TM, Speetrafleet , or DuraflectTM.
The method of combining in vivo testing and in vitro testing using an embodiment of the apparatus of the present invention as described, comprises any combination of the following:
1. A value of one or more analytes in a biological sample obtained from a patient, is obtained by applying one or more calibration algorithms to the order derivative of absorbance obtained from a biological sample obtained from the patient, in a sample vessel, at one or more wavelengths of a standard set of wavelengths.
2. One or more parameters is obtained from one or more sets of order derivative of absorbances obtained by applying one or more calibration algorithms to the one or more sets of order derivative of absorbance obtained from the body part of the same patient, wherein the one or more sets of order derivative of absorbances are obtained at one or more wavelengths of a standard set of wavelengths.
By "Sets of Order Derivative of Absorbances" it is meant absorbances obtained sequentially with optional pre-processing (e.g., calculating the first derivative of the absorbance)A set of absorbance can be the absorbance at one or more wavelengths obtained at the peak and through of a pulse. A set of absorbance can also be the absorbance at one or more wavelengths obtained with and without manipulation of the body part, for example, with and without squeezing blood out of the body part exposed to the EMR. It should be understood that these are only examples of "sets" of absorbance measurement, and should not be considered limiting in any way with respect to the scope of the present invention. The one or more wavelengths are selected from the wavelength range of 300nm to 2500 nm, and the order derivative of absorbance is optionally selected from the group consisting of, zero order, first order, second order, and third order.
3. The one or more parameters (in vivo testing) are optionally the same as or different from the one or more analytes(in vitro testing), and the one or more parameters are optionally used adjunctively with the one or more analytes. The one or more parameters may be _ _ .._.._.

calculated from the values obtained from the one or more analytes obtained from the biological sample.
4. Part of the in vivo testing is performed by applying a calibration algorithm to the absorbance for the body part at two or more wavelengths; wherein the calibration algorithm is a linear equaticin containing a constant plus one or more terms, wherein each of the one or more terms is an independent variable multiplied by a constant, and wherein each of the independent variable is the ratio: of absorbanees at two different wavelengths.
5. Part of the in vitro testing is performed by applying a calibration algorithm to an order derivative of the absorbance at one or more wavelengths of the biological sample in the sample vessel, and wherein part of the in vivo testing is performed by applying a calibration algorithm to an order derivative of the absorbance for the body part, at one or more wavelengths.
6. Some examples, which should not be considered limiting in any way, for using the in vitro analyte values and using the calculated parameters from the in vitro analytes are:
confirming the results of the in vivo testing, assessing the integrity of the results of the in vivo testing; correcting the results of the in vivo testing, and any combination thereof.

7. The in vitro,analyte could be different from the analyte or parameter measured in vivo, and also, the analyte measurement in vitro does not necessarily have to be the one that is used to calculate the in vivo parameter that is monitored.
It should also be understood that the apparatus of the present invention is not considered to be restricted to any particular use, and hence could also be used for either in vitro testing alone or in vivo testing alone.
All citations are herein incorporated by reference.
While the invention has been particularly shown and described with reference to certain embodiments, it will be understood by those skilled in the art that var ions other changes in form and detail may be made without departing from the spirit and scope of the invention.

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Claims

I claim:

1. An apparatus for combining in vivo testing and in vitro testing, comprising:
a. one or more sources of electromagnetic radiation (EMR);
f. one or more photodetectors;
g. one or more slots in the host system of said apparatus for a sample vessel for in vitro testing of a biological sample taken from a patient;
h. one or more receptors for a body part of said patient for in vivo testing, wherein one or more receptor is located in the host system of said apparatus, or one or more remote receptors are connected to the host system of said apparatus, or a combination thereof;
and i. electronics.

2. The apparatus according to claim l, wherein said apparatus further comprises a computer processor and software.

3. The apparatus according to claim l, wherein the one or more remote receptors are connected to said host system by a method selected from the group consisting of, a wireless method, one or more electrical wires, one or more fiber optic cables, and any combination thereof.

4. The apparatus according to claim 3, wherein one of said one or more remote receptors comprises, one or more light emitting diodes (LED's), one or more photodetectors, electronics, a transmitter, and said host system further comprises a receiver that is compatible with said transmitter.

5. The apparatus according to claim l, wherein said one or more receptors are shaped to accept said body part, and said one or more receptors is adapted to allow EMR
to enter said body part at a first surface of said body part, and said one or more receptors are also adapted to allow passage of at least some of the EMR, wherein the emerging EMR
emerges from a second surface of said body part, and wherein said second surface is the same as or different from said first surface.

6. The apparatus according to claim l, wherein said one or more receptors are shaped to accept said body part, and said one or more receptors are adapted to allow EMR
to enter said body part at the front surface of said body part, and said one or more receptors are also adapted to allow passage of at least some of said EMR, wherein the emerging EMR
emerges from the back side of said body part, to be reflected off a reflective surface in said one or more receptors adjacent to said backside of said body part, and the reflected EMR is collected either at said front surface or a different surface of said body part.

7. The apparatus according to claim 1, wherein said slot is adapted to allow EMR to enter a front side of said slot housing said sample vessel, and the transmitted EMR is collected at the back side of said slot.

8. The apparatus according to claim l, wherein said slot, is adapted to allow EMR to enter a front side of said slot housing said sample vessel, and the transmitted EMR is reflected off a reflective surface located at either the back side of said slot, or the side of said sample vessel adjacent to said back side of said slot, and the reflected EMR
is collected at said front side of said slot.

9. The apparatus according to claim l, wherein said sample vessel is selected from the group consisting of, a cuvette, a sample tab, a pipette tip, tubing, labeled test tubes, unlabeled test tubes, blood bag tubing, any transparent sample container, any translucent sample container, and a flow-through cuvette.

10. The apparatus according to claim l, wherein said sample vessel contains one or more reagents.

11. The apparatus according to claim 1, wherein said sample vessel is either a cuvette or a sample tab, and said cuvette or said sample tab contains one or more reagents.

12. The apparatus according to claim l, wherein said sample vessel is a sample tab, and said slot is designed to accept said sample tab in a horizontal direction.

13. The apparatus according to claim l, wherein said one or more sources of EMR is selected from the group consisting of, a tungsten lamp, one or more light emitting diodes (LED's), one or more lasers, and any combination thereof.

14. The apparatus according to claim 1, wherein said one or more photodetectors is selected from the group consisting of, a single photo diode, an array of photo diodes, an array of charged coupled detectors, and any combination thereof.

15. The apparatus according to claim l, wherein said vessel is a sample tab comprising of a base plate with a sample well and a cover, wherein at least a portion of said base plate and at least a portion of said cover, is adapted to permit transmission of EMR
therethrough.

16. The apparatus according to claim 1, wherein said vessel is a sample tab comprising of a base plate with a sample well and a cover, wherein at least a portion of said base plate is adapted to permit transmission of EMR through said sample, and at least a portion of said cover is adapted to reflect EMR emerging from said sample, and wherein the reflected EMR is allowed to traverse the sample before leaving said sample tab at said base plate, or wherein at least a portion of said cover is adapted to permit transmission of EMR
through said sample, and at least a portion of said base plate is adapted to reflect EMR
emerging from said sample, and wherein the reflected EMR is allowed to traverse the sample before leaving said sample tab at said cover.

17. The apparatus according to claim l, wherein said biological sample is selected from the group consisting of whole blood, a pinprick capillary blood sample, serum, plasma, urine, cerebrospinal fluid, sputum, synovial fluid, lymphatic fluid, feces.

18. The apparatus according to claim 1, wherein said body part is selected from the group consisting of a finger, an ear lobe, a forearm, a web between two fingers, a fold of skin, or the surface of any body part.

19. The apparatus according to claim l, wherein said one or more sources of EMR provides EMR at one or more wavelengths selected from the wavelength range of 300nm to nm.

20. A method that combines in vivo testing (step a) and in vitro testing (step b) using the apparatus according to claim 1, wherein said in vitro testing is performed at least once, and said in vivo testing is performed as frequently as necessary for monitoring a patient depending on the clinical usefulness of such testing, comprising:
a. obtaining a value of one or more analytes in a biological sample obtained from said patient, by applying one or more calibration algorithm to the order derivative of absorbance obtained from said biological sample in a vessel, at one or more wavelengths of a standard set of wavelengths;
b. calculating one or more parameters from one or more sets of order derivative of absorbances obtained from said body part of said patient, wherein said one or more sets of order derivative of absorbances are obtained at one or more wavelengths of a standard set of wavelengths, and wherein said one or more parameters are the same as or different from said one or more analytes.

21. The method according to claim 20, wherein said one or more parameters are the same as or different from said one or more analytes, and said one or more in vivo parameters are used adjunctly with said one or more in vitro analytes.

22. The method according to claim 20, wherein said method further comprises, calculating said one or more parameters from the values obtained from said one or more analytes measured in said biological sample, for one or more purposes selected from the group consisting of, confirming the results of the in vivo testing, assessing the integrity of the results of the in vivo testing, correcting the results of the in vivo testing, and any combination thereof.

23. The method according to claim 20, wherein said value of one or more analytes measured in said biological sample, is used for one or more purposes selected from the group consisting of, confirming the results of the in vivo testing, assessing the integrity of the results of the in vivo testing, correcting the results of the in vivo testing, and any combination thereof.

24. The method according to claim 20, wherein part of said in vivo testing is performed by applying a calibration algorithm to the absorbance for said body part at two or more wavelengths, wherein said calibration algorithm is a linear equation containing a constant plus one or more terms, wherein each of said one or more terms is an independent variable multiplied by a constant, and wherein each of said independent variable is the ratio of absorbances at two different wavelengths.

25. The method according to claim 20, wherein part of said in vitro testing is performed by applying a calibration algorithm to an order derivative of the absorbance at one or more wavelengths of said biological sample in said sample vessel, and wherein part of said in vivo testing is performed by applying a calibration algorithm to an order derivative of the absorbance for said body part, at one or more wavelengths.

26. The method according to claim 20, wherein said body part is selected from the group consisting of, a finger, an ear lobe, a forearm, a web between two fingers, a fold of skin, mucous membrane, the surface of any body part.

27. The method according to claim 20, wherein said biological sample is selected from the group consisting of, whole blood, serum, plasma, urine, cerebrospinal fluid, sputum, synovial fluid, lymphatic fluid, feces.

28. The method according to claim 27, wherein said whole blood is a pinprick capillary blood sample.

29. The method according to claim 20, wherein said sample vessel is a cuvette or a sample tab.

30. The method according to claim 20, wherein said vessel contains one or more reagents, and an altered absorbance is obtained, after reaction between said biological sample and said one or more reagents.

31. The method according to claim 20, wherein said one or more parameters is selected from the group consisting of, the proportion of Hemoglobin-based blood substitute in its Methemoglobin form, proportion of hemoglobin in its Carboxy-Hemoglobin form, proportion of hemoglobin in its Methemoglobin form, hemoglobin oxygen saturation, a ratio of bilirubin concentration to biliverdin concentration, and any combination thereof.

32. The method according to claim 20, wherein said one or more wavelengths in step (a) and step (b), are selected from the wavelength range of 300nm to 2500 nm.

33. The method according to claim 20, wherein order derivative of absorbance is selected from the group consisting of, zero order, first order, second order, and third order.