Patent Application
20240241141
This application describes systems and methods for optical detection of hemoglobin variants, oxygen affinity, and deoxygenation, and particularly relates to a hemoglobin variant detection approach, which will allow rapid screening of hemoglobin disorders.
As blood flows through the lungs, pH rises with an increase in hemoglobin (Hb) oxygen affinity permitting oxygen loading onto Hb; while the blood pH drops in tissue capillaries with decreasing Hb oxygen affinity, facilitating oxygen offloading. The Bohr effects describes Hb's low oxygen affinity with decreased pH. A single point mutation that results in the substitution of valine for glutamic acid at the β-globin subunits of normal hemoglobin (HbA) yields sickle hemoglobin (HbS), which has an altered oxygen affinity. HbS has an abnormally increased Bohr effect that induces oxygen desaturation and can lead to HbS polymerization upon deoxygenation. It is this property of HbS that causes sickle cell disease (SCD).
The HbS oxygen affinity is characterized by an in vitro oxygen dissociation curve (ODC) right shift or a single metric measurement of increased P50 value, which is defined as the partial pressure of oxygen (pO2) at which 50% of the HbS is saturated with oxygen at temperature of 37° C. and pH of 7.40. However, the in vivo HbS oxygen affinity cannot be characterized solely by standardized environmental conditions since various additional parameters, such as decreased intercellular pH, HbS concentration, polymerization, and elevated 2,3-diphosphoglycerate (2-3DPG), impact the HbS oxygen affinity. Although it is unknown how much variance some of these factors induce in HbS affinity, it is well recognized that the influence of those factors relates to increasing HbS polymerization and decreasing blood pH.
Embodiments described herein relate to methods and systems of determining at least one of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, or the presence of hemoglobin variants in blood of a subject. The methods and systems take advantage of differing Hb oxygenation and deoxygenation optical absorption spectra. It was found that under specific deoxygenation and pH levels, the absorption spectra of HbS exhibit right peak wavelength shift (bathochromic shift) and reduction in optical density (hypochromic shifts) that differ from normal hemoglobin (HbA). Absorption spectra of sickle cell disease (SCD) samples had a larger bathochromic and hypochromic shift magnitude under deoxygenation at all pH levels compared to normal healthy blood containing HbA, where the higher the magnitude of the shift, the lower the oxygen affinity of HbS. The magnitude of bathochromic shift in SCD samples was significantly correlated to the percentage of HbS. HbS bathochromic shift was associated with HbS concentration, suggesting that the low oxygen affinity of HbS, is influenced by the reduced pH and polymerization, which is HbS concentration dependent. In addition, the utility of the method described herein was assessed by the changes in the rate of deoxygenation with oxygen hemoglobin modifying drugs. By direct measurement of oxygen affinity, the methods and systems described herein can add a dimension to measure HbS polymerization, which has clinical implications for evaluating emerging hemoglobin modifying therapies for sickle cell disease.
In some embodiments, a method of determining at least one of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, or the presence of hemoglobin variants in blood of a subject can include determining differences of absorption spectra of oxygenated and deoxygenated hemoglobin, red blood, and/or blood obtained from the subject. The determined absorption spectra differences can be compared to a control value, wherein the absorption spectra differences are indicative of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, or the presence of hemoglobin variants in the blood of the subject.
The absorption spectra of the oxygenated and deoxygenated hemoglobin can be measured at the same pH, for example, from about 6.5 to about 9.0, preferably about 6.8 to less than 7.35 or greater than about 7.45 to less than 8.5, or more preferably, about 6.86 or 8.0.
In some embodiments, the differences of absorption spectra are determined by generating a first optical absorption spectra of oxygenated hemoglobin, red blood, and/or blood obtained from the subject, generating a second optical absorption spectra of deoxygenated hemoglobin, red blood, and/or blood obtained from the subject, and comparing the first optical absorption spectra with the second optical absorption to determine differences of the absorption spectra.
In some embodiments, the differences of the absorption spectra include at least one of a bathochromic shift and/or hypochromic shift in peak wavelength from the first absorption spectra to the second absorption spectra.
In some embodiments, the magnitude of barochromic shift in peak wavelength is indicative of at least one of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, or presence and/or percentage of hemoglobin variants in the hemoglobin, red blood cells, or blood of the subject.
In some embodiments, an increase in magnitude of biochromatic shifty and/or hypochromic shift in peak wavelength is indicative of decreased hemoglobin oxygen affinity, increased hemoglobin deoxidation, or the subject having sickle cell disease.
In some embodiments, the differences of area under a curve of and/or full width at half maximum of peak wavelengths of the first absorption spectra and the second absorption spectra are indicative of anemia of the subject and homogeneity of hemoglobin in the subject.
In some embodiments, the hemoglobin, red blood cells, and/or blood can be deoxygenated by mixing the hemoglobin, red blood cells, and/or blood with an amount of chemical deoxygenant effective to deplete oxygen from the hemoglobin. The chemical deoxygenant can include, for example, sodium metabisulfite.
In other embodiments, the hemoglobin, red blood cells, and/or blood can be deoxygenated by mixing the hemoglobin, red blood cells, and/or blood with an amount of enzymatic deoxygenant effective to deplete oxygen from the hemoglobin. The enzymatic deoxygenant can include, for example, EC-oxyrase.
In some embodiments, the at least one of a bathochromic shift and/or hypochromic shift in peak wavelength of an absorption spectra of hemoglobin, RBC, and/or blood from the subject can be used to detect the presence or quantity hemoglobin variants in the subject, where each hemoglobin variant has a bathochromic shift and/or hypochromic shift in peak wavelength that is unique to and can be used to detect and quantify the hemoglobin variant. The hemoglobin variant detected or quantified can be selected from HbSA, HbSS, HbSC, and HbA2.
In some embodiments, detection of HbSA hemoglobin variant diagnoses the subject as having a sickle cell trait.
In other embodiments, detection of HbSS hemoglobin variant diagnoses the subject as having a sickle cell disease.
In some embodiments, detection of HbSC hemoglobin variant diagnoses the subject as having a hemoglobin SC disease.
In some embodiments, detection of HbA2 hemoglobin variant diagnoses the subject as having thalassemia.
Other embodiments relate to a method of detecting hemoglobin variants in blood of a subject. The method can include determining an optical signature of hemoglobin, red blood cells, and/or blood obtained from the subject that has been deoxygenated, for example, by chemical or enzymatic deoxygenation, and that has a pH from about 6.5 to about 9.0, preferably about 6.8 to less than 7.35 or greater than about 7.45 to less than 8.5, or more preferably, about 6.86 or 8.0. The determined optical signature can then be compared to a control optical signature wherein differences between the determined optical signature and the control optical signature is indicative of hemoglobin variants.
In some embodiments, the optical signature is determined using UV-VIS light spectroscopy.
In some embodiments, the determined optical signature includes an absorption spectra of the deoxygenated hemoglobin, red blood cells, and/or blood.
In some embodiments, the control optical signature includes an absorption spectra of deoxygenated normal hemoglobin, red blood cells, and/or blood obtained at substantially the same pH as the optical signature of the hemoglobin, red blood cells, and/or blood obtained from the subject.
In some embodiments, the method further includes adding hemoglobin, red blood cells, and/or blood obtained from a subject to a pH buffer solution prior to determining the optical signature, wherein the hemoglobin, red blood cells, and/or blood added to the pH buffer solution undergoes a conformational change. The buffer solution can have a weak acidic or weak basic pH, preferably a pH of 6.86 or a pH of 8.0.
In some embodiments, the hemoglobin, red blood cells, and/or blood can be deoxygenated by mixing the hemoglobin, red blood cells, and/or blood with an amount of chemical deoxygenant effective to deplete oxygen from the hemoglobin. The chemical deoxygenant can include, for example, sodium metabisulfite.
In other embodiments, the hemoglobin, red blood cells, and/or blood can be deoxygenated by mixing the hemoglobin, red blood cells, and/or blood with an amount of enzymatic deoxygenant effective to deplete oxygen from the hemoglobin. The enzymatic deoxygenant can include, for example, EC-oxyrase.
In some embodiments, the optical signature, e.g., at least one of a bathochromic shift and/or hypochromic shift in peak wavelength of an absorption spectra of hemoglobin, RBC, and/or blood from the subject, can be used to detect the presence or quantity hemoglobin variants in the subject, where each hemoglobin variant has an optical signature that is unique to and can be used to detect and quantify the hemoglobin variant. The hemoglobin variant detected or quantified can be selected from HbSA, HbSS, HbSC, and HbA2.
In some embodiments, detection of HbSA hemoglobin variant diagnoses the subject as having a sickle cell trait.
In other embodiments, detection of HbSS hemoglobin variant diagnoses the subject as having a sickle cell disease.
In some embodiments, detection of HbSC hemoglobin variant diagnoses the subject as having a hemoglobin SC disease.
In some embodiments, detection of HbA2 hemoglobin variant diagnoses the subject as having thalassemia.
Still other embodiments described herein relate to a system for of determining at least one of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, or the presence of hemoglobin variants in blood of a subject. The system includes a UV-VIS spectrometer that is configured to determine an optical signature of hemoglobin, red blood cells, and/or blood obtained from the subject that has been deoxygenated and a processor for comparing the determined optical signature to a control optical signature wherein differences between the determined optical signature and the control optical signature is indicative of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, and/or the presence of hemoglobin variants in the blood of the subject.
In some embodiments, the processor is configured to determine differences of absorption spectra of oxygenated and deoxygenated hemoglobin, red blood, and/or blood obtained from the subject and compare the determined absorption spectra differences to a control value. The absorption spectra differences are indicative of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, and/or the presence of hemoglobin variants in the blood of the subject.
The absorption spectra of the oxygenated and deoxygenated hemoglobin can be measured at the same pH, for example, from about 6.5 to about 9.0, preferably about 6.8 to less than 7.35 or greater than about 7.45 to less than 8.5, or more preferably, about 6.86 or 8.0.
In some embodiments, the processor is configured to determine differences of absorption spectra by comparing a first optical absorption spectrum of oxygenated hemoglobin, red blood cells, and/or blood with a second optical absorption spectrum of deoxygenated hemoglobin, red blood cells, and/or blood.
In some embodiments, the processor is configured to determine differences in at least one of a bathochromic shift and/or hypochromic shift in peak wavelength from the first absorption spectra to the second absorption spectra.
In some embodiments, the magnitude of barochromic shift in peak wavelength is indicative of at least one of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, or presence and/or percentage of hemoglobin variants in the hemoglobin, red blood cells, and/or blood of the subject.
In some embodiments, an increase in magnitude of biochromatic shift and/or hypochromic shift in peak wavelength is indicative of decreased hemoglobin oxygen affinity, increased hemoglobin deoxidation, and/or the subject having sickle cell disease.
In some embodiments, the processor is configured to determine differences of area under a curve of and/or full width half maximum of peak wavelengths of the first absorption spectra and the second absorption spectra to determine anemia of the subject and homogeneity of hemoglobin in the subject.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an”, and “the” are not intended to refer to only a singular entity but also plural entities and also includes the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific aspects of the invention, but their usage does not delimit the invention, except as outlined in the claims.
Throughout the description, where compositions are described as having, including, or comprising, specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the compositions and methods described herein remains operable. Moreover, two or more steps or actions can be conducted simultaneously.
As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only” and the like in connection with the recitation of claim elements, or the use of a “negative” limitation. “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.
The term “patient” or “subject”, as used herein, is a human or animal and need not be hospitalized. For example, out-patients, persons in nursing homes are “patients.” A patient may comprise any age of a human or non-human animal and therefore includes both adult and juveniles (i.e., children). It is not intended that the term “patient” connote a need for medical treatment, therefore, a patient may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies.
The term “sample” as used herein is used in its broadest sense and includes environmental and biological samples. Environmental samples include material from the environment such as soil and water. Biological samples may be animal, including, human, fluid (e.g., blood, plasma and serum), solid (e.g., stool), tissue, liquid foods (e.g., milk), and solid foods (e.g., vegetables). A biological sample may comprise a cell, tissue extract, body fluid, chromosomes or extrachromosomal elements isolated from a cell, genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), cDNA (in solution or bound to a solid support) and the like.
Embodiments described herein relate to methods and systems of determining at least one of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, or the presence of hemoglobin variants in blood of a subject. We found that differing hemoglobin (Hb) oxygenation and deoxygenation optical absorption spectra determined using light absorption spectroscopy can be used to distinguish hemoglobin variants based on their oxygen retention capability. When an optical beam strikes a solution containing deoxygenated hemoglobin, light is absorbed, and the intrinsic hemoglobin molecules interact with light photons energy to produce distinct optical signatures of absorption shifts that are unique to each hemoglobin variant under investigation. We found that under specific deoxygenation and pH levels, the absorption spectra of HbS exhibit right peak wavelength shift (bathochromic shift) and reduction in optical density (hypochromic shifts) that differ from normal hemoglobin (HbA). Absorption spectra of sickle cell disease (SCD) samples had a larger bathochromic and hypochromic shift magnitude under deoxygenation at all pH levels compared to normal healthy blood containing HbA, where the higher the magnitude of the shift, the lower the oxygen affinity of HbS. The magnitude of bathochromic shift in SCD samples was significantly correlated to the percentage of HbS. HbS bathochromic shift was associated to HbS concentration, suggesting that the low oxygen affinity of HbS, is influenced by the reduced pH and polymerization which is HbS concentration dependent. In addition, the method described herein can be used to assess changes in the rate of deoxygenation with oxygen hemoglobin modifying drugs. By direct measurement of oxygen affinity, the methods and systems described herein adds a dimension to measure HbS polymerization, which has clinical implications for evaluating emerging hemoglobin modifying therapies for sickle cell disease.
Accordingly, in some embodiments a method of determining at least one of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, or the presence of hemoglobin variants in blood of a subject can include determining differences of absorption spectra of oxygenated and deoxygenated hemoglobin, red blood, and/or blood obtained from the subject. The determined absorption spectra differences can be compared to a control value, wherein the absorption spectra differences are indicative of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, or the presence of hemoglobin variants in the blood of the subject.
The absorption spectra of the oxygenated and deoxygenated hemoglobin can be measured at the same pH, for example, from about 6.5 to about 9.0, preferably about 6.8 to less than 7.35 or greater than about 7.45 to less than 8.5, or more preferably, about 6.86 or 8.0.
In some embodiments, the differences of absorption spectra are determined by generating a first optical absorption spectra of oxygenated hemoglobin, red blood, and/or blood obtained from the subject, generating a second optical absorption spectra of deoxygenated hemoglobin, red blood, and/or blood obtained from the subject, and comparing the first optical absorption spectra with the second optical absorption to determine differences of the absorption spectra.
In some embodiments, the differences of the absorption spectra include at least one of a bathochromic shift and/or hypochromic shift in peak wavelength from the first absorption spectra to the second absorption spectra.
In some embodiments, the magnitude of barochromic shift in peak wavelength is indicative of at least one hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, or presence and/or percentage of hemoglobin variants in the hemoglobin, red blood cells, or blood of the subject.
In some embodiments, an increase in magnitude of biochromatic shifty and/or hypochromic shift in peak wavelength is indicative of decreased hemoglobin oxygen affinity, increased hemoglobin deoxidation, or the subject having sickle cell disease.
In some embodiments, the differences of area under a curve of and/or full width at half maximum of peak wavelengths of the first absorption spectra and the second absorption spectra are indicative of anemia of the subject and homogeneity of hemoglobin in the subject.
In some embodiments, the hemoglobin, red blood cells, and/or blood can be deoxygenated by mixing the hemoglobin, red blood cells, and/or blood with an amount of chemical deoxygenant effective to deplete oxygen from the hemoglobin. The chemical deoxygenant can include, for example, sodium metabisulfite.
In other embodiments, the hemoglobin, red blood cells, and/or blood can be deoxygenated by mixing the hemoglobin, red blood cells, and/or blood with an amount of enzymatic deoxygenant effective to deplete oxygen from the hemoglobin. The enzymatic deoxygenant can include, for example, EC-oxyrase.
In some embodiments, the hemoglobin variant is selected from HbSA, HbSS, HbSC, and HbA2.
In the method at step 12, a sample of hemoglobin, red blood cells, or blood can be obtained from a subject. The sample can include whole blood, isolated red blood cells (RBCs), and purified hemoglobin lysed RBCs.
At step 14, the hemoglobin, red blood cells, and/or blood can be mixed with a buffer solution to provide a hemoglobin, red blood cell, or blood suspension with a pH that amplifies the Bohr effect of hemoglobin (Hb) including Hb variants, such as HbS. Any change in pH beyond the physiological range of 7.35 to 7.45 amplifies this Hb Bohr effect.
In some embodiments, the pH of the hemoglobin, red blood cell, or blood suspension can be adjusted with the buffer from a physiological blood pH of about 7.35 to 7.45 to a pH less or greater than physiological pH in a range of about 6.5 to about 9.0. For example, the hemoglobin, red blood cell, or blood suspension can be adjusted with the buffer from physiological blood pH to about 6.8 to less than 7.35 or greater than about 7.45 to less than 8.5, or preferably, about 6.86 or 8.0.
By way of example, whole blood, RBCs, and/or purified Hb can be mixed with pH 6.86 buffer solution and incubated for a duration of time at room temperature. The pH of the buffer suspensions can be checked after addition to the whole blood, RBCs, or Hb.
Following mixing of the Hb, RBCs, or blood with the pH buffer, at step 16, samples of the pH adjusted Hb, RBCs, or blood suspension can be oxygenated and deoxygenated to provide oxygenated and deoxygenated samples of the pH buffered Hb, RBCs, or blood suspension. For example, all samples can initially exposed to ambient air. The oxygen partial pressure (PO2) which is 21% of atmospheric pressure (773 mmHg), can then normalized to 162 mmHg PO2 during oxygenation to provide oxygenated samples of the Hb, RBCs, or blood suspension.
Samples of the Hb, RBCs, or blood suspension can be deoxygenated chemically or enzymatically using, for example, sodium metabisulphite (Na2S2O5), sodium dithionite (Na2S2O4), or EC-oxyrase. For example, predetermined amounts of sodium metabisulphite can be mixed with the prepared samples of the Hb, RBCs, or blood suspension to provide deoxygenated samples with a gradual reduction in the oxygen partial pressure and deoxygenation levels.
At step 18, optical absorption spectra of the oxygenated and the deoxygenated Hb, RBC, and/or blood samples can be generated using a UV-VIS. The optical absorption spectra can include a generated first optical absorption spectrum of oxygenated samples of the Hb, RBCs, and/or blood suspension and a generated second optical absorption spectrum of deoxygenated Hb, RBCs, and/or blood suspension. The spectral rang of the absorption spectra can be from about 300 nm to about 800 nm with a resolution of, for example, about 1 nm or 2 nm. For example, during sample testing of either oxygenated and deoxygenated samples, samples can be analyzed in microplate wells using a spectroscopy microplate reader, such as Petromax Me2, (Molecular devices, San Jose, CA), over a spectral range of about 350 nm to about 750 nm, with a wavelength resolution of 2 nm and at a customized microplate well reading setting and at room temperature.
By way of example, fully oxygenated samples were analyzed, to obtain reference signatures or control signature or control optical absorption spectra that included a Soret band (e.g., 380-480 nm) and two peaks in Q-band (e.g., 560-580 nm). All oxygenated samples had their highest absorption oxygenated peak at 414 nm. Following that, the deoxygenated samples were analyzed across the same spectral range as the oxygenated samples, but with Na2S2O5 concentrations varying, for example, from 0.039 to 0.092 M. The concentration of Na2S2O5 used was corresponding to a decrease from 100-0 (mmHg) of pO2 in samples.
In some embodiments, absorbance measurements are conducted at a substantially constant temperature. As such, the temperature during absorbance measurement changes by 5° C. or less, such as by 4.5° C. or less, such as by 4° C. or less, such as by 3.5° C. or less, such as by 3° C. or less, such as by 2.5° C. or less, such as by 2° C. or less, such as by 1.5° C. or less, such as 1° C. or less, such as by 0.5° C. or less, such as by 0.1° C. or less, such as by 0.05° C. or less, such as by 0.01° C. or less, such as by 0.005° C., such as by 0.001° C., such as by 0.0001° C., such as by 0.00001° C. or less and including by 0.000001° C. or less.
Following generation of the optical absorption spectra of the oxygenated and the deoxygenated samples of Hb, RBCs, and/or blood, at step 20, the optical absorption spectra of the Hb, RBCs, and/or blood can be compared to determine differences in the absorption spectra.
In some embodiments, the differences of the absorption spectra include at least one of a bathochromic shift and/or hypochromic shift in peak wavelength from the first absorption spectra to the second absorption spectra, differences of area under a curve of the first absorption spectra and the second absorption spectra, and/or differences of the full width at half maximum of peak wavelengths of the first absorption spectra and the second absorption spectra.
At step 22, the differences of the absorption spectra can be compared to a control value to determine at least one of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, or the presence of hemoglobin variants in blood of a subject.
A “control value” or “appropriate standard” is a standard, parameter, value or level indicative of a known outcome, status or result (e.g., a known disease or condition status). A control value or appropriate can be determined (e.g., determined in parallel with a test measurement) or can be pre-existing (e.g., a historical value, etc.). For example, a control value or appropriate standard may be a bathochromic shift and/or hypochromic shift in peak wavelength, differences of area under a curve, and/or differences of the full width at half maximum of peak wavelengths obtained from a subject known to have a sickle cell disease, or a subject identified as being disease-free. In the former case, a lack of a difference between the measured differences in adsorption spectra and the differences in absorption spectra of an appropriate standard may be indicative of a subject having a disease or condition. Whereas in the latter case, the presence of a difference between the measured differences of absorption spectra and the differences of absorption spectra of the control value or appropriate standard may be indicative of a subject having a disease or condition.
The magnitude of a difference between a parameter, level or value the absorption spectra that is indicative of outcome, status or result may vary. For example, a significant difference that indicates a known outcome, status or result may be detected when the level of a parameter, level or value is at least 1%, at least 5%, at least 10%, at least 25%, at least 50%, at least 100%, at least 250%, at least 500%, or at least 1000% higher, or lower, than the appropriate standard. Similarly, a significant difference may be detected when a parameter, level or value is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, or more higher, or lower, than the level of the appropriate standard. Significant differences may be identified by using an appropriate statistical test. Tests for statistical significance are well known in the art and are exemplified in Applied Statistics for Engineers and Scientists by Petruccelli, Chen and Nandram Reprint Ed. Prentice Hall (1999).
In some embodiments, the magnitude of barochromic shift in peak wavelength compared to a control value or shift is indicative of at least one of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, or presence and/or percentage of hemoglobin variants in the hemoglobin, red blood cells, and/or blood of the subject. An increase in magnitude of biochromatic shift and/or hypochromic shift in peak wavelength compared to a control value or shift can be indicative of decreased hemoglobin oxygen affinity, increased hemoglobin deoxidation, or the subject having sickle cell disease.
For example, both oxygenated HbS and HbA purified hemoglobin exhibited 3 distinct peaks at 414 nm and two-valley peaks at 540 and 576 nm. The maximum 414 nm peak observed in oxy-HbS and oxy-HbA shifted to different extents upon deoxygenation such that the shift of HbS was greater (424 nm and 418 nm). It was noted that the trends of changes in the wavelength with deoxygenation was similar to the trend of deoxygenation with Na2S2O5 indicating that bathochromic shift is a surrogate marker for deoxygenation. At 162 mm Hg pO2, both normal and sickle whole blood had a peak wavelength of 414 nm. The reduction in pO2 from 162 to 10 mmHg in both normal and sickle whole blood caused an increase in shift and quasi-equilibrium was observed at 28 mmHg pO2.
The variations of bathochromic shift shown for purified hemoglobin was also observable for RBCs and whole blood. RBCs and whole blood from normal and SCD patients also exhibited the three wavelengths that were present in purified hemoglobin. From 162 to 10 mmHg pO2, decrease in PO2 induced a rise in both normal and sickle samples peak wavelength for both RBCs and whole blood, followed by a plateau from 20−10 mmHg. At 28 mmHg pO2, bathochromic shifts between normal and sickle RBCs (p=0.001) and normal and sickle whole blood (p=0.001) were significantly different. Compared to bathochromic shift of purified hemoglobin for both HbA and HbS at pH 6.86, RBCs had the largest magnitude of bathochromic shift, followed by purified hemoglobin and then whole blood samples. Sickle RBCs and whole blood had a larger bathochromic shift compared to normal RBCs and whole blood.
In some embodiments, the differences of area under a curve of and/or full width at half maximum of peak wavelengths of the first absorption spectra and the second absorption spectra are indicative of anemia of the subject and homogeneity of hemoglobin in the subject.
In some embodiments, the at least one of a bathochromic shift and/or hypochromic shift in peak wavelength of an absorption spectra of hemoglobin, RBC, and/or blood from the subject can be used to detect the presence or quantity hemoglobin variants in the subject, where each hemoglobin variant has a bathochromic shift and/or hypochromic shift in peak wavelength that is unique to and can be used to detect and quantify the hemoglobin variant. The hemoglobin variant detected or quantified can be selected from HbSA, HbSS, HbSC, and HbA2.
In some embodiments, detection of HbSA hemoglobin variant diagnoses the subject as having a sickle cell trait.
In other embodiments, detection of HbSS hemoglobin variant diagnoses the subject as having a sickle cell disease.
In some embodiments, detection of HbSC hemoglobin variant diagnoses the subject as having a hemoglobin SC disease.
In some embodiments, detection of HbA2 hemoglobin variant diagnoses the subject as having thalassemia.
Still other embodiments described herein relate to a system for determining at least one of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, or the presence of hemoglobin variants in blood of a subject.
In some embodiments, the processor 34 is configured to determine differences of absorption spectra of oxygenated and chemically deoxygenated hemoglobin, red blood, and/or blood obtained from the subject and compare the determined absorption spectra differences to a control value. The absorption spectra differences are indicative of hemoglobin oxygen affinity, rate of hemoglobin deoxygenation, or the presence of hemoglobin variants in the blood of the subject.
The processor 34 typically receives and processes optical measurements that are performed by the UV-VIS spectrometer 32. Further typically, the processor 34 controls the acquisition of optical measurements that are performed by the UV-VIS spectrometer. The processor 34 communicates with a memory 36. A user (e.g., a laboratory technician) sends instructions to the computer processor via a user interface 38. For some applications, the user interface includes a keyboard, a mouse, a joystick, a touchscreen device (such as a smartphone or a tablet computer), a touchpad, a trackball, a voice-command interface, and/or other types of user interfaces that are known in the art. Typically, the computer processor generates an output via an output device 40. Further typically, the output device includes a display, such as a monitor, and the output includes an output that is displayed on the display. For some applications, the processor generates an output on a different type of visual, text, graphics, tactile, audio, and/or video output device, e.g., speakers, headphones, a smartphone, or a tablet computer. For some applications, user interface 36 acts as both an input interface and an output interface, i.e., it acts as an input/output interface. For some applications, the processor generates an output on a computer-readable medium (e.g., a non-transitory computer-readable medium), such as a disk, or a portable USB drive, and/or generates an output on a printer.
In some embodiments, the temperature of the system may be controlled by a temperature control subsystem (not shown), which measures the system temperature and if necessary, controls the ambient conditions to maintain a desired system temperature. Temperature subsystems may include any convenient temperature control protocol, including, but not limited to heat sinks, fans, exhaust pumps, vents, refrigeration, coolants, heat exchanges, Peltier or resistive heating elements, among other types of temperature control protocols.
In some embodiments, the UV-VIS spectrometer can include broadband light source (such as a Tungsten Halogen bulb), monochromator that selects certain wavelengths from that broadband light source, a computer than can command the monochromator to select certain wavelengths, a collimating stage that accepts a sample and a detector that characterizes light intensity after it has passed through the sample cell.
In some embodiments, the memory has instructions stored thereon, which when executed by the processor, cause the system to irradiate the sample with the desired wavelengths of light, determine a measured intensity of light at the desired wavelengths, and calculate the absorbance until a spectrum of absorbance vs wavelength covering the desired wavelength region.
In some embodiments, the processor is configured to determine bathochromic shift and/or hypochromic shift in peak wavelength, differences of area under a curve, and/or differences of the full width at half maximum of peak wavelengths.
In some embodiments, a computer readable storage medium may be employed on one or more components of the system having a display and operator input device. Also provided are non-transitory computer readable storage media. Such media can be, for example, a CD-ROM, a USB drive, a floppy disk, or a hard drive. In some cases, the medium comprises instructions stored thereon for separating an absorption spectrum into a Rayleigh scattering contribution and an absorption contribution. In some cases, the instructions comprise: an algorithm for measuring an absorption spectrum (ii) an algorithm for generating a fit spectrum by fitting the absorption spectrum to a power function (iii) an algorithm for generating a difference spectrum by subtracting the fit spectrum from the absorption spectrum (iv) an algorithm for generating an adjusted spectrum by selecting points from the absorption spectrum for wavelengths wherein the difference spectrum is less than or equal to zero points from the fit spectrum for wavelengths wherein the difference spectrum is greater than zero (v) an algorithm for repeating steps (ii)-(iv) zero or more times, wherein the most recent adjusted spectrum is used in place of the absorption spectrum if the steps are repeated.
In some embodiments, the system described herein can employ supervised machine learning. In some embodiments, supervised machine learning can detect difference in the generated or measured absorption spectra. For example, supervised machine learning can detect changes or differences in differences of a bathochromic shift and/or hypochromic shift in peak wavelength from generated or measured absorption spectra. In some embodiments, supervised machine learning can detect changes such as differences of area under a curve of and/or full width at half maximum of peak wavelengths of absorption spectra. In some cases, supervised machine learning can be used to classify samples Hb variant or concentration as well as hemoglobin oxygen affinity or rate of hemoglobin deoxygenation.
This Example describes a new rapid optical diagnostic approach and, rate of deoxygenation and hemoglobin oxygen affinity measurement method. We exploited the differing oxygenation and deoxygenation spectra of Hb to determine the difference in the oxygen affinity and Bohr effect between HbA and HbS in purified hemoglobin, RBCs and in whole blood. Optical absorption spectra of HbA are affected by the oxygenation-deoxygenation dynamics and to any alteration in the Hb confirmation structure. HbA's oxygenated (Oxy-HbA) and deoxygenated (Deoxy-HbA) optical spectra have been vital in measurement of physiological parameters including hemoglobin concentration, noninvasive blood oxygen saturation levels and pulse oximetry. The absorption spectra of deoxygenated and oxygenated hemoglobin can be utilized to monitor hemoglobin's rate of deoxygenation and oxygen affinity. In the presence of certain hemoglobinopathies, the absorption spectra of HbA were found to shift in peak wavelength.
We show that while it has been assumed that the absorption spectral of HbA and HbS are identical, under specific deoxygenation levels and pH, the absorption spectra of HbA and HbS are distinct. HbS exhibited spectra peak wavelength shift to the right (bathochromic shift) and reduction optical density (hypochromic shift) in purified hemoglobin, RBCs, and whole blood that differ from HbA. Leveraging this newly discovered property, initially we used this method to detect HbS. We then applied the bathochromic shifts to evaluate oxygen affinity from whole blood. We found that the magnitude of bathochromic shift was a concomitate to the oxygen affinity. With HbS the spectral bathochromic shift was associated to HbS concentration, suggesting that the low oxygen affinity of HbS, is influenced by the reduced pH and polymerization which is an HbS concentration dependent mechanism. We demonstrated the utility of using our method to measure the rate deoxygenation by describing the kinetics of deoxygenation in whole blood sample with hemoglobin oxygen modifying treatment. We provide an optical approach for in vitro and in vivo diagnostic approaches for sickle hemoglobin, and oxygen affinity and rate of deoxygenation measurement and demonstrate its clinical significance by evaluating the hemoglobin modifying therapies for sickle cell disease both by direct inhibition of polymerization and by increasing the oxygen affinity.
Buffer solutions of pH 6.86, 7.2, 7.4, 8.0 and 10.0 were purchased from Fisher Scientific (Pittsburgh, PA). All buffer solutions were stored at room temperature. Sodium metabisulfite (Na2S2O5) was purchased from Sigma Aldrich (St Louis, Mo) to be used for chemical deoxygenation. Nunc polystyrene 90 microwell plate non treated surface with flat bottom and lid were purchased from Thermo Fisher Scientific (Waltham, MA).
Blood samples were collected from de-identified healthy donors and SCD patients as part of standard clinical care. All participants in the study provided written informed consent. When blood samples were collected, they were kept at 4 degrees Celsius and processed within 6 hours. Samples were collected in EDTA-containing vacutainer tubes and separated into two groups: Normal samples from healthy donors and SS samples from SCD. Hemoglobin profiles of samples were verified using the reference standard HPLC (VARIANT II, Bio-Rad Laboratories, Inc Hercules, California). The results were given as a percentage of HbS, A2, F, and the remainder was considered HbA since the values were normalized to 100%. Intracellular hemoglobin concentration for all the samples was measured under nomoi with CBC (Hema vet 950FS, Hematology System, Draw Scientific Inc; Miami, Florida).
Three independent sample preparations were performed to test the different hypotheses. 1) whole blood, 2) isolated red blood cells (RBC) and 3) purified hemoglobin (lysed samples).
75 μl of whole blood was mixed with 22 ml of pH 6.86 buffers and incubated for 10 minutes at room temperature before deoxygenation. Each microplate well used for analysis contained 12 μl of cells suspension that were diluted 300-fold from whole blood diluted in a buffer solution.
To create Vox+ samples 75 μl of undiluted blood was combined with 67 mg/mL voxelotor (Selleck chem) in 100% dimethyl sulfoxide (DMSO) (Sigma-Aldrich) to a final concentration of 30 μmol/L voxelotor that was determined based on pervious publication. The Vox+ samples used in this investigation were incubated for 1 hour with voxelotor at 37° C. temperature.
Whole blood was centrifuged at 500 g for 10 minutes in a micro centrifuge (model 21r; Thermo Scientific, Waltham, MA). The plasma and buffy coat were removed via aspiration. The RBCs were then washed three times with phosphate buffered saline (PBS) at pH 7.4. (Gibco, Thermo scientific, Waltham, MA).
45 μL of the washed RBC suspension was diluted with a pH 6.86 buffer solution, until the baseline hematocrit of 0.2% was achieved. Prior to deoxygenation, RBCs were incubated for 10 minutes in a pH 6.86 buffer solution at room temperature.
Lysed samples were prepared as follows. Hemolysis was carried out by sonication for 30 second (Hemet Health; Portland, OR). Following hemolysis, the lysed cell suspension was added to various pH buffers solutions and centrifuged at 2000 g for 1 hour. Three hundred μl of the cell lysate was removed and the supernatant was discarded. Cell lysates were further diluted in 700 μl of the specific pH buffer solution and centrifuged at 2000 g for 1 hour. The 1 ml mixture of pH buffer solution and cell lysate were then filtered through a 0.02 um Millipore membrane.
The hemoglobin concentration in the lysed sample was determined using the hemoglobin cyanide (HiCN) method and measured with a cuvette spectrophotometer at 540 nm. Each of the purified lysed samples was diluted with different pH solutions (6.86, 7.2, 7.4, 8.0, and 10.0) to achieve a hemoglobin concentration of 4.36 μM, which had been determined as the baseline for lysed sample analysis.
The pH of the buffer solutions was checked before and after additional to the prepared samples with an acumen AE150 pH meter (Fisher Scientific Waltham, MA). Using the same pH meter, we also measured the pH of a mixture of buffer, whole blood samples before and after additional of Na2S2O5 (
All samples were initially exposed to ambient air for 600 seconds. The oxygen partial pressure (PO2) which is 21% of atmospheric pressure (773 mmHg), was then normalized to 162 mmHg PO2 during oxygenation. (
During sample testing of either oxygenated and deoxygenated samples, samples were analyzed in three microplate wells using a spectroscopy microplate reader Petromax Me2, (Molecular devices, San Jose, CA), over a spectral range of 350-750 nm, with a wavelength resolution of 2 nm and at a customized microplate well reading setting and at room temperature. Each acquisition lasted 270 seconds and the microplate was shaken for 5 seconds by the plate reader before each read.
The hypochromic and bathochromic shift were obtained every 30 seconds for 600 seconds and used to determine the time course of deoxygenation of whole blood sample (
Oxygenated and deoxygenated spectra were obtained and processed in Softmax Pro 6.3. Individual peak, intensity and the peak wavelength shifts were identified from the spectra through an automated peak and intensity search (
We used minitab software (Release 2021, Version 20; Minitab) for the statistical analysis. Mann Whitney U-test was performed to compare spectral variables between the normal and sickle groups. Pearson's correlation analysis was used to assess correlation between spectral variables, P<0.05 was considered statistically significant. Levene's test was performed to determine the homogeneity of variance between samples treated with voxelotor and those not treated. A paired t-test was performed to compare paired groups before and after voxelotor treatment and at the different temperature analyzed. Unless otherwise noted, results were expressed as means±standard error of the mean (SEM). Full set of 201 intensities from 350-750 nm (UV-Visible range) at 20% pO2 and the four optical variables (peak wavelength shift, intensity, FWHM and area under the peak) data from normal and sickle blood sample were pooled and subjected to two-dimensional principal components analysis (PCA) using R Studio (Release 2021; 4.1.1) independently. The first and second principal components were PC1 and PC2, respectively. The two components were then clustered and visualized to represent the sickle and normal samples, respectively.
Sodium Metabisulphite (Na2S2O5) Induces Whole Blood Oxygen Partial Pressure (pO2) Decrease in Dose and pH Dependent Manner
We used Sodium metabisulphite (Na2S2O5) to deoxygenate hemoglobin and blood. To quantify the effect of Na2S2O5 on deoxygenation (
Under all Na2S2O5 concentrations, the pO2 of both normal and sickle whole blood decreased with decrease in pH (
Purified Sickle Hemoglobin's Bathochromic Peak Wavelength Shifts More than Normal Hemoglobin Upon Deoxygenation
Both oxygenated HbS and HbA purified hemoglobin exhibited 3 distinct peaks at 414 nm and two-valley peaks at 540 and 576 nm (
At all pH values, fully oxygenated HbA and HbS had the same peak wavelengths of 414 nm (
RBCs and Whole Blood Samples from Patients with SCD Demonstrate Increased Optical Absorption Bathochromic Shift Compared to Samples from Normal Donors
For the analysis of RBCs and whole blood samples, we focused our analyses on pH 6.86 because the highest magnitude of peak wavelength difference between HbA and HbS purified hemoglobin was obtained at this pH value. The variations of bathochromic shift shown for purified hemoglobin was also observable for RBCs (
Characteristics of Whole Blood Optical Absorption Measure Demonstrated Association with Hemoglobin Levels and Percentages of Sickle Hemoglobin
Aside from obtaining the measurable optical variables and examining their confounding effects, it was also critical to fully comprehend the association between absorption bathochromic shift and intensity, with HbS fraction and hemoglobin concentration. Sickle whole blood bathochromic shifts were highly associated with the changes in percentage of hemoglobin S (HbS %) obtained using High Performance Liquid Chromatography (HPLC) for each whole blood sample at pH 6.86 and pO2 20% (
Principal Component Analysis (PCA) Differentiates Normal from Sickle Whole Blood Samples Based on Measured Optical Absorption Variables and Deoxygenated Spectra Intensity
The two principal components obtained accounted for 98.5% variability in the original full spectrum peak intensity data set, with the first component accounting for 77.96% of the total variation (
The magnitude of hypochromic shift was found to correlate to the magnitude of bathochromic shift between 400 and 430 nm wavelength. As the magnitude of bathochromic shift increased, the magnitude of hypochromic shift increased. The time course of deoxygenation or rate of deoxygenation was obtained from assessing the hypochromic and bathochromic shift every 30 seconds interval for 600 seconds (
The repeatability of bathochromic shift was established by comparing variations between two users in 20 experiments using the same whole blood samples at approximately 20 mmHg pO2 (
We describe a rapid optical method that can detect HbS, anemia and measures Hb oxygen affinity, rate of deoxygenation and Hb concentration for whole blood samples, determined at a controlled deoxygenation levels and pH. We found that the optical absorption spectral of HbA and HbS differed under certain deoxygenation and pH circumstances, based on actual spectral bathochromic shifts and hypochromic shifts. The bathochromic shifts of HbA and HbS purified hemoglobin's were significantly different at pH 6.86, and no bathochromic shift appeared at pH 10.0, revealing that under more alkaline conditions above pH 8.5, the Bohr effect does not hold true, for HbS and the oxygen affinity of HbS is similar to that HbA irrespective of the hypoxia levels. These findings provide a clear explanation and mechanism behind the attempts of previous investigators to treat sickle cell disease crises by alkalizing the blood.
We show that the oxygen affinity of Hb in whole blood can be measured using optical bathochromic shifts. SCD samples had a larger bathochromic and hypochromic shift magnitude under deoxygenation at all pH levels compared to normal healthy blood containing HbA, where the higher the magnitude of the shift, the lower the oxygen affinity of HbS. The magnitude of bathochromic shift in SCD samples significantly correlated to the percentage of HbS. This explains that the lowered oxygen affinity of HbS is caused by polymerization (HbS concentration dependent process) and decreased pH. Low pH causes an increase in hydrogen ions binding to histidine amino acids such as β-146 and α-122, displacing oxygen and promoting the formation of salt bridges that stabilize hemoglobin in the deoxygenated state (
We also have found that shifted peak intensity is positively correlated with the hemoglobin concentration and the bathochromic shift had a negative correlation with the area under the peak. However, no correlations are related with FHWM, but the results were statistically significant. These results revealed that other than the detection of HbS and assessment of oxygen affinity, this method can be used to optically determine the hemoglobin concentration and homogeneity of the sample as well as detection of anemia, a common condition in patients with SCD.
Although we have focused mainly on assessment of oxygen affinity, the developed method can also be used to study the kinetics of whole blood deoxygenation. We demonstrated the effect of an oxygen modifying drug, voxelotor that has recently been approved for treatment of SCD. We found a significant difference in the rate of deoxygenation with SCD whole blood samples treated with voxelotor and samples that were not treated. Although voxelotor could reduce the rate of deoxygenation for SCD samples, deoxygenation rate was still lower than for normal healthy patients (
While methods of detection of HbA and measurement of the oxygen affinity and rate of deoxygenation were conducted at room temperature, this method was also tested at an increased temperature of 37° C. We found that the magnitude of bathochromic shift was elevated at 37 C for both normal and sickle samples compared to room temperature (
This example provides support for using optical absorption parameters to differentiate hemoglobin variants, and motivates investigation to understanding HbS deoxygenation response dynamics and single cell RBC assessment that is critical in the recent genetic approach treatment for SCD. We provide evidence that could be used to improve the diagnostic accuracy of systems that use the current absorption spectrophotometer and are affected by variant testing. We show the influence of pH (Bohr effect) using a ubiquitous absorption method and provide convincing evidence that once the pH becomes more acidic and in the initial alkaline ranges, absorption peak wavelength shifts were detected, and these shifts were associated with HbS profile in the sickle cell blood sample. We also demonstrated how we could test the effectiveness of treatment for sickle cell disease by assessing the alteration in the rate of deoxygenation. We show that the magnitude of bathochromic shift relates to the pH which was also was dependent on the concentration of HbS.
The finding of a correlation between bathochromic shift in sickle whole blood samples and hemoglobin S percentages suggests that bathochromic shift can be utilized to estimate the profile of hemoglobin S in a sample. Our Principal component analysis (PCA) algorithm for the entire intensity spectrum and the four variables clearly showed distinguishable clusters of normal and sickle whole blood samples. The PCA results revealed that, while we originally clustered sickle blood from normal samples in this study, the assay may also be utilized to identify other hemoglobin variants based on their altered oxygen affinity, as shown by the acquired optical variables. These could include common hemoglobin variants with altered oxygen affinity for which no point-of-care diagnostic technology exists.
The effect of pH and thus increases Bohr effect was shown with the optical absorption peak for both HbS and HbA shifting under different pH conditions. We revealed that when the pH is lower than 7.4, the magnitude of the bathochromic and hypochromic shift for both HbS and HbA is greatest, indicating rapid oxygen dissociation from hemoglobin. However, as pH is increased to 8.0 and then to 10.0 the magnitude of bathochromic shift decreases as compared to pH of 6.86. The bathochromic shift at pH 7.4 or higher must have been contributed by the increased levels of 2,3 DPG leading to changes in the Donna equilibrium and thus lowering the pH. On another hand no bathochromic or hypochromic shift were observed at pH of 10.0
Considering the effect of pH on subsequent hemoglobin structural changes has been studied in the Bohr effect using the p50. In comparison to HbA, any change in pH beyond the physiological range of 7.35 to 7.45 amplifies the HbS Bohr effect, resulting in increased deoxygenation.
We discovered that when hemoglobin or RBC from a blood sample is placed in a pH buffer solution of 6.86, or 8.0, hemoglobin molecules undergo a conformational change that is based on the type of hemoglobin variant. The alternated hemoglobin variant molecules in a concentration range (4.36 μM) or 2% hematocrit are then chemically deoxygenated by adding sodium metabisulfite in a range (15-25 mg) to deplete oxygen from hemoglobin molecule and there after exposed to an optical beam of light in the UV-Visible wavelength range. The absorbed light by the hemoglobin-buffer solution yields unique optical signatures of absorption shifts and a reduction in the optical density that are unique to every hemoglobin variant under investigation. The shift and intensity ratios could then be used to identify hemoglobin variant and their oxygen bind capability.
We have recently used a validated bench top absorption spectroscopy analyzer to perform this proof of concept on nearly 47 patient blood samples (23 normal AA,23 hemoglobin variant SS, 2 S beta thalassemia and 2 delta beta thalassemia) (
From the above description of the invention, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications within the skill of the art are intended to be covered by the appended claims. All references, publications, and patents cited in the present application are herein incorporated by reference in their entirety.
This application claims priority from U.S. Provisional Application No. 63/191,469 filed May 21, 2011, the subject matter of which is incorporated herein by reference in its entirety.
This invention was made with government support under HL133574 awarded by The National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/030561 | 5/23/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63191469 | May 2021 | US |
