WO2023238005A1

WO2023238005A1 - A method for screening hemoglobinopathies by globin chain analysis using electrospray tandem quadrupole mass spectrometry.

Info

Publication number: WO2023238005A1
Application number: PCT/IB2023/055767
Authority: WO
Inventors: Dr Deepalakshmi P D
Original assignee: Neuberg Anand Academy Of Laborato Ry Medicine Private Limited
Priority date: 2022-06-06
Filing date: 2023-06-05
Publication date: 2023-12-14

Abstract

The present invention relates to a method for screening hemoglobinopathies in blood sample by analyzing expression levels of intact globin chains of hemoglobin using electrospray tandem quadrupole mass spectrometry and a relative ion quantification model. The method includes obtaining mass spectra comprising mass/charge ratios (m/z) of the intact globin chains by mass analyzing the blood sample ions, selecting at least three representative mass/charge ratios (m/z) of subunits of the intact globin chains in multiple reaction monitoring (MRM) mode by a third quadrupole, summing ion intensities of the subunits of the selected intact globin chains, calculating ratios of the summed ion intensities between the subunits of the selected intact globin chains, and obtaining a score of 0-10 by summing at least five different calculated ratios. The score of 0-4 is indicative of normal expression levels and a score of 5-10 is indicative of abnormal expression levels of the globin chains.

Description

A METHOD FOR SCREENING HEMOGLOBINOPATHIES BY GLOBIN CHAIN ANALYSIS USING ELECTROSPRAY TANDEM QUADRUPOLE MASS SPECTROMETRY

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from prior Indian provisional patent application No. 202241032403 and filed on 06/06/2022. The entire collective teachings thereof are herein incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

[001] The present invention is in the technical field of tandem Quadrupole Mass Spectrometry, and particularly the present invention relates to a screening method to detect hemoglobinopathies by measuring the mass of the intact globin chains using electrospray tandem quadrupole mass spectrometry.

BACKGROUND OF THE INVENTION

[002] Hemoglobin is a tetrameric protein, which has four heme (iron and protoporphyrin) molecules bound to it. It is responsible for physiological oxygen transport. The major composition of normal human adult blood hemoglobin consists of two a chains and 2 P chains and normal fetal hemoglobin consists of two a chains and 2 y chains. A small % of 6 chains are also present. Hemoglobin disorders occur because of mutations, which include point mutations, insertions, and deletions in the globin chains. Hemoglobin disorders or hemoglobinopathies mainly arise either due to the disruption in the ratio of a and P chains present in the hemoglobin or due to the presence of structurally abnormal Hb corpuscles. The former results in Thalassemia and the later results in blood disorders such as sickle-cell anemia.

[003] Haematological parameters such as Hb, RBC count, Mean Corpuscular Volume (MCV), Mean Corpuscular Hb (MCH), peripheral blood smear does provide definite information about the hemoglobin status. But sophisticated techniques such as CE-HPLC and Capillary Electrophoresis (CE) are needed to detect Hb variants and relative quantitation of these variants. Relative expressions of the globin proteins present in the Hb are essential to detect hemoglobin disorders such as P thalassemia, sickle-cell anemia etc. The limited selectivity of CE-HPLC and CE techniques, necessitates the assistance of molecular analysis, in addition to the above said hyphenated- separation and quantitation techniques, to confirm the presence and extent of severity in cases of diseases like thalassemia, to arrive at the treatment regimen. Also, to arrive at a definite conclusion, several other clinical parameters and patient family history too should be considered before one can confirm any blood disorder or hemoglobinopathy.

[004] The current techniques to screen for hemoglobinopathies are HPLC and CE that exploit the differences in the physico-chemical properties of the functional hemoglobin protein (tetramers). HPLC separates Hb and variants based on the protein’s retention behavior onto the stationary phase of the column. The retention time of the protein onto the column, percentage of peak and peak shape is compared to the retention time present in the database to identify hemoglobinopathies.

[005] CE is an electrophoretic technique, where the charge difference of different variants of Hb migrate at different rates under an electric field. The migration of Hb is divided into 15 migration zones. Along with migration position, Hb variant percentage and characteristic CE pattern, form the basis for possible identification of hemoglobinopathies. The drawbacks associated with HPLC and CE includes the necessity of additional costly molecular techniques.

[006] Mass-spectrometry is a versatile technique that can be used to detect and confirm hemoglobinopathies with much accuracy and precision. But a high-resolution mass- spectrometric technique is needed for such measurements which cannot be used for mass-screening and routine lab-checkups.

[007] EP3186640 and WO20180224462 disclosed a rapid screening of whole blood samples, pin prick and blood spot cards, MALDI-ToF Mass spectrometry to detect hemoglobinopathies. W02020201734 disclosed a method of newborn screening ("NBS") comprising, directing ultrasonic energy or ultrasonic waves into a metabolite or analyte sample derived from a newborn, neonate or infant so as to cause a mist of charged sample droplets or sample ions to be ejected. The charged sample droplets or sample ions are then mass analysed and a determination is made as to whether or not one or more first metabolites or analytes indicative of a disorder or inborn error are present in the sample. W02006082389 disclosed a screening method to detect peptide variants by mass spectrometry. W02004090552 revealed a screening method using mass spectrometry, particularly where the ionisation technique produces a multiple-charged spectrum which is used to detect variant peptides, polypeptides and proteins that cause or indicative of disease. Ricardo j. Pais et al came out with a screening method for multiple blood abnormalities using an automated tool for MALDI-ToF spectrometry analysis. Yvonne A. Daniel et al discussed specific detection of clinically significant haemoglobinopathies using electrospray mass spectrometry-mass spectrometry. Jae-Seok Kim and others worked out on the diagnosis of hemoglobinopathy and P-thalassemia by 21-Tesla Fourier transform ion cyclotron resonance mass spectrometry. Yvonne Daniel et al worked out on a Newborn Sickle Cell Disease Screening Using Electrospray Tandem Mass Spectrometry. Yingying Wang et al developed a UPLC-MS/MS determination of linezolid and heme in plasma of infected patients and correlation analysis.

[008] The prior art revealed the use of highly sophisticated mass-spectrometry techniques that are costly and also the techniques dealt with digested globin or peptide variants to diagnose hemoglobinopathies which may not be sufficient enough to get at the true picture of hemoglobinopathy since they estimate certain targeted fragments.

[009] Moreover, these techniques needed highly skilled personnel and more time to arrive at the diagnosis. Keeping these aspects in mind, the current invention “A Method For Screening Hemoglobinopathies By Globin Chains Analysis Using Electrospray Tandem Quadrupole Mass Spectrometry” is taken up to come out with a cost-effective and less- time consuming, mass -screening method to detect hemoglobinopathies.

SUMMARY OF THE INVENTION

[010] The first aspect of the present invention provides a method for screening hemoglobinopathies in a blood sample by analyzing expression levels of intact globin chains of hemoglobin using electrospray tandem quadrupole mass spectrometry and a computing device configured to implement a relative ion quantification model. The method includes (i) ionizing the blood sample in a source region of the electrospray tandem quadrupole mass spectrometry, (ii) obtaining mass spectra comprising mass/charge ratios (m/z) of the intact globin chains by analyzing the blood sample ions; and (iii) computing a scoring function to relatively quantify expression levels of the intact globin chains using the computing device that implements the relative ion quantification model by (a) selecting at least three representative mass/charge ratios (m/z) of subunits of the intact globin chains in multiple reaction monitoring (MRM) mode by a third quadrupole, (b) summing ion intensities of the subunits of the selected intact globin chU"¹

subunits of the selected intact globin chains, (d) providing a score for each calculated ratio between 0-2, and (e) obtaining a score of 0-10 by summing at least five different calculated ratios, wherein the score of 0-4 is indicative of normal expression levels of the globin chains and a score of 5-10 is indicative of abnormal expression levels of the globin chains.

[Oi l] According to an embodiment, the method includes selectively detecting the at least three representative mass/charge ratio (m/z) of the subunits in the multiple reaction monitoring (MRM) mode with a collision energy of 3 to 10 eV.

[012] According to another embodiment, for each calculated ratio a reference range is set. Any value outside the reference range for the ratio is considered abnormal for the ratio.

[013] According to yet another embodiment, the score is obtained using the at least five different calculated ratios using a cumulative scoring method.

[014] According to yet another embodiment, the mass spectra comprise expression levels of the intact globin chains in their monomeric state. Each intact globin chain is the subunit of the Hemoglobin. The subunits are a, P, 6, Gy, Ay and modified P (HbS).

[015] According to yet another embodiment, the biological sample is Dried Blood spot (DBS), whole blood spotted onto Dried blood spot (DBS), or blood in EDTA.

[016] According to yet another embodiment, the blood sample is hemo lysed with water and diluted with an MS solvent comprising acetonitrile and acidified water.

[017] According to yet another embodiment, the third quadrupole scans the blood sample ions at unit resolution with a scan time of 1-10 mseconds.

[018] According to yet another embodiment, the hemoglobinopathies comprise Sickle Cell Disease (SCD), a-thalassemia, and P-thalassemia.

[019] The second aspect of the invention provides a system for screening hemoglobinopathies in a blood sample by analyzing expression levels of intact globin chains of hemoglobin, the system comprising an electrospray tandem quadrupole mass spectrometry and a computing device configured to implement a relative ion quantification model for performing a method. The method includes (i) ionizing the blood sample in a source region of the electro spray tandem quadrupole mass spectrometry, (ii) obtaining mass spectra comprising mass/charge ratios (m/z) of the intact globin chains by analyzing the blood sample ions, (iii) computing a scoring function to relatively quantify expression levels of the intact globin chains using the computing device that implements the relative ion quantification model by (a) selecting at least three representative mass/charge ratios (m/z) of subunits of the intact globin chains in multiple reaction monitoring (MRM) mode by a third quadrupole, (b) summing ion intensities of the subunits of the selected intact globin chains, (c) calculating ratios of the summed ion intensities between the subunits of the selected intact globin chains, (d) providing a score for each calculated ratio between 0-2, (e) obtaining a score of 0-10 by summing at least five different calculated ratios. The score of 0-4 is indicative of normal expression levels of the globin chains and a score of 5-10 is indicative of abnormal expression levels of the globin chains.

[020] Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.

BRIEF DESCRIPTION OF THE DRAWINGS

[021] Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below.

[022] FIGS. 1A-1B depicts MRM of representative globin chains from commercially available Human Hb and Sickle Hb (HbS), according to the aspects of the present invention;

[023] FIGS. 2A-2B depicts MRM of globin chains from commercially available reference standards, according to the aspects of the present invention;

[024] FIGS. 3A-3B illustrate flow diagrams of a method for screening hemoglobinopathies in a blood sample by analyzing expression levels of intact globin chains of hemoglobin using electrospray tandem quadrupole mass spectrometry and a computing device configured to implement a relative ion quantification model, according to the aspects of the present invention;

[025] FIG 4 is a block diagram illustrating the details of a computing device / digital processing system, according to the aspects of the present invention. [026] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

DETAILED DESCRIPTION OF THE INVENTION

[027] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[028] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.

[029] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

[030] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.

[031] Example embodiments of the present invention are described with reference to the accompanying figures.

[032] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

[033] DEFINITIONS [034] The term “hemoglobinopathies” refers to a group of disorders passed down through families (inherited) in which there is abnormal production or structure of the hemoglobin molecule.

[035] The term “sample” refers to any sample of a biological nature that requires analysis. For example, samples may include proteins or peptides extracted from tissues, body fluids, cells of an animal, plant, fungus, bacteria or other microorganisms. It can also include peptides that are chemically synthesised.

[036] The term "protein variant" refers to a member of a set of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences.

[037] The term “Electrospray ionization” refers to an Electrospray ionization (ESI) technique to generate ions for mass spectrometry using electro spray by applying a high voltage to a liquid to produce an aerosol.

[038] The term “multiple reaction monitoring (MRM)” refers to a highly specific and sensitive label-free technique for quantifying targeted protein/peptides.

[039] The term “quadrupole” refers to a component of the mass spectrometer responsible for selecting sample ions based on their mass-to-charge ratio (m/z).

[040] The term “mass spectrum” refers to the m/z ratios of the ions present in a sample plotted against their intensities.

[041] The term “collision cell” refers to a device used in mass spectrometry to remove interfering ions through ion/neutral reactions and to provide structural information of the molecules.

[042] The term “triple quadrupole mass spectrometer (TQMS)” refers to a tandem mass spectrometer made up of two quadrupole mass analyzers, with a (non-mass-resolving) radio frequency-only quadrupole between them, acting as a collision cell for collision- induced dissociation (CID) to fragment the selected precursors ions, and to generate fragment ions.

[043] The term “Cation-exchange high-performance liquid chromatography (CE-HPLC)” refers to a widely used laboratory test to detect variant hemoglobins for the diagnosis of thalassemia syndromes.

[044] The term “cumulative scoring” refers to a method of scoring whereby points or scores accumulated on individual items or subtests are tallied, and the higher the total sum, the higher the individual is presumed to be on the ability, trait, or other characteristic being measured. A. EMBODIMENTS OF THE INVENTION:

[045] The first aspect of the present invention provides a method for screening hemoglobinopathies in a blood sample by analyzing expression levels of intact globin chains of hemoglobin using electrospray tandem quadrupole mass spectrometry and a computing device configured to implement a relative ion quantification model. The method includes (i) ionizing the blood sample in a source region of the electrospray tandem quadrupole mass spectrometry, (ii) obtaining mass spectra comprising mass/charge ratios (m/z) of the intact globin chains by analyzing the blood sample ions; and (iii) computing a scoring function to relatively quantify expression levels of the intact globin chains using the computing device that implements the relative ion quantification model by (a) selecting at least three representative mass/charge ratios (m/z) of subunits of the intact globin chains in multiple reaction monitoring (MRM) mode by a third quadrupole, (b) summing ion intensities of the subunits of the selected intact globin chains, (c) calculating ratios of the summed ion intensities between the subunits of the selected intact globin chains, (d) providing a score for each calculated ratio between 0-2, and (e) obtaining a score of 0-10 by summing at least five different calculated ratios, wherein the score of 0-4 is indicative of normal expression levels of the globin chains and a score of 5-10 is indicative of abnormal expression levels of the globin chains.

[046] According to an embodiment, the method includes selectively detecting the at least three representative mass/charge ratio (m/z) of the subunits in the multiple reaction monitoring (MRM) mode with a collision energy of 3 to 10 eV.

[047] According to another embodiment, for each calculated ratio a reference range is set. Any value outside the reference range for the ratio is considered abnormal for the ratio.

[048] According to yet another embodiment, the score is obtained using the at least five different calculated ratios using a cumulative scoring method.

[049] According to yet another embodiment, the mass spectra comprise expression levels of the intact globin chains in their monomeric state. Each intact globin chain is the subunit of the Hemoglobin. The subunits are a, P, 6, Gy, Ay and modified P (HbS).

[050] According to yet another embodiment, the biological sample is Dried Blood spot (DBS), whole blood spotted onto Dried blood spot (DBS), or blood in EDTA.

[051] According to yet another embodiment, the blood sample is hemo lysed with water and diluted

[052] According to yet another embodiment, the third quadrupole scans the blood sample ions at unit resolution with a scan time of 1-10 mseconds.

[053] According to yet another embodiment, the hemoglobinopathies comprise Sickle Cell Disease (SCD), a-thalassemia, and P-thalassemia.

[054] The second aspect of the invention provides a system for screening hemoglobinopathies in a blood sample by analyzing expression levels of intact globin chains of hemoglobin, the system comprising an electrospray tandem quadrupole mass spectrometry and a computing device configured to implement a relative ion quantification model for performing a method. The method includes (i) ionizing the blood sample in a source region of the electro spray tandem quadrupole mass spectrometry, (ii) obtaining mass spectra comprising mass/charge ratios (m/z) of the intact globin chains by analyzing the blood sample ions, (iii) computing a scoring function to relatively quantify expression levels of the intact globin chains using the computing device that implements the relative ion quantification model by (a) selecting at least three representative mass/charge ratios (m/z) of subunits of the intact globin chains in multiple reaction monitoring (MRM) mode by a third quadrupole, (b) summing ion intensities of the subunits of the selected intact globin chains, (c) calculating ratios of the summed ion intensities between the subunits of the selected intact globin chains, (d) providing a score for each calculated ratio between 0-2, (e) obtaining a score of 0-10 by summing at least five different calculated ratios. The score of 0-4 is indicative of normal expression levels of the globin chains and a score of 5-10 is indicative of abnormal expression levels of the globin chains.

[055] B. METHODOLOGY:

[056] The invention provides a simple screening tool for hemoglobinopathies based on the intact mass to charge ratio (m/z) of the globin chains using low-resolution triple quadrupole mass spectrometry. The invention provides a relative quantification of various subunits which suggests abnormality in their expression levels, thereby supporting the current diagnosis of hemoglobinopathies using CE-HPLC and CE.

[057] The mass spectrometer (MS) provides a mass spectrum of all the intact globin chains. At least three representative m/z of individual globin chains are selectively monitored / detected in the MS by a method called multiple reaction monitoring (MRM) with a collision energy of 3 to 10 eV. The ion intensities of the subunits of the selected intact globin chains are summed. Ratios of the summed ion intensities between the subunits of the selected intact globin chains are calculated. At least five different ratios were used and scored for individual ratios. A cumulative scoring system was devised with a score of 0-10, 0 being indicative of normal and 10 being indicative of abnormal synthesis of globin chains. A full scan analysis may be acquired simultaneously to identify any 5 known variants that differ > lODa from the wild type other than the clinically significant variants of Hb (HbS). The ratio is applicable to ages greater than one year and no differences between genders. The interpretation serves as a screening tool for hemoglobinopathies based on normal or abnormal expression of globin chains. In FIGS.1A-1B, m/z ratio of one peak for each protein is shown. Six subunits of the Hb 10 were scanned by quadrupole (QI), namely, a, P, 6,^Gy,^Ay and ^sp and detected by quadrupole (Q3)Two more similar sets at different m/z are measured. The intensity of the peak in FIG. 1A is that of the standard Human Hb whereas the peak in FIG. IB is that of the Hb Sickle as represented by the intensity of 932.4m/z which is absent in the normal sample.

15 [058] Table 1. Ratio, individual scoring, cumulative scoring, INDEX and interpretation of the Hb standards (Human Hb and Hb Sickle). Hb is considered normal and Hb Sickle is considered abnormal.

[059] To gain more confidence in the analysis, due to non-availability of quality control 20 samples for MS, a particular process was followed involving injections of blank, process blank, reference standards with different injection volumes, before injection of any samples.

[060] In FIGS.2A-2B, m/z ratio of one peak for each protein is shown. Each peak indicates a subunit of the globin chain according to embodiments of the present invention. The intensity of 6 chain is slightly higher in P thal when compared to §P thal Two more similar sets at different m/z are measured. The intensity of the peak in FIG. 2 A is P thal and §P thal in FIG. 2B. Though the peaks are similar visually, there is difference when calculating the intensity of the peaks and ratios of different proteins according to the 5 method of the present invention.

[061] Table 2: Ratio, individual scoring, cumulative scoring, INDEX and interpretation of the reference standards (P thal and §P thal) and few abnormal samples eg., P thal, §P thal, HbS and a thal.

[062] INDEX 0-4 indicates normal synthesis and 5-10 indicates abnormal synthesis of globin chains.

[063] In FIGS. 3A-3B, the method includes ionizing the blood sample in a source region of the electrospray tandem quadrupole mass spectrometry at step 302, obtaining mass 5 spectra comprising mass/charge ratios (m/z) of the intact globin chains by analyzing the blood sample ions at step 304 and computing a scoring function to relatively quantify expression levels of the intact globin chains using the computing device that implements the relative ion quantification model at step 306 by (a) selecting at least three representative nr""'-'¹ - reaction monitoring (MRM) mode by a third quadrupole, (b) summing ion intensities of the subunits of the selected intact globin chains, (c) calculating ratios of the summed ion intensities between the subunits of the selected intact globin chains, (d) providing a score for each calculated ratio between 0-2, (d) obtaining a score of 0-10 by summing at least five different calculated ratios, wherein the score of 0-4 is indicative of normal expression levels of the globin chains and a score of 5-10 is indicative of abnormal expression levels of the globin chains.

[064] The present invention is illustrated in further details by the following non-limiting examples.

[065] C. EXAMPLE EMBODIMENTS:

[066] The method includes measuring m/z ratio of the major subunits of globin chains viz., a, P, 6,^Gy,^Ay and modified P chain. ^Gy,^Ay both are y chains that differ by one aminoacid. SP is a mutated P chain that results in sickle cell anemia. The mass spectra of the six subunits (i.e.) a, P, 6,^Gy,^Ay and modified P chain are obtained with three peaks for each subunit. Intensities of 18 m/z mass spectral peaks are obtained in total. The intensities of 3 peaks of a subunit are summed to obtain a total of 6 intensities. Ratio is calculated between the summed intensities of subunits, (i.e.) summed intensity of subunit 1/ summed intensity of subunit 2 or summed intensity of subunit 1/ summed intensity of subunit 3 or summed intensity of subunit 1/ summed intensity of subunit 4+summed intensity of subunit 5. A range is set for each ratio. If the value is within the range, a score of 0 is given. If values are outside the range, a score of 1 or 2 is given. The scores of all 5 ratios (cumulative score) are summed to provide the INDEX. The index with a value of 0-4 is normal and 5-10 is considered abnormal.

[067] The current invention is a cost-effective method, which provides information on the mass to charge (m/z) ratio of intact subunits of Hb. The ratio of subunits of hemoglobin aids in the diagnosis of different disorders of hemoglobin. At least five ratios are used to create a multi- scoring system. There is no requirement to purchase any kits for the analysis. Minimal sample preparation, less instrument time, analysis that can be automated, and an objective interpretation are the hallmarks of the invention.

[068] An alternate two-tier method is proposed which provides mass to charge ratio of various subunits of hemoglobin using MS. MRM method scans for select ions and provides ion intensity while the full scan analysis of the globin chains provide information on the mass of the intact proteins upon deconvolution. The method includes measuring m/z in the range 600-1500 and the deconvolution is performed to arrive at the mass of the globin chain.

[069] Mass spectra of Hb provide information about the expressed proteins in their monomeric state.

[070] The instant invention discloses a new method to check the performance of ESIMS in the absence of proper calibrant or reference standards before the injection of the samples.

[071] The invention provides information about the expression of globin chains, which provides a simple screening method to identify abnormal synthesis of Hb.

[072] The invention can quickly identify possible sickle cell disorders.

[073] The invention can screen unknown Hb variants with improved selectivity.

[074] The invention can be optimized to screen newborns for possible sickle cell disorders and thalassemia.

[075] Minimal amount of sample (whole blood or dried blood spot) is required.

[076] Minimal sample preparation, simple method and hence can be broadly applied to other laboratories.

[077] The invention can be used for population screening of hemoglobinopathies.

[078] It is a cost-effective, high throughput screening tool for identifying hemoglobinopathies.

[079] D. HARDWARE

[080] Computing device / Digital Processing System

[081] FIG 4 is a block diagram illustrating the details of a computing device / digital processing system in which various aspects of the present invention are operative by execution of appropriate execution modules, firmware or hardware components.

[082] Digital processing system may correspond to each of user system: local system or remote and server noted above. Digital processing system may contain one or more processors (such as a central processing unit (CPU)), random access memory (RAM), secondary memory, graphics controller (GPU), primary display unit, network interfaces like (WLAN), and input interfaces (not shown).

[083] CPU executes instructions stored in RAM to provide several features of the present invention. CPU may contain multiple processing units, with each processing unit potentially being designed for a specific task. Alternatively, CPU may contain only a single general purpose processing unit. RAM may receive instructions from secondary/system memory. [084] Graphics controller (GPU) generates display signals (e.g., in RGB format) to primary display unit based on data/instructions received from CPU. Primary display unit contains a display screen (e.g. monitor, touchscreen) to display the images defined by the display signals. Input interfaces may correspond to a keyboard, a pointing device (e.g., touch-pad, mouse), a touchscreen, etc. which enable the various inputs to be provided. Network interface provides connectivity to a network (e.g., using Internet Protocol), and may be used to communicate with other connected systems. Network interface may provide such connectivity over a wire (in the case of TCP/IP based communication) or wirelessly (in the case of WIFI, Bluetooth based communication).

[085] Secondary memory may contain hard drive (mass storage), flash memory, and removable storage drive. Secondary memory may store the data (e.g., the specific requests sent, the responses received, etc.) and executable modules, which enable the digital processing system to provide several features in accordance with the present invention.

[086] Some or all of the data and instructions may be provided on a removable storage unit (SD card), and the data and instructions may be read and provided by removable storage drive to CPU. Floppy drive, magnetic tape drive, CD-ROM drive, DVD Drive, Flash memory, removable memory chip (PCMCIA Card, EPROM) are other examples of such removable storage drive.

[087] Removable storage unit may be implemented using storage format compatible with removable storage drive such that removable storage drive can read the data and instructions. Thus, removable storage unit includes a computer readable storage medium having stored therein executable modules and/or data. However, the computer (or machine, in general) readable storage medium can be in other forms (e.g., non-removable, random access, etc.). CPU may retrieve the executable modules, and execute them to provide various features of the present invention described above.

[088] ADVANTAGES OF THE INVENTION OVER EXISTING TECHNOLOGY

[089] Less volume (few drops of blood) of sample is required for analysis.

[090] Minimal sample preparation time is required.

[091] Direct sample introduction (Flow Injection Analysis, FIA mode) without the use of LC.

[092] No requirements for any commercial kits.

[093] Data acquisition time is limited to one minute per sample.

[094] Simple calculations using excel to interpret the results. [095] Does not require an expert for analysis and interpretation.

[096] Sample type can be whole blood in EDTA tubes or in dried blood spot.

[097] DBS provides easy storage and transport of samples between laboratories and/or from collection center to the lab.

[098] USES, APPLICATIONS AND BENEFITS OF THE INVENTION

[099] As will be appreciated by a person skilled in the art the present invention provides following advantages:

[0100] The invention can be easily translated into clinical laboratories.

[0101] The invention can be introduced in clinical labs as an alternate supporting method to Complete Blood Count (CBC) and/or CE.

[0102] A quick method to perform population-level screening to identify people at risk to provide counseling.

[0103] The method can be optimized to screen for sickle cell disorders in newborn babies and adults.

[0104] The invention can screen for unknown variants that must be correlated with clinical findings and supported with other diagnostic tests.

[0105] A new method to check the performance of ESIMS in the absence of calibrant or reference materials can be applied to other areas of research, in academia and industries.

[0106] BEST MODE TO PRACTICE

[0107] Best mode to practice the invention is through commercialization of this invention through the distributors, e-commerce sites, and marketing.

[0108] It will be available to use after IP generation and regulatory clearance. The applicant will collaborate with the industry partner for manufacturing and marketing of our product at reasonable cost. It may be available in the national and international market. The product may be launched in various designs & sizes.

[0109] Merely for illustration, only representative number/type of graph, chart, block, and sub-block diagrams were shown. Many environments often contain many more block and sub-block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.

[0110] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles. [0111] While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

[0112] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0113] It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.

[0114] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMS What is claimed is:

1. A method for screening hemoglobinopathies in a blood sample by analyzing expression levels of intact globin chains of hemoglobin using electrospray tandem quadrupole mass spectrometry and a computing device configured to implement a relative ion quantification model, wherein the method comprises:

(i) ionizing the blood sample in a source region of the electrospray tandem quadrupole mass spectrometry;

(ii) obtaining mass spectra comprising mass/charge ratios (m/z) of the intact globin chains by analyzing the blood sample ions; and

(iii) computing a scoring function to relatively quantify expression levels of the intact globin chains using the computing device that implements the relative ion quantification model by a. selecting at least three representative mass/charge ratios (m/z) of subunits of the intact globin chains in multiple reaction monitoring (MRM) mode by a third quadrupole; b. summing ion intensities of the subunits of the selected intact globin chains; c. calculating ratios of the summed ion intensities between the subunits of the selected intact globin chains; d. providing a score for each calculated ratio between 0-2; and e. obtaining a score of 0-10 by summing at least five different calculated ratios, wherein the score of 0-4 is indicative of normal expression levels of the globin chains and a score of 5-10 is indicative of abnormal expression levels of the globin chains.

2. The method of claim 1, wherein the method includes selectively detecting the at least three representative mass/charge ratio (m/z) of the subunits in the multiple reaction monitoring (MRM) mode with a collision energy of 3 to 10 eV.

3. The method of claim 1, wherein for each calculated ratio a reference range is set, wherein any value outside the reference range for the ratio is considered abnormal for the ratio.

4. The method of claim 1 , wherein the score is obtained using the at least five different calculated ratios using a cumulative scoring method.

5. The method of claim 1 , wherein the mass spectra comprise expression levels of the intact globin chains in their monomeric state, wherein each intact globin chain is the subunit of the Hemoglobin, wherein the subunits are a, P, 5, Gy, Ay and modified P (HbS).

6. The method of claim 1, wherein the biological sample is Dried Blood spot (DBS), whole blood spotted onto Dried blood spot (DBS), or blood in EDTA.

7. The method of claim 1, wherein the blood sample is hemolysed with water and diluted with an MS solvent comprising acetonitrile and acidified water.

8. The method of claim 1, wherein the third quadrupole scans the blood sample ions at unit resolution with a scan time of 1-10 mseconds.

9. The method of claim 1, wherein the hemoglobinopathies comprise Sickle Cell Disease (SCD), a-thalassemia, and P-thalassemia.

10. A system for screening hemoglobinopathies in a blood sample by analyzing expression levels of intact globin chains of hemoglobin, the system comprising an electrospray tandem quadrupole mass spectrometry and a computing device configured to implement a relative ion quantification model for performing a method, the method comprising: