Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
  • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
  • G01N33/721—Haemoglobin

Definitions

• the method describes rapid screening of whole blood samples, pin prick and blood spot cards, subjected to MALDI - ToF Mass spectrometry following:
• the spectra is generated using preferably sinapinic acid as matrix and characteristic spectra are indicative of the presence of an hemoglobinopathy and can screen/diagnose all sickle cell diseases, alpha and beta Thalassemias.
• Hemoglobinopathies are the largest group of inherited human monogenic disorders worldwide and are due to mutations in one or more of the genes coding for the globin proteins that form the ⁇ 2 ⁇ 2 hetero-tetrameric complex that binds oxygen in red blood cells - hemoglobin.
• the resulting structural alteration in the patients (Hb) can be clinically mild, even occult, to being the cause of severe chronic morbidity and even neonatal death.
• hemoglobinopathies are the most common genetic diseases; with reported carrier rates of 1-11 % for ⁇ -thalassaemia, 1-58 % for a-thalassaemia and 0.3-30 % for sickle cell trait.
• the most common hemoglobinopathies are sickle cell trait, Hb D and Hb E. It was estimated that, in the year 2000, with a population of 1000 million and a birth rate of 25 per thousand, there would be about 45 million carriers and about 15,000 infants born in India each year with hemoglobinopathies.
• Hemoglobin itself should be a heterotetrameric complex containing two of alpha gene globin protein products associated with two beta gene globin proteins.
• human ⁇ -globin locus is composed of five genes located on a short region of chromosome 11, responsible for the creation of the beta parts of Hemoglobin. This locus contains not only the gene for the classic expressed beta ( ⁇ ) globin gene but also delta ( ⁇ ), gamma-A (Ay), gamma-G (Gy), and epsilon globin (£).
• the human a- globin gene cluster is found on chromosome 16 and contain expressed globin genes Alpha 1 (ctl) and Alpha 2 (ct2) and Zeta ( ⁇ ) (see figure 1) but a new a-globin gene has recently been identified termed Hb Mu ( ⁇ ).
• HbF ⁇ 2 ⁇ 2
• HbA2 ⁇ 2 (HbA2) ⁇ 2 ⁇ 2 (HbA2) and trace amounts of HbF (see figure 1).
• the various hemoglobinopathies identified were named as characteristics of the hetero-tetrameric hemoglobin complex rather than the feature of an isolated globin proteins.
• the hemoglobin Hb S is where globin gene ⁇ has a specific amino acid change mutation 6Glu-Val at position 6 of the ⁇ globin protein and is termed 5 ⁇ ; and the heterotetrameric Hb is ct 2 5 ⁇ Sickle cell trait being a complex of normal ⁇ and 5 ⁇ within the red cells constituent hemoglobin molecules as a result of hetrozygosity and is often unnoticed.
• Hemoglobin C is a structural variant of normal hemoglobin (HbA) caused by an amino acid substitution at position 6 of the ⁇ -globin chain ⁇ 6Glu-Lys). It is one of the most prevalent abnormal hemoglobin mutations globally alongside hemoglobin S, which occurs at the same position (HbS; ⁇ - ⁇ ), and hemoglobin E (HbE, ⁇ 2661 ⁇ - ⁇ ). In HbC heterozygote individuals (AC), this trait is asymptomatic. Homozygosity (CC) causes clinically mild haemolytic anaemia, due to the reduced solubility of the red blood cells which can lead to crystal formation.
• HbC is mainly of clinical significance when inherited in combination with HbS (sickle-hemoglobin C disease).
• the Thalassemias arise because of a mutation that prevents the expression of either the ⁇ -globin gene ( ⁇ -thalassemia) or the alpha-globin genes (a-thalassemia).
• ⁇ -thalassemia the ⁇ -globin gene
• a-thalassemia the alpha-globin genes
• ⁇ -Thalassemia there is reduced synthesis of ⁇ globin ⁇ -thalassemia
• °-thalassaemia Clinically mild forms of ⁇ - thalassaemia are called thalassemia intermedia.
• tetrameric-a hemoglobin is structurally unstable, with a tendency to denature upon oxidation, filling the cytoplasm and cell membrane with precipitated ⁇ -globin chains, free heme, porphyrins and iron, which further propagate ROS production.
• Erythroid cells have proteolytic pathways to degrade excess free a- globin, but these pathways can be overwhelmed.
• Cis-type a 0 thalassemia trait tends to be found in individuals of Asian descent, while trans-type a + thalassemia tends to run in individuals of African descent.
• a mother is a carrier of a 0 thai assemia, her pregnancy is at risk for Bart's hydrops foetalis syndrome, while the worst possible outcome of a pregnancy of a mother with a + thalassemia is a much milder condition, Hb-H disease.
• HbH is not stable and thermally labile, patients are anaemic and there is splenomegaly.
• Hemoglobin Constant Spring mutation An unusual case of the silent carrier state is the individual who carries the Hemoglobin Constant Spring mutation. This is an abnormal elongated a- globin due to a termination codon mutation. Individuals who have this mutation have normal red blood cell indices, but can have children who have HbH-Constant Spring disease if the other parent has a- thalassemia trait.
• the physicochemical characteristics and molecular genetic of specific globin proteins/genes is the bases of the molecular testing for the hemoglobinopathies.
• the molecular masses of a globin is 15,128 and ⁇ globin 15,868; a difference of some 740 da.
• homo tetrameric complexes of Hb and their hemes (60,000 to 68,000 Da) and derivative trimers (approx. 47,500 Da) and dimers (30,000 to 35,000 Da), of a or ⁇ globin will vary in mass by 2960, 2220 and 1480 Da respectively.
• mass resolutions are within the scope of MALDI-ToF mass spectrometry.
• MALDI-Tof mass spectrometry dissociates such hemoglobin complexes into the free monomers.
• Formalin treatment usually "fixes" a protein by forming internal crosslinks via lysine residue side chains forming a methyl bridge with a free hydrogen of an adjacent amide bond.
• formalin will only crosslink between proteins if they are naturally in very close proximity to each other and the correct amino acid side groups align in close proximity. This is the case for the tetrameric complex of Hemoglobin.
• a formalin solution such as citrate buffered formal saline will "fix" the Hb complex such that it no longer dissociated and is resolved by MALDI-ToF MS as characteristic m/z of the tetra, tri and dimeric complexes.
• MALDI-ToF MS mass pattern characteristic of any particular thalassemia can then be mapped and used as a diagnostic/screening tool for Thalassaemias as described here.
• the present application provides a method of detecting a hemoglobinopathy comprising subjecting a blood sample obtained from a subject to direct mass spectral analysis.
• Direct mass spectral analysis means that the data generated from the mass spectral analysis is used in the method, and not the inferred mass of the components present in the sample.
• Hemoglobinopathy refers to any condition caused by a genetic mutation which results in the abnormal expression or structure of one of the globin molecules in the hemoglobin molecule.
• hemoglobinopathies include, but are not limited to Sickle cell anaemia, a-thalassemia, ⁇ -thalassemia, HbAG, HbA /Enfield, HbH, HbAF, HbS, HbC, HbE, HbD-Punjab, HbO- Arab, HbG-Philadelphia, Hb Constant Spring, Hb Hasharon, Hb Korle-Bu, Hb Lepore, HbM, and Hb Kansas.
• Preferred hemoglobinopathies are sickle cell anaemia, a-thalassemia, ⁇ -thalassemia, HbC, HbE, HbAG, HbA/Enfield, HbH, and HbAF.
• the blood sample can be a whole blood sample collected using conventional phlebotomy methods.
• the sample can be obtained through venipuncture or as a pin prick sample, such as a finger stick or heel prick.
• the blood sample may be a dried blood spot captured on filter paper or other suitable blood spot capture material.
• the blood sample can be an untreated sample.
• the blood sample may be diluted or processed (concentrated, filtered, etc.).
• the blood sample is preferably mixed with either a lysing agent to lyse the red blood cells or initially a crosslinking agent such as citrated formalin for up to 24 hours prior to lysis.
• a lysing agent to lyse the red blood cells or initially a crosslinking agent such as citrated formalin for up to 24 hours prior to lysis.
• the lysing agent can be mixed with the sample at a suitable concentration such as 1/1 (i.e. 1 part blood to 1 part lysing agent), 1/5, 1/10 or 1/20 or greater.
• the blood sample is a dried blood spot
• the blood spot capture material on which the sample is dried can be placed in either a lysing agent or citrated formalin to reconstitute the sample.
• the blood spot can be reconstituted in a suitable buffer prior to formalin fixation followed by lysis or direct lysis.
• Suitable buffers and other protein crosslinking agents are known to the skilled person.
• a preferred lysing agent is deion
• the preferred crosslinking agent is citrate buffered formal saline.
• the sample is preferably mixed with the formalin containing agent at a concentration of 1/5 (i.e. 1 part blood to 5 part citrated formal saline).
• the sample is allowed to react for a suitable period to allow the red blood cells' globin molecules to become fixed.
• the mixture can be left for 4, 5, 6, 7, 8, 10, 12, 16, 20, 24 hours or more.
• the sample is preferably left for a minimum of 6 hours
• the blood sample is diluted, preferably after lysis.
• the dilution step effectively purifies the Hb from other components of blood for mass spectral analysis as Hb is the most abundant protein.
• the blood sample may be diluted 1/10 (i.e. one part sample in 10 parts diluent), 1/166, 1/333, 1/500, 1/1000, 1/2000, 1/2500, 1/8000 or more. Most preferably the sample is diluted 1/2000 i.e. one part blood sample in 2000 parts diluent.
• the diluent is 0.1%
• the blood sample is not processed between fixation, lysis and dilution.
• the blood sample is only lysed and diluted ;or fixed ,lysed and diluted.
• processing includes concentrating the proteins of interest e.g. Hb; isolating Hb for example by HPLC or treatment with a chemical agent to disrupt or break intramolecular bonds.
• the sample is preferably not treated with a reducing agent. More preferably the sample is not treated with dithiothrietol (DTT).
• DTT dithiothrietol
• the method may comprise comparing the spectra patterns resulting from said direct mass spectral analysis of a sample to mass spectral patterns obtained from direct mass spectral analysis of a blood sample from a normal healthy control to determine whether said patterns from said sample are indicative of a hemoglobinopathy.
• a "normal healthy control” is a subject who does not have a hemoglobinopathy.
• the differences in the patterns of mass spectra are determined by an automated quantitative method that can distinguish between a mass spectrum of a blood sample from a normal healthy control and the mass spectral pattern of a blood sample from a subject with a hemoglobinopathy.
• an "automated quantitative method” refers to the processing of the direct output data from a mass spectrometer to which the sample was subjected by a computer software program.
• Y axis in these spectra is an indicator of "relative strength" of mass peak within the spectra, but not between mass peaks in one sample versus another sample.
• normalisation needs to render Y axis value comparable between sample spectra.
• the spectra obtained from the direct mass spectral analysis is preferably normalised.
• the spectra is subjected to data processing which results in a normalised statistically determined index of relative proportion of mass spectra. This converts the qualitative mass spectra into a quantitative value. Normalization is the process of producing a data structure to reduce repetition and inconsistencies of data.
• Typical normalisation methods include percentage of total area at a given point, Square difference and ratio of differences. The percentage difference is calculated as
• Percentage difference (Yl-Yref/ Y ref X 100%) Wherein Y ref is the minimum Y value of the spectra, and Yl is Y value for each point.
• the ratio difference is calculated as
• Ratio Difference ( Ratio 1-Ratio 2) .
• the spectral model is created by a method of data processing which results in a normalised statistically determined index of relative proportion of mass spectra within a set range. This renders all spectra comparable such that the median and centile variability at any given mass value can be modelled.
• the range is between about 6,000-700,000 m/z.
• the range examined is 6,000 - 17000 m/z, more preferably 7,500-16,200m/z.
• the single charged and/or double charged molecules of red cell globins can be measured.
• the spectra at the mass/charge range of 15000 m/z to 16200 m/z is examined.
• the range examined is 30,000 - 700,000 m/z
• a normalised statistically determined index of relative proportion of mass spectra within a given range can be calculated from using the total area under the curve of mass spectra. This can then be used to calculate the relative intensity.
• the area under the curve of mass spectra is calculated by dividing the mass spectra into a plurality of bins of a given number of m/z.
• Bin has its usual statistical meaning, for example, of being one of a series of ranges of numerical value into which data are sorted in statistical analysis.
• the bins can be 100m/z, 50m/z, 25m/z, lOm/z or 5m/z in size. The smaller the size of the bin used, the more refined the method.
• the relative intensity (Y Axis value) can be calculated by the "square of difference” method and therefore a comparable Y value given for every bin.
• the minimum Y value of the spectra (Y ref) was subtracted from the Y value at every bin and the difference was squared.
• the formula used to calculate square of difference (yl-yref) 2 and the calculated square of difference was then named as "relative intensity”.
• the relative intensity at each mass bin in a sample can be captured using commercially available statistical tests such as MATLAB ® , Stats DirectTM and Origin 8TM.
• the levels of the globins present can be determined by measuring the relative height of the peaks corresponding to the various globins.
• the range is between about 6,000-700,000 m/z.
• the range examined is 5,000-8,200m/z, preferably 6,000 - 17000 m/z, more preferably 7,500-16,200m/z.
• the spectra at the mass/charge range of 5,000-8,200m/z or 6000 to 8100m/z, more preferably 7550 to 8100 m/z or 7,550 to 8,200 m/z is examined.
• the spectra obtained from the direct mass spectral analysis of the sample is compared to a reference spectral model of expected mass between about 6,000-700,000 m/z or 6,000-
• the "reference spectral model” is the expected mass within a set range, determined from statistical analysis of a collection of blood samples from normal healthy controls.
• the range is between about 6,000-700,000 m/z.
• the range examined is 6,000 - 17000 m/z, preferably 7,500-16,200m/z, more preferably 5,000-8,200 m/z .
• the spectra at the mass/charge range of 5,000-8,200m/z or 6000 to 8100m/z, more preferably 7550 to 8100 m/z or 7,550 to 8,200 m/z is examined.
• the spectral model of expected mass between about 5,000-8,200m/z or 6,000 -8,100 m/z is determined from statistical analysis of a collection of blood samples from normal healthy controls.
• the spectral model is created by a method of data processing which results in a normalised statistically determined index of relative proportion of mass spectra within a set range. This renders all spectra comparable such that the median and centile variability at any given mass value can be modelled.
• the range is between about 6,000-700,000 m/z. More preferably the range is 5,000-8,200m/z or 6000 to 8100m/z, most preferably 7550 to 8100 m/z or 7,550 to 8,200 m/z.
• the spectra obtained from the direct mass spectral analysis of the sample is compared to a disease model, of expected mass between about 6,000-300,000 m/z determined from statistical analysis of a collection blood samples from subjects with a hemoglobinopathy.
• a parallel "disease" model is generated from normalised statistically determined index of relative proportion of mass spectra within a set range is created from blood samples obtained from a subject known to have a hemoglobinopathy.
• the range is between about 6,000- 700,000 m/z. More preferably the range is 5,000-8,200m/z or 6000 to 8100m/z, most preferably 7550 to 8100 m/z or 7,550 to 8,200 m/z.
• the spectral value of samples obtained from normal healthy controls and those from subjects suffering from a hemoglobinopathy are compared.
• the presence of a hemoglobinopathy causes a change in the pattern of the peaks in the normalised spectra, due to a shift in mass caused by the mutation within the globin proteins.
• Subjects with sickle cell anaemia have a peak for S at 7920 m/z clearly resolved from ⁇ -globin at 7934 m/z and 0 approx. 7933 m/z. Thus the presence of a peak for S at 7920 m/z is indicative of sickle cell anaemia.
• HbAG there is a mutation at a68Asn-Lys. This causes additional peaks at 7612 and 7645 m/z that may represent fetal alpha- globins ⁇ and the newly discovered ⁇ . There may also be an elevation of Gy at 7993 m/z. Thus the presence of peaks at 7612 and 7645 m/z, optionally together with an elevation of Gy at 7993 m/z can be indicative of HbAG.
• HbA/Enfield is a mutation of a89His-Glu. Subjects with this condition have additional peaks at 7612 and 7645 m/z that may represent fetal alpha- globins ⁇ and the newly discovered ⁇ -globin. There may also be an elevation of ⁇ at 7963 and Gy at 7993 m/z. Thus the presence of peaks at 7612 and 7645 m/z, optionally together with an elevation of ⁇ at 7963 and Gy at 7993 m/z can be indicative of HbA/Enfield.
• the ratio of normalized spectral value of samples obtained from normal healthy controls and those from subjects suffering from a hemoglobinopathy can be compared and statistical analysis carried out so that various measures e.g. mean, standard deviation, skewness, upper and lower quartile, median, kurtosis as well as 95th and 5th centile can be calculated.
• the difference in relative intensity at each mass bin between samples obtained from normal healthy controls and those from subjects suffering from a hemoglobinopathy can be captured using commercially available statistical tests such as MATLAB ® , Stats DirectTM and Origin 8TM.
• the reference spectral model and the disease model are then compared by plotting in order to identify 'hot spots' i.e. points of difference between the two models. This may be a decrease or increase in the size of a peak, or the appearance of a peak.
• the points of difference can then be used to determine the presence of a hemoglobinopathy. Preferably this is done by using a suitable algorithm.
• the analysis of the mass spectra can be easily calculated using a suitable computer software program.
• a computer can also be programmed with the suitable algorithm in order to provide an indication of the presence of a hemoglobinopathy.
• the mass spectral analysis carried out is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS).
• MALDI-ToF MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
• the relative percentage abundance of the tetramers, pentamers and trimer present compared to the dimers can be utilised to diagnose the presence of a hemoglobinopathy.
• tetramer There are two forms of tetramer present - the S-tetramer which contain only the globin proteins and the L-tetramer which contains the heme as well as the globins.
• Also described is a method of detecting a hemoglobinopathy comprising:
• the spectra patterns resulting from the analysis are compared to mass spectral patterns obtained from a blood sample from a normal healthy control.
• the verb "comprise” has its normal dictionary meaning, to denote nonexclusive inclusion. That is, use of the word “comprise” (or any of its derivatives) to include one feature or more, does not exclude the possibility of also including further features.
• the word “preferable” (or any of its derivates) indicates one feature or more that is preferred but not essential.
• Figure 1 shows a Simplified Diagrammatic representation of the functionally expressed Beta and Alpha-globin genes found in humans (omitted are the known pseudogenes within the gene clusters).
• Figure 2 shows a spectra of HbA whole blood
• FIG. 3 shows a spectra of HbAS
• FIG. 4 shows a spectra of HbAC
• FIG. 5 shows a spectra of HbAC
• FIG. 7 shows a spectra of HbF
• FIG. 8 shows a spectra of HbH
• Figure 9 shows a spectra of HbAG
• Figure 10 shows a spectra of HbA/Kenya
• Figure 11 shows tabulated Comparison of spectra for phenotypic normal and abnormal samples
• Figure 12 shows the mass spectral pattern between 30,000 to 70,000 m/z of formalin fixed purified HbA and formalin fixed whole blood HbA
• Figure 13 shows a spectra of formalin fixed Sickle cell disease (SSD) blood sample
• Figure 14 shows tabulated Comparison of formalin fixed spectra for phenotypic normal and abnormal samples
• the optimal dilution for whole blood is between 1/1000 and 1/2000 in either ddH 2 0 or 0.1% TFA in ddH20 after an initial lysis of sample with ddH 2 0 (1:1 v/v).
• This dilutional step effectively purifies the Hb from other components of blood for mass spectral analysis as Hb is the most abundant protein.
• the dilution in ddH20 (or 0.1% TFA/ddH2O) dissociates the constituent globin proteins for resolved analysis by MALDI-ToF Mass spectrometry.
• the optimal matrices are sinnapinic acid (SA), ferulic acid (FA) and alpha 4- cyano
• hydroxycinnamic acid (CHCA).
• Sinapinic acid being the preferred matrix, is mixed or used as pre - coating layer to a mixed drop of 1/1000 to 1/8000 diluted sample (optimal 1/2000).
• the ions were accelerated by a 20 kV electrical field down a 1.2 m linear tube and detected by a micro-channel plate detector at a sampling rate of 500 MHz. Spectra were generated by summing 20-30 laser shots. A positive linear mode with delayed extraction was used in order to acquire the spectra.
• HbAS sickle cell trait
• Heriditary Persistant fetal hemoglobin blood sample, HbAF showed a single normal and SA adducted ⁇ -globin peaks.
• the intensity of ⁇ -globin at 7934 m/z was markedly reduced and Ay at 8005 mz was markedly elevated.
• Baseline elevation of ⁇ and Gy globins at 7965 and 7996 m/z was also seen,
• Blood sample from HbH revealed a peak showed a single normal and SA adducted ⁇ -globin peaks at 7564 and 7667 m/z.
• ⁇ -globin was evident at 7934 m/z and baseline elevation of Gy and Ay globins at 7996 & 8017 m/z was evident.
• HbAG is caused by the mutation in a globin at a68Asn-Lys. An a-globin was seen at 7564 m/z, SA adducted a at 7667 m/z but additional peaks at 7612 (unknown) and 7645 m/z (glycated a-globin) were seen.
• the ⁇ globin at 7933 m/z was accompanied with an elevation of Gy at 7993 m/z HbA/Kenya phenotype is a a fusion of Gy and ⁇ showed similarly An ⁇ -globin was seen at 7564 m/z, SA adducted a at 7667 m/z but additional peaks at 7961 m/z, coinciding with the peak for ⁇ at 7963 m/z, probably represent the fusion ⁇ globin.
• the ⁇ globin at 7933 m/z was accompanied with an elevation of Gy at 7993 m/z.
• a further 9 phenotypically normal blood samples and 9 phenotypically abnormal blood samples were examined.
• a feature of all hemoglobinopathies and carriers is elevated proportions of ⁇ -globin, ⁇ -(G and A) globins and marker m/z 8088 (possibly ⁇ -globin), compared to non-affected individuals.
• beta-thalassemia and beta-thalassemia trait samples were characterized by elevated aberrant peaks at 8088 m/z (possible ⁇ -globin), moderate elevated ⁇ -globins and reduced ⁇ / ⁇ globin ratio. This pattern was similar for HbE disease but a more marked elevation of ⁇ -globin was seen.
• the optimal matrices are sinnapinic acid (SA), ferulic acid (FA) and alpha 4- cyano
• hydroxycinnamic acid (CHCA).
• Sinapinic acid being the preferred matrix is mixed or used as pre - coating layer to a mixed drop of 1/1000 to 1/8000 diluted sample (optimal 1/2000).
• the ions were accelerated by a 20 kV electrical field down a 1.2 m linear tube and detected by a micro-channel plate detector at a sampling rate of 500 MHz.
• Spectra were generated by summing 20-30 laser shots.
• a positive linear mode with delayed extraction was used in order to acquire the spectra.
• a mass spectral region of between 30,000 and 70,000m/z was collected and analysed. These are characterised both in respect to centroid mass assignment and relative peak intensity either as comparative normalised peak height or normalised peak area in the spectral range examined.
• peaks corresponding to separated monomeric globins dimers, trimers and tetramers and complexes such as pentamers are found.
• Individual peaks or mass distribution of broad peaks corresponding to compositional dimers, trimers, tetramers and larger complex correspond to the relative mass representations of the various globin pairing, i.e. for dimers, ⁇ , act, ⁇ , ⁇ , ⁇ , aGy etc.
• the large complex mass distribution within the broad peaks represent relative composition of trimeric, tetrameric, pentameric and other complex combinations of the individual globins.
• a normal purified adult HbA sample reveals peaks for a- globin and ⁇ -globin with other globins found in HbA2 and HbF ( ⁇ , Gy and Ay) not detected.
• Specific oligomeric pairing of globins were detected dimers central maxima 31396 m/z corresponding to ⁇ - ⁇ globin; trimers central maxima of 47308 m/z corresponding to ⁇ and ⁇ ; tetramers central maxima 62858 m/z
• a further 9 phenotypically normal blood samples and 9 phenotypically abnormal blood samples were examined.
• a feature of all the hemoglobinopathies is an altered profile in the relative Intensity ratios of formalin fixed dimeric globins to tetramer/oligomers (figure 13). This is most dramatic in sickle cell disease (see figure 14) but also seen in beta- and alpha-thallasemia's including trait which have double the ratio of tetramer/oligomers compared to unaffected.
• Pin prick and blot spots can be rapidly screened for sickle cell and other potential
• hemoglobinothies are characterised by detection of elevated m/z signal from fetal globins. These fetal globins may be expressed, under stressed conditions, in an attempt to compensate for defective a and ⁇ globin gene expression and act as a biomarker of hemoglobinopathy. However, if fixed by formalin or other suitable crosslinking agent, and then lysed the constituent tetrameric, dimeric pairing and any other oligomeric grouping of the globins (exampled are trimers and pentamers) are revealed and can indicate excessive and unusual a or ⁇ globin oligomeric pairing characteristic of a and ⁇ Thalassemias.

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