Comparative Effectiveness of eConsent: Systematic Review.

BACKGROUND: Providing informed consent means agreeing to participate in a clinical trial and having understood what is involved. Flawed informed consent processes, including missing dates and signatures, are common regulatory audit findings. Electronic consent (eConsent) uses digital technologies to enable the consenting process. It aims to improve participant comprehension and engagement with study information and to address data quality concerns. OBJECTIVE: This systematic literature review aimed to assess the effectiveness of eConsent in terms of patient comprehension, acceptability, usability, and study enrollment and retention rates, as well as the effects of eConsent on the time patients took to perform the consenting process ("cycle time") and on-site workload in comparison with traditional paper-based consenting. METHODS: The systematic review was conducted and reported in accordance with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Ovid Embase and Ovid MEDLINE were systematically searched for publications reporting original, comparative data on the effectiveness of eConsent in terms of patient comprehension, acceptability, usability, enrollment and retention rates, cycle time, and site workload. The methodological validity of the studies that compared outcomes for comprehension, acceptability, and usability across paper consent and eConsent was assessed. Study methodologies were categorized as having "high" validity if comprehensive assessments were performed using established instruments. RESULTS: Overall, 37 publications describing 35 studies (13,281 participants) were included. All studies comparing eConsenting and paper-based consenting for comprehension (20/35, 57% of the studies; 10 with "high" validity), acceptability (8/35, 23% of the studies; 1 with "high" validity), and usability (5/35, 14% of the studies; 1 with "high" validity) reported significantly better results with eConsent, better results but without significance testing, or no significant differences in overall results. None of the studies reported better results with paper than with eConsent. Among the "high" validity studies, 6 studies on comprehension reported significantly better understanding of at least some concepts, the study on acceptability reported statistically significant higher satisfaction scores, and the study on usability reported statistically significant higher usability scores with eConsent than with paper (P<.05 for all). Cycle times were increased with eConsent, potentially reflecting greater patient engagement with the content. Data on enrollment and retention were limited. Comparative data from site staff and other study researchers indicated the potential for reduced workload and lower administrative burden with eConsent. CONCLUSIONS: This systematic review showed that compared with patients using paper-based consenting, patients using eConsent had a better understanding of the clinical trial information, showed greater engagement with content, and rated the consenting process as more acceptable and usable. eConsent solutions thus have the potential to enhance understanding, acceptability, and usability of the consenting process while inherently being able to address data quality concerns, including those related to flawed consenting processes.

Results from the "Me & My Heart" (eMocial) Study: a Randomized Evaluation of a New Smartphone-Based Support Tool to Increase Therapy Adherence of Patients with Acute Coronary Syndrome.

PURPOSE: This study evaluated whether patient support, administered via an electronic device-based app, increased adherence to treatment and lifestyle changes in patients with acute coronary syndrome (ACS) treated with ticagrelor in routine clinical practice. METHODS: Patients (aged ≥ 18 years) with diagnosed ACS treated with ticagrelor co-administered with low-dose acetylsalicylic acid were randomized into an active group (with support tool app for medication intake reminders and motivational messages) and a control group (without support tool app), and observed for 48 weeks (ClinicalTrials.gov Identifier: NCT02615704). Patients were asked to complete the 36-item Short-Form Health Survey (SF-36) and Lifestyle Changes Questionnaire (LSQ), and were assessed for blood pressure and body mass index (BMI) at baseline (visit 1) and at the end of the study (visit 2). Medication adherence was measured using the Brilique Adherence Questionnaire (BAQ). RESULTS: Patients (N = 676) were randomized to an active (n = 342) or a control (n = 334) group. BAQ data were available for 174 patients in the active group and 174 patients in the control group. Over the 48-week period, mean (standard deviation) adherence for the active and control groups was 96.4% (13.2%) and 91.5% (23.1%), respectively (effect of app intervention, p < 0.05). There were no significant differences in blood pressure and BMI between visits. General improvements in SF-36 and LSQ scores were observed for both groups. CONCLUSION: The patient support tool app was associated with significant improvements in patient-reported treatment adherence compared with a data collection app alone in patients prescribed ticagrelor for ACS.

Analysis of Use and Outcomes of the Balance Digital Disease Management Tool for Patients with Type 2 Diabetes.

Management of type 2 diabetes mellitus (T2DM) is a serious medical need for millions of patients and clinicians worldwide. Numerous smartphone apps for T2DM management are available. Due to their global accessibility, computing power and cellular connectivity, the pervasiveness of mobile phones now provide an opportunity for non-invasive Digital Therapeutics that have the potential to manage disease by modifying patient behavior as new modality for disease management and intervention. However, this novel approach has yet to be tested in large clinical studies. The BALANCE clinical study was designed to evaluate mobile phone App usage in a large multi-center clinical trial and its impact on T2DM outcomes. It included a digital aid for the management of, blood glucose, diet, physical activity, and medication adherence. Overall, patient use of the BALANCE-App was low (21% of significant patients users), and it diminished over time. BALANCE showed no effect on HbA1c or weight, what is consistent with other smartphone apps for T2DM which were tested on large clinical trials. Nevertheless, post-hoc subgroup analysis showed women using the App significantly achieved a significant reduction in HbA1c and weight.Clinical relevance Suitability of Digital Therapeutics, at least in the form of smartphone apps, for T2DM is under question. The low use indicates need for a strong focus in patient acceptability and patient engagement in the design process.

Design and rationale for the "Me & My Heart" (eMocial) study: A randomized evaluation of a new smartphone-based support tool to increase therapy adherence of patients with acute coronary syndrome.

A novel smartphone-based patient support tool was developed to increase the adherence to antiplatelet therapy and lifestyle changes in patients after coronary angioplasty for acute coronary syndrome (ACS). The eMocial study (ClinicalTrials.gov Identifier: NCT02615704) investigates whether an electronic support tool will improve adherence to comedication and lifestyle changes in ACS patients. The primary hypothesis of this trial is that an electronic support tool can increase adherence to comedication (primary endpoint) thereby supporting positive lifestyle changes (secondary endpoints). Patients hospitalized with ACS (ST elevation myocardial infarction [STEMI], non-ST elevation myocardial infarction [NSTEMI], or unstable angina pectoris) and treated with ticagrelor coadministered with low-dose acetylsalicylic acid will be randomized 1:1 to an active group receiving the patient support tool via a smartphone-based application or to a control group without the patient support tool. Patient questionnaires to evaluate lifestyle changes and quality of life will be used at baseline and at the end of the 48-week observation phase. Patients are asked to fill out questionnaires to determine their adherence, treatment attitudes, health-care utilization and risk factors on a monthly basis. The study was started in February 2016 and the completion date is scheduled for October 2019. For final analysis 664 patients are expected be available. Preliminary baseline demographics were unstable angina pectoris (13.7%), NSTEMI (49.9%), STEMI (36.4%), male gender (86.3%), and diabetes mellitus (17.6%). Our study could significantly help to understand how inadequate adherence to antiplatelet therapy in ACS patients could be improved with a smartphone-based application.

Patient-Centered Interventions to Improve Adherence to Statins: A Narrative Synthesis of Systematically Identified Studies.

Poor adherence to statins increases cardiovascular disease risk. We systematically identified 32 controlled studies that assessed patient-centered interventions designed to improve statin adherence. The limited number of studies and variation in study characteristics precluded strict quality criteria or meta-analysis. Cognitive education or behavioural counselling delivered face-to-face multiple times consistently improved statin adherence compared with control groups (7/8 and 3/3 studies, respectively). None of four studies using medication reminders and/or adherence feedback alone reported significantly improved statin adherence. Single interventions that improved statin adherence but were not conducted face-to-face included cognitive education in the form of genetic test results (two studies) and cognitive education via a website (one study). Similar mean adherence measures were reported for 17 intervention arms and were thus compared in a sub-analysis: 8 showed significantly improved statin adherence, but effect sizes were modest (+7 to +22 % points). In three of these studies, statin adherence improved despite already being high in the control group (82-89 vs. 57-69 % in the other studies). These three studies were the only studies in this sub-analysis to include cognitive education delivered face-to-face multiple times (plus other interventions). In summary, the most consistently effective interventions for improving adherence to statins have modest effects and are resource-intensive. Research is needed to determine whether modern communications, particularly mobile health platforms (recently shown to improve medication adherence in other chronic diseases), can replicate or even enhance the successful elements of these interventions while using less time and fewer resources.

Terbutaline enantiomer separation and quantification by complexation and field asymmetric ion mobility spectrometry-tandem mass spectrometry.

Recently, we introduced a new approach to chiral separation and analysis of amino acids by chiral complexation and electrospray high-field asymmetric waveform ion mobility spectrometry coupled to mass spectrometry (ESI-FAIMS-MS). In the present work, we extended this approach to the separation of the drug compound terbutaline. Terbutaline enantiomers were complexed with metal ions and an amino acid to form diastereomeric complexes of the type [M(II)(L-Ref)2((+)/(-)-A)-H](+), where M(II) is a divalent metal ion, L-Ref is an amino acid in its L-form, and A is the terbutaline analyte. When metal and reference compound were suitably chosen, these complexes were separable by FAIMS. We also detected and characterized larger clusters that were transmitted at distinct FAIMS compensation voltages (CV), disturbing data analysis by disintegrating after the FAIMS separation and forming complexes of the same composition [M(II)(L-Ref)2((+)/(-)-A)-H](+), thus giving rise to additional peaks in the FAIMS CV spectra. This undesired phenomenon could be largely avoided by adjusting the mass spectrometer skimmer voltages in such a way that said larger clusters remained intact. In the quantitative part of the present work, we achieved a limit of detection of 0.10% (-)-terbutaline in a sample of (+)-terbutaline. The limit of detection and analysis time per sample compared favorably to literature values for chiral terbutaline separation by HPLC and CE.

An objective comparison of pre-processing methods for enhancement of liquid chromatography-mass spectrometry data.

Four data pre-processing methods have been applied with different settings to data sets obtained from the analysis of a pharmaceutical drug and its degradation products by liquid chromatography-mass spectrometry (LC-MS). The methods compared were the frequently used component detection algorithm (CODA) and three kinds of digital filters--matched filtration (MF), Gaussian second derivative (GSD) and Savitzky-Golay. The aim was to evaluate the performance and robustness of these methods for extracted ion chromatogram (XIC), total ion chromatogram (TIC) and base peak chromatogram (BPC) in the presence of different types of noise. In accordance with theory, the best improvements in signal-to-noise ratio (S/N) of the XICs were obtained with MF under the ideal case with random white noise. However, when highly coloured noise was present, it was found that no improvements in XIC S/N could be obtained with any of the pre-processing methods studied. GSD and CODA did, however, improve the S/N for both TIC and BPC. GSD and CODA also significantly reduced the background in the spectral domain, thereby facilitating the interpretation of the mass spectra. Another advantage associated with CODA and to some extent also with GSD is their data reduction ability.

An investigation of peak-broadening effects arising when combining CE with MS.

In this study, peak-broadening effects caused by nebulizing gas flow and lack of temperature control have been investigated for separation capillaries with three different inner diameters. The study was performed with serial UV/ESI-MS detection in an effort to distinguish between peak broadening arising in the separation and peak broadening arising in the ion source. The nebulizing gas was found to significantly affect both migration time and separation efficiency when using capillaries with 50 and 75 microm id. If the nebulizing gas is on during injection, the injection volume increases to such an extent that significant peak broadening is induced. Reducing the id to 25 microm minimizes the parabolic flow induced by the nebulizing gas. Results indicate that the nebulizing gas pressure can be optimized to minimize peak broadening in the ion source. A decrease in detection sensitivity, possibly related to the orthogonal design of the interface, was observed when the nebulizing gas pressure was increased. A tapered capillary tip was found to provide superior separation efficiency as well as sensitivity.

Continuous full filling capillary electrochromatography: chromatographic performance and reproducibility.

Continuous full filling capillary electrochromatography with nanoparticles as pseudostationary phase interfaced with electrospray ionisation mass spectrometric detection was used for reversed phase separations with very high separation efficiency. Several batches of nanoparticles were synthesised and their electrochromatographic performance were evaluated. Different parameters, such as repeatability, reproducibility, limit of detection, and peak asymmetry, were investigated yielding excellent results. The stability of the system over wide pH ranges and over time was found to be excellent. Very high separation efficiencies with over 1.1 million theoretical plates per metre were obtained. The limit of detection for the investigated dialkyl phthalates was approximately 1.0 micromol L(-1), corresponding to 3-5 fmol injected. After preparation, nanoparticle suspensions could be used without further treatment for at least an entire working day with maintained chromatographic qualities.

Continuous full filling capillary electrochromatography: nanoparticle synthesis and evaluation.

Reversed phase continuous full filling capillary electrochromatography with electrospray ionisation mass spectrometric detection was performed with highly sulphated poly[styrene-co-(lauryl methacrylate)-co-(divinylbenzene)] nanoparticles. The nanoparticles that contained a hydrophobic core and a hydrophilic surface were prepared in a one step synthesis using soap free emulsion polymerisation. By changing the concentration of monomers, the polymerisation temperature, and the polarity of the dispersive phase, the size of the nanoparticles could be controlled. With the optimised conditions, nanoparticles with an average size of 157 nm were obtained. These nanoparticles were dispersed in the background electrolyte and used for reversed phase continuous full filling. An orthogonal electrospray ionisation interface was used to separate the eluting nanoparticles from the eluting analytes prior to mass spectrometry detection. Compared to previous studies on reversed phase continuous full filling, the retention, the separation efficiency, and the resolution of a homologous series of dialkyl phthalates were greatly improved.

Enantiomer separation of amino acids by complexation with chiral reference compounds and high-field asymmetric waveform ion mobility spectrometry: preliminary results and possible limitations.

We present a new method for separation of enantiomers with high-field asymmetric waveform ion mobility spectrometry (FAIMS), coupled to mass spectrometric detection. Upon addition of an appropriate chiral reference compound to the analyte solution and subsequent ionization of the solution by electrospray ionization, analyte enantiomers formed diastereomeric complexes, which were potentially separable by FAIMS. The methodology being developed is intended to be general, but here amino acid analytes are specifically considered. In the examples presented herein, six pairs of amino acid enantiomers were successfully separated as metal-bound trimeric complexes of the form [MII(L-Ref)2(D/L-A)-H]+, where MII is a divalent metal ion, L-Ref is an amino acid in its L form acting as chiral reference compound, and A is the amino acid analyte. For example, D- and L-tryptophan were separated in FAIMS as [NiII(L-Asn)2(D-Trp)-H]+ and [NiII(L-Asn)2(L-Trp)-H]+. As FAIMS separation typically takes place over a time scale of only a few hundred milliseconds, the presented separation method opens new possibilities for rapid analysis of one analyte enantiomer in the presence of the other enantiomer. Preliminary quantification results are presented, which suggest that fast and sensitive quantitative chiral analyses can be performed with FAIMS. Method limitations are discussed in terms of diverse phenomena, which are not yet understood.

Nanoparticle-based continuous full filling capillary electrochromatography/electrospray ionization-mass spectrometry for separation of neutral compounds.

Highly efficient reversed-phase capillary electrochromatography (CEC) separations (plate numbers up to 700 000/m), with electrospray ionization mass spectrometry detection were achieved utilizing novel dextran-coated polymer nanoparticles as a pseudostationary phase. A continuous full filling (CFF) technique in which nanoparticles are continuously introduced into the capillary was employed for separation of neutral analytes (dialkyl phthalates), utilizing an orthogonal electrospray interface to prevent nanoparticles from entering the mass spectrometer. CFF-CEC benefits from that an entirely fresh column is employed for every analysis, avoiding carryover effects associated with stationary-phase contamination. The highly efficient separations obtained were accomplished by optimizing the organic modifier concentration in the electrolyte and by using a high nanoparticle concentration (5 mg/mL), to improve interparticle mass transfer and gain sufficient retention. Nanoparticles, with an average diameter of 600 nm, were prepared by polymerization of methacrylic acid and trimethylolpropane trimethacrylate, which in turn were coated with dextran. These nanoparticles formed stable suspensions in electrolytes having broad ranges of polarities, enabling straightforward optimization of the reversed-phase conditions.

Derivatization using dimethylamine for tandem mass spectrometric structure analysis of enzymatically and acidically depolymerized methyl cellulose.

Structure analysis of partially depolymerized methyl cellulose was performed by nanoelectrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) and by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). Dimethylamine (DMA) was used for the first time as a reducing end derivatization reagent for oligosaccharides. This is an attractive reagent since it could be easily removed from the reaction mixture. Most important it also introduces a basic functional group that increased the sensitivity in both MALDI and nano-ESI. Depolymerization was made in two ways: one by the cellulose selective endoglucanase 5A from Bacillus agaradhaerens (Ba Cel5A) and the other by trifluoroacetic acid. The DMA derivatives formed both protonated and sodiated molecules in nano-ESI and MALDI. Tandem MS of protonated molecules yielded predominantly Y fragments from which the distribution of the substituents in the oligomers could be measured. Fragments obtained in tandem MS of sodiated molecules provided information regarding the positions of the substituents within the anhydroglucose units (AGUs). It was found that Ba Cel5A could cleave glucosidic bonds also if the AGU on the reducing side of the bond was fully methylated. The combination of DMA derivatization and tandem MS was demonstrated as a tool for the characterization of endoglucanase selectivity.

A chemometric study of active parameters and their interaction effects in a nebulized sheath-liquid electrospray interface for capillary electrophoresis-mass spectrometry.

A chemometrics approach has been used for evaluating the effect of four experimental parameters when coupling capillary electrophoresis (CE) to electrospray ionization-mass spectrometry (ESI-MS). Electrospray voltage, sheath-liquid flow rate, nebulizing gas flow rate, and spray needle position in respect to the MS orifice were varied according to a full factorial design. In addition to main effects, two interaction effects could be identified as significant when measuring the peak intensity of the analytes, from a sample mixture containing peptides and pharmaceuticals. The first interaction effects, between the nebulizing gas flow rate and the sheath-liquid flow rate, and the second interaction effect, between the nebulizing gas flow rate and the spray position, could further explain the impact that these variables have on the spray performance. The number of theoretical plates and the baseline noise were also measured. The sheath-liquid flow was found to significantly affect the separation efficiency, while the noise level mainly was controlled by the nebulizing gas flow. The same factorial design was also used for a CE capillary with lower internal diameter (ID) and the effects of the same variables were compared on those capillaries using equal injection volume for both capillaries. Similar trends were obtained in both capillaries but capillary ID was shown to be a significant variable when evaluating both capillaries in a single model. It was found that a capillary with 25 microm ID provided improved CE-MS performance over than corresponding 50 microm ID capillary. Enhanced sensitivity was obtained using the narrow-bore capillary, and at lower sheath-liquid flow rate the 25 microm ID capillary also gave rise to more efficient peaks.

Protein pre-fractionation in detergent-polymer aqueous two-phase systems for facilitated proteomic studies of membrane proteins.

Pre-fractionation of a complex mixture of proteins increases the resolution in analytical separations of proteins from cells, tissues or organisms. Here we demonstrate a novel method for pre-fractionation of membrane proteins by a detergent-based aqueous two-phase system. Membrane proteins are strongly under-represented in proteomic studies based on two-dimensional electrophoresis (2-DE). As a model system, we have isolated mitochondria from the yeast Saccharomyces cerevisiae. Mitochondrial proteins were fractionated in an aqueous two-phase system consisting of the polymer poly(ethylene glycol) and either of two commonly used non-ionic detergents, Triton X-114 or dodecyl maltoside (DDM). Soluble proteins partitioned mainly to the polymer phase while membrane proteins were enriched in the detergent phase, as identified from one-dimensional electrophoresis (1-DE) and/or 2-DE followed by mass spectrometric analysis. Pre-fractionation was further enhanced by addition of an anionic detergent, sodium dodecyl sulfate, or a chaotropic salt, NaClO4, and by raising the pH in the system. The two-phase system pre-fractionation was furthermore combined with an alternative two-dimensional high-resolution separation method, namely ion-exchange chromatography and 1-DE. By this approach a larger number of membrane proteins could be identified compared to separation with conventional 2-DE. Thus, pre-fractionation of complex protein mixtures using the aqueous two-phase systems developed here will help to disclose larger proportions of membrane proteins in different proteomes.

Direct coupling of micellar electrokinetic chromatography to mass spectrometry using a volatile buffer system based on perfluorooctanoic acid and ammonia.

Direct coupling of micellar electrokinetic chromatography (MEKC) to mass spectrometry (MS) without employing partial filling is considered to be a challenge. One way of solving the problem would be the use of an MS-compatible surfactant. In the present study, the applicability of a series of surfactants (sodium dodecyl sulfate (SDS), lauric acid, cholic acid and perfluorated carboxylic acids) have been investigated both in terms of separation performance and MS compatibility. It was found that a MEKC system based on perfluorooctanoic acid (PFOA) and ammonia gave excellent results. The separation performance of the suggested system is comparable to the one obtained with standard systems based on SDS and sodium borate buffer although the selectivity is different. The electrospray ionization MS signal of the analytes is not seriously suppressed even at a PFOA concentration of 100 mM. Clusters are formed but their intensities are relatively low and comparable to those obtained with acetic acid. PFOA is volatile enough to allow long-term use, 30 h of continuous use has been recorded without any signs of decreasing performance. After use residual PFOA is easily removed from the ion-source (no memory effects). Furthermore, quantitation of trace impurities is possible at 25 ppb level when employing selected ion monitoring.

Nanoparticles as pseudostationary phase in capillary electrochromatography/ESI-MS.

A novel technique that uses polymer nanoparticles as pseudostationary phase in capillary electrochromatography with electrospray ionization mass spectrometry detection is described. A continuous full filling technique in which the nanoparticles were suspended in the entire electrolyte volume as well as a conventional partial filling technique is presented. No nanoparticles entered the mass spectrometer, which was fitted with an orthogonal electrospray interface, despite the continuous flow of nanoparticles into the interface. Nanoparticles (average diameter 160 nm) were prepared from methacrylic acid, methyl methacrylate, and trimethylolpropane trimethacrylate by utilizing a precipitation polymerization technique. Salbutamol, nortriptyline, and diphenhydramine were used as analytes. The interaction between analytes and nanoparticles was found to be predominantly ionic.

Application of shielding boronate affinity chromatography in the study of the glycation pattern of haemoglobin.

Human haemoglobin (Hb) may appear in a number of glycated species. The glycation pattern of Hb using shielding boronate affinity chromatography (SBAC) has been studied in the present work. SBAC is a novel separation technique, which eliminates nonspecific boronate-protein interactions by introducing a so-called shielding reagent. Two samples from Bio-Rad (Lyphochek)--one from normal persons' blood with relatively low HbA(1c) level (HbL) and the other from diabetic patients' blood with an elevated HbA(1c) level (HbH)--were used for the investigation. Glycated Hb (GHb) was separated from nonglycated Hb species using Tris as the shielding reagent. Two eluted peaks, eluted peak 1 (E1) and eluted peak 2 (E2), were obtained using a linear gradient elution with Tris. Several bands were observed on isoelectric focusing gel, which showed the same migration positions as Hb adducts, such as HbA(0), which is major Hb component containing two alpha chains and two beta chains; HbA(1c), which is post-translational glycation on the N-terminus of the beta chains of HbA(0); Foetal Hb (HbF), consisting of two alpha chains and two gamma chains; and glutathione Hb (also called HbSSG), which is the result from thiol-disulphide interchain exchange during oxidation of the thiol groups of Hb. In both HbL and HbH samples, E2 exhibited slightly higher amounts of HbF than E1. Electrospray-ionisation mass spectrometry showed that: (1) HbL-E1 was glycated with single glucose on both alpha and beta chains while no observable glycated chains were present in HbL-E2; (2) both HbH-E1 and HbH-E2 were glycated with single glucoses on both alpha and beta chains, however, compared with HbH-E1, HbH-E2 showed a higher relative intensity of the glycated beta chain and lower relative intensity of the glycated alpha chain; and (3) the degree of glycation increased with increasing glycation level of the sample. The amount of HbA(1c) presented in the eluted peaks was further determined using enzymatic digestion of glycated Hb by endoproteinase Glu-C and the subsequent separation and analysis of the digested peptides by reversed-phase high-performance liquid chromatography and capillary electrophoresis. The values of HbA(1c)/HbA(0) of the eluted peaks, i.e. HbL-E1, HbL-E2, HbH-E1 and HbH-E2, were 0.27, 0.19, 0.50 and 0.43, respectively. In both HbL and HbH samples, E1 contained higher amounts of HbA(1c) than E2. This study demonstrates the structural heterogeneity of GHb as well as the possibility of using SBAC to detect glycated species of Hb.