Hemox Analyzer Manual

Hemox Analyzer Manual

This article has been cited by other articles in PMC. Abstract Introduction Hemoglobin (Hb) is a critical molecule necessary for all vertebrates to maintain aerobic metabolism. We have developed a novel screening assay based upon the spectral changes observed during Hb deoxygenation and termed it the oxygen dissociation assay (ODA). Methodology ODA allows for the quantitation of oxygenated Hb at given time points during Hb deoxygenation on a 96-well plate. Results A correlation ( R 2 ) of 0.7 indicated that the ODA has the potential to screen and identify potent exogenous Hb modifiers. In addition, it allows for concurrent comparison of compounds, concentrations, buffers, or pHs on the level of Hb oxygenation. The TCS Hemox Analyzer, which is the most advanced instrumentation currently deployed, measures the oxygen partial pressure and concurrently measures Hb saturation with a Clark oxygen electrode and spectrophotometry, respectively. These factors are limiting when using OECs for drug discovery. To overcome these drawbacks, researchers have attempted to develop a variety of miniaturization techniques. Materials and methods Compounds Phytic acid (also known as inositol hexaphosphate) was purchased from MilliporeSigma, Burlington, MA, USA and efaproxiral was purchased from BePharm Ltd, Shanghai, China. All other compounds including reference compounds, tucaresol, voxelotor (formerly known as GBT440), and GBT1118, were synthesized at Global Blood Therapeutics, South San Francisco, CA, USA. 5, 7, 13 For all studies herein, compounds were solubilized in 100% dimethyl sulfoxide (DMSO) at concentrations ranging from 10 to 100 mM. Hb was purified from individual donor red blood cells (RBCs) by gel filtration and DE-52 anion exchange chromatography. 14 Samples of the eluted fractions were run on a Tris-glycine 12% acrylamide native gel where they were separated according to their isoelectric point (pI) allowing for their identification.

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In between each measurement, the plate was shaken in a figure 8 for 1 minute at 300 rpm. Data analysis was performed using Excel’s LINEST function on the wavelengths ranging from 380 to 700 nm. The Hb spectrum at time zero was used as the reference for 100% oxy Hb, while the average spectra of Hb with 15 mM sodium dithionite (Merck Millipore, Burlington, MA, USA), a reducing agent, was used as the reference for 100% deoxy Hb (spectra not shown). The results were then expressed as %oxy Hb. 5 For the data presented within this manuscript, the issue of methemoglobin was resolved by a visual inspection of all spectra. Purified Hb samples were then oxygenated with compressed air for 10 minutes within the Hemox Analyzer. After oxygenation, the samples were deoxygenated with compressed N 2 and OECs were collected during deoxygenation as previously described. 11 From the OECs, p50 values were derived using TCS Hemox Analysis Software. After incubation, data collection and data analysis proceeded as in the standard ODA. The experiments were conducted using 50 mM potassium phosphate buffer at pH 7.4. Voxelotor or tucaresol was diluted from a 10 mM stock solution (in 100% DMSO). Following the incubation, data collection and data analysis proceeded as in the standard ODA. As the maximal activity for tucaresol and voxelotor is not equivalent, the data were normalized to the maximal %oxy Hb value. This normalization allowed for discerning the contributions of HSA binding for different compounds. Results Assay design All instruments used to determine the oxygen affinity of Hb rely on the spectral difference between oxy Hb and deoxy Hb. The first step in validating the ODA was to monitor the spectral change from oxy Hb to deoxy Hb during the 2-hour deoxygenation process. After samples are incubated with Hb in 96-well half-area plates, they are placed in the spectrophotometer. The spectrophotometer is set up with a gas inlet that allows for the reading chamber to be filled with N 2.

When using a BMG SPECTROstar Nano, the microplate chamber can be filled with N 2 directly ( Figure 1A ). As the chamber fills, each well within the 96-well plate is exposed to diminishing levels of O 2 ( Figure 1B ). This change in O 2 levels alters the equilibrium between the well and the local atmosphere driving O 2 out of the buffer. As oxy Hb releases O 2 to the buffer, a spectral shift can be measured ( Figure 1C ). Humidity was measured using a hygrometer placed up against the vent port in the spectrophotometer. Open in a separate window Figure 1 Schematics and principles of the oxygen dissociation assay. Notes: ( A ) The flow of N 2 within the spectrophotometer for the ODA. ( B ) The equilibrium for liganded Hb at room air and how it changes during the 2-hour deoxygenation. ( C ) While N 2 is flowing across the top of each well, the sample deoxygenation is monitored by Hb spectra changes that are captured in an orthogonal direction to the gas flow. Abbreviations: Hb, hemoglobin; ODA, oxygen dissociation assay. Figure 2A shows the spectral measurements of Hb at the beginning and end of the 2-hour deoxygenation process. Similarly, in the Q-band region, the double peaks of oxy Hb (541, 577 nm) transition toward a single peak at 555 nm, which is also characteristic of deoxy Hb. Data analysis was performed using Excel’s LINEST function on the wavelengths ranging from 380 to 700 nm, as described in the Materials and methods. The results were then expressed as %oxy Hb. The above analysis was conducted on the two spectra from Figure 2A as well as the 18 spectra in between and the results were plotted against time in Figure 2B. As expected the percent of oxy Hb decreased over time, with Hb proceeding from 100 %oxy Hb to 6.46 %oxy Hb. Open in a separate window Figure 2 Spectra of Hb over 2 hours of deoxygenation. After 2 hours, the peak shifts toward 430 and 555 nm (respectively), indicating that Hb shifts to a significantly deoxygenated state (6.46 %oxy Hb).

( B ) Using Excel’s LINEST function, the 20 spectra collected over the 2-hour period of deoxygenation were analyzed and plotted against time to quantitate the level of Hb deoxygenation (data collected in triplicate). All experiments were conducted in phosphate buffer. Abbreviations: Hb, hemoglobin; oxy Hb, oxygenated Hb. Figure 3A shows representative OECs of GBT1118, 7 an analog of voxelotor, 5, 13 and phytic acid, 17 a potent analog of 2,3-DPG, in comparison to Hb alone. GBT1118 has a left-shifted OEC and a lower p50 value while phytic acid has a right-shifted OEC and a higher p50 in comparison to Hb control ( Table 1 ). Figure 3B shows the %oxy Hb values over time for phytic acid and GBT1118 in the ODA. The data indicate that GBT1118 delays the transition from oxy Hb to deoxy Hb while phytic acid accelerates the transition. Based upon data from Figure 3B, left-shifting compounds seem to be best evaluated at longer deoxygenation times (108 minutes). On the other hand, right-shifting compounds (such as phytic acid) seem to be better differentiated at earlier times (30 minutes). Open in a separate window Figure 3 OECs and oxygen dissociation assay are highly correlated. GBT1118 shows a leftward curve shift and lowers the p50. The deoxygenation of Hb solution over time in the presence of GBT1118, which stabilizes the oxygenated state of Hb, and phytic acid, which sustains the deoxygenated state of Hb. Abbreviations: Hb, hemoglobin; ODA, oxygen dissociation assay; OECs, oxygen equilibrium curves. Abbreviations: Hb, hemoglobin; p50, the partial pressure of O 2 at which Hb is 50% saturated with O 2. Based upon the changes observed with reference compounds, a large-scale comparison was performed to establish how left-shifters (compounds with left-shifted OECs compared to the Hb control) and right-shifters (compounds with right-shifted OECs compared to the Hb control) behaved in the TCS Hemox Analyzer and ODA. A correlation ( R 2 ) of 0.

70 was obtained for both left-shifter (with %oxy Hb measured at 108 minutes) and right-shifter compounds (with %oxy Hb measured at 30 minutes) indicating that this is a relevant primary screening assay to filter out inactive compounds as well as to rank compounds based on Hb modifying potency. The robustness of the assay was determined with a variety of parameters. However, in the Hb modifier field, standard dose responses in triplicate have been hindered by the limitation of the instrumentation. Figure 4 shows examples of ODA dose response of two compounds, GBT1118 7 (a left-shifter, Figure 4A ) and phytic acid 17 (a right-shifter, Figure 4B ), previously described in the literature. Open in a separate window Figure 4 Dose responses obtained using the oxygen dissociation assay. Notes: ( A ) Dose response of GBT1118, a “left-shifting” Hb modifier collected in triplicate using the ODA; %oxy Hb values were collected at 108 minutes. ( B ) Dose response of phytic acid, a “right-shifting” Hb modifier using the ODA; %oxy Hb values were collected at 30 minutes. All experiments were conducted in triplicate in TES buffer. Abbreviations: Hb, hemoglobin; ODA, oxygen dissociation assay; oxy Hb, oxygenated Hb; EC 50, half maximal effective concentration. The combination of the increasing CO 2 and increasing lactic acid levels lowers the pH of the oxygen-starved tissues. Figure 5A shows how Hb behaves over time in the ODA. As expected, Hb at pH 6.8 is more rapidly deoxygenated than Hb at pH 7.4, while Hb at pH 8 sustains the oxy Hb state for a longer time frame. Figure 5B shows that GBT1118 maintains a similar % of Hb in the oxy Hb state regardless of pH, which is ideal for a compound designed to maintain a portion of Hb in the oxy Hb state in order to delay HbS polymerization and prevent sickling in SCD patients.

13 Even though there was a response to the changing pH, GBT1118 is able to maintain an approximately equal portion of Hb in the oxy Hb state, thereby ensuring that it will be effective at delaying HbS polymerization even in metabolically active tissues. Open in a separate window Figure 5 Hb-O 2 affinity is pH dependent. As the pH of the buffer is decreased, a decrease in %oxy Hb can be observed for any given time. The assay was conducted using phosphate buffer. ( B ) Analysis of GBT1118 at various pHs using the ODA. Experiments were conducted in triplicate in TES buffer. Abbreviations: Hb, hemoglobin; ODA, oxygen dissociation assay; oxy Hb, oxygenated Hb. Table 2 The effect of pH on the p50s of purified Hb in the presence of Hb modifiers pH. Albumin vs Hb binding Finally, the ODA can be used to screen and select Hb modifiers with appropriate protein binding profiles. Depending on the desired output, the compound can be preincubated with either Hb or albumin first and then the second protein added. In Figure 6, the samples were preincubated with albumin for 1 hour and then Hb was added to the system followed by a subsequent 1-hour incubation. The data in Figure 6 indicate that compared to tucaresol, a compound known to have high affinity for plasma proteins, 20 voxelotor has a tenfold greater affinity for Hb over albumin. Open in a separate window Figure 6 Albumin vs Hb binding. Abbreviations: HSA, human serum albumin; Hb, hemoglobin; oxy Hb, oxygenated Hb. The TCS Hemox Analyzer is a well-established instrument convenient for clinical samples. However, it requires a significant amount of time and compound in order to conduct SAR studies. These conditions include, but are not limited to: pH, buffers, and coincubation with proteins such as HSA. As shown in the voxelotor vs tucaresol experiment, measuring HSA effects could prove predictive of pharmacokinetic parameters or RBC:plasma partitioning.

We also showed the ability of the ODA to be adapted to characterize compounds with respect to pH and albumin binding sensitivity. One of the major challenges in developing ODA was determining the optimal Hb concentration. Many screening assays use protein concentrations within the pico- to nano molar range in an effort to minimize compound needs and compound solubility issues. However, at such concentrations, the Hb tetramer dissociates to dimers making lower Hb concentrations a non-optimal choice for a screening campaign. As detailed above, the ODA examines the ability of compounds to modulate the release of O 2 from Hb under various conditions. This is exemplified by the ODA experiments at various pHs. To conduct a similar experiment with the Hemox Analyzer, the data collection alone would take 2 weeks, which would add day-to-day variability to the data. Finally, one of the strengths of the ODA is its adaptability, allowing researchers to use the assay for both screening and characterizing Hb modifiers. Similarly, the albumin vs Hb assay can aid in rank-ordering compounds for in vivo studies. Conclusion This novel assay (ODA) is simple, reproducible, and adaptable and requires no special reagents. It has been evaluated against the TCS Hemox Analyzer with over 500 compounds and displayed a correlation of 0.7 for both right and left-shifting Hb modifiers. In addition, the ODA platform has been used to characterize various compounds 5 including those discussed in this article: GBT1118, phytic acid, voxelotor, tucaresol, and efaproxiral. Footnotes Disclosure Mira P Patel, Vincent Siu, Abel Silva-Garcia, Qing Xu, Zhe Li, and Donna Oksenberg are employees, and shareholders, of Global Blood Therapeutics, Inc. The authors report no other conflicts of interest in this work. References 1. Safo MK, Ahmed MH, Ghatge MS, Boyiri T. Hemoglobin-ligand binding: understanding Hb function and allostery on atomic level. Oxygen transport by hemoglobin.

Myo-inositol trispyrophosphate: a novel allosteric effector of hemoglobin with high permeation selectivity across the red blood cell plasma membrane. Insights into the solution structure of human deoxyhemoglobin in the absence and presence of an allosteric effector. GBT440 Increases haemoglobin oxygen affinity, reduces sickling and prolongs RBC half-life in a murine model of sickle cell disease. GBT1118, a potent allosteric modifier of hemoglobin oxygen affinity increases tolerance to severe hypoxia in mice. Kister J, Wajcman H. Oxygen equilibrium measurements of human red blood cells. Guarnone R, Centenara E, Barosi G. Performance characteristics of hemox-analyzer for assessment of the hemoglobin dissociation curve. Nakagawa A, Lui FE, Wassaf D, et al. Identification of a small molecule that increases hemoglobin oxygen affinity and reduces SS erythrocyte sickling. Metcalf B, Chuang C, Dufu K, et al. Discovery of GBT440, an orally bioavailable R state stabilizer of sickle cell hemoglobin. Goldberg MA, Husson MA, Bunn HF. Participation of hemoglobins A and F in polymerization of aickle hemoglobin. Griffon N, Baudin V, Dieryck W, et al. Tetramer-dimer equilibrium of oxyhemoglobin mutants determined from auto-oxidation rates. Fylaktakidou KC, Lehn JM, Greferath R, Nicolau C. Inositol tripyrophosphate: a new membrane permeant allosteric effector of haemoglobin. Kavanagh BD, Khandelwal SR, Schmidt-Ullrich RK, et al. A Phase I study of RSR13, a radiation-enhancing hemoglobin modifier: tolerance of repeated intravenous doses and correlation of pharmacokinetics with pharmacodynamics. Rolan PE, Parker JE, Gray SJ, et al. Lehrer-Graiwer J, Howard J, Hemmaway CJ, et al. Rolan PE, Mercer AJ, Wootton R, Posner J. Pharmacokinetics and pharmacodynamics of tucaresol, an antisickling agent, in healthy volunteers. Aprahamian M, Bour G, Akladios CY, et al. Raykov Z, Grekova SP, Bour G, et al.

Myo-inositol trispyrophosphate-mediated hypoxia reversion controls pancreatic cancer in rodents and enhances gemcitabine efficacy. The recording can be performed in the association or dissociation modes, utilizing fresh whole blood or hemolysate.As the healthcare industry embraces value-based care, hospitals and other provider organizations are relying on digitization to better follow patients through their healthcare journey -- and recoup each and every reimbursement dollar. For more insight from Jeffrey Goldstein, MD, MS, Senior Healthcare Specialist and Daniel Colling, BSC, RN, Global Lead for Clinical and Print Workflow Solutions at HP inc.To protect patient health information, hospitals and healthcare organizations need to be sure they are incorporating often overlooked endpoint technologies -- namely, printers -- in their healthcare IT security plans. For more insight from Pamela Dill, Senior Security Advisor at HP Inc.Gains made in the areas of staffing, overhead, and payer reimbursements can contribute to more effective recruitment and retention as well as boost financial performance in the long run. Download This Research Please only use one option at a time.Boolean operators define the relationships between Terms or Phrases. Please note that Boolean operators must be all uppercase.It will be used if there is no Boolean operator between two terms. For example:The - character is synonymous with using NOT. By using our services, you agree to our use of cookies. For the best chance of winning, increase your maximum bid.Please check your email account for more details.You can contact the auctioneer on 617-227-6553 for more information.To be sure to win, log in for the live auction broadcast on Aug 05, 2014 10am ET or increase your max bid. We have thousands of new lots everyday, start a new search.Please register now so you are approved to bid when auction starts.

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Lots not paid for and removed within the time allowed herein may be resold at public or private sale without further notice, and any deficiency, together with all expenses and charges of resale including legal fees, will be charged to the defaulting purchaser. The party or parties who submit a Successful Bid for the Tangible Personal Property shall have until September 5, 2014 (the “Removal Date”) to remove the Tangible Personal Property from the Premises. Lots not paid for and removed within the time allowed herein may be resold at public or private sale without further notice, and any deficiency, together with all expenses and charges of resale including legal fees, will be charged to the defaulting purchaser. Methemoglobin levels are often included in blood gas measurements. In normal physiological conditions, methemoglobin is converted to hemoglobin by enzyme cytochrome b5 reductase (also known as methemoglobin reductase or b5R). Congenital methemoglobinemia, an autosomal recessive disorder, is most commonly due to a cytochrome b5 reductase (b5R) deficiency. Methemoglobinemia can also be caused by various mutations of globin genes, known as hemoglobins M, inherited as an autosomal dominant phenotype. Acquired methemoglobinemia is usually caused by exposure to oxidizing substances or drugs, including nitrates and sulfa-containing antibiotics. Acute methemoglobinemia is a medical emergency and early recognition is critical because it can be life-threatening. It usually does not cause polycythemia unless it has been chronic for weeks, allowing a compensatory increase of erythropoiesis responding to tissue hypoxia to take place. Patients with chronic methemoglobinemia can be asymptomatic to minimally symptomatic. View chapter Purchase book Read full chapter URL: A worldwide yearly survey of new data in adverse drug reactions A. Nobili,. R.

Latini, in Side Effects of Drugs Annual, 2015 Hematologic Methaemoglobinaemia has been attributed to nicorandil in an elderly patient with chronic stable angina. A 75-year-old male patient on treatment with nicorandil at therapeutic dosage for chronic stable angina consulted his physician because he noticed that his fingernails had developed a bluish tinge. No specific therapy was undertaken, but nicorandil was omitted from the drug regime. Six weeks later the methaemoglobinaemia had decreased to 3.7% and the bluish discoloration was not perceptible. There was no history of familial methemoglobinemia, but in one case the mother had been taking phenacetin before labor and this was most probably the cause. In five of the six other cases the methemoglobinemia was thought to be due to the use of disinfecting solutions or ointments containing triclocarban that had been used as a vaginal disinfectant in four cases and as a powder for the umbilicus in one case. The authors advised against the use of triclocarban in maternity units and by those dealing with neonates. View chapter Purchase book Read full chapter URL: Side Effects of Drugs Annual 32 Stephan A. Schug, Hari Krshnan, in Side Effects of Drugs Annual, 2010 Hematologic Methemoglobinemia is a common adverse effect of prilocaine (see also benzocaine above). Only two children were given methylthioninium chloride (methylene blue). No cause other than lidocaine could be identified. There was no correlation between the dose of lidocaine and the occurrence of methemoglobinemia. Most of these mutations occur within the ?-globin chain where ?1 and ?2 chains contact. This change impairs intramolecular rotation or 2,3-biphosphoglycerate (BPG) binding making hemoglobin unable to transition from high-oxygen-affinity to low-oxygen-affinity states thus causing tissue hypoxia and compensatory polycythemia.

Hemoglobin electrophoresis is insufficient to identify hemoglobin structural defects and some hemoglobin mutants will be missed. The only reliable screening test is P50 measurement either by the Hemox-Analyzer or calculated from venous blood gases. Often the mutation is identified by sequencing globin genes. Treatment Phlebotomy is usually not beneficial because it causes decrease in exercise tolerance. 2,3-BPG Deficiency 2,3-bisphosphoglycerate (BPG), also known as 2,3-DPG, promotes hemoglobin transition from a high-oxygen-affinity state to a low-oxygen-affinity state. 2,3-BPG binds to the central compartment of the hemoglobin tetramer changing its conformation and shifting the oxygen disassociation curve to the right. The deficiency is created by ineffective bisphosphoglyceratemutase (BPGM), a red cell enzyme of the early glycolytic pathway that converts 1,3-BPG to 2,3-BPG. Mutations of BPGM are extremely rare and are typically autosomal recessive. Diagnosis is confirmed by establishing a decreased P50 and excluding other hemoglobin mutants and by establishing a decreased 2,3-BPG quantity and BPGM enzyme activity. Treatment Phlebotomy is usually not beneficial because it causes decrease in exercise tolerance. Methemoglobinemia Methemoglobinemia is usually considered when the patient is cyanotic with low oxygen saturation by pulse oximetry, yet PaO 2 levels are normal. In normal physiological conditions the body will reduce the methemoglobin to a level of View chapter Purchase book Read full chapter URL: Hematology Steven E. McKenzie MD, PhD, in Pediatric Secrets (Fifth Edition), 2011 18 A 14-month-old child presents symptoms including marked cyanosis, lethargy, and normal oxygen saturation by pulse oximetry after drinking from a neighbor's well. What is the likely diagnosis. Methemoglobinemia should always be considered when a patient presents symptoms of cyanosis without demonstrable respiratory or cardiac disease.

Methemoglobin is produced by the oxidation of ferrous iron in hemoglobin into ferric iron. Methemoglobin cannot transport oxygen. Normally, it constitutes less than 2% of circulating hemoglobin. Oxidant toxins (e.g., antimalarial drugs, nitrates in food or well water) can dramatically increase the concentration. Patients with cyanosis as a result of methemoglobinemia can have normal oxygen saturation as measured by pulse oximetry because the oximeter operates by measuring only hemoglobin that is available for saturation. View chapter Purchase book Read full chapter URL: Hemoglobin Variants Associated With Hemolytic Anemia, Altered Oxygen Affinity, and Methemoglobinemias Edward J. Benz Jr., Benjamin L. Ebert, in Hematology (Seventh Edition), 2018 Diagnosis Methemoglobinemia should be suspected in patients with unexplained cyanosis. It is obviously a medical emergency when any patient has cyanosis and altered mental status; a Pa o 2 more normal than expected on the basis of the O 2 saturation should trigger a consideration of methemoglobinemia. The ingestion of nitrites as a suicidal gesture, especially in people knowledgeable with respect to chemistry, medicine, or pharmacology, should be considered. Exposure to nitrate-containing therapeutic compounds, e.g., in the setting of the intensive care unit, should also raise suspicion. Methemoglobinemia can be suspected from the brownish color of blood when it is drawn. Laboratory detection is simple; methemoglobin exhibits characteristic peaks of absorption at 630 and 502?nm, rendering it easily distinguishable from normal hemoglobin. Pulse oximetry, using a ratio of absorption at 660?nm and 940?nm, gives an inaccurate reading of 85% oxygen saturation for blood with 100% methemoglobin. The inherited M hemoglobin mutants are frequently detectable by altered electrophoretic mobility, especially if ferricyanide treatment in vitro is used to convert all the hemoglobin solution to methemoglobin.