Patent Application
20170176464
Point of Care (“POC”) and home testing for various blood analytes and other detectable metrics in bodily fluids is desirable for patient and doctor. One such analyte is A1C, a type of glycated hemoglobin. High levels of blood glucose cause over-glycation of proteins, including hemoglobin, throughout the body. Glycation of hemoglobin can occur at the amino termini of the alpha and beta chains, as well as other sites with free amino groups. Hemoglobin A undergoes a slow glycation with glucose that is dependent on the time-average concentration of glucose over the 120-day life span of red blood cells. The most prevalent and well-characterized species of glycated hemoglobin A is A1C, making up approximately 3% to 6% of the total hemoglobin in healthy individuals. The correlation of A1C and blood glucose levels make it a useful method of monitoring long-term blood glucose levels in people with diabetes. The mean (average) blood glucose level (MBG) is a function of the A1C levels and, therefore, is derivable.
Polymer Technology Systems, Inc. (PTS), manufactures the A1CNow®, a Point-of-Care device for measuring HbA1C. This test is an immunochemistry (antigen-antibody)-based system which performs well unless the patient has hemoglobin variances HbS and HbC which create false high results as much as 30%. It would be desirable to provide an assay that is not affected by these hemoglobin variances.
Provided herein are multiple methods for improving the performance of a system measuring HbA1C in respect to variants found in the population. In one embodiment, a system for determining a concentration of hemoglobin A1C includes a first lateral flow test strip, the first lateral flow test strip providing for a percent of HbA1C concentration and providing for the total amount of hemoglobin, an antibody-microparticle stripe on the first lateral flow test strip. The system further includes a conjugate stripe on the first lateral flow test strip; and a sample treatment buffer. In one alternative, the sample treatment buffer is strongly denaturing. Optionally, antibodies in the antibody-microparticle strip are covalently bound to microparticles. Alternatively, the microparticles include an aldehyde group. In one alternative, the sample treatment buffer includes sodium perchlorate as an oxidizing agent. Alternatively, the sample treatment buffer includes 50 mM Imidazole, pH 7.0, 1M LiSCN, 0.5M GuSCN, 1% Brij 35, 500 mM NaClO4. Optionally, the sample treatment buffer includes 50 mM Imidazole, pH 7.0, 1M LiSCN, 0.5M GuSCN, 1% Brij 35 and lithium perchlorate. In another alternative, the sample treatment buffer includes 50 mM Imidazole, pH 7.0, 1M LiSCN, 0.5M GuSCN, 1% Brij 35 and sodium nitrite. Optionally, the sample treatment buffer is 50 mM Imidazole, pH 7.0, 1M LiSCN, 0.5M GuSCN, 1% Brij 35, 2.0925 g/L K3Fe(CN)6. In one configuration, the sample treatment buffer includes STB057 50 mM Imidazole, pH 7.0, 1.5M NaSCN, 0.5M GuSCN, 1% Brij 35, 2.0925 g/L K3Fe(CN)6. In another configuration, the sample treatment buffer includes STB058 50 mM Imidazole, pH 7.0, 0.75M LiSCN, 0.75M NaSCN, 0.5M GuSCN, 1% Brij 35, 2.0925 g/L KFeCN. Optionally, the system further includes a second lateral flow test strip, the second lateral flow test strip providing for a percent of HbA1C concentration and providing for the total amount of hemoglobin, an antibody-microparticle stripe on the second lateral flow test strip.
In one embodiment, a method for determining a concentration of hemoglobin A1C includes providing a system including a first lateral flow test strip, the first lateral flow test strip providing for a percent of HbA1C concentration and providing for the total amount of hemoglobin, an antibody-microparticle stripe on the first lateral flow test strip; a conjugate stripe on the first lateral flow test strip; and a sample treatment buffer. The method further includes flowing a sample along the first and second lateral flow test strips. Optionally, the system further includes a sample treatment buffer; and the method further includes mixing the sample with the sample treatment buffer; flowing the sample to the microparticle stripe on the first lateral flow test strip; flowing the sample to the conjugate stripe; and measuring an optical feature of the first lateral flow test strips. In one configuration, the sample treatment buffer is strongly denaturing. Optionally, antibodies in the antibody-microparticle stripe are covalently bound to microparticles. In one alternative, the microparticles include an aldehyde group. In one alternative, the sample treatment buffer includes sodium perchlorate as an oxidizing agent. Alternatively, the sample treatment buffer includes 50 mM Imidazole, pH 7.0, 1M LiSCN, 0.5M GuSCN, 1% Brij 35, 500 mM NaClO4. In another embodiment, the sample treatment buffer includes 50 mM Imidazole, pH 7.0, 1M LiSCN, 0.5M GuSCN, 1% Brij 35, 2.0925 g/L K3Fe(CN)6. Alternatively, the sample treatment buffer includes STB057 50 mM Imidazole, pH 7.0, 1.5M NaSCN, 0.5M GuSCN, 1% Brij 35, 2.0925 g/L K3Fe(CN)6. In one configuration, the sample treatment buffer includes STB058 50 mM Imidazole, pH 7.0, 0.75M LiSCN, 0.75M NaSCN, 0.5M GuSCN, 1% Brij 35, 2.0925 g/L KFeCN.
In one embodiment, a system for determining a concentration of hemoglobin A1C includes a first lateral flow test strip, the first lateral flow test strip providing for a percent of HbA1C concentration and providing for the total amount of hemoglobin; an antibody-microparticle stripe on the first lateral flow test strip; a conjugate stripe on the first lateral flow test strip; and a sample treatment buffer, wherein the sample treatment buffer is strongly denaturing, and antibodies in the antibody-microparticle stripe are covalently bound to microparticles. Optionally, the microparticles include an aldehyde group. In one alternative, the sample treatment buffer includes sodium perchlorate as an oxidizing agent. Alternatively, the sample treatment buffer includes 50 mM Imidazole, pH 7.0, 1M LiSCN, 0.5M GuSCN, 1% Brij 35, 500 mM NaClO4. Alternatively, the sample treatment buffer includes STB058 50 mM Imidazole, pH 7.0, 0.75M LiSCN, 0.75M NaSCN, 0.5M GuSCN, 1% Brij 35, 2.0925 g/L KFeCN. Optionally, the antibody is monoclonal.
Certain terminology is used herein for convenience only and is not to be taken as a limitation on the embodiments of the systems and methods for interference correction from hemoglobin variants. In the drawings, the same reference letters are employed for designating the same elements throughout the several figures. In many embodiments, a sample treatment buffer that is strongly denaturing is used in combination with a microparticle that is strongly bound to an antibody. In many embodiments, this may be a covalent bond. Additionally, in many configurations, a sample treatment buffer is used that includes stability at room temperature. In many configurations, this is accomplished by configuring the oxidizing agent. This oxidizing agent may be sodium perchlorate.
Polymer Technology Systems, Inc. (PTS), manufactures the A1CNow®, a Point-of-Care device for measuring HbA1C. This test is an immunochemistry (antigen-antibody)-based system which performs well unless the patient has hemoglobin variances (HbS and HbC are the two most common variants) which create false high results as much as 30%. In many places herein, A1C bias is referenced. A1C bias relates to the absolute A1C basis in relation to the percentage of A1C as a unit. For example, a 6% A1C sample with 2% A1C bias Tosoh reference result is 6% A1C, and the A1CNow® result is 8% A1C. In the art, the Tosoh G8 HPLC Analyzer (merely referred to as “Tosoh” herein) utilizes the Gold Standard Ion-Exchange method of HbA1C measurement and, therefore, many times is used as a reference. Other highly regarded testing methods/systems for HbA1C, especially those including hemoglobin variants, include the Trinity (Primus) HPLC (affinity), which has been used as the reference value assignment method for our HbS and HbC samples. At multiple points herein, the bias of the system or test is referred to. Generally, bias refers to the relative % bias (percent bias). For example, for a sample with a Tosoh value of 6% A1C and the A1CNow® value is 8% A1C, the bias percentage would be: % Bias=33% (A1C bias=2% A1C).
The occurrence of variants may greatly affect the use and reliability of A1C testing. For instance, Hb variants affect approximately 10% of African-Americans (10% African-Americans carry either HbS or HbC) and 2% Hispanic Americans. In the United States, HbS is the most common variant, HbC is the second most common variant, and HbE is the third most common variant. NGSP has put a lot of emphasis recently on the requirement to eliminate the interference of Hb variants. The current acceptance limit for Hb variants is <±7% relative bias (it was ±10% in 2012 and before). The FDA has requested that Hb variant interference for diagnosis/screening indication be eliminated.
There are at least five methodologies for solving the issues that variants pose to the accuracy of Hb testing. Typical systems used for Hb detection include the A1CNow+® and the A1CNow® systems, and those described in US Patent Application Publication No. 20050227370. A first approach calls for a stronger sample treatment buffer (STB) while using the current microparticles and current Ab (antibody). Currently, a polyclonal antibody is used. As is known, polyclonal antibodies typically have lower specificity than monoclonal antibodies. The result of using a stronger STB, as show in Table 1, is that for two of the variants, the bias is significantly reduced. One issue that arises, however, is that a strong STB tends to reduce the signal. It is theorized that this is due to the antibody detaching from the microparticles. Typically, a competitive assay or sandwich assay is used in the determination of Hb; however, other assay techniques may be used in alternatives.
A second approach includes using an antibody with a higher specificity. This involves switching to a monoclonal antibody. This approach also yielded some results, reducing the basis for both HbC and HbS somewhat. However, the reduction in bias appeared to be capped at approximately 7% to 8% for HbS.
A third alternative involves the usage of stronger microparticle-antibody bonding. Additionally, in most configurations, the usage of a stronger sample treatment buffer is used as well. This alternative is shown in alternative three of the chart of Table 1. Although in the previous alternative/approach 1 the usage of a stronger STB resulted in improved results, it is thought that the stronger STB resulted in the dissociation of the antibody with the microparticle. In order to reduce the occurrence of this dissociation, a stronger bond may be created between the antibody and the microparticle. In some embodiments, this strong bond is a covalent bond. In some alternatives, the beads are bound to antibodies using an aldehyde bond. The double bonded oxygen that is part of the aldehyde group is a proton acceptor, thereby making it conducive to forming a covalent bond with a proton donor. By binding an aldehyde group to the microparticle, a covalent bond may be formed between the antibody and the microparticle having an aldehyde group. In some alternatives, a ketone group might be used; however the covalent bonding properties will likely be reduced; therefore, the STB strength may correspondingly be reduced. In many embodiments, the microparticle is composed of latex, and the latex includes an aldehyde group.
A fourth approach includes the usage of a monoclonal antibody and a stronger STB. Standard coupling between the microparticle and the antibody is used. This approach resulted in a signal that was too low (too light). This approach appears to need stronger antibody-microparticle binding. This is thought to be because the combination of the stronger STB and the monoclonal antibody lead to too strong of a preference for dissociation between the antibody and the microparticle.
A fifth approach is thought to be optimal and generates improved results. The fifth approach replaces the traditional polyclonal antibody with a monoclonal antibody, utilizes a stronger STB, and utilizes a stronger bond between the antibody and the microparticle. In many embodiments, this may be an aldehyde bond. Based on the formulations used experimentally, it is believed that a somewhat weaker STB might be used in some embodiments. Alternatively, a bond of a different strength between the antibody and microparticle may be formed. It is believed that this system may be promising; however, adjustments to the assay must be made before it becomes viable.
The following describe some possible STBs. An exemplary STB that has stronger denaturing and cytotropic characteristics as compared to the standard buffer has been used previously. The standard STB used is 5 mM Imidazole, 2% Zwittergent, 2.0925 g/L KFeCN, 112 mM NaCl, 0.1% Surfynol, pH 7.5. An exemplary and stronger STB is 20 mM Glycine, 1M LiSCN, 1% Triton, 2.0925 g/L KFeCN, 0.5M GuSCN, pH 9. The use of the stronger STB alone reduces the HbC variant issues to acceptable levels; however, it does not have enough impact to reduce HbS variant issues to acceptable levels. Additionally, numerous oxidant replacements for the buffers provided are possible. In the STBs, typically an oxidant of K3Fe(CN)6 is used. Alternatively, sodium perchlorate, lithium perchlorate, or sodium nitrite may be used as well as other oxidants that may provide for the formation of methemoglobin.
Various STBs are possible for use as a strong STB. A strong STB was needed to function with the stronger Ab-MP coupling. A variety of STB and microparticle combinations were tested, as shown in Table 4 below. Additional STBs using different oxidizing agents are shown later in this disclosure.
Using a new Aldehyde MP to have stronger Ab-MP coupling is such that the system can tolerate a much stronger buffer for sample treatment (without Ab detachment). The use of different stronger buffers to denature and present the antigen better for interference reduction was achieved by the combination shown.
The buffers tested are as follows:
Before the introduction of the embodiments of a combination of stronger STB and Ab-MP coupling, the system had about 30% positive bias when testing with Hb variant samples.
The new system can significantly reduce the bias caused by Hb variants.
STB058 gave good results.
In various embodiments, the formulation may include a replacement oxidizing agent that replaces the K3Fe(CN)6. Sodium perchlorate, lithium perchlorate, or sodium nitrite may be used as well as other oxidants that may provide for the formation of methemoglobin.
Table 5 below summarizes the results of testing these oxidizing agent substitutes. Some of the concentrations of the oxidizing agents that do not meet specifications are 500 mM LiClO4, 1M NaClO4, and 1M LiClO4. Note the delta R and the underlined values that indicate the results may not meet useful specifications.
0.3504
0.3142
0.4342
0.1591
Additionally, the oxidizing agent sodium perchlorate was tested for sample treatment kinetics and red blood cell lysis kinetics. The results are shown in Table 6 below and
0.1311
0.1168
0.1206
0.1276
0.1250
0.1231
0.1219
Table 9 and
Additionally, in some embodiments, sodium nitrite may be used as an oxidizing agent.
In the embodiments discussed, various optimizations may be conducted. The goal of many of the embodiments is to provide a more strongly denaturing STB while also providing a more strongly bound antibody-microparticle combination. In some configurations discussed, alternative oxidizing agents are provided for the STB in order to increase the shelf life of the product at room temperature.
In many embodiments, a hemoglobin lateral flow assay 210 may be created. The conjugate(s) described above will be coated on the area in zone 1230 as shown in
Embodiments of systems and methods described herein provide for differentiation between glycated and unglycated hemoglobin (called “total hemoglobin” or “total Hb”). In many configurations, the glycated hemoglobin (A1C) is removed from the “total hemoglobin” via an affinity method (usually chromatographically; like boronate method); then that hemoglobin would result in a differentiated hemoglobin sample (called “differentiated hemoglobin” or “diff Hb”) as expressed by the following equation 1:
“diff Hb”=total Hb−glycated HbA1C (1)
Thus, if one can quantify the level of “differentiated hemoglobin (diff Hb),” then percent glycated Hb easily can be computed by equation 2:
% glycated HbA1C=[(total Hb−diff-Hb)/total Hb]*100 (2)
In many embodiments, a premix step may be included. Typically, a sample is exposed to a premix step with a buffer solution (referred to as the sample treatment buffer). After a premix, the sample and buffer solution are applied to a test strip. The test strip includes an antibody-microparticle zone and a capture zone for capturing antibodies that have not reacted with the sample. Typically, after the lateral flow of the sample, the flow of the microparticles is measured using an optical meter.
While specific embodiments have been described in detail in the foregoing detailed description and illustrated in the accompanying drawings, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and the broad inventive concepts thereof. It is understood, therefore, that the scope of this disclosure is not limited to the particular examples and implementations disclosed herein but is intended to cover modifications within the spirit and scope thereof as defined by the appended claims and any and all equivalents thereof.
This application claims the benefit of Provisional Application No. 62/263,478 filed on Dec. 4, 2015 titled “Systems and Methods For Interference Correction From Hemoglobin Variants” and of Provisional Application No. 62/322,687 filed on Apr. 14, 2016 titled “Systems and Methods For Interference Correction From Hemoglobin Variants.”
| Number | Date | Country | |
|---|---|---|---|
| 62263478 | Dec 2015 | US | |
| 62322687 | Apr 2016 | US |
