Point of Care Tests for Plasma Clotting Factors

This document includes the following sections:
1. Executive Summary
2. Introduction and Review of Clotting Factors
3. Summary of Components for the Test
4. Description of the Test
5. Summary of Data Supporting the Test
6. References

1. Executive Summary

A lateral flow Point of Care (POC) test for blood clotting factors may be developed using a lateral flow test device, with which the patient can routinely measure blood clotting factors at home. Sampling is performed using a bloodstick or lance, and a dosed amount of whole blood is applied to a lateral flow device. The device will measure the amount of clotting factor present by a visual display. An optional reader may be used for storing an electronic record of the result.

Presented below are commercially available examples of each envisioned component, along with representative data to indicate feasibility. A description is given of how the lateral flow device is adapted for quantitation.

2. Introduction and Review of Clotting Factors

Hemophilia A
Hemophilia A is a hereditary bleeding disorder caused by a lack of blood clotting factor VIII. Hemophilia A is caused by an inherited X-linked recessive trait. Males who inherit the trait will have hemophilia A. Children of such males will all inherit a defective X chromosome. Females who inherit one defective X chromosome are carriers of hemophilia A. Females who are homozygous (both X chromosomes defective) will have hemophilia A. In about 30% of cases, there is no family history of the disorder and the condition is the result of a spontaneous gene mutation.

Standard treatment involves replacing the missing clotting factor with factor VIII concentrates isolated from human plasma. Mild hemophilia may be treated with desmopressin (DDAVP), a synthetic hormone that helps the body release factor VIII that is stored within the lining of blood vessels. Commercially available and approved replacement factors include Advate and Kogenate FS, which are recombinant Factor VIII concentrates.

Hemophilia B
Hemophilia B is a hereditary bleeding disorder similar to hemophilia A, but is caused by a lack of blood clotting factor IX. Inheritance of hemophilia B is also an X-linked recessive trait. Treatment includes replacing the defective clotting factor with human factor IX concentrate such as Mononine, which is isolated from human plasma.

On January 17, 2013, the FDA approved Octaplas, a pooled (human) blood product for the replacement of coagulation factors in certain medical conditions where patients have insufficient levels. Versions of Octaplas have been marketed outside the US since 1992. It does not appear indicated for hemophilia, although it did increase levels of Factors VIII and IX in clinical trials.

Current Assays
There are many commercially available clotting factor tests currently on the market. These typically require plasma or serum rather than whole blood, and require a licensed laboratory for the necessary instrumentation. These are based on technologies including coagulation time, immunoassays, and colorimetric (chromogenic) assays.

The immunoassays are of particular relevance to the proposed test, since they indicate that the antibodies required for implementing the test are available. For example, Affinity Biologicals (www.affinitybiologicals.com) offers ELISA lab tests for Factors VIII and IX. These use goat anti-human Factor VIII (hFVIII) coupled with an anti-goat/HRPO conjugate. Affinity Biologicals also sells the antibodies separately. These include sheep anti-hFVIII, sheep anti-hFIX, and goat anti-hFIX.

3. Summary of Components for the Test

The proposed test is not limited to a single start-to-finish device. For each step of the procedure, there are several commercially available choices. The component purpose will be described, followed by examples of marketed devices that fulfill that purpose. The assay as envisioned by this proposal consists of:

1. Bloodstick or lancing device
2. Blood sampling method
3. Lateral flow devices
4. Plasticware
5. Test Strips
6. Antibodies
7. Optional Readers for Quantitation
8. Quantitative Lateral Flow Assays

Bloodstick or Lancing Device
This proposal calls for a method of testing the concentration levels of clotting factors in the blood on a routine basis. The preferred test must require no technical skills such that it may be performed at home. Typical methods for at-home blood collection for diabetics include lancets and other bloodstick devices. Blood must be drawn to measure the concentration of clotting factors. The standard method for a limited volume of blood is a controlled needlestick or lancet. One example is that used by the OraQuick Advance Rapid HIV-1/2 Antibody test. OraQuick uses a lance for performing the needlestick, then a loop to collect a dose of whole blood. The OraQuick requires but does not include the lancet itself. Lancets are available from Becton Dickinson, MediPoint, or even from Amazon.

Blood Sampling Device
The OraQuick HIV-1/2 kit mentioned above includes a Specimen Collection Loop. The Nipro True2go blood glucose test uses a lancet for fingertip blood. The Nipro TRUEtest test strip has a C-shaped edge that collects the dosed blood volume (0.5 microliter blood sample). Hopkins et al. (2011) compared five different devices for transferring constant volumes of blood, including a loop similar to the OraQuick device.

One option is a calibrated capillary tube (Mylar-wrapped, lithium heparin-coated capillary tubes calibrated to 20 μl (Drummond Scientific, Broomall, PA) are used to collect capillary fingertip whole blood (Laderman et al., 2008). The capillary tube will collect the specific volume of blood, and an eyedropper squeeze bulb will expel it onto the test device. Drummond sells capillaries for volumes ranging from 0.25 to 200 μl.

Lateral Flow Devices
There are many lateral flow devices commercially available for numerous medical tests. The basic components are outlined in many patents and publications, including an introductory guide from Millipore (Millipore LF guide.pdf).

There are many patents and patent applications for lateral flow devices. One family for example, with Auric Enterprises LLC (now BioAssay Works LLC) as the assignee, includes five applications and three issued patents.

1 20110091906 Immuno gold lateral flow assay
2 20090246804 Immuno gold lateral flow assay
3 20080188009 Immuno gold lateral flow assay
4 20070087451 Immuno-gold lateral flow assay
5 20070087450 Immuno-gold lateral flow assay

The three patents that have issued are:
1 8,153,444 Immuno gold lateral flow assay, issued April, 2012.
2 7,910,381 Immuno gold lateral flow assay
3 7,344,893 Immuno-gold lateral flow assay

Inverness (now Alere Inc.) also has some fundamental patents in the lateral flow assay field, and sells assays for lactoferrin, a marker of fecal leukocytes and an indicator of intestinal inflammation. They also sell lateral flow assays for respiratory syncytial virus, malaria, hCG for pregnancy detection, and others.

Quidel Corp. also produces rapid diagnostics but appears geared for the physician’s office rather than home use.

A review from 2009 by Wong and Tse (2009) provides a summary of lateral flow devices including the development history, market trends, device assembly, antibodies, colloidal gold and other labels, handheld readers, and quantitation.

Many OEM companies will design and manufacture the desired plastic cassette components. Companies will also collaborate to design and manufacture the entire device. Examples are BioAssay Works, Aptech Services LLC, Detekt Biomedical LLC, and Diagnostic Consulting Network (www.dcndx.com).

Test strips
Companies such a Millipore and Whatman (now part of GE Healthcare) sell chromatography paper and other solid support media that may be used for the actual lateral flow. The Millipore LF guide (mentioned above and attached) outlines the flow characteristics of the different available media. Briefly, the tradeoff is speed for accuracy. A faster assay will also tend to give a smear rather than a single sharp band. The user is urged to try several different types of chromatography supports to determine the desired balance.

According to the GE Healthcare website, the LF1 membrane may be used for lateral-flow assays and works well with one drop of whole blood. Because a test to be run at home should use whole blood (instead of plasma or serum), one may want to use a Blood Separation Membrane such as type FR with a running buffer so the assay will need only 5 μL blood. The Blood Separation Membrane will remove red blood cells that otherwise may clog the lateral flow membrane. Another option for removal of red blood cells is the Vivid Plasma Separation Membrane from Pall Corporation.

As noted above, Affinity Biologicals sells antibodies to human clotting factors as well as complete assays. Other available anti-Factor VIII and anti-Factor IX sources include Millipore. The Sigma Aldrich clotting factor antibody page may be found at http://tinyurl.com/az355pp. QED Bioscience, Inc., sells anti-Factor VIII but not anti-Factor IX. Santa Cruz Biotechnology, Inc., however, sells antibodies to both Factor IX and Factor VIII.

Positive Controls
Assays for Factors VIII and IX should have a Positive Control run with each test. While there are recombinant factors available, a more cost-effective control is a human-sourced plasma such as those available from Affinity Biologicals, or a normal human plasma sourced from a blood products company such as Blood Centers of America and BloodCenter of Wisconsin.

Optional Readers for Quantitation
Several companies sell readers that can detect and quantify the results of lateral flow assays. Examples are:
• Detekt Biomedical LLC (Austin, TX) offers complete industrial design, electronics, and software prototyping. http://www.idetekt.com/custom_services.htm
• BioMedomics.com will develop a specific OEM assay as a disposable test strip and a reader platform optimized for that assay. They are also developing advanced reader platforms.
• Concile GmbH produces a reader for the InfectCheck CRP assay as described below.

Quantitative Lateral Flow Assays
TechNote 303 from Bangs Laboratories describes Lateral Flow Tests technology along with vendors of assay components. They note that semi-quantitative tests in a research setting often use serial dilutions (and hence multiple strips) of a sample to determine the analyte titer.

An Amgen Foundation poster by University of Washington scientists James Clements, Elain Fu, and Paul Yager presented a semi-quantitative assay format (openwetware.org/images/3/33/Clements-AmgenPoster.pdf). This work was followed up by Fu et al. (2011), in which they used a gold enhancement (GE) solution (Nanoprobes, Yaphank, NY) as the signal amplification reagent.

Rohrman et al. (2012) describe a quantitative assay for HIV-1 RNA, illustrated by their Figure 2:

Leung et al. (2008) describe the InfectCheck CRP barcode-style lateral flow assay for semi-quantitative detection of C-reactive protein. Their semi-quantitation distinguishes a low or mild inflammation with CRP levels 10-25 μg/mL), severe (>25-50 μg/mL), and very severe inflammation (>50-100 μg/mL). The InfectCheck CRP assay can allow self-testing by interested individuals without an expensive reading device. This seems available only through http://www.concile.de/en/home/ with optional reader. The lowest CRP level is twice the highest blood clotting factor level presented in the Challenge, so a more sensitive assay will be required.

4. Description of the Test

The proposed test provides for two separate lateral flow immunoassays, one for quantitating Factor IX and a separate (and more sensitive) test for Factor VIII. Both assays will collect a dosed amount of patient blood from a lancet or other bloodsticking device. The dosed amount of blood will be placed on the appropriate device’s sample port. The assay will run within a few minutes and provide a visible display.

Examples of rapid lateral flow immunoassays have been provided above in descriptions of the proposed components. Some, such as Laderman et al. (2008), use two antigens to capture a patient sample antibody (if present). Their Figure 1 illustrates a system similar to that proposed by this proposal, although this proposal will instead use two antibodies to capture each clotting factor.

There will in fact be two separate proposed tests, one for each clotting factor, since a given patient will far more likely be lacking either Factor VIII or Factor IX but not both. If a market survey indicates the need for testing patients for both factors simultaneously, a multichannel (and hence multiplex) device may be designed.

Each device will have multiple lanes. At least one lane will transport the patient blood sample across the lateral flow membrane for contact with the detection components such as antibodies. Other lanes may contain Positive and Negative Controls to ensure proper assay performance.

The amount of Factor present within the dosed amount of blood can be read visually from the display port, or from a small reader device. In principle, the reader should be optional although it may be medically important to store the numerical values for trending purposes.

The envisioned test involves a membrane that has anti-Factor (VIII or IX as appropriate) immobilized on its surface. Unlike standard lateral flow assays, the antibody is not present in only a single line to detect presence of analyte. Instead, antibody is coated over a long area of surface. This may be one continuous coating of antibody or several strips of antibody interspersed with uncoated areas. These areas may be instead coated with antibodies that bind substances within patient samples that might interfere with the desired detection (such as interfering substances).

The general procedure for an assay is:
1. A patient lances a fingertip with a sterile lancet (or equivalent).
2. The patient collects a measured amount of blood from the resulting blood droplet, using a capillary or other constant volume device.
3. The patient applies the measured volume of blood to the lateral flow device’s sample input port.
4. Optional: The sample input port may contain a blood separation membrane that will remove red blood cells.
5. Optional: The patient may add a buffer to the sample input port to aid in reagent mixing.
6. The patient ensures the reagent mix (labeled antibodies, nonionic detergent, and buffer) is added to the sample, initiating reaction and chromatographic detection. This may be by adding drops of reagent mix, by physically starting release of the mix that is stored within the lateral flow device’s cassette, or mixing may begin automatically with blood sample addition.
7. The blood sample/reagent mix will migrate across the solid matrix. Within a few minutes, the liquid will reach a region visible to the user and containing another antibody anchored to the membrane. Factor that is held between a labeled antibody and an anchored antibody will be detected at its appropriate location.
8. Reaction mix migrates across the entire length of the lateral flow cassette, drawn by an absorbent pad at the end farthest from the sample input port.
9. The reaction is complete when the entire membrane is covered with reaction mix. The result may be read visually or by placing the cassette within a portable reader. The reader may detect visible or fluorescent signal.

As reaction mix containing Factor/labeled antibody complex travels across the membrane, immobilized anti-Factor pulls the complex out of solution. Once all of the Factor present in the sample has been pulled out of solution, no further signal will be generated. Ideally, the concentration of Factor in the patient can be read by the length of travel for detectable signal.

The immobilized antibody can be spotted onto the solid phase membrane in stripes, in a constant concentration, or in gradients of concentration. The latter may be used to emphasize breakpoints in Factor concentration and to de-emphasize wide concentrations that have no medical distinction. For example, for Factor VIII, the desired detection ranges are:

o Severe disease = <1% of ‘normal ‘plasma concentration
o Mild disease = 1-5% ‘normal ‘plasmas concentration
o Moderate disease = 5-40% = ‘normal ‘plasma concentration
o FVIII ‘normal ‘plasma concentration = 100 ng/ml (0.3 nM)

Therefore, the device may stretch out the 0-10% range to allow clear distinction between Severe, Mild, and Moderate Disease. The 10-35% range might be contracted (by increasing the amount of antibody locally) since any reading in this range has the same interpretation. Likewise, there may be no medical benefit to detecting Factor amounts greater than 50% (50 ng/mL).

Figure 1 illustrates the basic detection principle. The solvent front indicates the reaction mix (green) as it migrates across the entire lateral flow device membrane.

 Figure 1: Travel of Solvent and Color Development

Figure 1: Travel of Solvent and Color Development

The travel of the Factor (VIII or IX as appropriate) is indicated in blue. Antibodies anchored on the membrane pull Factors out of solution until no more remains to be detected (indicated as Extent of Factor Travel).

Figure 2 shows the decrease of Factor/labeled antibody complex signal intensity as complex is pulled out of solution as a function of time and distance traveled.

 Figure 2: Decrease of Detectable Factor with Migration

Figure 2: Decrease of Detectable Factor with Migration

Figure 3 indicates options for coating the device membrane with anti-Factor antibody.

Figure 3: Linear and Nonlinear Coating of Immobilized Anti-Factor

Figure 3: Linear and Nonlinear Coating of Immobilized Anti-Factor

Panel A shows the standard antibody coating pattern of a single concentration. Thus decrease in Factor with distance will be linear. This may not be ideal, since certain concentration ranges are of more medical interest than others. Panel B shows a nonlinear coating of antibody that stretches the range across which the first 5% (relative to normal range) of Factors are removed. This allows visual discernment of travel above and below the 5% threshold. Panel C shows an option of coating a larger dose of antibodies after the travel zone of 10% since any reading in the 10-35% range will be flagged as ‘Moderate disease.’ A shallower gradient covering the 35-45% range allows discernment of the 40% ‘Moderate disease’ threshold.

Figure 4 shows how these gradients might appear after run on patients with the indicated Factor levels (as % of normal range).

Figure 4: Lateral Flow Results for Different Levels of Disease Severity

Figure 4: Lateral Flow Results for Different Levels of Disease Severity

A patient with severe disease (<1% normal range) may result in color visible in only the first region (or none). Mild disease will show detectable Factor in two regions. Moderate disease will show detectable Factor in three regions, and a normal range of Factor will be detected in all parts of the readout area.

5. Summary of Data Supporting the Test

Examples of small bloodstick volumes and commercially available blood collection devices were provided above. Examples of lateral flow assays including patents were also presented above. Here we present examples that indicate the application to blood clotting factors will be successful.

Examples of lateral flow assay sensitivity
Quantitation of nucleic acids was demonstrated by Rohrman et al. (2012) where the result could be assessed visually or by analysis of digital images (see their Figure 2) with a sensitivity down to about 50 copies of HIV-1 gag RNA.

The most common “standard” technology type used a combination of colloidal gold label dried in a conjugate pad and nitrocellulose membrane lateral flow medium. Gordon and Michel (2008) published a survey of lateral flow assay sensitivity reports. They found the lowest detection limit using this standard format was 30 pM (Fernandez-Sanchez et al., 2005). We note normal range quoted by the literature is 0.3 nM (300 pM). Thus the desired 5% of normal is at the lower range of prior art sensitivity.

Gordon and Michel (2008) also reported other assay formats that drove sensitivity as low as 10-16 (Ahn-Yoon et al., 2003). These methods require more expensive reagents and/or detection equipment.

One feasible method for boosting sensitivity, however, is described by Chen and Wu (2012) to detect presence of genetically modified food. The enhanced detection is based on creating a branched antibody (Figure 1) to amplify the assay signal. Their sensitivity (Figure 3) reaches below 100 pg/mL of Cry1Ab protein (60-70 kDa) with a Limit of Detection of 10 pg/mL. Since human Factor VIII has a molecular size of 250 kDa, such an assay could have the sensitivity of [~70 kDa/250 kDa =] ~4 pg Factor VIII per mL blood. The Challenge states an interest in detecting 1-5% of the FVIII ‘normal ‘plasma concentration of 100 ng/mL, or 1-5 ng/mL. This indicates that sensitivity required for blood clotting factors is well within reach. The Chen and Wu (2012) method required a fluorescence detector, although an amplified chromophoric solution could also be employed.

The assay described by Fu et al. (2011) increased sensitivity about four-fold compared to a commercially available dipstick assay. As mentioned above, they incorporated a gold enhancement solution as a signal amplification reagent. They also performed assay quantitation by analyzing images of the completed assay, with results illustrated in their Figure 4.

Examples of Quantitation
The InfectCheck CRP assay mentioned above provides semi-quantitation (see package insert; page 2 is in English) by visual inspection. Precise quantitation is achieved with the optional reader.

Calculation of Antibody Loading for Proposed Quantitation
The Millipore LF guide presents the following as a basis for calculating whether sufficient antibodies can be bound to a lateral flow membrane to detect the blood clotting factors:

The loading capacity of a protein on a given surface area depends on the protein’s compactness of structure and its Stokes radius (effective diameter). For IgG, the approximate loading capacity is 1 μg/cm2. Multiplying the loading capacity of IgG (1 μg/cm2) by the surface area ratio of the membrane (50 – 200) produces an approximate IgG binding capacity of 50 – 200 μg/cm2. In a typical test strip, the test line is 1 mm high. If the strip is also 1 cm wide, the amount of capture reagent that can be bound is 5 – 20 μg (0.1 cm width x 1cm long=0.1cm2). This is 10–100 times greater than required for most assays.

How many micrograms of anti-Factor antibodies are needed to bind 20 μL blood’s worth of Factor? An IgG antibody has a molecular size of about 150,000 kDa.
Antibody (IgG) ~150,000 μg/μMol so 5 μg/150,000 μg/μMol = 3.33 x 10-5 μmol
= 3.33 x 10-11 moles.

20 x 10-6 L times 0.3 x 10-9 Mol/L = 6 x 10-15 moles Factor VIII
Factor IX ~ 300 x FVIII = 1800 x 10-15 moles = 1.8 x 10-12 moles Factor IX

Therefore, more than sufficient anti-Factor antibodies can be bound to solid phase membrane to deplete the sample.

6. References

1. Ahn-Yoon S, DeCory TR, Baeumner AJ, Durst RA. Ganglioside-liposome immunoassay for the ultrasensitive detection of cholera toxin. Anal Chem 2003;75:2256-2261.
2. Chen C and Wu J. A Fast and Sensitive Quantitative Lateral Flow Immunoassay for Cry1Ab Based on a Novel Signal Amplification Conjugate. Sensors 2012, 12, 11684-11696.
3. Fernandez-Sanchez C, McNeil CJ, Rawson K, Nilsson O, Leung HY, Gnanapragasam V. One-step immunostrip test for the simultaneous detection of free and total prostate specific antigen in serum. J Immunol Methods 2005;307:1-12.
4. Fu E, Liang T, Houghtaling J, Ramachandran S, Ramsey SA, Lutz B, Yager P. Enhanced sensitivity of lateral flow tests using a two-dimensional paper network format. Anal Chem. 2011 Oct 15;83(20):7941-7946.
5. Gordon J and Michel G. Analytical Sensitivity Limits for Lateral Flow Immunoassays. Clinical Chemistry July 2008 vol. 54 no. 7 1250-1251.
6. Hopkins H, Oyibo W, Luchavez J, Mationg ML, Asiimwe C, Albertini A, González IJ, Gatton ML, Bell D. Blood transfer devices for malaria rapid diagnostic tests: evaluation of accuracy, safety and ease of use. Malar J. 2011 Feb 8;10(1):30-38.
7. Leung W, Chan CP, Rainer TH, Ip M, Cautherley GW, Renneberg R. InfectCheck CRP barcode-style lateral flow assay for semi-quantitative detection of C-reactive protein in distinguishing between bacterial and viral infections. J Immunol Methods. 2008 Jul 20;336(1):30-36.
8. Rohrman BA, Leautaud V, Molyneux E, Richards-Kortum RR. A lateral flow assay for quantitative detection of amplified HIV-1 RNA. PLoS One. 2012;7(9):e45611. doi: 10.1371/journal.pone.0045611. Epub 2012 Sep 21.
9. Wong RC and Tse HY, eds. Lateral Flow Immunoassay. Humana Press, 2009.

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