A portable device to detect circulating tumor cells in blood sample with colorable test strip

Abstract

A portable device for early cancer tumor detecting using the advantages of Ep-CAM antigen properties (Epithelial Cell Adhesion Molecule) which adhere to cancer cells and ability to adhere SPIONs. In this invention we calculate the concentration of ferric ions which have to adhere to the cancer cells throw the antigen. We add the antigen mixed with SPIONs to a blood sample and enriches, incubate and then inject it into the device to process it and produce a residual for test. A special Test strip designed for this device consists of two reagents potassium ferrocyanide and potassium thiocyanate. The purpose of using two -reagents test strip is to increase the integrity of the test. If the sample contain cancer tumor cells, then the antigen will stick to this cell with the ferric ions and the device contain a removable filter unit with a micro pore size to prevent tumor cells from passing throw it. If the sample contain cancer cell, the all-ferric ions will stick to it and not passing through the filter. The residual will not contain any ferric ions and the test strip will act nothing. If the sample is free of cancer cell, then the antigen will find nothing to stick with and consequently the ferric ions which will pass through the filter to precipitate in the residual. The test strip will change color according to reaction with ferric ions which is indication of free-cancer sample.

Description

[0001] A portable device to detect Circulating Tumor Cells in blood sample with colorable test strip

[0002] Technical Field

[0003] Discovering tumor cell in stage 0 with 10 cm3blood sample with test strip changes its color according to the presence or absence of tumor cells

[0004] Background

[0005] Circulating Tumor Cells (CTCs) are sheds spreading into the vascular or lymphatic from primary tumor and are carried around the body through blood circulation.

[0006] “CTCs” is a very early biomarker of tumor formation in Epithelial stage which can extravasate and become a seed for tumors in other organs.

[0007] Liquid biopsy is the ideal way to detect the CTCs in blood samples. It is non- invasive method and could be repeated several times for: a) periodical checks b) pursuing medical progresses c) follow up recovery during convalescence period.

[0008] For the prior art, there are many techniques like ScreenCell®, Parsortix™, Galleri® and university of Missouri Tech but the only FDA approved device for MCED device is CELLSEARCH®(Fig 2-A) and because of its superiority in this field, we will consider it only previous art.

[0009] Critical prior art considerations

[0010] Cell Search® Limitations

[0011] Device avoids CellSearch®’s limitations:

[0012] No reliance on cytokeratin staining

[0013] 20x lower cost (target <$50 vs. $1,000)

[0014] 95% recovery rate at 5 CTCs / 7.5mL blood

[0015] 2. Differentiation from ScreenCell

[0016] Novelty lies in: Magnetic enrichment vs. ScreenCell’s size-only filtration • Colorimetric quantitation vs. manual microscopy

[0017] 3. University of Missouri Tech

[0018] Complementary: This device captures CTCs, while Missouri’s miRNA detection could be combined for molecular analysis.

[0019] As shown in fig (1), the steps start with liquid biopsy from the patient followed by enrichment with antigen-antibody named Ep-CAM (Epithelial Cell Adhesion Molecule) which -as name indicates- phenomenally adheres to the CTCs in Epithelial stage. The latest studies support the Ep-CAM effectiveness and efficiency of adhering most of metastasis in epithelial stage as shown in the next table: The test package contains the antigen mixed with SPIONs (Superparamagnetic Iron Nanoparticles) which has the ability to adhere to the antigen; the antigen adheres to the CTCs and SPIONs adhere to the antigen.

[0020] The “enrichment” process is an incubation of the blood sample with antigen. The mixture is then exposed to electromagnetic field (0.5T) to impower the magneticity of the ferric beads -which in this Nano form- can obtain it (magneticity) eightfold more than any other form. The next step is “isolation” which uses a normal magnet to attract the ferric beads that are attached to the antigen which consequently is attached to CTCs.

[0021] Now the device could easily fish the CTCs from the sample. The device will dye the collected CTCs and then operate many processes (DEP array- genome amplifier - RNA) and analyses these data to identify the cancer tumor type. This device is effective but with some disadvantages: very high cost (approx. 1000 USD), only CELL SEARCH® lab devices needed, time consuming process and special materials needed. More importantly, negative results cost are substantial for early detection with the tumor plus the processes that follow.

[0022] Disclosure of invention

[0023] The main principal of this invention is providing point of care (POC) for a large number of users with a low cost and portable mobile system mainly for TUMOR EARLY DETECTION.

[0024] The device depends on Test Strip system with color indication to give only three results, positive, negative and not sure without any referring to the type of the cancer tumor. The device contains a removable filter unit that will detain the CTCs which allows the patient of “not sure” and “positive” results to check it in pathology lab to identify the tumor type. Blood sample preparation:

[0025] Drawing 10 cm3of blood in EDTA tube to prevent coagulation and dilute it with an equal volume of PBS (Phosphate-Buffer Saline).

[0026] 1- Centrifuging for 10 min in 2000-3000 rpm.

[0027] 2- Separating the “Buffy Coat” which is containing the CTCs.

[0028] To explain the necessity of 10 cm3of blood; It is primarily due to the rare presence of the CTCs cells in blood-could be only one cell in 7.5 cm3- in stage 0 , therefore for more safety we will draw 10cm

[0029] Adding the antigen (HumanCD326 (Ep-CAM) Capture Beads for Flow Detection from Immunostep® Co. SKU:326CB-25) to the “buffy Coat” which contains the SPIONs and start the incubation process for 30-60 min at room temperature as shown in Fig (2-B) the CTCs (2) will attract the antigen (3) while the antigen attracts the ferric nano particles (4) and to complete incubating we must expose this cell (2+3+4) to magnetic field (5) in vibrator for at least 10 min.

[0030] 3- Preparing a syringe containing the incubated mixture to be injected into the device.

[0031] The device description:

[0032] As shown in Fig (3), the device (6) is with dimensions of 13><6x6cm (L, W and H) with three main holes. The first hole is to receive the removable filter (Disposable polycarbonate membrane filter with 0.5-1 pm pores) (7), the second is for the collecting drawer(8) which containing - the drawer- two slot holes one for collecting the test residual (9) and the other (10) to insert the test strip in it.

[0033] The third hole (11) is for Hydrochloride acid container (12). there is a DC input (13) and on / off switch (14). As long as we use an acid, we need a mixing tank (15) with high acidity resistivity material like PTFE (polytetrafluoroethylene).

[0034] There is a microcontroller board (16) to operate a simple task. The main function of the controller is to control the fluid flow throw the filter and the valves (17, 18) via microcontroller-regulated peristaltic pump maintaining flow rate < Iml / min to prevent CTC lysing during filtration and to provide washing cycle after every test.

[0035] To operate the device,

[0036] 1- Insert the filter tightly in the hole, insert the drawer to the end in its slot and carefully insert the HCI acid container in its hole.

[0037] 2- Fix the injection containing the sample in the filter inlet.

[0038] 3- Push START button (14) and wait until finish buzzer, in meanwhile, the controller will open the valve (18) to allow passing a calculated amount of acid and close it to mix with the sample while valve 17 is closed till the reaction finished (5 seconds) and then controller will open valve (17) to free the sample in the drawer through the hole (9).

[0039] 4- Pick-up the drawer (8) and insert the test strip in its hole (10) and wait for 5 seconds and then remove it from the drawer.

[0040] Test Strip preparation:

[0041] The preparation of test strip will need some special process because it contains two elements of reagents for accuracy and visual verification

[0042] As shown in Fig (4), the base plastic strip (18) will carry two arrays of different reagents (19) and (20) with another plastic cover (21) with holes exactly over the pieces of reagents to concentrate the mixture in defined spots.

[0043] The square pieces of reagents (19 & 20) are essentially made of filter paper dipped in chemical materials and dried well and then catted into small square pieces and organized as shown in Fig (5).

[0044] The chemical preparation of dual reagents material:

[0045] The first array of reagents containing potassium thiocyanate (KSCN) dissolved in sterilized waterand left to dry completely Fig (5).

[0046] The second is containing potassium ferrocyanide, (K4[Fe(CN)e]) then we collect the dried filter papers and cutting them in square shape to array them as shown in figure to finally have a dual-reagent test strip. The main purpose of using these materials is their sensitivity for ferric ions (Fe3+), so, for potassium ferrocyanide reaction with ferric oxide give us a clear “Prussian Blue”and for potassium thiocyanate the result is “BloodRed” as shown in Fig (6 ,7). The sensitivity of the two compositions differ one from another -for very important reason will show later- as the next table show

[0047] The theory of operation:

[0048] The antigen used for this test (HumanCD326 (Ep-CAM) Capture Beads for Flow Detection from Immunostep®Co. SKU:326CB-25) containing around 6000 beads of nano ferric particles in size of 5nm (from the product data sheet) for each bead and as we explained before, if there are tumor cells, then the beads of iron will be collected (totally or partially) by the CTCs and the antigen in standard operation. So far we are on the CELLSEARCH® pattern for first step operations.

[0049] The size of CTCs is 6-12pm while the ferric beads are in range of 5-10 nm and the antigen are 3-7nm which means the CTCs is bigger in size than the ferric beads and antigen approx. 1000 times.

[0050] The removable filter used is pore size <lpm which means it will detain the CTC from passing throw it, meanwhile the ferric beads and antigen will pass.

[0051] If the sample is positive and containing tumor cells, so, the all beads attached to the antigen will stick and adhere to the CTCs and in this case, the filter will prevent the CTCs with its load from passing.

[0052] Consequently, the mixture in the collecting tank is free of ferric ions and the acid will not react with anything in this mix.

[0053] The potassium thiocyanate and ferrocyanide react only with ferric ions Fe3+to produce red and blue color respectively. Since, the mix produced from the device not containing any ferric ions, there will be no reaction when inserting the test strip which will remain blank.

[0054] Now if the sample is free of tumor cells (negative), so the antigen will not find any cells to attached with and the nano size ferric beads will pass solely or with the antigen throw.

[0055] The Hydrochloric acid will oxidize the ferric ions to make sure of changing any Fe2to Fe3+and when we insert the test strip, the ferric ions will react with potassium thiocyanate and ferrocyanide to coloring the strip with red and blue according to the chemical reactions as follow:

[0056] Now, the device could differentiate between cancer tumor samples and free of tumor sample with conclusive colorimetric instant method, the defected sample will give nothing on the test strip while the good sample will give colored (red and blue) test strip

[0057] Execution problems & solution:

[0058] 1 -During laboratory experiments, we start with only one reagent (potassium ferrocyanide) and when noticed that -in positive cases- the antigen doesn’t catch all nano-particles beads with a leakage which gave false negative results, so, to increase the test reliability we design the test strip to measure the concentration of ferric ions.

[0059] With experiments, the concentration should be expressed in degreed level meter to calculate every zone and what its indication. The reason of using two reagents with different sensitivity degree divided in ladder steps shape is to separate every different colormatic result to be translated into a decision. In addition, the results are interpretable via smartphone camera using RGB thresholding algorithm for the uncertain results.

[0060] For example, the concentration will appear clearly on the potassium Thiocyanate parts as red color, it will start very bright in first pieces and eventually degrade and fade, piece after piece while the potassium ferrocyanide will not respond which means a very low ferric concentration because of the super sensitivity of thiocyanate (0.05ppm) which could be interpreted as positive result with leakage.

[0061] On the other hand, bright red and blue along all pieces in both reagents zone means a high concentration of ferric ions which means sure negative results while degraded red and degraded blue means “not sure” results.

[0062] The using of two reagents divided in parts along the test strip allow the test to measure any tiny changes to raise the integrity of the test and with more lab experiments and statistical, we can estimate the stage of metastasis according to the test strip multi-color results.

[0063] 2- The reagents may react with other blood components to give a false result, so, we use the buffy coat to avoid the Fe atom in RBC Hemoglobin which could hemolyzed with HCL acid, also other component like Zink could react with test strip, so, we use a smaller pore size filter to ensure unwanted elements not pass (approx. 0.45pm pore size) .Also using Potassium thiocyanate is very important because it doesn’t react with any of the blood components except Ferric ions to give the red color.

Claims

Claims1. A point-of-care portable device comprising: (i) a bufferless filtration chamber, (ii) pH-stabilized HC1 reservoir (0.1-0.5M), (iii) laminated test strip with KSCN / K ,| l e( C\ ] reagents.

2. A portable device as in claim 1 comprising of a mixer tank to mix the blood sample with chemicals to produce a testable liquid to discover absence or presence of SPIONs which refers indirectly to cancer cell in the sample.

3. As in claim 2, the anti-Ep-CAM-conjugated SPIONsexist in test package in preparing the sample with the ability to adhere with cancer tumor.

4. As in claim 3, the SPIONs will be magnetized to stick to the antigen to consist a new composition of tumor cell, antigen and ferric ion all gather.

5. As in claim 1, the device contains a removable filter "Disposable polycarbonate membrane filter with 0.5-1 pm pores, configured to retain CTCs (>6 pm) while allowing unbound nanoparticles to pass” and compatible with fluorescence in Situ hybridization (FISH) and RT-PCR analysisRationale: Pore size critical for size-based CTC enrichmentto detaining the tumor cells if the sample contain cancer.

6. As in claim 5, the filter could be removed in case of positive to cancer to analyzed in pathology lab to identify the type and stage of tumor.

7. As in claim 1, the test strip comprised of two reagent materials with different degree of sensitivity to give an accurate result. A ladder-type test strip comprising:(a) Potassium thiocyanate (KSCN) zones with detection threshold of 0.05 ppm Fe3+,(b) Potassium ferrocyanide (K4[Fe(CN)e]) zones with threshold of 0.5 ppm Fe3+, wherein color gradients across zones correlate inversely with CTC presencewherein test strip results are interpretable via smartphone camera using RGB thresholding algorithms.Rationale: Quantifiable thresholds address false negatives / positives.

8. As in claim 1, the device containing a microcontroller - regulated peristaltic pump maintaining flow rate <1 ml / min to prevent CTC lysing during filtration ioRationale: Flow control for CTC viabilityIndependent ClaimsA portable CTC detection system comprising: a) Ep-CAM-conjugated SPIONs (5-10nm) for magnetic enrichment b) Disposable 1pm pore filter cartridge c) Ladder-type test strip with KSCN / K4[Fe(CN)e] zones d) Microcontroller regulating flow rate <lmL / minA method for CTC detection a) Centrifuge lOmL blood at 2, 000-3, 000g for buffy coat isolation b) Incubate with SPIONs under 0.5T magnetic field c) Filter through 1pm membrane d) React filtrate with HC1 and detect Fe3+via colorimetric strip

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