Test device and method for determining hematocrit and / or fibrinogen in a blood sample

Abstract

The present invention relates to a method for determining a hematocrit level in a blood sample comprising the steps of a ) providing a lateral flow device comprising a blood separation pad, b ) applying a blood sample on the blood separation pad of the lateral flow device, and c ) determining the hematocrit level by comparing the ratio of red blood cells travel distance to the total travel distance on the blood separation pad with a reference ratio.

Description

[0001] TEST DEVICE AND METHOD FOR DETERMINING HEMATOCRIT AND / OR FIBRINOGEN IN A BLOOD SAMPLE

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of blood analysis .

[0004] BACKGROUND ART

[0005] Lateral flow assays ( LFAs ) are rapid, inexpensive , and easy-to-use without the need of any additional equipment since all reagents are stored within membranes on the device itsel f . The origin of lateral flow tests goes back to the 1950s when paper-based dipstick tests for glucose measurements in urine were developed and in parallel , latex agglutination assays and radioimmunoassay, pioneers of the detection mechanism, emerged . The first LEA test , like it is known today, was developed in the 1980s for analyzing the presence of human chorionic gonadotropin (hCG) to confirm pregnancy . Since then, lateral flow tests have become success ful analytical devices for point-of-care testing in various application fields , ranging from the detection of disease biomarkers , pathogenic bacteria, viruses , mycotoxins to chemical contaminants like veterinary drug residues or pesticides . Since the SARS-CoV-2 outbreak in 2019 , LFAs are one of the most used diagnostic tools because they have been established as a key measure to control and monitor virus spread and implement quarantine measures .

[0006] Fibrinogen plays an important role in maj or hemorrhage . During a sustained traumatic inj ury, f ibrinogen-supplementa- tion could decrease the risk of mortality in trauma patients . Therefore , accurate and rapid tests determining the fibrinogen concentration are needed, especially in emergency settings .

[0007] So far, developed PoC devices for the detection of fibrinogen mainly rely on coagulation . As an example , thrombin dried in the sample application port of a paper-based device is released after blood application and results in fibrin formation . Consequently, the distance the blood travelled depends on the fibrinogen concentration . In a similar approach, the paper strip is placed in a reservoir with blue dye and the elution height depends on the fibrinogen concentration . In order to detect fibrinogen directly from blood, it is proposed mixing blood with thrombin, placing the droplet on a slide and allowing the reaction to happen . After that , a paper strip is added and the wicking length measured since the wicking length depends on fibrin formation, which further depends on the fibrinogen concentration . Another approach used screen printed paper electrodes to separate blood by dielectrophoretic force and after that, to measure the fibrinogen concentration based on a resistance change after thrombin application and thus , fibrin formation .

[0008] Hematocrit may af fect the results obtained by fibrinogen measurements from blood . A high hematocrit means a lower amount of plasma which passes the detection zone . Hence , it is advantageous to determine the hematocrit in the sample as well .

[0009] Methods and devices for determining the hematocrit level in a blood sample are well known in the art . Usually, blood is separated into erythrocytes and plasma through a membrane . The hematocrit level correlates with the travel distance of the erythrocytes and is correlated to a calibration curve ( e . g . JP 2019- 174145 A and US 2010 / 0203578 Al ) .

[0010] It is an obj ect of the present invention to provide a method for determining the hematocrit level in a blood sample .

[0011] SUMMARY OF THE INVENTION

[0012] The present invention relates to a method for determining a hematocrit level in a blood sample comprising the steps of a ) providing a lateral flow device comprising a blood separation pad, b ) applying a blood sample on the blood separation pad of the lateral flow device , and c ) determining the hematocrit level by comparing the ratio of red blood cells travel distance to the total travel distance on the blood separation pad with a reference ratio .

[0013] Blood applied / dropped directly or via a sample loading pad on a blood separation pad spreads through the pad from the application / dropping position. Thereby, erythrocytes (i.e. red blood cells) are separated from plasma and other blood components. Since erythrocytes show a red colour and plasma and other blood components have another colour the running fronts of the erythrocytes and of the plasma and other blood components are visible on the blood separation pad. Of course, the colour of the blood separation pad shall not interfere with the colours of the different blood components.

[0014] The running front of the blood components on the blood separation pad allows to determine the travel distance of the erythrocytes and the plasma on the blood separation pad from the application / dropping position after a predetermined time. The travel distance of the plasma from the application / dropping position after the same predetermined time is the total travel distance.

[0015] The ratio of the travel distance of the erythrocytes of a blood sample applied on a blood separation pad ( IRBC) to the total travel distance (i.e. running front of the plasma) ( I O- tai) correlates with the hematocrit level of the blood sample. Due to this correlation, the ratio IRBc / l otai obtained from a blood sample indicates its hematocrit level.

[0016] The correlation between the ratio IRBc / l otai and the hematocrit level has to be determined for each blood separation pad with a reference method, since the spreading velocity of blood components within a blood separation pad depends on the material of the blood separation pad. Of course, the applied blood volume and the running time have to be identical, too. A reference method to determine the correlation between the IRBC / I O- tai ratio and the hematocrit level involves a hematology analyzer or is the microhematocrit method (CLSI (2000) Procedure for Determining Packed Cell Volume by the Microhematocrit Method; Approved Standard (3rd edn) CLSI document H7-A3 [ISBN 1-56238-413-9] ) . Once the blood separation pad specific IRBc / l otai ratio has been determined and correlated to the hematocrit level determined with a reference method, said ratio can be used as a reference ratio in the method of the present invention. This method ensures comparability between individuals and with other hematocrit measurements carried out in the laboratory, which further simpli fies the interpretation by medical personnel .

[0017] Another aspect of the present invention relates to a method for determining fibrinogen in a blood sample and / or whether a blood sample comprises more or less than 1 . 5 mg / ml fibrinogen comprising the steps of a ) providing a lateral flow device comprising a blood sample loading pad fluidly connected to an optional conj ugate pad, a detection pad and an absorbance pad in series , wherein the conj ugate pad comprises labelled fibrinogen-binding molecules and the detection pad comprises at least two areas separated from each other comprising fibrinogen-binding molecules immobili zed thereto , b ) applying a blood sample on the blood sample loading pad of the lateral flow device , and c ) determining the presence of fibrinogen in the blood sample , when labelled fibrinogen-binding molecules bind to at least one of said at least two areas and / or the presence of more than 1 . 5 mg / ml fibrinogen in the blood sample , when labelled fibrinogen-binding molecules bind to at least two of said at least two areas .

[0018] Low levels of fibrinogen in blood result in various disorders like prolonged and / or spontaneous bleeding, easy bruising, especially after an inj ury or surgery and pregnancy complications . People having low fibrinogen levels are also more susceptible to free- floating clots that block blood vessels since fibrin, a breakdown product of fibrinogen, is not present to inhibit the formation of internal clots . Fibrinogen levels may drop as a result of traumatic inj uries and blood loss , liver diseases , leukemia, medication or genetic disorders . Current guidelines recommend fibrinogen replacement when fibrinogen levels drop below 1 . 5 mg / ml . I f such a drop occurs in patients plasma products are commonly used to increase fibrinogen levels in acute settings . The method of the present invention allows determining fibrinogen in a blood sample , in particular it allows determining whether a blood sample comprises more or less than 1 . 5 mg / ml fibrinogen . Hence , the method of the present invention is helpful to identi fy patients needing the supplement of fibrinogen .

[0019] The method of the present invention for determining fibrinogen in a blood sample is particularly advantageous since it allows to determine the presence of fibrinogen in a blood sample and in addition thereto it is possible to determine whether the level of fibrinogen within the blood sample is higher or lower than 1 . 5 mg / ml , which is the information which is required to decide whether a patient requires medication to increase the fibrinogen level .

[0020] A further aspect of the present invention relates to a device for determining a hematocrit level in a blood sample and for determining fibrinogen in a blood sample and / or whether a blood sample comprises more than 1 . 5 mg / ml fibrinogen comprising a blood separation pad or a first sample loading pad upstream of and fluidly connected to the blood separation pad and a second blood sample loading pad, wherein the second blood sample loading pad is fluidly connected to a conj ugate pad, a detection pad and an absorbance pad in series , wherein the conj ugate pad comprises labelled fibrinogen-binding molecules and the detection pad comprises at least two areas separated from each other comprising fibrinogen-binding molecules immobili zed thereto .

[0021] The device of the present invention allows to determine the hematocrit level and the fibrinogen level and / or whether a blood sample comprises more than 1 . 5 mg / ml fibrinogen in a blood sample . The measurement of both blood parameters on the same device ( simultaneously or in series ) is important since hematocrit may af fect the results of the determination of fibrinogen concentration in a blood sample . Because , i f the same volume of blood is applied, but the hematocrit di f fers , variations in the plasma volume passing the detection zone occur . A lower plasma volume means that less fibrinogen molecules pass the detection zone compared to a higher plasma zone, although the fibrinogen concentration is the same . Therefore, the hematocrit needs to be taken into account when measuring the fibrinogen concentration . Another aspect of the present invention relates to a method for determining a hematocrit level in a blood sample and for determining fibrinogen in a blood sample and / or whether a blood sample comprises more than 1 . 5 mg / ml fibrinogen comprising the step of a ) applying a blood sample on a blood separation pad or a first sample loading pad upstream of and fluidly connected to the blood separation pad and a second blood sample loading pad of the device of the present invention and b ) determining the hematocrit level by comparing the ratio of red blood cells travel distance to the total travel distance on the blood separation pad with a reference ratio and / or the presence of fibrinogen in the blood sample .

[0022] A further aspect of the present invention relates to the use of a device of the present invention for determining a hematocrit level in a blood sample and for determining fibrinogen in a blood sample and / or whether a blood sample comprises more than 1 . 5 mg / ml fibrinogen

[0023] A further aspect of the present invention relates to a data processing device comprising means for carrying out step c ) of the method of the present invention or step b ) of the method of the present invention .

[0024] Another aspect of the present invention relates to a computer program comprising instructions which, when the program is executed by a computer, cause the computer to carry out step c ) of the method of the present invention or step b ) of the method of the present invention .

[0025] BRIEF DESCRIPTION OF THE FIGURES

[0026] Fig . 1 shows the hematocrit test of the present invention . A) Schematic for the used method to characteri ze dif ferent sample pads . B) Plasma distance of di f ferent sample pads ( GF, GX, GR, LF1 ) (width = 3mm) after application of 20 pl blood (n = 10 ) . C ) Schematic of the working principle of the hematocrit PoC test ( 1RBC = travel length of red blood cells ; Itotai = travel length of red blood cells and plasma ) . D) Correlation between hematocrit PoC test and hematocrit determined by hematology analyzer. E) Hematocrit of blood samples determined with developed PoC hematocrit test (PoC) and hematology analyzer (Lab) .

[0027] Fig. 2 shows the characterization of detection pads' properties with buffer (Hank's Balanced Salt Solution) and plasma. A) Schematic of used method for characterization. Flow profile of different detection pads (AE99, RP, FP) of B) buffer and C) plasma. D) Comparison of travel distance after 35s (width = 3 mm) . E) Effect of membrane width (detection pad: RP) . F) Effect of blocking with bovine serum albumin (BSA) prepared in water (H2O) or Phosphate Buffered Saline (PBS) (detection pad: RP, buffer only) .

[0028] Fig. 1-3 shows A) a schematic for the used method to test the effectiveness of the conjugation of gold nanoparticles (AuNP) with antibodies. If AuNP are modified with antibodies (AB) , the aggregation of AuNP in presence of NaCl is prevented. B) Spectrum scan of bare (blue) and antibody modified (red) AuNP in presence of NaCl . C) Effect of pH of conjugation buffer to the conjugation of antibodies with AuNP. D) Schematic for the used method to study release properties of conjugate pads. Conjugate pad is saturated with AuNP and an image is taken after the pad is dried. Next, stored conjugates are released and imaged again after the pad dried. The intensity difference describes the AuNP release. E) Conjugate release of different conjugate pads (F5, ST17, ST14) and conjugate release buffer (BB: 5 mM borate buffer pH 9 with 10% sucrose, PBST: phosphate buffered saline with 0.05% TWEEN® 20 and 10% sucrose) . (C, E) Statistical significance by Welch t-test *p<0.033, **p<0.002, ***p<0.001 (n = 9 (C) , n = 15 (E) from 3 independent experiments) .

[0029] Fig. 4 shows A) an ELISA with two different capture antibodies (clone KT9 and AB05-1F11) . B) Images of LEA tested with different fibrinogen concentrations. The second line (indicated with arrow) is used to determine the signal intensity. If the line appears, the fibrinogen concentration is below the threshold of 1.5 mg / ml. C) LEA tested with different fibrinogen concentrations. Line indicates threshold of 1.5 mg / ml. (n > 6 from 3 independent experiments ) . D) LFA tested with di fferent dilutions of reference plasma . Line indicates threshold of 1 . 5 mg / ml . (n = 6 from 2 independent experiments ) .

[0030] DESCRIPTION OF EMBODIMENTS

[0031] "Blood" , as used herein, which is used as a sample to determine a hematocrit level and / or the fibrinogen level refers always to whole blood . Hence , in the context of the determination of a hematorcrit level and the fibrinogen level , blood can be used interchangeable with whole blood .

[0032] The travel distance of the red blood cells and the plasma fraction of the blood sample can be determined using a scale and / or electronic means . I f electronic means are used these means are also able to discriminate between the running front of the red blood cells and the plasma . In case of the use of a camera to record a picture of the device of the present invention showing the running fronts , the device may comprise one or more reference points or a scale as well to facilitate the electronic determination of the travel distance .

[0033] The blood separation pad is made of an absorptive material having a void volume allowing the spread of blood and its components through the respective material . The void volume within the blood separation pad allows passing red blood cells and the plasma fraction with di f ferent velocities so that the running fronts of at least these two components can be made visible on the blood separation pad . Such blood separation pads are well known to the person skilled in the art .

[0034] According to a preferred embodiment of the present invention the blood separation pad comprises a porous material , preferably a fibrous material and / or a thermoplastic material .

[0035] According to another preferred embodiment of the present invention the fibrous material is a glass fiber material , more preferably bound glass fiber, and / or the thermoplastic material is polysul fone .

[0036] According to a further preferred embodiment of the present invention the porous material has a void volume of lx to 3x, preferably lx to 2 . 5x, more preferably 1 . 5x to 2 . 5x, more preferably 2x to 2 . 5x . According to a preferred embodiment of the present invention the blood separation pad has a thickness of 25 to 300 pm, preferably of 50 to 250 pm .

[0037] According to another preferred embodiment of the present invention the blood separation pad has a length ranging from 1 to 100 mm, preferably from 2 to 80 mm, more preferably from 3 to 60 mm, more preferably from 3 to 55 mm .

[0038] According to another preferred embodiment of the present invention the blood separation pad has a width ranging from 1 to 10 mm, preferably from 2 to 5 mm .

[0039] According to a particular preferred embodiment of the present invention the blood separation pad comprises an anticoagulant and / or the blood sample is treated before step b ) with an anticoagulant .

[0040] In order to avoid coagulation of the blood sample applied on the blood separation pad the blood separation pad itsel f comprises or the blood sample is treated before the blood application with an anticoagulant . I f no anticoagulant is present during the hematocrit level determination within the blood separation pad, there is a risk that the blood coagulates so that the test does not work .

[0041] According to another preferred embodiment of the present invention the anticoagulant is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA) , a citrate , heparin, hirudin and a direct thrombin inhibitor .

[0042] According to a further preferred embodiment of the present invention 5 to 100 pl , preferably 5 to 80 pl , more preferably 10 to 50 pl , more preferably 15 to 30 pl , of the blood sample are applied on the blood the blood separation pad or a sample loading pad upstream of and fluidly connected to the blood separation pad .

[0043] The device of the present invention to be used in the hematocrit level determination and in particular its blood separation pad is designed to allow the application of a relatively small amount of blood . Hence , the blood volume applied on the blood separation pad may range from 5 to 200 pl , preferably from 5 to 100 pl , more preferably from 10 to 50 pl , more preferably from 10 to 40 pl , more preferably from 10 to 30 pl , more preferably from 15 to 25 pl , more preferably from 18 to 22 pl , blood .

[0044] According to a preferred embodiment of the present invention the lateral flow device further comprises an optional blood sample loading pad fluidly connected to an optional conj ugate pad and a detection pad in series for determining fibrinogen in the blood sample and / or whether the blood sample comprises more or less than 1 . 5 mg / ml fibrinogen, wherein a part of the blood sample is applied to the optional sample loading pad .

[0045] In order to determine simultaneously or in series the fibrinogen in the blood sample and / or whether the blood sample comprises more or less than 1 . 5 mg / ml fibrinogen in the blood sample part of the blood sample is applied to an optional sample loading pad which is fluidly connected to further means for determining fibrinogen in a sample .

[0046] According to another preferred embodiment of the present invention the conj ugate pad comprises labelled fibrinogen- binding molecules and the detection pad comprises at least two areas separated from each other comprising fibrinogen-binding molecules immobili zed thereto .

[0047] Another aspect of the present invention relates to a method for determining fibrinogen in a blood sample and / or whether a blood sample comprises more or less than 1 . 5 mg / ml fibrinogen comprising the steps of a ) providing a lateral flow device comprising a blood sample loading pad fluidly connected to an optional conj ugate pad, a detection pad and an absorbance pad in series , wherein the conj ugate pad comprises labelled fibrinogen-binding molecules and the detection pad comprises at least two areas separated from each other comprising fibrinogen- binding molecules immobili zed thereto , b ) applying a blood sample on the blood sample loading pad of the lateral flow device , and c ) determining the presence of fibrinogen in the blood sample , when labelled fibrinogen-binding molecules bind to at least one of said at least two areas and / or the presence of more than 1.5 mg / ml fibrinogen in the blood sample, when labelled fibrinogen-binding molecules bind to at least two of said at least two areas.

[0048] The materials used in the pads of the device (i.e. blood sample loading pad, conjugate pad, detection pad and absorbance pad) are known in the art and used in lateral flow devices known in the art (Bahadir EB et al. TrAC - Trends in Analytical Chemistry 82 (2016) : 286-306; https: / / doi.Org / 10.1016 / .trac.2016.06.006) .

[0049] According to a preferred embodiment of the present invention fibrinogen-binding molecules are added to the blood sample before applying said sample to the sample loading pad. The fibrinogen-binding molecules are required for the readout of the performed test. The fibrinogen-binding molecules may be pre-labelled to enable an optical readout that can be analysed visually (e.g. by comparing with reference intensity lines) or by imaging the lines and automated line intensity analysis. Alternatively, the fibrinogen-binding molecules are labelled in a separate step for the optical readout.

[0050] According to another preferred embodiment of the present invention fibrinogen-binding molecules are antibodies or functional fragments thereof or apatmers or any other molecule binding to fibrinogen.

[0051] According to a further preferred embodiment of the present invention fibrinogen-binding molecules of the conjugate pad are labelled with gold nanoparticles, coloured latex beads, carbon nanoparticles, selenium nanoparticles, silver nanoparticles, quantum dots, organic f luorophores , polystyrene or poly (methyl methacrylate) (PMMA) particles, radioactive labels or chemiluminescent labels.

[0052] According to a preferred embodiment of the present invention the areas of the at least two areas closest to the conjugation pad comprises fibrinogen-binding molecules in a sufficient amount to bind approx. 1 mg to approx. 1.5 mg fibrinogen per ml blood applied to the blood sample loading pad.

[0053] According to a particular preferred embodiment of the present invention the detection pad comprises a control area upstream to the at least two areas , wherein the control area comprises immobili zed molecules binding to the fibrinogen- binding molecules or to fibrinogen .

[0054] According to another preferred embodiment of the present invention the immobili zed molecules binding to the fibrinogen- binding molecules are antibodies ( e . g . fibrinogen-binding mouse antibodies ) or functional fragments thereof .

[0055] Another aspect of the present invention relates to a device for determining a hematocrit level in a blood sample and for determining fibrinogen in a blood sample and / or whether a blood sample comprises more than 1 . 5 mg / ml fibrinogen comprising a blood separation pad or a first sample loading pad upstream of and fluidly connected to the blood separation pad and a second blood sample loading pad, wherein the second blood sample loading pad is fluidly connected to a conj ugate pad, a detection pad and an absorbance pad in series , wherein the conj ugate pad comprises labelled fibrinogen-binding molecules and the detection pad comprises at least two areas separated from each other comprising fibrinogen-binding molecules immobili zed thereto .

[0056] According to a preferred embodiment of the present invention the detection pad comprises a control area upstream to the at least two areas , wherein the control area comprises immobili zed molecules binding to the fibrinogen-binding molecules ( e . g immobili zed is an anti-mouse antibody) or fibrinogen .

[0057] Another aspect of the present invention relates to a method for determining a hematocrit level in a blood sample and for determining fibrinogen in a blood sample and / or whether a blood sample comprises more than 1 . 5 mg / ml fibrinogen comprising the step of a ) applying a blood sample on a blood separation pad or a first sample loading pad upstream of and fluidly connected to the blood separation pad and a second blood sample loading pad of the device of claim 19 or 20 and b ) determining the hematocrit level by comparing the ratio of red blood cells travel distance to the total travel distance on the blood separation pad with a reference ratio and / or the presence of fibrinogen in the blood sample .

[0058] A further aspect of the present invention relates to the use of a device of the present invention for determining a hematocrit level in a blood sample and for determining fibrinogen in a blood sample and / or whether a blood sample comprises more than 1 . 5 mg / ml fibrinogen

[0059] A further aspect of the present invention relates to a data processing device comprising means for carrying out step c ) of the method of the present invention or step b ) of the method of the present invention .

[0060] Step c ) of the method of the present invention can be carried out using a data processing device . The data processing device used herein uses data obtained, for instance, by a camera for determining the hematocrit level by comparing the ratio of red blood cells travel distance to the total travel distance on the blood separation pad with a reference ratio and / or by determining the presence of fibrinogen in the blood sample , when labelled fibrinogen-binding molecules bind to at least one of said at least two areas and / or the presence of more than 1 . 5 mg / ml fibrinogen in the blood sample , when labelled fibrinogen-binding molecules bind to at least two of said at least two areas . The data processing device executes a computer program / sof tware for determining these levels as described above for the manual determination .

[0061] Another aspect of the present invention relates to a computer program comprising instructions which, when the program is executed by a computer, cause the computer to carry out step c ) of the method of the present invention or step b ) of the method of the present invention .

[0062] The present invention is shown in more details in the following examples , however, without being restricted thereto .

[0063] EXAMPLE

[0064] Ma terial s

[0065] Phosphate buffered saline ( PBS ) , Hank' s Balanced Salt Solution (HBSS ) , 40 nm gold nanoparticles (AuNP ) , borate buf fer (0.5M, pH 9) , bovine serum albumin (BSA) and TWEEN® 20 and sucrose were purchased from Merck. 40 nm AuNP modified with streptavidin were obtained from Abeam. Cytiva provided us Whatman™ blood separator LEI, Whatman™ nitrocellulose membranes Immunopore FP, Immunopore RP, AE99 and the conjugate pads Whatman™ Standard 14 (ST14) , Standard 17 (ST17) and Fusion 5 (Fusion 5) . In addition, Pall provided the Vivid™ plasma separation membranes GF, GX and GR. The adhesive tape ARflow 90469® were procured from Adhesive Research. The antihuman fibrinogen monoclonal antibodies were either purchased from Thermo Fisher Scientific Inc., Bio-Rad Laboratories, Inc. or Absea Biotechnology Ltd. Fibrinogen was procured from CSL Behring and STA® - QUALI-CLOT I from Stage.

[0066] Whole blood sample preparation

[0067] To obtain plasma from whole blood samples, blood was centrifuged at 2500x g for 15 minutes at room temperature. After that, the plasma fraction was collected, and a second centrifugation step was carried out by applying 2500x g for 10 minutes. Remaining blood was split into Eppendorf tubes and centrifuged at lOOx g for 15 minutes. Blood samples with different hematocrit values were prepared by combining erythrocytes and pure plasma. In addition, the hematocrit values have been characterized by a cell counter OX-360 (Balio Diagnostics, Bidart) provided by the Ludwig Boltzmann Institute for Traumatology .

[0068] Detection pad characterization

[0069] Three nitrocellulose membranes (Immunopore FP, Immunopore RP, AE99) were prepared with three different widths (5 x 30, 3 x 50, 2 x 75 mm) . The membrane strips were immobilized on an adhesive tape (ARflow 90469®) . For determining the flow profile, 10 pl buffer (HBSS) or plasma was dropped on the membrane and a video was recorded for later data analysis using open-source image processing software FIJI.

[0070] For the blocking buffer, bovine serum albumin (0.1 and 1 w / v%) was dissolved in PBS and distilled water, respectively. Membrane strips of Immunopore RP with a width of 3 mm were immersed in the blocking solution for 15 minutes. After that, the strips were washed twice in PBS and water. The strips were dried overnight and then immobilized on ARflow 90469® before adding 10 gl HBSS. A video was recorded and data analyzed using FIJI .

[0071] Conjugation of gold nanoparticles with antibodies

[0072] For the conjugation of 200 gl gold nanoparticles (OD1) , 8 gl 0. IM borate buffer with pH 9 or PBS with pH 7.4 were added. Subsequently, 4 gl human fibrinogen monoclonal antibody with a concentration of 0.1, 0.5 or 1 mg / ml was added. This mix was incubated on a ThermoMixer C (Eppendorf SE) for 30 min at 700 rpm, followed by 15 minutes of centrifugation (1400 RCF) . The supernatant was discarded and gold nanoparticles were resuspended in 0.1% BSA w / v in PBS. A sample of gold nanoparticles was mixed with an equal amount of 10% w / v sodium chloride (NaCl) and absorbance was measured (spectrum scan with a step width of 5 nm) .

[0073] Conjugate pad characterization

[0074] Three different conjugate pads (ST14, ST17, Fusion 5) were prepared with a biopsy puncher (diameter 3 mm) . Commercially available gold nanoparticles conjugated with streptavidin were used for the release studies. The conjugates were centrifuged (1400 RCF, 10 min) and resuspended in PBST (PBS with 0.05% TWEEN® 20) or borate buffer (pH 9, 5 mM) with 10% sucrose, respectively, to obtain an optical density of 5 (OD5) . Then, each pad was wicked with 3 gl OD5 conjugate solution and dried overnight at room temperature. The next day, a test strip was assembled and release initiated with 10 gl HBSS. Images were taken before and after releasing the conjugates (as soon as pads were dried) . The intensity of the pads was analyzed with FIJI .

[0075] Sample pad characterization and PoC hematocrit test

[0076] Four blood separation pads (GF, GX, GR - Vivid™ plasma separation membrane from Pall; LF1 - Whatman™ blood separator from Cytiva) with a size of 3 x 55 mm were prepared using a paper cutter (Novus Dahle GmbH) and the strips immobilized on an adhesive tape, ARflow 90469®. For the blood uptake time, 20 gl blood were added with a pipette and the timer started after the pipette was emptied. As soon as no excess blood was visible anymore, the timer was stopped. To determine the plasma generation capability of the different pads, 20 pl blood was applied on the strip and images were taken as soon as the flow stopped. For the PoC hematocrit test, LF1 strips with a size of 3 x 30 mm were placed on an adhesive ARflow 90469®. To test the samples with different hematocrit values, 10 pl of each sample were applied onto the LF1 membrane using heparinized capillaries (Servoprax GmbH) . The length of red blood cell area and plasma were analyzed with FIJI.

[0077] ELISA

[0078] A high binding microplate (Greiner Bio-One International GmbH) was coated with 2 pg / ml capture antibody overnight and then blocked with 1% BSA. A dilution series of fibrinogen was incubated, followed by the incubation of 4 pg / ml biotinylated detection antibody. Then, streptavidin-HRP is incubated in order to analyze the presence of fibrinogen enzymatically. HRP converts 3, 3 ', 5, 5 ' -Tetramethylbenzidine and the absorbance was measured with the plate reader EnSpire 2300 (Perkin Elmer Inc.) . Between all incubation steps, wells were washed multiple times.

[0079] LEA preparation and testing

[0080] Fibrinogen capture antibodies were dispensed on the nitrocellulose membrane using the AD1520™ Aspirate Dispense System (BioDot, Inc.) . After that, the membrane was dried at 37°C for 1 hour, then blocked in 1% BSA for 15 min and washed twice in PBST. Finally, the membrane was dried for 1 hour at 37 °C and stored in the fridge until further use. For the LFA assembly, strips with a width of 2.5 mm were prepared and placed on an adhesive. On one end, conjugate pad with stored AuNP conjugates and the sample pad was added and on the other end, the absorbance pad was attached. For testing, different fibrinogen dilutions (2.5, 2, 1.5, 1, 0.5, 0.25 mg / ml) were prepared in PBS and reference plasma (STA® - QUALI-CLOT I) with a known fibrinogen concentration of 2.475 mg / ml was diluted with PBS. For the LFA, 10 pl sample (fibrinogen dilution or reference plasma) was added to an Eppendorf tube containing 20 pl PBS and after that applied to the LFA strip. For quantitative analysis, the LFA strips were imaged using a Molecular Imager® ChemiDoc™ XRS System (Bio-Rad Laboratories, Inc.) with the Image Lab Software. The line intensity was analyzed with FIJI.

[0081] Results

[0082] Hematocrit test

[0083] Pad material was tested to determine the pad' s ability to retain red blood cells, which is key for efficient fibrinogen labeling and detection. In a comparative study, a glass fiber separator (LF1) and three polysulfone membranes (GX, GF, GR) , varying in the void volume were analyzed.

[0084] The two membrane types differ in their separation mechanism since LF1 separates horizontally, whereas GX, GF and GR separate vertically. Figure 1A shows a schematic drawing of the method used. 20 pl blood are added and the plasma extraction was studied by measuring the running distance of the plasma. Besides determining the efficiency of the separation, the determined running distance of the plasma was used to adjust the pad sizes accordingly. Comparing the polysulfone membranes, GR generates the most plasma and is comparable to LF1 (Figure IB) .

[0085] In addition, the area covered with red blood cells is 44 + 2.6% for LF1 and 46 + 2.5% for GR, indicating a very efficient separation since the hematocrit of the blood sample was determined to be 41%.

[0086] Although GR and LF1 are comparable in the separation efficiency, the LF1 was used for the hematocrit determination since it separates horizontally and thus blood and plasma is clearly visible, whereas the GR needs to be flipped to see the distance of the plasma. To determine the hematocrit, the same analysis method as applied in glass capillary hematocrit measurements was used, in which the capillary is filled with blood and the ratio of red blood cells determined after centrifugation. In the present PoC hematocrit test, blood was added to the sample pad, separated by the blood separation membrane and the ratio of red blood cells (IRBC) and total travel distance (Itotai) determined, as depicted in Figure 1C. This approach was characterized by measuring samples with different hematocrit and comparing it to the hematocrit values obtained by a hematology analyzer. As shown in Figure ID, the obtained linear correlation allows to determine the hematocrit accurately. For validation, the hematocrit of 4 unknown blood samples was measured and compared to the results obtained by using a standard laboratory method. The determined accuracy was of 95%.

[0087] Fibrinogen Lateral Flow Assay

[0088] Assay Design

[0089] To perform the test efficiently and rapidly with a fingerpick, a sample volume of 20 pl and a time-to-result of 10 minutes is aimed. The detection threshold should be 1.5 mg / ml because lower fibrinogen concentrations are critical for the patients and require an immediate change of the treatment.

[0090] In general, LFAs usually consist of 4 different pads facilitating distinct functions: (1) sample pad for sample application and preparation, (2) conjugate pad for storage of labeled detection reagents, (3) detection pad with immobilized capture probes for test and control line and (4) absorbance pad for soaking excess liquid. Thus, a set of membranes for each pad is selected in regard of sample matrix requirements. As an example, blood needs a membrane capable of separating red blood cells as well as a detection pad that is suitable for plasma. In addition, different membrane widths of the nitrocellulose membrane were studied since it effects the flow behavior. Furthermore, the assay format, sandwich or competitive, needs to be selected. A sandwich assay is more common for big molecules like antibodies or protein, whereas a competitive assay is used for small molecules or peptides. Fibrinogen has a molecular weight of 340 kDa, thus, a sandwich assay is preferred. Besides a LFA for the detection of fibrinogen, it is advantageous to integrate a method to determine the hematocrit of the blood sample. If the hematocrit is determined additionally, the result of the fibrinogen LFA is more accurate. Furthermore, the hematocrit gives valuable information about the patient's condition. To keep the applicability for the enduser in mind, the LFA and the hematocrit test can be integrated on the same device. Blood is applied and when the tests are finished, the results may be analyzed using a PoC reader (e.g. smartphone) .

[0091] Detection pad selection and flow modulation in nitrocellulose membrane

[0092] To immobilize capture probes at the detection pad, a nitrocellulose membrane is most used since it binds proteins efficiently. Three different nitrocellulose membrane, two with a plastic backing (RP and FP) and one unbacked (AE99) , were characterized with buffer and plasma by studying the effect of i) membrane composition, ii) geometry and iii) blocking solutions on the flow profile. Aliquots of 10 pl sample were used to simulate approximate plasma volume generated after red blood cell separation when 20 pl blood samples are applied. It is important to note that in the current testing set up, hydrostatic sample loading over a period of 5 sec is used instead of capillary forces present in the final LFA assembly (Figure 2A) . Results of the flow study are shown in Figures 2B and 2C where the distances of the flow front is determined visually every 10 seconds. While non-linear flow rates reaching a plateau after 60 sec is observed for all nitrocellulose membrane, higher flow rates are present for the unbacked membrane AE99. A direct flow rate comparison between buffer and plasma samples revealed that the flow rates of plasma is generally slower independent of the employed membranes (Figure 2D) , allegeable by the higher viscosity of plasma. The plasma travel distance of AE99 is 1.6- and 1.7-fold increased compared to RP and FP, respectively, although it is important to consider that AE99 is an unbacked membrane and was immobilized on a hydrophilic adhesive, which affects the flow rate. RP showed the lowest standard deviation (Figure 2D; 17.9 + 0.19 mm for RP compared to 28.1 + 3.56 mm for AE99 and 16.4 + 0.68 mm for FP) . To gain a deeper understanding of other factors modulating flow behavior such as geometry, the three nitrocellulose membrane types were prepared with 2, 3 and 5 mm width, respectively. The results demonstrate (Figure 2E) that there is a linear correlation between membrane width and flow rate. This means, in case of small sample volumes, such as in fingerpick applications, a deliberate reduction of the membrane width can be used to adjust total length of the assembled membranes within the LEA device. Since narrower test strips also facilitate a faster flow profile, the interaction time between the analyte and the immunocon ugate is reduced. The addition of blocking agents such as bovine serum albumin (BSA) is a commonly used for membrane pretreatment to avoid unspecific adsorption. RP nitrocellulose membrane is soaked in solutions with increasing BSA concentrations of 0 % - 0.1% - 1% and the travel distances after 35 sec was recorded. Figure 2F reveals that the blocking decreases the speed of the flow 35% and 43% for 0.1% and 1% BSA in water respectively. In contrast, BSA dissolved in PBS reduces the flow rate even more (59% and 64% for 0.1% and 1% BSA) , indicating that the salt has an additional blocking effect. This points out that pre-treatment of the membrane can not only be used to reduce unspecific adsorption, it is also a way to modulate the flow profile.

[0093] Conjugation of gold nanoparticles and conjugate pad release

[0094] Gold nanoparticles (AuNP) are widely used in LFAs for an optical read-out and need to be modified with detection antibodies beforehand. AuNP aggregates in the presence of a high salt concentration result in a color shift from red to blue. However, if antibodies or proteins adsorbed to the gold nanoparticles, aggregation is not possible anymore. Therefore, absorption spectra of bare (blue trace) and fibrinogen antibody decorated (red trace) 40 nm AuNP were recorded in presence of sodium chloride, exhibiting an absorption maximum of 530 nm of the none-aggregated nanoparticles (see Figure 3B) . Since the conjugation efficiency improves when the pH is close or slightly above the isoelectric point of the protein, different pH values were tested. Here, pH 7.4 (PBS) and pH 9 (borate buffer) were compared and it seems that more stable conjugates can be obtained with pH 9 (Figure 3C) .

[0095] Conjugate pad materials were compared regarding efficient release of AuNP conjugates as soon as the sample enters the pad. In total two glass fiber pads (ST14 and ST17) and a proprietary material Fusion 5 (F5) were tested using commercially available gold nanoparticle streptavidin conjugates as low- cost model conjugate. For that, the conjugates within the conjugate pad were dried and then the intensity of the conjugate pad before and after the release was analyzed (Figure 3D) . In addition, borate buffer (BB) and PBS with TWEEN® 20 (PBST) , both supplemented with 10% sucrose since carbohydrates are added to increase antibody stability and efficient conjugate release, were compared. As shown in Figure 3E, the highest intensity difference (before and after the release) was observed for ST17, and PBST releases the conjugates significantly better than the borate buffer in all cases (1.4-fold higher release for ST17 ) .

[0096] Fibrinogen LFA development

[0097] Suitable antibody pairs for the sandwich assay were tested using ELISA. Two different capture antibodies with a biotinylated detection antibody were tested. As depicted in Fig. 4A, both antibody pairs tested gave good results, however, AB05- 1F11 resulted in slightly higher absorbance values. By transferring the assay to the LFA, it was observed that blocking of the membrane is advantageous since fibrinogen may bind to the membrane and AuNP may be captured immediately after the release from the conjugate pad. However, blocking of the membrane decreased the flow rate and due to the abundance of fibrinogen, the membrane was clogged at high fibrinogen concentration. Consequently, the sample was diluted 3-fold prior to sample application in order to lower the protein concentration and ensure proper fluid flow. Furthermore, for high fibrinogen concentrations, lower band intensities were observed (Fig. 4B) , however, if sample and conjugate were added separately, the line appeared. This indicates that due to the high fibrinogen concentration, released AuNP conjugates as well as the immobilized capture antibody are saturated with fibrinogen. It seems that fibrinogen without conjugate travels faster than the conjugate and thus saturates the capture antibody. This hypothesis is supported by the fact that the intensity of the line depends on the line's position. Lines close to the conjugate pad, result in a higher intensity, compared to lines further downstream. Thus, two identical lines were integrated in the LFA to observe a line close to the conjugate pad when fibrinogen is present, and use the second line do determine the fibrinogen concentration due to the concentration dependent line intensity decrease. First, the LFA was tested with different fibrinogen dilutions and, as depicted in Figure 4C, fibrinogen concentrations of 0.25 and 0.5 mg / ml were below the set threshold of 1.5 mg / ml, whereas, 1 mg / ml was still in the threshold's range. Since an increase in matrix complexity results in signal intensity changes, the LFA was validated with reference plasma, which has a known fibrinogen concentration of 2.5 mg / ml. It was determined that 0.25, 0.5 and 1 mg / ml fibrinogen were below the threshold and proved the applicability of the developed LFA. Thus, the developed LFA can be used to determine fibrinogen concentrations below a set threshold of 1.5 mg / ml using imaging and data analysis, which should be integrated in an application for a PoC reader in a next step.

Claims

CLAIMS :1 . A method for determining a hematocrit level in a blood sample comprising the steps of a ) providing a lateral flow device comprising a blood separation pad, b ) applying a blood sample on the blood separation pad of the lateral flow device , and c ) determining the hematocrit level by comparing the ratio of red blood cells travel distance to the total travel distance on the blood separation pad with a reference ratio .2 . The method according to claim 1 , wherein the blood separation pad comprises a porous material , preferably a fibrous material and / or a thermoplastic material .3 . The method according to claim 2 , wherein the fibrous material is a glass fiber material , more preferably bound glass fiber, and / or the thermoplastic material is polysulfone .4 . The method according to claim 2 or 3 , wherein the porous material has a void volume of lx to 3x, preferably lx to 2 . 5x, more preferably 1 . 5x to 2 . 5x, more preferably 2x to 2 . 5x .5 . The method according to any one of claims 1 to 4 , wherein the blood separation pad has a thickness of 25 to 300 pm, preferably of 50 to 250 pm .6 . The method according to any one of claims 1 to 5, wherein the blood separation pad has a length ranging from 1 to 100 mm, preferably from 2 to 80 mm, more preferably from 3 to 60 mm, more preferably from 3 to 55 mm .7 . The method according to any one of claims 1 to 6, wherein the blood separation pad comprises an anticoagulant and / or the blood sample is treated before step b ) with an anticoagulant .8 . The method according to claim 7 , wherein the anticoagulant is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA) , a citrate , heparin, hirudin and a direct thrombin inhibitor .9 . The method according to any one of claims 1 to 8 , wherein 5 to 100 pl , preferably 5 to 80 pl , more preferably 10 to 50 pl , more preferably 15 to 30 pl , of the blood sample are applied on the blood the blood separation pad or a sample loading pad upstream of and fluidly connected to the blood separation pad .10 . The method according to any one of claims 1 to 9 , wherein the lateral flow device further comprises an optional blood sample loading pad fluidly connected to an optional conj ugate pad and a detection pad in series for determining fibrinogen in the blood sample and / or whether the blood sample comprises more or less than 1 . 5 mg / ml fibrinogen, wherein a part of the blood sample is applied to the optional sample loading pad .11 . The method according to claim 10 , wherein the conj ugate pad comprises labelled fibrinogen-binding molecules and the detection pad comprises at least two areas separated from each other comprising fibrinogen-binding molecules immobili zed thereto .12 . A device for determining a hematocrit level in a blood sample and for determining fibrinogen in a blood sample and / or whether a blood sample comprises more than 1 . 5 mg / ml fibrinogen comprising a blood separation pad or a first sample loading pad upstream of and fluidly connected to the blood separation pad and a second blood sample loading pad, wherein the second blood sample loading pad is fluidly connected to a conj ugate pad, a detection pad and an absorbance pad in series , wherein the conj ugate pad comprises labelled fibrinogen-binding molecules and the detection pad comprises at least two areas separated from each other comprising fibrinogen-binding molecules immobili zed thereto .13 . Device according to claim 12 , wherein the detection pad comprises a control area upstream to the at least two areas , wherein the control area comprises immobili zed molecules binding to the fibrinogen-binding molecules .14 . Method for determining a hematocrit level in a blood sample and for determining fibrinogen in a blood sample and / or whether a blood sample comprises more than 1 . 5 mg / ml fibrinogen comprising the step of a ) applying a blood sample on a blood separation pad or a first sample loading pad upstream of and fluidly connected to the blood separation pad and a second blood sample loading pad of the device of claim 12 or 13 and b ) determining the hematocrit level by comparing the ratio of red blood cells travel distance to the total travel distance on the blood separation pad with a reference ratio and / or the presence of fibrinogen in the blood s amp 1 e .15 . Use of a device of claim 12 or 13 for determining a hematocrit level in a blood sample and for determining fibrinogen in a blood sample and / or whether a blood sample comprises more than 1 . 5 mg / ml fibrinogen .16 . A method for determining fibrinogen in a blood sample and / or whether a blood sample comprises more or less than 1 . 5 mg / ml fibrinogen comprising the steps of a ) providing a lateral flow device comprising a blood sample loading pad fluidly connected to an optional conj ugate pad, a detection pad and an absorbance pad in series , wherein the conj ugate pad comprises labelled fibrinogen-binding molecules and the detection pad comprises at least two areas separated from each other comprising fibrinogen-binding molecules immobili zed thereto , b ) applying a blood sample on the blood sample loading pad of the lateral flow device , andc) determining the presence of fibrinogen in the blood sample, when labelled fibrinogen-binding molecules bind to at least one of said at least two areas and / or the presence of more than 1.5 mg / ml fibrinogen in the blood sample, when labelled fibrinogen-binding molecules bind to at least two of said at least two areas.

17. The method according to claim 16, wherein fibrinogen-binding molecules are added to the blood sample before applying said sample to the sample loading pad.

18. The method according to claim 16 or 17, wherein fibrinogen-binding molecules are antibodies or functional fragments thereof or apatmers .

19. The method according to any one of claims 16 to 18, wherein fibrinogen-binding molecules of the conjugate pad are labelled with gold nanoparticles, coloured latex beads, carbon nanoparticles, selenium nanoparticles, silver nanoparticles, quantum dots, organic f luorophores , polystyrene or poly (methyl methacrylate) (PMMA) particles, radioactive labels or chemiluminescent labels.

20. The method according to any one of claims 16 to 19, wherein the areas of the at least two areas closest to the conjugation pad comprises fibrinogen-binding molecules in a sufficient amount to bind approx. 1 mg to approx. 1.5 mg fibrinogen per ml blood applied to the blood sample loading pad.

21. The method according to any one of claims 16 to 20, wherein the detection pad comprises a control area upstream to the at least two areas, wherein the control area comprises immobilized molecules binding to the fibrinogen-binding molecules or fibrinogen.

22. The method according to claim 21, wherein the immobilized molecules binding to the fibrinogen-binding molecules are antibodies or functional fragments thereof.

23. A data processing device comprising means for carrying out step c) of the method of claim 1 or 16 or step b) of the method of claim 14.

24. A computer program comprising instructions which, when the program is executed by a computer, cause the computer to carry out step c) of the method of claim 1 or 16 or step b) of the method of claim 14.

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