WO2020157375A1

WO2020157375A1 - Sampling and assay kit and method for sampling a biological sample

Info

Publication number: WO2020157375A1
Application number: PCT/FI2020/050030
Authority: WO
Inventors: Sanna KAUSTARA; Juha Vartiainen
Original assignee: Aidian Oy
Priority date: 2019-01-31
Filing date: 2020-01-17
Publication date: 2020-08-06
Also published as: EP3917398A1

Abstract

The present invention is directed to a sampling and assay kit comprising a receptacle and a sample holder, the receptacle being for holding a predetermined volume of an aqueous liquid; the sample holder comprising a sampler; and a body portion, which holds the sampler and is insertable within the receptacle, wherein the body portion comprises projection means, wherein the receptacle has an internal projection such that when the body portion is inserted in the receptacle, the projection means of the body portion rests on said internal projection, so as to cause positioning of the sampler within the receptacle at a predetermined position, and wherein said predetermined position is such that, in use, the sampler is above said liquid so that the sampler is not in contact with the surface of said liquid.

Description

Sampling and assay kit and method for sampling a biological sample

FIELD OF THE INVENTION

This invention relates to the field of diagnostic techniques and sample collection. The invention provides a sampling and assay kit with improved reaction management as well as a method for controlled sampling of a biological sample. Particularly, the invention provides a novel sampling and assay kit for use in clinical tests on biological fluids, such as blood, said kit comprising a receptacle and a separate sampler holder which is insertable within said receptacle to a predetermined position.

BACKGROUND OF THE INVENTION

Typically, in diagnostic tests, biological fluid or tissue sample is collected and mixed with a buffer in a suitable receptacle in order to store the sample or for immediate analysis. For the latter purpose, a reagent in liquid or solid form may subsequently be added to the receptacle. For a quantitative assay, it is known in the art to provide sampling and assay kits which include sample receptacles with premeasured amounts of the reagent and the buffer. Also known in the art are sampling kits comprising an integrated sample-taking device with which errors in the precise sample dosing are substantially prevented. The analysis of the sample typically utilises an analyser equipment using optical measurement techniques.

WO 2015/177004 discloses a sampling and assay kit comprising a cuvette for holding a quantity of a buffer solution and a sample holder including a capillary tube for obtaining a sample. The sample holder includes a body portion which holds the capillary tube. The body portion when inserted in the cuvette is effective to position the capillary tube within the cuvette at a predetermined position above the end of the cuvette. In use of the sampling and assay kit, the sample holder is placed in the cuvette so that a sample within the capillary tube dilutes to the buffer solution in the cuvette. In order to avoid contamination of the sample, the sample holder is placed in the cuvette right after the collection of the sample from a patient. The subsequent measurement of the sample is based on a photometric, such as turbidimetric method. The cuvette is inserted in a test apparatus arranged to direct a beam of light, or radiation through the analyte present in the measuring zone in the cuvette. The amount of light or radiation passing through the analyte is measured by a detector and used to provide an analysis of the analyte. US 2012/0214251 discloses a test set for a photometric measuring device, consisting of a mixing container which receives a first reagent fluid in its interior and a closing element which is removable from its filling opening, and a dosing container which contains a reagent second fluid in a sealed hollow chamber, with the dosing container being insertable in a sealing manner into the filling opening of the mixing container, with the second fluid being conveyed into the interior of the mixing container by pressing a sealing plunger of the dosing container and being mixed with the same. For the purpose of simplifying the input of the sample, the dosing container comprises an integrated sample-taking device, such as a capillary, which after the insertion of the dosing container in the filling opening of the mixing container is in contact with the first fluid present in the mixing container. US 2012/0214251 also discloses a photometric measuring method including the steps of taking a sample fluid by means of a capillary fixed to the dosing container; inserting the dosing container into a mixing container; mixing of a first reagent fluid present in said mixing container with the sample fluid by shaking the mixing container; mixing a second reagent fluid with the previously obtained mix; and measuring the chemical reaction in a photometric analyzer.

US 5833630 discloses a sample collecting device comprising a receptacle (a cuvet) and a sample holder comprising a sampler (a capillary tube) and a capillary holder, which holds the sample holder when it is placed on the receptacle. The sample holder comprises a

circumferential rim by the aid of which, the sample holder rests on the rim of the receptacle. However, the sample is contacted with the reagent liquid in a receptacle by manually pressing a cap structure and thus the disclosed collecting device is not suitable for automated analysis of the sample.

EP1712180 discloses another sample collecting kit with a pressing button for manually contacting a collected sample with a reagent liquid.

In many diagnostic tests, a known problem is the deterioration of the sample before the actual analysis can be performed, i.e. there is an uncontrollable delay between the sample collection step and the start of the analysis step. Usually, this problem is handled by conditioning the sample by bringing it into contact with a suitable buffer and instructing the user to perform the analysis as soon as possible after the sample collection. However, this is not always practical and in some cases difficult to arrange. A further problem is that in some tests, particularly those based on reaction rate measurements, the time point for initiation of the reaction must be controllable. The present invention is directed to a novel sampling and assay kit which addresses these problems of the prior art and provides improved reaction control, particularly improved sample management, with a special combination of a receptacle and a separate sample holder which is insertable within said receptacle to a predetermined position.

SUMMARY OF THE INVENTION

In the present invention, the inventors surprisingly found that in assay kits comprising a test receptacle, such as a cuvette, holding a predetermined volume of an aqueous liquid, the presence of said liquid adds sufficient humidity inside the receptacle to prevent deterioration such as drying of the sample placed above the surface level of said liquid within said receptacle. The present invention thus renders possible improved sample management as the sample can be stored safely for a period of time within the test receptacle before performing the assay and the time point for mixing the stored sample with the assay reagents can easily be controlled without conditioning the sample.

In order to achieve the effects of the present invention, one aspect of the invention is to provide a sampling and assay kit comprising a receptacle and a sample holder: the receptacle being for holding a predetermined volume of an aqueous liquid; the sample holder comprising: a sampler; and a body portion, which holds the sampler and is insertable within the receptacle, wherein the body portion comprises projection means, wherein the receptacle has an internal projection such that when the body portion is inserted in the receptacle, the projection means of the body portion rests on said internal projection, so as to cause positioning of the sampler within the receptacle at a predetermined position, and wherein said predetermined position is such that, in use, the sampler is above said aqueous liquid so that the sampler is not in contact with the surface of said liquid.

Another aspect of the invention is to provide a method for sampling a biological sample with a sampling and assay kit, wherein said kit comprises a receptacle and a sample holder: the receptacle holds a predetermined volume of an aqueous liquid; the sample holder comprising: a sampler; and a body portion, which holds the sampler and is insertable within the receptacle, wherein the body portion comprises projection means, wherein the receptacle has an internal projection such that when the body portion is inserted in the receptacle, the projection means of the body portion rests on said internal projection, so as to cause positioning of the sampler within the receptacle at a predetermined position, and wherein said predetermined position is such that, in use, the sampler is above said liquid so that the sampler is not in contact with the surface of said liquid, the method comprising the steps of:

- collecting a sample with the sampler of said sample holder; and

- inserting the sample holder into the receptacle so that the sampler rests above the surface of a liquid in said receptacle.

A further aspect of the invention is to provide a use of a sampler holder for sampling a biological sample, wherein said sample holder comprises: a sampler; and a body portion, which holds the sampler and is insertable within a receptacle, wherein the body portion comprises projection means, wherein the receptacle has an internal projection such that when the body portion is inserted in the receptacle, the projection means of the body portion rests on said internal projection, so as to cause positioning of the sampler within the receptacle at a predetermined position, and wherein said predetermined position is such that, in use, the sampler is above a predetermined volume of an aqueous liquid present in said receptacle so that the sampler is not in contact with the surface of said liquid.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an example of the sample holder of the present invention comprising a body part 1 in which a capillary tube 5 is mounted. The top of the body part 1 is formed with two projections in the form of shoulders 3A, 3B whose function is to place the sample holder into a receptacle such as a cuvette to a predetermined position. An indented area 4 is defined between the shoulders 3A, 3B. The sample holder also comprises opening 2. It will be appreciated that the capillary 5 may be molded with the body part 1 as a single part, as an alternative to being mounted in the body part 1.

Figure 2 shows the sample holder of Fig. 1 after it has been inserted in the cuvette 6. The cuvette 6 is prefilled with an aqueous liquid 8. The cuvette 6 has an inwardly facing ledge 9 on which, in use, the shoulder portions 3A, 3B of the sample holder of Fig. 1 rest, so as to maintain the sample holder of Fig. 1 accurately in position in the cuvette 6, with the end of the capillary tube 5 being positioned above the liquid 8 within the cuvette 6 to enable at least short-term storage of the sample before the sample is mixed with the liquid 8. The measuring zone 7 for photometric measurement of the sample mix is at the base of the cuvette.

Figure 3. Comparison of immunoturbidimetric detection of HbAlc from whole blood in the known system of WO 2015/177004 and the present invention to demonstrate the effect of the delay between insertion of the blood filled sample holder into the cuvette and the start of the analysis step. The skeletal box-plot of the results shows median as a bold line, 1st to 3rd quartile as a box and whiskers with end caps extending to the minimum and maximum. The 0 % difference is indicated with a line. For the known system and the present invention multiple samples were measured in replicates at 0 s and 300 s and averages of the replicates were calculated. Relative differences of averages at 0 s and 300 s were used to generate the plot. The present invention shows significant improvement in the reaction control. The median relative difference between 0 s and 300 s measurements with known system is 8.0 % (95 %

Cl: 7.2 %-10.1 %) whereas for the present invention the median is 0.1 % (95 % Cl: -1.2 %- 1.5 %). The corresponding interquartile ranges are 5.1 % and 2.5 %. There also is a statistically significant difference between the means (p < 0.0001, with 5 % confidence level).

Figure 4. Stability of whole blood samples for the immunoturbidimetric detection of HbAlc in an assay system according to the present invention, wherein the samples were stored before analysis within a cuvette in a sample holder above a latex solution present in said cuvette. Three patient samples were used in repeated measurements at different time points. Every time point measurement result was divided by the measurement result for the same sample at time point zero to calculate the relative difference. The obtained relative differences (in %) were averaged for each time point. The error bars show the standard deviation of these averages. For the samples stored in the cuvette, the quality of the results is acceptable up to the 7200 s time point and the variation is minor. Figure 5. Stability of whole blood samples for the immunoturbidimetric detection of HbAlc, wherein the samples were stored outside the cuvette before analysis in a sample holder placed horizontally on a hard surface in ambient conditions. Three patient samples were used in repeated measurements at different time points. Every time point measurement result was divided by the measurement result for the same sample at time point zero to calculate the relative difference. The obtained relative differences (in %) were averaged for each time point. The error bars show the standard deviation of these averages. The variation between sample measurement results is vast and exceeds the ±10 % range already at the 300 s time point. The average difference fluctuates over time from over ±10% to under -10%. For the samples stored outside of a cuvette, the quality of the results is not acceptable at any of the measured time points where t > 0 s.

Figure 6. Comparison of immunoturbidimetric detection of HbAlc from whole blood in the known system of WO 2015/177004, the present invention and an alternative procedure (see Example 4 below) to demonstrate the effect of the delay between insertion of the blood filled sample holder into the cuvette and the start of the analysis step. The skeletal box-plot of the results shows median as a bold line, 1st to 3rd quartile as a box and whiskers with end caps extending to the minimum and maximum. The 0 % difference is indicated with a line. The results show that the present invention is superior compared to the known system and alternative procedure in terms of reaction initiation control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention enables improved sample management by providing a combination of a receptacle and a separate sampler holder which is insertable within said receptacle to a position above a predetermined volume of an aqueous liquid such as a buffer or reagent, present in said receptacle. The presence of said liquid provides sufficient humidity inside the receptacle to prevent drying of the sample collected and stored by said sampler holder. The separation of the liquid and the sample within said receptacle further enables improved control of the assay to be performed as the starting time of the assay reaction can be calculated from the exact time point when the sample is contacted with the liquid present in the receptacle. Preferably, the contact between the sample and the liquid is achieved by mechanical movement or agitation of the receptacle, this contacting step being preferably automated and carried out in a test apparatus in a controlled sequence with a subsequent analysis step. Due to the controlled separation of the sample and the assay reagents, the present invention allows employment of various assay chemistries, e.g., such with two or even more separate reagents, and particularly assays dependent on measuring enzyme or binding kinetics.

Accordingly, the present invention is directed to a sampling and assay kit comprising a receptacle, preferably a test tube or cuvette, and a sample holder: the receptacle being for holding a predetermined volume of an aqueous liquid, preferably a buffer or a first reagent required for the assay; the sample holder comprising: a sampler; and a body portion, which holds the sampler and is insertable within the receptacle, wherein the body portion comprises projection means, wherein the receptacle has an internal projection such that when the body portion is inserted in the receptacle, the projection means of the body portion rests on said internal projection, so as to cause positioning of the sampler within the receptacle at a predetermined position, and wherein said predetermined position is such that, in use, the sampler is above said liquid so that the sampler is not in contact with the surface of said liquid, i.e. the sampler does not reach to the surface of said liquid at the base of the receptacle. In order to ensure that the sampler remains above the liquid level until the analysis step is initiated, the receptacle is preferably set and/or kept essentially upright (as shown in Figure 2) although it is appreciated that slight variation in the angle of the central longitudinal axis of the receptacle is acceptable (at least +/- 10-20 degrees and preferably even +/- 30 degrees). The receptacle can also be placed in a suitable tube or cuvette rack providing supporting structures that maintain the essentially vertical position of the receptacle.

In a preferred embodiment, the receptacle is prefilled with the predetermined volume of liquid such as any suitable buffer solution. More preferably, the buffer solution might be tris buffer, Good’s buffer or phosphate buffer. The buffer is preferably hypotonic. The buffer may contain some reagents, for example a haemo lysing compound or latex particles, the particle diameter preferably being 50 to 200 nm; and the particle concentration preferably being 0.05% to 0.2% (w/v). The prefilled receptacle is preferably provided with a foil seal closing the open end of the receptacle. The volume of the receptacle may be in the range of 2-5 ml, more preferably about 3 ml. Said predetermined volume of liquid is preferably in the range of 500-1000 mΐ, more preferably 800-1000 mΐ, and most preferably about 900 mΐ.

In another preferred embodiment, said sampler of the sample holder is a capillary tube or capillary passage which is particularly suitable for blood samples. More preferably, the capillary tube is open on both ends and volume of the capillary tube is between 0.5 mΐ and 50 mΐ, preferably between 1 mΐ and 50 mΐ, more preferably between 1 mΐ and 20 mΐ. Another example of a suitable sampler is a swab.

In another preferred embodiment, the sampling and assay kit further comprises a stopper (i.e. a cap or plug) for the kit, wherein said stopper should be insertable into the receptacle in the space above the body portion of the sample holder within said receptacle. The stopper may also comprise an externally threaded portion which acts as a seal and maintains the stopper in place at the top of the receptacle.

In a more preferred embodiment, the stopper comprises a chamber for holding a reagent, a lid, and a plunger device effective to open the lid, wherein said body portion of the sample holder comprises an indented portion that enables opening of the lid so as to enable ejection of reagent held within the chamber into the receptacle in a space above the body portion. An example of such stopper is disclosed in WO 2015/177004.

Although the predetermined volume of the liquid is able to maintain sufficient humidity in an open receptacle to protect the sample from drying, it is clear that, in use, the stopper closing the receptacle is able to maintain high humidity inside the receptacle for a longer time and/or more consistently.

The sampling and assay kit of the present invention is preferably for assaying of biological samples, such as bodily fluid samples including, e.g., blood samples, saliva samples and urine samples. Measurement of the sample is based on photometric, preferably spectrophotometric methods, known in the art (see, e.g., WO 2015/177004). In the method, the receptacle such as cuvette is inserted in a test apparatus arranged to direct a beam of light, or radiation through the analyte present in the measuring zone in the base of the receptacle. The amount of light or radiation passing through the analyte is measured by a detector and used to provide an analysis of the analyte. The test apparatus may also comprise means to move, turn and/or agitate the inserted receptacle, preferably in order to mix a sample with a reagent or buffer required for the analysis. In a preferred embodiment, the test apparatus comprises means to carry out and automate the step of mixing a sample in the sampler (inserted to the

predetermined position within the receptacle) with a liquid in said receptacle and the subsequent step of measuring an analyte in the mixed sample. In a more preferred

embodiment, the step of mixing is automated and carried out in a test apparatus in a controlled sequence with a subsequent measuring step.

The present invention is further directed to a method for sampling a biological sample with a sampling and assay kit, wherein said kit comprises a receptacle and a sample holder: the receptacle holds a predetermined volume of an aqueous liquid; the sample holder comprising: a sampler; and a body portion, which holds the sampler and is insertable within the receptacle, wherein the body portion comprises projection means, wherein the receptacle has an internal projection such that when the body portion is inserted in the receptacle, the projection means of the body portion rests on said internal projection, so as to cause positioning of the sampler within the receptacle at a predetermined position, and wherein said predetermined position is such that, in use, the sampler is above said liquid so that the sampler is not in contact with the surface of said liquid, the method comprising the steps of:

- collecting a sample with the sampler of said sample holder;

Preferably, an essentially vertical, i.e. upright, position of the receptacle is maintained in the latter step of the method. This can be done by placing said receptacle in a tube or cuvette rack providing supporting structures that maintain the essentially vertical position of the receptacle. The sample may be stored this way before the analysis step at least 5, 12, 30, 60 or 120 minutes.

Preferably, the method comprises a further step of contacting the sampler with said liquid allowing the sample to dilute from the sampler into said liquid. The contact between the sampler and the liquid is preferably achieved by mechanical or manual movement or agitation of the receptacle. In a more preferred embodiment, the step of contacting (i.e. mixing) is automated and carried out in a controlled sequence with a subsequent measuring step.

Accordingly, the method preferably comprises a second further step of measuring the mixed sample in said receptacle.

In a preferred embodiment, said method is an immunoturbidimetric detection of hemoglobin Ale (HbAlc), wherein the amount of latex bound HbAlc over time is measured from a whole blood sample. In the detection, the whole blood sample is mixed with a buffer containing latex particles as defined above. Before the mixing step, the detection preferably comprises a step of closing the receptacle with a stopper (i.e. a cap), said stopper preferably comprising a chamber for holding a second reagent comprising an anti-HbAlc antibody.

The present invention is further described in the following examples, which are not intended to limit the scope of the invention.

EXAMPLES

Commercially available reagents for immunoturbidimetric detection of HbAlc (as described in EP 2 947 458) were used to demonstrate the invention. In the used measurement system, 1 pL of whole blood sample collected with a sample holder is added into a cuvette containing 900 pL of latex reagent. The cuvette is closed with a cap. The cap contains 100 pL of antibody reagent in a recommended concentration where the reagent has been freeze-dried. The sealed receptacle is inserted into a semi-automated QuikRead go instrument (Orion Diagnostica, Espoo, Finland). In the instrument, the latex reagent and the sample are mixed within the cuvette and then incubated for 150 seconds. During this process haemoglobin is released from erythrocytes and is bound to the surface of the latex particles present.

Thereafter the antibody reagent is released from the cap and mixed with the latex solution and the mix is incubated for 120 seconds. The antibody binds specifically to HbAlc and causes aggregation of formed antibody-latex complexes. The aggregation is detected by measuring absorbance during the aggregation reaction at 658 nm. For all measurements, fresh left-over whole blood samples were used across a HbAlc range of 25-120 mmo 1/mol.

In a previously known system as described in WO 2015/177004, the sample is applied into a cuvette with a sample holder including a capillary tube, the tube end contacting with the liquid present in the cuvette. Consequently, the contact allows the sample to flow out from the capillary tube thus initiating the first incubation step before the cuvette is inserted into an instrument for the analysis step. This approach thus causes high variation in the initiation time between separate measurements since the time used by the user for the steps following the sample collection such as the closure of the cuvette and insertion of the cuvette into the instrument is difficult to standardize, measure or control.

Example 1. Improved reaction control with amended sample management

Fresh whole blood samples (n=62) were measured with the known measurement system where the capillary tube end is in contact with the liquid in the cuvette. The following protocols were used with three replicates for each time point per sample:

1. A sample was collected with a sample holder, immediately placed into a cuvette, which was closed with a cap and immediately inserted into a QuikRead go instrument and immediately measured. These results are noted herein as“0 s” results.

2. A sample was collected with a sample holder and immediately placed into a cuvette. The cuvette was then closed with a cap and inserted into a QuikRead go instrument and measured 300 seconds after the sample holder was placed into the cuvette to obtain“300 s” results.

Similar protocols were also performed with the assay kit of the present invention so that the sample was not contacted with the latex solution present in the cuvette until the cuvette was inserted into the QuikRead go instrument. The protocols were repeated (n=19) and four replicates per sample and time point were used.

Comparison of the results is presented in Figure 3 as a box plot. The skeletal box-plot of the results shows the median as a bold line, 1st to 3rd quartile as a box and whiskers with end caps extending to the minimum and maximum. The relative difference was calculated from averaged 0 s and 300 s results. The 0 % difference between results is indicated with a line.

The median for relative difference of the known system is 8.0 %, (95 % Cl: 7.2 %-10.1 %) and for the present invention 0.1 % (95 % Cl: -1.2 %-1.5 %).

The interquartile ranges are 5.1 % (known system) and 2.5 % (present invention). The present invention shows significant improvement in the reaction control, (p < 0.0001 between means, 5 % significance level). The confidence interval is equally distributed around 0 % and the interquartile range (1st to 3rd) representing that the variation between samples in the present invention is 50 % lower than with the known system. Additionally, the differences between minimum and maximum values are vast between the known system and present invention.

The results are also presented in Table 1.

Table 1. Results of comparison of the known system (n=62) and the present invention (n=19). The results are presented as relative difference (%) between averaged 0 s and 300 s results.

The present invention is superior compared to the known system in terms of reaction initiation control. However, it could be argued that similar results can be achieved if the sample holder in the known system is stored out of the receptacle. This is not the case (see the following example). Example 2 Stability of a whole blood sample in a sample holder

Stability of whole blood samples (n=3) in a sample holder was studied by storing the samples in said sample holder for varying periods of times in two different conditions: in a cuvette of the present invention or placing the sample holder horizontally on a surface not inside a cuvette, and comparing the results to an average of two immediate measurements performed on the same sample. The following protocols were used:

1. A sample was collected with a sample holder, immediately placed into a cuvette according to the present invention so that the sample was not in contact with the latex solution present in said cuvette. The cuvette was then closed with a cap and inserted into a QuikRead go instrument and immediately measured. These results are noted herein as“0 s” results. Two replicate measurements per sample were conducted.

2. A sample was collected with a sample holder and immediately placed into a cuvette according to the present invention so that the sample was not in contact with the latex solution present in said cuvette. The cuvette was then closed with a cap and inserted into a QuikRead go instrument and measured 300, 720, 1400, 3600 or 7200 seconds after the sample holder was placed into the cuvette to obtain“300 s”,“720 s”,“1400 s”,“3600 s” or“7200 s” results, respectively.

3. A sample was collected with a sample holder according to the present invention, stored in a horizontal position on a hard surface in ambient conditions, and placed into a cuvette within seconds before the intended measurement. The cuvette was then immediately closed with a cap and inserted into a QuikRead go instrument and measured to obtain measurement results at 300, 720, 1400, 3600 or 7200 seconds after the sample was collected into the sample holder.

The results were normalized to the 0 s measurements of the same samples. Average of three samples were plotted and the results are presented in Figure 4 for the sample stored in the receptacle in the sample holder. The results for the sample stored in a horizontal position on a hard surface are presented in Figure 5. The dots for time points represent the average of the three samples with error bars of one standard deviation of the averages. A relative range of ±10 % representing clinically acceptable range compared to the 0 s measurements is presented with lines. The quality of the measurement results with sample holders stored in the cuvette of the present invention is acceptable in all the measured time points up to 7200 s whereas the results with sample holders stored in horizontal position exceed the ±10 % limit already at 300 s time point showing high variation between samples. The results for the sample stability measurements are shown in Table 2.

Table 2. Measurement results for whole blood samples in a sample holder stored before the analysis step in the cuvette of the present invention or alternatively at a horizontal position outside a cuvette. The results are presented as the relative difference (%) of time points compared to 0 s measurements. SD=standard deviation.

Example 3. Stability of a whole blood sample in a closed or open cuvette

The effect of storing a whole blood sample within a closed or open cuvette in a sample holder was studied with control samples (n=2), commercially available for HbAlc. Since the quality of results with sample holders stored in horizontal position outside of a cuvette was not acceptable at 300 s time point (see Example 2), approaches using a closed or open cuvette for sample storage were also studied. The following protocols were used:

1. A control sample was collected with a sample holder, immediately placed into a cuvette according to the present invention so that the sample was not in contact with the latex solution present in said cuvette. The cuvette was then closed with a cap, inserted into a QuikRead go instrument, and immediately measured. These results are noted herein as“0 s” results. Six replicate measurements per control sample were conducted.

2. A sample was collected with a sample holder and immediately placed into a cuvette according to the present invention so that the sample was not in contact with the latex solution present in said cuvette. The cuvette was then closed with a cap and inserted into a QuikRead go instrument and measured 300 seconds after the sample holder was placed into the cuvette to obtain“Closed 300 s” results. Three replicate measurements per control sample were conducted. 3. A sample was collected with a sample holder and immediately placed into a cuvette according to the present invention so that the sample was not in contact with the latex solution present in said cuvette. The cuvette was then left open until closed with a cap within seconds before the intended measurement and inserted into a QuikRead go instrument and measured 300 seconds after the sample holder was placed into the cuvette to obtain“Open 300 s” results. Three replicate measurements per control sample were conducted.

For each control measurement point, an average result was calculated. The open and closed 300 s time point average results were then divided by the average result for the control sample at 0 s time point to calculate the relative difference (in %). Both of the“open” and“closed” samples show invariable results within acceptable range of ±10 % compared to the 0 s measurement, i.e. an open cuvette of the present invention protects the sample from drying as well as a closed cuvette. Results for the closed and open cuvette comparison measurements are shown in Table 3.

Table 3. Measurement results for the comparison of closed and open cuvette when storing a sample holder before the analysis step of the present invention. The results are presented as the relative difference (%) of 300 s time point compared to 0 s measurement with two samples.

Example 4 Improved reaction control with amended sample management

1. A sample was collected with a sample holder, immediately placed into a cuvette, which was closed with a cap and immediately inserted into a QuikRead go instrument and immediately measured. These results are noted herein as“0 s” results. 2. A sample was collected with a sample holder and immediately placed into a cuvette. The cuvette was then closed with a cap and inserted into a QuikRead go instrument and measured 300 seconds after the sample holder was placed into the cuvette to obtain“300 s” results.

Similar protocols were also performed with the assay kit of the present invention so that the sample was not contacted with the latex solution present in the cuvette until the cuvette was inserted into the QuikRead go instrument. The protocols were repeated (n=19) and four replicates per sample and time point were used. As an alternative procedure to improve the sample management, the following protocols were performed, as a comparison with the present invention and known system. For the alternative procedure (n=62) three replicates for each time point sample was measured.

1. A sample was collected with a sample holder, immediately placed into a cuvette, which was closed with a cap, following a brisk manual shaking of the cuvette until the capillary of the sample collector was visually empty (there was no blood seen in the capillary). Shaking time for all the samples was 10-15 s. The cuvette was inserted into a QuikRead go instrument immediately after the shaking was finished. These results are noted herein as“0 s” results.

2. A sample was collected with a sample holder and immediately placed into a cuvette, which was closed with a cap, following a brisk manual shaking of the cuvette until the capillary of the sample collector was visually empty (there was no blood seen in the capillary). Shaking time for all the samples was 10-15 s. The cuvette was inserted into a QuikRead go instrument and measured 300 seconds after the sample holder was placed into the cuvette to obtain“300 s” results.

The median for relative difference of the known system is 8.0 % (95 % Cl: 7.2 %-10.1 %), for the alternative procedure 4.6 % (95 % Cl: 4.0 %-5.1 %) and for the present invention 0.1 % (95 % Cl: -1.2 %-1.5 %).

The interquartile ranges are 5.1 % (known system), 2.7 % (alternative procedure) and 2.5 % (present invention). The present invention shows significant improvement in the reaction control, (p < 0.0001 between means, 5 % significance level) compared to the known system. The confidence interval is equally distributed around 0 % and the interquartile range (1st to 3rd) representing that the variation between samples in the present invention is 50 % lower than with the known system. The alternative procedure also indicates lower variation than the known system, but the median is high in addition to the confidence interval being high.

Additionally, the differences between minimum and maximum values are vast between the known system and present invention and between the alternative procedure and present invention. The results are also presented in Table 4 and Figure 6.

Table 4. Results of comparison of the known system (n=62), alternative procedure (n=62) and the present invention (n=19). The results are presented as relative difference (%) between averaged 0 s and 300 s results.

The present invention is superior compared to the known system and alternative procedure in terms of reaction initiation control. The superiority indicates that the alternative procedure does not solve the issue causing poor reaction control and that the present invention is needed.

REFERENCES

WO 2015/177004 US 2012/0214251 EP 2 947 458 US 5833630 EP1712180

Claims

1. A sampling and assay kit comprising a receptacle and a sample holder: the receptacle being for holding a predetermined volume of an aqueous liquid; the sample holder comprising: a sampler; and a body portion, which holds the sampler and is insertable within the receptacle, wherein the body portion comprises projection means, wherein the receptacle has an internal projection such that when the body portion is inserted in the receptacle, the projection means of the body portion rests on said internal projection, so as to cause positioning of the sampler within the receptacle at a predetermined position, characterized in that said predetermined position is such that, in use, the sampler is above said liquid so that the sampler is not in contact with the surface of said liquid.

2. The sampling and assay kit according to claim 1, wherein said receptacle holds a predetermined volume of said liquid.

3. The sampling and assay kit according to claim 1 or 2, wherein said liquid is a buffer solution.

4. The sampling and assay kit according to any of claim 1-3, wherein said sampler is a capillary tube.

5. The sampling and assay kit according to claim 4, wherein the volume of the capillary tube is between 0.5 mΐ and 50 mΐ.

6. The sampling and assay kit according to any one of the preceding claims further comprising a stopper.

7. The sampling and assay kit according to claim 6, wherein said stopper is insertable into the receptacle in the space above the body portion within said receptacle.

8. The sampling and assay kit according to claim 6 or 7, wherein, in use, said receptacle holds a predetermined volume of a buffer solution.

9. The sampling and assay kit according to any of claims 6-8, wherein the stopper comprises an externally threaded portion which acts as a seal and maintains the stopper in place at the top of the receptacle.

10. The sampling and assay kit according to any one of claims 6-9, wherein said stopper comprises a chamber for holding a reagent, a lid, and a plunger device effective to open the lid, wherein said body portion comprises an indented portion that enables opening of the lid so as to enable ejection of reagent held within the chamber into the receptacle in a space above the body portion.

11. The sampling and assay kit according to any one of the preceding claims, wherein said kit is for a blood sample.

12. The sampling and assay kit according to any one of the preceding claims, wherein said receptacle is a cuvette.

13. The sampling and assay kit according to any one of the preceding claims, wherein said sample holder is arranged in said receptacle so that, in use, the presence of said aqueous liquid provides sufficient humidity inside the receptacle to prevent deterioration such as drying of a sample in said sampler placed within said receptacle above the surface level of said liquid.

14. A method for sampling a biological sample with a sampling and assay kit, wherein said kit comprises a receptacle and a sample holder: the receptacle holds a predetermined volume of an aqueous liquid; the sample holder comprising: a sampler; and a body portion, which holds the sampler and is insertable within the receptacle, wherein the body portion comprises projection means, wherein the receptacle has an internal projection such that when the body portion is inserted in the receptacle, the projection means of the body portion rests on said internal projection, so as to cause positioning of the sampler within the receptacle at a predetermined position, characterized in that said predetermined position is such that, in use, the sampler is above said liquid so that the sampler is not in contact with the surface of said liquid, the method comprising the steps of: - collecting a sample with the sampler of said sample holder; and

- inserting the sample holder into the receptacle so that the sampler rests above the surface of a liquid in said receptacle at a predetermined position.

15. The method according to claim 14 comprising a further step of inserting a stopper in the receptacle.

16. The method according to claim 14 or 15 comprising a further step of contacting the sampler with said liquid allowing the sample to dilute from the sampler into said liquid.

17. Use of a sampler holder for sampling a biological sample, wherein said sample holder comprises: a sampler; and a body portion, which holds the sampler and is insertable within a receptacle, wherein the body portion comprises projection means, wherein the receptacle has an internal projection such that when the body portion is inserted in the receptacle, the projection means of the body portion rests on said internal projection, so as to cause positioning of the sampler within the receptacle at a predetermined position, characterized in that said predetermined position is such that, in use, the sampler is above a predetermined volume of an aqueous liquid present in said receptacle so that the sampler is not in contact with the surface of said liquid.

18. The use according to claim 17, wherein the sampler is contacted with said liquid allowing the sample to dilute from the sampler into said liquid to initiate an analysis step.