A method for determining run errors for an analysis-method for quantifying a plurality of analytes in a plurality of biological samples

Abstract

A quality control method (100) for determining run errors for an analysis-method (10) for quantifying a plurality of analytes in a plurality of biological samples.

Description

[0001] A METHOD FOR DETERMINING RUN ERRORS FOR AN ANALYSIS-METHOD FOR QUANTIFYING A

[0002] PLURALITY OF ANALYTES IN A PLURALITY OF BIOLOGICAL SAMPLES

[0003] Technical field

[0004] The present disclosure relates to a method for determining run errors for an analysismethod for quantifying a plurality of analytes in a plurality of biological samples.

[0005] Background art

[0006] Modern proteomics methods require the ability to detect a large number of different proteins (or protein complexes) in a small sample volume. To achieve this, multiplex analysis must be performed. Common methods by which multiplex detection of proteins in a sample may be achieved include dual-recognition immunoassays. Dual-recognition immunoassays build on a concept developed by Ulf Landegren and co-workers and described i.a. in Fredriksson et al., Nature Biotechnology, vol. 20, 2002, pp. 473-477 and WQ01 / 61037.

[0007] Dual-recognition immunoassay methods include Proximity Extension Assay (PEA), commercially available from Olink Proteomics AB (Uppsala, Sweden). Various aspects of PEA is further described in WO 03 / 044231, WO 2004 / 094456, WO 2005 / 123963, WO 2006 / 137932, WO 2013 / 113699, WO 2021 / 191442, WO 2021 / 191448, WO 2021 / 191449, WO 2021 / 191450, and WO 2022 / 112300; Assarsson et al., PLoS 1, 2014, 9, 4, e95192; Lundberg et al., Molecular & Cellular Proteomics 10:10.1074 / mcp.M110.004978, 1-10, 2011; and Wik et al., 2021, Mol Cell Proteomics 20, 100168, all incorporated herein by reference in their entirety.

[0008] The Proximity Ligation Assay (PLA) methodology has primarily been used in combination with Rolling Circle Amplification (RCA) for detection of protein-protein interactions and post- translational modifications and is commercially available from Merck under the trademark Duolink®. A PLA-based method for multiplex detection of proteins is described in WO 2021 / 113290.

[0009] PEA and PLA are dual-recognition assays, which rely on the principle of "proximity probing". In these methods an analyte is detected by the binding of multiple (i.e. two or more, generally two or three) probes, which when brought into proximity by binding to the analyte (hence "proximity probes") allow a signal to be generated. Typically, at least one of the proximity probes comprises a nucleic acid domain (or moiety) linked to the analyte-binding domain (or moiety) of the probe, and generation of the signal involves an interaction between the nucleic acid moieties and / or a further functional moiety which is carried by the other probe(s). Thus, signal generation is dependent on an interaction between the probes (more particularly between the nucleic acid or other functional moieties / domains carried by them) and hence only occurs when the necessary probes have bound to the analyte, thereby lending improved specificity to the detection system.

[0010] In PEA, nucleic acid moieties linked to the analyte-binding domains of a probe pair hybridise to one another when the probes are in close proximity (i.e. when bound to the same target molecule), and are then extended using a nucleic acid polymerase. The extension product forms a reporter nucleic acid, detection of which demonstrates the presence of a particular analyte (the analyte bound by the relevant probe pair) in a sample of interest. In PLA, nucleic acid moieties linked to the analyte-binding domains of a probe pair come into proximity when the probes of the probe pair bind their target, and may be ligated together, or alternatively they may together template the ligation of separately added oligonucleotides, which are able to hybridise to the nucleic acid domains when they are in proximity. The ligation product is then amplified, acting as a reporter nucleic acid. Multiplex analyte detection using PEA or PLA may be achieved by including one or more unique barcode sequences in the nucleic acid moiety of each probe. Oligonucleotides comprising a barcode sequence unique to a specific sample may further be added to the respective sample and incorporated into all reporter molecules generated from that sample. A reporter nucleic acid molecule corresponding to a particular analyte, and optionally a particular sample, may then be identified by the barcode sequences it contains. The methods of the present invention find particular utility in multiplex PEA and PLA methods.

[0011] Panels of proximity assays, as described above, are commercially available from Olink Proteomics AB (Uppsala, Sweden) under the trademark Olink® Target, Olink® Focus, Olink® Explore, and Olink® Flex. These are panels of up to 92 assays (Olink® Target, Olink® Focus and Olink® Flex) or up to ~3,000 assays split over eight different panels (Olink® Explore). A panel may further be divided into a number, typically four, of "abundance blocks" and the samples may be diluted based on their predicted abundance prior to being incubated with the assay probes of the respective abundance block. Each panel generally includes assays for proteins that have known functions within certain biological or physiological areas, pathways or organs in the body, such as inflammation, organ-specific proteins, cardiovascular, neurology etc. It is also possible for a user to select a specific combination of protein assays that are of particular interest to create a tailor-made panel. Proximity based assays produce a number of reporter molecules with a certain barcode set-up, which number correlates to the amount of protein with the corresponding barcode. This number can be quantified by either quantitative Polymerase Chain Reaction (qPCR) or sequencing, preferably Next Generation Sequencing (NGS). The qPCR produces a Ct value that corresponds to the amount of protein in the sample and NGS produces an actual number, called "counts", of reporter molecules and the counts correlate with the amount of protein in the sample.

[0012] Based on Ct values and counts, it is possible to calculate both absolute and relative amounts of protein in the sample. Olink uses an arbitrary relative quantification unit called NPX that may be calculated with software adapted for use with the above panels. A measure of relative protein quantity, such as NPX, can be calculated from counts as described in Wik et al., cited above.

[0013] It is further known to perform Quality Control (QC) on data produced from proximitybased assays, as e.g. described in Wik et al., cited above. To facilitate QC, test reagents (also known as internal controls) and standardized samples (also known as external controls) are included in the protocol. The test reagents used for QC are an incubation test reagent and an amplification test reagent. The incubation test reagent comprises PEA probes measuring a fixed concentration of nonhuman green fluorescent protein (GFP), added to each sample. The amplification test reagent consists of a synthetic double-stranded DNA template and is used in QC to monitor the PCR steps in the protocol. Standardized samples used for QC comprise a blank sample (buffer only) run in triplicate, and standard samples, run in duplicate.

[0014] A standard sample is pooled plasma samples run in duplicates on each plate. These are used as standardized sample to estimate inter and intra precision for each assay.

[0015] A blank sample (also known as negative controls) comprises buffer run as a normal sample. Blank samples are used to monitor any background noise generated when DNA-tags come in close proximity without prior binding to the appropriate protein. The blank sample set the background levels for each protein assay and are used to calculate the limit of detection (LOD).

[0016] A test reagent is a reagent used to identify substances contained within a test sample.

[0017] A test plate sample comprises pooled plasma originating from healthy blood donors. Standard samples are used in data normalization to compensate potential variation between runs and plates. The QC assessment is performed at two levels; run QC and sample QC. At the run QC level each of the abundance blocks for each panel and sample plate should fulfil the following criteria: (i) mean absolute deviation (MAD) in test reagents may not exceed a certain threshold of relative protein quantity (such as 0.3 NPX) and (ii) deviation on the sample QC level is allowed for a maximum of one out of six samples. Further, in each panel, the median of at least 90% of the assays in test plate sample and blank samples must be in the accepted range from predefined values set during validation. Apart from run QC, the performance of each sample is assessed individually by the test reagents that should be within a predefined range of relative protein quantities (such as ±0.3 NPX) from the median level across the abundance block. Additionally, the mean assay count for a sample may not be below 500 counts. Abundance blocks and samples that do not fulfil their respective QC criteria will receive a QC warning. Another QC metric is precision evaluated as the coefficient of variation (CV). CV is a measure of technical variation for individual assays both within a plate (intra-CV) and across multiple plates (inter-CV) and may be calculated as described in Wik et al. It is possible to set upper limits on intra-CV and inter-CV among all assays as additional QC criteria.

[0018] However, when receiving a QC warning it is not certain what caused the possibly poor performance. There is thus a need for an improved quality control of this type of analysis methods.

[0019] Summary

[0020] It is an object of the present disclosure to mitigate, alleviate or eliminate one or more of the above-identified deficiencies and disadvantages in the prior art and solves at least the above mentioned problem.

[0021] According to a first aspect there is provided a quality control method for determining run errors for an analysis-method for quantifying a plurality of analytes in a plurality of biological samples, wherein the analysis-method comprises: providing a plurality of samples in a plurality of reaction containers of a plate, wherein each sample is identified as a biological sample or a standardized sample, wherein a first plurality samples in a first set of reaction containers are identified as biological samples and a second plurality of samples in a second set of reaction containers are identified as standardized samples, wherein the samples identified as biological samples are samples under investigation and wherein the samples identified as standardized samples are further identified as either a standard sample, or a blank sample. The method further comprises adding, to each of the plurality of samples, predetermined amounts of at least one test reagent; and adding assay reagents to each of the plurality of samples, wherein the assay reagents are configured to generate nucleic acid reporter molecules in an amount correlated to the amounts of analyte or test reagent in the plurality of samples, wherein each nucleic acid reporter molecule pertains to an analyzed sample among the plurality of samples and comprises identification sequences, the identification sequences comprises a first identification sequence identifying the reaction container of the analyzed sample and a second identification sequence uniquely identifying one of: the analyte or a test reagent from the one or more test reagents.

[0022] The quality control method comprises obtaining using the identification sequences of the nucleic acid reporter molecules, for each reaction container, a set of counts, the set of counts comprising a count for the amount of analyte reporter molecules detected in the reaction container; and for each test reagent of the at least one test reagent detected in the reaction container, a count for the amount of the test reagent specific reporter molecules corresponding to the test reagent.

[0023] The quality control method further comprises determining, for all reaction containers comprising a sample identified as a standardized sample, a mean count of the set of counts of the test reagent specific reporter molecules corresponding to any of the one or more test reagents, and upon a number of reaction containers comprising a sample identified as standardized sample, in which the count forthe amount of analyte reporter molecules detected in the reaction container exceeds the mean count, exceeds a second threshold, determining a run error for the plate.

[0024] This embodiment involves calculating the mean (e.g., average, or median) count of the test reagent-specific reporter molecules for all reaction containers containing standardized samples. This mean count represents the mean number of reporter molecules detected for each test reagent across all standardized samples.

[0025] Next, each reaction container identified a standardized sample is examined to determine if the count of analyte reporter molecules exceeds the calculated mean count. Specifically, the method identifies the reaction containers where the count of analyte reporter molecules is higher than the mean count. The number of these reaction containers is then compared to a predefined second threshold. If the number of reaction containers with counts exceeding the mean count surpasses this second threshold, a run error is determined for the entire plate. When performing the analysis method, the plate is inserted into the device that carries out the analysis. Each sample in the reaction containers of the plate is identified as either a biological sample or a standardized sample based on the correct orientation of the plate in the device. However, human error may result in the plate being inserted incorrectly, such as being rotated 180 degrees. This can lead to misidentification, where some samples intended to be biological samples are identified as standardized samples and vice versa. An advantage of the QC method is its ability to detect such errors, ensuring the integrity and accuracy of the analysis by verifying that samples are correctly identified regardless of the orientation of the plate. This reduces the risk of inaccurate results due to misidentification and enhances the reliability of the overall analytical process. When a plate-level run error is detected, it suggests that the results from the entire plate may be unreliable, necessitating a re-run of the analysis for all samples on that plate.

[0026] The nucleic acid reporter molecules include both test reagent-specific reporter molecules and analyte reporter molecules. Each nucleic acid reporter molecule is equipped with identification sequences that enable precise identification and quantification. The first identification sequence is used to identify the specific reaction container of the analyzed sample, ensuring that the data correlates accurately to the correct sample location. The second identification sequence is used to uniquely identifying one of: the analyte or a test reagent from the one or more test reagents, ensuring that the data correlates accurately to the amount of analyte and test reagent(s) of the sample. This system allows for the accurate detection and measurement of both test reagents and analytes in each sample, facilitating robust quality control and error detection throughout the analysis process. By focusing on the raw count of the test reagent-specific reporter molecules corresponding to the test reagent in the QC method, known reference values can be used to verify the quality and accuracy of the analysis method.

[0027] In some examples, the method further comprises determining for each reaction container comprising a sample identified as standardized sample, a combined count of the set of counts determined for the reaction container, wherein upon the combined count for the reaction container is below a first threshold, determining a run error for the reaction container.

[0028] By focusing on the raw count of the test reagent-specific reporter molecules corresponding to the test reagent in the QC method, known reference values can be used to verify the quality and accuracy of the analysis method. In this example, if a standardized sample's total count falls below a set threshold, it may be flagged as being a run-error. This flag indicates potential technical issues, resulting in insufficient data for reliable analysis. This QC step ensures only samples with adequate content are considered for further analysis. The threshold may be established based on the known reference values and specific implementation requirements.

[0029] A run error can occur at either the reaction container level or the plate level, each representing different scopes of issues in the quality control process. A run error of a reaction container occurs when a specific reaction container (well) has issues. It indicates a problem with an individual sample or reagent addition in that particular container. This type of error flags that specific reaction container for re-evaluation or exclusion from analysis. When a large enough amount of reaction container has been flagged with a run error, this may lead to a run error being determined for the entire plate. Run error on a plate level occurs when multiple reaction containers on a plate exhibit errors, indicating a broader issue affecting the entire plate. This can be due to systemic problems such as incorrect plate insertion, widespread reagent failure, or instrument malfunction.

[0030] In some examples, the method further comprises determining for each reaction container comprising a sample identified as standardized sample, and for each test reagent among the one or more test reagent, whether a count for the amount of test reagent specific reporter molecules of the reaction container is below a test reagent specific threshold, wherein upon the count is below the test reagent specific threshold, determining a run error for the reaction container.

[0031] Advantageously, the amount of each test reagent, identified via the raw count of the reagent-specific reporter molecules, can be verified against known reference values. This ensures that the addition of the test reagent has been successfully accomplished. This QC step ensures that only samples with adequate content are considered for further analysis.

[0032] In some examples, the method further comprises determining, for each reaction container comprising a sample identified as standard samples, and for each test reagent among the one or more test reagent, whether a fraction of the count for the amount of test reagent specific reporter molecules of the reaction container to the combined count of the reaction container is outside a test reagent specific range, wherein upon the fraction is outside the test reagent specific range, determining a run error for the reaction container.

[0033] The aim of identifying deviating plate controls may be to ensure the reliability and accuracy of the assay results. By flagging samples with internal control deviations, the method helps to identify and correct underlying technical issues that could compromise data quality. Again, using the raw count of the reagent-specific reporter molecules, the QC checks may be improved, since the known reference values can be used to verify the quality and accuracy of the analysis method.

[0034] The purpose of formal plate quality control as exemplified above is to identify samples, block-plates or assays that are severely affected by technical errors, such as reagent contaminations, operation mistakes, and instrument failures. The method provides for obtaining more detailed information about the quality of the performed assay and the reasons for any poor performance.

[0035] According to some embodiments, the test reagents comprise incubation test reagents, extension test reagents, and amplification test reagents. In one embodiment, the test reagents comprise incubation control, extension control, and amplification control as known in the art and described e.g. in Wik et al., supra. The term extension test reagent is to be interpreted as two paired oligonucleotides coupled to the same antibody molecule, thereby being in constant proximity.

[0036] According to some embodiments, the incubation test reagents comprise of one or more non-human antigens, such asphycoerythrin and green fluorescent protein, with matching proximity probes.

[0037] According to some embodiments, the extension test reagents is made by antibodies conjugated to a set of single stranded oligonucleotides capable of at least partial hybridization.

[0038] According to some embodiments, the amplification test reagents comprise synthetic double-stranded nucleic acid molecules.

[0039] According to some embodiments, the method further comprises determining the type of run error.

[0040] According to some embodiments, the method further comprises determining and actions to be taken in response to determining a run error for the plate and / or a run error for a reaction container.

[0041] According to some embodiments, the method further comprises an initial step of initiating, wherein the step of initiating the method comprises receiving a quality control warning from the analysis-method indicating that the method should be performed. According to a second aspect of the invention, the above object is achieved by a computer program product comprising a non-transitory computer-readable storage medium having thereon a computer program comprising program instructions, the computer program being loadable into a processor and configured to cause the processor to perform the method according to the first aspect.

[0042] According to a third aspect of the invention, the above object is achieved by system comprising, one or more processors; and one or more non-transitory computer-readable media storing instructions executable by the one or more processors, wherein the instructions, when executed, cause the system to perform the method according to the first aspect.

[0043] The present disclosure will become apparent from the detailed description given below. The detailed description and specific examples disclose preferred embodiments of the disclosure by way of illustration only. Those skilled in the art understand from guidance in the detailed description that changes and modifications may be made within the scope of the disclosure.

[0044] Hence, it is to be understood that the herein disclosed disclosure is not limited to the particular component parts of the device described or steps of the methods described since such device and method may vary. It is also to be understood that the terminology used herein is for purpose of describing particular embodiments only and is not intended to be limiting. It should be noted that, as used in the specification and the appended claim, the articles "a", "an", "the", and "said" are intended to mean that there are one or more of the elements unless the context explicitly dictates otherwise. Thus, for example, reference to "a unit" or "the unit" may include several devices, and the like. Furthermore, the words "comprising", "including", "containing" and similar wordings does not exclude other elements or steps.

[0045] Brief descriptions of the drawings

[0046] The above objects, as well as additional objects, features and advantages of the present disclosure, will be more fully appreciated by reference to the following illustrative and nonlimiting detailed description of example embodiments of the present disclosure, when taken in conjunction with the accompanying drawings.

[0047] Figure 1 shows schematically a flowchart of the method according to an embodiment of the present disclosure. Figure 2 shows schematically a top view of the plate from which the method, according to an embodiment of the present disclosure, obtains measurements.

[0048] Figure 3 show schematically a data processing unit comprising a computer program product.

[0049] Terms and abbreviations

[0050] All terms used herein are intended to have the general meaning ascribed to them by the person skilled in the art of multiplex protein detection, and in particular affinity-based proteomics technologies.

[0051] Detailed description

[0052] The present disclosure will now be described with reference to the accompanying drawings, in which preferred example embodiments of the disclosure are shown. The disclosure may, however, be embodied in otherforms and should not be construed as limited to the herein disclosed embodiments. The disclosed embodiments are provided to fully convey the scope of the disclosure to the skilled person.

[0053] The analysis method to which the present invention is to be applied is performed on biological samples, usually of human origin. For quality control purposes, each run of the method also includes standardized samples. The analysis results from standardized samples shall be as expected from the constitution of these samples and these results are used as described herein to assess, or control, the quality of the specific run of the analysis method. Standardized samples as used herein are of two types, standard samples and blank samples. Standard samples are prepared to reflect a biological sample with a normal content of the analytes to be analysed by the analysis method. They are in general prepared by pooling biological samples of the same type as those being analyzed, from a number of healthy individuals. Blank samples do not contain any of the analytes to be tested for in the analysis method, and usually contain only buffer. Standardized samples are sometimes also referred to in the art as "external controls".

[0054] The present invention also makes use of one or more test reagents. Test reagents are reagents that are specifically constructed to produce specific reporter nucleic acid molecules when added to the biological and standardized samples in the analysis method. Test reagents as used in the present invention are known in the art and sometimes referred to as "internal controls".

[0055] The present invention relates to a method for determining whether an error has occurred in a certain run of the analysis method (a "run error"), based on the data patterns obtained by the analysis method from the standardized samples and the test reagents. The present invention also allows for connecting specific data patterns to specific errors, thereby simplifying identification of error sources.

[0056] Fig. 1 shows a flowchart of analysis-method 10 and method 100 according to the first aspect of this disclosure. Fig.l shows a method 100 for determining at least one run error for an analysis-method 10 for quantifying a plurality of analytes in a plurality of biological samples. The analysis-method 10 comprises the step of adding S12 a plurality of biological samples in a first set of reaction containers (e.g. Al-All, ...,H1-H11 as shown in Fig.2) of a plate 200 and adding a plurality of standardized samples in a second set of reaction containers (e.g. A12-H12 as shown in Fig.2) of the plate 200. The biological samples are samples under investigation and the standardized samples are of a type selected from at least a standard sample, and a blank sample. Thereby, there is provided a plurality of samples in a plurality of reaction containers of a plate, wherein each sample is identified as a biological sample or a standardized sample, wherein a first plurality samples in a first set of reaction containers (Al-All, ...,H1-H11) are identified as biological samples and a second plurality of samples in a second set of reaction containers (A12-H12) are identified as standardized samples. However, as discussed, if the plate 200 is inserted into the analysis device incorrectly, such as in the wrong direction, a sample identified as a biological sample may actually be a standardized sample, and vice versa.

[0057] The analysis-method 10 further comprises the step of adding S16, to each biological sample and to each standardized sample, predetermined amounts of at least one test reagent.

[0058] Analysis-method 10 further comprises the step of adding S14 assay reagents to the biological samples and to the plurality of the standardized samples. The assay reagent is configured to generate a nucleic acid reporter molecules in an amount correlated to the amount of analyte and test reagent in the biological sample and the standardized sample. Each nucleic acid reporter molecule pertains to an analyzed sample among the plurality of samples and comprises identification sequences. The identification sequences comprise a first identification sequence identifying the reaction container of the analyzed sample and a second identification sequence uniquely identifying one of: the analyte or a test reagent from the one or more test reagents. The assay reagents are thus designed to generate nucleic acid reporter molecules in quantities that correspond to the amounts of analyte and test reagent present in each sample. Each nucleic acid reporter molecule contains identification sequences which have two main purposes. The first identification sequence identifies the reaction container (well) that holds the analyzed sample, ensuring that the data collected is accurately associated with the correct sample location on the plate. The second identification sequence uniquely identifies either the analyte or a specific test reagent from among the multiple test reagents used. Such identification aids in determining the type and quantity of each substance within the sample. This process allows for accurate tracking and differentiation of samples and reagents, ensuring that results are correctly attributed to the appropriate sample and reagent. By correlating the amount of nucleic acid reporter molecules to the amount of analyte and test reagent, the method provides reliable quantification of the substances present in the samples. The identification sequences also enhance data integrity by preventing mix-ups between samples and reagents, which is especially important in high-throughput settings where many samples are processed simultaneously. Advantageously, identification data of known amounts of test reagent is used for quality control purposes. As such, known reference values can be utilized to verify the quality and accuracy of the analysis method.

[0059] Finally, the analysis-method 10 comprising the step of detecting S18 the amount of reporter molecules carrying identification sequences such that obtaining a count value for each unique combination of the first and second identification sequences may be possible in a quality control method 100 described below. In this context, a "unique combination" refers to the pairing of two specific identification sequences within each nucleic acid reporter molecule. The first identification sequence identifies the specific reaction container from which the sample originates, ensuring that data can be accurately traced back to its source. The second identification sequence uniquely identifies eitherthe analyte orthe specific test reagent present in the sample. Together, these sequences create a unique identifier for each data point, ensuring that the counts obtained can be precisely attributed to the correct sample and its corresponding amount of reagent or analyte.

[0060] When the analysis-method 10 has been performed, a quality control warning may or may not be received. If a quality control warning is received, the quality control method 100 aim to determining the cause of said quality control warning.

[0061] The method 100 comprises the step of obtaining S110 using the identification sequences of the nucleic acid reporter molecules, for each reaction container, a set of counts, the set of counts comprising a count for the amount of analyte reporter molecules detected S18 in the reaction container; and for each test reagent of the at least one test reagent detected in the reaction container, a count for the amount of the test reagent specific reporter molecules corresponding to the test reagent. As described above, each nucleic acid reporter molecule has identification sequences that indicate the specific reaction container and whether it corresponds to an analyte or a test reagent. The system then counts (obtains) the nucleic acid reporter molecules, providing a set of counts that includes both the count of analyte reporter molecules and the count of test reagent-specific reporter molecules for each sample. These counts are used in the quality control method 100 as discussed below to ensure that the data is reliable and that the test reagents were properly added. The method 100 than determining which of a set of criteria are fulfilled. The criteria are based on parameters, which correlate to certain issues.

[0062] The method 100 may comprise determining S118, for all reaction containers comprising a sample identified as a standardized sample, a mean count of the set of counts of the test reagent specific reporter molecules corresponding to any of the one or more test reagents, and upon a number of reaction containers comprising a sample identified as standardized sample, in which the count for the amount of analyte reporter molecules detected in the reaction container exceeds the mean count, exceeds a second threshold, determining a run error for the plate.

[0063] When an incorrect sample plate is used, or if a sample plate is not oriented correctly, the negative controls (standard sample) or blanks may not be in their expected positions (wells). As a result, the expected positions for negative controls or blanks may be occupied by other types of samples. The median count of internal controls in a block for a negative control or blank sample can be calculated using the formula: medianint_count_biock[a],s=median(counto,s) where a represents the type of internal control (e.g., test reagents, extension test reagents, and / or amplification test reagents) and s represents the type (negative control, empty). In other examples, mean may be used instead of median.

[0064] A protein assay identified as a negative control or empty sample is considered to have received an unexpectedly high number of counts if the amount of analyte reporter molecules detected in the reaction container exceeds the mean count. If enough such protein assays have such an unexpectedly amount of analyte reporter molecules (i.e., over the second threshold, which may for example be 1, 5, 7, 10, etc.), a run error for the plate is determined. For example, if the mean count of test reagent-specific reporter molecules across all standardized samples is 1000, and a reaction container identified as a standardized sample shows 1500 analyte reporter molecules, this exceeds the mean count. If the second threshold is set to 5 and there are 6 reaction containers where the analyte reporter molecules exceed 1000, this number exceeds the threshold. Consequently, a run error is determined for the plate.

[0065] The method may further comprise determining (S112) for each reaction container comprising a sample identified as standardized sample, a combined count of the set of counts determined for the reaction container, wherein upon the combined count for the reaction container is below a first threshold, determining a run error for the reaction container. Example of the first threshold may be 2000, 4000, 5600, 10000, etc.

[0066] The method may further comprise determining S114 for each reaction container comprising a sample identified as standardized sample, and for each test reagent among the one or more test reagent, whether a count for the amount of test reagent specific reporter molecules of the reaction container is below a test reagent specific threshold, wherein upon the count is below the test reagent specific threshold, determining a run error for the reaction container For example, if the counts for any of the test reagents, e.g., incubation-, extension- and amplification test reagents, is less than 150, a run error for the reaction container may be is determined In another example, if the counts for any of the test reagents in container comprises standardized sample is greater than 150, and less than or equal to 500, 1000, and 500 for incubation-, extension- and amplification test reagent respectively, run error for the reaction container may be is determined

[0067] In yet another example, if the counts for any of the test reagents in container comprises biological samples is greater than 150, and less than or equal to 500, 1000, and 500 for incubation-, extension- and amplification test reagent respectively, run error for the reaction container may be is determined. The correlated issue causing the problem may be low test reagent levels.

[0068] The method may comprise determining S116, for each reaction container comprising a sample identified as standard samples, and for each test reagent among the one or more test reagent, whether a fraction of the count for the amount of test reagent specific reporter molecules of the reaction container to the combined count of the reaction container is outside a test reagent specific range, wherein upon the fraction is outside the test reagent specific range, determining a run error for the reaction container The correlated issue causing the problem may be that there is a sample instead of a blank sample in the reaction container. It should be noted that the quality control method 100 can include any of the steps S112- S118, either individually or in combination. For instance, in some cases, the quality control method 100 may consist of just one of the steps S112-S118. In other cases, the method may include two or more of these steps, such as step S118 followed by step S112 and step S116, or step S112 followed by step S116.The method 100 may then comprise determining S160 at least one run error for one or more reaction containers and / or the plate as described above, depending on which of the QC checks (and how many) that have failed.

[0069] The method 100 further comprises the step of determining S170, on basis on QC checks that failed, the type of run error caused the quality control warning.

[0070] The method 100 further comprises determining S180 on basis on which QC checks that have failed or not failed, or, on basis on the type of the determined run error, which corresponding actions to be taken. For example, if an incorrect sample plate is used or a sample plate is not facing the right direction, then blank sample will not be at the expected positions and the expected positions for blank sample will be samples of other types.

[0071] The method 100 may further comprise the step of initiating S105, which precedes the other steps. The step of initiating 105 is triggered when receiving the quality control warning from the analysis-method 10 indicating that the method 100 may be performed.

[0072] Fig. 2 depicts schematically a well plate 200 comprising 96 wells illustrated by a circle. The plate has the dimensions of 8 samples in a column (A-H) and 12 samples in a row (1-12).

[0073] Fig. 3 depicts schematically a data processing unit comprising a computer program product for determining a position and an orientation. Fig. 3 depicts a data processing unit 310 comprising a computer program product comprising a non-transitory computer-readable storage medium 312. The non-transitory computer-readable storage medium 312 having thereon a computer program comprising program instructions. The computer program is loadable into a data processing unit 310 and is configured to cause a processor 311 to carry out the method 100 for determining run errors for an analysis-method 10 for quantifying a plurality of analytes in a plurality of biological samples with the description of fig. 1.

[0074] The relevant predetermined thresholds and ranges discussed herein depend on the specific set-up of assay reagents, test reagents, and assay conditions used in any particular embodiment of the analysis method to which the present method is applied and may readily be set by the skilled practitioner. The person skilled in the art realizes that the present disclosure is not limited to the preferred embodiments described above. The person skilled in the art further realizes that modifications and variations are possible within the scope of the appended claims. Additionally, variations to the disclosed embodiments can be understood and effected by the skilled person in practicing the claimed disclosure, from a study of the drawings, the disclosure, and the appended claims. All prior art documents referenced herein are in their entirety incorporated by reference in the present disclosure. Furthermore, the co-pending European patent application 23185080.1 related to other quality control methods is also incorporated by reference in its entirety.

[0075] The present invention is further illustrated in the below examples. These examples are illustrative of the invention and shall not be construed as limiting the invention, which is as described in the appended claims.

[0076] Example 1: Plate QC

[0077] The Quality Control (QC) is performed directly on Counts, on one block per 96 samples. A "block" in this regard is an abundance block as described in Wik et al., supra. The Plate QC are divided into three parts, Sample QC, Block QC and Assay QC.

[0078] Sample QC

[0079] 1. Total number of counts per sample per block

[0080] Each sample and external control should have enough number of counts in a block for being proceeded with other steps in QC. Failure of a sample at this stage might indicate that index (i.e. identification sequence identifying reaction container) was not properly added into the sample.

[0081] 2. Number of counts of internal controls (a.k.a. test reagents)

[0082] Low counts in any of the internal controls might be an indication of a technical error in the workflow for the corresponding sample.

[0083] - Each sample and external control should have a minimum number of counts for any of the internal controls, otherwise they consider as missing the internal controls and fail.

[0084] - If the minimum number of counts are detected for each of the internal controls in samples and external controls, but still they are not considered as enough, sample gets QC warnings while external Controls fail QC. Block QC

[0085] To assess if the data in the whole block can pass QC, the corresponded Plate and Negative controls should pass some QC criteria. This step is performed only on the Plate / Negative controls that pass Sample QC step and checks if the block has been affected by any technical errors. The failed block requires to be rerun.

[0086] 1. Deviating Plate Control

[0087] Deviation of internal control's counts from the expected ranges in Plate Controls might be an indicator of different technical errors. A plate control fails this QC step if the fraction of counts of all internal control to total counts in logarithmic scale, deviates positively or negatively from the reference values. The reference ranges are block specific and kit lot related.

[0088] The data in a block passes QC if more than half of the plate controls in the corresponding block passes this QC step (ex: 3 out of 5 plate controls need to pass QC).

[0089] 2. Unexpected signal in negative Control (blank sample)

[0090] Detection of high number of counts for many assays, relative to the counts of internal controls, in any Negative Controls might be an indicatorthat signals are from other sample types or that not from a pure buffer. A Negative Control fails this step if many assays get higher number of counts in comparison with the counts of all internal controls. The data in a block passes QC if enough number of Negative Control pass this QC step (ex: 1 out of 2 Negative Controls)

[0091] Assay QC

[0092] Detection of high number of counts for any assay, relative to the internal controls, in any of the Negative Controls is considered as unexpected signal. This QC step is performed in Negative Controls that pass Sample and Block QC steps. Assays get QC warnings if they get high number of counts compared to the internal controls commonly in all Negative Controls that pass Sample and Block QC.

[0093] Only datapoints that pass the Plate QC will be passed on for further data processing.

Claims

CLAIMS1. A quality control method (100) for determining run errors for an analysis-method (10) for quantifying a plurality of analytes in a plurality of biological samples, wherein the analysis-method (10) comprises: providing a plurality of samples in a plurality of reaction containers of a plate, wherein each sample is identified as a biological sample or a standardized sample, wherein a first plurality samples in a first set of reaction containers (Al-All, ...,H1-H11) are identified as biological samples and a second plurality of samples in a second set of reaction containers (A12- H12) are identified as standardized samples, wherein the samples identified as biological samples are samples under investigation and wherein the samples identified as standardized samples are further identified as either a standard sample, or a blank sample; adding (S16), to each of the plurality of samples, predetermined amounts of at least one test reagent; adding (S14) assay reagents to each of the plurality of samples, wherein the assay reagents are configured to generate nucleic acid reporter molecules in an amount correlated to the amounts of analyte or test reagent in the plurality of samples, wherein each nucleic acid reporter molecule pertains to an analyzed sample among the plurality of samples and comprises identification sequences, the identification sequences comprises a first identification sequence identifying the reaction container of the analyzed sample and a second identification sequence uniquely identifying one of: the analyte or a test reagent from the one or more test reagents; wherein the quality control method (100) comprises: obtaining (S110) using the identification sequences of the nucleic acid reporter molecules, for each reaction container, a set of counts, the set of counts comprising a count for the amount of analyte reporter molecules detected (S18) in the reaction container; and for each test reagent of the at least one test reagent detected in the reaction container, a count for the amount of the test reagent specific reporter molecules corresponding to the test reagent; and determining (S118), for all reaction containers comprising a sample identified as a standardized sample, a mean count of the set of counts of the test reagent specific reporter molecules corresponding to any of the one or more test reagents, and upon a number of reaction containers comprising a sample identified as standardized sample, in which the countfor the amount of analyte reporter molecules detected in the reaction container exceeds the mean count, exceeds a second threshold, determining a run error for the plate.

2. The method of claim 1, further comprising: determining (S112) for each reaction container comprising a sample identified as standardized sample, a combined count of the set of counts determined for the reaction container, wherein upon the combined count for the reaction container is below a first threshold, determining a run error for the reaction container.

3. The method of any one of the preceding claims, further comprising: determining (S114) for each reaction container comprising a sample identified as standardized sample, and for each test reagent among the one or more test reagent, whether a count for the amount of test reagent specific reporter molecules of the reaction container is below a test reagent specific threshold, wherein upon the count is below the test reagent specific threshold, determining a run error for the reaction container.

4. The method of any one of the preceding claims, further comprising: determining (S116), for each reaction container comprising a sample identified as standard samples, and for each test reagent among the one or more test reagent, whether a fraction of the count for the amount of test reagent specific reporter molecules of the reaction container to the combined count of the reaction container is outside a test reagent specific range, wherein upon the fraction is outside the test reagent specific range, determining a run error for the reaction container.

5. The method (100) according to any of the preceding claims, wherein the test reagents comprise incubation test reagents, extension test reagents, and amplification test reagents.

6. The method (100) according to claim 5, wherein the incubation test reagents contain one or more non-human antigens, such as phycoerythrin (PE) and green fluorescent protein (GFP).

7. The method (100) according to any of claim 5-6, wherein the extension test reagents contain antibodies conjugated to a set of single-stranded oligonucleotides capable of at least partial hybridisation.

8. The method (100) according to any of clam 5-9, wherein the amplification test reagents comprise a synthetic double-stranded nucleic acid molecules.

9. The method (100) according to any of the preceding claims, wherein the method (100) further comprises determining (S170) a type of run error.

10. The method (100) according to any of the preceding claims, wherein the method (100) further comprises determining (S180) an actions to be taken in response to determining a run error for the plate and / or a run error for a reaction container.

11. The method (100) according to any of the preceding claims, wherein the method (100) further comprises an initial step of initiating (S105), wherein initiating (105) comprising receiving a quality control warning from the analysis-method (10) indicating that the method (100) should be performed.

12. A computer program product (300) comprising a non-transitory computer- readable storage medium (312) having thereon a computer program comprising program instructions, the computer program being loadable into a processor (314) and configured to cause the processor (314) to perform the method (100) according to any one of claims 1-11.

13. A system comprising, one or more processors; and one or more non-transitory computer-readable media storing instructions executable by the one or more processors, wherein the instructions, when executed, cause the system to perform the method (100) according to any one of claims 1-11.

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