Methods, compositions, and systems for using pooled donor samples for clinical testing.

Abstract

Methods, compositions, and systems for preparing and using pooled biological samples for clinical testing are disclosed. In certain embodiments, the methods and systems may be used to pool donors that provide basophils for clinical testing of chronic spontaneous urticaria.

Description

Technical Field

[0001]

[0001] Methods, compositions, and systems for creating and using pooled biological samples for clinical testing are disclosed.

Background Art

[0002]

[0002] Many diseases require the detection of abnormal expression of biomarkers (e.g., proteins, cytokines) using flow cytometry or other cellular methods. For example, chronic spontaneous urticaria (CSU) is a common skin disease that can be characterized by IgG autoantibodies directed against IgE and IgE receptors on the surface of mast cells or basophils.

[0003]

[0003] Chronic spontaneous urticaria (CSU) is a disorder in which hives occur without an identifiable trigger and persist for more than 6 weeks (Zuberbier et al., The international EAACI / GA 2LEN / EuroGuiDerm / APAAACI guideline for the definition, classification, diagnosis, and management of urticaria, Allergy, 2022;77(3):734-766; Kolkhir et al., Autoimmune chronic spontaneous urticaria, J.Allergy Clin.Immunol., 2022, 149(6):1819-1831). CSUs are thought to arise from the pathogenic activation of mast cells and basophils, which release histamine and other inflammatory mediators into them. Based on experimental and clinical evidence, a significant proportion of CSU cases are recognized to have an autoimmune etiology (Konstantinou et al., EAACI taskforce position paper: evidence for autoimmune urticaria and proposal for defining diagnostic criteria, Allergy, 2013, 68:27-36; Kolkhir et al., Autoimmune chronic spontaneous urticaria: what we know and what we do not know, J. Allergy Clin. Immunol., 2017; 139:1772-81; Bracken et al., Autoimmune Theories of Chronic Spontaneous Urticaria, Front. Immunol., 2019; 10:627).The autoimmune nature of CSUs is further supported by the autologous serum skin test (ASST), an in vivo assay of mast cell activation induced by intradermal injection of the patient's serum into itself (Sahiner et al., Chronic urticaria: etiology and natural course in children, Int. Arch. Allergy Immunol., 2011, 156:224-30; Hide et al., Autoantibodies against the high-affinity IgE receptor as a cause of histamine release in chronic urticaria, N. Engl. J. Med., 1993; 328(22):1599-604; Sabroe et al., 1999, The autologous serum skin test: a screening test for auto-antibodies in chronic idiopathic urticaria, Br. J. Dermatol., 1999, 140:446-52; Konstantinou et al. al., EAACI / GA(2)LEN task force consensus report: the autologous serum skin test in urticaria, Allergy, 2009, 64:1256-68). Nearly 50% of patients with CSU develop a wheal-red reaction at the injection site when inoculated with their own serum.Unfortunately, positive ASST results are not unique to patients with chronic idiopathic urticaria (CSU), but are observed in a substantial proportion of patients with allergic or non-allergic rhinitis, multidrug allergy syndrome, and even in healthy controls (Taskapan et al., Evaluation of autologous serum skin test results in patients with chronic idiopathic urticaria, allergic / non-allergic asthma or rhinitis and healthy people, Clin Exp Dermatol., 2008, 33(6):754-8). The usefulness of ASST is further limited by the fact that it cannot be used in patients treated with antihistamines, a very common condition for patients with CSU in clinical practice (D'Auria et al., Basophil activation test in children with autoimmune chronic spontaneous urticaria: Is it ready for clinical practice? Immunobiology, 2019, Jan;224:30-33).

[0004]

[0004] Historically, laboratory researchers have used the tendency of donor basophils to release histamine upon exposure to CSU patient serum in the basophil histamine release assay (BHRA) for the in vitro evaluation of CSU (Konstantinou et al., 2013; Schoeppke et al., Biomarkers and clinical characteristics of autoimmune chronic spontaneous urticaria: Results of the PURIST Study, Allergy, 2019, 74(12):2427-2436; Hoffmann et al., The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease, Allergy, 2015, 70(11):1393-405).More recently, flow cytometry has been employed to measure the expression of activation-related cell surface antigens, including CD63, in the more easily automated indirect basophil activation assay (BAT) (Konstantinou et al., 2013; D'Auria et al., 2019; Schoeppke et al., 2019; Irinyi et al., Extended diagnostic value of autologous serum skin test and basophil CD63 expression assay in chronic urticaria, Br.J.Dermatol., 2013, 168:656-8; Frezzolini et al., Serum induced basophil CD63 expression by means of a tricolor flow cytometric method for the in vitro diagnosis of chronic urticaria, Allergy, 2006, 61:1071-72006; Szegedi et al., Significant correlation between the CD63 assay and the histamine release assay in chronic urticaria,Br.J.Dermatol.,2006,155(1):67-752006;De Swerdt et al.,Detection of basophil-activating IgG autoantibodies in chronic idiopathic urticaria by induction of CD 63,J.Allergy Clin.Immunol.,2005,116:662-7).

[0005]

[0005] However, current assay protocols require donors who can provide three readily available basophils with high activation in response to anti-FcεRI stimulation, which can be difficult for both clinical laboratories and blood donors. For example, numerous studies have shown that positive indirect BAT results are more frequent in CSU patients than in non-CSU controls (Monino-Romero et al., Positive Basophil Tests Are Linked to High Disease Activity and Other Features of Autoimmune Chronic Spontaneous Urticaria: A Systematic Review, J. Allergy Clin. Immunol. Pract., 2023: S2213-2198(23)00603-7; Marcelino et al., What Basophil Testing Tells Us About CSU Patients - Results of the CORSA Study, Front. Immunol., 2021, 12: 742470; HOssein et al., The CD63 basophil activation test as a diagnostic tool for assessing autoimmunity in patients with chronic spontaneous urticaria, Eur. J. Dermatol., 2019, 29(6): 614-618; Irinyi et al.,2013;Netchiporouk et al.,Positive CD63 basophil activation tests are common in children with chronic spontaneous urticaria and linked to high disease activity,Int.Arch.Allergy Immunol.,2016,171:81-82016;De Swerdt et al.,2005).Furthermore, positive results from the BAT test have been suggested to be associated not only with high disease activity in both adults and children, but also as a predictor of how quickly patients respond to treatment (Netchiporouk et al., 2016; Gericke et al., Serum autoreactivity predicts time to response to omalizumab therapy in chronic spontaneous urticaria, J. Allergy Clin. Immunol., 2017, 139:1059-61; Curto-Barredo et al., Basophil activation test identifies the patients with chronic spontaneous urticaria suffering the most active disease, Immun. Inflamm. Dis., 2016, 4:441-5).

[0006]

[0006] One of the challenges in performing indirect BAT is the variability in the response of donor basophils in the assay. Some donor basophils are highly reactive to pathological serum samples, while others are not. Conversely, some donor basophils are nonspecifically activated by healthy control serum. In general laboratory practice, this variability in donor reactivity is taken into account by testing patient samples with multiple donors in separate experiments (Gentinetta et al., 2011). If activation varies for individual donors, arbitrary rules are established to evaluate the results (Marcelino et al., What Basophil Testing Tells Us About CSU Patients-Results of the CORSA Study, Front.Immunol., 2021, 12:742470; D'Auria et al., 2019).

[0007]

[0007] Therefore, it is necessary to develop a more robust and reliable testing method. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Zuberbier et al.,The international EAACI / GA2LEN / EuroGuiDerm / APAAACI guideline for the definition, classification, diagnosis, and management of urticaria,Allergy,2022;77(3):734-766 [Non-Patent Document 2] Kolkhir et al.,Autoimmune chronic spontaneous urticaria,J.Allergy Clin.Immunol.,2022,149(6):1819-1831 [Non-Patent Document 3] Konstantinou et al.,EAACI taskforce position paper:evidence for autoimmune urticaria and proposal for defining criteria diagnostic,Allergy,2013,68:27-36 [Non-Patent Document 4] Kolkhir et al.,Autoimmune chronic spontaneous urticaria:what we know and what we do not know,J.Allergy Clin.Immunol.,2017;139:1772-81 [Non-Patent Document 5] Bracken et al.,Autoimmune Theories of Chronic Spontaneous Urticaria,Front.Immunol.,2019;10:627 [Non-Patent Document 6] Sahiner et al.,Chronic urticaria:etiology and natural course in children,Int.Arch.Allergy Immunol.,2011,156:224-30 [Non-Patent Document 7] Hide et al., Autoantibodies against the high-affinity IgE receptor as a cause of histamine release in chronic urticaria, N.Engl.J.Med., 1993;328(22):1599-604 [Non-Patent Document 8] Sabroe et al.,1999,The autologous serum skin test:a screening test for auto-antibodies in chronic idiopathic urticaria,Br.J.Dermatol.,1999,140:446-52 [Non-Patent Document 9] Konstantinou et al.,EAACI / GA(2)LEN task force consensus report:the autologous serum skin test in urticaria,Allergy,2009,64:1256-68 [Non-Patent Document 10] Taskapan et al.,Evaluation of autologous serum skin test results in patients with chronic idiopathic urticaria,allergic / non-allergic asthma or rhinitis and healthy people,Clin Exp Dermatol.,2008,33(6):754-8 [Non-Patent Document 11] D'Auria et al.,Basophil activation test in children with autoimmune chronic spontaneous urticaria:Is it ready for clinical practice? Immunobiology,2019,Jan;224:30-33

Outdoor Tools 12

Outdoor Content13

Outdoor Tools 14

Outdoor Tools 15

Outdoor Content 16

Non - Patent Document 17

Non - Patent Document 18

Non - Patent Document 19

Non - Patent Document 20

Non - Patent Document 21

[0009]

[0008] Methods and systems for preparing pooled biological samples that can be used to test subjects for a given indication are disclosed. In one embodiment, a method for pooling biological samples for use in a test is disclosed, which includes screening multiple biological samples for responses indicating the presence of a given indication and selecting multiple samples that provide responses exceeding a first predetermined threshold to generate a pool. In one embodiment, the method may be used to pool donors that provide basophils from routine CBC testing in order to provide a consistent, reliable, and clinically valid assay.

[0010]

[0009] Methods are also disclosed for using pooled samples to test whether a subject, e.g., a patient, has a given target indication. Accordingly, methods are disclosed for testing for a response indicative of the presence of a given indication, the methods comprising generating a plurality of pooled samples from a sample from a subject that provide a response exceeding a first predetermined threshold and optionally do not exhibit a non-specific response, and adding a portion of the sample from the subject to the pooled samples to determine whether the samples generate a response.

[0011]

[0010] Compositions, kits, systems, and computer program products for performing any of the disclosed methods or steps of the disclosed methods are also disclosed.

[0012]

[0011] The present disclosure may be better understood by reference to the following non-limiting figures.

Brief Description of the Drawings

[0013] [Figure 1] A method for generating and using a donor pool for patient testing, according to one embodiment of the present disclosure, is shown. [Figure 2] A diagram showing a system for generating and using a donor pool for patient testing, according to one embodiment of the present disclosure. [Figure 3] A diagram showing an exemplary computing device according to various embodiments of the present disclosure. [Figure 4A] Representative gating of cells with uptake set to 1000 basophil events, according to various embodiments of the present disclosure, is shown. A diagram showing basophil gating. [Figure 4B] Representative gating of cells with uptake set to 1000 basophil events, according to various embodiments of the present disclosure, is shown. A diagram showing background control gating for activated basophils (shown as black circles). [Figure 4C]This figure shows typical gating of cells with uptake set to 1000 basophil events, according to various embodiments of the present disclosure. It also shows anti-FcεRI controlled gating for activated basophils (shown as black circles). [Figure 4D] This figure shows typical gating of cells in which uptake is set to a 1000 basophil event, according to various embodiments of the present disclosure. It also shows low-response serum pooling gating for activated basophils (shown as black circles). [Figure 4E] This figure shows typical gating of cells in which uptake is set to a 1000 basophil event, according to various embodiments of the present disclosure. It also shows the medium-reaction serum pool gating for activated basophils (shown as black circles). [Figure 4F] This figure shows typical gating of cells in which uptake is set to a 1000 basophil event, according to various embodiments of the present disclosure. It also shows high-reactivity serum pooling gating for activated basophils (shown as black circles). [Figure 5A] The distribution of %CD63+ basophils during the donor screening period in one embodiment of the present disclosure is shown, representing %CD63+ basophils in screening BATs for a total of 463 donors processed for 15 days over a 4-month period. Selected donors for the CSU-BAT pool are indicated by the word "present" on the cell population (dark purple circles), and excluded donors are indicated by the word "absent" on the cell population (light blue circles). [Figure 5B] A further depiction of donor screening results providing basophils, as shown in Figure 5A, according to one embodiment of the present disclosure, is shown. The line plots show %CD63+ basophils in the screening PD-BAT for a total of 463 donors treated for 15 days. Donors selected for the CSU-BAT pool are shown as dark (purple) shading, and excluded donors are shown as lighter blue hatched lines. [Figure 6]This figure shows the reproducibility and accuracy of PD-BAT according to one embodiment of the present disclosure. The mean of activated basophils (n=4) across three levels of serum pooling (low-response serum pool (LSP), medium-response serum pool (ISP), and high-response serum pool (HSP)) across the kit control and donor pools providing 10 different basophils demonstrates the consistency of PD-BAT. Error bars represent one standard deviation from the mean. Background (orange), low-response serum pool (LSP) (gray), medium-response serum pool (ISP) (blue), high-response serum pool (HSP) (purple), and anti-FcεRI positive control (red) are shown. [Figure 7] This shows the serum temperature stability of the highly reactive serum pool. Ambient temperature (blue) and refrigerated temperature (green) conditions of the HSP were tested on days 0, 1, 3, 7, and 14. [Figure 8] This figure shows the evaluation of pooled whole blood stability according to one embodiment of the present disclosure. Basophil donors were screened and selected. Basophil activation was analyzed at 0, 2, 6, and 24 hours for background, anti-FcεRI, LSP, ISP, and HSP. The standard deviations ranged from 0.22 (HSP) to 2.23 (anti-FcεRI and HSP). [Figure 9A] This figure shows the distribution of serum used to determine the technical cutoff. It depicts the distribution of results for 142 samples with total IgE < 80 IU / L according to one embodiment of this disclosure. Two outliers were removed by Chauvenet exclusion. The central 95% was set as the technical cutoff. [Figure 9B] The distribution of serum used to determine the technical cutoff is shown. The validation of the threshold in separate analyses of 145 samples with a BHRA negative result of less than 2.2 CU Index® (after excluding Chauvenet outliers) is depicted, where the upper limit of the reference interval was determined to be 11.52% (10.19–13.02, 95% CI). [Figure 10]This figure shows a method comparison between CU Index® and PD-BAT according to one embodiment of the present disclosure. The quantitative method comparison was completed via EP Evaluator (n=343). The regression analysis-Passing-Bablok gradient is 0.814. The correlation coefficient is 0.6144. The PD-BAT technical cutoff (10.61) (horizontal red line), the CU Index® technical cutoff (above 10 CU Index® units) (BHRA technical cutoff) (vertical yellow line), and the Passing-Bablok regression analysis (dashed blue line) are shown. [Figure 11] Figures 11A, 11B, and 11C show 69 serum samples from CSU patients analyzed using a pooled donor method and three individual donors according to one embodiment of the present disclosure. Donor 1 is shown in Figure 11A. Donor 2 is shown in Figure 11B. Donor 3 is shown in Figure 11C. The black dashed lines represent linear regressions, respectively (R=0.85; R=0.93; R=0.59 for Donors 1, 2, and 3, respectively). The vertical and horizontal lines represent the 10.61% CD63+ cutoff. [Figure 12] This figure shows the PD-BAT comparability of results from two runs according to one embodiment of the present disclosure. The two results are plotted against the pairwise mean. Donor pool 1 (R=0.997) (purple triangle); green circle, Donor pool 2 (R=0.998) (green circle); PD-BAT technical cutoff (horizontal and vertical red lines). [Modes for carrying out the invention]

[0014]

[0024] The disclosed subject matter is described more fully below with reference to the accompanying description and drawings, which illustrate some, though not all, embodiments, of the disclosed subject matter. The disclosed subject matter can be embodied in many different forms and should not be construed as being limited to the embodiments described herein. Similar numbers refer to similar elements throughout. Certain terms are adopted herein, but they are used only in a general and descriptive sense and not for limiting purposes. Furthermore, any reference referred to as “incorporated by reference herein” is understood to be incorporated in its entirety.

[0015]

[0025] definition The following terms are expected to be readily understood by those skilled in the art, but their definitions are provided for the convenience of describing the subject matter now disclosed. Other definitions are found throughout this specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the field to which the subject matter described herein belongs.

[0016]

[0026] Although the numerical ranges and parameters defining the broad scope of this disclosure are approximations, the numerical values ​​described in specific examples are reported as accurately as possible. However, any numerical value inherently contains certain errors that inevitably arise from the standard deviation found in each test measurement. Furthermore, all ranges disclosed herein are understood to encompass all subranges contained therein. For example, a range described as "1 to 10" should be considered to encompass all subranges between (and including) the minimum value of 1 and the maximum value of 10; that is, all subranges starting from a minimum value greater than or equal to 1, e.g., 1 to 6.1, and ending from a maximum value less than or equal to 10, e.g., 5.5 to 10.

[0017]

[0027] Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise. Where used herein, the terms “comprises” and / or “comprising” include embodiments of “consisting of” and / or “consisting essentially of,” identifying the presence of the described features, integers, processes, operations, elements, and / or components, but not excluding the presence or addition of one or more other features, integers, processes, operations, elements, components, and / or groups thereof. Where used herein, the terms “and / or” include any and all combinations of one or more of the items listed in relation. Where used herein, phrases such as “between X and Y” and “about between X and Y” should be interpreted as including X and Y. Where used herein, phrases such as “about X to Y” mean “about X to about Y.”

[0018]

[0028] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art in which the invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having the meaning consistent with their meanings in the context of this specification and related art, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. Well-known functions or configurations may not be described in detail for the sake of brevity and / or clarity.

[0019]

[0029] Terms such as "first," "second," etc., may be used herein to describe various elements or components, but it will be understood that these elements or components should not be limited by these terms. Furthermore, the order of operations (or processes) is not limited to the order presented in the claims or drawings unless otherwise specifically indicated. The term "programmatically" means that an action is performed using computer programs and / or software, a processor, or an ASIC. The term "electronic" and its derivatives refer to automated or semi-automated actions performed using devices having electrical circuits and / or modules, rather than through mental processes, and typically refer to actions that are performed programmatically.

[0020]

[0030] The terms "automated" and "automatic" mean that an operation can be performed with minimal or no manual intervention or input. The term "semi-automated" refers to allowing some input or activation from the operator, but where calculation, intake, purification, and other processes are performed electronically, typically programmatically, without requiring manual input.

[0021]

[0031] The term "approximately" refers to ±10% (mean or average) of a specified value or number.

[0022]

[0032] As used herein, “pool” includes multiple (i.e., >1) samples.

[0023]

[0033] As used herein, “a given threshold” may include a range, or a lower limit (e.g., a quantity that determines the lower limit of a range), or an upper limit (e.g., a quantity that determines the upper limit of a range). For example, a given threshold may be a range between two units, for example, between 2.00 and 4.00, and such a range includes 2.00 and 4.00 and all values ​​in between. A given threshold may also include values ​​less than (or equal to and / or less than) the unit value (upper limit). For example, a given threshold may be a range of 2.00 or less. This given threshold includes all values ​​between 0 and 2.00 and / or equal to it. A given threshold may also include values ​​greater than (or equal to and / or greater than) the unit value (lower limit). For example, a given threshold may be a range of 4.00 or more. This given threshold includes all values ​​equal to or greater than the lower limit of 4.00. Unless otherwise stated, a given threshold includes positive (>0) values.

[0024]

[0034] As used herein, repeatability (or intraassay precision) describes the degree of agreement between the results of consecutive measurements of the same analyte performed under the same measurement conditions. Intraassay repeatability is a measure of variability when the same sample is analyzed during a single analytical run.

[0025]

[0035] As used herein, reproducibility (or inter-assay precision) describes the degree of agreement between the results of consecutive measurements of the same analyte performed under the same measurement conditions. Inter-assay repeatability is a measure of variability when the same sample is analyzed during two or more runs.

[0026]

[0036] The terms “sample,” “patient sample,” “biological sample,” or “specimen” may be used interchangeably herein. Where used herein, a biological sample for use in a disclosed pool is different from a biological sample, such as a patient sample or a sample from an object that can be evaluated for a given indication of the intended purpose. In some cases, a biological sample used in a pool (or pools) is referred to as a “donor sample” or “donor biological sample.”

[0027]

[0037] Non-limiting examples of samples that may be used to generate and / or test pools using the disclosed methods and systems include blood or blood products (e.g., serum, plasma, etc.). In some cases, such blood products may be used directly. The term “blood” includes whole blood, blood products, or any blood fraction of blood, e.g., serum, plasma, buffy coat, etc., as conventionally defined. Other samples may include urine, nasal swabs, liquid biopsy samples, skin swabs, lesion swabs, or combinations thereof.

[0028]

[0038] The terms “individual,” “patient,” or “subject” are used broadly to refer to individuals that provide samples for testing or analysis, and / or biological samples that may be used in pools (e.g., donor biological samples). “Individual,” “patient,” or “subject” from which a sample is taken, obtained, and / or provided includes any warm-blooded mammalian subject, such as humans and / or animals.

[0029]

[0039] The term “specified indication” is used to describe a medical condition such as a disease and / or a predisposition to the disease. Such a disease may result from autoantibodies or other congenital medical conditions.

[0030]

[0040] The term "response indicating a specified indication" refers to a biological response that can be measured and used to diagnose the presence or predisposition to a specified indication. In certain cases, the response may be of a cellular nature, such as, but not limited to, basophil activation. Abbreviation ASST, autologous serum skin test BAT, Basophil Activation Test BHRA, basophil histamine release assay CBC, complete blood count CCR3, CC motif chemokine receptor 3 CD63, Differentiation Cluster 63 CSU, Chronic Spontaneous Urticaria CSU-BAT, an indirect basophil activation test using flow cytometry with a single donor. CV%, coefficient of variation EDTA, ethylenediaminetetraacetic acid FcεRI, high affinity IgE receptor FSC, forward scatter HSP, highly reactive serum pool IgE, immunoglobulin E ISP, intermediate reaction low serum pool LSP, low-response serum pool mAB; monoclonal antibody NSP, Negative serum pool PD-BAT, pooled donor basophil activation test by flow cytometry RBC, red blood cell SD, standard deviation SSC, side scatter

[0031]

[0041] method A method for preparing and using pooled biological samples for use in a laboratory and / or clinical laboratory is disclosed. In certain embodiments, the method may include screening a plurality of biological samples for responses indicating the presence of a predetermined indication, and selecting a subset of such biological samples that provide responses exceeding a first predetermined threshold in order to generate a pool.

[0032]

[0042] The pooled biological samples may be further screened to remove samples with nonspecific responses. Therefore, in certain embodiments, the method may include screening multiple biological samples to determine whether a sample has a nonspecific response exceeding a predetermined threshold, and removing samples with nonspecific responses from the pool.

[0033]

[0043] For example, a pooled sample may be screened to include samples that have a response above a certain threshold for a positive control. For instance, if the pool is to be used to measure basophil activation using the detection of an activation-triggered anti-FcεRI mAb and CD63 as a marker of basophil degranulation, the positive control is an anti-FcεRI mAb (e.g., with an appropriate stimulation buffer / mix), and the donors selected for the pool have a response at or above a first predetermined threshold. In one embodiment, donor samples showing a low response (less than 40% activation) to the anti-FcεRI mAb control are excluded from the pool.

[0034]

[0044] Samples evaluated for inclusion in the pool may also be screened to exclude donors exhibiting a nonspecific response. Thus, samples screened for use in the pool may be evaluated for responses occurring upon exposure to reagents expected to provide a negative control or a low response. Negative controls may include the addition of buffers and other assay reagents, such as the low-response serum pool (LSP) control disclosed herein. In one embodiment, a predetermined threshold used to determine a nonspecific response is different from a first predetermined threshold. In certain embodiments, the two thresholds are the same. In one embodiment, donor samples exhibiting nonspecific activation (>4.6%) with the LSP control are excluded from the pool.

[0035]

[0045] The method may further include removing samples that do not contain cell populations typically found in biological samples used for pooling. For example, if the pool is used to screen for basophil activation as discussed herein, biological samples that do not contain cell types, such as lymphocytes, monocytes, or granulocytes, may be excluded. In certain embodiments, if the pool is used to measure basophil activation as disclosed herein, biological samples may be excluded from the pool if the basophil population cannot be clearly distinguished between SSC and CCR3, in order to prevent inappropriate gating and results.

[0036]

[0046] The biological sample may vary depending on the desired response. In certain embodiments, the biological sample used for pooling is blood. For example, in certain embodiments, a blood sample collected for whole blood cell count (CBC) screening may be used. Such a sample may be used after storage. In certain embodiments, individual donor blood is stored at room temperature for up to 24 hours after collection. This assay screens only blood collected within 24 hours. After the blood donor has been screened, individual aliquots of blood are stored in a refrigerator for up to 24 hours, if refrigerated (4°C), before the blood is pooled. In one embodiment, the blood sample is processed to provide leukocytes. In one embodiment, such leukocytes include basophils. Alternatively, other samples (e.g., plasma, serum, saliva) may be used for pooling.

[0037]

[0047] Pooled samples may be useful for cell-based tests, such as, but not limited to, flow cytometry. For example, in some cases, pooled blood samples are used to test patient serum samples for diseases caused by abnormal basophilic activation. Thus, in certain embodiments, samples evaluated for inclusion in the pool are screened for basophilic activation. In one embodiment, samples evaluated for inclusion in the pool are screened for basophilic activation using the detection of an anti-FcεRI mAb as an activation trigger and CD63 as a marker of basophilic degranulation. In one embodiment, a given indication is an autoantibody disease. In one embodiment, the method may then further include using pooled samples to test patient serum for a basophilic response showing chronic spontaneous urticaria.

[0038]

[0048] In one embodiment, a pooled sample is used to evaluate the response resulting from exposure of a subject to a separate sample. For example, in one embodiment, the pooled sample is used to evaluate basophil activation that occurs in the pooled sample upon exposure of the pooled sample to a biological sample from the test subject. In one embodiment, the biological sample from the test subject is serum.

[0039]

[0049] The pool size may depend on the test being performed and the availability of samples for pooling. In certain embodiments, the pool may contain at least 10, or at least 20, or at least 30 biological samples. Alternatively, more or fewer samples may be used. The pooled samples are derived from a larger collection of samples. For example, in certain embodiments, the pool may contain about 50% or more of the samples to be screened for pooling. Alternatively, a larger or smaller proportion of the samples available for screening may be used.

[0040]

[0050] Sample pooling allows for the development of robust tests for various indications, as pooled samples may provide more consistent responses than individual samples. In certain embodiments, the technical cutoff for pooled samples showing a positive response indicating the presence of a given indication is defined using the 95% confidence interval (CI) of multiple normal samples. Thus, as shown in Examples 2 and 3, sample pooling for use in testing can provide a significant improvement over the use of individual samples.

[0041]

[0051] In one embodiment, the accuracy of the pool is evaluated by comparing the results with known quality control (QC) samples. In one embodiment, known quality control includes (i) biological samples that produce a high response above a first predetermined QC threshold (i.e., high QC), (ii) biological samples that produce an intermediate response above a second predetermined QC threshold but below the first QC threshold (i.e., intermediate QC), and (iii) biological samples that produce a low response below a third predetermined QC threshold that is lower than the second QC threshold (i.e., low QC). The thresholds used for QC may differ from the thresholds used for evaluating high responders to a positive anti-FcεRI mAb control and the thresholds used to determine nonspecific responses. Alternatively, some of the thresholds may be the same.

[0042]

[0052] In one embodiment, the high-response, intermediate-response, and low-response quality control controls include serum. In a particular embodiment, the high-response, intermediate-response, and low-response quality control controls include a pool of samples. For example, high QC may include pooled serum that produces a high response above a first predetermined QC threshold (i.e., high-response serum pool; HSP), intermediate QC may include pooled serum that produces an intermediate response above a second predetermined QC threshold but below the first QC threshold (i.e., intermediate-response serum pool; ISP), and low QC may include pooled serum that produces a low response below a third predetermined QC threshold that is lower than the second QC threshold (i.e., low-response serum pool; LSP). The thresholds used for QC may differ from the thresholds used for evaluating high responders to a positive anti-FcεRI mAb control and the thresholds used to determine nonspecific responses. Alternatively, some of the thresholds may be the same.

[0043]

[0053] Quality control controls may also include known positive controls and / or known negative controls. For example, if the pool is to be used to measure basophil activation using an anti-FcεRI mAb as an activation trigger and the detection of CD63 as a marker of basophil degranulation, the positive control would be an anti-FcεRI mAb and a stimulating buffer, as disclosed herein. Negative controls may include the addition of a buffer and / or other assay reagents.

[0044]

[0054] A predetermined QC threshold used in the QC pool can be based on a predetermined reference value related to the response being measured. For example, in a particular embodiment, high QC (e.g., HSP) may produce a response that is 3.0 standard deviations (SD) or more above a defined technical cutoff for the response being measured. Intermediate QC (e.g., ISP) may produce a response that is 1 to 3 SD below the technical cutoff for the response being measured. Low QC (e.g., LSP) may produce a response that is more than 3 SD below the technical cutoff for the response being measured.

[0045]

[0055] For example, to evaluate basophil activation, a low-response serum pool (LSP) quality control control may include a pool of previously screened and stored serum with results of 0–4.67% activated basophils, i.e., approximately 3 standard deviations (SD) below the reference interval of 10.61 (PD-BAT technical cutoff). In certain embodiments, an intermediate-response serum pool (ISP) quality control control may include a pool of previously screened and stored serum with results of 4.67–8.63% activated basophils, i.e., approximately 1–3 SD below the reference interval of 10.61 (PD-BAT technical cutoff). In certain embodiments, a high-response serum pool control (HSP) may include a pool of previously screened and stored serum with results of greater than 16.55% activated basophils, i.e., approximately 3 SD above the reference interval of 10.61 (PD-BAT technical cutoff). These serum control pool sub-aliquots can be stored at -20°C for future use.

[0046]

[0056] Also disclosed is a method for using pooled samples to test whether a subject, for example a patient, has an indication for a particular purpose. Thus, a method for testing for a response indicating the presence of a particular indication is disclosed, the method comprising the steps of: generating a plurality of pooled samples from a subject that provide a response exceeding a first predetermined threshold and that do not show a nonspecific response at arbitrary selection; and adding a portion of the sample from the subject to the pooled samples to determine whether the sample produces a response.

[0047]

[0057] The pooled samples used in the test may be screened to remove samples that do not respond to a positive control and / or have a nonspecific response. Therefore, in certain embodiments, the method may include screening multiple samples to determine whether a sample has a nonspecific response exceeding a predetermined threshold, and removing samples with a nonspecific response from the pool.

[0048]

[0058] For example, pooled samples may be screened to remove samples that have a response below a specific threshold for a positive control. For instance, if the pool is to be used to measure basophil activation using the detection of an anti-FcεRI mAb as an activation trigger and CD63 as a marker of basophil degranulation, the positive control is an anti-FcεRI mAb and stimulation buffer as disclosed herein, and the donor selected for the pool has a response at or above a first predetermined threshold. In one embodiment, donor samples showing a low response (less than 40% activation) to the anti-FcεRI mAb control are excluded from the pool.

[0049]

[0059] The negative control may include the addition of a buffer and other assay reagents, such as a low-response serum pool (LSP) control disclosed herein. In one embodiment, a predetermined threshold used to determine a nonspecific response is different from a first predetermined threshold. In certain embodiments, the two thresholds are the same. In one embodiment, donor samples showing nonspecific activation (>4.6%) by the LSP control are excluded from the pool.

[0050]

[0060] The method may further include removing samples that do not contain cell populations typically found in biological samples used for pooling. For example, biological samples that do not contain cell types such as lymphocytes, monocytes, or granulocytes may be excluded. In certain embodiments, when the pool is used to measure basophil activation as disclosed herein, biological samples may be excluded from the pool if the basophil population cannot be clearly distinguished between SSCs and CCR3s in order to prevent inappropriate gating and results.

[0051]

[0061] Depending on the desired response, the biological sample used to generate the pool may vary. In certain embodiments, the biological sample used for pooling is blood. For example, in certain embodiments, a blood sample collected for whole blood cell count (CBC) screening may be used. Such samples may be used after storage. For example, individual donor blood is stored at room temperature for up to 24 hours after collection. This assay screens only blood collected within 24 hours. After the blood donor has been screened, individual aliquots of blood are stored in a refrigerator for up to 24 hours, if refrigerated (4°C), before the blood is pooled. In one embodiment, the blood sample is processed to provide leukocytes. In one embodiment, such leukocytes include basophils. Alternatively, other samples (e.g., plasma, serum, saliva) may be used for pooling.

[0052]

[0062] Samples from the test subject (e.g., a patient) are separate from the samples used in the pool. In one embodiment, the biological sample from the test subject is serum. Alternatively, other types of patient samples may be tested using the pool.

[0053]

[0063] Pooled samples may be useful for cell-based tests, such as, but not limited to, flow cytometry. For example, in some cases, pooled blood samples are used to test patient serum samples for diseases caused by abnormal basophilic activation. Thus, in certain embodiments, samples evaluated for inclusion in the pool are screened for basophilic activation. In one embodiment, samples evaluated for inclusion in the pool are screened for basophilic activation using an activation trigger anti-FcεRI mAb and detection of CD63 as a marker of basophilic degranulation. In one embodiment, a given indication is an autoantibody disease. In one embodiment, the method may include using pooled samples to test patient serum for a basophilic response showing chronic spontaneous urticaria.

[0054]

[0064] The pool size may depend on the test being performed and the availability of samples for pooling. In certain embodiments, the pool used for the test may contain at least 10, or at least 20, or at least 30 biological samples. Alternatively, more or fewer samples may be used. The pooled samples are derived from a larger collection of samples. For example, in certain embodiments, the pool may contain about 50% or more of the samples to be screened for pooling. Alternatively, a larger or smaller proportion of the samples available for screening may be used.

[0055]

[0065] As shown in Examples 2 and 3 of this specification, sample pooling allows for the development of robust tests for various indications, as pooled samples may provide a more consistent response than individual samples. In certain embodiments, the technical cutoff for pooled samples showing a positive response indicating the presence of a given indication is defined using the 95% CI of multiple normal samples.

[0056]

[0066] As discussed above, the accuracy of the pool used in the test can be evaluated by comparing the results using patient samples with known quality control (QC) samples. In one embodiment, known quality control controls include (i) biological samples that produce a high response above a first predetermined QC threshold (high QC), (ii) biological samples that produce an intermediate response above a second predetermined QC threshold but below the first threshold (intermediate QC), and (iii) biological samples that produce a low response below a third predetermined QC threshold that is lower than the second threshold (low QC). The thresholds used for QC may differ from the thresholds used for evaluating high responders to a positive anti-FcεRI mAb control and the thresholds used to determine nonspecific responses.

[0057]

[0067] In one embodiment, the high-response, intermediate-response, and low-response quality control controls include serum. In a particular embodiment, the high-response, intermediate-response, and low-response quality control controls include a pool of samples. For example, high QC may include pooled serum that produces a high response above a first predetermined QC threshold (i.e., high-response serum pool; HSP), intermediate QC may include pooled serum that produces an intermediate response above a second predetermined QC threshold but below the first QC threshold (i.e., intermediate-response serum pool; ISP), and low QC may include pooled serum that produces a low response below a third predetermined QC threshold that is lower than the second QC threshold (i.e., low-response serum pool; LSP). The quality control controls may also include known positive controls and / or known negative controls. For example, if the pool is to be used to measure basophil activation using the detection of an anti-FcεRI mAb as an activation trigger and CD63 as a marker of basophil degranulation, then the positive control is an anti-FcεRI mAb. The negative control may include the addition of buffer and other assay reagents.

[0058]

[0068] The predetermined QC thresholds used in the QC pool can be based on predetermined reference values ​​related to the response being measured. In certain embodiments, high QC may produce a response that is 3.0 standard deviations (SD) or more above the defined technical cutoff for the response being measured. Intermediate QC may produce a response that is 1 to 3 SD below the technical cutoff for the response being measured. Low QC may produce a response that is more than 3 SD below the technical cutoff for the response being measured.

[0059]

[0069] For example, to evaluate basophil activation in a sample from a subject (e.g., a patient), a low-response serum pool (LSP) quality control control (LSP) may include a pool of previously screened and stored serum with results of 0–4.67% activated basophils, i.e., approximately 3 standard deviations (SD) below the reference interval of 10.61 (PD-BAT technical cutoff). In certain embodiments, an intermediate-response serum pool (ISP) quality control control may include a pool of previously screened and stored serum with results of 4.67–8.63% activated basophils, i.e., approximately 1–3 SD below the reference interval of 10.61 (PD-BAT technical cutoff). In certain embodiments, a high-response serum pool control (HSP) may include a pool of previously screened and stored serum with results of greater than 16.55% activated basophils, i.e., approximately 3 SD above the reference interval of 10.61 (PD-BAT technical cutoff). These serum control pool sub-aliquots can be stored at -20°C for future use.

[0060]

[0070] Figure 1 shows a diagram of method 100 of the present disclosure. As shown, the method may include a step 102 of obtaining a biological sample from each of a plurality of donors. In one embodiment, blood or a blood product (e.g., serum) is used. The method may further include a step 104 of dispensing portions of each donor sample into reaction vessels and adding reagents in order to evaluate the individual donor samples for a response indicating a predetermined indication. For example, each tube may be to which reagents may be added to evaluate whether the donor sample has a sufficiently high response to a positive control and / or whether it has a nonspecific response when evaluated using a negative or low-response control. If the response being measured is basophil activation, the reagents may include anti-FcεRI as a positive control, as well as an LSP master mix and / or buffer for separating the donor samples into two separate negative controls (i.e., for evaluating donor samples that produce nonspecific basophil activation).

[0061]

[0071] Next, the method may include a step 106 of isolating cells from a donor sample and a step 108 of evaluating the cells for a response indicating a predetermined indication. As disclosed herein, in certain embodiments, the method may include separate steps of heating incubation at 37°C to stimulate basophil activation in response to a stimulant (i.e., anti-FcεRI, or LSP), lysing of erythrocytes (RBCs), centrifugation and removal of supernatant, and resuspension of pelleted leukocytes (WBCs) containing basophils. The sample may then be taken up in a flow cytometer and gated as disclosed herein. As disclosed herein, if the predetermined indication is chronic spontaneous urticaria, the response being measured is basophil activation measured using an activation-triggered anti-FcεRI mAb, which is detected as CD63+ expression.

[0062]

[0072] In one embodiment, each donor sample is evaluated to determine whether the response is specific or not 109. If the response is not specific, the donor sample is not used in pool 110. If the response is specific, the donor sample is selected for use in pool 112.

[0063]

[0073] For example, for basophil activation testing, donors with high anti-FcεRI mAb stimulation and who are highly unresponsive to or do not show high background in the low-response serum pool (LSP) may be included in the pool. Conversely, in one embodiment, donors showing nonspecific activation (>4.6%) by the low-response serum pool (LSP; i.e., low-QC pool) are excluded from use in the pool. Also, donors showing a low response (<40%) to an anti-FcεRI mAb positive control are excluded from use in the pool. Donors may also be excluded if one of the following populations: lymphocytes, monocytes, or granulocytes, is missing in the SSC singlet plot. Donors may also be excluded if they do not have at least 300 basophil events within 200 seconds of uptake, and / or if the basophil population is not clearly distinguishable between SSC and CCR3 to prevent inappropriate gating and outcomes. Once selected for use in the pool, multiple donor samples (e.g., about 16-20) are selected for inclusion in the pool. The selected donor samples may be stored (e.g., refrigerated overnight) before being pooled and used in the assay of patient samples.

[0064]

[0074] Next, each aliquot of the selected donor sample is pooled for use in an assay to test the patient sample to determine whether the patient sample produces a response indicating a given indication 114. At this point, a master mix is ​​prepared using the pooled donor samples 116. This master mix can then be used to evaluate the patient sample, as well as appropriate quality control controls (e.g., LSP, ISP, HSP, background, and positive controls) for the response being measured 118. This screening may include steps of heat incubation to stimulate a basophilic response, lysis of red blood cells (RBCs) in the pooled donor sample, centrifugation and removal of the supernatant, and resuspension of pelleted white blood cells (WBCs) containing basophils. The sample can then be taken up in a flow cytometer and gated. The results can then be reported to the subject and / or their healthcare provider 120.

[0065]

[0075] Compositions and kits Compositions and kits for carrying out the disclosed methods are disclosed. Accordingly, in one embodiment, a composition is disclosed comprising a pool of biological samples for screening samples from a subject for a predetermined indication, wherein the pool comprises a number of biological samples, each exhibiting a response above a first predetermined threshold and optionally not exhibiting a nonspecific response, wherein the response indicates the presence of the predetermined indication. In one embodiment, the composition comprises individually packaged pooled donor biological samples.

[0066]

[0076] Also disclosed is a kit comprising a composition for screening samples from subjects for a predetermined indication, wherein the pool comprises a plurality of biological samples, each exhibiting a response exceeding a first predetermined threshold and optionally not exhibiting a nonspecific response, wherein the response indicates the presence of the predetermined indication, and instructions for use. In one embodiment, the composition used in the kit comprises individually packaged / filled pooled donor biological samples.

[0067]

[0077] Pooled samples used for either the composition and / or its kit can be screened to remove samples exhibiting a nonspecific response. Thus, in a particular embodiment, multiple samples used in the pool are screened to determine whether the sample exhibits a nonspecific response exceeding a predetermined threshold, and samples showing a nonspecific response are not used from the pool.

[0068]

[0078] For example, pooled samples may be screened to remove samples that have a response below a specific threshold for a positive control. In one embodiment, if the pool is to be used to measure basophil activation using the detection of an activation-triggered anti-FcεRI mAb and CD63 as a marker of basophil degranulation, the positive control is the anti-FcεRI mAb and stimulation buffer, and the donors selected for the pool have a response at or above a first predetermined threshold. In one embodiment, donor samples showing a low response (less than 40% activation) to the anti-FcεRI mAb control are excluded from the pool.

[0069]

[0079] The negative control may include the addition of a buffer and other assay reagents such as a low-response serum pool (LSP) control disclosed herein. In one embodiment, a predetermined threshold used to determine a nonspecific response is different from a first predetermined threshold. In a particular embodiment, the two thresholds are the same. In a particular embodiment, the two thresholds are the same. In one embodiment, donor samples showing nonspecific activation (>4.6%) by the LSP control are excluded from the pool.

[0070]

[0080] In certain embodiments, samples that do not contain cell populations typically found in biological samples are excluded from the pool. For example, if the pool is used to screen for basophil activation as discussed herein, biological samples that do not contain cell types, such as lymphocytes, monocytes, or granulocytes, may be excluded. In certain embodiments, if the pool is used to measure basophil activation as disclosed herein, biological samples may be excluded from the pool if the basophil population cannot be clearly distinguished between SSC and CCR3, in order to prevent inappropriate gating and results.

[0071]

[0081] Depending on the desired response, the biological sample used for pooling the composition and kit may vary. In certain embodiments, the biological sample used for pooling is blood. For example, in certain embodiments, a blood sample collected for whole blood cell count (CBC) screening may be used. Such samples may be used after storage. For example, individual donor blood is stored at room temperature for up to 24 hours after collection. This assay screens only blood collected within 24 hours. After the blood donor has been screened, individual aliquots of blood are stored in a refrigerator for up to 24 hours, if refrigerated (4°C), before the blood is pooled. In one embodiment, the blood sample is processed to provide leukocytes. In one embodiment, such leukocytes include basophils. Alternatively, other samples (e.g., plasma, serum, saliva) may be used for pooling.

[0072]

[0082] Pooled samples may be useful for cell-based tests, such as, but not limited to, flow cytometry. For example, in some cases, pooled blood samples of the disclosed compositions and kits may be used to test patient serum samples for diseases caused by abnormal basophilic activation. Thus, in certain embodiments, samples evaluated for inclusion in the pool are screened for basophilic activation. In one embodiment, samples evaluated for inclusion in the pool are screened for basophilic activation using the detection of an anti-FcεRI mAb as an activation trigger and CD63 as a marker of basophilic degranulation. In one embodiment, a given indication is an autoantibody disease. In one embodiment, pooled samples of the compositions and kits may be used to test patient serum for a basophilic response showing chronic spontaneous urticaria.

[0073]

[0083] In one embodiment, a pooled sample of the composition and / or kit is used to evaluate the response resulting from exposure of a subject to a separate sample. For example, in one embodiment, the pooled sample is used to evaluate basophil activation that occurs in the pooled sample upon exposure of the test subject to a biological sample. In one embodiment, the biological sample from the test subject is serum.

[0074]

[0084] The pool size of the composition and / or kit may depend on the test being performed and the availability of samples for pooling. In certain embodiments, the pool may contain at least 10, or at least 20, or at least 30 biological samples. Alternatively, more or fewer samples may be used. The pooled samples may be derived from a larger collection of samples. For example, in certain embodiments, the pool may contain about 50% or more of the samples to be screened for pooling. Alternatively, a larger or smaller proportion of the samples available for screening may be used.

[0075]

[0085] Sample pooling allows for the development of robust tests for various indications, as pooled samples may provide a more consistent response than individual samples. In certain embodiments, the technical cutoff for pooled samples used in the composition and / or kit as those showing a positive response indicating the presence of a given indication is defined using the 95% confidence interval (CI) of multiple normal samples.

[0076]

[0086] In one embodiment, the accuracy of the pool of compositions and / or kits is evaluated by comparing the results with known quality control (QC) samples. In one embodiment, known quality control controls include (i) biological samples that produce a high response above a first predetermined QC threshold (high QC), (ii) biological samples that produce an intermediate response above a second predetermined QC threshold but below the first threshold (intermediate QC), and (iii) biological samples that produce a low response below a third predetermined QC threshold that is lower than the second threshold (low QC). The thresholds used for QC may differ from the thresholds used for evaluating high responders to a positive anti-FcεRI mAb control and the thresholds used to determine nonspecific responses.

[0077]

[0087] In one embodiment, the high-response, intermediate-response, and low-response quality control controls include serum. In a particular embodiment, the high-response, intermediate-response, and low-response quality control controls include a pool of samples. For example, high QC may include pooled serum that produces a high response (HSP) above a first predetermined QC threshold, intermediate (QC) may include pooled serum that produces an intermediate response (ISP) above a second predetermined QC threshold but below the first threshold, and low QC may include pooled serum that produces a low response (LSP) below or below a third predetermined QC threshold that is lower than the second threshold. The quality control controls may also include known positive controls and / or known negative controls. For example, if the pool is used to measure basophil activation using the detection of an anti-FcεRI mAb as an activation trigger and CD63 as a marker of basophil degranulation, the positive control would be the anti-FcεRI mAb and the stimulation buffer. The negative control may include the addition of the buffer and other assay reagents.

[0078]

[0088] The predetermined thresholds used in the QC pool can be based on predetermined reference values ​​related to the response being measured. For example, in a particular embodiment, high QC may produce a response that is 3.0 standard deviations (SD) or more above a defined technical cutoff for the response being measured. Intermediate QC may produce a response that is 1 to 3 SD below the technical cutoff for the response being measured. Low QC may produce a response that is more than 3 SD below the technical cutoff for the response being measured.

[0079]

[0089] For example, for compositions and / or kits used to evaluate basophil activation, a low-response serum pool (LSP) quality control control (LSP) may include a pool of previously screened and stored serum having results of 0–4.67% activated basophils, i.e., approximately 3 standard deviations (SD) below the reference interval of 10.61 (PD-BAT technical cutoff). In certain embodiments, an intermediate-response serum pool (ISP) quality control control may include a pool of previously screened and stored serum having results of 4.67–8.63% activated basophils, i.e., approximately 1–3 SD below the reference interval of 10.61 (PD-BAT technical cutoff). In certain embodiments, a high-response serum pool control (HSP) may include a pool of previously screened and stored serum having results of greater than 16.55% activated basophils, i.e., approximately 3 SD above the reference interval of 10.61 (PD-BAT technical cutoff). These serum control pool sub-aliquots can be stored at -20°C for future use.

[0080]

[0090] system Furthermore, systems for carrying out the disclosed methods and / or for using any of the disclosed compositions and kits are also disclosed. The disclosed systems may include specific stations (physical locations) and / or components (e.g., experimental reagents). In some embodiments, the stations and / or components are separate from each other. In some embodiments, the stations and / or components are combined or identical.

[0081]

[0091] Accordingly, a system for generating a pool of biological samples for use in a test is disclosed, the system comprising components or stations for screening multiple biological samples for a response indicating the presence of a given indication, and components or stations for selecting multiple samples that provide a response exceeding a first predetermined threshold in order to generate a pool. The system may further comprise components or stations for evaluating biological samples from a subject for a response, the biological samples from the subject being separate from any of the biological samples used to generate the pool, and used to determine whether a sample produces a response and to quantify the level of response compared to the pool.

[0082]

[0092] In certain embodiments, the system may further include stations and / or components for screening a plurality of samples to determine whether a sample has a nonspecific response, and stations and / or components for removing samples having a nonspecific response from the pool, as disclosed herein. In one embodiment, a predetermined threshold used to determine the nonspecific response is different from a predetermined threshold used to generate the pool. In certain embodiments, the two thresholds are the same.

[0083]

[0093] For example, the system may include stations and / or components for screening pooled samples to select samples that have a response exceeding a predetermined threshold to a positive control, and / or to remove samples with a nonspecific response. Thus, the system may have stations and / or components for adding positive and negative controls (e.g., buffer or LSP) to specific biological samples being screened for pooling. For example, if the pool is used to measure basophil activation using the detection of an anti-FcεRI mAb as an activation trigger and CD63 as a marker of basophil degranulation, the system may include components for adding an anti-FcεRI mAb as a positive control and components for determining donors with a nonspecific response (e.g., to a negative control or LSP).

[0084]

[0094] The system may further include components for selecting such donors for the pool. In one embodiment, a predetermined threshold used to determine a nonspecific response is different from a first predetermined threshold. In a particular embodiment, the two thresholds are the same. In one embodiment, donor samples showing a low response (less than 40% activation) to an anti-FcεRI mAb control are excluded from the pool, and / or donor samples showing nonspecific activation (greater than 4.6%) to an LSP control are excluded from the pool. The system may further include components and / or stations for evaluating whether a biological sample being evaluated for inclusion in the pool does not contain cell populations typically found in biological samples used for pooling. For example, if the pool is used to screen for basophil activation as discussed herein, biological samples that do not contain cell types, e.g., lymphocytes, monocytes, or granulocytes, may be excluded. In a particular embodiment, if the pool is used to measure basophil activation as disclosed herein, biological samples may be excluded from the pool if the basophil population cannot be clearly distinguished between SSC and CCR3, in order to prevent inappropriate gating and results.

[0085]

[0095] Depending on the desired response, the biological sample used for pooling may vary. In certain embodiments, the biological sample used for pooling is blood. For example, in certain embodiments, blood samples collected for whole blood cell count (CBC) screening may be used. Such samples may be used after storage. For example, individual donor blood is stored at room temperature for up to 24 hours after collection. This assay screens only blood collected within 24 hours. After the blood donor has been screened, individual aliquots of blood are stored in a refrigerator for up to 24 hours, if refrigerated (4°C), before the blood is pooled. In one embodiment, the blood sample is processed to provide leukocytes. In one embodiment, such leukocytes include basophils. Alternatively, other samples (e.g., plasma, serum, saliva) may be used for pooling.

[0086]

[0096] Pooled samples may be useful for cell-based tests, such as, but not limited to, flow cytometry. For example, in some cases, pooled blood samples are used to test patient serum samples for diseases caused by abnormal basophilic activation. Thus, in certain embodiments, samples evaluated for inclusion in the pool are screened for basophilic activation. In one embodiment, samples evaluated for inclusion in the pool are screened for basophilic activation using the detection of an anti-FcεRI mAb as an activation trigger and CD63 as a marker of basophilic degranulation. In one embodiment, a given indication is an autoantibody disease. In one embodiment, the method may then further include using pooled samples to test patient serum for a basophilic response showing chronic spontaneous urticaria.

[0087]

[0097] In one embodiment, the system may include a station or component for evaluating the response resulting from exposure of a subject to a separate sample using a pooled sample. For example, in one embodiment, the pooled sample is used to evaluate basophil activation that occurs in the pooled sample upon exposure of the test subject to a biological sample. In one embodiment, the biological sample from the test subject is serum.

[0088]

[0098] The pool size used in the disclosed system may depend on the test being performed and the availability of samples for pooling. In certain embodiments, the pool may contain at least 10, or at least 20, or at least 30 biological samples. Alternatively, more or fewer samples may be used. The pooled samples are derived from a larger collection of samples. For example, in certain embodiments, the pool may contain about 50% or more of the samples to be screened for pooling. Alternatively, a larger or smaller proportion of the samples available for screening may be used.

[0089]

[0099] Sample pooling allows for the development of robust tests for various indications, as pooled samples may provide more consistent responses than individual samples. In certain embodiments, the system may include stations and / or components for evaluating the technical cutoff of pooled samples as those showing a positive response indicating the presence of a given indication, defined using the 95% confidence intervals (CIs) of multiple normal samples.

[0090]

[0100] In one embodiment, the accuracy of the pool is evaluated by comparing the results with known quality control (QC) samples. Thus, the disclosed system may include such QC samples. In one embodiment, known quality control controls include (i) biological samples that produce a high response above a first predetermined QC threshold (high QC), (ii) biological samples that produce an intermediate response above a second predetermined QC threshold but below the first threshold (intermediate QC), and (iii) biological samples that produce a low response below a third predetermined QC threshold that is lower than the second threshold (low QC). The thresholds used for QC may differ from the thresholds used for evaluating high responders to a positive anti-FcεRI mAb control and the thresholds used to determine nonspecific responses.

[0091]

[0101] In one embodiment, the high-response, intermediate-response, and low-response quality control controls include serum. In a particular embodiment, the high-response, intermediate-response, and low-response quality control controls include a pool of samples. For example, high QC may include pooled serum (HSP) that produces a high response above a first predetermined QC threshold, intermediate QC may include pooled serum (ISP) that produces an intermediate response above a second predetermined QC threshold but below the first QC threshold, and low QC may include pooled serum (LSP) that produces a low response below a third predetermined QC threshold that is lower than the second QC threshold. The quality control controls may also include known positive controls and / or known negative controls. For example, if the pool is to be used to measure basophil activation using the detection of an anti-FcεRI mAb as an activation trigger and CD63 as a marker of basophil degranulation, the positive control would be an anti-FcεRI mAb and a stimulating buffer, as disclosed herein. The negative control may include the addition of a buffer and other assay reagents.

[0092]

[0102] The predetermined QC thresholds used in the QC pool can be based on predetermined reference values ​​related to the response being measured. For example, in a particular embodiment, high QC may produce a response that is 3.0 standard deviations (SD) or more above a defined technical cutoff for the response being measured. Intermediate QC may produce a response that is 1 to 3 SD below the technical cutoff for the response being measured. Low QC may produce a response that is more than 3 SD below the technical cutoff for the response being measured.

[0093]

[0103] For example, to evaluate basophil activation, a low-response serum pool (LSP) quality control control (LSP) may include a pool of previously screened and stored serum with results of 0–4.67% activated basophils, i.e., approximately 3 standard deviations (SD) below the reference interval of 10.61 (PD-BAT technical cutoff). In certain embodiments, an intermediate-response serum pool (ISP) quality control control may include a pool of previously screened and stored serum with results of 4.67–8.63% activated basophils, i.e., approximately 1–3 SD below the reference interval of 10.61 (PD-BAT technical cutoff). In certain embodiments, a high-response serum pool control (HSP) may include a pool of previously screened and stored serum with results of greater than 16.55% activated basophils, i.e., approximately 3 SD above the reference interval of 10.61 (PD-BAT technical cutoff). These serum control pool sub-aliquots can be stored at -20°C for future use in the system.

[0094]

[0104] In one embodiment, the system for testing samples can be automated. For example, in certain embodiments, the system may include a computer program product tangibly embodied in a non-temporary machine-readable storage medium, which includes instructions configured to execute any of the system's components or stations.

[0095]

[0105] Figure 2 shows a diagram of system 200 of the present disclosure. As shown, the system may include a station and / or component 202 for evaluating donor biological samples for inclusion in a pool. In one embodiment, blood or blood products (e.g., serum) are used. The system may further include a station and / or component 204 for processing donor samples. This station and / or component may include reagents to be added to measure a response indicating a predetermined indication. For example, a portion of each donor sample may be dispensed into a reaction vessel, and reagents may be added to evaluate individual donor samples for a response indicating a predetermined indication. For example, each tube may be to which reagents may be added to evaluate whether the donor sample has a sufficiently high response to a positive control and / or whether it has a nonspecific response when evaluated using a negative or low-response control. If the response being measured is basophil activation, the reagents may include anti-FcεRI as a positive control, as well as an LSP master mix and / or buffer for separating the donor samples into two separate negative controls (i.e., for evaluating donor samples that produce nonspecific basophil activation). The system may also include stations and / or components for isolating cells from each donor sample. In certain embodiments, as disclosed herein, components and / or stations may be included for lysing, centrifugation and removal of supernatant of red blood cells (RBCs), and for resuspending pelleted leukocytes (WBCs) including basophils.

[0096]

[0106] The system may further include stations and / or components 206 for evaluating cells for a response indicating a given indication. For example, in certain embodiments, cells are evaluated by flow cytometry. Thus, a sample is taken up in a flow cytometer and may be gated as disclosed herein. As disclosed herein, if the given indication is chronic spontaneous urticaria, the response being measured is basophil activation measured using an activated trigger anti-FcεRI mAb, which is detected as CD63+ expression.

[0097]

[0107] The system may also include stations and / or components for evaluating whether a donor sample should be used in pool 208. As disclosed herein, such evaluation may include assessing the ability of donor cells to produce a response when exposed to a positive control and / or a negative control such as a buffer or a low-response serum pool (LSP). As disclosed herein, if a given indication is chronic spontaneous urticaria, the response being measured is basophil activation measured using an activated trigger anti-FcεRI mAb, which is detected as CD63+ expression. If the response to the positive control does not exceed a certain threshold, or if the response to the background or LSP exceeds a certain threshold (i.e., is not specific), the donor sample is not used in one or more pools. For example, in certain embodiments, for the basophil activation test, donors with high FcεRI stimulation and who do not respond highly to LSP are selected for inclusion in the pool. Thus, donors may be excluded if they are non-responders, i.e., any donor with less than 10-40% activated basophils relative to the FcεRI control. In the SSC singlet plot, a donor may be excluded if one of the following populations is missing: lymphocytes, monocytes, or granulocytes. A donor may also be excluded if they do not have at least 300 basophilic events within 200 seconds of uptake.

[0098]

[0108] The system may include a station and / or component 212 for preparing a pool of selected donor samples to be used to evaluate responses produced by separate patient samples. Thus, the system may also include a station and / or component for evaluating samples from another subject (i.e., a patient seeking diagnosis) for a given indication using the established pool 212. As disclosed herein, in a particular embodiment, the given indication is chronic spontaneous urticaria. Thus, in such an embodiment, the station may include a component and / or multiple components for evaluating biological samples from a subject for responses, the biological samples from the subject being separate from any of the biological samples used to generate the pool, and used to determine whether the sample produces a response and to quantify the level of the response.

[0099]

[0109] The station may further include components and / or reagents for comparing results obtained from patient samples with positive controls, negative controls, and high, medium, and low QC controls (e.g., HSP, ISP, and LSP) disclosed herein. The system may also include stations and / or components for reporting results to the patient and / or their healthcare provider 216.

[0100]

[0110] As illustrated in Figure 2, any station and / or component of the system may be automated, robotically controlled, and / or controlled at least partially by computer 300 and / or programmable software. Thus, the system may include a computer program product tangibly embodied in a non-temporary machine-readable storage medium, which includes instructions configured to execute the system or any part of the system (e.g., a station or component) and / or one or more steps of the method described in any of the disclosed embodiments. In some embodiments, a system is provided, which includes one or more data processors and a non-temporary computer-readable storage medium, which, when executed on the one or more data processors, causes the one or more data processors to execute some or all of the methods or processes disclosed herein and / or any part of the system disclosed herein.

[0101]

[0111] Also disclosed is a computer program product tangibly embodied on a non-temporary machine-readable storage medium, which includes instructions configured to cause one or more data processors to perform any of the components of the system or any of the steps of the disclosed method. Any suitable computer-readable medium may be used, including hard disks, CD-ROMs, optical storage devices, or magnetic storage devices.

[0102]

[0112] Accordingly, in one embodiment, a computer program product tangibly embodied in a non-temporary machine-readable storage medium is disclosed, which includes instructions configured to screen a plurality of biological samples for responses indicating the presence of a predetermined indication, and to select a plurality of samples that provide responses exceeding a first predetermined threshold in order to generate a pool.

[0103]

[0113] Also disclosed is a computer program product tangibly embodied in a non-temporary machine-readable storage medium, which includes instructions configured to perform testing for a response indicating the presence of a predetermined indication in a sample from a subject, wherein the instructions are configured to generate a pool of multiple biological samples, each pool providing a response above a predetermined threshold indicating the presence of a predetermined indication in the subject and optionally not providing a nonspecific response, and to add a portion of the sample from the subject to each of the pools of (a) in order to determine whether the sample produces the response.

[0104]

[0114] Also disclosed is a computer program product tangibly embodied in a non-temporary machine-readable storage medium, which includes instructions configured to run a system comprising components or stations for screening multiple biological samples for responses indicating the presence of a predetermined indication, and components or stations for selecting multiple samples that provide responses exceeding a first predetermined threshold in order to generate a pool. In certain embodiments, the computer program product further includes instructions configured to run a system comprising components or stations for testing samples from a subject for responses indicating the presence of a predetermined indication, using the pool.

[0105]

[0115] Systems and computer products may perform any of the methods disclosed herein. One or more embodiments described herein may be implemented using program modules, engines, or components. Program modules, engines, or components may include programs, subroutines, parts of programs, software components, or hardware components capable of performing one or more of the described tasks or functions. As used herein, a module or component may reside on a hardware component independently of other modules or components. Alternatively, a module or component may be a shared element or process of another module, program, or machine.

[0106]

[0116] Figure 3 shows a block diagram of an analytical system 300 used for the detection and / or quantification of progesterone metabolites. As shown in Figure 3, modules, engines, or components (e.g., programs, code, or instructions) executable by one or more processors may be used to implement various subsystems of the analytical instrument system according to various embodiments. The modules, engines, or components may be stored in a non-temporary computer medium. If necessary, one or more of the modules, engines, or components may be loaded into system memory (e.g., RAM) and executed by one or more processors of the analytical instrument system. The example shown in Figure 3 illustrates modules, engines, or components for carrying out the methods of the present disclosure or for running any of the systems of the present disclosure.

[0107]

[0117] Accordingly, Figure 3 shows an exemplary computing device 300 suitable for use in the systems and methods of the present disclosure. The exemplary computing device 300 includes a processor 305 that communicates with a memory 310 and other components of the computing device 300 using one or more communication buses 315. The processor 305 is configured to execute processor-executable instructions stored in the memory 310 to perform one or more methods for detecting progesterone metabolite levels or to operate one or more stations, according to different examples such as those in Figures 1-2 and 4-14, or those disclosed elsewhere in this specification. In this example, the memory 310 may store processor-executable instructions 325 that can analyze results 320 with respect to a sample, as described herein.

[0108]

[0118] The computing device 300 in this example may also include one or more user input devices 330, such as a keyboard, mouse, touchscreen, or microphone, to accept user input. The computing device 300 may also include a display 335 for providing visual output to the user, such as a user interface. The computing device 300 may also include a communication interface 340. In some examples, the communication interface 340 may enable communication using one or more networks, including a local area network ("LAN"), a wide area network ("WAN") such as the Internet, a metropolitan area network ("MAN"), a point-to-point connection, or a peer-to-peer connection. Communication with other devices may be achieved using any suitable network protocol. For example, one suitable network protocol may include the Internet Protocol ("IP"), Transmission Control Protocol ("TCP"), User Datagram Protocol ("UDP"), or a combination thereof, such as TCP / IP or UDP / IP.

[0109]

[0119] Computer-usable or computer-readable media may be, but are not limited to, electronic, magnetic, optical, electromagnetic, infrared, or semiconductor systems, apparatus, devices, or propagation media. More specific examples (a non-exhaustive list) of computer-readable media include electrical connections with one or more wires, portable computer diskettes, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM or flash memory), optical fibers, and portable compact disk read-only memory (CD-ROM). Computer-usable or computer-readable media may also be paper or other suitable media on which a program is printed, and the program may be electronically captured, for example, by optical scanning of the paper or other medium, then compiled, interpreted, or processed in an appropriate manner as necessary, and then stored in computer memory.

[0110]

[0120] Computer program code for performing the operations of the Disclosure may be written in an object-oriented programming language such as Java 7, Smalltalk, Python, LabVIEW, C++, or Visual Basic. However, computer program code for performing the operations of the Disclosure may also be written in a traditional structured programming language such as the C programming language or assembly language. The program code can run entirely on the user's computer, partially on the user's computer, as a standalone software package, partially on the user's computer, partially on a remote computer, or entirely on a remote computer. In the latter scenario, the remote computer may be connected to the user's computer via a local area network (LAN) or wide area network (WAN), or it may be connected to an external computer (for example, via the Internet using an Internet service provider). [Examples]

[0111]

[0121] Example 1 Test Principle: The basophil activation test ("BAT") is a flow cytometry-based functional assay that stimulates live cells in fresh whole blood and evaluates IgE crosslinking, providing a more accurate indicator of allergy than measuring the concentration of allergen-specific IgE. (Iqbal K, Bhargava K, Skov PS, Falkencrone S, Grattan CE. A Positive Serum Basophil Histamine Release Assay Is a Marker for Ciclosporin-Responsiveness in Patients With Chronic Spontaneous Urticaria. Clin Transl Allergy (2012) 2:19. doi:10.1186 / 2045-7022-2-19; ​​Santos AF, Douiri A, Becares N, et al. Basophil activation test discriminates between allergy and tolerance in peanut-sensitized children. J Allergy Clin) (Immunol. 2014;134(3):645-652). Basophil activation testing in chronic urticaria assesses basophil activation in a whole blood pool upon patient serum (allergen) stimulation. The BAT assay uses staining, lysis, washing methods and a two-antibody panel. The CCR3 (eosinophil and basophil marker) antibody used in this flow cytometry-based assay selects basophils, and CD63 (activation marker) is used to determine basophil degranulation. However, conventional methods for basophil activation testing require donors providing three readily available basophils that show high activation against anti-FcεRI, which can be challenging for both clinical laboratories and blood donors. This study defines an approach to provide a consistent, reliable, and clinically validated assay for commercial laboratories by pooling basophil donors from routine CBC testing. This CU assay takes two days to complete.On day 1, 32 or more basophil donors (ELISA basophil IgG > 0.2; i.e., 0.2 and 0.3, with 0.1 added as needed) were screened for basophil activation using an activation-triggered anti-FcεRI mAb. The following day, non-responders were excluded, and the 20 highest CD63+ responders to anti-FcεRI mAb were selected, their background < 4%, and their response to the low-response serum pool was also < 4%. Given the sufficient results in the low-response serum pool, background control was deemed unnecessary.

[0112]

[0122] Next, CSU patient serum was screened using pooled blood. The accuracy of the assay was determined using low-response, intermediate-response, and high-response serum pools. Furthermore, the Chauvenet method was used for outlier removal, and the technical cutoff for pooled blood in the assay was defined using the median 95% of 142 normal serum patients with total IgE (<80). Definition: CSU = Chronic Spontaneous Urticaria BAT = Basophil Activation Test ASST = Autologous Serum Skin Test IgE = Immunoglobulin E CD63 = Differentiated Cluster 63 CCR3 = CC motif receptor 3 RBC = Red blood cell FSC=forward scatter SSC=side scatter FDA = Food and Drug Administration SOP = Standard Operating Procedure reagent: stimulation buffer Staining reagents (antibody cocktails) Anti-FcεRI mAb stimulation control Washing buffer Dissolving reagents

[0113]

[0123] In-house Low-Reactivity Serum Pool Control (LSP): To prepare an in-house low-activity serum pool control (LSP), previously screened and stored serum with activated basophils less than 4.67% was pooled. Individual serums pooled for the low-activity control showed results 3 standard deviations below the technical cutoff of 10.61. The total volume of each individual serum was mixed in a 250 mL sterile bottle. 5 mL and 1.2 mL sub-aliquots were prepared and stored at -20°C.

[0114]

[0124] In-house Intermediate Reaction Serum Pool Control (ISP): To prepare an in-house intermediate reaction serum pool control (ISP), previously screened and stored serums containing 4.67–8.63% activated basophils were pooled. Individual serums pooled for the intermediate reaction control were located 1–3 standard deviations below the technical cutoff of 10.61. The total volume of each individual serum was mixed in a 250 mL sterile bottle. 5 mL and 1.95 mL sub-aliquots were prepared and stored at -20°C.

[0115]

[0125] In-house High-Reactivity Serum Pool Control (HSP): To prepare an in-house high-activity serum pool control (HSP), previously screened and stored serums showing activated basophils exceeding 16.55% were pooled. Individual serums pooled for the high-activity serum pool control showed results exceeding the reference interval by more than 3 standard deviations. The total volume of each serum was mixed in a 250 mL sterile bottle. 5.0 mL and 250 μL sub-aliquots were prepared and stored at -20°C.

[0116]

[0126] Parallel Testing: To maintain the accuracy of laboratory reports when new lot numbers and / or reagent shipments are used for patient testing, parallel reagent checks (lot-to-lot comparisons) may be performed and documented before reporting patient results with the new lot and / or shipment. This involves testing the same patient samples and / or quality control (QC) materials with reagents from the current and new lot and / or shipment to compare results for similarity.

[0117]

[0127] Quality Control: To ensure that all reagents and procedures function correctly, quality control was performed on low-reactive serum pools, intermediate-low-reactive serum pools, and high-reactive serum pools for each batch of samples. Expression levels must be within the expected outcome parameters for the lot.

[0118]

[0128] Sample processing protocol for day 1: Patient samples may be prepared manually as described in detail herein. Alternatively, patient samples may be processed by automated liquid handling. 420 μL aliquots of the working lysis reagent, working low-reactivity serum pool lot, and FcεRI stimulating control were brought to room temperature. Next, the stimulating buffer was prepared (by thawing frozen aliquots or by reconstituting lyophilized buffer with nuclease-free water). Next, the samples were prepared. Two tubes (three tubes if using a background control) were used for each basophil-providing donor. Then, selected basophil-providing donor samples were collected using samples from lavender EDTA blood collection tubes collected the previous day with an absolute basophil count of 0.20 × 10⁻³ / μL to 0.30 × 10⁻³ / μL. Next, the whole blood of the basophil-providing donors was mixed inverted, 50 μL of patient whole blood was added to the corresponding tube, the volume was dispensed to the bottom of the tube rather than the wall, and the tube was covered with aluminum foil or Parafilm.

[0119]

[0129] At this point, a control master mix is ​​created.

[0120]

[0130] For the FcεRI stimulation control, 3.675 mL of stimulation buffer was transferred to a conical tube of appropriate size, 0.875 mL of FcεRI stimulation control was added, and 700 μL of staining reagent was added. The tube was sealed and covered to avoid exposure. The amounts used for each FcεRI stimulation test were 105 μL of stimulation buffer, 25 μL of FcεRI stimulation control, and 20 μL of staining reagent.

[0121]

[0131] For the in-house control in the low-reactivity serum pool, 2.8 mL of stimulation buffer was transferred to an appropriately sized conical tube, and 1.75 mL of the in-house control in the low-reactivity serum pool and 700 μL of staining reagent were added. The tube was sealed and covered to avoid exposure. The amount used for each FcεRI stimulation test was 80 μL of stimulation buffer, 50 μL of the in-house control in the low-reactivity serum pool, and 20 μL of staining reagent.

[0122]

[0132] At this point, 150 μL of the corresponding master mix was added to each tube. The tubes were briefly vortexed to mix, covered with aluminum foil, and placed in a 37°C water bath for 20 minutes. After incubation, 1.5 mL of lysis reagent was added to each tube, and the tubes were incubated at room temperature for 10 minutes. After gentle vortexing, the tubes were centrifuged at 500xg for 5 minutes at room temperature with the brake set to 2. The supernatant was then decanted, and the tubes were aspirated onto an absorbent pad. Next, 300 μL of cold wash buffer was added to each tube, and the tubes were gently vortexed to resuspend the cell pellet.

[0123]

[0133] If immediate incorporation was not possible, the tubes were incubated in the dark at room temperature for 30 minutes, and if the patient sample assay was to be performed the following day, they were stored overnight at 2-8°C.

[0124]

[0134] Sample acquisition: During sample acquisition, cell collection was confirmed in the SSC singlet plot. At least 500 basophil events were acquired per tube. Acquisition times may vary between donors.

[0125]

[0135] Furthermore, it was confirmed that 500 basophils were taken up from each processed sample. For any sample that did not take up basophils, an attempt was made to re-incorporate the tube if the sample volume was not exhausted. If the sample volume was exhausted, the sample had to be reprocessed. If a donor did not have at least 300 basophil events after 200 seconds of incorporation, that donor was excluded from pool selection.

[0126]

[0136] Gating and batch analysis: After batch ingestion is complete, chronic urticaria reports are evaluated for each donor to ensure that each donor is properly gated and does not contain cells from adjacent population clusters.

[0127]

[0137] FcεRI control tubes were selected because all gating was performed on positive control tubes. On the reporting worksheet, the SSC singlet gate was tightened to identify doublets, and the time-dot plot was adjusted to ensure that the lower left corner (50 SSC-H × 50 SSC-A region) was not gated out, as the target cells may be present in this region. On the SSC singlet plot, it was confirmed that the total leukocyte gate did not exclude lymphocyte and monocyte regions.

[0128]

[0138] To assist in gate placement, we used contour plots along with histograms to adjust gating on the basophil plot. We placed the Act on the histogram plot. We positioned the basophil gate on the positive peak, and then adjusted the quadrant gate at the same CD63-A position. The placement of that quadrant gate was close to the third decade.

[0129]

[0139] Selection of donors who provide basophils for whole blood pooling. After completing gating, 20 donors were selected for the day 2 procedure (i.e., patient sample assay). Donors who were unresponsive to anti-FcεRI stimulation (i.e., less than 10% activated basophils compared to the FcεRI control) were excluded. Furthermore, donors were excluded if they had spontaneous basophil activation that reflected a high percentage of activated basophils in any background and / or LSP control. In addition, donors were excluded if one of the following populations: lymphocytes, monocytes, or granulocytes, was missing in the SSC singlet plot. Donors were also excluded if they did not have at least 300 basophil events within 200 seconds of uptake. Table 1 shows an example of donor screening for basophils. [Table 1]

[0130]

[0140] Sample processing protocol for day 2 Patient specimens can be processed manually, as disclosed in detail herein. Alternatively, patient specimens can be processed by automated liquid handling.

[0131]

[0141] All patient serum, reagents, and controls (LSP, ISP, and HSP; FcεRI-stimulated controls) were brought to room temperature, and the following steps were performed.

[0132]

[0142] Label the batch sets of 12 x 75 mm tubes as follows: • Tube / well 1: Background control • Tube / well 2: FcεRI stimulation control • Tube / well 3: Low-response serum pool • Tube / well 4: Intermediate reaction serum pool • Tube / well 5: High-reactivity serum pool • Tube / well 6-XX: Patient serum

[0133]

[0143] The donors providing the selected basophils were removed from the refrigerator at least 5 minutes before pooling the donors, and the tubes were mixed by inverting them (e.g., 5-7 times). The donors providing the basophils to be pooled were examined to confirm that there was no hemolysis in the selected donors. If hemolysis was present, the next best donor that met the criteria for inclusion in the pool was selected.

[0134]

[0144] 50 μL of patient serum was added to the corresponding tubes (tube / well #6 and beyond).

[0135]

[0145] Whole blood (approximately 0.5–1.0 mL) from each selected donor was mixed by inverting each donor tube before transfer, and then transferred to a labeled conical tube for basophil pooling. After pooling blood from all 20 donors, the tubes were inverted and mixed. Next, the calculated whole blood pool volume was transferred to a conical tube for master mix and mixed by pipetting. Next, the calculated volume of staining reagent (antibody) was transferred and mixed by pipetting. Finally, 150 μL of the pooled donor master mix was transferred to each tube and mixed by briefly vortexing the tubes.

[0136]

[0146] Next, the tubes were transferred to a 37°C water bath and incubated for 20 minutes. After incubation in the water bath, 1.5 mL of lysis solution was added, and the tubes were incubated in the dark at room temperature for 10 minutes. Then, the tubes were gently vortexed for 1 second, and the brake was set to 2, and the tubes were centrifuged at 500xg for 5 minutes at room temperature. After decanting the supernatant, 300 μL of cold wash buffer was added to each tube, and the tubes were gently vortexed to resuspend the cell pellet.

[0137]

[0147] If ingestion was not performed immediately, the tubes were incubated in the dark at room temperature for 30 minutes, and then stored at 2–8°C for up to 72 hours.

[0138]

[0148] Sample acquisition During sample acquisition, cells were collected in the SSC singlet plot, and it was confirmed that 1000 basophil events were taken up per tube.

[0139]

[0149] Furthermore, it was confirmed that 1000 basophils were incorporated into each processed sample. For any sample in which no basophils were incorporated, if the sample volume was not exhausted, an attempt was made to re-incorporate the basophils into the tube. If the sample volume was exhausted, the sample was reprocessed.

[0140]

[0150] Gating and batch analysis on day 2 Gating was the same as on day 1 for "all events," time, SSC singlet, and total leukocyte plot.

[0141]

[0151] HSP control and anti-FcεRI control tubes were used to gate the basophil plots. High-reactive serum pool (HSP) controls had both characteristic positive and negative CD63 populations, while anti-FcεRI controls may have had a clearly defined positive population, and the negative population may not have been as defined.

[0142]

[0152] HSP control tubes / wells were selected. The "activated basophils" gate was adjusted to gate CD63+ on the histogram plot, and then the anti-FcεRI control was clicked to confirm the gating of the histogram. Gating typically occurs in the third decade (10 3 It's close to a notch.

[0143]

[0153] In anti-FcεRI control tubes, gating on the basophil scatter plot was adjusted to closely match the basophil histogram as much as possible. Attention was paid to the position of the histogram gate (decade number and notch) when adjusting the gate on the scatter plot.

[0144]

[0154] To verify that the gating configuration on the scatter plot is similar to that of a histogram gate, we compared the percentage values ​​of activated basophils and activated basophils relative to their parent (% Parent) in the runpointer statistics.

[0145]

[0155] report For chronic urticaria, BAT-negative results: <10.61%; positive results: ≥10.61%.

[0146]

[0156] Example 2 method Thirty basophil donors (ELISA basophil IgG > 0.2) were screened for basophil activation using an anti-FcεRI mAb, which is an activation trigger. The following day, CD63 + The 20 most responsive donors were selected, non-responders were excluded, and a pool of 20 donor blood samples was prepared. CSU patient serum was then screened against the pooled blood. Representative flow cytometry gating for background control, anti-FcεRI control, low-response serum pool, intermediate-response serum pool, and high-response serum pool are shown in Figures 4A–4F. Assay precision was determined using the low-response, intermediate-response, and high-response serum pools. Furthermore, the Chauvenet method was used for outlier exclusion, and a technical cutoff for pooled blood for the assay was defined based on the 95% confidence interval (CI) of 120 normal serum patients with total IgE (<80).

[0147]

[0157] result Precision was demonstrated over 10 days for donor pools providing 10 different basophils. The background of the pooled basophils was set to 3.8%–4.0% based on the natural separation between activated and inactivated basophils in multiple donor pools. Over 10 days and across 10 experiments, the mean of the pool activated by anti-FcεRI mAb was 86.3% (CV 4.3%), demonstrating precision for low-response, medium-response, and high-response serum pools.

[0148]

[0158] conclusion BAT is considered an in vitro surrogate indicator of a patient's allergic reaction and therefore supports the diagnosis of CSU. This example demonstrates a robust laboratory method using a pooled basophil-providing donor for the expansion of BAT testing in commercial laboratories. A comparison of the pooled donor method and the individual donor method disclosed herein is described in further detail in Example 3 and shown in Figures 11A, 11B, and 11C. Standardization of this assay with clinically validated results will significantly advance the patient diagnosis of CSU.

[0149]

[0159] Example 3 material and method Basophil activation by stimulation or patient serum The amount of CD63 present on the surface of donor basophils was determined by flow cytometry. Flow CAST® (BUHLMANN Laboratories AG) was adapted for use in CSU patients as previously described (Gentinetta et al., 2011). Roughly, 50 μL of donor blood was mixed with 50 μL of patient serum (or other stimulant), 100 μL of stimulation buffer containing IL-3, and 20 μL of antibody mixture (containing fluorescein isothiocyanate-labeled anti-CD63 antibody and phycoerythrin-labeled anti-CCR3 antibody). Basophil activation and staining were achieved by incubation at 37°C for 20 minutes. Anti-FcεRI antibody (50 μL) was used as a positive control, and 50 μL of stimulation buffer was used as a background control. Red blood cells were lysed in the dark at ambient temperature for 10 minutes. The samples were centrifuged at ambient temperature, 500xg for 5 minutes on a low brake setting, decanted, and then the pellet was resuspended in 300 μL of cold wash buffer containing 0.1% formaldehyde for cell fixation. If sample uptake was not complete immediately after processing, the samples were incubated in the dark at room temperature for 30 minutes to fix the cells, and then stored in the dark at 2–8°C for up to 3 days.

[0150]

[0160] Flow cytometry data acquisition and analysis Basophils were identified by CCR3 expression. At least 1000 basophil events were included for basophil screening. For analysis, time plots were used to observe the sample flow across all events. Lateral scattering singlet plots were created from all events to identify doublets. From the lateral scattering singlets, the gate was applied to total leukocytes. From the total leukocyte gate, CCR3+ basophils were identified. Following the basophil gate, the activation of basophils (CCR3+CD63+) was analyzed using a double fluorescent dye gate. For analysis of basophil donor screening (day 1), an anti-FcεRI stimulated control from each donor was used for gating. For analysis of patient serum screening (day 2), gating was established using the minimum between the activated and inactivated basophil populations with an anti-FcεRI stimulated control, with minimal adjustment for the CD63+ population using a highly reactive serum pool (HSP) control. Data analysis of flow cytometry screening results was performed using the ggplot2 package (Wickham 2016) in R, version 4.2.5 (R Core Team).

[0151]

[0161] Selection of basophil donors and pooling production Deidentified EDTA whole blood samples with basophil absolute counts in the range of 0.2–0.3 × 10⁻³ / μL, submitted to a large reference laboratory for complete blood cell count (CBC), were selected within 24 hours of collection. Donor cell reactivity was tested by incubation with 1) stimulation buffer (background), 2) anti-FcεRI mAb, and 3) the low-response serum pool (LSP) of the present invention. See Section 2.5 for details of pool production specifications. Donors showing high baseline activation (>4%) with background control, nonspecific activation (>4%) with LSP, or low response (<40%) to anti-FcεRI control were excluded from the donor pool.

[0152]

[0162] Furthermore, in the SSC singlet plot, donors were excluded if any of the following populations were missing: lymphocytes, monocytes, or granulocytes. To prevent inappropriate gating and outcomes, donors providing basophils were excluded if the basophil population could not be clearly distinguished between SSC and CCR3. More than 30 potential donors were screened for each test batch, and 20 best-performing donors (best anti-FcεRI mAb response meeting other criteria) were pooled and used for PD-BAT testing of patient serum.

[0153]

[0163] Patient serological testing using PD-BAT Each batch of the test was processed with controls (background, anti-FcεRI stimulation, and low-response serum pool (LSP), intermediate-response serum pool (ISP), and high-response serum pool (HSP)). Batch testing was initiated within 10 minutes of donor pool preparation. If flow cytometry uptake was not completed after processing, the samples were incubated in a wash buffer containing 0.1% formaldehyde in the dark at room temperature for 30 minutes to fix the cells, and stored at 2–8°C for up to 3 days. At least 1000 basophil events were captured for serological testing.

[0154]

[0164] Preparation of serum pooled controls Serums showing results above the PD-BAT technical cutoff by more than 3.0 standard deviations (SD) were pooled for the high-response serum pool (HSP). Serums showing results more than 3 standard deviations (SD) below the technical cutoff were pooled to create the low-response serum pool (LSP). Serums tested between 1 and 3 SD below the technical cutoff were pooled to form the intermediate-response serum pool (ISP). Sub-aliquots of these serum control pools were stored at -20°C for future use.

[0155]

[0165] Reproducibility and accuracy PD-BAT measurements were performed in quadruple series for background, anti-FcεRI, LSP, ISP, and HSP. These internal control serum pools were stored as frozen aliquots before being re-equilibrated to room temperature prior to daily testing. PD-BAT measurements were performed separately in quadruple series for 10 days. Intra-assay and inter-assay reproducibility were calculated for the five materials.

[0156]

[0166] PD-BAT pooled whole blood stability The stability of pooled whole blood was assessed by testing the background, anti-FcεRI control, LSP, ISP, and HSP at baseline and 2, 4, 6, and 24 hours after donor pool production. Pooled whole blood was stored at 2–8°C between time points.

[0157]

[0167] Stability analysis of treated samples for PD-BAT Control (background, anti-FcεRI control, LSP, ISP, HSP) and serum samples were processed for incorporation at baseline and every 24 hours up to 120 hours after processing. Processed samples were stored in the dark at 2–8°C.

[0158]

[0168] Serum temperature stability for PD-BAT The stability of results produced from serum samples stored under three temperature conditions (ambient, refrigerated, and frozen) was evaluated for PD-BAT using LSP, ISP, and HSP. Isochronization tests were performed on serum pools at 14, 7, 3, 1, and 0 days (baseline) for ambient and refrigerated (2–8°C) conditions. For freeze stability testing, serum pools were tested at baseline and at each interval up to 71 days.

[0159]

[0169] Stability studies of reagents and key components The performance of staining reagents stored under dark conditions was evaluated for up to 3 days. For each staining reagent condition and time point, controls (background, anti-FcεRI control, ISP, LSP, HSP) and 19 serum samples were tested with the staining reagent. The anti-FcεRI stimulated control was reconstituted, refrigerated for 1 and 7 days, and its stability was compared to baseline.

[0160]

[0170] Determination of PD-BAT reference intervals and technical cutoffs To reduce the likelihood of including atopic individuals in the reference population, a total of 144 serum samples with total IgE (ThermoFisher ImmunoCAP®) levels less than 80 IU / mL were selected for technical cutoff determination (Chang et al., Analysis of total immunoglobulin E and specific immunoglobulin E of 3,721 patients with allergic disease, Biomed Rep., 2015, Jul;3(4):573-577.doi:10.3892 / br.2015.455.Epub 2015 Apr 29.PMID:26171168;PMCID:PMC4486958). After removing outliers using Chauvenet's criteria, reference intervals were generated by transformed parametric analysis using EP Evaluator® (Data Innovations, Burlington, Vermont, USA). The clinical cutoff was validated using 157 additional samples that showed results below the detection limit of the basophil histamine release assay (CU Index®, Eurofins Viracor, Lenexa, KS).

[0161]

[0171] BAT trials by individual donors The FlowCAST® protocol, in accordance with the Instructions for Use (IFU), was performed to screen for healthy basophil donors. Selection criteria for three basophil donors included unresponsiveness to anti-FcεRI (<10% CD63 activation), high anti-FcεRI response (>65% CD63 activation), high basophil count (over 1000 cells per 50 μL of whole blood), and low basophil activation (<10% CD63 activation) relative to a negative serum pool (NSP). A negative serum pool, serving a similar purpose to the LSP (low-response serum pool), contained serum from seemingly healthy individuals and played a role in excluding donors with spontaneous activation. Furthermore, the leukocyte population should be separated into three distinct populations (lymphocytes, monocytes, and granulocytes) on an FSC / SSC dot plot.

[0162]

[0172] Comparison of methods for individual donor CSU-BAT versus PD-BAT A set of 69 CSU patient samples was analyzed using both the conventional method (using three individual donors) and the PD-BAT method for BAT testing. BUHLMANN Laboratories AG performed the conventional method, while Labcorp performed the PD-BAT testing.

[0163]

[0173] Comparison of BHRA with PD-BAT 343 samples tested using the CU-Index® were also tested using the PD-BAT assay. The results were compared using the EP Evaluator® statistical model (qualitative method comparison, quantitative method comparison).

[0164]

[0174] result Donor screening results In the initial stages of the study, potential basophil donors were screened to ensure the robustness of the inventors' pooled donor basophil activation test (PD-BAT). This screening process aimed to identify responsive donors while excluding donors with high background activation. The results were based on the gating strategy using positive controls and HSPs described above. Of the screened potential donors, 1.1% produced over 4% CD63+ activation with the addition of stimulation buffer alone (elevated background activation). 6.4% produced over 4% CD63+ activation to low-response serum pooled LSPs (non-specific response). 7.1% were unable to produce over 10% CD63+ activation to the positive control anti-FcεRI (non-responders). 18.5% were unable to produce over 40% CD63+ activation to the positive control anti-FcεRI (low-responders). As a result of the combination of criteria used, 13.7% of the 463 donors screened were excluded from use in donor pool creation across all experiments (Figures 5A and 5B).

[0165]

[0175] Reproducibility and accuracy The evaluation of reproducibility and precision in the PD-BAT method is crucial for its reliability in clinical applications. The results, as shown in Figure 6 and summarized in Table 2, highlight remarkable intra-assay and inter-assay comparability. In Table 2, intra-assay results were calculated as 10 replicates of the assay for each sample. Inter-assay results were obtained by combining replicates of each sample tested across 10 different pools. Specifically, the intra-assay coefficient of variation (CV) was low, at 1.92% for anti-FcεRI and 4.57% for HSP. Similarly, the inter-assay coefficient of variation (CV) remained low, at 3.44% for anti-FcεRI and 9.56% for HSP. In particular, the CVs for background, LSP, and ISP were high, as expected, as any changes were amplified because they are derived from smaller values. Furthermore, 99% confidence intervals (±3 standard deviations) were found for positive controls and internal serum controls, further supporting the reliability of the findings. [Table 2]

[0166]

[0176] Serum stability The stability of high-reactivity serum pools (HSPs) at baseline and over intervals of up to 14 days was evaluated under room temperature (20–23°C) and refrigerated (4–8°C) storage conditions (Figure 7). Recovery rates for samples stored at room temperature decreased by 17.1% within 24 hours and by more than 20% with longer storage periods. Refrigerated samples remained stable for 14 days (i.e., decreased by less than 20%). Freezing stability was demonstrated in an inter-assay precision test (Figure 6) in which no degradation of results occurred during the course of an experiment in which 10 assays were performed over 71 days for both controls and pools. These findings highlight the importance of timely serum testing for the successful clinical application of PD-BAT.

[0167]

[0177] Stability of pooled whole blood donors A whole blood pool was prepared and used to test the serum pool and controls by PD-BAT at multiple time points up to 24 hours (Figure 8). HSP recovery rates decreased by 8.5% at 2 hours and 35.7% at 6 hours. LSP recovery rates increased from 3.6% at baseline to 11.3% at 24 hours. These results indicate that donor pools should be used for testing promptly after preparation (within 2 hours).

[0168]

[0178] Stability analysis of processed samples PD-BAT test samples processed up to the point of loading into the flow cytometer remained stable for up to 72 hours when stored under refrigerated conditions (Table 3). In Table 3, control and 20 serum samples were processed and analyzed at 0 (baseline), 24, 48, 72, 96, and 120 hours. To determine the stability of the processed samples, changes in activated basophil readout from baseline were evaluated. Substantial changes were observed beyond 72 hours, particularly in the sample background, highlighting the importance of timely analysis after processing.

[0169]

[0179] Stability of important reagents Key reagents (including anti-FcεRI control antibody, CCR3-PE, and CD63-FITC staining reagents) were reconstituted and used for the baseline test. The anti-FcεRI control was evaluated after a 7-day refrigerated storage period. The staining reagents were evaluated after a 3-day period at room temperature (20–23°C) in the dark. Analysis revealed consistent category agreement across controls and sera tested under both conditions (Tables 4 and 5). In Table 4, the stability of the staining reagents was tested at baseline and after 3 days of storage in the dark and under ambient conditions. Basophil activation was measured for 5 controls and 20 sera samples at each time point (n=1). In Table 5, the stability of the stimulated controls was tested at baseline and after 1 day and 7 days of storage at 2–8°C. Basophil activation was measured for 5 controls and 20 sera samples at each time point (n=1). [Table 3] [Table 4] [Table 5]

[0170]

[0180] Determination of the reference interval The upper limit of the reference interval, determined from a population of 142 individuals with total IgE < 80 IU / L, was determined to be 10.61% (9.79–11.49, 95% CI) (Figure 9A). This threshold was validated in separate analyses of 145 samples with BHRA negative results < 2.2 CU Index® (after outlier exclusion using the Chauvenet criterion), and the upper limit of the reference interval was determined to be 11.52% (10.19–13.02, 95% CI) (Figure 9B).

[0171]

[0181] Therefore, Figure 9A shows the determination of the reference interval. A total of 144 low IgE (<80 IU / mL) serum samples were screened via PD-BAT, and two samples were excluded by the Chauvenet method. Figure 9A shows the distribution of the 142 low IgE serum samples used to obtain the reference interval. Conversion parametric analysis by EP Evaluator provides a reference interval of 10.61% activated basophils. Mean, 5.79% CD63+; standard deviation, 1.98; median, 5.34; range, 2.15–11.24. Figure 9B shows the validation of the reference interval. A total of 157 strongly negative (CU Index® <2.2) BHRA serum samples were screened via PD-BAT, and 12 samples were excluded by the Chauvenet method. Figure 9B shows the distribution of the 145 serum samples used to validate the reference interval. Parametric analysis using the EP Evaluator provides a reference interval for 11.52% activated basophils. Mean, 4.71%; standard deviation, 2.47; median, 4.0; range, 1.3–11.9. Establishing a robust reference interval provides a crucial basis for the application of PD-BAT.

[0172]

[0182] Comparison of basophil histamine release assay and PD-BAT A comparison of results obtained from 343 samples tested with CU-Index and PD-BAT revealed an overall percent agreement of 79.9% (Figure 10, Table 6). In these experiments, serum extending to the BHRA analytical spectrum was screened with PD-BAT, and the results were analyzed via EP Evaluator. The PD-BAT technical cutoff (10.61) (horizontal red line) and the CU Index® technical cutoff (greater than 10 CU Index® units) (vertical yellow line) are shown. A Passing-Bablok linear regression gradient of 0.814 (dotted line), explaining the proportional and systematic differences between the two methods, suggests a moderate linear relationship between BHRA and PD-BAT. 28% of the patients tested had strongly positive results for CU Index® (greater than 50 CU Index® units). Of these strongly positive samples, 75 serums (78.1%) were positive by PD-BAT. In summary, methodological comparisons between donor pooling for BAT and BHRA, conducted across a range of sample values, show substantial agreement. This highlights that the PD-BAT method yields results similar to established assays. [Table 6]

[0173]

[0183] Methodological comparison for individual donor CSU BAT A key aspect of this study focused on comparing the pooled donor basophil activation test (PD-BAT) with the conventional method using individual donors for the chronic spontaneous urticaria basophil activation test (CSU-BAT). Nine healthy blood donors were pre-screened for use in the CSU-BAT (using individual donors). Three of these were selected based on high CD63+ responses to anti-FcεRI (77.3%, 77.5%, and 93.0%, respectively) and low responses to a normal serum pool (CD63+ 3.6%, 2.8%, and 6.6%, respectively). The CSU-BAT was performed on the three donors using 69 serum samples from patients suspected of having CSU, and the results were compared to the PD-BAT (average of results from two runs) (Figures 11A, 11B, and 11C show the results for donors 1, 2, and 3, respectively). Correlation coefficients showed variability among donors. In particular, donors 1 and 2 met the PD-BAT criteria for inclusion in the final pool and showed moderate correlations of 0.85 and 0.92 with the PD-BAT results. However, donor 3 had a lower correlation of 0.59 with the average PD-BAT result.

[0174]

[0184] To analyze methodological agreement, the mean of duplicate PD-BAT trials (on the x-axis) is considered the "true" value. Table 7 shows that the positive predictive value, as well as the positive and negative agreement rates, are highly dependent on the individual basophil-providing donor. In Table 7, TP: true positive, TN: true negative, FP: false positive, FN: false negative, PPA: positive agreement rate, NPA: negative agreement rate, PPV: positive predictive value, NPV: negative predictive value. [Table 7]

[0175]

[0185] Sixty-nine samples were tested twice using PD-BAT with two separate donor pools providing basophils, demonstrating consistency between measurements (Figure 12).

[0176]

[0186] Consideration The application of basophil activation assays to evaluate patients with CSU using both flow cytometry and histamine release approaches is challenging due to intrinsic variability in individual donors. This variability has been observed in previous screening processes, where some screened donors were found to perform below optimal in assays of characterized control and patient serum pools (Figures 5A and 5B). To address this, some laboratories test patient serum samples with multiple donors and use arbitrary criteria to determine positive patient outcomes (Gentinetta et al., 2011; Marcelino et al., 2021; D'Auria et al., 2019). However, even with pre-screening to eliminate non-responders and donors exhibiting elevated background activation, results obtained using individual donors have been found inconsistent in many patients tested (Figures 11A, 11B, and 11C, Table 7). By pooling multiple donors that meet defined performance specifications, it was possible to achieve more consistent batch-to-batch performance for flow cytometry-based assays (Figure 12).

[0177]

[0187] In addition to improving assay performance, the donor pooling approach helped improve the efficiency of instrument and reagent use. Pre-selecting donors with relatively high basophil counts reduced flow cytometer usage time, which helped reduce the time required to count the required number of basophils compared to individual donors with lower basophil counts. Pre-screening and pooling approaches require more effort and reagents when used in small batch trials. Pre-screening 30 candidate donors would require conducting 90 individual trials for background, LSP, and anti-FcεRI controls. However, in actual patient trials, only one donor pool needs to be tested, whereas conventional methods required testing at least three individual donors (Gentinetta et al., 2011; Marcelino et al., 2021; D'Auria et al., 2019).

[0178]

[0188] An interesting observation in this study concerns changes in the stability of pooled whole blood, particularly those occurring 2 hours after the pooling process, which may be due to mixed lymphocyte reactions (MLR). MLR is the phenomenon in which T lymphocytes from one individual react with major histocompatibility complex (MHC) antigens from another individual. In the context of this study, mixed lymphocyte reactions in donor blood pools may contribute to a rapid decrease in activation levels, as observed particularly in highly reactive serum pools. For optimal results, it was found that sample testing should be initiated within 2 hours of pool formation. However, once the sample and donor mixing process is complete and the samples have been processed (stimulation, staining, lysis, and washing), actual sample uptake can be delayed by up to 3 days.

[0179]

[0189] The moderate correlation observed between PD-BAT and CU-Index® (Figure 10) is consistent with the results of other studies comparing histamine release-based methods with flow cytometry activation marker-based methods (Schoepke et al. 2019). The data in this example demonstrate excellent inter-assay comparability of results obtained by PD-BAT (Figures 6 and 12), which is thought to be due to the consistent performance of multiple donor pools. The lack of direct comparability of results may be partly related to the inconsistent performance of individual donors in CSU-BAT, particularly donor 3 (Figure 11C), as observed in donor 3. Donor 3 would not have been selected for the PD-BAT assay as it did not meet the criteria required for inclusion in the pool. Unsurprisingly, this donor also had a poor correlation with the mean PD-BAT result. These findings highlight PD-BAT's ability to minimize donor-related variability and ensure reliable and consistent results.

[0180]

[0190] Example 4 - Embodiment This disclosure may be better understood by referring to the following non-limiting embodiments.

[0181]

[0191] A.1 A method for pooling biological samples for use in a test, comprising screening multiple biological samples for responses indicating the presence of a given indication, and selecting multiple samples that provide responses exceeding a first predetermined threshold in order to generate a pool.

[0182]

[0192] A.2 The method according to any of the above or below embodiments of the method, further comprising screening multiple biological samples to determine whether a sample has a nonspecific response exceeding another predetermined threshold, and removing the sample having a nonspecific response from the pool.

[0183]

[0193] A.3 The method according to any of the above or below embodiments of the method, wherein the biological sample is blood.

[0184]

[0194] A.4 The method according to any of the preceding or following method embodiments, wherein the pool contains at least 10, or at least 20, or at least 30 biological samples.

[0185]

[0195] A.5 The method according to any of the above or below embodiments of the method, wherein the response is basophil activation.

[0186]

[0196] A.6 The method according to either of the above or below embodiments of the method, wherein basophil activation is evaluated using CD63 as a marker for basophil degranulation and activation by an anti-FcεRI mAb.

[0187]

[0197] A.7 The method according to any of the embodiments described above or below, wherein the specified indication is chronic spontaneous urticaria.

[0188]

[0198] A.8 The method of any of the above or below embodiment of the method, further comprising evaluating the accuracy of the pool by comparing the results with (i) a first pool of additional samples that produce a high response above a first predetermined quality control (QC) threshold, (ii) a second pool of additional samples that produce a moderate response at a second predetermined QC threshold, and (iii) a third pool of additional biological samples that produce a low response at a third predetermined QC threshold.

[0189]

[0199] A.9 The method according to any of the above or below embodiments of the method, wherein the additional biological sample is serum.

[0190]

[0200] A.10 The method according to any of the preceding or following method embodiments, wherein the technical cutoff for pooled samples showing a positive response indicating the presence of a specified indication is defined using the 95% confidence index (CI) of a plurality of normal samples.

[0191]

[0201] A.11 Accordingly, a method for testing for a response indicating the presence of a predetermined indication, comprising the steps of: generating a plurality of pooled samples from a subject that provide a response exceeding a first predetermined threshold and that do not show a nonspecific response at arbitrary selection; and adding a portion of the sample from the subject to the pooled samples of (a) to determine whether the sample generates a response.

[0192]

[0202] A.12 The method according to any of the above or below embodiments of the method, wherein the pooled sample is blood.

[0193]

[0203] A.13 The method according to any of the above or below embodiments of the method, wherein the pooled sample comprises at least 10, or at least 20, or at least 30 biological samples.

[0194]

[0204] A.14 The method according to any of the above or below embodiments of the method, wherein the response is basophil activation.

[0195]

[0205] A.15 The method according to any of the above or below embodiments of the method, wherein basophil activation is evaluated using CD63 as a marker for basophil degranulation and activation by an anti-FcεRI mAb.

[0196]

[0206] A.16 The method according to any of the embodiments of the method described above or below, wherein the specified indication is chronic spontaneous urticaria.

[0197]

[0207] A.17 The method according to any of the above or below embodiments of the method, wherein the sample from the subject is serum.

[0198]

[0208] A.18 The method of any of the above or below method embodiments, further comprising evaluating the accuracy of a pool by comparing the results with (i) a first pool of additional serum samples that produce a high response above a first predetermined quality control (QC) threshold, (ii) a second pool of additional serum samples that produce a moderate response at a second predetermined QC threshold, and (iii) a third pool of additional serum samples that produce a low response at a third predetermined QC threshold.

[0199]

[0209] A.19 The method according to either of the preceding or subsequent method embodiments, wherein the technical cutoff for pooled samples showing a positive response indicating the presence of a specified indication is defined using the 95% CI of a plurality of normal samples.

[0200]

[0210] B.1 Compositions or kits for carrying out any of the disclosed method embodiments or for using any of the disclosed system or computer program embodiments.

[0201]

[0211] B.2 A composition or kit comprising a pool of biological samples for screening samples from subjects for a specified indication, wherein the pool comprises a plurality of biological samples, each exhibiting a response exceeding a first predetermined threshold and optionally not exhibiting a nonspecific response, the response indicating the presence of the specified indication and optionally comprising instructions for use.

[0202]

[0212] B.3 A composition or kit according to any of the above or below compositions or kit embodiments, wherein the specified indication is an autoantibody disease.

[0203]

[0213] B.4 The composition or kit according to any of the composition or kit embodiments described above or below, wherein the disease is chronic spontaneous urticaria (CSU).

[0204]

[0214] B.5 The composition or kit according to any of the composition or kit embodiments described above or below, wherein the biological sample in the pool is a whole blood sample.

[0205]

[0215] B.6 A composition or kit according to any of the composition or kit embodiments described above or below, wherein the pool comprises at least 10, at least 20, or at least 30 biological samples.

[0206]

[0216] B.7 A composition or kit according to any of the above or below embodiments of a composition or kit, wherein the response is basophil activation.

[0207]

[0217] B.8 A composition or kit according to any of the above or below embodiments of a composition or kit, wherein basophil activation is evaluated using anti-FcεRI mAb activation of CD63 as a marker of basophil degranulation.

[0208]

[0218] C.1 A system for carrying out any of the previously disclosed method embodiments, or for using any of the previously disclosed composition or kit embodiments.

[0209]

[0219] C.2 A system for generating a pool of biological samples for use in a test, comprising components or stations for screening multiple biological samples for a response indicating the presence of a predetermined indication, and components or stations for selecting the multiple samples that provide a response exceeding a first predetermined threshold in order to generate a pool.

[0210]

[0220] C.3 A system according to any of the system embodiments described above or below, further comprising components or stations for evaluating a biological sample from a subject for response, wherein the biological sample from the subject is separate from any other biological sample used to generate a pool, and is used to determine whether the sample generates a response and to quantify the level of response compared to the pool.

[0211]

[0221] C.4 The system according to any of the system embodiments described above or below, further comprising components or stations for screening multiple biological samples to determine whether a sample has a nonspecific response, and for removing biological samples having a nonspecific response from inclusion in a pool.

[0212]

[0222] C.5 The system according to any of the system embodiments described above or below, wherein the biological sample screened for inclusion in the pool is blood.

[0213]

[0223] C.6 The system according to any of the system embodiments described above or below, wherein the pool contains at least 10, or at least 20, or at least 30 biological samples.

[0214]

[0224] C.7 A system according to any of the system embodiments described above or below, wherein the response is basophil activation.

[0215]

[0225] C.8 A system according to either of the above or below system embodiments, wherein basophil activation is evaluated using CD63 as a marker for basophil degranulation and activation by an anti-FcεRI mAb.

[0216]

[0226] C.9 The system according to any of the system embodiments described above or below, wherein the specified indication is chronic spontaneous urticaria.

[0217]

[0227] C.10 The system according to any of the system embodiments described above or below, further comprising stations and / or components for evaluating the accuracy of the pool.

[0218]

[0228] C.11 A station and / or component for evaluating the accuracy of a pool, wherein the result is compared with (i) a first pool of additional biological samples that generates a high response above a first predetermined quality control (QC) threshold, (ii) a second pool of additional biological samples that generates a medium response at a second predetermined QC threshold, and (iii) a third pool of additional biological samples that generates a low response at a third predetermined QC threshold, the system according to any of the foregoing or following system embodiments.

[0219]

[0229] C.12 The system according to any of the foregoing or following system embodiments, wherein the additional biological sample is serum.

[0220]

[0230] C.13 The system according to any of the foregoing or following system embodiments, wherein the technical cut-off for the pooled samples, as being indicative of a positive response indicating the presence of a predetermined indication, is defined using the 95% CI of a plurality of normal samples.

[0221]

[0231] C.14 The system according to any of the foregoing or following system embodiments, further comprising a computer program product tangibly embodied in a non-transitory machine-readable storage medium including instructions configured to execute any of the components or stations of the system.

[0222]

[0232] D.1 A computer program product tangibly embodied in a non-transitory machine-readable storage medium including instructions configured to perform any of the methods of the foregoing method embodiments, or to use any of the compositions of the foregoing composition embodiments, or to execute any of the stations and / or components according to any of the system embodiments.

[0223]

[0233] D.2 A computer program product tangibly embodied in a non-temporary machine-readable storage medium, comprising instructions configured to screen multiple biological samples for responses indicating the presence of a predetermined indication and to select multiple samples that provide responses exceeding a first predetermined threshold in order to generate a pool.

[0224]

[0234] D.3 A computer program product tangibly embodied in a non-temporary machine-readable storage medium, comprising instructions configured to generate a plurality of pooled samples that provide a response exceeding a first predetermined threshold and which, at arbitrary selection, do not exhibit a nonspecific response, and to add a portion of the sample from a subject to the pooled samples to determine whether or not the sample generates a response.

[0225]

[0235] D.4 A computer program product tangibly embodied in a non-temporary machine-readable storage medium, comprising instructions configured to cause a system to run which includes components or stations for screening multiple biological samples for responses indicating the presence of a predetermined indication, and components or stations for selecting multiple samples that provide responses exceeding a first predetermined threshold in order to generate a pool.

[0226]

[0236] A computer program product according to either of the computer program embodiments described above or below, further comprising instructions configured to run a system that includes components or stations for testing for the presence of a predetermined indication in a sample from a subject using a pool.

Claims

1. A method for pooling biological samples for use in a test, (a) A step of screening multiple biological samples for responses indicating the presence of a specified indication, (b) A step of selecting a plurality of biological samples that provide the response exceeding a first predetermined threshold in order to generate the pool, Methods that include...

2. The method according to claim 1, (a) A step of screening the plurality of biological samples to determine whether the samples have nonspecific responses exceeding different predetermined thresholds, (b) A step of removing a sample having a nonspecific response from the pool Methods that include...

3. The method according to claims 1 to 2, wherein the biological sample is blood.

4. The method according to claims 1 to 3, wherein the pool comprises at least 10, or at least 20, or at least 30 biological samples.

5. The method according to claims 1 to 4, wherein the response is basophil activation.

6. The method according to claim 5, wherein the basophil activation is evaluated using anti-FcεRI mAb activation of CD63 as a marker of basophil degranulation.

7. The method according to claims 1 to 6, wherein the prescribed indication is chronic spontaneous urticaria.

8. The method according to claims 1 to 7, further comprising evaluating the accuracy of the pools by comparing the results with (i) a first pool of additional samples that produce a high response above a first predetermined quality control (QC) threshold, (ii) a second pool of additional samples that produce a moderate response at a second predetermined QC threshold, and (iii) a third pool of additional biological samples that produce a low response at a third predetermined QC threshold.

9. The method according to claim 8, wherein the additional biological sample is serum.

10. The method according to claims 1 to 9, wherein the technical cutoff of the pooled samples as showing a positive response indicating the presence of a predetermined indication is defined using the 95% CI of a plurality of normal samples.

11. A method for testing for a response indicating the presence of a specified indication, wherein in a sample from a subject, (a) A step of generating a plurality of pooled samples that provide the response exceeding a first predetermined threshold and that do not exhibit a nonspecific response at an optional rate, (b) A step of adding a portion of the sample from the subject to the pooled sample in (a) in order to determine whether the sample produces the response, Methods that include...

12. The method according to claim 11, wherein the pooled sample is blood.

13. The method according to claims 11 to 12, wherein the pooled sample comprises at least 10, or at least 20, or at least 30 biological samples.

14. The method according to claims 11 to 13, wherein the response is basophil activation.

15. The method according to claim 14, wherein the basophil activation is evaluated using anti-FcεRI mAb activation of CD63 as a marker of basophil degranulation.

16. The method according to claims 11 to 15, wherein the prescribed indication is chronic spontaneous urticaria.

17. The method according to claims 11 to 16, wherein the sample from the subject is serum.

18. The method according to claims 11 to 17, further comprising evaluating the accuracy of the pool by comparing the results with (i) a first pool of additional serum samples that produce a high response above a first predetermined quality control (QC) threshold, (ii) a second pool of additional serum samples that produce a moderate response at a second predetermined QC threshold, and (iii) a third pool of additional serum samples that produce a low response at a third predetermined QC threshold.

19. The method according to claims 11 to 18, wherein the technical cutoff of the pooled samples as showing a positive response indicating the presence of a predetermined indication is defined using the 95% CI of a plurality of normal samples.

20. A composition comprising a pool of biological samples for screening samples from a subject for a predetermined indication, wherein the pool comprises a plurality of biological samples, each exhibiting a response exceeding a first predetermined threshold and, at arbitrary selection, not exhibiting a nonspecific response, and the response indicates the presence of the predetermined indication.

21. The composition according to claim 20, wherein the predetermined indication is an autoantibody disease.

22. The composition according to claim 20 or 21, wherein the disease is chronic spontaneous urticaria (CSU).

23. The composition according to claims 20 to 22, wherein the biological sample in the pool is a whole blood sample.

24. The composition according to claims 20 to 23, wherein the pool comprises at least 10, at least 20, or at least 30 biological samples.

25. The composition according to claims 20 to 24, wherein the response is basophil activation.

26. The composition according to claims 20 to 25, wherein the basophil activation is evaluated using anti-FcεRI mAb activation of CD63 as a marker of basophil degranulation.

27. A system for generating a pool of biological samples for use in testing, (a) Components or stations for screening multiple biological samples for responses indicating the presence of a specified indication, and (b) Components or stations for selecting the plurality of samples that provide the response above a first predetermined threshold in order to generate the pool A system that includes this.

28. A system according to claim 27, wherein the system further comprises components or stations for evaluating a biological sample from a subject for the response, the biological sample from the subject being separate from any of the biological samples used to generate the pool, and used to determine whether the sample generates a response and to quantify the level of the response in comparison to the pool.

29. The system according to claim 27 or 28, further comprising components or stations for screening the plurality of biological samples to determine whether the samples have a nonspecific response, and for removing the biological samples having a nonspecific response from inclusion in the pool.

30. The system according to claims 27 to 29, wherein the biological sample screened for inclusion in the pool is blood.

31. The system according to claims 27 to 30, wherein the pool comprises at least 10, or at least 20, or at least 30 biological samples.

32. The system according to claims 27 to 31, wherein the response is basophil activation.

33. The system according to claims 27 to 32, wherein the basophil activation is evaluated using anti-FcεRI mAb activation of CD63 as a marker of basophil degranulation.

34. The system according to claims 27 to 33, wherein the predetermined indication is chronic spontaneous urticaria.

35. The system according to claims 27 to 34, further comprising a station and / or components for evaluating the accuracy of the pool.

36. The system according to claim 35, wherein the station and / or component for evaluating the accuracy of the pools includes a station or component for comparing the results with (i) a first pool of additional biological samples that produce a high response above a first predetermined QC threshold, (ii) a second pool of additional biological samples that produce a moderate response at a second predetermined (QC) threshold, and (iii) a third pool of additional biological samples that produce a low response at a third predetermined QC threshold.

37. The system according to claim 35, wherein the additional biological sample is serum.

38. The system according to claims 27 to 37, wherein the technical cutoff of the pooled samples as showing a positive response indicating the presence of a predetermined indication is defined using the 95% CI of a plurality of normal samples.

39. The system according to claims 27 to 38, further comprising a computer program product tangibly embodied in a non-temporary machine-readable storage medium, which includes instructions configured to execute any of the components or stations of the system.

40. A computer program product tangibly embodied in a non-temporary machine-readable storage medium, comprising instructions configured to screen multiple biological samples for responses indicating the presence of a predetermined indication, and to select multiple samples that provide responses exceeding a first predetermined threshold in order to generate a pool.

41. (a) Components or stations for screening multiple biological samples for responses indicating the presence of a specified indication, and (b) Components or stations for selecting a number of samples that provide a response above a first predetermined threshold in order to generate a pool. A computer program product tangibly embodied in a non-temporary, machine-readable storage medium, including instructions configured to run a system that includes [a specific system].

42. The computer program product according to claim 41, further comprising instructions configured to cause a system to run using the pool, which includes components or stations for testing for a response indicating the presence of the predetermined indication in a sample from a subject.

Images