Assay Interference Research Articles

Evaluation of the relationship between the 14-3-3\u03b5 protein and LvRab11 in the shrimp Litopenaeus vannamei during WSSV infection

The 14-3-3 proteins interact with a wide variety of cellular proteins for many diverse functions in biological processes. In this study, a yeast two-hybrid assay revealed that two 14-3-3ε isoforms (14-3-3ES and 14-3-3EL) interacted with Rab11 in the white shrimp Litopenaeus vannamei (LvRab11). The interaction of 14-3-3ε and LvRab11 was confirmed by a GST pull-down assay. The LvRab11 open reading frame was 645 bp long, encoding a protein of 214 amino acids. Possible complexes of 14-3-3ε isoforms and LvRab11 were elucidated by in silico analysis, in which LvRab11 showed a better binding energy score with 14-3-3EL than with 14-3-3ES. In shrimp challenged with the white spot syndrome virus (WSSV), the mRNA expression levels of LvRab11 and 14-3-3ε were significantly upregulated at 48 h after challenge. To determine whether LvRab11 and binding between 14-3-3ε and LvRab11 are active against WSSV infection, an in vivo neutralization assay and RNA interference were performed. The results of in vivo neutralization showed that LvRab11 and complexes of 14-3-3ε/LvRab11 delayed mortality in shrimp challenged with WSSV. Interestingly, in the RNAi experiments, the silencing effect of LvRab11 in WSSV-infected shrimp resulted in decreased ie-1 mRNA expression and WSSV copy number. Whereas suppression of complex 14-3-3ε/LvRab11 increased WSSV replication. This study has suggested two functions of LvRab11 in shrimp innate immunity; (1) at the early stage of WSSV infection, LvRab11 might play an important role in WSSV infection processes and (2) at the late stage of infection, the 14-3-3ε/LvRab11 interaction acquires functions that are involved in immune response against WSSV invasion.

Antibody Interference in Thyroid Assays: a case report

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Lithium heparin interference in the Abbott enzymatic creatinine assay: the significance of under-filled tubes.

Spuriously high results using the Abbott Architect enzymatic creatinine assay were noted to be particularly associated with very small sample volumes. This led us to query the effect of under-filling lithium heparin tubes on the measured enzymatic creatinine result. Blood was provided by 5 laboratory personnel and then decanted into 5 x1.2mL Sarstedt S-Monovette tubes, giving final blood volumes of 200, 400, 600, 800 and 1200μL. Plasma was analysed using Abbott Architect Jaffe, enzymatic creatinine, Beckman Coulter (AU500) enzymatic creatinine and Roche (Cobas c702) enzymatic creatinine assays. Saline was also added to Sarstedt 1.2mL and Teklab 2mL tubes and analysed using the Abbott Jaffe and enzymatic creatinine methods. Increasing degrees of under-fill were associated with greater over-estimation of creatinine using the Abbott enzymatic assay, but no difference was noted using Jaffe methodology on the same platform or enzymatic assays provided by Roche or Beckman. On average, creatinine was 40.6% (+27.7μmol/L) higher when only 200μL of blood was present in the tube. Small volumes of saline added to lithium heparin tubes measured significant creatinine concentrations using the Abbott enzymatic method. Lithium heparin directly interferes in the Abbott Architect enzymatic creatinine assay. Under-filling lithium heparin tubes can lead to clinically significant over-estimation of creatinine results by this assay. Users of this assay should be aware of the potential for spurious results in small sample volumes collected into lithium heparin tubes and implement robust procedures for identifying and reporting results on these samples.

Evaluation of interference from hemoglobin C, D, E and S traits on measurements of hemoglobin A1c by fifteen methods

BackgroundHemoglobin C, D Punjab, E or S trait can interfere with hemoglobin A1c (HbA1c) results. We assessed whether they affect results obtained with 15 current assay methods. MethodsHemoglobin AA (HbAA), HbAC, HbAD Punjab, HbAE and HbAS samples were analyzed on 2 enzymatic, 4 ion-exchange HPLC and 9 immunoassay methods. Trinity Premier Hb9210 boronate affinity HPLC was the comparative method. An overall test of coincidence of least-squared linear regression lines was performed to determine if HbA1c results were statistically significantly different from those of HbAA samples. Clinically significant interference was defined as >6% difference from HbAA at 6 or 9% HbA1c compared to Premier Hb9210 using Deming regression. ResultsAll methods showed statistically significant effects for one or more variants. Clinically significant effects were observed for the Tosoh G11 variant mode (HbAD), Roche b 101 (HbAC and HbAE) and Siemens DCA Vantage (HbAE and HbAS). All other methods (Beckman Coulter B93009 and B00389 on DxC700AU, and Unicel DxC, Ortho Clinical Vitros 5.1, Roche cobas c 513, Siemens Dimension RxL and Vista, and Enzymatic on Advia and Atellica, Tosoh G8 5.24 and 5.28, and GX) showed no clinically significant differences. ConclusionsA few methods showed interference from one or more variants. Laboratories need to be aware of potential HbA1c assay interferences.

Analytical and Clinical Considerations in Implementing the Roche Elecsys Troponin T Gen 5 STAT Assay.

To evaluate the analytical and clinical performance characteristics of the fifth-generation troponin T reagent. Troponin T was measured in 2,332 paired serum and plasma samples from emergency department and hospital patients using the fourth- and fifth-generation reagents. Testing was repeated after recentrifugation to determine the frequency of analytical outliers and percentage of patients with elevated values for each assay. We conducted separate experiments to determine the effects of biotin and hemolysis interference, as well as measure interinstrument variability, for fifth-generation troponin T. Analytic outliers occurred more frequently using the fifth-generation reagent (3.4%) compared with the fourth-generation reagent (1.0%). The frequency of elevated troponin T above the 99th percentile upper reference limit was 26% for the fourth-generation reagent and 52% for the fifth-generation reagent. Clinically significant assay interference by biotin was observed at 20 ng/mL, but hemolysis interference was not observed until an H index of 150. Instrument-to-instrument variability between e411 and e601/602 instrument platforms is predicted to confound clinical interpretation of troponin changes. Analytical outliers and instrument-to-instrument variability are the two analytical variables most likely to confound interpretation of changes in fifth-generation troponin T results over time.

Addressing Compound Reactivity and Aggregation Assay Interferences: Case Studies of Biochemical High-Throughput Screening Campaigns Benefiting from the National Institutes of Health Assay Guidance Manual Guidelines.

Compound-dependent assay interferences represent a continued burden in drug and chemical probe discovery. The open-source National Institutes of Health/National Center for Advancing Translational Sciences (NIH/NCATS) Assay Guidance Manual (AGM) established an "Assay Artifacts and Interferences" section to address different sources of artifacts and interferences in biological assays. In addition to the frequent introduction of new chapters in this important topic area, older chapters are periodically updated by experts from academia, industry, and government to include new technologies and practices. Section chapters describe many best practices for mitigating and identifying compound-dependent assay interferences. Using two previously reported biochemical high-throughput screening campaigns for small-molecule inhibitors of the epigenetic targets Rtt109 and NSD2, the authors review best practices and direct readers to high-yield resources in the AGM and elsewhere for the mitigation and identification of compound-dependent reactivity and aggregation assay interferences.

45-Year-Old Woman With Fever and Malaise

45-Year-Old Woman With Fever and Malaise

Flow cytometric myeloma measurable residual disease testing in the era of targeted therapies.

Therapies in myeloma are rapidly advancing with a host of new targeted therapies coming to market. While these drugs offer significant survival benefits and better side-effect profiles compared with conventional chemotherapeutics, they raise significant difficulties in monitoring post-therapy disease status by flow cytometry due to assay interference and/or selection of phenotypically different sub-clones. The principal culprit, anti-CD38 monoclonal antibodies, limits the ability to detect plasma cells based on classical CD38/CD45 gating. Other markers, such as CD138, are known to be suboptimal by flow cytometry. Various techniques have been proposed to overcome this problem. The most promising of these techniques has been the marker VS38c, a monoclonal antibody targeting an endoplasmic reticulum protein which has shown high sensitivity for plasma cells. Alternative techniques for gating plasma cells, while variably effective in the near term are already the subject of several targeted therapies rendering their usefulness limited in the longer term. Likewise, future targets of these therapies may render present aberrancy markers ineffective in MRD testing. These therapies pose challenges that must be overcome with new markers and novel panels in order for flow cytometric MRD testing to remain relevant.

A Novel Laboratory Assay for Monitoring Unfractionated Heparin in Patients Taking Direct Oral Anticoagulants Prior to Hospital Admission

Introduction: As conditions like atrial fibrillation and venous thromboembolism (VTE) become more prevalent in the United States, the use of direct oral anticoagulants (DOACs) is increasing. Patients presenting to the hospital with indications of thrombosis are often started on unfractionated heparin (UFH) infusion, which has traditionally been monitored using laboratory based PTT and anti-Xa assays. While monitoring UFH with anti-Xa assays have been shown to correlate with better outcomes than dose adjustments guided by PTT, anti-Xa assays are susceptible to interferences from DOACs taken prior to admission and can result in inappropriate dose adjustments that can negatively impact patient care.Methods: We derived a novel assay, termed corrected heparin (CH), using quantified values from a chromogenic anti-Xa assay with heparin calibrators prior to and following heparinase treatment to detect any residual DOAC still present within the patient sample. We retrospectively assessed 79 adult patients admitted to our institution who had their UFH infusion monitored using the CH assay due to known DOAC use. These patients were continually monitored by the CH assay and the PTT (unreported) if they had detectable DOAC levels (>0.1 IU/mL by anti-Xa). Of the patients included in the study, 92% were administered apixaban, while 8% were administered rivaroxaban prior to admission and all received UFH while admitted.Results: To assess linearity of anti-Xa assay for this purpose, patient samples with both UFH and apixaban present were diluted with drug-free plasma and displayed good correlation (R2=0.999) throughout the linear range of the assay. Of the 79 patients, 53 patients reached the therapeutic range by the PTT and CH assays, 17 reached the therapeutic range by the CH assay only, 2 reached the therapeutic range by PTT only, and 2 never reached the therapeutic range with either assay. The average time to therapeutic dosing level for the CH assay was 9 hours after initiation of UFH infusion. Using the CH assay, we were able to determine how long the DOAC persisted and was detected by the anti-Xa assay. At 12 hours after the last DOAC dose, more than 80% of patients still had detectable DOAC levels capable of interfering with the anti-Xa assays. The majority of patients (80%) still had measurable anti-Xa assay interference from the DOAC until 72 hours after the last dose. We evaluated the role of kidney function as a predictor for prolonged interference in the anti-Xa assay however, clearance rates of apixaban were difficult to predict based on kidney function status alone.Conclusions: Our CH assay has provided clinicians with an assay that accounts for the presence of residual DOAC and allows patients to reach therapeutic heparin levels faster. Our study also highlights the delayed clearance of commonly prescribed DOACs, which can be longer than expected and independent of kidney function status resulting in prolonged interference in anti-Xa assays that can lead to improper dose adjustments and potentially negative outcomes for patients on UFH infusion. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Development of a cell‐free protein synthesis protocol to rapidly screenL‐asparaginaseproteoforms by enzymatic activity

AbstractBACKGROUNDCell‐free protein synthesis (CFPS) technology has emerged as a powerful tool for a variety of biotechnological applications, including the expression of different classes of biopharmaceutical products. L‐Asparaginase (E.C. Number: 3.5.1.1, L‐asparagine amidohydrolase) (L‐ASNase) is an important biopharmaceutical used to treat leukemia, but expression of multiple proteoforms in CFPS systems and rapid characterization using standard colorimetric methods has not yet been fully exploited. Herein, recombinant expression and characterization of an L‐ASNase fromErwinia chrysanthemi(Erwinase) using a new CFPS protocol is reported.RESULTSExpression and quantification of the enzymatic activity of a soluble his‐tagged L‐ASNase directly from a CFPS reaction was successfully achieved. Purification of the protein was not required in order to assess its biological activity. Activity of L‐ASNase was significantly higher than the control reaction (7.07 ± 0.68 U mL–1vs. 1.83 ± 0.14 U mL–1, respectively). Expression of a mutant Erwinase proteoform – V293M – was also achieved and it presented a similar enzymatic activity. No significant loss in L‐ASNase enzymatic activity was noticed after removal of cyclic AMP, spermidine, transfer RNA, T7 RNA polymerase and, especially, ammonium acetate (a common interference in ASNase enzymatic assays) from the CFPS reaction.CONCLUSIONThe protocol developed in this work will facilitate the screening of novel clinically‐relevant L‐ASNase proteoforms. © 2021 Society of Chemical Industry (SCI).

Long Non-coding RNA MSTRG.24008.1 Regulates the Regeneration of the Sciatic Nerve via the miR-331-3p-NLRP3/MAL Axis.

Peripheral nerve injury (PNI) is a common clinical problem, which can cause severe disability and dramatically affect a patient’s quality of life. Neural regeneration after PNI is a complex biological process that involves a variety of signaling pathways and genes. Emerging studies demonstrated that long non-coding RNAs (lncRNAs) were abnormally expressed after PNI and played pivotal roles in peripheral nerve regeneration. Based on the rat sciatic nerve injury model, we found that the expression levels of several lncRNAs were increased significantly in the sciatic nerve after injury. Software prediction prompted us to focus on one up-regulated lncRNA, MSTRG.24008.1. Dual-luciferase reporter assay, RNA pull-down assay and RNA interference approach verified that MSTRG.24008.1 regulated neuroregeneration via the miR-331-3p/nucleotide-binding oligomerization domain-like pyrin domain containing 3 (NLRP3)/myelin and lymphocyte protein (MAL) axis in vitro. Subsequently, we performed gastrocnemius muscle gravity and sciatic functional index experiments to evaluate the recovery of injured sciatic nerves after MSTRG.24008.1 siRNA interference in vivo. In conclusion, knockdown of MSTRG.24008.1 promotes the regeneration of the sciatic nerve via the miR-331-3p/NLRP3/MAL axis, which may provide a new strategy to evaluate and repair injured peripheral nerves clinically.

Rapid Point of Care Diagnostic Test for the Detection of Rare Metabolic Disorders

Rapid Point of Care Diagnostic Test for the Detection of Rare Metabolic Disorders

Spurious Thyroid Function Test Results due to Biotin Interference: a Report of Three Cases and a Literature Review

Biotin (vitamin B7) is a water-soluble vitamin used as a co-enzyme for carboxylases essential for human metabolism. The high affinity to streptavidin makes biotin an important substance in immunoassays. Excessive biotin intake due to over-the counter supplements has become problematic because of the effects on laboratory test results. There have been no reports of biotin-induced thyroid immunoassay interference in Korea. We report three patients with papillary thyroid cancer who showed false thyrotoxicosis on follow-up laboratory examinations with a literature review. The patients’ medical history should be thoroughly questioned and patients should be informed to curtail consuming biotin before laboratory tests to avoid assay interference. Non-biotinylated assays can be considered if it is impossible to withhold the supplements. These methods will prevent physicians from making incorrect decisions that could result in an inappropriate treatment for their patients.

Abstract #1038067: Clinical Hypothyroidism Masked Biochemically due to Biotin Supplements???

Biotin( Vitamin B7) is widely available as an over the counter supplement for skin, hair and nail health. Many immunoassays use the biotin-streptavidin interaction as an immobilizing system, in which biotin can be readily incorporated into a hormone. Ingestion of high doses of biotin has been reported to cause abnormal results in thyroid function tests by this assay interference. Hereby we describe the case of a patient with clinical hypothyroidism but biochemically hyperthyroid due to biotin supplementation.

Biotin Interference in Assays for Thyroid Hormones, Thyrotropin and Thyroglobulin.

Background: Biotin has been reported to interfere with several commonly used laboratory assays resulting in misleading values and possible erroneous diagnosis and treatment. This report describes a prospective study of possible biotin interference in thyroid-related laboratory assays, with a comparison of different commonly used assay platforms. Materials and Methods: Thirteen adult subjects (mean age 45 ± 13 years old) were administered biotin 10 mg/day for eight days. Blood specimens were collected at three time points on day 1 and on day 8 (baseline, two, and five hours after biotin ingestion). Thyrotropin (TSH), free triiodothyronine (fT3), free thyroxine (fT4), total triiodothyronine (TT3), total thyroxine (TT4), thyroxine binding globulin (TBG), and thyroglobulin (Tg) levels were analyzed with four different platforms: Abbott Architect, Roche Cobas 6000, Siemens IMMULITE 2000, and liquid chromatography with tandem mass spectrometry (LC-MS/MS). TSH, fT3, fT4, TT3, and TT4 were measured with Abbott Architect and Roche Cobas 6000. fT3, fT4, TT3, and TT4 were also measured by LC-MS/MS. Tg was measured by Siemens IMMULITE 2000. TBG was assessed with Siemens IMMULITE 2000. Results: Significant changes in TSH, fT4, and TT3 measurements were observed after biotin exposure when the Roche Cobas 6000 platform was used. Biotin intake resulted in a falsely lower Tg level when measurements were performed with Siemens IMMULITE 2000. At the time points examined, maximal biotin interference was observed two hours after biotin exposure both on day 1 and day 8. Conclusions: A daily dose of 10 mg was shown to interfere with specific assays for TSH, fT4, TT3, and Tg. Physicians must be aware of the potential risk of erroneous test results in subjects taking biotin supplements. Altered test results for TSH and Tg can be particularly problematic in patients requiring careful titration of levothyroxine therapy such as those with thyroid cancer.

A toolbox for studying respiratory viral infections using air-liquid interface cultures of human airway epithelial cells

Submerged cultures of primary human airway epithelial cells or human airway epithelial cell lines have been a mainstay of airway epithelial biology research for decades due to their robust in vitro proliferative capacity, relatively low maintenance culture conditions, and clinically translatable results to nasal or bronchial brushings. With the development and improvement of air-liquid interface (ALI) cultures of human airway epithelial cells, such cultures have been considered superior to immortalized cell lines and primary cell monolayers, as such cultures effectively recapitulate in vivo epithelial architecture and cell types. Although ALI culture growth protocols are well-established and widely available, many researchers have avoided their use, as ALI cultures not only take longer to grow but also present technical challenges and limitations that make in vitro intracellular and structural assays taxing. Challenges arise relating to their complex structure, requirements for air exposure, the constraints of transwell growth apparatus, and interference in assays caused by mucus secretion. Although few publications briefly describe technical adaptations for some assays, there is still considerable trial and error required for researchers to establish consistent and reliable assay adaptations, often becoming a deterrent for pursuing mechanistic investigation. We have created a user-friendly toolbox detailing comprehensive protocols for numerous techniques and assay adaptations, particularly focusing on respiratory virus infections. By expanding the repertoire of ALI culture-adapted in vitro assays, we hope to facilitate the widespread adoption of this valuable culture system for mechanistic investigations of respiratory viral infections or other epithelial-pathogen models.

Discordant Thyroid Function Tests in Patients With HIV: A Case Report and Discussion of Diagnostic Approach

Background: TFT patterns correlate directly with clinical symptoms in most patients with thyroid disease. However, some patients have TFTs which are at odds to clinical findings, posing a diagnostic challenge. Such cases warrant careful evaluation with a structured, rational approach to avoid misdiagnosis and mistreatment. Clinical Case: A 48-year-old female liver transplant recipient with HIV presented with an asymptomatic multinodular goiter, low TSH and low/normal fT4. Her medications were nadolol, Bactrim, ursodiol, azithromycin, tacrolimus, efavirenz, emtricitabine, tenofovir and zolpidem. Thyroid US showed 3 dominant nodules with benign pathology on FNA. Uptake scan showed diffuse increased uptake consistent with Grave’s disease. She tried MMI for one year, then opted for RAI ablation and was started on LT4 with titration of dose. fT4 remained low/normal regardless of receiving LT4, MMI or no therapy over the following eight years. TSH was appropriately suppressed from 1.42 to 0.160 (0.45 - 4.500 mIU/L) when LT4 weekly dose was increased from 175 mcg to 350 mcg. On lower dose LT4 (150 mcg to 250 mcg weekly) her fT4 stayed low, 0.50 - 0.74 (0.82 - 1.777mg/dL) with normal TSH of 0.73 - 1.42. She remained clinically euthyroid throughout. Serial yearly thyroid US showed no significant change. Pituitary hormonal work-up (IGF-1, FSH, LH, ACTH, estradiol and cortisol) was appropriate for age. fT4 by direct dialysis assay was low with normal TSH. tT4, T3 uptake, rT3, TBG and TPO Ab were normal. HAMA antibodies were negative. LT4 was stopped and she remains clinically euthyroid with low fT4 0.56 and normal TSH 1.73. Conclusion: The pattern of low fT4 with normal TSH is uncommon. Potential causes that were systematically evaluated include non-thyroidal illness, central hypothyroidism and assay interference. Normal pituitary function and appropriate TSH suppression while on LT4 ruled out central hypothyroidism. Her overall profile suggested fT4 assay interference. Commonly known interference factors were ruled out; biotin, HAMA Ab, pregnancy, renal failure, TBG excess, familial dysalbuminemic hyperthyroxiemia and trans-thyretin-associated hyperthyroxidemia. Direct dialysis is the gold standard fT4 assay but does not detect agents displacing T4 from TBG. It is less commonly recognized that low fT4 with normal TSH has been described amongst clinically euthyroid persons with HIV, at a prevalence of 1.3%–6.8%. Low fT4 is associated with didanosine, stavudine, and ritonavir, but not her anti-virals. The significance of low fT4 in patients with HIV is unclear, because they do not have a higher rate of hypothyroid symptoms. In the absence of clinical hypothyroidism, such as in this patient, LT4 treatment is not recommended. This case uniquely spans eight years and therefore offers valuable insight into the appropriate work-up, management and long-term natural history of low fT4 in persons with HIV.

Optimization of hemolysis, icterus and lipemia interference thresholds for 35 clinical chemistry assays

ObjectivesInterference of chemistry assays by hemolysis, icterus and lipemia (HIL) was investigated on the Abbott Alinity c system. We sought to empirically establish optimized HIL index thresholds for the purposes of reporting HIL interference in a hospital laboratory and advising clinicians on the interpretation of laboratory results in the presence of hemolysis, icterus or lipemia. MethodsHIL index values measured by spectrophotometry were compared with concentrations of hemoglobin, bilirubin and Intralipid. HIL interference of 35 Abbott Alinity chemistry assays was subsequently investigated by pairwise comparison of test results in pooled serum or plasma with those in test preparations spiked with hemolysate, bilirubin or Intralipid. Data generated from the interference experiments were critically assessed according to assay-specific acceptance criteria adapted from multiple sources, and optimized thresholds for HIL indices were established. ResultsCorrelations between HIL index values and their corresponding concentrations of hemoglobin, bilirubin and Intralipid were, in general, very good within the ranges of interferent concentrations tested. Hemolysis significantly affected 12 of 35 assays, whereas bilirubin and Intralipid interfered with four and three assays, respectively. Both the direction and magnitude of Intralipid interference with the direct bilirubin assay were dependent on the concentrations of the analyte. ConclusionsHIL interference of the Abbott Alinity clinical chemistry assays investigated in this study was not uncommon. At present, there are no universally accepted criteria for defining significant assay interference for clinical practice. In establishing acceptance criteria for defining assay interference, each assay should be assessed according to both analytical criteria and clinical relevance.

High-Throughput Label-Free Biochemical Assays Using Infrared Matrix-Assisted Desorption Electrospray Ionization Mass Spectrometry.

Mass spectrometry (MS) can provide high sensitivity and specificity for biochemical assays without the requirement of labels, eliminating the risk of assay interference. However, its use had been limited to lower-throughput assays due to the need for chromatography to overcome ion suppression from the sample matrix. Direct analysis without chromatography has the potential for high throughput if sensitivity is sufficient despite the presence of a matrix. Here, we report and demonstrate a novel direct analysis high-throughput MS system based on infrared matrix-assisted desorption electrospray ionization (IR-MALDESI) that has a potential acquisition rate of 33 spectra/s. We show the development of biochemical assays in standard buffers for wild-type isocitrate dehydrogenase 1 (IDH1), diacylglycerol kinase zeta (DGKζ), and p300 histone acetyltransferase (P300) to demonstrate the suitability of this system for a broad range of high-throughput lead discovery assays. A proof-of-concept pilot screen of ∼3k compounds is also shown for IDH1 and compared to a previously reported fluorescence-based assay. We were able to obtain reliable data at a speed amenable for high-throughput screening of large-scale compound libraries.

Brief Guide: Experimental Strategies for High-Quality Hit Selection from Small-Molecule Screening Campaigns.

Small-molecule screening is a powerful approach to identify modulators of either specific biological targets or cellular pathways with phenotypic endpoints. A myriad of assay technologies are available to assess the activity of enzymes, monitor protein–protein interactions, measure transcription factor activity in reporter assays, or detect cellular features and activities using high-content imaging. A common challenge during small-molecule screening is, however, the presence of hit compounds generating assay interference, thereby producing false-positive hits. Thus, efforts are needed to uncover such interferences to prioritize high-quality hits for further analysis. This process encompasses (1) computational approaches to flag undesirable compounds, and (2) the use of experimental approaches like counter, orthogonal, and cellular fitness screens to identify and eliminate artifacts. In this brief guide, we provide an overview for first-time users, highlighting experimental screening strategies to prioritize high-quality bioactive hits from high-throughput screening/high-content screening (HTS/HCS) campaigns.