Beyond the Hydrashift assay: the utility of isoelectric focusing for therapeutic antibody and paraprotein detection
-
Rieke Reiter
Abstract
Objectives
In serum immunofixation electrophoresis, therapeutic monoclonal antibodies (tmAbs) can be misinterpreted as paraproteins. Strategies to circumvent these interferences include the use of anti-tmAb antibodies, which led to the development of the Hydrashift assay. However, this assay is exclusively available for the Sebia platform and limited to specific tmAbs, such as daratumumab (DmAb) and isatuximab (ImAb). Furthermore, we observed some cases in which complete shifting of tmAbs by the Hydrashift Assay was questionable. This study aimed to explore isoelectric focusing (IEF) as an alternative, widely applicable method for clinical laboratories.
Methods
First, the neutralization capacity of the DmAb and ImAb Hydrashift assay was assessed by testing undiluted tmAb samples. Second, DmAb and ImAb were spiked into paraprotein-containing samples in clinically relevant concentrations and analyzed by the Hydrashift assay and IEF. Presence of unshifted tmAb and identification of tmAb and paraproteins were examined in these samples and routinely collected patient samples.
Results
The Hydrashift assay reliably shifted up to 0.5 g/L DmAb and up to 1.5 g/L ImAb. IEF was found to be equally effective as the Hydrashift assay in identifying antibody interferences and detecting paraproteins. Additionally, IEF successfully identified two pseudo-monoclonal gammopathies as oligoclonal and detected other therapeutic antibodies.
Conclusions
Insufficient neutralization capacity of the Hydrashift Assay was not observed within the clinically relevant range of investigated tmAb concentrations. IEF offers a simple and flexible alternative to the Hydrashift assay for distinguishing tmAbs from paraproteins. Its advantages include broader applicability to various tmAbs and independency from specific commercial platforms.
-
Research ethics: The Ethics Commission of the Philipps-University of Marburg deemed the study exempt from review (Faculty of Medicine, 23–64 BO).
-
Informed consent: Not applicable.
-
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission. Rieke Reiter (Conceptualization – Supporting, Formal analysis, Investigation, Methodology – Equal, Project administration – Equal, Resources – Lead, Validation – Lead, Visualization, Writing – original draft – Equal, Writing – review & editing – Equal), Christoph Mann (Resources – Supporting, Writing – review & editing – Supporting), Wolfgang Andreas Nockher (Conceptualization – Lead, Methodology – Equal, Project administration – Equal, Supervision, Validation – Supporting, Writing – original draft – Equal, Writing – review & editing – Equal).
-
Use of Large Language Models, AI and Machine Learning Tools: None declared.
-
Conflict of interest: The authors state no conflict of interest.
-
Research funding: None declared.
-
Data availability: The data that support the findings of this study are available from the corresponding author, WAN, upon reasonable request.
References
1. Kyle, RA, Gertz, MA, Witzig, TE, Lust, JA, Lacy, MQ, Dispenzieri, A, et al.. Review of 1027 patients with newly diagnosed multiple myeloma. Mayo Clin Proc 2003;78:21–3. https://doi.org/10.4065/78.1.21.Suche in Google Scholar PubMed
2. Rajkumar, SV, Dimopoulos, MA, Palumbo, A, Blade, J, Merlini, G, Mateos, MV, et al.. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncol 2014;15:e538–48. https://doi.org/10.1016/s1470-2045(14)70442-5.Suche in Google Scholar
3. Caillon, H, Irimia, A, Simon, JS, Axel, A, Sasser, K, Scullion, MJ, et al.. Overcoming the interference of daratumumab with immunofixation electrophoresis (IFE) using an industry-developed Dira test: Hydrashift 2/4 Daratumumab. Blood 2016;128:2063. https://doi.org/10.1182/blood.v128.22.2063.2063.Suche in Google Scholar
4. McCudden, C, Axel, A, Slaets, D, Frans, S, Bald, J, Schecter, J, et al.. Assessing clinical response in multiple myeloma (MM) patients treated with monoclonal antibodies (mAbs): validation of a daratumumab IFE reflex assay (DIRA). J Clin Oncol 2015;A8590.10.1200/jco.2015.33.15_suppl.8590Suche in Google Scholar
5. Kirchhoff, DC, Murata, K, Thoren, KL. Use of a daratumumab-Specific immunofixation assay to assess possible immunotherapy interference at a major cancer center: our experience and recommendations. J Appl Lab Med 2021;6:1476–83. https://doi.org/10.1093/jalm/jfab055.Suche in Google Scholar PubMed PubMed Central
6. Lee, HW, Kim, SM, Park, HD. Evaluating the Hydrashift 2/4 Daratumumab assay: a powerful approach to assess treatment response in multiple myeloma. Clin Chem Lab Med 2024;62:2223–32. https://doi.org/10.1515/cclm-2024-0416.Suche in Google Scholar PubMed
7. Cornell, FN, McLachlan, R. Isoelectric focusing in the investigation of gammopathies. Sydney: Australian Association of Clinical Biochemists; 1985:31–7 pp.Suche in Google Scholar
8. Kim, K, Phelps, MA. Clinical pharmacokinetics and pharmacodynamics of daratumumab. Clin Pharmacokinet 2023;62:789–806. https://doi.org/10.1007/s40262-023-01240-8.Suche in Google Scholar PubMed PubMed Central
9. Xu, XS, Dimopoulos, MA, Sonneveld, P, Ho, PJ, Belch, A, Leiba, M, et al.. Pharmacokinetics and exposure–response analyses of daratumumab in combination therapy regimens for patients with multiple myeloma. Adv Ther 2018;35:1859–72. https://doi.org/10.1007/s12325-018-0815-9.Suche in Google Scholar PubMed PubMed Central
10. European Medicines Agency. Darzalex – Daratumumab. https://www.ema.europa.eu/en/medicines/human/EPAR/darzalex [Accessed March 2024].Suche in Google Scholar
11. Martin, T, Strickland, S, Glenn, M, Charpentier, E, Guillemin, H, Hsu, K, et al.. Phase I trial of isatuximab monotherapy in the treatment of refractory multiple myeloma. Blood Cancer J 2019;9:41. https://doi.org/10.1038/s41408-019-0198-4.Suche in Google Scholar PubMed PubMed Central
12. Sunami, K, Suzuki, K, Ri, M, Matsumoto, M, Shimazaki, C, Asaoku, H, et al.. Isatuximab monotherapy in relapsed/refractory multiple myeloma: a Japanese, multicenter, phase 1/2, safety and efficacy study. Cancer Sci 2020;111:4526–39. https://doi.org/10.1111/cas.14657.Suche in Google Scholar PubMed PubMed Central
13. Simonelli, M, Garralda, E, Eskens, F, Gil-Martin, M, Yen, CJ, Obermannova, R, et al.. Isatuximab plus atezolizumab in patients with advanced solid tumors: results from a phase I/II, open-label, multicenter study. ESMO Open 2022;7:100562. https://doi.org/10.1016/j.esmoop.2022.100562.Suche in Google Scholar PubMed PubMed Central
14. Mikhael, J, Richter, J, Vij, R, Cole, C, Zonder, J, Kaufman, JL, et al.. A dose-finding phase 2 study of single agent isatuximab (anti-CD38 mAb) in relapsed/refractory multiple myeloma. Leukemia 2020;34:3298–309. https://doi.org/10.1038/s41375-020-0857-2.Suche in Google Scholar PubMed PubMed Central
15. Thoren, KL, Pianko, MJ, Maakaroun, Y, Landgren, CO, Ramanathan, LV. Distingushing drug from disease by use of the Hydrashift 2/4 Daratumumab assay. J App Lab Med 2019;3:857–63. https://doi.org/10.1373/jalm.2018.026476.Suche in Google Scholar PubMed PubMed Central
16. HYDRASHIFT 2/4 daratumumab (20 tests) Ref. 4639, package insert, version 22/5. Sebia, Lisses, France.Suche in Google Scholar
17. Wheeler, RD, Costa, MV, Crichlow, A, Willis, F, Reyal, Y, Linstead, SE, et al.. Case report: interference from isatuximab on serum protein electrophoresis prevented demonstration of complete remission in a myeloma patient. Ann Clin Biochem 2022;59:144–8. https://doi.org/10.1177/00045632211062080.Suche in Google Scholar PubMed
Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2025-0352).
© 2025 Walter de Gruyter GmbH, Berlin/Boston
Artikel in diesem Heft
- Frontmatter
- Editorial
- Macroprolactinaemia – some progress but still an ongoing problem
- Review
- Understanding the circulating forms of cardiac troponin: insights for clinical practice
- Opinion Papers
- New insights in preanalytical quality
- IFCC recommendations for internal quality control practice: a missed opportunity
- Genetics and Molecular Diagnostics
- Evaluation of error detection and treatment recommendations in nucleic acid test reports using ChatGPT models
- General Clinical Chemistry and Laboratory Medicine
- Pre-analytical phase errors constitute the vast majority of errors in clinical laboratory testing
- Improving the efficiency of quality control in clinical laboratory with an integrated PBRTQC system based on patient risk
- IgA-type macroprolactin among 130 patients with macroprolactinemia
- Prevalence and re-evaluation of macroprolactinemia in hyperprolactinemic patients: a retrospective study in the Turkish population
- Defining dried blood spot diameter: implications for measurement and specimen rejection rates
- Screening primary aldosteronism by plasma aldosterone-to-angiotensin II ratio
- Assessment of serum free light chain measurements in a large Chinese chronic kidney disease cohort: a multicenter real-world study
- Beyond the Hydrashift assay: the utility of isoelectric focusing for therapeutic antibody and paraprotein detection
- Direct screening and quantification of monoclonal immunoglobulins in serum using MALDI-TOF mass spectrometry without antibody enrichment
- Effect of long-term frozen storage on stability of kappa free light chain index
- Impact of renal function impairment on kappa free light chain index
- Standardization challenges in antipsychotic drug monitoring: insights from a national survey in Chinese TDM practices
- Potential coeliac disease in children: a single-center experience
- Vitamin D metabolome in preterm infants: insights into postnatal metabolism
- Candidate Reference Measurement Procedures and Materials
- Development of commutable candidate certified reference materials from protein solutions: concept and application to human insulin
- Reference Values and Biological Variations
- Biological variation of serum cholinesterase activity in healthy subjects
- Hematology and Coagulation
- Diagnostic performance of morphological analysis and red blood cell parameter-based algorithms in the routine laboratory screening of heterozygous haemoglobinopathies
- Cancer Diagnostics
- Promising protein biomarkers for early gastric cancer: clinical performance of combined detection
- Infectious Diseases
- The accuracy of presepsin in diagnosing neonatal late-onset sepsis in critically ill neonates: a prospective study
- Corrigendum
- The Unholy Grail of cancer screening: or is it just about the Benjamins?
- Letters to the Editor
- Analytical validation of hemolysis detection on GEM Premier 7000
- Reconciling reference ranges and clinical decision limits: the case of thyroid stimulating hormone
- Contradictory definitions give rise to demands for a right to unambiguous definitions
- Biomarkers to measure the need and the effectiveness of therapeutic supplementation: a critical issue
Artikel in diesem Heft
- Frontmatter
- Editorial
- Macroprolactinaemia – some progress but still an ongoing problem
- Review
- Understanding the circulating forms of cardiac troponin: insights for clinical practice
- Opinion Papers
- New insights in preanalytical quality
- IFCC recommendations for internal quality control practice: a missed opportunity
- Genetics and Molecular Diagnostics
- Evaluation of error detection and treatment recommendations in nucleic acid test reports using ChatGPT models
- General Clinical Chemistry and Laboratory Medicine
- Pre-analytical phase errors constitute the vast majority of errors in clinical laboratory testing
- Improving the efficiency of quality control in clinical laboratory with an integrated PBRTQC system based on patient risk
- IgA-type macroprolactin among 130 patients with macroprolactinemia
- Prevalence and re-evaluation of macroprolactinemia in hyperprolactinemic patients: a retrospective study in the Turkish population
- Defining dried blood spot diameter: implications for measurement and specimen rejection rates
- Screening primary aldosteronism by plasma aldosterone-to-angiotensin II ratio
- Assessment of serum free light chain measurements in a large Chinese chronic kidney disease cohort: a multicenter real-world study
- Beyond the Hydrashift assay: the utility of isoelectric focusing for therapeutic antibody and paraprotein detection
- Direct screening and quantification of monoclonal immunoglobulins in serum using MALDI-TOF mass spectrometry without antibody enrichment
- Effect of long-term frozen storage on stability of kappa free light chain index
- Impact of renal function impairment on kappa free light chain index
- Standardization challenges in antipsychotic drug monitoring: insights from a national survey in Chinese TDM practices
- Potential coeliac disease in children: a single-center experience
- Vitamin D metabolome in preterm infants: insights into postnatal metabolism
- Candidate Reference Measurement Procedures and Materials
- Development of commutable candidate certified reference materials from protein solutions: concept and application to human insulin
- Reference Values and Biological Variations
- Biological variation of serum cholinesterase activity in healthy subjects
- Hematology and Coagulation
- Diagnostic performance of morphological analysis and red blood cell parameter-based algorithms in the routine laboratory screening of heterozygous haemoglobinopathies
- Cancer Diagnostics
- Promising protein biomarkers for early gastric cancer: clinical performance of combined detection
- Infectious Diseases
- The accuracy of presepsin in diagnosing neonatal late-onset sepsis in critically ill neonates: a prospective study
- Corrigendum
- The Unholy Grail of cancer screening: or is it just about the Benjamins?
- Letters to the Editor
- Analytical validation of hemolysis detection on GEM Premier 7000
- Reconciling reference ranges and clinical decision limits: the case of thyroid stimulating hormone
- Contradictory definitions give rise to demands for a right to unambiguous definitions
- Biomarkers to measure the need and the effectiveness of therapeutic supplementation: a critical issue
