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Updated: Jan 17, 2026
A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood
Published on: February 8, 2016
Ad hoc antibody modification of a validated flow cytometric immunophenotyping panel-recommendations and safeguards
Christopher J Groves1, Michael A Linden2, Ahmad Al-Attar3,4
1Translational Science and Innovation Lab, IQVIA Laboratories, Durham, North Carolina, USA.
Insights
Clinical laboratories can make minor, validated antibody panel modifications for flow cytometry immunophenotyping in rare cases. This guidance ensures assay integrity and optimizes patient care during unforeseen situations.
Area of Science:
- Clinical laboratory science
- Immunology
- Hematology
Background:
- Flow cytometry immunophenotyping is crucial for timely clinical diagnostics.
- Validated antibody panels ensure consistent and accurate results in routine laboratory settings.
- Unforeseen clinical scenarios or supply chain issues may require panel modifications.
Purpose of the Study:
- To provide guidance on performing minor, ad hoc modifications to validated antibody panels for flow cytometry immunophenotyping.
- To ensure assay integrity and reliable results when adapting panels for rare clinical situations.
- To optimize patient care by enabling laboratories to address unique diagnostic challenges.
Main Methods:
- The manuscript outlines best practices for minor antibody panel modifications, such as adding or substituting one to two antibodies.
- Key considerations include assessing impacts on fluorescence compensation, antibody binding, and assay sensitivity.
- Recommendations cover limitations, examples, troubleshooting, and documentation for ad hoc changes.
Main Results:
- Minor modifications can be performed while maintaining assay integrity for rare clinical situations.
- Careful assessment of impacts on assay performance is essential for reliable results.
- Ad hoc modifications are not substitutes for full validation protocols (e.g., CLSI H62).
Conclusions:
- Laboratories can adapt validated flow cytometry panels for unique immunophenotypes or supply issues with minor modifications.
- Proper guidance and documentation mitigate risks associated with ad hoc panel changes.
- These modifications support timely and accurate patient diagnosis in challenging clinical scenarios.
Abstract:
Immunophenotyping by flow cytometry is a valuable test providing important information in a timely manner. In clinical laboratories, it is performed using validated antibody panels designed to ensure consistent and accurate results. However, unforeseen situations, such as unique or unusual immunophenotypes, or supply chain issues, may necessitate ad hoc modifications to these panels. This manuscript provides guidance for performing minor modifications, such as substituting or adding one or two antibodies, while maintaining the integrity of the assay. These modifications are intended for rare clinical situations and are not substitutes for the full validation protocols outlined in CLSI H62. An example of this would be a patient with a rare, but not uncommon, situation in which a B cell lymphoma lacks expression of CD19, CD20, and surface light chains, such that the lineage of the neoplastic cells cannot be determined without a straightforward addition or substitution of another marker into a laboratory's available panel. The recommendations and best practices herein aim to optimize patient care by allowing laboratories to adapt to unique clinical scenarios without compromising assay performance and are not a way to permanently modify the assay. Key considerations include assessing the impact on fluorescence compensation, antibody binding, assay sensitivity, and overall assay performance. The manuscript provides limitations for the extent of modifications, examples, and troubleshooting strategies to ensure reliable results when ad hoc changes are made. Proper documentation with review and approval by laboratory medical directors is recommended to mitigate risks associated with these modifications.
