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Updated: Aug 10, 2026
A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood
Published on: February 8, 2016
An automatable format for accurate immunohematology testing by flow cytometry
John D Roback1, Sheilagh Barclay, Christopher D Hillyer
1Transfusion Medicine Program, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA. jroback@emory.edu
Insights
Flow cytometry (FC) offers accurate and cost-effective immunohematology testing, improving upon traditional methods for blood group typing and antibody detection. This automated technique enhances transfusion safety and efficiency.
Area of Science:
- Immunohematology
- Blood Transfusion Medicine
- Clinical Laboratory Science
Background:
- Current immunohematology methods face limitations in cost, throughput, and automation adaptability.
- Existing automated systems struggle to accommodate diverse transfusion-related tests.
Purpose of the Study:
- To evaluate a novel flow cytometry (FC) technique for immunohematology testing.
- To compare FC accuracy against established methods like column agglutination technology (CAT) and standard tube tests.
- To assess FC's suitability for challenging clinical samples.
Main Methods:
- Clinical samples were tested for ABO group, D type, and RBC alloantibodies using FC, CAT, and tube methods.
- FC involved antibody staining of RBCs, initially in tubes and later in microtiter filter plates.
- Challenging samples included those with rouleaux, autoantibodies, mixed-field reactions, and weak antibodies.
Main Results:
- FC achieved 99.1% accuracy for ABO/D typing of 222 samples, surpassing CAT (91.9%) and tube methods (95.0%).
- FC detected 99.5% of clinically relevant RBC alloantibodies in 239 samples, outperforming CAT (98.9%) and LISS-IAT (94.7%).
- The FC filter plate technique correctly identified ABO/D in 95.4% of 109 samples and detected antibodies in 98.3%.
Conclusions:
- Optimized FC methods demonstrate accuracy comparable to standard immunohematology techniques.
- FC with filter plates enables rapid, cost-effective, and automated testing for patient and donor samples.
- This FC workstation can automate additional pretransfusion assays, enhancing laboratory efficiency.
Background:
Current immunohematology testing methods have limitations including cost, throughput, and adaptability to automation. Furthermore, current automated and semiautomated workstations cannot accommodate many other tests relevant to blood transfusion.
Study Design And Methods:
Authentic clinical samples from hospitalized patients were tested for ABO group, D type, and presence of RBC alloantibodies by column agglutination technology (CAT), standard tube methods, and a recently developed flow cytometry (FC) technique. Included were challenging samples with rouleaux, autoantibodies, mixed-field reactions, and weak antibodies. Antibody staining of RBCs for FC was initially performed in test tubes and subsequently in microtiter filter plates interfaced with a vacuum manifold.
Results:
When antibody staining was performed in tubes, FC testing determined the correct ABO group and D type for 99.1 percent of 222 clinical samples, as compared to accuracies of 91.9 percent for CAT and 95.0 percent for standard tube testing. FC testing also detected 99.5 percent of clinically relevant RBC alloantibodies in 239 patient samples, as compared to 98.9 percent for CAT and 94.7 percent for LISS-IAT. Using the FC filter plate technique, 104 of 109 samples (95.4%) were correctly typed for ABO and D (the remaining five samples were read as "no type determined" due to RBC and serum testing discrepancies), and RBC alloantibodies of the IgG and IgM classes were correctly identified in 98.3 percent of samples.
Conclusions:
Optimized FC testing methods that are comparable in accuracy to standard CAT and tube methods are described. When used with filter plates, this methodology should allow rapid and cost-effective immunohematology testing of both patient and donor samples in an automated workstation format. The same workstation should support automation of other pretransfusion assays that can be analyzed by FC.
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