Name: a semi-automated approach to preparing antibody cocktails for immunophenotypic analysis of human peripheral blood
Uploaded: 2016-02-08T15:00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood at JoVE.com

We thank Hannah Puzas for assistance with system design and configuration, and Kevin Khovananth for technical advice. Funding for this work was provided by The Hearst Foundations and the Providence Portland Medical Foundation.

<ol>
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YK, ILG, TLM, WLM, DPH, and KSB: No competing financial interests. ANT is an employee of Beckman Coulter, Inc. Publication of this video article is sponsored by Beckman Coulter, Inc.

Immunophenotyping of peripheral blood is critically important for gaining insights into individual responses to immunotherapy. The challenge lies in assay standardization to control experiment-to-experiment variability1,14. One main source of variability lies in the human manipulation of samples. Therefore, it is conceivable that partial or full automation of sample processing will facilitate a dramatic reduction in experiment-to-experiment variability1,14. In this protocol, we report our successful effort to minimize assay variability by introducing an automated liquid handler equipped with 2D barcode reader for sample staining in whole blood immunophenotyping.
In design, our method is versatile and can monitor other immune subsets by adding additional &#34;Base Cocktails&#34; to define them. Such subsets include T helper cells, T regulatory cells, B cells, NK cells, dendritic cells, and monocytes (manuscript in preparation)18. We adapted the consortium&#39;s recommended antibody panels by introducing additional markers1. Cell populations were identified using &#34;Base Cocktail&#34; conjugated to fluorochromes with minimal emission into the brilliant violet 421 (BV421), phycoerythrin (PE), and allophycocyanin (APC) detectors, as we wanted to reserve these channels for the detection of inducible antigens. This feature not only ensures the highest sensitivity to monitor activation status of immune cells but also enables flexible accommodation of new activation markers as these three fluorochromes are most frequently conjugated with antibodies against inducible markers. Therefore, our method is not only suitable for cocktail preparation for whole blood immunophenotyping but is also useful for staining other samples including peripheral blood mononuclear cells and cells recovered from disaggregated tissues (e.g., tumor).
Successful introduction of our method requires special attention to steps in labware preparation, and programing and execution of the method. Preparation of the 2D barcode tubes includes labeling with human-readable labels and transferring of the antibodies to the designated tubes. To prevent the introduction of errors, we recommend doing this with two people. Whenever changing spreadsheets, investigators should verify whether the method works properly. Choosing appropriate pipetting methods during programing ensures successful liquid transfer. LLS enables pipetting without getting precipitated debris from the bottom of antibody tubes, for which we use either P50 or P200 conductive tips. LLS may not be a good option for P1000 tips as LLS is more sensitive with P1000 tips and sometimes falsely triggered by presence of bubbles. Heights in labware definition should be adjusted for each instrument as suboptimal liquid transfer could occur, for example, if there is not enough space between the edge of tips and the bottom of tubes. As shown in Figure 12, if the &#34;Base Cocktail&#34; and/or &#34;Activation Cocktail&#34; are not vortexed well, it may result in suboptimal staining.
We thought about using lyophilized reagents for cell staining as an alternative approach to reduce variability related with reagent dispensing19. However, polymer conjugates of brilliant violet dyes are known to interact with each other causing non-specific signals. As such, adding more than two polymer conjugates (e.g., BV421 and BV650 etc.) in lyophilized reagents may cause non-specific signaling (e.g., increase of BV650 background signal in BV421+ population)20. Moreover, lyophilized reagents lack flexibility for including new staining. They are usually more expensive and require a bulk order. For those reasons, we chose to use the automated liquid handler equipped with the 2D barcode tubes. Although it takes time to set up and entails an upfront investment to purchase the instrument, in the long run such factors will be compensated for by the increased productivity and reproducibility of assays. In fact, a few groups previously reported the successful integration of the automated liquid handler into their workflow of immunophenotyping or similar applications21,22. Automated solutions for flow cytometry analysis are also available from commercial sources (FACS SPA III, Automated Cocktail Preparation Workstation, and FlowStainer). This further indicates there is a great need for automated cocktail preparation for immunophenotyping.
After mastering this technique, we envision that the development of fully automated whole blood immunophenotyping will further reduce experiment-to-experiment variability and might make whole blood immunophenotyping feasible even in a multicenter clinical trial setting23. We have already begun using a lyse wash assistant to automate the lysis and washing steps. We also envision that automated determination of the volume of antibody in the 2D barcode tubes and the tracking of reagent dispensing will greatly improve inventory of antibodies and the quality control over our method, respectively.

Immunophenotyping of human peripheral blood determines quantitative and qualitative changes in immune cell subsets1. It provides insight into mechanisms of action and resistance, aids discovery of predictive biomarkers, and facilitates the development of combination immunotherapies. Therefore, the validation and standardization of immunophenotyping is an area of considerable interest to academic researchers, clinical laboratories, and industries.
Although immunophenotyping of peripheral blood by flow cytometric methods is currently used for clinical management of hematological malignancies2,3 and human immunodeficiency virus (HIV) infection4,5, reliable immunophenotyping of peripheral blood for immunotherapy demands specific considerations in the validation and standardization because it requires more extensive coverage over immune cell subsets and activation/inhibitory receptors1,6-8. Successful immunophenotyping requires careful standardization to minimize experiment-to-experiment variability related to instrument setup and the cell staining procedure9,10. While standardization of instrument settings for flow cytometry is well established to address the former concern9,11,12, it remains unclear how to minimize variability related to cell staining without restricting coverage of immune cell subsets and their activation status.
As the number of antigens to be detected increases, so too does the opportunity for error and variability owing to suboptimal reagent dispensing and cross-contamination. Methods for the phenoptypic analysis of anticoagulated whole blood were established in the late 1980&#39;s for use in clinical laboratory assays. These same methods serve the needs of the research laboratory for sample preparation. Importantly, the antibody cocktails used in the clinical laboratory are typically less complex and available pre-titered and pre-mixed from the manufacturer. Only a single transfer of the pre-mixed antibody cocktail is required. In the research setting, antibody cocktails of 10-16 antibodies are typical. Each cocktail must be validated for stability by the laboratory or prepared fresh before each assay. Preparing multiple antibody cocktails for a sample might entail the pipetting of 50-80 individual antibodies, a task that is both tedious and error prone.
Automation of cocktail preparation has several advantages over manual cocktail preparation such as fewer errors, increased accuracy of pipetting, and possibly even decreased reagent wastage. Here, we report the successful introduction of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into the cell-staining process of immunophenotyping to minimize variability related to sample preparation.

<table border="0" cellpadding="0" cellspacing="0" width="1546">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:613px;">BD LSRFortessa</td>
			<td style="width:188px;">BD Biosciences</td>
			<td style="width:116px;"></td>
			<td style="width:629px;">Flow cytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NXp Span-8 Laboratory Automation Workstation&#160;</td>
			<td>Beckman Coulter</td>
			<td>A31839</td>
			<td>Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insert, Tube, 11 mm, White, for 1.5 mL Microfuge Tubes (case of 25)</td>
			<td>Beckman Coulter</td>
			<td>373696</td>
			<td>Accessary for 24-Position Tube Rack to accommodate microfuge tubes.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LLS kit</td>
			<td>Beckman Coulter</td>
			<td>719262</td>
			<td>Attach bottom of the deck in Laboratory Automation Workstation to enable liquid level sensing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-Position Tube Rack</td>
			<td>Beckman Coulter</td>
			<td>373661</td>
			<td>Tube rack to hold microfuge tubes for antibody cocktails or blood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BIOHAZARD AUTOCLAVE BAGS 12x24 IN. RED</td>
			<td>LabMart</td>
			<td>M111416</td>
			<td>Disposable autoclave bags for the Laboratory Automation Workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VisionMate High Speed 2D Barcode Reader</td>
			<td>Thermo Scientific</td>
			<td>AB-1850</td>
			<td>2D barcode reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8-Channel Handheld Screw Cap Capper/Decapper</td>
			<td>Thermo Scientific</td>
			<td>4105MAT</td>
			<td>For de-capping and capping 2D barcode tubes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>02-681-335</td>
			<td>For making cocktail in or supplying blood speciment to the Laboratory Automation Workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CoolRack XT CFT24</td>
			<td>biocision</td>
			<td>BCS-534</td>
			<td>To hold sample tubes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.0 mL Matrix ScrewTop Amber Tubes in Latch Racks</td>
			<td>Thermo Scientific</td>
			<td>3741AMB</td>
			<td>2D barcode tubes to store fluorescent dye-cojugated antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biomek Span-8 P1000 Tips, Conductive, Pre-sterile with Barrier, 1025 &#181;L</td>
			<td>Beckman Coulter</td>
			<td>987925</td>
			<td>Tips for Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biomek Span-8 P50 Tips, Conductive, Pre-sterile with Barrier, 50 &#181;L (Case of 10 racks)</td>
			<td>Beckman Coulter</td>
			<td>B01091</td>
			<td>Tips for Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biomek Span-8 P250 Tips, Conductive, Pre-sterile with Barrier (Case of 10 racks)</td>
			<td>Beckman Coulter</td>
			<td>394627</td>
			<td>Tips for Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACS Lysing Solution 10X Concentrate</td>
			<td>BD Biosciences</td>
			<td>349202</td>
			<td>RBC lysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Round-Bottom Polystyrene Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-5</td>
			<td>Sample tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD45 FITC</td>
			<td>BD Biosciences</td>
			<td>347463</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD3 Alexa Fluor 700</td>
			<td>eBioscience</td>
			<td>56-0038-42</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD4 PerCPCy5.5</td>
			<td>BD Biosciences</td>
			<td>341654</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TCRgd BV650</td>
			<td>BD Biosciences</td>
			<td>564156</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TCRab BV786</td>
			<td>BD Biosciences</td>
			<td>563825</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD8 APC-H7</td>
			<td>BD Biosciences</td>
			<td>560179</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CCR7 PE-CF594</td>
			<td>BD Biosciences</td>
			<td>562381</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD45RA PE-Cy7</td>
			<td>BD Biosciences</td>
			<td>337167</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-HLA-DR BV421</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">562804</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD278(ICOS) PE</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">557802</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD38 APC</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">340439</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD279(PD1) BV421</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">562516</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD357(GITR) PE</td>
			<td>eBioscience</td>
			<td style="width:116px;">12-5875-42</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD137(41BB) APC</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">550890</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD69 BV421</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">562884</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD314(NKG2D) PE</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">557940</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD134(OX40) APC</td>
			<td style="width:188px;">BioLegend</td>
			<td>350008</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Vacutainer Plastic Blood Collection Tubes with Sodium Heparin: Conventional Stopper</td>
			<td>Fisher Scientific</td>
			<td>02-689-6</td>
			<td>For collecting blood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin, Bovine Serum, Fraction V, RIA and ELISA Grade</td>
			<td>EMD Millipore</td>
			<td>126593</td>
			<td>For flow wash buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>S8032</td>
			<td>For flow wash buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin, sodium salt</td>
			<td>Affimetrix</td>
			<td>16920</td>
			<td>For flow wash buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#8217;s Balanced Salt Solution (HBSS), 1X, with Calcium, Magnesium, without Phenol Red</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30588.02</td>
			<td>For flow wash buffer</td>
		</tr>
	</tbody>
</table>

a semi-automated approach to preparing antibody cocktails for immunophenotypic analysis of human peripheral blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Watch this Scientific Journal Video about A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood at JoVE.com

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Research

JoVE Journal

Immunology and Infection

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Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

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An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

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The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Analysis of Human Natural Killer Cell Metabolism

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to ...