PerFix-nc (No Centrifuge Assay): A new intracellular no-wash assay for whole blood analysisFabrice Malergue, Valerie Mallet, Laeticia Khemici, Caroline Scifo, Sylvain Monseaux, Tewfik Miloud, Emmanuel Gautherot, Felix A. Montero-JulianBeckman Coulter Life Science, Inc., Global Assay and Applications Development, Marseille, France

INTRODUCTIONBackground: The vast majority of Flow Cytometry applications target cell surface proteins, even if they represent about 10% of the protein repertoire only. Therefore, numerous available monoclonal antibodies targeting intracellular antigens remained poorly used in Flow Cytometry. This is primarily due to the technical complexity associated with intracellular staining methodologies, particularly when concomitant extracellular analysis is needed.

Commercially available staining techniques are based on the same principle: Stain with surface markers/ Fix blood/ Remove fixative / Add permeabilization buffer / Add intracellular markers / Wash one or two times. These methods are time and labour consuming (total duration is two to three hours), and require at least two mandatory centrifugation steps, making the protocols difficult to automate.

Here we present a new commercially available method, named PerFix-nc*, based on the use of a new detergent and new fixative procedure allowing simultaneous intra- and extra-cellular staining. The single-step staining together with the absence of washing strongly improves the work flow, i.e. 45 min instead of 2 to 3 hours.

MATERIALS & METHODSMaterials: Several antibodies targeting various specificities and conjugated to different fluorochromes were tested on whole blood samples using Perfix-nc*. For cell surface marker, Versalyse was used as method of reference. For intracel-lular markers, Intraprep (Beckman Coulter) and Fix&Perm (Invitrogen) were used as methods of reference. Samples were acquired on Gallios* cytometer and data were analyzed with Kaluza* software (Beckman Coulter).

Methods: PerFix-nc STRAIGHT procedure:

RESULTSFigure 1. Workflow and hands-on time comparison:

Figure 2. Surface markers:

Figure 3. Cell recovery:

Figure 3. Dye compatibility:

Figure 4. Repeatability:

Figure 4. Examples:

SUMMARY

The novel PerFix-nc kit brings excellent robustness, repeatability and cell recovery. It is compatible with most commercially available fluorochromes and tandem dyes. The work load is reduced (less than 15 min), the workflow is shorter (45 min) and can easily be automated. The surface staining is largely unaffected, whereas the intracellular staining is frequently improved. The method is versatile, and accommodates various optimizations depending on the application: one kit for all intracellular markers.

*PerFix-nc, Gallios and Kaluza are for Research Use Only. Not for use in diagnostic procedures. Krome Orange, Gallios and Kaluza are trademarks of Beckman Coulter, Inc. Beckman Coulter, VersaLyse and the stylized logo are registered trade marks of Beckman Coulter, Inc and are registered in the USPTO. Pacific Blue is a trademark of Molecular Probes, Inc. Alexa Fluor is a registered trademarks of Molecular Probes, Inc.

Optional final wash: To further increase S/N and/or concentrate the cells, it is possible here to centrifuge the tube and resuspend the cells in the same R3 buffer.

(As expected on a normal whole blood sample, TdT is negative on all leukocyte subpopulations.)

§ Dr Bernard Drénou, Dr Agathe Debliquis, Hematology Laboratory,Emile Muller Hospital, Mulhouse, France.

Optional final wash: To further increase S/N and/or concentrate the cells, it is possible here to centrifuge the tube and resuspend the cells in the same R3 buffer.

Optional final wash: To further increase S/N and/or concentrate the cells, it is possible here to centrifuge the tube and resuspend the cells in the same R3 buffer.

(As expected on a normal whole blood sample, TdT is negative on all leukocyte subpopulations.)

§ Dr Bernard Drénou, Dr Agathe Debliquis, Hematology Laboratory,Emile Muller Hospital, Mulhouse, France.

Optional final wash: To further increase S/N and/or concentrate the cells, it is possible here to centrifuge the tube and resuspend the cells in the same R3 buffer.

Optional final wash: To further increase S/N and/or concentrate the cells, it is possible here to centrifuge the tube and resuspend the cells in the same R3 buffer.

(As expected on a normal whole blood sample, TdT is negative on all leukocyte subpopulations.)

§ Dr Bernard Drénou, Dr Agathe Debliquis, Hematology Laboratory,Emile Muller Hospital, Mulhouse, France.

Optional final wash: To further increase S/N and/or concentrate the cells, it is possible here to centrifuge the tube and resuspend the cells in the same R3 buffer.

Optional final wash: To further increase S/N and/or concentrate the cells, it is possible here to centrifuge the tube and resuspend the cells in the same R3 buffer.

(As expected on a normal whole blood sample, TdT is negative on all leukocyte subpopulations.)

§ Dr Bernard Drénou, Dr Agathe Debliquis, Hematology Laboratory,Emile Muller Hospital, Mulhouse, France.

Optional final wash: To further increase S/N and/or concentrate the cells, it is possible here to centrifuge the tube and resuspend the cells in the same R3 buffer.

Optional final wash: To further increase S/N and/or concentrate the cells, it is possible here to centrifuge the tube and resuspend the cells in the same R3 buffer.

(As expected on a normal whole blood sample, TdT is negative on all leukocyte subpopulations.)

§ Dr Bernard Drénou, Dr Agathe Debliquis, Hematology Laboratory,Emile Muller Hospital, Mulhouse, France.

Optional final wash: To further increase S/N and/or concentrate the cells, it is possible here to centrifuge the tube and resuspend the cells in the same R3 buffer.

Optional final wash: To further increase S/N and/or concentrate the cells, it is possible here to centrifuge the tube and resuspend the cells in the same R3 buffer.

(As expected on a normal whole blood sample, TdT is negative on all leukocyte subpopulations.)

§ Dr Bernard Drénou, Dr Agathe Debliquis, Hematology Laboratory,Emile Muller Hospital, Mulhouse, France.

Optional final wash: To further increase S/N and/or concentrate the cells, it is possible here to centrifuge the tube and resuspend the cells in the same R3 buffer.

Optional final wash: To further increase S/N and/or concentrate the cells, it is possible here to centrifuge the tube and resuspend the cells in the same R3 buffer.

(As expected on a normal whole blood sample, TdT is negative on all leukocyte subpopulations.)

§ Dr Bernard Drénou, Dr Agathe Debliquis, Hematology Laboratory,Emile Muller Hospital, Mulhouse, France.

Optional final wash: To further increase S/N and/or concentrate the cells, it is possible here to centrifuge the tube and resuspend the cells in the same R3 buffer.