Comparison of anti-human CD45 clones

CD45, also known as leukocyte common antigen (LCA), is a type I transmembrane protein tyrosine phosphatase expressed exclusively on the surface of all nucleated hematopoietic cells, except mature erythrocytes and platelets. It plays a central role in immune cell signaling, particularly in regulating antigen receptor-mediated activation in T and B lymphocytes. Here we present our comparison data of four anti-human CD45 clones commonly used in Flow cytometry.
Encoded by the PTPRC gene, CD45 exists in multiple isoforms resulting from alternative splicing of exons 4, 5, and 6 (often referred to as A, B, and C), producing cell type- and activation state-specific variants such as CD45RA, CD45RB, and CD45RO. These isoforms serve as important markers for distinguishing naive versus memory T cells, among other subsets.

Functionally, CD45 is crucial for modulating signal transduction by dephosphorylating Src family kinases, such as Lck and Fyn in T cells, thus enabling T-cell receptor (TCR) signaling. Due to its broad expression across leukocyte lineages and its involvement in immune regulation, CD45 is widely used as a pan-leukocyte marker in flow cytometry and immunohistochemistry.

Alterations in CD45 expression or function have been associated with immunodeficiencies, autoimmune diseases, and hematological malignancies, making it not only a vital biological molecule but also a potential diagnostic and therapeutic target.

We analyzed the staining profile and other staining parameters of four anti-human CD45 (pan) clones - 2D1, HI30, LT45 and MEM-28 on peripheral whole blood of two healthy blood donors. Antibody conjugates with Pacific Orange™ were titrated at 5 concentrations (16, 4, 1, 0.25 and 0.06 µg/ml). In Fig. 1, Staining profiles at 1 µg/ml are shown. In Fig. 2, five-point titration curves for MFI (lymphocytes) and Stain index (positive – lymphocytes; negative – CD45 negative blood debris) of all four clones are presented.

According our data, human lymphocytes have the best MFI and separation of blood debris using HI30, closely followed by LT45 and finished by a pair of clones with similar staining parameters – MEM-28 and 2D1. Yet all four clones are capable to distinguish lymphocytes, from blood debris and also basophils, have the same specificity and ensure sufficient separation between CD45 positive and negative events, even when labeled with lower-intensity dyes like Pacific Orange™.