Characteristic immunophenotyping artefact seen in patients with anti‐mouse immunoglobulin antibodies

Abstract

Patients treated with murine monoclonal antibodies develop anti-mouse phocytes for immunophenotyping. immunoglobulin antibodies which can interfere with flow cytometry of lymphocytes, giving rise to high levels of nonspecific staining. This artefact can be avoided by using separated, washed lymKey terms: Cytometry, monoclonal antibodies, human immunodeficiency virus, anti-idiotypic antibodiesThe use of murine monoclonal antibodies is increasing in all fields of medicine. They are now used for in vivo diagnosis and therapy in many situations, and the development of human antibodies against both the constant and variable regions of mouse immunoglobulin has been well documented (1,2).We have been investigating the possible role of antiidiotypic antibodies against anti-CD4 monoclonal antibodies in the therapy of human immunodeficiency virus infection, the results of which will be the subject of 3 separate report. As part of the follow-up of patients immunized with six intramuscular injections of 1 mg of mti-Leu3a, a murine anti-CD4 monoclonal antibody, Ne have seen a characteristic artefact when attempting ;o enumerate T-cell subsets by flow cytometry.Flow cytometry was performed using a Becton-Dicknson FACScan cytometer, with either the Immune Monitoring Kit (IMK) panel of fluorochrome-conjugated mouse monoclonal antibodies and software, or Simulset 2-Colour antibodies and software (Bectonlickinson, San Jose, California). EDTA-anticoaguIated blood was stained and then lysed using BectonIlickinson FACS lysing solution followed by washing once in phosphate buffered saline, according to the inanufacturer's instructions. All the patients in this study have developed antibodies directed against murine immunoglobulins, demonstrable to high titre by ELISA. Coincident with the development of anti-mouse immunoglobulin antibodies, an increase in non-specific staining of up to 100% of all lymphocytes was seen, rendering immunophenotyping of lysed whole blood impossible (Fig. la,b). This characteristic artefact, which we believe is due to formation of immune complexes between the patients' anti-mouse-immunoglobulin antibodies and the monoclonal antibodies used for staining, is not due to circulating mouse Ig and it persists for a t least 6 months after immunization, in spite of considerable waning of anti-mouse Ig antibody titres as determined by ELISA. Forward and side scatter were not affected.We have found that separation of peripheral blood mononuclear cells by centrifugation over Ficoll-Paque (Pharmacia, Sweden) followed by washing the cells twice in RPMI medium is sufficient to remove this artefact and to allow immunophenotyping to be performed (Fig. lc,d).Since the therapeutic and diagnostic use of monoclonal antibodies in conditions such as HIV infection and haematological malignancies can only increase, this artefact will certainly be seen more commonly.