Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


Hydroxychloroquine Can Interfere with Flow Cytometric Analysis of Blood Cells from Patients Taking This Medication.

Fraser1,  Louise, Mitchell1,  Graham H., Sangle2,  Shirish, Choong2,  Lee Meng, Ellis1,  Richard, Spencer1,  Jo, D'Cruz3,  David P.

Department of Immunobiology, King's College London
Lupus Research Unit, St Thomas' Hospital
St Thomas Hospital, London, United Kingdom

Background:

Immunological investigation of blood cells from patients with systemic lupus erythematosus (SLE) often involves flow cytometry. Quinoline ring-containing compounds are known to fluoresce. Therefore we asked whether the drug hydroxychloroquine (HCQ) might fluoresce and whether this might interfere with flow cytometric analysis of blood cells from patients taking this drug.

Methods:

Blood cells from 13 patients with SLE were studied. They were all female, age range 19 to 71 years (average 45 years) with range of disease duration between 3 and 30 years (average 14 years). 9 patients were taking HCQ, and 5 taking steroids. In addition 3 disease controls and 10 healthy controls were studied. Peripheral blood lymphocytes were analysed by flow cytometry, either with no stain or with a standard assay for expression of kappa and lambda light chains by B cells, which uses FITC, PE and APC channels. Data were analysed with Flowjo software. All the patients studied fulfilled the ACR classification criteria. The study was approved by the St Thomas Hospital Ethics committee and all subjects gave written informed consent.

Results:

Blood cells from patients taking HCQ showed fluorescence on FITC, PE and APC channels when no fluorochrome conjugated antibodies were added to the assays. This was apparent when cells were sampled from any of the gates placed based on forward scatter and side scatter including the lymphocyte gate, and identified approximately 20% of cells as distinct populations Figure.

Blood cells from all individuals studied were assayed for kappa and lambda light chain expression on CD19 B cells by flow cytometry. The assays were standardized using blood from healthy control individuals and the percentage of B cells expressing each light chain determined accordingly. We observed that the fluorescence caused a shift so that cells appeared artifactually in the top right hand quadrant of the flow cytometric analysis of bloods from patients taking HCQ.

Conclusion:

HCQ appears to have a broad spectrum of fluorescent excitation and emission which can interfere with flow cytometric analysis of blood cells from patients taking this medication. It is important when analyzing bloods from patients taking this drug that each sample is standardized according to its own isotype matched fluorescent control antibody.

Figure. A. Analysis of healthy control blood. Lymphocytes (first column) were selected for expression of CD19 (second column) and the ratio of kappa: lambda light chain expression observed (third column).

B. When the process was repeated with blood from a SLE patient taking HCQ, the number of cells in the apparent CD19 gate increased. The profile of kappa and lambda expression was skewed (circles).

C. When cells from patients taking HCQ underwent the same procedure fluorescence in the FITC (second column), APC and PE channels was observed.

To cite this abstract, please use the following information:
Fraser, Louise, Mitchell, Graham H., Sangle, Shirish, Choong, Lee Meng, Ellis, Richard, Spencer, Jo, et al; Hydroxychloroquine Can Interfere with Flow Cytometric Analysis of Blood Cells from Patients Taking This Medication. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :463
DOI: 10.1002/art.28232

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