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Published on: March 28, 2014
Detection by flow cytometry of antibodies against surface and intracellular granulocyte antigens
A Wikman1, I Olsson, A Shanwellt
1Department of Laboratory Medicine, Karolinska Hospital, Stockholm, Sweden. Agneta.Wikman@ks.se
Insights
Flow cytometry (FC) effectively detects granulocyte antibodies, distinguishing between surface and intracellular targets. This method offers a reliable alternative to traditional immunofluorescence tests for routine leukocyte antibody screening.
Area of Science:
- Immunology
- Hematology
- Cell Biology
Background:
- Detection of granulocyte antibodies is crucial for diagnosing and managing transfusion reactions and autoimmune disorders.
- Traditional methods like granulocyte immunofluorescence test (GIFT) and indirect immunofluorescence test (IIF) have limitations in specificity and ease of use.
Purpose of the Study:
- To evaluate flow cytometry (FC) as a sensitive and routine method for detecting granulocyte antibodies.
- To differentiate between antibodies targeting surface-expressed versus intracellularly expressed antigens on granulocytes.
Main Methods:
- Analysis of 10 positive and 10 negative serum samples from GIFT, and 10 positive samples from IIF, against healthy donor leukocytes using FC.
- Utilized unpermeabilized and permeabilized cells to distinguish between surface and intracellular antigen binding.
Main Results:
- FC results largely corresponded with GIFT, with minor discrepancies in four samples.
- All IIF-positive sera were also positive by FC, with some showing both intracellular and surface antigen reactivity.
- FC demonstrated the ability to detect both HLA and granulocyte-specific antibodies.
Conclusions:
- Flow cytometry is a readily performable and reliable technique for routine detection of antibodies against leukocyte antigens.
- FC allows for the specific identification of antibodies targeting surface and intracellular antigens.
- Screening for granulocyte-specific antibodies can be efficiently performed using pooled granulocytes from three donors.
Abstract:
A flow cytometric technique was used to detect granulocyte antibodies, with attention to the distinction between antibodies directed against surface and intracellularly expressed antigens. Ten serum samples with positive results and 10 with negative results detected by the granulocyte immunofluorescence test (GIFT), together with 10 positive serum samples detected by an indirect immunofluorescence test (IIF) were analyzed against leukocytes from healthy blood donors tested by flow cytometry (FC). Unpermeabilized and permeabilized cells were used to allow identification of surface and intracellular binding, respectively. The results of testing by GIFT corresponded with those by FC, with the exception of the results for four sera: one serum sample was FC negative, GIFT positive, and three samples were FC positive, GIFT negative. The IIF-positive sera were all FC positive, analyzed against permeabilized granulocytes, and one serum was also positive against non-permeabilized granulocytes. Analysis by FC is a readily performed technique, which can be used for the routine detection of antibodies against leukocyte antigens. Screening for granulocyte-specific antibodies can be carried out with pools of granulocytes from three donors. Analysis by FC allows detection of both HLA antibodies and granulocyte-specific antibodies and by using both unpermeabilized and permeabilized cells, antibodies against surface and intracellular antigens, respectively, can be identified.
