Proof of concept - Detection of erythrocyte-bound immunoglobulin G (IgG) in an anemic foal using flow cytometry

Aim of the study Immune-mediated hemolytic anemia (IMHA) is a rare but potentially lethal disease in horses. The direct antiglobulin test (DAT, “Coombs’ test”) is used to detect erythrocyte-bound antibodies (EBA) or complement proteins on erythrocytes indicative of IMHA. Typically, well plate-based agglutination assays are used. Hypothetically, EBA can also be detected via flow cytometry. The aim of the study was to test the practical feasibility of detecting EBA in horse samples by flow cytometric assay as possible tool for routine diagnostics.

Clinical case & methods A Haflinger foal developed fever and lethargy 3 weeks after birth. After one week of symptomatic therapy leading to no improvement in symptoms, blood samples were sent for routine-diagnostic work-up including complete blood count, protein electrophoresis, clinical chemistry and DAT. Additionally, for flow cytometric analysis, an erythrocyte suspension was prepared from EDTA blood, which was incubated with FITC-conjugated antibodies (anti-equine IgG), washed and resuspended. The resuspended sample was measured using flow cytometry (AttuneNxT, Thermofisher, Darmstadt). Negative controls were prepared with left-over material from DAT negative routine samples.

Results The red blood count showed a severe anemia (hematocrit 0.11 L/L, erythrocyte count 2.3 T/L, hemoglobin 35 g/L). Also, leukocytosis (14.8 G/L), hyperbilirubinemia and increased α-globulins were detected. The DAT was positive for EBA.

DAT result was confirmed by flow cytometric analysis, because IgG coated 24% of erythrocytes (preliminary cutoff for unspecific binding set to 10%). Negative controls showed values below 1%; a questionable range was defined for 1–10 %.

Conclusions & limits Flow cytometric analysis of antibodies bound to equine erythrocytes delivered results consistent with those of the standard assay. For establishment in routine diagnostics clinically healthy and anemic horses are currently analyzed to validate the method and define reliable reference ranges. Sample collection of DAT positive samples is our time-limiting factor.

Publication History

Article published online:
07 March 2024