Simultaneous detection of IgG, IgM, IgA complexes and C3d attached to erythrocytes by flow cytometry

Alzate, Maria A.; Manrique, L. G.; Bolaños, Natalia I.; Duarte, Mónica; Coral-Alvarado, Paola; González, John Mario

doi:10.1111/ijlh.12297

Cited by 7 publications

(7 citation statements)

References 16 publications

(43 reference statements)

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“…In a very recent article, concomitant detection of IgG, C3d, IgM and IgA was described. 19 This study, however, does not give insight into IgM detection by FACS due to lack of samples positive for IgM. In our hands, DAT by FACS gave very good agreement with the traditional DAT, performed using the column technique, but no true improvement in IgM detection.…”

Section: Discussionmentioning

confidence: 48%

Complement deposition in autoimmune hemolytic anemia is a footprint for difficult-to-detect IgM autoantibodies

et al. 2015

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© F e r r a t a S t o r t i F o u n d a t i o nwhich leads to remission in 50% of patients. 7 Rituximab has also been seen to be a successful treatment for IgGmediated AIHA, 8 and -despite its high cost and side effects -is recommended as second-line therapy in steroid-refractory AIHA. Cold (IgM-mediated) AIHA usually does not respond to prednisone. In some cases hemolysis can be prevented by protection from cold, but otherwise the therapeutic anti-CD20 antibody rituximab seems to be a promising strategy for treatment of this group of patients, showing a response rate of around 50%. 9,10 In general, patients with mixed AIHA initially respond well to steroids, but usually go on to develop chronic hemolysis. 11,12 To determine the optimal therapy, it is crucial to identify the causative RBC autoantibodies correctly, and to evaluate the presence of anti-RBC IgM autoantibodies in AIHA. However, with the current diagnostic techniques, it can be challenging to detect IgM because of its frequently low avidity. Moreover, a distinction is not routinely made between IgG and IgM in the IAT and can be difficult. We, therefore, studied alternative methods to detect autoantibodies in AIHA, focusing on the detection of IgM. First, we adapted the DAT and IAT to a fluorescence-activated cell sorting (FACS)-based assay, since a similar strategy had previously been shown to be successful for the detection of IgG and C3 on RBC.13-16 Secondly, we studied a method for detecting IgM based on the presence of deposited complement factors on patients' RBC. It is known that IgM activates complement more efficiently than IgG and it has been shown that if a patient's serum induces hemolysis in vitro, this is often caused by anti- RBC IgM 17 . Instigated by this, we studied whether the presence or extent of complement C3 deposition, as routinely measured in the DAT, can indicate the presence of anti-RBC IgM by separating IgG and IgM in the sera from AIHA patients and subsequently testing which fractions of the samples activated complement. Methods Patients' materialFor the FACS-based IAT and DAT studies, anonymized residual samples from patients with AIHA (34 and 50 patients, respectively) were used. The samples were collected in 2013 and 2014 and had been sent in for diagnostic tests to the Dutch reference laboratory for immunohematology and had a positive DAT for at least C3d and positivity in the hemolysin test. For the fractionation/depletion experiments, 16 additional samples were used that were positive for at least C3d in the DAT. Details can be found in Online Supplementary Table S1. The presence of clinical hemolysis was confirmed using the following parameters, if available: low hemoglobin, high lactate dehydrogenase, decreased haptoglobin, increased indirect bilirubin or a confirmed diagnosis by the treating physician.Fractionation of serum or eluate from patients with autoimmune hemolytic anemia AIHA patients' serum, recalcified plasma or eluate was filtered through a 0.22 μm filter before loading 0.5 mL on a 10/30...

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Section: Discussionmentioning

confidence: 48%

Complement deposition in autoimmune hemolytic anemia is a footprint for difficult-to-detect IgM autoantibodies

et al. 2015

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“…In this test, we opted to choose a cutoff with a high sensitivity to ensure the test will correctly identify 100% of all patients, as there are few testing options available to immune‐mediated DAT(–) patients. A few groups have used flow cytometry to diagnose AIHA in both DAT(–) and DAT‐positive patients . A recent study by Fayek and colleagues established a fluorescence cutoff with a sensitivity and specificity of 100%.…”

Section: Discussionmentioning

confidence: 99%

Western immunoblotting as a new tool for investigating direct antiglobulin test–negative autoimmune hemolytic anemias

et al. 2015

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WB analysis is more sensitive than conventional test tube DAT or elution analysis. Our assay confirms: 1) previous studies showing normal RBCs are sensitized with IgG, perhaps due to natural autoantibody to senescence; 2) that some normal RBCs have increased levels of IgG without signs of disease; and 3) that WB distinguishes between non-immune- and immune-mediated hemolytic anemia in DAT(-) patients and may be useful for clinical diagnosis.

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“…Immunoglobulin complexes were formed in vitro based on a previously described protocol by Alzate et al [11], with some variations, such as the use of umbilical erythrocytes and the titration of sera for the complement assays. For complement deposition and detection, preformed IgA, IgG and IgM complexes were exposed to different dilutions of normal serum or complementdepleted serum (56 C for 30 min), starting at a 1:2 dilution ratio in 1Â PBS.…”

Section: In Vitro Immune Complex Formationmentioning

confidence: 99%

“…Recently, flow cytometry is applied to detect immune complexes on the surface of erythrocytes. This technique allows for the detection of immune complexes in patients with autoimmune hemolytic anemia, including some with a negative Coombs test [10,11,12]. A direct Coombs test on umbilical cord blood has been used as a predictor of CONTACT John M. Gonz alez, MD, PhD johgonza@uniandes.edu.co Grupo de Ciencias B asicas M edicas, School of Medicine, Universidad de los Andes, Carrera 1 #18A-10, area code 111711, Bogot a D.C., Colombia ß 2016 Universidad de los Andes, Colombia.…”

Section: Introductionmentioning

confidence: 99%

“…In general, the evaluation of immune complexes has focused primarily on their pathological role; however, little is known about the physiological properties of immune complexes that are transported by erythrocytes. Our group standardized a flow cytometry protocol that allows for the multiparametric and simultaneous detection of different isotyopes of immunoglobulin complexes along with C3d deposition on erythrocytes [11].…”

Section: Introductionmentioning

confidence: 99%

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Delivery route determines the presence of immune complexes on umbilical cord erythrocytes

Lima

Franco

Sarmiento

et al. 2016

The Journal of Maternal-Fetal & Neonatal Medicine

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Simultaneous detection of IgG, IgM, IgA complexes and C3d attached to erythrocytes by flow cytometry

Abstract: Flow cytometry is a consistent method for identifying the presence of IgG, IgM, IgA immune complexes and C3d attached to erythrocytes and can be helpful for understanding the mechanisms involved in AIHA pathogenesis.

Cited by 7 publications

References 16 publications

Complement deposition in autoimmune hemolytic anemia is a footprint for difficult-to-detect IgM autoantibodies

Complement deposition in autoimmune hemolytic anemia is a footprint for difficult-to-detect IgM autoantibodies

Western immunoblotting as a new tool for investigating direct antiglobulin test–negative autoimmune hemolytic anemias

Delivery route determines the presence of immune complexes on umbilical cord erythrocytes

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