Abstract:Background
Both M and N alleles encode antigens on Glycophorin A (GPA), a red blood cell (RBC) surface sialoglycoprotein. Interaction between RBC GPA and leukocyte surface lectins may downregulate their activation. The current study investigates if RBC autoantibodies against GPA, such as auto‐anti‐M/N, prime an activated phenotype in peripheral blood leukocytes.
Methods
Leukocyte activation was assessed in whole blood from patients with auto‐anti‐GPA (anti‐M/N) and compared to those with allo‐anti‐M/N and heal… Show more
“…As shown previously, 7 , 8 whole blood-derived leukocytes are activated upon separation from RBCs. In light of the immunosuppressive properties of RBC transfusions in chronically transfused patients, 11 we evaluated if leukocyte activation could be reversed in vitro by suspending them with aliquots of RBCs either coated or not coated with anti-GPA.…”
Section: Methodssupporting
confidence: 70%
“… 14 , 15 , 16 CD63 expression from isolated monocytes separated from whole blood served as the reference activated control group, and we have previously shown that CD63 is significantly increased in isolated monocytes from buffy coats compared with monocytes in whole blood. 8 CD63 expression in isolated monocytes was suppressed by 26% after incubation with autologous RBCs ( n = 7 ) at a 1000:1 ratio ( P = .0464) ( Figure 4 A). Figure 4.…”
Section: Resultsmentioning
confidence: 97%
“…Samples incubated with RBCs were gated as previously described. 8 For TF-1 experiments, the gating scheme was as follows: (1) FSC-A vs FSC-H to exclude doublets and large aggregates. (2) 7AAD vs FSC-H to exclude dead cells.…”
Section: Methodsmentioning
confidence: 99%
“… 6 The interaction between RBC surface GPA and lectin Siglec-9 maintains neutrophil and monocyte quiescence. 7 , 8 …”
Glycophorin A (GPA), a red blood cell (RBC) surface glycoprotein, can maintain peripheral blood leukocyte quiescence through interaction with a sialic acid-binding Ig-like lectin (Siglec-9). Under inflammatory conditions such as sickle cell disease (SCD), RBCs' GPA undergo structural changes affecting this interaction. Peripheral blood samples from SCD patients pre- and post-RBC transfusion were probed for neutrophil and monocyte activation markers and analyzed by FACS; RBCs were purified and tested by FACS for Siglec-9 binding and GPA expression, and incubated ex vivo with cultured endothelial cells to evaluate their effect on barrier function. Activated leukocytes from healthy subjects (HS) were co-incubated with healthy RBCs (RBCH), with GPA-altered RBCs, or with GPA-overexpressing (OE) cells, and analyzed by FACS. Monocyte CD63 and neutrophil CD66b from SCD patients at baseline were increased 47% and 27%, respectively, as compared to HS (p=0.0017, p=0.0162). Post-transfusion, these markers were suppressed by 22% and 17% (p=0.0084, p=0.0633), respectively. GPA expression on RBCSCD was 38% higher (p=0.0291) with decreased Siglec-9 binding compared to RBCH (0.0266). Monocyte CD63 and neutrophil CD66b was suppressed by 26% and 10%, respectively, following incubation with RBCH, (p=0.0464, p=0.0388) and GPA-OE cells, but not with GPA-altered RBCs. Endothelial barrier dysfunction following LPS challenge was restored fully with exposure to RBCH, but not with RBCSCD from patients in pain crisis, nor with RBCH with altered GPA. In conclusion, RBCSCD, prior to transfusion, do not effectively maintain the quiescence of leukocytes and endothelium, but quiescence is restored through RBC transfusion likely by re-established GPA-Siglec-9 interactions.
“…As shown previously, 7 , 8 whole blood-derived leukocytes are activated upon separation from RBCs. In light of the immunosuppressive properties of RBC transfusions in chronically transfused patients, 11 we evaluated if leukocyte activation could be reversed in vitro by suspending them with aliquots of RBCs either coated or not coated with anti-GPA.…”
Section: Methodssupporting
confidence: 70%
“… 14 , 15 , 16 CD63 expression from isolated monocytes separated from whole blood served as the reference activated control group, and we have previously shown that CD63 is significantly increased in isolated monocytes from buffy coats compared with monocytes in whole blood. 8 CD63 expression in isolated monocytes was suppressed by 26% after incubation with autologous RBCs ( n = 7 ) at a 1000:1 ratio ( P = .0464) ( Figure 4 A). Figure 4.…”
Section: Resultsmentioning
confidence: 97%
“…Samples incubated with RBCs were gated as previously described. 8 For TF-1 experiments, the gating scheme was as follows: (1) FSC-A vs FSC-H to exclude doublets and large aggregates. (2) 7AAD vs FSC-H to exclude dead cells.…”
Section: Methodsmentioning
confidence: 99%
“… 6 The interaction between RBC surface GPA and lectin Siglec-9 maintains neutrophil and monocyte quiescence. 7 , 8 …”
Glycophorin A (GPA), a red blood cell (RBC) surface glycoprotein, can maintain peripheral blood leukocyte quiescence through interaction with a sialic acid-binding Ig-like lectin (Siglec-9). Under inflammatory conditions such as sickle cell disease (SCD), RBCs' GPA undergo structural changes affecting this interaction. Peripheral blood samples from SCD patients pre- and post-RBC transfusion were probed for neutrophil and monocyte activation markers and analyzed by FACS; RBCs were purified and tested by FACS for Siglec-9 binding and GPA expression, and incubated ex vivo with cultured endothelial cells to evaluate their effect on barrier function. Activated leukocytes from healthy subjects (HS) were co-incubated with healthy RBCs (RBCH), with GPA-altered RBCs, or with GPA-overexpressing (OE) cells, and analyzed by FACS. Monocyte CD63 and neutrophil CD66b from SCD patients at baseline were increased 47% and 27%, respectively, as compared to HS (p=0.0017, p=0.0162). Post-transfusion, these markers were suppressed by 22% and 17% (p=0.0084, p=0.0633), respectively. GPA expression on RBCSCD was 38% higher (p=0.0291) with decreased Siglec-9 binding compared to RBCH (0.0266). Monocyte CD63 and neutrophil CD66b was suppressed by 26% and 10%, respectively, following incubation with RBCH, (p=0.0464, p=0.0388) and GPA-OE cells, but not with GPA-altered RBCs. Endothelial barrier dysfunction following LPS challenge was restored fully with exposure to RBCH, but not with RBCSCD from patients in pain crisis, nor with RBCH with altered GPA. In conclusion, RBCSCD, prior to transfusion, do not effectively maintain the quiescence of leukocytes and endothelium, but quiescence is restored through RBC transfusion likely by re-established GPA-Siglec-9 interactions.
“…Characterizing cell populations or immunophenotyping is a prevalent method for disease diagnosis from surface biomarkers using microfluidic arrays for label-free phenotyping [416] and interaction between RBC Glycophorin and leukocyte surface lectins activation [417].Other studies related to drug delivery assess the interaction mapping between RBCs and drug carriers via image analysis [418], capture and release of circulating tumor cells [419], drug-induced erythrocyte deformation [420], antibodies and biotinlabeled RBC effect on posttransfusion survival [421], shear-induced encapsulation in drug delivery [422], and anti-tumor applications incorporating RBCs and nanoparticles [423]. For COVID-19 related research, RBCs, as oxygen carriers, were analyzed for respiratory issues in COVID-19 patients for RBC deformability/morphology effect on tissues oxygen supply [424][425][426][427][428].…”
Section: Antibody Binding and Drug Discovery Applicationsmentioning
The utilizations of microfluidic chips for single RBC (red blood cell) studies have attracted great interests in recent years to filter, trap, analyze, and release single erythrocytes for various applications. Researchers in this field have highlighted the vast potential in developing micro devices for industrial and academia usages, including lab-on-a-chip and organ-on-a-chip systems. This article critically reviews the current state-of-the-art and recent advances of microfluidics for single RBC analyses, including integrated sensors and microfluidic platforms for microscopic/tomographic/spectroscopic single RBC analyses, trapping arrays (including bifurcating channels), dielectrophoretic and agglutination/aggregation studies, as well as clinical implications covering cancer, sepsis, prenatal, and Sickle Cell diseases. Microfluidics based RBC microarrays, sorting/counting and trapping techniques (including acoustic, dielectrophoretic, hydrodynamic, magnetic, and optical techniques) are also reviewed. Lastly, organs on chips, multi-organ chips, and drug discovery involving single RBC are described. The limitations and drawbacks of each technology are addressed and future prospects are discussed.
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