Elevated Complement (C) and IgG Bound Microparticles (MP) in Immune Thrombocytopenic Purpura (ITP) and Hemolytic Anemia (HA).
Authors: Jy Wenche , Horstman Lawrence L , Lin Andrew , Bidot Carlos , Ahn Yeon-Soong 
Published: October 2019

Abstract
Abstract 1313
Poster Board I-337

Background
Cell derived microparticles (MP) are shed during cell activation and apotosis and MP profiles reflect the status of cell disturbances in various forms of pancytopenias. It was demonstrated that when antibodies fix complement (C) on the membrane, red cells evade C mediated lysis by shedding MP with bound C. We analyzed C and IgG bound to MP to gain insight on the underlying mechanisms of cell destruction. We measured C1q fragment and IgG on cell-derived (MP) in plasmas of patients with ITP, hemolytic anemias (HA) and thrombosis (TBS).

Methods
(1) Patient population. Consenting patients consisted of 18 TBS, 14 ITP and 6 HA (5 AIHA and 1 TTP), as well as 20 normal controls (NC). (2) Flow cytometry. MP were centrifuged from 1 mL fresh (not frozen) platelet-poor plasma (PPP), washed twice with saline, resuspended in 100 μL, then incubated with fluorescent mAb to C1q and IgG, then analyzed by flow cytometry. In addition, MP in the PPP were analyzed for MP from RBC (RMP) using marker glycophorin A, and MP from platelets (PMP) by CD42b. Values were considered elevated if >2SD above the mean of NC. Control values were (mean ±SD per μL): C1q+ MP = 536 ±151; IgG+ MP = 5,542 ±2,081; RMP = 823 ±246; PMP = 7,520 ±2,084.

Results
We observed significant elevation of C1q+ and IgG+ MP in patients with ITP (2-3 fold) and hemolytic anemias (6-10 fold) but not in those with thrombosis. These findings indicate that complement mediated cell destruction or disturbance in these disorders is frequent. (1). The ITP group consisted of 2 subgroups, one of which had elevated C1q+ and IgG+ MP, the other not, and these subgroups also differed in RMP. Specifically, six ITP with high C1q+ MP also had high RMP (1,899 ±682 /μL) and PMP (16,602 ±4,216 /μL) while those with normal C1q+ MP had normal RMP (504 ±186 /μL) and PMP (3,472 ±1196 /μL). This suggests that platelet destruction in ITP can proceed via C in some but not all cases, probably depending on the autoantibodies. We have previously reported high RMP in ITP. These findings suggest subclinical C mediated hemolysis in a subset of ITP. (2) In HA patients, all 6 had elevated C1q+ MP (3,934 ±1,419 /μL, p<0.001) as compared to normal controls (536 ±151 /μL). The mean in HA was nearly 6-fold greater than NC. The HA group also had higher IgG+ MP, with mean counts about 10-fold higher than NC (61,531 ±20,733 vs. 5,542 ±2,081 /μL, p<0.001). Furthermore, the HA patients also had elevated RMP (2,191 ±635 /μL, p<0.01). This suggests that C-mediated destruction of RBC is a major mechanism in HA. (3) Linear regression analysis showed that C1q+ MP is well correlated with IgG+ MP (R = 0.84, p<0.0001) and RMP (R = 0.79, p<0.001). (4) The TBS group did not show higher levels of any of the measures assayed.

It is widely believed that phagocytosis is the mechanism of cell destruction in ITP and AIHA. Our findings support the concept that complement (C) -mediated platelet or red cell destruction play an important role and is common in these disorders. Assay of C and IgG on MP can provide new insight to underlying mechanisms of immune mediated platelet and red cell destruction.

Disclosures
No relevant conflicts of interest to declare.