Microparticles for diagnosis of graft-versus-host disease after allogeneic stem transplantation.

BACKGROUND: The differentiation between acute graft-versus-host disease (aGvHD) and infection is still a clinical challenge in patients after allogeneic hematopoietic stem-cell transplantation (HSCT). Definitive diagnosis is based on histologic findings, but a simple blood test for differentiation is missing. METHODS: In a prospective study, we measured the plasma levels of erythrocyte-derived microparticles (EryMP) in 19 recipients during HSCT. Microparticles were isolated by differential centrifugation, double stained for glycophorin A (CD235) and annexin V, and analyzed by flow cytometry. RESULTS: Eight patients developed aGvHD (42%), 15 patients developed infectious complications (79%), and two patients developed microangiopathic hemolytic anemia (11%). The levels of EryMP, as measured before conditioning therapy (535 × 10(6)/L in median), were not affected by total body irradiation, high-dose chemotherapy, or in vivo T-cell depletion. EryMP levels were unaffected in uncomplicated patients during aplasia (522 × 10(6)/L in median; P=0.394) or after engraftment (480 × 10(6)/L in median; P = 0.594) and in patients with infectious complications or sepsis (586 × 10(6)/L in median; P = 0.606). In contrast, in patients who developed aGvHD after HSCT, a 1.7-fold increase in the plasma levels of EryMP was observed (880 ×1 0(6)/L in median; P<0.001 compared with the time before therapy and P = 0.015 compared with patients with infections or sepsis). CONCLUSION: Increased plasma levels of EryMP are present in patients who develop aGvHD but not in patients who develop infection or sepsis after HSCT. Therefore, EryMP are a potential, novel, blood marker that may be helpful in the diagnosis of this common complication after HSCT.

Surveillance of megakaryocytic function by measurement of CD61-exposing microparticles in allogeneic hematopoietic stem cell recipients.

Increasing evidence suggests that circulating microparticles (MP) exposing CD61 originate predominantly from megakaryocytes. Dramatic changes in megakaryocytic homeostasis are regularly observed following allogeneic hematopoietic stem cell transplantation (HSCT) and associated with transplantation-associated complications. We studied MP plasma levels prospectively in healthy subjects (n = 10) and allogeneic HSCT recipients (n = 19) twice weekly from the start of conditioning therapy up to day 30. A total of 224 measurement points were evaluated. MP were isolated, double-stained with annexin V and anti-CD61, and analyzed by flow cytometry. In uncomplicated HSCT, we found a correlation between platelet and CD61-exposing MP count, which resulted in a constant ratio of MP per platelet. The ratio was increased in patients with active hematological malignancies before transplantation and normalized during conditioning therapy. After take, the MP ratio increased, whereas infections and microangiopathic hemolytic anemia did not affect the ratio. In patients with GvHD, a decreased MP ratio was observed depending on the grade of GvHD, possibly indicating megakaryocytic damage. The MP ratio was able to discriminate between toxic, septic, and GvHD-induced hyperbilirubinemia. We first describe CD61+ MP levels during allogeneic HSCT and postulate that the MP ratio might be a useful biomarker for the surveillance of megakaryocytes during HSCT.

Cellular origin of platelet-derived microparticles in vivo.

INTRODUCTION: Microparticles (MP), presumably of platelet origin, are the most abundant microparticles in blood. To which extent such MP may also directly originate from megakaryocytes, however, is unknown. During hematopoietic stem cell transplantation, patients undergo total body irradiation which leads to an irreversible destruction of hematopoiesis. MATERIAL AND METHODS: We studied the levels of "platelet-derived" MP (PMP) in 13 patients before and after total body irradiation with 12 Gy (4 Gy for 3 days, dose rate 4.5 cGy/min). PMP were isolated and double-stained with annexin V and anti-CD61. In 6 patients, we additionally analyzed MP exposing P-selectin or CD63. RESULTS: PMP rapidly declined upon total body irradiation, which was 2.4-fold faster than platelet disappearance. In contrast, the kinetics of MP exposing P-selectin or CD63 was comparable to platelets. CONCLUSIONS: Since CD61-positive MP disappear faster than platelets or MP exposing P-selectin or CD63, our data indicate that MP exposing P-selectin or CD63 are likely to originate from platelets, whereas at least a major fraction of CD61-exposing MP is likely to originate from megakaryocytes in vivo.