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.

Gender-specific and menstrual cycle dependent differences in circulating microparticles.

In comparison to age-matched men, young women are at increased risk to suffer from venous thromboembolism (VTE). Some risk factors of inherited and acquired thrombophilia are known, but approximately 30% of the overall risk remains unexplained. Recently, a role for microparticles (MP) in coagulation has been suggested. We investigated, if gender- and menstrual cycle-specific differences in circulating MP exist. Platelet- and endothelial cell-derived microparticles (PMP, EMP) and subpopulations thereof were evaluated flow-cytometrically in healthy women (n = 27) in different phases of their menstrual cycles (follicular phase: n = 14, luteal phase: n = 13) and in healthy men (n = 18). Additionally, D-dimer levels were determined. Compared to men, women had elevated numbers of annexin V-binding MP (p = 0.007), PMP (CD61; p = 0.013), P-selectin-exposing PMP (p = 0.002) and E-selectin-exposing EMP (p = 0.009). During the luteal phase, women had strongly elevated concentrations of MP, PMP, P-selectin- and CD63-exposing PMP as well as E-selectin-exposing EMP (p = 0.001, p < 0.001, p = 0.004, p = 0.003, and p < 0.001, respectively), and the ratio of P-selectin-exposing PMP/platelet increased more than three-fold as compared to men (p = 0.01). When different phases of the menstrual cycle were analysed, MP (annexin V; p = 0.025), PMP (CD61: p < 0.001; CD63: p = 0.015) and E-Selectin-positive EMP (p = 0.006) were all increased in the luteal phase. Although D-dimer concentrations in women were increased compared to men (p = 0 = 0.006), no menstrual cycle-specific differences were observed. In summary, circulating MP and subpopulations thereof are increased in women when compared to men, and this increase seems to be modulated by the menstrual cycle. Therefore, circulating MP may be an additional risk factor contributing to the hitherto unexplained procoagulatory state of young women.

Endothelial cell-derived microparticles in allogeneic hematopoietic stem cell recipients.

BACKGROUND: Alterations of microparticles derived from different cell types are described in a number of diseases associated with inflammation and hemostatic disorders. METHODS: In this prospective study, we firstly analyzed endothelial cell derived microparticles (EMP) in 19 hematopoietic stem cell recipients. Cultured human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor-alpha (TNF-alpha) served as positive controls. EMP were analyzed by fluorescent activated cell sorting (FACS), detecting the particels via expression of CD62 (E-selectin) and anionic phospholipids binding to annexin V. RESULTS: EMP were not significantly influenced by conditioning regimens with non-myeloablative chemotherapy and 4 Gy total body irradiation (TBI) or by myeloablative regimens containing 12 Gy TBI. During acute graft versus host disease (aGVHD), significantly higher levels of EMP were detected than in patients without aGVHD (18.5/microl s=10.1 vs. 14.6/microl SD = 11.5; P = 0.004) while infectious complications did not alter EMP levels significantly. Immunosuppressive therapy with corticosteroids tendentially elevated EMP levels. HUVEC treated with TNF-alpha 1 ng/ml, 10 ng/ml and 100 ng/ml released significantly more EMP than unstimulated cultures (30.0/microl ss = 13.6 vs. 126.8/microl SD = 66.9, P = 0.032 / vs. 683.3/microl SD = 349.9; P = 0.03 / vs. 489.3 s = 184.4; P = 0.013). CONCLUSIONS: Elevation of EMP during aGVHD might express severe endothelial cell injury within this complication after hematopoietic stem cell transplantation and might serve as a diagnostic test for early differentiation of aGVHD from other transplanted related complications.

Evaluation of C-reactive protein, interleukin-6, and procalcitonin levels in allogeneic hematopoietic stem cell recipients.

BACKGROUND: Prompt detection of transplant-related complications (TRC) as infections, acute graft-versus-host disease (aGVHD), microangiopathic hemolytic anemia, or veno-occlusive disease following allogeneic hematopoietic stem cell transplantation (HSCT) is essential. PATIENTS AND METHODS: We conducted a prospective trial on clinical significance of C-reactive protein (CRP), interleukin-6 (IL-6) and procalcitonin (PCT) serum levels in TRC. A total of 350 stem cell recipients were admitted. CRP, IL-6 and PCT were analyzed prior to conditioning and weekly until 8 wk after HSCT. TRC were recorded weekly throughout the study. RESULTS: CRP (4.4 mg/dL vs. 12.8 mg/dL; P < 0.001), IL-6 (93 ng/mL vs. 1.138 ng/mL; P < 0.001) and PCT (0.8 ng/dL vs. 5.7 ng/dL; P < 0.001) were increased in infectious complications. Only PCT differentiated between infection and other TRC. Exclusive aGVHD did not increase CRP (4.4 mg/dL vs. 5.7 mg/dL; n.s.), IL-6 (93 ng/mL vs. 153 ng/mL; n.s.) and PCT (0.8 ng/dL vs. 0.8 ng/dL; n.s.). CRP (6.1 mg/dL vs. 3.1 mg/dL; P < 0.001) and IL-6 (295 ng/mL vs. 122 ng/mL; P = 0.001) were decreased during steroid therapy, but not PCT (2.3 ng/dL vs. 2.0 ng/dL; n.s.). CONCLUSION: Our study confirmed CRP, IL-6 and PCT serum levels as helpful markers for TRC. PCT can differentiate infection from GVHD despite steroid therapy. Further trials are needed focusing on the identification of patients who benefit from early risk stratification.

Diagnosis of hepatic veno-occlusive disease by plasminogen activator inhibitor-1 plasma antigen levels: a prospective analysis in 350 allogeneic hematopoietic stem cell recipients.

BACKGROUND: Veno-occlusive disease (VOD) is one of the most serious complications following allogeneic hematopoietic stem cell transplantation (HSCT) and is associated with a high mortality. We conducted a large trial in order to investigate the value of plasminogen activator inhibitor-1 (PAI-1) plasma antigen levels in VOD patients as PAI-1 has been described as a possible diagnostic marker of VOD. METHODS: In all, 350 stem cell recipients were included in our study. PAI-1 levels were analyzed prior to conditioning therapy and then weekly until eight weeks after HSCT. Transplantation-related complications (TRC) including VOD, microangiopathic hemolytic anemia (MAHA), and graft-versus-host disease (GVHD) were recorded weekly throughout the study. RESULTS: Maximum PAI-1 antigen levels were increased in all patients with VOD (n=15; mean 248 ng/ml; 95% CI 183-314 ng/ml). Maximum PAI-1 levels above 120 ng/ml showed a sensitivity of 100% and a specificity of 30.6% for VOD after HSCT. CONCLUSION: Our study underlines that maximum PAI-1 plasma antigen levels not exceeding 120 ng/ml have a strong negative predictive value in the diagnosis of VOD and thus represent a helpful non-invasive tool for exclusion of VOD after HSCT.

Peripheral dendritic cell chimerism in allogeneic hematopoietic stem cell recipients.

BACKGROUND: Relapse and graft-versus-host disease (GVHD) represent major causes of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). Although leukocyte and T-cell chimerism analyses are performed routinely suggesting a predictive value on the patients outcome, little is known about chimerism of dendritic cells (DC) representing strong initiators of immune responses. METHODS: In this prospective study, peripheral DC1 (CD11c+) and DC2 (CD123+) chimerism was determined in hematopoetic stem cell recipients. DCs were isolated from peripheral blood by fluorescence activated cell sorting. Chimerism analyses were performed by fluorescent in situ hybridization or by polymerase chain reaction-based typing of short tandem repeats. RESULTS: At time of engraftment, DC chimerism analyses showed complete chimerism in 76.3% (DC1)/79.5% (DC2), mixed chimerism (MC) in 21.0% (DC1)/17.9% (DC2) and no chimerism in 2.7% (DC1)/2.6% (DC2) of the patients. Peripheral DC chimerism had no significant effect on relapse-free or overall survival. Although acute GVHD was observed more often in patients with MC for DC1/DC2 and chronic GVHD occurred more often in patients with MC for DC2, there was no statistically significant correlation. CONCLUSIONS: Although DCs as antigen presenting cells are supposed to have an impact on the induction of GVHD, there was no significant correlation between incidence of GVHD and DC chimerism after HSCT.

Transforming growth factor beta-1 released from platelets contributes to hypercoagulability in veno-occlusive disease following hematopoetic stem cell transplantation.

BACKGROUND: Hepatic veno-occlusive disease (VOD) is one of the most disastrous complications after allogeneic hematopoetic stem cell transplantation (HSCT). Thrombocytopenia with refractoriness to platelet transfusions suggests an increased platelet consumption in these patients. Interactions between platelets and endothelial cells might contribute to the hypercoagulable state at the sinusoidal endothelium as a central mechanism in the pathogenesis of VOD. STUDY DESIGN: The influence of activated platelets on cultured human endothelial cells was investigated in vitro. We focused on the release of plasminogen activator inhibitor-1 (PAI-1) from endothelial cells which has earlier been found to be significantly elevated in plasma of VOD patients. Endothelial cells isolated from human umbilical cords (HUVEC) were incubated with activated platelets. The release of PAI-1 in the presence or absence of specific antibodies was determined by ELISA technique. Tissue factor (TF) expression on endothelial cells was observed by flowcytometric analysis. RESULTS: HUVEC incubated with activated platelets were found to release significantly more PAI-1 compared to untreated cultures. The endothelial PAI-1-secretion after incubation of HUVEC with activated platelets was completely inhibited by an IgG monoclonal antibody against human transforming growth factor beta-1 (TGF beta-1). In contrast, PAI-1 production was not suppressed after inhibition of HUVEC-platelet-interaction by an IgG monoclonal antibody against CD154 (CD40L) expressed on the surface of activated platelets. An increased release of PAI-1 and an increased expression of tissue factor (TF) on the endothelial cell surface were observed after stimulation with TGF beta-1. CONCLUSION: TGF beta-1 released from activated platelets contributes to the hemostatic imbalance at the sinusoidal endothelium in patients with hepatic VOD by increase of endothelial cell PAI-1 production and TF expression. As a potent profibrotic cytokine, TGF beta-1 might further be involved in phlebosclerosis and sinusoidal fibrosis occurring in VOD.