J. Damoiseaux

Impaired thrombin generation and fibrin clot formation in patients with dilutional coagulopathy during major surgery

Patients subjected to haemodilution during surgery are at increased risk of bleeding. We hypothesised that, in the acquired dilutional coagulopathy, insufficient haemostasis is due to either insufficient thrombin generation or insufficient fibrin clot formation. In tissue factor-activated plasmas from patients with coagulation deficiency, we measured time curves of thrombin generation and fibrin clot formation (thromboelastography). Investigated were in study A: 10 patients treated with vitamin K antagonist and five healthy subjects; in study B: 30 patients undergoing cardiopulmonary bypass (CPB) surgery and infused with on average 2,000 ml crystalloids and colloids (no major bleeding); in study C: 58 patients undergoing major general surgery, and transfused with >5,000 ml crystalloids, colloids and red cell concentrates, who experienced major bleeding and were post-transfused with fresh frozen plasma. The treatment with vitamin K antagonist led to a progressive reduction in thrombin generation but not fibrin clot formation. In CPB patients, plasma factor levels post-surgery were 53-60% of normal. This was accompanied by moderate reduction in both haemostatic processes. In plasmas from patients undergoing major surgery, factor levels were 38-41% of normal, and these levels increased after plasma transfusion. Taking preset thresholds for normal thrombin generation and fibrin clot formation, at least one of these processes was low in 88-93% of the patients with (persistent) bleeding, but only in 40-53% of the patients without bleeding. In conclusion, the ability of thrombin generation and fibrin clot formation is independently reduced in acquired dilutional coagulopathy, while minimal levels of both are required for adequate haemostasis.

Evaluation of a new fluorescent-enzyme immuno-assay for diagnosis and follow-up of ANCA-associated vasculitis

In this study we have evaluated a new, fully automated fluorescent-enzyme immuno-assay (FEIA) for detection and quantification of anti-PR3 and anti-MPO ANCA in diagnosis and follow-up of ANCA-associated small vessel vasculitis (AAV). PR3- and MPO-ANCA were determined by FEIA technology in (1) sera of 87 consecutive patients with biopsy-proven, pauci-immune necrotizing crescentic glomerulonephritis (NCGN) and 72 controls; (2) 120 sera (60 patients with Wegener’s granulomatosis and 60 controls) that were previously used in a multicentre comparison of direct and capture ELISAs for PR3-ANCA; (3) in samples preceding relapse in 23 PR3-AAV patients with and 23 matched PR3-AAV patients without relapse for prediction of relapses. PR3- and/or MPO-ANCA detection in pauci-immune NCGN by FEIA revealed an overall sensitivity of 82.8%. The FEIA specificity was 96% and 100% for PR3- and MPO-ANCA, respectively. The overall sensitivity of MPO- and PR3-ANCA could be increased to 88.5% by lowering the cut-off values without affecting the specificity (ROC-curve analysis), which is similar to a multistep ANCA procedure that combines indirect immunofluorescence with direct and capture ELISAs. The sensitivity for Wegener’s granulomatosis (WG) of the PR3-ANCA FEIA (60%) was more comparable to direct ELISAs (64%) than to capture ELISAs (74%). A rise of 100% in ANCA level as measured by FEIA appeared optimal (ROC-curve) for prediction of relapses and such a rise was observed in 26 patients. In 18 of these 26 patients the rise was followed by a relapse (PPV 69%), whereas in 15 of the 20 patients without a rise no relapse was observed (NPV 75%). In conclusion, detection of PR3- and MPO-ANCA by FEIA has excellent performance in terms of diagnosis of AAV patients. Furthermore, detection of rises in PR3-ANCA by FEIA for prediction of relapses gives results comparable to other techniques.

Evaluation of a Novel Line-Blot Immunoassay for the Detection of Antibodies to Extractable Nuclear Antigens

Abstract: We have evaluated the performance of a novel line‐blot immunoassay (LIA; Mikrogen) and compared results with those obtained by CIE (in‐house), ELISA (Pharmacia Diagnostics), and FEIA (Pharmacia Diagnostics). Sera from systemic lupus erythematosus (SLE) patients (n= 123), systemic sclerosis patients (n= 25), and healthy controls (n= 40) were analyzed for the presence of antibodies to RNP, Sm, SSA, SSB, CENP‐B, Scl‐70, and Jo‐1. Reading of LIA results, as compared with a cutoff control, was performed by automatic analysis of the test strips. Because LIA enables recognition of separate subunits of RNP (68, A, and C), Sm (B and D), and SSA (52 and 60), at least two of the RNP antigens and either one of the Sm or SSA antigens should be detected for considering the test RNP, Sm, or SSA‐positive, respectively. LIA had the highest sensitivity in patients with autoimmune connective tissue diseases: 131 specificities (not PO, PCNA, or histones), as compared with ELISA (121), FEIA (119), and CIE (80). However, LIA revealed three positive reactions in healthy controls; other assays were completely negative. LIA is better than CIE, but similar to ELISA and FEIA, in terms of detecting systemic sclerosis‐associated antibodies (CENP‐B and Scl‐70). Furthermore, LIA had the highest sensitivity (17.9%) for the SLE‐specific anti‐Sm antibodies, as compared with ELISA (11.4%), CIE (8.1%), and FEIA (5.7%). Finally, anti‐SSA antibodies were far more prevalent by LIA in the systemic sclerosis samples because of anti‐SSA52 reactivity. The clinical relevance of the latter finding remains to be determined. In conclusion, LIA is suitable for routine evaluation of autoantibodies to extractable nuclear antigens.

Evaluation of the FIDIS vasculitis multiplex immunoassay for diagnosis and follow-up of ANCA-associated vasculitis and Goodpasture's disease

Abstract:  We have evaluated a new‐multiplex immunoassay (FIDIS Vasculitis) for simultaneous detection and quantification of anti‐MPO, ‐PR3, and ‐glomerular basement membrane (GBM) antibodies in diagnosis and follow‐up of ANCA‐associated vasculitides (AAV) and Goodpastures disease. ANCA were determined in sera of (a) 87 consecutive patients with biopsy‐proven pauci‐immune NCGN and 72 controls; (b) 9 patients with Goodpastures disease; and (c) 60 WG patients and 60 controls, previously used in a multicenter comparison of direct and capture ELISA for PR3‐ANCA. Finally, for prediction of relapses, PR3‐ANCA was measured in samples preceding relapse in 23 PR3‐AAV patients and in 23 matched PR3‐AAV patients without relapse. The relative sensitivity of the FIDIS Vasculitis assay was 97.4% for MPO‐ANCA and 92.3% for PR3‐ANCA; specificity was 100% and 97.2%, respectively. Evaluation of the anti‐GBM antibody detection revealed a sensitivity of 100% and a specificity of 99.6%. The sensitivity for WG of the PR3‐ANCA detection (71.6%) approached the performance of capture ELISA (74%), although at the cost of specificity (96.7% versus 100%). For prediction of relapses a rise of 50% in ANCA level by FIDIS Vasculitis appeared optimal (ROC curve) for prediction of relapses. However, as compared to capture ELISA, both positive (63% versus 76%) and negative (68% versus 72%) predictive values were reduced. In conclusion, simultaneous detection of anti‐MPO, ‐PR3, and ‐GBM antibodies in the multiplex FIDIS Vasculitis assay has excellent performance in terms of diagnosis of patients with AAV or Goodpastures disease. However, detection of rises in PR3‐ANCA for prediction of relapses gives less optimal results when compared to capture ELISA.

Type II cryoglobulinemia is not associated with hepatitis C infection: the Dutch experience.

Abstract:  Mixed cryoglobulinemia (MC) are cryoprecipitable immunocomplexes. In type II MC, a combination of polyclonal and monoclonal immunoglobulins is found, whereas in type III a combination of polyclonal immunoglobulins is detected. MC is usually associated with hepatitis C (HCV) infection as has been found in studies that have been performed in countries with a high prevalence of HCV. Because HCV has an extremely low prevalence in the Netherlands (<0.1% of the population), we wondered whether HCV is also associated with MC in our regional referral center. To answer this question, we tested consecutive patients with type II MC for HCV antibodies and for HCV‐mRNA by polymerase chain reaction (PCR). Between January 2000 and June 2005, 22 patients tested positive for type II MC. Seven patients had essential MC, 2 patients had MC due to a lymphoproliferative disease, 10 patients had MC in the context of a systemic autoimmune disease, and 3 patients had MC without a clear diagnosis. HCV antibodies were not detected in any of the 22 patients. Also, all samples tested negative for HCV‐mRNA. During follow‐up none of these patients developed an HCV infection. In summary, the estimated occurrence of HCV in 60–90% of patients with MC is not found in our region where MC is only infrequently associated with HCV. In a substantial proportion of our patients a really “essential MC” is observed. A search for yet unknown etiological factors is clearly needed in these patients, who frequently have severe renal involvement warranting aggressive immunosuppressive therapy.

Prevalence of anti-endothelial cell antibodies in idiopathic pulmonary arterial hypertension

To the Editors: Pulmonary arterial hypertension (PAH) is a rare disease often resulting in right-sided heart failure and premature death 1. PAH is idiopathic (IPAH), heritable, or related to conditions such as connective tissue diseases (CTD) 2. IPAH prognosis remains poor despite improved patient survival with current treatment options. Therefore, elucidating the pathophysiology of IPAH is important for the discovery of novel therapeutic approaches. Inflammation and immune reactivity have been implicated in the pathophysiology of IPAH. In 2005, Tamby et al. 3 described the presence of anti-endothelial cell antibodies (AECA) in IPAH, pointing at the involvement of humoral immunity. AECA are a heterogeneous family of auto-antibodies capable of reacting with different endothelial cell (EC) structures 4. In PAH, pulmonary EC dysfunction is considered a key player in the initiation and progression of the disease 5. Systemic sclerosis (SSc) is a paradigm of autoimmune PAH, as 10–15% of these patients develop PAH 6. Interestingly, in SSc, immunoglobuliln (Ig)G AECA targeting cell-surface antigens have indeed been shown to induce EC dysfunction 7, 8. Thus, IgG AECA-induced endothelial dysfunction may be the initiating event in IPAH. Whereas IgG auto-antibodies are considered pathogenic in various autoimmune diseases, IgM auto-antibodies have been proposed to be protective in these diseases 9. Regarding a pathophysiological role of AECA, it is important to identify whether AECA are reactive with EC-surface antigens. To corroborate and further investigate the role of the humoral immune system in IPAH, we aimed to study the prevalence of IgG, as well as IgM AECA targeting cell-surface EC antigens using a cell-based ELISA with viable human umbilical vein EC (HUVEC). We screened five study cohorts for the presence of AECA. 1) 29 IPAH patients; mean pulmonary artery …

Haptoglobin Phenotype May Alter Endothelial Progenitor Cell Cluster Formation in Cerebral Small Vessel Disease

Cerebral small vessel disease results in silent ischemic lesions (SIL) among which is leukoaraiosis. In this process, endothelial damage is probably involved. Endothelial progenitor cells (EPC), are involved in endothelial repair. By restoring the damaged endothelium, EPC could mitigate SIL and cerebral small vessel disease. Haptoglobin 1-1, one of three phenotypes of haptoglobin, relates to SIL and may therefore attenuate the endothelial repair by EPC. Our aim was to quantify EPC number and function and to assess haptoglobin phenotype and its effect on EPC function in patients with a high prevalence of SIL: lacunar stroke patients. We assessed EPC In 42 lacunar stroke patients and 18 controls by flow cytometry and culture with fetal calf serum, patient and control serum. We determined haptoglobin phenotype and cultured EPC with the three different haptoglobin phenotypes. We found that EPC cluster counts were lower in patients (96.9 clusters/well +/- 83.4 (mean +/- SD)), especially in those with SIL (85.0 +/- 64.3), than in controls (174.4 +/- 112.2). Cluster formation was inhibited by patient serum, especially by SIL patient serum, but not by control serum. Patients with haptoglobin 1-1 had less clusters in culture, and when haptoglobin 1-1 was added to EPC cultures, cluster numbers were lower than with the other haptoglobin phenotypes. We conclude that lacunar stroke patients, especially those with SIL, have impaired EPC cluster formation, which may point at decreased endothelial repair potential. The haptoglobin 1-1 phenotype is likely a causative factor in this impairment.

Cross-Reactivity of IgM and IgG Anticardiolipin Antibodies with Oxidized-Low Density Lipoproteins

Abstract: Anticardiolipin antibodies (aCLAs) and antibodies to oxidized‐low density lipoproteins (oxLDL) are associated with two distinct diseases: the antiphospholipid syndrome and atherosclerosis. Because both diseases may be apparent in patients with systemic lupus erythematosus (SLE), it is important to establish the relationship between these two types of antibodies. In the present study, we examined whether sera containing IgM and/or IgG aCLAs also react with LDL that has been oxidized by conjugation with malondialdehyde (MDA‐LDL) or by incubation with copper ions (Cu‐LDL). Results revealed a clear correlation between IgM aCLAs and IgM anti‐MDA‐LDL antibodies, and a weak correlation between IgG aCLAs and IgG anti‐Cu‐LDL antibodies. Cross‐reactivity between both antibodies seemed to be limited. Because aCLAs are heterogeneous, only a minor subset of these antibodies may cross‐react with oxLDL. Therefore, identification of both antibodies may be relevant for determination of the prognosis of accelerated atherosclerosis in SLE patients.

Functional implications of IgG anti-endothelial cell antibodies in pulmonary arterial hypertension

Abstract The objective of this study was to research the functionality of anti-endothelial cell antibodies (AECA) in pulmonary arterial hypertension (PAH) by assessing the effects of IgG from AECA-positive PAH patients on the induction of adhesion molecules on human umbilical vein endothelial cells (HUVECs) and on the production of pro-inflammatory cytokines and chemokines by HUVECs. To achieve this purified IgG from 28 PAH patients were included. IgG from systemic sclerosis (SSc) (n = 58) and systemic lupus erythematosus (SLE) (n = 16) patients without PAH were included as disease controls. Intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression on HUVECs, incubated with patient IgG, were quantified by flow cytometry. Production of interleukin (IL)-1β, -6, -8, and CC chemokine ligand 2 (CCL2) by HUVECs, incubated with patient IgG, were quantified by multiplex flow cytometry. Our results showed that IgG from AECA-positive PAH, SSc and SLE patients induced significantly higher expression of ICAM-1, VCAM-1, and E-selectin and production of IL-6, -8, and CCL2 compared to IgG from AECA-negative patients and IgG from healthy controls. Like in SLE and SSc, IgG from AECA-positive PAH patients can activate endothelial cells to a pro-adhesive and pro-inflammatory state. Therefore, IgG AECA could play a pathogenic role by inducing inflammatory injury of vascular endothelium which is considered a key player in the initiation and progression of PAH.

Anti‐oxidized low‐density lipoprotein antibodies in myeloperoxidase–positive vasculitis patients preferentially recognize hypochlorite‐modified low density lipoproteins

Many patients surviving vasculitis are prone to accelerated atherosclerosis and often have enhanced levels of antibodies to oxidized low‐density lipoprotein (oxLDL). To measure anti‐oxLDL antibodies, oxidation of LDL is achieved with copper (Cu) or malondialdehyde (MDA). Because, in vivo, LDL may be oxidized with myeloperoxidase (MPO) or its product hypochlorite, we measured anti‐hypochlorite LDL antibodies in patients with vasculitis, haemodialysis patients and healthy controls. A newly developed enzyme‐linked immunosorbent assay (ELISA) was used to detect antibodies to oxLDL as modified by hypochlorite. Results are compared with data obtained by standard LDL oxidation using MDA–LDL or Cu–LDL as substrate. Results were compared between anti‐neutrophil cytoplasmic antibodies (ANCA)‐associated vasculitis (AAV) patients (n = 93), haemodialysis (HD) patients (n = 59) and healthy controls (HC; n = 43). Furthermore, patients with MPO–ANCA‐associated vasculitis (n = 47) were compared with patients with proteinase 3 (PR3)–ANCA associated vasculitis (n = 46). Optimal cut‐off points were determined by receiver operator characteristic (ROC) curve analysis. Anti‐oxLDL antibodies are enhanced in AAV patients (MDA–LDL and hypochlorite–LDL) and in HD patients (hypochlorite–LDL), when compared to HC. Furthermore, patients with MPO–ANCA‐associated vasculitis had higher levels of antibodies to hypochlorite–LDL than patients with PR3–ANCA‐associated vasculitis. Our newly developed assay, in which hypochlorite–LDL is used as substrate, seems a more sensitive assay than traditional assays to measure oxLDL antibodies. Furthermore, our results suggest that enhanced MPO‐mediated LDL oxidation occurs in patients with MPO–ANCA.