"Macro transcobalamin causing raised vitamin B12: Case-based laboratory investigation".

Determination of plasma vitamin B12 (B12) is a frequently requested laboratory analysis, mainly employed to establish B12 deficiency. However, an increased level of B12 is a common unexpected finding that may be related to an increased concentration of one of the B12 binding proteins, haptocorrin or transcobalamin. This paper describes the extensive laboratory evaluation of a patient with an elevated level of plasma B12 with various well-established assays. Initial studies suggested the presence of a macromolecule consisting of haptocorrin bound B12. Specific determinations of the B12-binding proteins revealed normal amounts of haptocorrin but a markedly increase in both total and B12 saturated transcobalamin (holo-TC). The results are in accord with the presence of macro-transcobalamin. These experiments reveal that determination of the nature of the B12-macromolecules is troublesome due to differences in assays applied to measure these proteins. In addition, this publication creates awareness of macro-holo-TC as a cause of an unexplained increased B12 level.

INR vs. thrombin generation assays for guiding VKA reversal: a retrospective comparison.

BACKGROUND: Prothrombin complex concentrate (PCC) is used to reverse vitamin K antagonist (VKA)-induced anticoagulation. Prothrombin time-derived international normalized ratio (INR) measurements are widely used in determining the required PCC dose, but this approach requires reappraisal. The aim of the present study was to determine the added value of the thrombin generation assay (TGA) compared with the INR in guidance of VKA reversal by PCC. METHODS: In an open, observational study, INR and TGA measurements were carried out on plasma samples from phenprocoumon-treated patients receiving VKA reversal. Following both analytical methods, PCC dosing correlates were calculated and compared retrospectively. Alternatively, in vitro PCC spiking experiments were performed. RESULTS: As expected, an exponential relationship between PCC dose and INR was found. For the TGA parameters peak thrombin and endogenous thrombin potential (ETP), however, this relationship was found to be linear throughout the full therapeutic range. Additional computational analysis showed a positive correlation (r²=0.7) between the initial INR and PCC dose required for a target INR of 2.1, which was completely lost at a lower target INR. In contrast, a positive correlation (r²=0.8) between initial ETP as well as peak height and PCC dose required to obtain parameter normalization was found. These correlates appeared useful for calculating PCC dose. CONCLUSIONS: Our results support the current debate questioning the rationale for the use of the INR in the management of anticoagulation by VKA. Compared with INR, TGA-based calculations may enable a more accurate PCC dosing regimen for patients requiring VKA reversal.

Comparison of the direct antiglobulin test and the eluate technique for diagnosing haemolytic disease of the newborn.

OBJECTIVE: The direct antiglobulin test (DAT) is an important tool for identification of haemolytic disease of the newborn (HDN) caused by erythrocyte immunization. Although this test has been used for decades, accurate insights into its diagnostic properties and optimal use in the diagnosis of HDN are limited. We aimed to gain more insight into the diagnostic properties of the DAT for HDN by comparing it with erythrocyte eluate screening. DESIGN AND METHODS: DAT and erythrocyte eluate screening was performed in umbilical cord blood of neonates obtained from 317 consecutive deliveries. Clinical jaundice was scored 4-6 days after delivery for the determination of HDN. RESULTS: In 21 neonates a positive DAT and in 61 neonates a positive eluate screening was found, while only 4 cases of HDN were observed. For the overall population the positive predictive value (PPV) and specificity of the DAT for HDN were 10% and 93% respectively and in the population of neonates with abnormal post-partum jaundice population the PPV and specificity were both 100%. The DAT missed two cases of HDN. These missed cases were, however, positive in the erythrocyte eluate screening. CONCLUSION: The detection of clinically irrelevant ABO immunization limits the specificity of the DAT and eluate for HDN in ABO-incompatible pregnancies. For optimal use, the DAT should be requested only in cases of jaundice and be interpreted in the context of ABO-incompatibility. Finally, a negative DAT does not rule out HDN. When clinical suspicion is high, an eluate should be added following a negative DAT.

[Thalassaemia diagnostics]. / De diagnostiek van thalassemieën.

The thalassaemias are characterised by quantitative aberrations in the production of the globin chains that make up haemoglobin, and are a subgroup of the haemoglobinopathies. In this LabQuiz we show how thalassaemia carrier status can be indicated in the results of regular laboratory tests, and discuss the laboratory diagnostics that can confirm or rule out thalassaemia. In these two cases we will present a man of Moroccan descent, and two brothers of Filipino descent, all with anaemia and microcytosis. We show it is possible to differentiate between iron-deficiency anaemia and thalassaemia carrier status on the basis of a complete blood count and measurement of ferritin levels, and which laboratory diagnostics can be subsequently performed in order to confirm a suspicion of thalassaemia. The background section discusses the properties and pitfalls of routine laboratory diagnostics for the thalassaemias, and thalassaemia diagnostics in the Dutch newborn screening programme.

[The diagnostic value of NT-proBNP in heart failure: in depth analysis]. / De diagnostische waarde van NT-proBNP bij hartfalen.

A 77-year-old man with dyspnoea was suspected to have a decompensatio cordis by the general practitioner. A diuretic was prescribed. Additional radiological and laboratory investigation (e.g. natriuretic peptides and D-dimers) showed pulmonary embolism instead of heart failure. A second patient, a woman aged 79 years, with a history of leukaemic mantle cell lymphoma, was treated with poly-chemotherapy (R-CHOP), after which remission was achieved. Four years later the lymphoma recurred and R-CHOP treatment was started. However this was without success, after which R-CHOP treatment was repeated. Subsequently the patient developed dyspnoea and pneumonia. Following additional radiological and laboratory investigation (e.g. natriuretic peptides) the patient was finally diagnosed with doxorubicin-induced heart failure. Based upon these case studies, the role of brain-natriuretic peptides in the differential diagnostic work-up of dyspnoea is highlighted. Test performance, correlation with disease, monitoring, prognostics, differential diagnostic power, reference values and pitfalls of brain natriuretic peptides are discussed.

Introduction of a new cobalamin (vitamin B12) assay: lessons from a flawed validation study.

BACKGROUND: Plasma vitamin B12 [cobalamin (Cbl)] concentrations are usually measured as a screening marker for vitamin B12 deficiency. Siemens Healthcare Diagnostics has introduced Cbl assays for various platforms, i.e., the immulite (IML) 2000 and 2500. In our laboratories, regular validation studies for the IML 2500 were conducted and showed acceptable quality specifications. After the introduction of the IML 2500 Cbl assay, clinicians in the department of internal medicine reported an increased frequency of patients with Cbl-concentrations less than 148 pmol/L. METHODS: In order to investigate this claim from the clinicians, we retrospectively analyzed the internal and external quality control (QC) of the Cbl assay. In addition, the monthly patient means for the Cbl assay were analyzed both before and after the introduction of the new Cbl assay. RESULTS: No abnormalities were found in the internal and external QCs. However, the monthly patient means for the Cbl assay showed a statistically significant decrease in cobalamin concentrations. Siemens acknowledged the problems and formulated a new Cbl assay, which was subsequently validated in our laboratories and showed equivocal Cbl results when compared to the IML 2000 Cbl assay. CONCLUSIONS: We report a flawed validation study conducted by the manufacturer that resulted in an undetected analytical problem in the IML 2500 Cbl assay, its subsequent introduction on the market, the final recognition of the poor performance of the assay by our clinicians, and the eventual resolution by the manufacturer. Hence, it emphasizes the utmost importance for thorough comparison between assays over the entire measurement range, even when both assays are produced by the same manufacturer.

Standardised assessment of membrane proteinase 3 expression. Analysis in ANCA-associated vasculitis and controls.

OBJECTIVES: Increased numbers of neutrophils expressing proteinase 3 on their membrane (mPR3) have been reported in anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) and are suggested to be involved in AAV immunopathogenesis. In most studies, neutrophils were analysed for mPR3 expression without priming with TNFalpha, suggesting that mPR3 expression on neutrophils is dependent on other priming events, such as isolation procedures . These priming events can be variable. Therefore, we analysed mPR3 expression on neutrophils before and after priming with TNFalpha to assess whether standardised assessment of mPR3 expression requires priming. Using neutrophils before and after priming with TNFalpha, we assessed percentages of mPR3(+) neutrophils in patients with AAV and in disease and healthy controls. METHODS: Neutrophils from patients with PR3-AAV and MPO-AAV, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), and from healthy controls were analysed before and after priming with TNFalpha for mPR3 expression. RESULTS: 42% of all individuals analysed showed minimal expression for mPR3 on all neutrophils before priming with TNFalpha, whereas after priming a clear mPR3(+) subset was observed next to mPR3(-) neutrophils, corresponding to bimodal mPR3 expression. In patients with PR3-AAV or MPO-AAV, the percentage of mPR3(+) neutrophils after priming with TNFalpha was significantly increased (p<0.01 and p<0.05, respectively) compared with healthy controls. Percentages of mPR3(+) PMN were also increased in patients with SLE (p<0.01) but not in RA. CONCLUSION: Standardised assessment of proteinase 3 on the membrane of neutrophils requires priming with TNFalpha. Percentages of mPR3(+) PMN are increased in AAV and SLE, but not in RA.

Abundance of the long pentraxin PTX3 at sites of leukocytoclastic lesions in patients with small-vessel vasculitis.

OBJECTIVE: The prototypical tissue pentraxin PTX3 inhibits phagocytosis of late apoptotic polymorphonuclear leukocytes (PMNs) by macrophages. Levels of PTX3 parallel disease activity in small-vessel vasculitis. Small-vessel vasculitis is often characterized by leukocytoclasia, a phenomenon of accumulation of nuclear remnants from unscavenged PMNs in or near the vessel wall. We therefore hypothesized that PTX3 accumulates at sites of leukocytoclastic vasculitis and, as such, is a key factor for the induction of leukocytoclasis. METHODS: We examined skin biopsy samples from 13 patients with small-vessel vasculitis and from 4 healthy and 3 inflammatory skin disease controls. Biopsy tissues, characterized histopathologically as leukocytoclastic vasculitis, were studied for the presence of PTX3 using rabbit anti-PTX3 polyclonal antibodies. Sections were scored morphometrically for leukocytoclastic infiltrates in conjunction with PTX3 staining. Morphometric scores were expressed as percentages of staining of the total tissue area. RESULTS: Biopsy specimens from patients with leukocytoclastic vasculitis revealed an abundant up-regulation of PTX3 at sites of leukocytoclastic infiltrates. Significantly more PTX3 was found in tissues from the 13 patients with vasculitis (mean +/- SEM 48.9 +/- 6.1%) than in tissues from the 7 controls (4.5 +/- 2.7%) (P = 0.0003). PTX3 was localized around vessels, as well as spread diffusely throughout the tissue. CONCLUSION: PTX3 is abundantly present at sites of leukocytoclastic infiltrates in patients with small-vessel vasculitis, but not in controls. Since PTX3 inhibits phagocytosis of late apoptotic PMNs by macrophages and is strongly up-regulated at sites of leukocytoclastic infiltration, PTX3 is a candidate factor in the phenomenon of leukocytoclasia in small-vessel vasculitis.

Activation, apoptosis, and clearance of neutrophils in Wegener's granulomatosis.

Wegener's granulomatosis (WG) is strongly associated with the presence of antineutrophil cytoplasmic autoantibodies (ANCAs). Within WG these ANCAs are usually (80-90%) directed against the azurophilic enzyme proteinase 3, the so called PR3-ANCA. A pathophysiological role for these autoantibodies, supported by numerous in vitro and in vivo studies, is specifically based on their capacity to bind and activate neutrophils and potentially may damage vessels. In this review, the pathogenic potential of different developmental stages of the neutrophil in the pathogenesis of WG is discussed. After release from the bone marrow into the circulation, neutrophils can be primed by TNFalpha and become attached to locally activated endothelium. Once attached to the endothelium, ANCAs can fully activate these primed neutrophils. In this activation process, the degree of activation after stimulation with PR3-ANCAs associates with the level of PR3 expression on the membrane of the neutrophil. Following activation, infiltrated neutrophils become apoptotic with further membrane expression of PR3. In WG patients, clearance of apoptotic neutrophils can be disturbed due to the opsonization of PR3-expressing apoptotic neutrophils with PR3-ANCAs, thereby perpetuating inflammation by the release of proinflammatory cytokines during clearance; or it may favor autoimmunity by PR3 presentation in an inflammatory environment. Furthermore, the presence of ANCAs and the release of the vessel-related pentraxin PTX3 may lead to the persistence of late apoptotic neutrophils in tissues, thereby inducing leukocytoclastic lesions that are characteristic in patients with WG. All together, alive neutrophils as well as apoptotic neutrophils play a key role in different inflammatory phenomena seen in patients suffering from WG.

Human anti-neutrophil cytoplasm autoantibodies to proteinase 3 (PR3-ANCA) bind to neutrophils.

BACKGROUND: Recently, the in vivo pathogenic role of anti-neutrophil cytoplasm autoantibodies (ANCA) in ANCA-associated vasculitis has been challenged by Abdel-Salam et al. In their report, they observed that ANCA directed against proteinase 3 (PR3) cannot bind to their target autoantigen PR3 on circulating neutrophils (PMN). Here we present evidence that human PR3-ANCA do specifically bind to PMN that express PR3 on their membrane. METHODS: PMN were isolated from donors showing bimodal membrane PR3 expression on their PMN (N= 3). TNFalpha-primed PMN or PMA-stimulated PMN were incubated with serum or plasma from PR3-ANCA-positive patients with Wegener's granulomatosis (WG) (N= 8) or healthy controls (N= 8). Binding of IgG in serum or plasma samples to PMN was assessed by indirect immunofluorescence. RESULTS: Binding of IgG in undiluted plasma or serum from PR3-ANCA-positive WG-patients to PMN was significantly increased compared to plasma or serum from healthy controls. Dilution of plasma and serum showed concentration-dependent binding of IgG. Double staining for PR3 and IgG demonstrated that IgG in plasma or serum from PR3-ANCA-positive patients only bound to those PMN that expressed PR3, and not to PMN that lacked PR3 expression on their membrane. CONCLUSION: PR3-ANCA in undiluted serum or plasma from PR3-ANCA-positive WG patients bind to TNFalpha- primed and PMA-stimulated PMN that express PR3 on their membrane. Therefore, the hypothesis that PR3-ANCA can bind and activate primed PMN is still the most attractive explanation for the contribution of PR3-ANCA to the pathogenesis of Wegener's granulomatosis.

The prototypic tissue pentraxin PTX3, in contrast to the short pentraxin serum amyloid P, inhibits phagocytosis of late apoptotic neutrophils by macrophages.

OBJECTIVE: Phagocytosis of apoptotic cells can be facilitated by complement components and short pentraxins, such as serum amyloid P (SAP). In contrast, the long pentraxin PTX3 was shown to inhibit phagocytosis of apoptotic Jurkat cells by dendritic cells and to bind late apoptotic polymorphonuclear leukocytes (PMNs). Recently, levels of the pentraxin PTX3 were shown to parallel disease activity in small-vessel vasculitis, which is often characterized by leukocytoclasia, a persistence of leukocyte remnants in the vessel wall. We undertook this study to test our hypothesis that PTX3 inhibits phagocytosis of late apoptotic PMNs by macrophages, thereby leading to their accumulation in the vessel wall. METHODS: Macrophages were allowed to phagocytose late apoptotic or secondary necrotic PMNs that were incubated with or without PTX3 for 30 minutes prior to phagocytosis. Phagocytosis was allowed to occur in the presence of 30% normal human serum with or without SAP and with or without depletion of complement. To discriminate between an inhibitory effect of PTX3 on binding and the internalization of apoptotic PMNs into macrophages, internalization was blocked by cytochalasin B. RESULTS: SAP and complement were both necessary for effective in vitro phagocytosis. In contrast, PTX3 inhibited phagocytosis in a dose-dependent manner, from 11% inhibition at 6.25 microg/ml to almost complete inhibition at 100 microg/ml. Furthermore, PTX3 partly affected binding of apoptotic PMNs to macrophages. CONCLUSION: PTX3, in contrast to SAP and complement, inhibits phagocytosis of late apoptotic PMNs by monocyte-derived macrophages in a dose-dependent manner. Therefore, PTX3 can play a role in the development of leukocytoclasia by affecting the clearance of apoptotic PMNs, thereby inducing their accumulation in the vessel wall.

Constitutive membrane expression of proteinase 3 (PR3) and neutrophil activation by anti-PR3 antibodies.

Antineutrophil cytoplasm autoantibodies with specificity for proteinase 3 (PR3) are thought to play a major role in the pathogenesis of Wegener's granulomatosis (WG), presumably by their potential to activate neutrophils. In patients with WG, high expression of PR3 on the surface of nonprimed neutrophils is associated with an increased incidence and rate of relapse. In this study, we analyzed the functional significance of constitutive PR3 expression for neutrophil activation as induced by anti-PR3 antibody. Therefore, primed and nonprimed neutrophils were stimulated with the monoclonal anti-PR3 antibody PR3G-3. Activation was measured as actin polymerization by the phalloidin assay as an early, detectable activation event and oxidative burst by the dihydrorhodamine assay, as a late, detectable activation event. In contrast to the oxidative burst, we found that anti-PR3 antibody-induced actin polymerization could be triggered in neutrophils without priming with tumor necrosis factor alpha (TNF-alpha). In addition, a correlation was found between the level of PR3 expression on the surface of these nonprimed neutrophils and the degree of actin polymerization. However, after priming with TNF-alpha, no correlation was found between membrane expression of PR3 and the level of actin polymerization or respiratory burst as induced by anti-PR3 antibody. These data suggest that the presence of PR3 on the surface of nonprimed neutrophils has consequences for their susceptibility to the initial activation step by anti-PR3 antibodies. These data may be relevant in view of the observed relation between membrane expression of PR3 on nonprimed neutrophils of patients with WG and their susceptibility for relapses.