Cerebrospinal fluid analyses for the diagnosis of subarachnoid haemorrhage and experience from a Swedish study. What method is preferable when diagnosing a subarachnoid haemorrhage?

Subarachnoid haemorrhage (SAH) has a high mortality and morbidity rate. Early SAH diagnosis allows the early treatment of a ruptured cerebral aneurysm, which improves the prognosis. Diagnostic cerebrospinal fluid (CSF) analyses may be performed after a negative computed tomography scan, but the precise analytical methods to be used have been debated. Here, we summarize the scientific evidence for different CSF methods for SAH diagnosis and describe their implementation in different countries. The principle literature search was conducted using PubMed and Scopus with the search items "cerebrospinal fluid", "subarachnoid haemorrhage", and "diagnosis". CSF analyses for SAH include visual examination, red blood cell counts, spectrophotometry for oxyhaemoglobin or bilirubin determination, CSF cytology, and ferritin measurement. The methods vary in availability and performance. There is a consensus that spectrophotometry has the highest diagnostic performance, but both oxyhaemoglobin and bilirubin determinations are susceptible to important confounding factors. Visual inspection of CSF for xanthochromia is still frequently used for diagnosis of SAH, but it is advised against because spectrophotometry has a superior diagnostic accuracy. A positive finding of CSF bilirubin is a strong indicator of an intracranial bleeding, whereas a positive finding of CSF oxyhaemoglobin may indicate an intracranial bleeding or a traumatic tap. Where spectrophotometry is not available, the combination of CSF cytology for erythrophages or siderophages and ferritin is a promising alternative.

Automated Analysis of Cerebrospinal Fluid Cells Using Commercially Available Blood Cell Analysis Devices-A Critical Appraisal.

The analysis of cells in the cerebrospinal fluid (CSF) is a routine procedure that is usually performed manually using the Fuchs-Rosenthal chamber and cell microscopy for cell counting and differentiation. In order to reduce the requirement for manual assessment, automated analyses by devices mainly used for blood cell analysis have been also used for CSF samples. Here, we summarize the current state of investigations using these automated devices and critically review their limitations. Despite technical improvements, the lower limit for reliable leukocyte counts in the CSF is still at approximately 20 cells/µL, to be validated depending on the device. Since the critical range for clinical decisions is in the range of 5-30 cells/µL this implies that cell numbers < 30/µL require a manual confirmation. Moreover, the lower limit of reliable erythrocyte detection by automated devices is at approximately 1000/µL. However, even low erythrocyte numbers may be of clinical importance. In contrast, heavily hemorrhagic samples from neurosurgery may be counted automatically at an acceptable precision more quickly. Finally, cell differentiation by automated devices provides only a rough orientation for lymphocytes, granulocytes and monocytes. Other diagnostically important cell types such as tumor cells, siderophages, blasts and others are not reliably detected. Thus, although the automation may give a gross estimate sufficient for the emergency room situation, each CSF requires a manual microscopy for cytological evaluation for the final report. In conclusion, although automated analysis of CSF cells may provide a first orientation of the cell profile in an individual sample, an additional manual cell count and a microscopic cytology are still required and represent the gold standard.

CSF and serum levels of soluble fractalkine (CX3CL1) in inflammatory diseases of the nervous system.

The new CX(3)C-chemokine fractalkine (CX(3)CL1) was measured by Western blot in the cerebrospinal fluid (CSF) and serum of patients with inflammatory diseases of the peripheral and central nervous system (Bell's palsy, BP; Guillain-Barré Syndrome, GBS; multiple sclerosis, MS; viral meningitis, VM; bacterial meningitis, BM) and patients with noninflammatory neurological diseases (controls). In controls, fractalkine was detectable at low concentrations in the CSF and, at much higher levels, in serum. In all inflammatory neurological diseases under study, CSF fractalkine levels were significantly (p<0.01) increased vs. controls (BM>>GBS>VM>MS>BP>controls). In serum, fractalkine levels were significantly increased only in MS patients. The fractalkine CSF/serum ratios (a measure of the chemotactic gradient) were significantly elevated in BM, VM and GBS; furthermore, they tended to be increased in BP and to be decreased in MS. The elevated fractalkine CSF/serum ratios in diseases without CSF pleocytosis (GBS, BP) and a lack of correlation between fractalkine levels and CSF leukocyte counts suggested that soluble fractalkine is not a major chemokine in the CSF. There was no evidence of significant intrathecal production of fractalkine as the mean fractalkine indices (fractalkine CSF/serum ratio:albumin CSF/serum ratio) were <1 in all inflammatory diseases and not significantly elevated vs. controls.

Establishment of age-dependent reference values for IgA subclasses.

BACKGROUND: Recently, subclass-specific antisera have been introduced for application in a nephelometric assay. The aim of this study was to establish age-dependent reference values for serum concentrations of the two IgA subclasses in children and adults. METHODS: Serum levels of IgA1 and IgA2 were measured by automated immunonephelometry in samples from 235 clinically healthy children between 6 months and 18 years of age and 36 healthy adults. RESULTS: Both IgA1 and IgA2 were detectable in all samples, and both IgA1 and IgA2 increased with increasing age. In adults, the mean value for IgA1 is 1.46 g/l for IgA2 0.21 g/l and for total IgA 1.94 g/l. Individual IgA2 values correlate significantly (p < 0.0001) with IgA1 values (r(2) = 0.5433). In addition, there was a highly significant (p < 0.0001) correlation (r(2) = 0.9530) between the measured total IgA and the sum of the two IgA subclasses indicating that immunonephelometry using highly specific polyclonal antisera might be superior to other methods. CONCLUSIONS: These results and the availability of age-dependent reference values make it worthwhile to reassess the role of IgA subclasses in immunodeficiency and autoimmune diseases where conventional methods have led to conflicting results.

[Diagnosing disorders of iron metabolism. Begin with ferritin]. / Beginnen Sie mit dem Ferritin.

Diagnosing disorders of iron metabolism the concentration of the iron storing protein ferritin reflects the body's iron reserves much better than does serum iron concentrations or transferring saturation. Merely in the event of acute phase reactions is the validity of the ferretin level compromised. This applies in particular to the redistribution of iron in anemia caused by inflammatory conditions or malignancies, as also, though less markedly, to functional iron deficiency in renal anemia. Here, an additional diagnostic work-up, in particular when EPO/iron therapy is applied. Iron overload should be recognized already in the latent state before organ damage occurs. Clinically and chemically confirmed iron overload that cannot be ascribed to hematological disease, iron replacement of transfusions, should prompt a molecular-biological analysis of hemochromatosis-associated genetic defects.

Usefulness of antibody index assessment in cerebrospinal fluid from patients negative for total-IgG oligoclonal bands.

BACKGROUND: Testing for cerebrospinal fluid (CSF)-restricted oligoclonal bands (OCB) by isoelectric focusing is used to detect intrathecally produced total IgG. By contrast, antibody indices (AI) are assessed to test for intrathecally produced antigen-specific IgG. A number of previous cases reports have suggested that AI testing might be more sensitive than OCB testing in detecting intrathecal IgG synthesis. FINDINGS: Here we report on 21 patients with positive AI for either herpes simplex virus, varicella zoster virus, cytomegalovirus, measles virus, rubella virus, or Borrelia burgdorferi in the absence of total-IgG OCB and, accordingly, in the presence of a normal total-IgG CSF/serum ratio. CONCLUSION: Our findings indicate that AI testing should not generally be omitted in OCB-negative patients and provide a rationale for systematic and prospective studies on the comparative sensitivity and specificity of AI and total-IgG OCB testing in infectious and other diseases of the CNS.

The intrathecal, polyspecific antiviral immune response: Specific for MS or a general marker of CNS autoimmunity?

BACKGROUND: 80-100% of patients with multiple sclerosis (MS) display a polyspecific, intrathecal humoral immune response against a broad panel of viral agents including antibodies to measles, rubella and varicella zoster virus as its three most abundant components (called MRZ reaction [MRZR]). However, a positive MRZR reaction can also be found in some patients with CNS vasculitis, another rare autoimmune condition, raising the question whether this marker is really of high specificity for MS as previously claimed or whether it just represents a non-specific marker of CNS autoimmunity. Besides MS and CNS vasculitis, paraneoplastic neurological disorders (PND) represent the best recognized models of CNS autoimmunity. OBJECTIVE: To investigate MRZR for the first time in patients with PND. PATIENTS AND METHODS: Forty-two patients with MS and 34 with PND were compared in this study. The intrathecal synthesis of antibodies against measles, rubella, and varicella zoster virus was detected by calculation of the respective antibody indices (AI). RESULTS: A positive MRZ reaction as defined by a combination of at least two positive AIs was present in 37/42 patients with MS, but in none of the patients with PND (p < 0.0001). Median AI values differed significantly between groups (p < 0.0005). CONCLUSIONS: Our results confirm that MRZR is not a general marker of CNS autoimmunity. Taking into account the very rarity of CNS vasculitis as well the lack of MRZR positivity in infectious inflammatory CNS conditions as previously demonstrated, MRZR might indeed be a promising marker of MS. Further investigations on MRZR in more rare autoimmune conditions, which were not available for analysis in this study, are now warranted to refine further the specificity of this parameter.

Intravenous immunoglobulins contain naturally occurring antibodies that mimic antineutrophil cytoplasmic antibodies and activate neutrophils in a TNFalpha-dependent and Fc-receptor-independent way.

Intravenous immunoglobulin (IVIg) preparations are increasingly used for therapy of several neuroimmunologic diseases. IVIg therapy is considered safe, although serious side effects like aseptic meningitis, cerebral vasospasm, or ischemic encephalopathy have been reported. These side effects are frequently associated with neutrophilic pleocytosis in the cerebrospinal fluid (CSF), suggesting a neutrophil-mediated mechanism. To elucidate the potential role of neutrophil activation, we analyzed IVIg preparations from 5 different commercial sources for the presence of antineutrophil cytoplasmic antibody (ANCA)-like immunoglobulins against ethanol-fixed peripheral-blood neutrophils, purified human antigens, and a panel of human and nonhuman tissues. All IVIg batches tested (n = 13) contained atypical ANCAs (IgG titer up to 1:2048, IgA up to 1:512). Moreover, all preparations were capable of inducing hydrogen peroxide production in TNFalpha-primed human neutrophils, with a significant correlation (P < .005) between atypical ANCA titers in IVIg preparations and neutrophil activation. Fc-mediated binding and activation was ruled out by the use of IVIg-F(ab')(2) fragments. Our findings strongly suggest that in vivo activation of TNFalpha-primed neutrophils by atypical ANCAs of IVIg may contribute to the side effects of IVIg therapy and for the first time demonstrate that the activation of neutrophil granulocytes by IVIg occurs in an Fc receptor (FcR)-independent, hence antigen-dependent, way.

BAFF is elevated in serum of patients with Wegener's granulomatosis.

BAFF (B-cell activating factor of the TNF family) plays a crucial role in B-cell survival. Elevated BAFF serum levels have been linked to several autoimmune diseases in humans, and therapies targeting BAFF were successful in animal models of rheumatoid arthritis and systemic lupus erythematosus. Wagener's granulomatosis (WG), a chronic systemic vasculitis, is characterized by circulating autoantibodies (cANCA) targeting neutrophils, which can produce BAFF. To investigate whether BAFF is involved in WG pathology, BAFF serum levels were measured by ELISA in 46 WG patients and 62 healthy donors. We report the novel finding that in WG patients serum levels of BAFF were significantly increased (median 3.95 ng/ml, p=0.009) compared to healthy controls (median 2.38 ng/ml). The difference was even more pronounced when comparing controls with untreated WG patients (median 4.61 ng/ml, p=0.001). Treatment of WG patients with glucocorticoids was associated with lower BAFF levels. The serum BAFF level in treated WG patients was about the same as in the control group. We propose that BAFF might be a pathogenic factor in WG and that targeting BAFF may represent a new therapeutic strategy in a subset of chronically relapsing WG patients with elevated BAFF levels.

The presence of immunoglobulins in the gastric juice of patients infected with Helicobacter pylori is related to a reduced secretion of acid.

BACKGROUND: It has been reported that treatment with proton pump inhibitors (PPI) leads to partial elimination and suppression of Helicobacter pylori. In theory, since acid is known to denature immunoglobulins, this antibacterial activity of PPI may be due to a reduction in the acid output favouring humoral immunity. MATERIALS AND METHODS: We analysed prospectively fasting gastric juice in 54 consecutive patients attending upper endoscopy for pH and levels of IgG, IgA and IgM. In addition, two antral and two corpus biopsies were obtained and histologically examined for the presence of H. pylori. RESULTS: 41/54 patients were infected with H. pylori. Immunoglobulines in the gastric juice of these patients were found in 25/41 (IgG), 27/41 (IgA), and 29/41 (IgM) patients. There was a highly significant difference in the gastric pH when H. pylori infected patients with measurable IgG, IgA, or IgM were compared with those in whom no immunoglobulines were found (median pH: 6 vs. 2 in each group; p <.001) CONCLUSIONS: There is a close correlation between a high gastric pH and the presence of IgG, IgA, and IgM antibodies. Hence, it may be speculated that the efficacy of humoral immunity following H. pylori infection depends on a high pH such as resulting from PPI treatment.

IgG subclass concentrations in certified reference material 470 and reference values for children and adults determined with the binding site reagents.

BACKGROUND: There is currently no international reference preparation for IgG subclass (IgGSc) quantification. This situation has led to calibration differences among assays and a variety of reference interval values with consequential difficulties in comparing results. We therefore evaluated IgGSc concentrations in Certified Reference Material 470 (CRM 470). METHODS: Pure, polyclonal IgG1, -2, -3, and -4 were prepared from a large serum pool for use as primary standards. The IgG mass in each preparation was calculated from amino-acid analysis data. IgGSc concentrations were assessed in CRM 470 by nephelometry with modern analytical techniques, using these reference preparations. Subsequently, IgGSc concentrations were measured in 380 healthy individuals (250 males and 130 females), and age-dependent reference intervals were established. RESULTS: IgGSc concentrations in CRM 470 were as follows: IgG1, 5028 mg/L; IgG2, 3418 mg/L; IgG3, 579 mg/L, and IgG4, 381 mg/L, with a total IgG concentration of 9406 mg/L, 2.83% below the certified total IgG value of 9680 mg/L. Age-dependent percentile curves for the four IgGSc were constructed using a Box-Cox transformation. Maximum median values were as follows: IgG1, 6.02 g/L at 11 years; IgG2, 3.45 g/L at 31 years; IgG3, 0.63 g/L at 17 years; and IgG4, 0.48 g/L at 14 years. No significant sex-related differences were observed. CONCLUSIONS: The correlation between the summation of individual IgGSc and separate measurements of total IgG concentrations was good and supports the accuracy of the results. The results are based on The Binding Site assays and should not be considered appropriate for other assays unless so demonstrated.