Reference Intervals of Factor H and Factor H-Related Proteins in Healthy Children.

Complement is activated as part of the innate immune defense against invading pathogens. Also, it helps to remove apoptotic debris and immune complexes from the circulation. Impaired complement function due to aberrant plasma levels of complement proteins may be indicative for complement-mediated diseases or can be involved in susceptibility for infections. To determine whether plasma levels are abnormal, reference intervals (RIs) are used from adult healthy donors. Since many complement-mediated diseases have an onset during childhood, it is important to know whether these RIs can be extrapolated to children. RIs of Factor H (FH), the crucial fluid-phase regulator, and the FH-related proteins (FHRs), its homologous counterparts, are unknown in healthy children. While FH is measured to diagnose and monitor therapy of patients with atypical hemolytic uremic syndrome, recent studies also implicated increased plasma levels of FHRs in disease. Here, we investigated the levels of FH and FHRs in healthy children using recently developed specific ELISAs. We found that levels of FH, FHR-2, and FHR-3 were equal to those found in healthy adults. Levels of FHR-4A and FHR-5 were lower in children than in adults. However, only the FHR-5 levels associated with age. The RIs of these FH family proteins now serve to support the interpretation of plasma levels in prospective and retrospective studies that can be used for routine diagnostic and monitoring purposes including pediatric patient samples.

Analytical validation of the Hevylite assays for M-protein quantification.

BACKGROUND: The heavy/light chain (HLC) immunoassay quantifies the different heavy chain/light chain combinations of each immunoglobulin (Ig) class. This makes the HLC assay suited to quantify monoclonal immunoglobulins (M-protein) and for monitoring of patients with monoclonal gammopathies. This method is particularly advantageous for those samples in which electrophoretic quantification of the M-protein is not possible. METHODS: In this study we tested the analytical performance of the HLC assay in 166 routine clinical samples and in 27 samples derived from the Dutch external quality assessment (EQA) for M-protein diagnostics (74 participating laboratories). Analytical accuracy was assessed by verification that the sum of the HLC-pairs equaled total Ig concentration. Sensitivity of the HLC assay was determined in a direct method comparison with immunofixation electrophoresis (IFE). RESULTS: Comparison of HLC data with routine Ig diagnostics in 27 EQA samples showed very good correlation for both the quantification of polyclonal and monoclonal IgG, IgA and IgM (Pearson correlations [r] were 0.94, 0.99 and 0.99, respectively; slopes were 0.94, 1.07 and 0.98, respectively). The overall concordance between IFE and the HLC ratio was high (93%) with a Cohen κ coefficient of 0.84. Discrepancies between both assays were mainly caused by the higher sensitivity of IFE to detect monoclonality. CONCLUSIONS: We conclude that the HLC assay is an accurate method to quantify M-proteins that can improve monitoring of M-proteins in the beta fraction that cannot be quantified using electrophoretic techniques.

Pediatric reference intervals for immunoglobulin G and its subclasses with Siemens immunonephelometric assays.

OBJECTIVE: To determine the pediatric reference intervals for the Dade Behring IgG subclass reagents. DESIGN AND METHODS: Blood samples of healthy children and adolescents were analyzed to determine the level of immunoglobulin G (IgG) and the IgG subclass using the reagents of Dade Behring Marburg GmbH. RESULTS: The reference intervals categorized by age were reported from a total of 436 samples. There was an excellent correlation between the total IgG and the sum of the IgG subclasses. CONCLUSIONS: Our data provide the missing pediatric reference intervals and enable the use of the Dade Behring nephelometric IgG subclass reagents in children and adolescents.

Complement factor 7 gene mutations in relation to meningococcal infection and clinical recurrence of meningococcal disease.

Meningococcal disease is caused by Neisseria meningitidis which is associated with high morbidity and mortality. Recurrences of meningococcal infection have been observed in patients with terminal complement component defects, because of the inefficient formation of the lytic membrane attack complex (MAC), C5b-9. Complement component C7 is one of the five plasma proteins to form the MAC. The gene C7 may carry mutations that cause functional abnormalities or the mere absence of the C7 protein. More than 200 patients were screened for aberrant C7 protein by isoelectric focusing (C7 IEF). These were compared with patients in whom recurrent meningococcal infection had resulted in the diagnosis of complete C7 absence (C7Q0). A higher proportion of C7 IEF variants were found in meningitis cases compared to controls (p=0.03). In contrast to C7Q0 patients, recurrent meningococcal infection was never observed in C7 IEF cases. Whereas C7Q0 sera were defective in meningococcal serogroup B and W135 killing assays, the sera of patients with C7 IEF variants were only defective in complement-mediated killing when classical pathway activation by (endogenous) anti-meningococcal antibodies was blocked. Upon sequence analysis we characterized the genetic background of the C7*6 and C7*8 IEF pattern and identified three novel C7 gene mutations in 13 C7Q0 patients. In conclusion, C7 IEF variants can determine meningococcal killing in the early stage of infection when antibody-independent killing prevails. The results endorse the lack of clinical recurrences once antibodies are present, whereas in C7Q0 patients the anti-meningococcal antibodies may not suffice to protect from recurrent meningococcal infection.

Pediatric reference intervals for soluble transferrin receptor and transferrin receptor-ferritin index.

BACKGROUND: Recent studies showing an improved diagnosis of iron deficiency (ID) with soluble transferrin receptor (sTfR) and sTfR-ferritin index did not take into account the age-dependency of sTfR and ferritin. Moreover, there is a paucity of data on pediatric reference intervals for sTfR and sTfR-ferritin index. METHODS: A study cohort of 436 apparently healthy children was analyzed to establish reference intervals for ferritin, transferrin, sTfR and sTfR-ferritin index. To account for age-dependency, standard deviation scores (Z-scores) for these markers were calculated. The association between these parameters and C-reactive protein (CRP) was analyzed. RESULTS: The Z-scores of ferritin, transferrin and sTfR had a significant association with CRP, whereas the Z-score of sTfR-ferritin did not correlate with CRP. The reference intervals of these parameters were reported. CONCLUSION: Among the different markers of ID, the Z-scores of sTfR, transferrin and ferritin, but not sTfR-ferritin index, associate with the inflammatory status.

Functional C1-inhibitor diagnostics in hereditary angioedema: assay evaluation and recommendations.

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent episodes of potentially life-threatening angioedema. The most widespread underlying genetic deficiency is a heterozygous deficiency of the serine protease inhibitor C1 esterase inhibitor (C1-Inh). In addition to low C4 levels, the most important laboratory parameter for correct diagnosis of HAE or angioedema due to acquired C1-Inh deficiency is reduced C1-Inh function (fC1-Inh). No direct recommendations about the assays for fC1-Inh or sample handling conditions are available, although this would prove especially useful when a laboratory first starts to offer assays on fC1-Inh for HAE diagnosis. In the present study we evaluated the performance of fC1-Inh assays in the 15 different laboratories that are specialised in HAE diagnostics and assessed inter-laboratory variation with each laboratory using their own assays and standards. A double-blind survey was conducted using plasma/serum samples from healthy donors and HAE patients and the uniformity of HAE diagnosis was evaluated. It can be concluded that the diagnosis of fC1-Inh deficiency was made correctly in most cases in this survey. We can recommend the chromogenic assay for the determination of fC1-Inh, while the complex ELISA needs further investigation.

Mannan-binding lectin (MBL)-mediated opsonization is enhanced by the alternative pathway amplification loop.

The complement system is a humoral effector in the innate immune system. Three activation pathways exist in the complement system, known as the classical pathway, the lectin pathway and the alternative pathway. Dysfunction of lectin pathway activation is caused by MBL deficiency. MBL deficiency in a cohort of healthy Caucasian blood bank donors was investigated with MBL genotyping and MBL plasma concentration. Recognition of the yeast-derived zymosan by MBL was investigated with Western blot. The involvement of the alternative pathway amplification loop in enhancing MBL-mediated opsonization of zymosan was investigated in a novel opsonophagocytosis assay for flow cytometry. Sera deficient for MBL, factor D or properdin were tested, and purified MBL, factor D or properdin were used to recover opsonization. The optimal receiver-operator characteristic (ROC) cut-off value for dividing the Caucasian cohort in MBL-sufficient and MBL-deficient was calculated at 0.7 microg/ml. Thirty-eight percent of the group had concentrations below 0.7 microg/ml. Zymosan eluates opsonized with MBL-sufficient sera contain high oligomers of MBL, while eluates from MBL-deficient donors contained hardly any MBL. The MBL-, factor D- and properdin-deficient sera showed reduced opsonophagocytosis by human control neutrophils, as compared to normal MBL-sufficient sera. This reduction in opsonization was restored to normal levels by addition of purified MBL, factor D and properdin. The absence of opsonization in the factor D- and properdin-deficient sera, but presence in normal serum after blocking with anti-C1q-F(ab)2 and anti-MBL-F(ab)2, demonstrates the involvement of the amplification loop in MBL-initiated zymosan opsonization, even at very low serum concentrations (up to 3%, v/v). In conclusion, our data demonstrate that the MBL-mediated route of complement activation depends on the alternative pathway amplification loop for optimal opsonization of zymosan.