European proficiency study with control serum for the tumor marker CA 19-9 measured on different test systems.

BACKGROUND: Reliable and precise CA 19-9 testing is required for the long-term follow-up of patients with pancreatic carcinoma during therapy. The aim of this longitudinal proficiency study was to evaluate the comparability, linearity, and precision of CA 19-9 determinations performed in different laboratories using currently available test systems under routine conditions. METHODS: During the one year study period, 15 laboratories applied 7 different tests and included a liquid BIOREF control serum with pancreatic carcinoma derived CA 19-9 in their routine testing and quality control procedures. The results were collected centrally and evaluated statistically. RESULTS: The comparability of CA 19-9 results is limited especially when different tests are used, albeit, some tests show a good correlation: The CA 19-9 values obtained by different laboratories using different test systems vary up to a factor of 2. The precision of CA 19-9 determinations was acceptable in most laboratories with coefficients of variation ranging between very low 3.2% and high 17.8%. The imprecision was slightly increased when automatic dilution procedures of the analysers were used. CONCLUSIONS: The comparability of CA 19-9 test results must be improved. The precision is acceptable in most cases. In order to monitor key performance parameters, every laboratory should participate in external quality assessment schemes and should perform a routine internal quality control with a control serum independent from the test kit manufacturer.

Alpha-1-Fetoprotein (AFP): international proficiency study with different test systems.

BACKGROUND: The present proficiency study aimed to elucidate the comparability and reliability of test systems for the determination of AFP concentrations. METHODS: 25 laboratories using 8 different commercial test systems used liquid BIOREF-AFP control serum in their routine internal quality control over a period of one year. For statistical analysis the results were collected centrally. RESULTS: The statistical analysis of the test results revealed considerable variation for the different laboratories. The deviations of the mean values of different laboratories from the overall mean value varied between 0.1 and 26.1%, and for most of the laboratories the deviation was round about 10%. The precision of measured values in the individual laboratories was in most cases acceptable: Nevertheless, the coefficients of variation of the individual laboratories ranged from 13 to 16.1%. CONCLUSIONS: In conclusion, this study indicates that AFP results vary between different laboratories albeit an international standard for AFP is available. Therefore, every laboratory should participate in external ring studies and should use a quality control serum independent of the test kit manufacturer for the internal quality control.

Precision and long-term stability of different estradiol immunoassays assessed in a multi-center quality control study.

BACKGROUND: Because of the vast range of physiological relevant estradiol concentrations the requirements to be met by an estradiol assay are high. In the present study the performance of various commercially available estradiol assays was evaluated with regard to imprecision and long-term stability. METHODS: Precision and long-term stability of 7 commercially available estradiol immunoassays were assessed in a multi-centre quality control study based on the repeated measurement of liquid BIOREF estradiol control sera by 18 laboratories during a 14-month study period. RESULTS: The mean estradiol concentrations determined in 594 runs performed for each control level were 71 pg/ml, 349 pg/ml and 676 pg/ml. A high variation was found for the method specific mean values calculated from all results measured with the same method, which ranged between 32 - 90 pg/ml, 187 - 392 pg/ml and 373 - 790 pg/ml, resulting in a similar high inter-laboratory variation with coefficients of variation (CVs) of 25.0%, 16.7% and 17.5%. In contrast, the intra-laboratory variation of estradiol values as well as the variation of values measured with the same method were found to be considerably lower with coefficients of variation < 10% for most laboratories and methods; only the low control level was measured with CV values > 10% by the majority of laboratories and methods. For none of the laboratories a tendency was observed in the results from beginning to end of the 14 month study period indicating a high uniformity in assay production and a good long-term stability of the control material used. CONCLUSIONS: The present data demonstrate that also with the currently available estradiol immunoassays the comparability of results measured with different methods is limited. With most assays very low estradiol concentrations, as observed in postmenopausal women, can be determined only with a precision which is not adequate for clinical assessment.

Multicentre evaluation of performance of C-reactive protein analysis over a one-year period.

This communication deals with a longitudinal evaluation of C-reactive protein (CRP) analysis during a one-year period using a single lot of liquid control sera (3 levels) (BIOREF-CRP levels 1, 2 and 3) in different laboratories. A total of 652 sets of data were returned from 20 participating laboratories using 13 different reagent-measuring device combinations. The use of the control materials was defined in a standard operating procedure. Data was returned to the organizers on a monthly basis and questions could be asked or problems presented during the evaluation period. Although the performance of different reagents varied, the control materials were shown to be stable over the whole of the evaluation period when stored at 4-7 degrees C in a refrigerator/cold room. Typical problems were encountered, examples of which are presented here in graphical and tabular form.

External quality control program for inter-laboratory quality control.

BACKGROUND: The accuracy and precision of tumor marker testing is of high clinical importance requiring the implementation of effective quality control procedures in the diagnostic laboratory. An external quality control program including an inter-laboratory comparison could be of help in improving the quality of test results. MATERIALS AND METHODS: Laboratories using BIOREF reference material sent their routine internal quality control results of each monthly control period to the evaluation center for statistical analysis and inter-laboratory comparison. RESULTS: The inter-laboratory comparison showed that the mean values of test results obtained by different laboratories can vary considerably especially when different test kits are used. A comparison of test results of laboratories using the same test kit showed much better correlation but discrepancies of coefficients of variation between these laboratories were still observed. CONCLUSION: An external quality control program revealed problems in the accuracy and precision of tumor marker test results in the individual laboratory, which could serve as a basis for the improvement of test performance.

In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (FcgammaRII) phosphorylation.

Human B cells express four immunoglobulin G receptors, FcgammaRIIa, FcgammaRIIb1, FcgammaRIIb2, and FcgammaRIIc. Coligation of either FcgammaRII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcgammaRIIa/c and FcgammaIIb1 isoforms become phosphorylated. To identify the FcgammaRII-phosphorylating protein tyrosine kinase (PTK), we used the combination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different FcgammaRII isoforms as well as tyrosine exchange mutants, we show that each of the BCR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphorylation patterns with regard to the different FcgammaR isoforms and point mutants. While each PTK phosphorylated FcgammaRIIa/c, FcgammaRIIb1 was phosphorylated by Lyn and Blk whereas FcgammaRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcgammaRIIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosphorylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an FcgammaR- B-cell line transfected with wild-type or mutated FcgammaRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcgammaRIIa/c was sufficient to abolish FcgammaRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcgammaRIIa/c, with the ITAM being necessary for association. Comparison of the phosphorylation pattern of each PTK observed in vitro with the phosphorylation pattern observed in vivo suggests that Lyn is the most likely candidate for FcgammaRIIa/c and FcgammaRIIb1 phosphorylation in vivo.

Epitope mapping of new monoclonal antibodies recognizing distinct human FcRII (CD32) isoforms.

The class II Fc gamma receptors are widely distributed on cells of the immune system. Nevertheless, the exact cell type distribution of the FcRII isoforms is still unclear because of the lack of appropriate antibodies that discriminate between the various isoforms. In this study we describe the generation and characterization of three monoclonal antibodies (MAbs) raised against recombinant human FcRIIb2 as well as a synthetic peptide (amino acids 30-39) of this receptor. Analyses of the isoform specificity of these antibodies using ELISA and Western blots revealed that the MAbs II1A5 (mIgG1) and ID2.7 (mIgM) are pan FcRII antibodies recognizing all known FcRII isoforms. In contrast, the MAb II8D2 (mIgG1) specifically reacts with FcRIIb but not with FcRIIa. The observed antibody reactivities could be confirmed by examination of the exact epitopes using overlapping 15-mer peptides spanning the entire FcRIIb2. So far these antibodies are the only ones described that detect FcRII in Western blots. Moreover, they can be used to analyze the cellular FcRII isoform distribution at the protein level, which was otherwise not possible.

Tyrosine-containing sequence motifs of the human immunoglobulin G receptors FcRIIb1 and FcRIIb2 essential for endocytosis and regulation of calcium flux in B cells.

Human B cells express two closely related immunoglobulin G receptors, FcRIIb1 and FcRIIb2, which differ by a 19 amino acid insertion in the cytoplasmic tail of FcRIIb1. The cytoplasmic tails of both isoforms contain a conserved sequence motif (AENTITYSLL) essential for mediating endocytosis via FcRIIb2. Truncation of this motif abolished endocytosis, while replacement of tyrosine (Tyr273) in FcRIIb2 by phenylalanine had no effect on the amount and kinetics of ligand uptake. Co-cross-linking of FcRIIb1 or FcRIIb2 with the antigen receptor on B cells led to an abortive calcium signal. Neither isoform interfered with the early intracellular calcium mobilization, but both prevented the opening of a plasma membrane calcium channel essential for a sustained elevated intracellular calcium level. Modulation of calcium channel activity is mediated by the same sequence motif essential for endocytosis but requires the presence of Tyr292 in FcRIIb1 and Tyr273 in FcRIIb2. Co-cross-linking of FcRIIb1 with surface IgG is associated with tyrosine phosphorylation of Tyr292, whereas Tyr272 in FcRIIb2 was not phosphorylated. Thus, FcRIIb phosphorylation is probably not directly involved in the modulation of the calcium signal but may be essential for further diversification of signals transduced via the coexpressed isoforms FcRIIb1 and FcRIIb2.

Biological functions of human Fc gamma RIIa/Fc gamma RIIc in B cells.

The human immunoglobulin receptor IIa (FcRIIa) was transfected into the FcR- mouse B cell line IIA1.6 to study its role in mediating endocytosis of human IgG complexes as well as its possible function in inhibiting the attenuated increased calcium level in B cells after antigen receptor cross-linking. Using FcRIIa mutants with truncated cytoplasmic domains we show that the region within the cytoplasmic region necessary for endocytosis differs from that necessary for the abrogation of the elevated calcium level in B cells induced after antigen receptor cross-linking. Deletion of 14 amino acids at the carboxy terminus led to a slow internalization of FcRIIa bound human IgG, whereas the mutant lacking 30 amino acids completely failed to mediate IgG uptake. The mutant lacking 14 amino acids at the carboxy terminus is not tyrosine phosphorylated in the course of receptor-mediated IgG uptake. This suggests that phosphorylation of FcRIIa is not necessary to mediate this function. FcRIIa cross-linking leads to a rapid transient rise in the intracellular calcium concentration due to calcium release from intracellular stores. Using the FcRIIa mutants we could further show that the signal delivered by FcRIIa is strongly dependent on the last 14 amino acids of the cytoplasmic tail. Analyses of the tyrosine phosphorylation of FcRIIa revealed that the calcium release mediated by FcRIIa cross-linking is dependent on tyrosine phosphorylation. In contrast, inhibition of the antigen receptor (sIgG) induced rise in intracellular calcium concentration by FcRIIa-sIgG co-cross-linking was not impaired when 30 amino acids were deleted at the carboxy terminus. FcRIIa wild type was rapidly phosphorylated when co-cross-linked with sIgG, but phosphorylation is not a prerequisite to inhibit calcium influx induced by sIgG cross-linking. These results show that distinct regions within the cytoplasmic tail of FcRIIa are necessary for the various signals transmitted to the cell.