Quantitative Alterations in Complement Alternative Pathway and Related Genetic Analysis in Severe Phenotype Preeclampsia.

Background: Preeclampsia and hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome share many clinical and biologic features with thrombotic microangiopathy syndromes caused by complement abnormalities. Our hypothesis was that similar functional and genetic alterations in the complement alternative pathway (CAP) are present in these disorders of pregnancy. Methods: We conducted quantitative analysis of proteins involved in CAP using ELISA and nephelometry on prospectively collected blood samples from patients with severe phenotype preeclampsia (defined as delivery ≤34 weeks due to preeclampsia), HELLP syndrome, or eclampsia, and matched normotensive controls (n=25 in each arm) between 2011 and 2016. Sequencing was performed to interrogate 14 genes encoding CAP components. Results: Both groups were similar in age, gravidity, parity, marital status, and race. The study group had a higher BMI (mean±SD, 32±8 versus 25±4 kg/m2; P=0.002) and earlier gestational age at delivery (32.5±3.6 versus 40.3±1 weeks; P<0.001). Serologic studies demonstrated elevated Bb subunit (median [range], 1.2 [0.5-4.3] versus 0.6 [0.5-1] µg/ml; P<0.001), complement C5 concentration (28 [18-33] versus 24 [15-34] mg/dl; P=0.03), and sMAC (371 [167-761] versus 184 [112-249] ng/ml; P<0.001) concentrations in patients with preeclampsia. Two thirds of patients with preeclampsia had at least one nonsynonymous sequence variant in CAP genes. Conclusion: Patients with severe phenotype preeclampsia manifest functional alterations in CAP activation. Genetic variants in the CAP genes were detected in several patients, but a larger population study is necessary to fully evaluate genetic risk. Genetic screening and complement-targeted treatment may be useful in risk stratification and novel therapeutic approaches.

The impact of eculizumab on routine complement assays.

BACKGROUND: Eculizumab (ECU) blocks complement C5 cleavage, preventing the formation of C5a and the cytolytic effects of the membrane attack complex. The presence of ECU in blood impacts routine complement tests used to monitor treatment. METHODS: Residual serum samples with normal total complement (CH50) and residual citrate plasma with normal PT/APTT were spiked with ECU at varied concentrations ranging from 25 to 600 µg/mL. In addition, seventy-one samples from patients on ECU were obtained. Artificial and patient samples were analyzed for CH50 and C5 function (Wako Diagnostics), C5 concentration (Quidel), AH50 (Wieslab ELISA) and sMAC (Quidel). ECU concentration was measured by mass spectrometry for all patients. RESULTS: Complement blockage by ECU was evident in spiked artificial samples. At 25 µg/mL ECU, partial complement blockage was observed in CH50, AH50 and C5 function in serum. Complete blockage defined by undetectable AH50 (<10%) occurred at 100 µg/mL ECU. C5 concentrations remained the same regardless of ECU. sMAC results stayed around 81% of baseline in serum and 47% in citrate plasma with 50µg/mL ECU. Patient samples had ECU ranging from <5 to 1220 µg/mL. In all patients with ECU >100 µg/mL, C5 function was <29 U/mL. CONCLUSIONS: The spiked sera and patient samples showed complement blockage with CH50, AH50 and C5 function assays when ECU >100 µg/mL. CH50, AH50 or C5 function assays can serve as indicators for the pharmacodynamic effects of eculizumab. Allied to ECU concentration, laboratory studies may be helpful to tailor therapy.

Simultaneous phenotyping and quantification of α-1-antitrypsin by liquid chromatography-tandem mass spectrometry.

BACKGROUND: α-1-Antitrypsin (A1AT) deficiency results from a genetic disorder at 2 common loci. Diagnosis requires quantification of A1AT and subsequent identification of the specific variant. The current algorithm of laboratory testing for the diagnosis of A1AT deficiency uses a combination of quantification (nephelometry), genotyping, and/or phenotyping. We developed a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of A1AT and identification of the 2 most common deficiency alleles present in 95% of the patients with A1AT deficiency. METHODS: Serum samples (n = 40) were digested with trypsin, and appropriate ¹³C/¹5N-labeled standard peptides were added. We performed LC-MS/MS analysis with a 0.5- by 150-mm C18 column and H2O:acetonitrile:n-propanol:formic acid (A:98:1:1:0.2 and B:10:80:10:0.2; flow 12 µL/min) mobile phase in positive ion mode on a TSQ Quantum triple quadrupole MS system. We measured the A1AT concentration by comparison to a calibration curve and determined the phenotype by the presence or absence of variant peptides. We compared the results to the current phenotyping assay by isoelectric focusing (IEF) and the immunonephelometry quantitative assay. RESULTS: For A1AT allele detection, in 39 of 40 samples the LC-MS/MS results were identical to those obtained by IEF gel electrophoresis. The single discrepant result was rerun by IEF at a lower dilution, and the results were in concordance. The A1AT quantification by LC-MS/MS also compared favorably with nephelometry. CONCLUSIONS: The LC-MS/MS method correlates well with current phenotyping and nephelometric assays and has the potential to improve the laboratory diagnosis of genetic A1AT deficiency.

Urine protein electrophoresis and immunoelectrophoresis using unconcentrated or minimally concentrated urine samples.

Our objective was to evaluate a gel system that uses unconcentrated urine specimens for protein electrophoresis (PEL) and immunofixation electrophoresis (IFE) in patients with monoclonal gammopathies. For the study, 222 urine specimens were analyzed by our current PEL method (Helena Laboratories, Beaumont, TX) and by a system that recommends use of unconcentrated urine (Sebia, Norcross, GA). M protein concentrations were compared in the 43 cases with a measurable M spike. IFE was performed on 111 of the samples using both methods. There was a 97% concordance for detection of PEL abnormalities. The concordance for IFE was 98%. M protein concentrations by the 2 methods correlated well (r2=0.99; slope, 1.04). Cases with insufficient urine volumes for concentration (PEL, 7; IFE, 20) were analyzed in the Sebia gel system, and in 11 cases (PEL, 2; IFE, 9) an M protein was identified.High-resolution gel electrophoresis of urine using the Sebia system offers similar performance for detection, characterization, and quantification of M proteins when compared with our current gel system. Testing unconcentrated urine specimens will mean fewer sample rejections owing to insufficient sample volume.