Immage immunochemistry system

Blood samples were collected after an overnight fast and processed for further biochemical analysis. Plasma glucose was measured by enzymatic hexokinase method (Cobas 6000, Roche Diagnostics, Germany) and glycated haemoglobin (HbA1c) by turbidimetric inhibition immunoassay (Cobas 6000, Roche Diagnostics, Germany) this being a National Glycohaemoglobin Standardisation Programme (NGSP) certified method. Creatinine levels were measured using the standardized creatinine assay (Cobas 6000, Roche Diagnostics, Germany). Total cholesterol (TC), high density lipoprotein cholesterol (HDL-c), triglycerides (TG) and γ-glutamyltransferase (GGT) were estimated by enzymatic colorimetric methods (Cobas 6000, Roche Diagnostics). Low density lipoprotein cholesterol (LDL-c) was calculated using Friedewald’s formula [40 (link)]. Insulin was determined by a microparticle enzyme immunoassay (Axsym, Abbot). C-reactive protein (CRP) was measured by a high-sensitivity CRP assay, based on the highly sensitive Near Infrared Particle Immunoassay rate methodology (Immage® Immunochemistry System; Beckman Coulter), with a lower limit of detection of 0.2 mg/L.

Blood samples were drawn from venous puncture right after presentation to ICU and then sent to laboratory department for analysis. CRP levels were quantified by IMMAGE Immunochemistry System (Beckman Coulter, Inc., CA, USA) using nephelometry test. PCT levels were analyzed by serum electrochemiluminescent immunoassay using the mini-VIDAS system (Biomerieux SA, France). The CD64 index was measured using the Cytomics FC 500 MPL (Beckman Coulter, Inc., CA, USA). Specifically, 50 µL of EDTA anticoagulated whole blood was collected and 5 µL CD64-FITC, 5 µL CD33-PE, 5 µL CD45-ECD (Beckman Coulter, Inc., CA, USA) antibody were added to the sample. The sample was then mixed thoroughly and incubated at room temperature in the dark for 15 min. Erythrocyte lysin was then added and mixed and incubated at room temperature in the dark for 10 min. Finally, 500 µL PBS was added. Monocytes, neutrophils and lymphocytes were obtained by CD45/CD33-gating, and the median fluorescence intensity (MFI) of CD64 on the cells was analyzed. nCD64 index = (nCD64 MFI/lymCD64 MFI)/(mCD64 MFI/nCD64 MFI). All the tests were professionally performed by laboratory technicians according to manufacturer’s instructions.

Biochemical and genetic tests to diagnose AATD were performed at the Centre for Diagnosis of Inherited Alpha1‐Antitrypsin Deficiency at Fondazione IRCCS Policlinico San Matteo (Pavia, Italy) with the understanding and written consent of each subject. All methodologies were in accordance with the Declaration of Helsinki and were approved by the local ethics committee. The plasma levels of AAT and CRP were determined by a rate immune nephelometric method assay (Immage Immunochemistry System; Beckman‐Coulter, Milan, Italy). DNA was isolated from whole peripheral blood using a commercial extraction kit (DNA IQ System; Promega, Milan, Italy). Genotyping for detection of the S and Z variants was performed by PCR/RFLP, as previously described 45. The new mutation was identified by sequencing all coding exons (II‐V) of the SERPINA1 gene (RefSeq: NG_008290) as reported previously 46, using the CEQ 8800 genetic analysis System (Beckman Coulter). The phenotype of the new variant was determined by IEF analysis on plasma samples, comparing it with other plasma of known phenotype in a pH gradient of 4.2–4.9 (Multiphor II Electroforesis System; GE Healthcare BioScience, Milan, Italy), as previously described 46. The clinical data were obtained from direct observation of clinical charts and they are reported in an anonymized form.

Routine laboratory data were obtained using standard methods. K-F rings were examined under a slit lamp by a single ophthalmologist with rich experience. Serum CP levels were measured using the nephelometric method (normal range, 210–500 mg/L; Beckman Coulter, Immage® Immunochemistry System, Brea, CA, USA). Copper levels in the serum, urine, and liver were determined using flame atomic absorption spectrophotometry at a wavelength of 324.7 nm, as previously described. [9 (link),11 (link)] The ATP7B coding region and exon/intron boundaries were amplified using a polymerase chain reaction and sequenced in both directions, as previously described.[12 (link)]

High-sensitivity (hs)-CRP was measured using Rate Near Infrared Particle Immunoassay (Immage Immunochemistry System; Beckman Coulter, Brea, CA, USA). This method can accurately measure protein concentrations from 0.2–1440 mg/l with a within-run precision <5.0 %, a total precision <7.5 %, and a reliability coefficient of 0.995 [26 (link)].

Hematologic parameters were measured using a Siemens-ADVIA 120 whole blood autoanalyzer (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Blood Chemistry for renal and liver function was performed using the Siemens Advia 1800 Clinical Chemistry System (Siemens Healthcare Diagnostics, Tarrytown, NY, USA), whereas CRP concentrations were determined with immunonephelometry using a Beckman Coulter IMMAGE immunochemistry system (Beckman Coulter, Inc., Fullerton, CA, USA). The intra- and interassay coefficients of variation (CVs) were 3.5% and 7.0%, respectively.
For suPAR determination, plasma was isolated from EDTA-K₃ anticoagulated blood samples of neonates following centrifugation at 3000 g for 10 minutes and stored at −80°C until analysis. Plasma suPAR levels were determined using a commercial enzyme-linked immunosorbent assay (ELISA) (suPARnostic Standard kit; ViroGates A/S, Birkerød, Denmark). This assay utilizes a double monoclonal antibody that measures all circulating suPAR, including full-length and cleaved forms of the receptor. According to the standards provided by the manufacturer in ng/mL, a curve was constructed by us and the results were expressed as ng/mL, in a range between 0.6 and 20.0 ng/mL. The intra- and interassay CVs ranged from 1.3% to 4.7% and 1.7% to 5.1%, respectively, whereas the sensitivity limit was 0.1 ng/mL.

All blood samples were drawn on the same day that TCD was performed, and all blood sampling was performed following no less than 12 hours of fasting.
Hematological parameters were obtained using a CELL-DYN Ruby System hematology analyzer (Abbott Diagnostics, Lake Forest, Illinois, USA), and hemoglobin profiles were analyzed by high-performance liquid chromatography using an HPLC/Variant-II hemoglobin testing system (Bio-Rad, Hercules, California, USA).
Biochemical parameters, including lipid profile, total proteins and fractions, total bilirubin and fractions, lactate dehydrogenase (LDH), alanine transaminase (ALT) and aspartate transaminase (AST), renal profile, and iron, were determined using an automated A25 chemistry analyzer (Biosystems S.A., Barcelona, Catalunya, Spain). Ferritin levels were measured using Access 2 Immunochemistry System (Beckman Coulter Inc., Pasadena, California, USA). C-reactive protein, alpha-1 antitrypsin, and haptoglobin levels were measured using IMMAGE® Immunochemistry System (Beckman Coulter Inc., Pasadena, California, USA).