临床化学分析方法：Alpha1-Antitrypsin

Kaplan: Clinical Chemistry, 5th EditionClinical References - Methods of Analysisα1-AntitrypsinJohn Beilby iNames:α1-Antitrypsin, α1-protease inhibitorClinical significance: α1-Antitrypsin deficiency associated with lung and liver disease Molecular mass: 52,000 DChemical class:GlycoproteinMost common allelic types:PiM, PiS, PiZRefer to Chapter 52, Diseases of Genetic Origin, in the 5th edition of Clinical Chemistry: Theory, Analysis, Correlation.Students’ Quick Hyperlink Review•Principles of analysis and current usage•Reference and preferred methods•Specimen•Interferences•Alpha 1-antitrypsin reference intervals•Interpretation•Alpha 1-antitrypsin performance goals•References•Alpha 1-antitrypsin methods tablePrinciples of Analysis and Current UsageThe clinical importance of the measurement of α1-antitrypsin serum levels is to detect people with deficient states. Severe deficiency (OMIM #107400) is clinically under-recognized [1,2] and is associated with a substantially increased risk for the development of pulmonary emphysema by the 3rd and 4th decades of life. It is also associated with the risk of hepatic disease, cutaneous panniculitis, arterial aneurysm, bronchiectasis, and renal disease [3]. α1-Antitrypsin is responsible for approximately 90% of the trypsin-inhibitory capacity of serum [4,5]. It is a member of the serine protease inhibitor or serpin superfamily [6] and is also knowni Alpha1-antitrypsinPrevious and current authors of this method: First edition: Not doneMethods edition: Gayle JacksonSecond edition: Not updatedThird edition: Steven C. Kazmierczak Fourth edition: Gayle JacksonFifth edition: John Beilbyas α1-protease inhibitor because of its ability to inhibit a broad range of protease enzymes, including trypsin, chymotrypsin, pancreatic elastase, neutral proteases of polymorphonuclear leukocytes and macrophages, and a number of other enzymes [7]. It is a globular glycoprotein that is found in the α1 region of an agarose electrophoresis pattern at pH 8.6. It has a molecular mass of 52 kDa and consists of a 418 amino acid single polypeptide chain with four carbohydrate side chains. α1-Antitrypsin is found in serum and in a number of body fluids such as saliva, tears, lymph, semen, cervical mucus, synovial fluid, and human milk [7]. The half-life of exogenous α1-antitrypsin in serum is approximately a week, with catabolism taking place in the liver.Quantitationα1-Antitrypsin concentrations in serum are measured by immunochemical techniques. In the past, electro-immunodiffusion and radial immunodiffusion techniques were used. Today, automated immunoturbidimetric and immunonephelometric methods are widely used in laboratories. These quantitative methods determine α1-antitrypsin concentrations in humanserum or plasma by measuring the absorbance change due to light scattering caused by insolu complexes that form between anti-αble n concentration. 1-antitrypsin antibodies and α1-antitrypsin in the sample. The amount of light scattering is dependent upon the α1-antitrypsin concentration in the sample and can be quantified by comparison with calibrators of known α1-antitrypsiSerum ElectrophoresisOccasionally, α1-antitrypsin deficiency can be detected by close examination of the α1-globulin band on serum electrophoresis gels. Since α1-antitrypsin composes approximately 90% of the α1-globulin band, a severe deficiency results in the almost complete absence of α1-globulins. It should be noted that the densitometer scan might give a normal value for the α1-globulins, even though visual inspection clearly shows an absence of the band. Other α1-proteins such as α1-lipoprotein may stain to give a diffuse background, thus accounting for the discrepancy between the visual inspection and the scan results.Phenotypingα1-Antitrypsin deficiency is inherited as an autosomal co-dominant disorder, with more than 100 alleles identified [8]. The 12.2 kb gene is located on the long arm of chromosome 14 (14q31-32.3) and consists of seven exons and six introns. Different phenotypes are classified by a coding system in which the inherited alleles are usually letters that denote the migration of the molecule in an isoelectric pH gradient from A (for anodal variants) to Z (for slower migrating variants). The MM phenotype indicates individuals who are homozygous for the normal allele, and ZZ indicates that they are homozygous for the Z allele. The Z variant consists of a glutamine substitution for a lysine residue at codon 342, causing an α1-antitrypsin-deficient state and a dysfunctional protein [9]. The S variant is due to a substitution of valine for glutamine at codon 264, causing intracellular degradation of the protein [10]. Low serum concentrations of α1-antitrypsin must be confirmed by Pi (Protease Inhibitor) phenotyping [11].The gold standard for the identification of α1-antitrypsin variants is the phenotyping of serum samples by isoelectric focusing on thin-layer gels in a pH gradient [11]. This can be done on polyacrylamide or agarose gels. In this procedure, a stable, stationary pH gradient is obtained by the use of ampholytes which are polyamino-polycarboxylic acids. Since α1-antitrypsin proteinshave isoelectric points in the pH region from 4.2 to 4.65, ampholines with a pH range of 4 to 5 are used. This technique separates the various isoforms of α1-antitrypsin based on their migration in a pH gradient. Each isoform migrates to the position within the pH gradient at which the overall net charge of the protein is zero. The separated protein bands are visualized by a protein stain such as Coomassie blue R-250 [12]. Acid-starch gel electrophoresis followed by crossed antigen-antibody electrophoresis [13] has been used in the past (Table 1, Meth od 6). Isoelectric focusing is a technically challenging test and requires a highly skilled scientist to perform the test and interpret the results. Samples of known Pi types are not readily available and can only be found by testing many samples. In addition, severe deficiency is relatively uncommon. Screening for heterozygous α1-antitrypsin deficiency or for intermediate deficiencies does not seem worthwhile, because these phenotypes do not correlate statistically to respiratory problems [14].The medically important variants are those associated with α1-antitrypsin deficiency—namely, the S and Z variants and the rare null (non-production) variant. Approximately 6% of people of northern European descent carry the S gene, and 3% to 4% carry the Z variant [15]. GenotypingGenotyping can largely replace the isoelectric focusing technique. The molecular assay is readily automated using real time PCR, and the interpretation is straightforward, unlike the isoelectric focusing method. The PCR assay detects the most common disease-associated alleles, Z and S. An algorithm for quantitation, genotyping, and phenotyping of α1-antitrypsin has been described [16]. In this approach, α1-antitrypsin was quantitated and genotyped when the serum concentrations were less than 70 mg/dL (0.7g/L). If the results were concordant, they were released with an interpretative comment. If the quantitation and genotype results were discrepant, isoelectric focusing was performed, and all results were reported with an interpretative comment. Snyder et al. [16] demonstrated that 2% of all results were discordant. In another study, all samples without an observed S or Z variant and a serum concentration of less than 100 mg/dL (1.0g/L) were phenotyped [17]. Using this technique, Bornhorst et al. [18] identified 6.5% of patients at risk of developing α1-antitrypsin-related symptoms owing to the presence of rare deficiency alleles not detected by the genotyping assay. A variation on this approach was genotyping by direct sequencing all patient samples with α1-antitrypsinconcentrations less than 100 mg/dL (1.0g/L) [18]. This approach identified all deficient alleles and two new null alleles. Genotyping is quick and easy to perform but needs to be complemented by either phenotyping or direct sequencing so that disease associated variants are not missed.Reference and Preferred MethodsThere is no reference method for the determination of serum α1-antitrypsin levels. Automated immunoturbidimetric and immunonephelometric methods are currently used with the immunoturbidimetric group having the best imprecision (see performance goals below). Reference material for serum proteins, CRM 470 was released in 1993 and has resulted in an improvement in between-laboratory variability of serum protein results [19].SpecimenSerum samples are preferred, but plasma samples are acceptable for α1-antitrypsin concentration determination. Samples may be stored at 4°C for up to 7 days prior to testing. For long-term storage or for samples suspected of a severe deficiency of α1-antitrypsin, storage at −70°C is recommended.Serum or plasma samples may be used for phenotyping. Samples with α1-antitrypsin concentrations of 50% of normal or above may be stored on a long-term basis at −70°C. Storage at −20°C is acceptable for 1 or 2 weeks. Unfortunately, samples with severe α1-antitrypsin deficiency cannot prevent normal activation of intrinsic proteolytic enzymes and may become degraded rapidly, even if stored at less than −20°C. Bacterial contamination or improper storage results in altered migration rates of the bands with eventual loss of banding [20]. Samples suspected of a severe deficiency should be phenotyped as soon as possible after collection. InterferencesSome work has been published on assay interferences for α1-antitrypsin concentration determination. However, the results appear to be variable, depending on the technique used and the manufacturer of the instrumentation [21]. To identify the interferences associated with each method, the user should refer to the manufacturer’s kit insert.α1-Antitrypsin Reference IntervalIn 1995, fifteen professional societies and twelve diagnostic companies agreed on a procedure for uniform reporting of protein reference intervals [19]. The α1-antitrypsin reference interval was reported as 90 to 200 mg/dL (0.9 to 2.0g/L) for subjects with the MM genotype. Age- and gender-related differences in reference interval have been reported. Following birth, concentrations fall during the first 6 months but rise to adult concentrations by 1 year of age [22]. Females have higher concentrations than men across all ages [23].InterpretationA number of factors have been reported to increase α1-antitrypsin concentration, including inflammatory disorders, malignancy, trauma, increases in estrogen concentrations with puberty, pregnancy, or the use of the oral contraceptive pill [23]. However, values rarely increase more than fourfold. These factors can cause considerable overlap in measured concentration between mildly and moderately α1-antitrypsin-deficient subjects and concentrations in normal subjects [7].Severe α1-antitrypsin deficiency is a genetic disorder that affects about 1 in 2000 to 5000 individuals [1]. The World Health Organization guidelines recommend screening all patients with chronic obstructive pulmonary disease and adults and children with asthma [24]. Any α1-antitrypsin concentration less than 100 mg/dL (1.0g/L) should be genotyped or phenotyped to detect α1-antitrypsin variants. In the few cases where the genotype does not agree with the α1-antitrypsin concentration, the sample should be further investigated by phenotyping or DNA sequencing. Serum concentrations of α1-antitrypsin less than a protective threshold of 50mg/dL (0.5 g/L) by nephelometry are associated with an increasing risk of emphysema [25].Allelic types that result in deficiencies of serum α1-antitrypsin include PiS (mean plasma concentration, 60% to 70% of normal), PiZ (mean plasma concentration, 10% to 15% of normal), and Pi null [26]. The ANT concentration in severely deficient subjects rises only slightly in response to inflammatory disorders, malignancy, trauma, and increases in estrogen concentration.The prevalence of the various genotypes are approximately MM 91%, MS 6.1%, MZ 2.7%, SS 0.1%, SZ 0.1%, ZZ 0.02% and null-null. The risk of emphysema is 20% to 50% for the SS genotype, 80% to 100% for SZ, and 100% for the null-null genotype [1]. The null alleles do not produce α1-antitrypsin transcript, produce truncated protein, or produce unstable proteins that are essentially completely degraded before secretion. The risk of emphysema is greatly increased-antitrypsin deficiency if they smoke cigarettein a person with a severe αs.1α1-Antitrypsin Performance GoalsSurvey data from the 2007 College of American Pathologists Participant Summary Report show that 73% of laboratories use immunonephelometric techniques, and 27% use immunoturbidimetric techniques. The imprecision of the methods as determined by the coefficient of variation (CV%) at a mean α1-antitrypsin concentration of 53.9 mg/dL (0.54g/L) ranged from 4.3% to 6.7% for immunonephelometric techniques and from 2.6% to 5.8% for immunoturbidimetric techniques.Acceptable performance criteria (CLIA-88) for measurement of α1-antitrypsin require that laboratories be accurate to within ± 3SD of the peer group mean. The intra-individual variation of α1-antitrypsin in blood in healthy adults has been determined to be approximately 5.9%. Desirable specifications for analytical imprecision derived from studies of biological variation indicate an assay imprecision of no greater than 3.0% and a total error of no greater than 9.2% [27]. The immunonephelometric and immunoturbidimetric techniques are both within the desired performance criteria as defined by CLIA-88. However, only one immunoturbidimetric technique is within the analytical imprecision specification if biological variability defines required performance.References1. Stoller JK, Aboussouan LS. Alpha1-antitrypsin deficiency. Lancet 2005; 365: 2225-2236.2. Laurell CB, Eriksson, S. The electrophoretic alpha 1-globulin pattern of serum in alpha 1antitrypsin deficiency. Scand J Clin Lab Invest 1963; 25: 132-140.3. Mulgrew AT, Taggart CC, McElvaney NG. Alpha-1-antitrypsin deficiency: currentconcepts. Lung 2007; 185: 191-201.4. Crystal RG. The alpha 1-antitrypsin gene and its deficiency states. Trends Genet 1989; 5:411-417.5. Lisowska-Myjak B. AAT as a diagnostic tool. Clin Chim Acta 2005; 352: 1-13.6. Potempa J, Korzus E, Travis J. The serpin superfamily of proteinase inhibitors: structure,function, and regulation. J Biol Chem 1994; 269: 15957-15960.7. Morse JO. alpha1-antitrypsin deficiency (first of two parts). N Engl J Med 1978; 299:1045-1048.8. DeMeo DL, Silverman EK. Alpha1-antitrypsin deficiency. 2: genetic aspects of alpha(1)-antitrypsin deficiency: phenotypes and genetic modifiers of emphysema risk. Thorax2004; 59: 259-264.9. Yoshida A, Lieberman J, Gaidulis L, Ewing C. Molecular abnormality of human alpha1-antitrypsin variant (Pi-ZZ) associated with plasma activity deficiency. Proc Natl Acad Sci USA 1976; 73: 1324-1328.10. Owen MC, Carrell RW, Brennan SO. The abnormality of the S variant of human alpha1-antitrypsin. Biochim Biophys Acta 1976; 453: 257-261.11. Stoller JK, Snider GL, Brantly ML, Fallat RJ, Stockley RA, Turino GM et al. [AmericanThoracic Society/European Respiratory Society Statement: Standards for the diagnosisand management of individuals with alpha1-antitrypsin deficiency]. Pneumologie 2005;59: 36-68.12. Kueppers F. Determination of alpha1-antitrypsin phenotypes by isoelectric focusing inpolyacrylamide gels. J Lab Clin Med 1976; 88: 151-155.13. Lieberman J, Gaidulis L, Garoutte B, Mittman C. Identification and characteristics of thecommon alpha 1 -antitrypsin phenotypes. Chest 1972; 62: 557-564.14. Morse JO, Lebowitz MD, Knudson RJ, Burrows B. Relation of protease inhibitorphenotypes to obstructive lung diseases in a community. N Engl J Med 1977; 296: 1190-1194.15. de Serres FJ. Worldwide racial and ethnic distribution of alpha1-antitrypsin deficiency:summary of an analysis of published genetic epidemiologic surveys. Chest 2002; 122:1818-1829.16. Snyder MR, Katzmann JA, Butz ML, Wiley C, Yang P, Dawson DB et al. Diagnosis ofalpha1-antitrypsin deficiency: An algorithm of quantification, genotyping, andphenotyping. Clin Chem 2006; 52: 2236-2242.17. Bornhorst JA, Procter M, Meadows C, Ashwood ER, Mao R. Evaluation of an integrativediagnostic algorithm for the identification of people at risk for alpha1-antitrypsindeficiency. Am J Clin Pathol 2007; 128: 482-490.18. Prins J, van der Meijden BB, Kraaijenhagen RJ, Wielders JP. Inherited ChronicObstructive Pulmonary Disease: New Selective-Sequencing Workup for α1-AntitrypsinDeficiency Identifies two Previously Unidentified Null Alleles. Clin Chem 2008; 54:101-107.19. Dati F, Johnson AM, Whicher JT. The existing interim consensus reference ranges andthe future approach. Clin Chem Lab Med 2001; 39: 1134-1136.20. Ritchie RF, Smith R. Immunofixation. II. Application to typing of alpha1-antitrypsin atacid pH. Clin Chem 1976; 22: 1735-1737.21. Bossuyt X, Blanckaert N. Evaluation of interferences in rate and fixed-timenephelometric assays of specific serum proteins. Clin Chem 1999; 45: 62-67.22. Ward AM, White PA, Wild G. Reference ranges for serum alpha 1-antitrypsin. Arch DisChild 1985; 60: 261-262.23. Ritchie RF, Palomaki GE, Neveux LM, Navolotskaia O, Ledue TB, Craig WY.Reference distributions for the positive acute phase serum proteins, alpha1-acidglycoprotein (orosomucoid), alpha1-antitrypsin, and haptoglobin: a practical, simple, and clinically relevant approach in a large cohort. J Clin Lab Anal 2000; 14: 284-292.24. Alpha 1-antitrypsin deficiency: memorandum from a WHO meeting. Bull World HealthOrgan 1997; 75: 397-415.25. American Thoracic Society/European Respiratory Society Statement: Standards for theDiagnosis and Management of Individuals with Alpha-1 Antitrypsin Deficiency. Am J Respir Crit Care Med 2003; 168: 818-900.26. Eriksson S, Elzouki AN. Alpha 1-antitrypsin deficiency. Baillieres Clin Gastroenterol1998; 12: 257-273.27. Ricos C, Alvarez V, Cava F, Garcia-Lario JV, Hernandez A, Jimenez CV et al. Currentdatabases on biological variation: pros, cons and progress. Scand J Clin Lab Invest 1999;59: 491-500.Clinical References - Methods of Analysis 10-8α1-Antitrypsin: Methods Summary TableMethod 1: Electrophoresis; quantitative; estimation of α1-globulins by physical separation Principle of analysis: Proteins separate based on class (albumin, gamma globulins)Comments: Serum; good screen for severe α1-antitrypsin deficiency. The α1-globulinband is absent on the electrophoretogram.Method 2: Electroimmunodiffusion (EID); quantitative; quantitation by size ofimmunoprecipitate in gelPrinciple of analysis: Electrophoresis of α1-antitrypsin into antibody-containing agarose gel; height of gel pattern (rocket) proportional to α1-antitrypsin concentrationComments: Serum; kits not available commercially; mainly of historical interest Method 3: Radial immunodiffusion; quantitative; quantitation by immunoprecipitate in gel Principle of analysis:α1-Antitrypsin diffuses into gel containing antibody, forming aring-shaped immunoprecipitate; diameter of ring proportional to concentrationComments: Serum; commercial plates readily available, not widely used in diagnosticlaboratoriesMethod 4: Nephelometry or immunoturbidimetry; quantitative; quantitation byimmunoprecipitate in solutionPrinciple of analysis: Reaction of α1-antitrypsin with its specific antibody results inimmunoprecipitate, which has light-scattering properties; amount of light scatterproportional to α1- antitrypsin concentrationComments: Serum; commercial kits readily availableMethod 5: Electrophoresis of serum on acid starch gel, followed by antigen-antibody crossed electrophoresis; phenotyping; physical separation of α1-antitrypsin variants followed by immunoprecipitin reaction in agarosePrinciple of analysis: Two-step procedure:1. Separation of α1-antitrypsin variants on acid-starch gel by electrophoresis2. A second electrophoresis causes the separated variants to migrateperpendicularly to the first separation into agarose containing antibody to α1-antitrypsin;precipitin peaks form; the second electrophoresis is necessary for separation of the Z and S variantsComments: Serum; original, standard method for phenotyping; time consuming (1½days) and technically difficult; mainly of historical interestMethod 6: Isoelectric focusing; separation of α1-antitrypsin variants on agarose or polyacrylamide gel based on surface-property chargePrinciple of analysis:α1-antitrypsin variants are electrophoresed on polyacrylamide or agarose gels containing ampholines with a pH range of 4 to 5. The variants migrate under the electric field until they reach a pH equal to their isoelectric point.Method 7: Genotyping for the separation of α1-antitrypsin variantsPrinciple of analysis: DNA extracted from whole blood is subject to real-time PCRamplifying the S and Z alleles in separate tubes. The different primer design anddetection probes have been described in several published articles [16, 17].。