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vitro hemolysis on 24:1966-70. chemical values for serum. Clin Chem 1978; 5. Laessig RH, Hassemer DJ, Paskey TA, Schwartz TH. The effects of 0.1 and 1.0 percent erythrocytes and hemolysis on serum chemistry values. Am J Clin Pathol 1976;66:639-44. 6. Randall UG, Garcia-Webb P, Beilby JP. Interference by haemolysis, icterus and lipaemia in assays on the Beckman Synchron CX5 and methods for correction. Ann Cliii Biochem 199027:34552. 7. Fairbanks VF, Klee GO. Biochemical aspects of hematology. In: Tiets NW, ed. Textbook of clinical chemistry. Philadelphia: WB Saunders, 1986:1495-588. 8. Perlstein MT, Thibert RJ, Zak B. Bilirubun and hemoglobin alterations in several hydrogen peroxide generating procedures. Microchem J 197823:13-27. 9. Tietz NW, Pruden EL, Siggaard-Anderaen 0. Electrolytes. Op. cit. (ref. 7):1172-91. 10. Kreutzer HH, Penmngs AW, Punt JMHM, Verduin PA. Further studies on the determination of lipase activity. Cliii Chim Acta 1975;60:273-9. CLIN.CHEM.38/4, 577-580 (1992) Genetic Variants of a1-Antitrypsin and Hemoglobin Typed by Isoelectric Focusing in Preselected Narrow pH Gradients with PhastSystemTM Jan-Olof Jeppsson1 and Roland Einarsson2 In this method for automatically running and staining isoelectric focusing (IEF) gels, pre-made dehydrated polyacrylamide gels were rehydrated before assays run with the PhastSystem (Pharmacia LKB Biotechnology). The typing of genetic variants of hemoglobin and a1-antitrypsin in narrow pH gradients (pH 6.7-7.7 and 4.2-4.9, respectively) was simple, convenient, and reproducible. The clinically important variants of a1-antitrypsin (ZZ and SZ) were identified from serum or dried blood on filter paper. The fast screening of abnormal hemoglobin samples (HbS) for cases in clinical medicine was easily performed. The total analysis time for the phenotyping with conventional protein staining was -60 mm. Additional Keyphra.es: screening electroplioresis polyaciylamide gel . sickle cell disease gycohemogkbin Isoelectric focusing (JEF) of proteins in polyacrylamide gels has been used in various applications in clinical and forensic medicine to reveal detailed protein microheterogeneity not obtained with other techniques. Considerable time, effort, and skill are required to achieve acceptable and reproducible results with current techniques for JEF, which restricts the use of the technique in routine clinical settings. Many developments have been made in these areas, including the use of ultrathin gels to shorten the distance for heat transportation so that the voltage can be increased (for greater resolution), introduction of tailor-made ampholines with narrow pH gradients, and plastic backings to simplify the handling of the gels during staining and drying. Most of these new ideas for electrophoresis have been ‘Department of Clinical Chemistry, General Hospital, Lund University, 5-21401 MahnO, Sweden. 2Pharmacia Diagnostics AB, S-751 82 Uppeala, Sweden (author for correspondence). Received May 21, 1991; accepted February 3, 1992. integrated in PhastSystemm (Pharmacia LKB Biotechnology, Uppsala, Sweden), a dedicated system for horizontal electrophoresis and JEF in small gels, with automated fixation, staining, and destaining (1,2). Here we describe an IEF method performed with PhastSystem with a new dry polyacrylamide gel, which is soaked in a narrow-pH gradient solvent before use in analysis for genetic variants of a1-antitrypsin and hemoglobin. MaterIals and Methods Blood and serum samples for typing genetic variants of a1-antitrypsin and hemoglobin were obtained from the Department of Clinical Chemistry, General Hospital, MalmS, Sweden. PhastSystemand Gels The apparatus comprises electrophoresis and automated staining and destaining units, which can be programmed and operated independently (1,2). Dehydrated polyacrylamide gels were rehydrated before use by applying a droplet of ampholyte solution on a plastic surface and either placing the dry gel upsidedown on the droplet for 30 mm or using a reswelling casette (Pharmacia LKB Biotechnology). For typing of a1-antitrypsin, we applied 1.3 mL of Pharmalyte, 4.24.9, diluted 16-fold with water onto the plastic surface; for typing of hemoglobin, we applied 1.3 mL of Pharmalyte, 6.7-7.7, also diluted 16-fold with water. A spacer molecule (-alanine, 17 mmol/L) was added in some experiments to better separate the glycohemoglobin fraction from nonglycated hemoglobin (3). IEF of a1 -Antitrypsin Samples were prepared either from dried blood on filter paper or from serum, essentially as previously described (4, 5). Briefly, dried blood on filter paper (4-mm-diameter disc) was eluted in 15 pL of 1 mol/L glycine, pH 7.4, containing cysteine hydrochloride, 0.5 CLINICAL CHEMISTRY, Vol. 38, No. 4, 1992 577 For serum samples, we mixed 90 L of serum with 10 pL of cysteine hydrochloride (0.3 mol/L, pH 7.4). The blood and serum samples were incubated overnight at 4#{176}C. Cysteine eliminates the adducts bound to the active thiol group in a1-antitrypsin and thus increases the sharpness of the IEF patterns (4). This effect is especially pronounced for dried blood and old serum samples. Fresh serum can be run without pretreatment. Assay: Aliquots (3.6 L) of cysteine-reduced serum or eluted whole blood were applied to PhastSystem rehydrated polyacrylamide IEF gels according to the manufacturer’s instruction. IEF was carried out according to a modified electrophoresis program (Table 1) with PhastSystem. The time for separation was 30 mm. After electrophoresis the gels were fixed for 5 mm in trichloroacetic acid (200 g/L) and stained with either Coomassie Brilliant Blue (PhaatGel Blue R, 0.5 g/L) or automated silver staining (PhastGel Silver Kit), according to the methods given in the PhastSystem Owner’s Manual (1986). Gels were destained in methanol/acetic acid/water (3/1/6, by vol). The processed gel was treated with glycerol, 50 mi/L in 50 mLfL acetic acid solution, to prevent deforming of the gel. The stained gels were evaluated by visual comparison with a reference pattern. mol/L. IEF Samples were prepared according to a previously procedure (6). Briefly, blood samples were collected into EDTA-containing tubes or as capillary blood into heparinized microtubes. The blood cells were washed in isotonic saline, hemolyzed by shaking the cells in a mixture of water and Cd4 for 30 s, and then centrifuged. The clear supernate was diluted with water described Assay: concentration We applied of -5 g/L. 1 p.L of hemolysate to the rehy- drated polyacrylamide gels for IEF. The electrophoresis was performed according to the method given in Table 1. The protein precipitation and staining procedures were as described for a1-antitrypsin. Resufts and DiscussIon a1-Antitrypsin Pi-typing (protease inhibitor typing) of a1-antitrypsin was performed by IEF in a narrow pH gradient, 4.2-4.9. Figure 1 demonstrates a gel pattern with the typical microheterogeneity of some common a1-antitrypsin variants of clinical importance after Coomassie BrilTable 1. isoeiectrlc Focusing Analysis for a1-Antitrypsln (AT) and Hemoglobin (Hb) with Rehydrated Polyacrylamide Gels current, Step Prefocusing Sample application Separation AT Hb 578 Potential, 2 V 2000 200 Powei, w limp, mA 2.0 2.0 3.5 3.5 15 15 4 - - 4.48 6 - - 4.55 7 4.59 8 4.67 zz SZ FZ M1 Fig.1. Isoelectric focusing of variousa1-antltnjpslnPiphenotypes in the pH range4.2-4.9 after Coomassle Brilliant Bluestaining(anode CLINICAL CHEMISTRY, 5.0 7.5 3.5 5.0 y.axls Figure 1 also shows a schematic the position of the bands. The variants of particular clinical interest are the ZZ and SZ phenotypes, which are associated with a1-antitrypsin deficiency. These variants were easily detected with this reproducible narrow pH gradient, as were the heterozyliant Blue staining. drawing indicating gotes FZ and MZ. More than 75 different variants have been identified by IEF of serum (7). The protein separation can be further improved by hybrid IEF in Immobiline, with use of an ultra-narrow pH gradient (8). More than six subtypes of the M variant have been identified, three of which (Ml, M2, and M3) are clearly detected with the PhastSystem dry-gel technique. Furthermore, the typical homozygous patterns-two major bands and three minor ones-are easily detected (numbered 2-8 on Figure 1). These patterns are due to the variation of the content of sialic acid and the length of the polypeptide chain (9). We included in each exper- 75 iment a sample for heterozygote MZ (Figure 1) as a marker for phenotyping and for quality control. This heterozygote constitutes one of the most heterogenous 15 patterns, Duration, #{149}c V’h 15 15 Vol.38, No. 4, 1992 M1Z at top) Inthe schematic drawing, the open bass represent the Z-aIIele products. The anodal major Z-isoform is superimposed on the majorcathodal S-isoform.The isoelectric points and isoform number for the M Isoforms are given on the being a combination of the normal M-allele Z-allele products of the human a1 -Pi gene. The resolution on this ultrathin polyacrylamide gel is good, despite the very short focusing time (30 mm). Using dried blood on filter paper (Guthrie spots) in and the mutant 2000 2000 p1 4.42 M1 of Hemoglobin to a hemoglobin 0 N:o 450 550 combination with the sensitive silver-staining method can help identify low-a1-antitrypsin-concentration phenotypes (Pi ZZ or SZ) in future screening programs. The PhastSystem typing procedure is very simple and rapid compared with the hybrid IEF method based on miniaturized immobilized pH-gradient gel (8); the latter gives excellent resolution but is too complex and time consuming for screening purposes. An extensive review of the genetic, functional, and medical aspects of a1-antitrypsin was published recently (7), demonstrating the need for phenotyping. Hemoglobin Screening requested for abnormal hemoglobins is a frequently in the clinical laboratories (10). For procedure optimal separation, high-resolution IEF polyacrylamide gels in a narrow pH gradient have been used. More than 500 hemoglobin variants have been characterized with defined amino acid substitutions (11). The most common of these endemic variants is HbS, but HbC and HbE are also common. Many hemoglobin variants are identified in single families or only a few families. Figure 2 demonstrates the hemoglobin patterns obtained with PhastSystem high-resolution IEF in a nar- row pH gradient, 6.7-7.7; a schematic drawing is in- cluded for comparison. The lane to the left represents a normal hemolysate gel pattern with the major HbA1 fraction (pI 7.12) and the normal amounts of HbF and HbA2 (p1 7.42) fractions (-1% and -2-3%, respectively). Anodal to HbA1 is the glycated form of hemoglobin, HbA1C (normal concentration 4-5.3%). The most anodal protein fraction is HbA3, a gluthathione adduct, which increases with time and storage of the samples. The second lane (Thal) shows increased HbA2, which is typical for /3-thalassemia minor. The third lane demonstrates a sample from a diabetic patient with increased HbA1C and HbF, accidentally found in ion-exchange chromatography for quantification of glycohemoglobin (12). The last three lanes show the most common hemoglobin variants AE, AS, and AC. HbE and HbC are superimposed on the HbA2 fraction, E being slightly more anodal and C more cathodal than A2. The commonly encountered abnormal hemoglobins were well resolved in the actual pH gradient 6.7-7.7, and further gel electrophoresis in alkaline or acid buffers was not necessary (13). The small mobility differences, which depend on the amino acid substitutions, with the narrow can be detected pH gradient used. Electrofocusing in gels rehydrated in diluted and pre-made polyacrylamide defined Pharmalyte solutions gives reproducible and reliable results. Furthermore, the high field strength eliminates problems of diffusion and allows the extremely high resolution necessary for the typing procedure. --- In conclusion, the total time for analysis is very short because of the short separation time and because of the rapid and efficient staining and destaining in the development unit, which makes the typing on PhastSystem - suitable A clinical diagnosis. The techniques intended for screening purposes. Characterization of unusual or new variants, of course, requires specialized laboratories with knowledge of peptide mapping, polymerase chain reaction, and DNA sequence techniques. A1 The skillful technical assistance appreciated. - - for routine presented 0 p1 A1 7.12 F S - 7.42 A2 N Thai Diab AE AS AC Fig. 2. Isoelectnc focusing patterns of hemolysates of a control and patients’ samples in the pH range 6.7-7.7 after Coomassie five Brilliant Blue staining (anode at top) In the schematic drawing of the six different sample types (normal, p-thalassemia, diabetic, and three hemoglobin vanants),the isoelectricpoints and typeof hemoglobinproteinbandare shown on the y-axis are mainly of Anna Arnetorp is highly References 1. Olsaon I, Axio-Fredriksson U-B, Degerman M, Olsson B. Fast horizontal electrophoresis. I. Isoelectric focusing and polyacryl-. amide gel electrophoresis using PhastSystem’. Electrophoresis 1988;9:16-22. 2. Olsson I, Wheeler R, Johansson C, et al. Fast horizontal electrophoresis. II. Development of fast automated staining procedures using PhastSystem”. Electrophoresis 1988;9:22-7. 3. Jeppsson J-O, Franz#{233}n B, Nilsson K-O. Determination of the glycosylated hemoglobin fraction HbAIC in diabetes mellitus by thin-layer electrofocusung. Sci Tools 1978;25:69-72. 4. Jeppeson J-O, Franzen B. Typing of genetic variants of a1antitrypsun by electrofocusing. Clin Chem 1982;28:219-25. 5. Jeppason ,J-0, Sveger T. Typing of genetic variants of a1antitrypsin using dried blood from the Guthrie test. Scand J Cliii Lab Invest 1984;44:413-6. 6. Jeppsson J-O, KAilman I, Lindgren G, Fagerstam G. HbLinkoping (-36 Pro-Thr): a new hemoglobin mutant characterized by reversed-phase high-performance liquid chromatography. J Chromatogr 19&4;297:31-6. 7. Cox-Wilson D. a1-Antitrypsin deficiency. In: Scriver CHR, CLINICALCHEMISTRY,Vol.38, No.4, 1992 579 Beaudet AL, Sly XS, Valle D, eds. The metabolic basis of inherited disease, 6th ed. New York: McGraw Hill, 1989:2409-38. 8. Alonso A. Human alpha1-antitrypsin subtyping by hybrid isoelectric focusing in miniaturized polyacrylamide gel. Electrophoresis 1989;10:513-9. 9. Jeppsson J-O, LiIja H, Johansaon M. Isolation and characterization of two minor fractions of a1-antitrypsun by high performance liquid chromatofocusing. J Chromatogr 1985;327:173-7. 10. Basset P, Benzard Y, Garel MC, Rosa J. Isoelectric focusing of human hemoglobin: its application to screening, to the character- ization of 70 variants, and to the study of modified fractions of normal hemoglobuns. Blood 1978;51:971-82. 11. H’iiemin THJ. Policies of the International Hemoglobin Information Center. Hemoglobin 1991;15:139-245. 12. Jerntorp P, Sundkvist G, Fex G, Jeppsson J-O. Clinical utility of serum fructosamune in diabetes mellitus compared with hemoglobin A1. Clin Chim Acta 1988;175:135-42. 13. Huisman THJ, Jouxis JHP. The haemoglobinopathies, techniques of identification. New York: Marcel Dekker, 1977. CLIN. CHEM. 38/4, 580-584 (1992) Rapid Immunometric Measurement of C-Reactive Protein in Whole Blood Petter Urdal,’ Stig M. Borch,2 Sverre Landaas,’ May B. Krutnes,2 We examined an instrument-free test for C-reactive pro- tein (CAP) in whole blood. The NycoCard CRP Whole Blood test uses a ceil-solubilizing dilution liquid, a membrane-bound antibodythat binds CAP, and a gold-conjugated antibody for making visible the bound CRP. We obtained essentially identical dose-response curves in citrate-, hepann-, and EDTA-treated blood. CVs were 6.7-12.5% within series and 10.1-14.7% between series. The detection limit was 12 mg/L. lntralipidadded to blood increased measured CRP by 10-20%, whereas no change was seen with added bilirubin, added serum amyloid P component, or the presence of rheumatoid factor. In 234 patients’ blood samples the results of the NycoCard Whole Blood test correlated well (r = 0.96) with those of a turbidimetric serum method. The test allows reliable measurement of CAP from a small volume of whole blood (25 L) without using expensive equipment; it should be useful for decentralized testing in hospital departments, emergency units, and primary health care centers. AddItional Keyphraeee: gated antibody intermethod comparison - gold-conju- C-reactive protein (CRP) is one of the more characteristic acute-phase proteins and is considered a reliable indicator of disease activity in various clinical conditions (1). Its concentration in blood increases rapidly by as much as 1000-fold upon exposure to various inflammatory stimuli, decreasing rapidly when the stimulus declines, e.g., after effective antibacterial treatment (2). CRP has been used successfully for clinical diagnosis and monitoring of a variety of infections and diseases, including infections caused by bacteria, fungi, and viruses (3-7); intercurrent infections in leukemia (8,9) and ‘Department of Clinical Chemistry, Ullevtl Hospital, N-407 Oslo 4, Norway; 2Reaearch and Development Unit, Diagnostics Division, Nycomed Pharma AS; and 3Department of General Practice, University of Oslo, Norway. Received July 22, 1991;accepted February 6, 1992. 580 CLINICALCHEMISTRY,Vol.38, No.4, 1992 Geir O Gogstad,2 and Per Hjortdahl3 systemic lupus erythematosus (10); noninfectious inflammatory diseases such as rheumatoid arthritis (11); and diseases with cellular necrosis such as myocardial infarction (12,13). Measurements of CRP are especially useful in distinguishing viral from bacterial infections (3,5,14). Among the methods used to measure CRP in serum are radioimmunoassay (15); radial immunodiffusion (16); latex agglutination (17), which also is available in a quantitative microtiter version (18); lipid agglutination (19); turbidimetry (16, 20, 21); nephelometry (16); particle-enhanced turbidimetry (22); enzyme immunoassays (23, 24); and fluorescence polarimetry (25). These methods assay CRP in serum, either as instrument-based quantifications or as instrument-free, qualitative to semiquantitative agglutination tests. The acute nature of many diseases in which CRP is relevant for diagnosis and monitoring requires a rapid, easily interpreted, instrument-free, quantitative test that may be applied directly to whole blood. We present here a test with such characteristics. Materials and Methods Materials Bilirubin was obtained from Sigma Chemical Co. (St. Louis, MO), serum amyloid P component from Calbiochem Corp. (San Diego, CA), and Intralipid from KabiVitrum (Stockholm, Sweden). Reference Preparations We used the 1st International Standard (World for human CRP (National Institute for Biological Standards and Controls, London, U.K.) to establish the concentration of CRP in the reference preparations. These preparations contained CRP at <5 to 1000 mg/L of plasma: purified CRP (Scipac Ltd., Kent, U.K.) added to whole blood (anticoagulated with citrate, heparin, or EDTA) obtained from healthy donors. We similarly prepared controls containing CRP concentrations of <5,30, and 125 mg/L in plasma, using blood from one donor, and stored these at 4#{176}C. The controls were used for estimating within- and betweenHealth Organization)