Serum protein electrophoresis: Italian survey 1986

Transcription

1 Ann Clin Biochem 1989; 26: Serum protein electrophoresis: Italian survey 1986 F AGUZZI, C PETRINI'" and C GASPARRO From the Laboratorio Analisi Ospedali di Broni e Stradel/a, Pavia, and " Laboratorio di Biochimica Ospedale San Carlo Borromeo, Milano, Italy SUMMARY. In 1986 the Protein Commission of the Italian Society of Clinical Biochemistry (SIBIOC) carried out its second survey on the use of serum protein electrophoresis in Italian laboratories. Three serum samples plus a questionnaire were sent to the 253 laboratories which agreed to take part. The three samples had the following characteristics: Serum I: a 60 gil IgM-lambda monoclonal component (MC); Serum 2: an artificially split alpha-2 zone; Serum 3: a very faint lambda chain Me. These features were chosen to assess (a) the type of report; (b) the resolution quality of the electrophoretic technique; and (c) the laboratory capacity to detect a small Me. The most significant features revealed by the survey were: (a) the poor capacity of assessment of the small MC in Serum 3 (only detected by 23 1% of laboratories); (b) the discouraging tendency to delegate electrophoretic diagnostic interpretation to ward physicians (44-4% of participating laboratories provided only densitometric values and graphs). Serum protein electrophoresis (SPEP) is a widely requested test in many European countries, but the information that may be obtained and the clinical indication for use of the test are not always well known. There are essentially two ways of performing SPEP: the first is characterised by more or less automation and by reporting the results as densitometrically measured relative or absolute concentrations of the five classical zones. The second involves high-resolution separation and reporting as visual interpretation of qualitative and/or semi-quantitative alterations. 'Five-zones' electrophoresis (so-called 'microzone') is usually carried out on cellulose acetate as the support medium, whereas high-resolution electrophoresis utilises agarose gels, but each support medium may be used for either method. The difference lies in the belief that the first method is capable of providing clinically useful quantitative information by using densitometry, a view that has been questioned;' 3 while the second is based on the belief that the clinical usefulness of SPEP relies on qualitative changes, detected by visual inspection. Many workers believe that only visual inspection can detect small qualitative changes and attribute single zones to specific proteins.l-v' Correspondence: Dr C Petrini. Although the diagnostic value of SPEP is high in many clinical fields, the most important field is in the monoclonal gammopathies, because they are fairly common (» 3% in people aged over 60, with ranges of 0 28%-12% depending on the origin and selection of the population':") and malignant gammopathies need to be distinguished from monoclonal gammopathies of undetermined significance (MGUS). The most important differential criteria are based on the increase ofmonoclonal component (MC) concentration and the presence of Bence-Jones proteinuria!", Furthermore, the frequently unrecognised light chain disease often shows minimal bands on SPEP. Normal and pathological control serum samples are available from many companies for quantitative densitometry but not for the detection of qualitative alterations. In 1985 the Protein Commission of the SlBIOC took the initiative of sending some laboratories, chosen by the regional organisation of the SlBIOC, three samples of serum with qualitative changes (alpha-i-antitrypsin heterozygosity; alpha-2-zone splitting due to haptoglobin-haemoglobin complexes; a small MC).lo We now report on a second survey carried out in A letter was sent out to 600 hospital laboratories asking for preliminary participation: 253 (42%) replied and agreed to participate, and were sent three serum samples and a reply form. 249

2 250 Aguzzi, Petrini and Gasparro Materials and methods SERUM SAMPLES The three samples were sent in colour-coded test tubes and had the following characteristics (Fig. I): Serum sample 1 was obtained from plasma pheresis ofa Waldenstrom's macroglobulinaemia patient with a high IgM lambda MC (about 60 g/i densitometrically) and a second very small monomeric IgM lambda MC (this serum was kindly provided by Dr P Fornasari, Ospedale San Matteo, Pavia). Serum sample 2 was a pool of 5 similar serum samples in which the bands due to alpha-2 macroglobulin and haptoglobin were clearly recognisable in the alpha-2 zone. To enhance the splitting of the alpha-2 zone, some haemolysate was added until a faint pink colourwas obtained. Serum sample 3 was from a single apparently healthy volunteer, with a small quantity of free monoclonal light chains added (lambda type in three different states of aggregation obtained from a light chain disease urine). The resulting band was densitometrically determined at <0 7 g/i and was at the limit of visibility. The three samples were stabilised according to Milford Ward et al," and despatched in quantities of 300 JlL (Sera 2 and 3), and 500 JlL (Serum I). These three sampleswere chosen for the following reasons: Serum 1- the very obvious abnormality would test the ability of the laboratory to report it and to proceed with further investigations; Serum 2- the abnormality would be detectable only if a technique ofsufficient resolution was used and, furthermore, it would test the ability to recognise a not uncommon artefact; A B c o FIG. I. Electrophoretic patterns of: (A) Serum 2; (8) Serum I; (C) Serum 3; (0) Urine providing light chains added to Serum 3. Serum 3-the abnormality, very difficult to recognise, would test the ability ofthe laboratory to detect a small but potentially clinically important Me. THE REPLY FORM The form consisted oftwo parts: the first section instructed participants to treat the serum samples initially exactly according to their routine procedure. In a second step the samples had to be treated as if sent by the clinician with the enquiry 'monoclonal gammopathy?' The second section of the form sought to obtain details about the size of the hospital, the characteristics of the electrophoretic technique and the diagnostic aptitude of the laboratory's staff. Results The hospitals were divided into three groups based on the number of beds, and within each group the report of abnormalities in the three samples was recorded together with the following two characteristics: (a) whether the report was limited only to the densitometric numerical data; (b) whether the laboratory independently identified all the MCs it detected. These two approaches were chosen because we believe the former is characteristic of a totally 'passive atitude', while the latter reveals a tendency to consider the SPEP the first step of a diagnostic procedure. Table I shows that the results from the large and medium-sized hospitals were similar; those from the smaller ones being worse. The difference is even greater if related to the number of beds. The number of SPEP/year/bed performed was From the technical point of view the most interesting data concern the length of electrophoretic migration and the separation ofthe beta zone in two bands. The recognition ofthe abnormalities in samples 2 and 3 is correlated both with the length ofthe separation (Table 2) and with the presence of beta zone splitting (Table 3). Six laboratories (3'8%) gave a visual interpretation only, while the remaining 154(96'2%) used the densitometer, often visually checking the graph, or the cleared strip, or the wet (uncleared) strip or viewing the dried strip against a strong light source (for the supported cellulose acetate) (Table 4). The ability to recognise the abnormalities was very high, 100% for the MC, by visual interpretation, whereas among the checks the best results were obtainedby inspectingthe stripwhen

3 Serum protein electrophoresis in Italy 251 TABLE 1. Findings according to size of hospital. Of 160 laboratories, 138 (86'3%) reported the number of beds. Bed occupancy totalled in 1986 (44 I 19 in 1985) Number of beds <400 n (0/c,) > 1000 n (%) n (%) No. of laboratories 68 (49'2) Bed occupancy (17'1) Only densitometric 35 (51'4) Independently identified Me 31 (45'2) Abnormality reported: Serum I 32 (47) Serum 2 II (16) Serum 3 II (16) TABLE 2. Effect of length of separation Length of the separation (mm) < >30 n (%) n (%) n (%) Laboratories 24 (IS) 90 (56'3) 46 (28'8) Only densitometric 14 (58) 42 (47'0) 15 (33'0) Abnormality reported Serum I 8 (80) 48 (100'0) 31 (100'0) Serum 2 1 (10) 14 (29'1) 29 (93'5) Serum 3 I (10) 18 (37'5) 18 (58'0) The percentages regarding reporting of abnormalities refer to those laboratories that did not give a merely numerical report. TABLE 3. Splitting of the beta zone Splitting of the beta zone Absent Present n (%) n (%) Laboratories 98 (61'3) 62 (38'7) Only densitometric 56 (57'1) 15 (24'2) Abnormality reported Serum I 38 (90'5) 47 (100'0) Serum 2 6 (14'3) 30 (63-8) Serum 3 10 (23-8) 27 (57.4) The percentages regarding reporting of abnormalities refer to those laboratories that did not give a merely numerical report. 50 (36'2) 20 (14'6) (44'2) (38'6) 19 (38) 5 (25) 35 (70) 15 (75) 31 (62) 14 (70) 15 (39) 6 (30) 16 (32) 7 (35) wet or viewed against a light source, for supported and dried media. Fifty-eight laboratories (36'8%) performed identification tests for at least one of the abnormalities in the samples. It is important to note that 18 of these (31%), evidently relying on a negative outcome of the screening electrophoresis, did not investigate Serum 3. In Table 5, the data regarding the quality ofidentification are set out. Table 6 presents the influence ofthe techniques used on identificationquality. As it can be seen, it is impossible to evaluate the effectiveness of the immunochemical (quantitative) techniques and of the kappa/lambda ratio, while immunofixation gives slightly better results than immunoelectrophoresis, especially for Serum 3. Tables 7 and 8 show the results ofthe immunochemical quantification (from 33 laboratories) for the Ig in the three sera and the relative densitometric values for the albumin, alpha-i zone, alpha-2 zone ofserum 3 and for the gamma zone (nearly all MC) of Serum I. The scatter was very high, but it is noteworthy that the densitometric gamma zone quantification of Serum I was more precise than with the immunochemical evaluation. The capacity to detect the presence of MCs is directly shown by the survey's results; in addition the reply form proposed a series of questions aimed at investigating: (a) whether the presence ofqualitative changes is reported; (b) whether the MCs are identified independently of the request; (c) whether the MCs are then quantified; (d) whether, when an MC is discovered, urine is requested by the laboratory in order to test for Bence-Jones proteinuria; (e) whether a file of MCs is kept; (I) whether MC concentration is recorded in the file. The results are summarised in Table 9.

4 252 Aguzzi, Petrini and Gasparro TABLE 4. Methods of investigation Reading Total n Reporting of abnormalities Serum I Serum 2 Serum 3 n ('Yn) n (%) n (%) Visual 6 (H) Densitometric 154 (96' 3) Densitometric with visual check of: Scan 116 (72-5) Cleared strip 100 (62'5) Wet strip 47 (29'4) 6 (100'0) 5 (83'3) 6 (100'0) 81 (52'6) 31 (20'1) 31 (20'1) 59 (50'9) 22 (19'0) 25 (21'6) 53 (53'0) 16 (16'0) 18 (18'0) 36 (76'6) 22 (46'8) 18 (38' 3) TABLE 5. Identification tests. Fifty-eight laboratories (36'8 1.,) performed an identification test on at least one serum Reports Correct Partial Mistaken n (%) n (%) n (%) n (%) Serum I 58 (100'0) 52 (89'7) 6 (10'3) 0 (0) Serum 2 39 (67'2) 29 (74'0) 0 (0) 10 (25'6) Serum 3 40 (69'0) 26 (65'0) 5 (12'5) 9 (22-5) In Serum I, partial identification refers to a mistaken light chain or to the additional detection of an IgA or IgG MC; in Serum 3 it refers to the reply 'IgG lambda'. Mistaken identification invariably refers to the failure to identify the presence of abnormalities. IC = immunochemical quantification; IF = immunofixation; IEP = immunoelectrophoresis. Fifteen laboratories used more than one method: 10 IF + IEP; 2 K/lambda + IEP; I IC + K/lambda; I IF + K/lambda; I K/lambda+ IF + IEP. Discussion An evaluation of both technical and professional performance can be made. As far as the technique is concerned, the recommendations ofthe Protein Commission of the SIBIOC12 are confirmed: the best results are obtained with a longer electrophoretic separation, splitting the beta zone into two bands and practising visual inspection of strips. The densitometric values show very poor precision and should only be used for the quantification of MCs, where the immunochemical techniques, especially the RID, give very bad results. For the identification of MCs, the superiority of the immunofixation technique is confirmed. As far as professional considerations are con-

5 Serum protein electrophoresis in Italy 253 TABLE 8. Relative densitometric values (n = 144) Mean Range SD CV ;', Serum 3 Albumin , Alpha-I Alpha , Gamma Serum 1 Gamma TABLE 9. Diagnostic aptitude of participants Reported an abnormality in: Serum I Serum 2 Serum 3 Reported only numerical values Independently identified MCs Independently quantified MCs Independently searched for BJ Recorded MCs Recording MC concentration Reported on the question 'monoclonal gammopathyt La bora tories n (%) cerned, it is essential to stress that the only aim of the clinical laboratory is to give clinicians diagnostically useful information. This was not the case for the 71 laboratories (44-4%) that gave only the densitometric values and graphs, thus delegating the interpretation to ward physicians. This procedure, in addition to abdicating professional responsibility, is extremely inefficient because although Serum 1 was obviously abnormal, the abnormality of Serum 3, equally important from the clinical point of view, was detected only when the laboratory staff directly inspected the electrophoretic separation, since almost all densitometers produce graphs without small changes being identified. Fewer than 60% independently identify the detected MCs, 42% quantify them, and 35% request urine to test for Bence-Jones proteinuria. An MC file is kept by 58%, but only 42% record their concentration, the others therefore losing the possibility to recognise change in concentra- 61 (54' 3) (22'5) (23'1) (44'4) (57'5) (49'3) (35'0) (58'7) (41'8) (38'1) tion ofmcs, one of the clinically most important criteria for monitoring the evolution of malignancy. These data show scanty understanding of the diagnostic purpose of the SPEP, but it is even more discouraging that only 61 (38%) indicated their willingness to carry out further investigations after the first routine step in orderto reply to the precise question 'monoclonal gammopathy'. In our opinion this last point requires deep reflection, because, if not corrected, it can only lead to a loss of the laboratory staffs professional role and of the usefulness of their work. References 1 Jeppson JE, Laurell CB, Franzen B. Agarose gel electrophoresis. Clin Chem 1979; 25: Aguzzi F, Jayakar SD, Merlini G, Petrini C. Electrophoresis: cellulose acetate vs agarose gel, visual inspection vs densitometry. (letter) Clin Chem 1981; 27: Kahn SN, Strony LP. Imprecision of quantification of serum protein fractions by electrophoresis on cellulose acetate. Clin Chem 1986; 32: Whicher JT, Spence CEoSerum protein electrophoresis-an outmoded test? Ann Clin Biochem 1987;24: Fine JM, Lambin P, Deryicke C et at. Systematic survey of monoclonal gammopathies in the sera from blood donors. Transfusion 1979; 19: Axelsson V, Bachmann R, Hallen J. Frequency of pathological patterns (M components) in 6995 sera from an adult population. Acta Med Scand 1966; 179: Sinclair D, Sheehan T, Parrot DMV, Scott DI. The incidence of monoclonal gammopathy in a population over 45 years old determined by isoelectric focusing. Br J Haemato/1986; 64: Kyle RA. Monoclonal gammopathy ofunder determined significance (MGUS): a review. Clinics in Haematology 1982; II: Waldenstroem J. Studies on conditions associated with disturbed gammaglobulin formation (gammopathies). Harvey Lectures 1961; 56: Aguzzi F, Petrini C, Marchini MA. L'elettroforesi delle sieroproteine in Italia-Un'indagine preliminare. Biochim Clin 1986; 10: II Milford Ward A, White PAl, Thomson RA et at. Preparation of calibration material for specific protein assay (SPS-OI). Ann Clin Biochem 1984; 21: L'elettroforesi delle sieroproteine I e II. Raccomandazione provvisoria SIBIOC. Biochim Clin 1985; 9: Acceptedfor publication 20 June 1988