Dr. AnDreA CiApini and the interlAb SCientifiC teAm
protein electrophoresis and immunofixation: from the laboratory to the clinical practice
Dr. Andrea Ciapini and the Interlab Scientific Team
Protein electrophoresis and immunofixation: from the laboratory to the clinical practice
Preface Over the past decade, progress in the field of clinical biochemistry, and serum protein and urinary protein electrophoresis in particular, has been so dramatic that even the experts have been taken by surprise. The purpose of this book is to provide a general survey of the techniques and instrumentation, i.e. the methods and equipment currently available to clinical chemists who work in laboratories, for the purpose of obtaining analytical results. This book is addressed to all operators in the field of electrophoresis, who wish to acquire a more thorough and systematic understanding of the analytical problems posed by modern “electrophoresis�. It also caters for others working in this sector who wish to keep abreast of developments or resolve doubts; by reading this book, they will be stimulated to consider the different options for applying both traditional techniques and more recent ones. All the staff at Interlab have contributed to this book, but we are particularly indebted to Dr. Maria Scala Bernalda for giving us the benefit of her technical and scientific expertise.
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Table of Contents 1. Introduction. ........................................................................... page 1 2. Research on monoclonal gammopathies in the clinical laboratory......................................................................... page 3 2.1 EBM and EBLM...................................................... » 3 3.
The “right kind of” electrophoretic profile................... 3.1 Introduction to specific proteins............................. 3.2 The right approach to electrophoresis................... 3.3 Protein changes........................................................ 3.4 The specific proteins most often subject to visual inspection.................................................................. 3.5 Data expression........................................................ 3.6 Definition of a monoclonal component (MC)........ 3.7 The process of research for studying monoclonal components . ............................................................ 3.8 Monoclonal components in electrophoretic traces 3.9 Qualitative anomalies not attributable to MCs, but which simulate an MC...................................... 3.10 List of factors that can adversely affect the quality of electrophoretic findings....................................... 3.11 Is the Microgel system capable of producing the good quality serum proteins electrophoretic profiles (EPs), according to the requirements of the SIBioC(1) 05 committee?...............................
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9 9 10 11
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11 11 12
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13 13
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14
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14
» 15
(1) SIBioC - Società italiana di biochimica clinica e biologia molecolare = Italian Clinical Biochemistry and Molecular Biology Society
4.
Specific proteins identifiable........................................... page 4.1 Specific proteins: morphological appearance, position and function .............................................. » 4.2 Serum protein profile of the most frequently occurring specific proteins...................................... » 4.3 Serum protein profile of the least frequently occurring specific proteins...................................... »
21 21 26 27
5. Standardisation of the Microgel system ....................... page 29 5.1 The Microgel system as a means of standardisation » 29 6.
Immunoglobulins............................................................. page 6.1 Introduction............................................................. » 6.2 Cell bases.................................................................. » 6.3 Structural, immunochemical and functional bases » 6.4 Summary of the main physicochemical, structural and functional characteristics of the 5 classes of human immunoglobulins........................ »
7.
How polyclonal and monoclonal Igs are distributed in electrophoretic runs..................................................... page 87 7.1 Polyclonal Igs........................................................... » 87 7.2 Monoclonal Igs......................................................... » 88
8.
Where MCs can migrate in electrophoretic profiles..... page 8.1 Position..................................................................... » 8.2 Percentage in terms of position and frequency..... » 8.3 Prevalence according to isotype, age and concentration............................................................ »
9.
Monoclonal components in serum and urine................ page 97 9.1 Electrophoretic definition of a monoclonal component................................................................ » 97 9.2 Immunological definition of a monoclonal component................................................................ » 99
10. The concept of electrophoretic semeiotics, as applied to immunoglobulins......................................................... 10.1 Introduction............................................................. 10.2 Serum protein and urinary protein profile, as a means of identification of immunoglobulin related pathologies . ................................................ 10.3 Proteinurias: classification according to Boylan 10.4 Immunoglobulin electrophoretic profiles.............. VI
49 49 50 57 74
91 91 92 9
» 103 » 103 » 106 » 108 » 111
11. Homogeneous band typing methods.............................. page 121 11.1 List of methods and comments............................... » 121 12. Bence-Jones proteinuria.................................................. page 131 12.1 Classification of proteinurias according to type............................................................................ » 131 12.2 Persistent proteinurias............................................ » 131 12.3 Pathogenetic classification of proteinurias............ » 132 12.4 Qualitative methods used to identify the type of proteinuria........................................................... » 134 12.5 The choice of support.............................................. » 135 12.6 Urinary electrophoretic profile: specific proteins » 139 12.7 Technical and methodological requirements for the right approach to urinary protein profiling.... » 140 12.8 Free light chains in electrophoresis........................ » 140 12.9 Visual and qualitative interpretation..................... » 143 12.10 Visual inspection as the only possible means of interpretation........................................................... » 144 12.11 Methodological details........................................... » 147 12.12 Concentration of biological fluids........................ » 150 12.13 Bence-Jones protein size . ..................................... » 155 12.14 Highlighting of monoclonal components............. » 156 12.15 Bence-Jones antiprotein antisera......................... » 159 13. The clinical significance of the presence of MCs in serum and urine............................................................... page 167 13.1 Identification of a homogeneous MC band........... » 167 14. Criteria for the differentiation of MGUS and MM(2)... page 169 14.1 Differential criteria.................................................. » 169 14.2 Classification of monoclonal gammopathies......... » 170 15. Which antisera should be used for Bence-Jones protein typing?........................................................... page 1 73 15.1 The choice of antisera.............................................. » 173 (2) MGUS = Monoclonal Gammopathy of Undetermined Significance; MM = Multiple Myeloma
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16. Primary and secondary immunodeficiencies................ page 177 17. CAP (College of American Pathologists) guidelines and laboratory assessments concerning patients with monoclonal gammopathies.............................................. 17.1 Differential criteria and guidelines........................ 17.2 Follow-up of asymptomatic patients...................... 17.3 Follow-up of patients with hyperviscosity syndrome.......................................................................... 17.4 Urinary follow-up....................................................
» 183 » 183 » 189 » 190 » 191
18 Conclusions....................................................................... page 193 19 Bibliography..................................................................... page 195
1. Introduction The discovery of homogeneous immunoglobulin components, in electrophoretic profiles of serum and urinary proteins, is an increasingly common occurrence. Many factors can explain this situation: • The increasing use of high-resolution convection systems • Standardisation of buffers • Standardisation of methodologies • Monitoring of electrophoretic parameters • The use of sensitive stains • Visual observation of migrations • Routine requests for protein electrophoresis • The occurrence of new pathologies. Initially, the monoclonal immunoglobulin component (M-protein) was almost exclusively regarded as an actual or predictive sign of lymphoproliferative disorders, such as myelosis and Waldenström’s macroglobulinaemia. Electrophoresis with more than one M-protein component (oligoclonal profiles) was mainly described and taken into account in electrophoresis of cerebrospinal fluid proteins, as aid to diagnosis of multiple sclerosis.
In such cases, where an oligoclonal profile was reported, that was mainly based on the morphology of the trace, researchers being unable or unwilling to proceed with definite immunoidentification of the actual immunoglobulin situation of those homogeneous components. The reason for these methodological – and therefore symptomatological – uncertainties was undoubtedly the fact that immunoelectrophoresis was the only method available at the time. Over the last twenty years, the increasing use of immunofixation techniques, the sensitivity and resolutive power of which are much greater than in the case of immunoelectrophoresis, has made it possible to identify monoclonal/oligoclonal components, irrespective of the extent to which they are represented in the absolute and with respect to the remaining polyclonal immunoglobulins. Parallel with the refinement of protein immunoidentification techniques, clinicians have increasingly focused on the symptomatology of the possible diagnostic, evolutionary and predictive value of monoclonal/oligoclonal components in serum, body fluids and urine. Because we favour the immunofixation technique, whilst acknowledging the very important contribution made by immunoelectrophoresis, our aim in this book is to examine the salient features of monoclonal components, starting with protein electrophoretic profiles, - a way of discovering M-proteins components – and then place them in the right clinical perspective.
2. Research of monoclonal gammopathies in the clinical laboratory Clinical laboratory research on Monoclonal Gammopathies (MGs), based on the discovery and identification of M-protein or the more common but not recommended term monoclonal component, MC (MCs), is one of the most important contributions that has been made to clinical medicine to date. It has provided the inspiration for this book, the purpose of which is to follow this laboratory process which even Interlab – with its integrated tool-kit systems – recommends to users as an effective support for this research. The first question every laboratory has to answer is how to determine the efficacy and efficiency of the system used – in other words, how to optimise efficiency and reliability (EBM and EBLM). 2.1. EBM and EBLM Evidence Based Medicine (EBM): EBM has been defined as “the conscientious, judicious and explicit use of best evidence in making decisions about care of [individual] patients”.
This definition by Sackett et al. specifically includes the laboratory as an integral part of the physician’s decision-making process. EBM has also been defined as a life-long personal updating process. Both definitions are applicable to laboratory medicine, in which laboratory doctors support clinicians in the care of patients. It can be seen from the foregoing that medicine is a field in a continuous process of discovery, evolution and change. Therefore it is crucial to ensure that practice is based on the best available evidence, and that an opportunity is provided for the adoption of new procedures, the benefit of which has been demonstrated. The practice of EBM calls for the integration of individual clinical experience and the best clinical evidence obtainable from systematic research. Conversely, the concept of Evidence Based Laboratory Medicine (EBLM) needs to be broached with great care. It has been maintained, from various quarters, that laboratory medicine – if misused – is pointless and even counter-productive in terms of people’s health. The introduction of EBLM would enable one to achieve three results: 1) to improve the quality of laboratory tests; 2) to discourage proliferation of diagnostic procedures the efficacy of which has not been adequately demonstrated; 3) to adapt insurance companies and National Health Services to the requirement for documentary evidence that the cost of tests is proportional to their utility. In order for each laboratory test to be evaluated, one would need to have a “Gold Standard” test, i.e. the most reliable test in terms of efficacy and diagnostic efficiency, to act as a reference point. Such a reference point is often unavailable, as has been the case in the field of electrophoresis and immunofixation in particular. There is as yet no IF system capable of detecting all MCs. We therefore have to refer to the best possible documentary evidence, and in particular, the work of Alper and Johnson on immunofixation, published in 1975. The College of American Pathologists Conference XXXII, states the following in its “Guidelines for laboratory diagnosis and monitoring of monoclonal gammopathies”, Chicago, III, May 29-31, 1999 states :
“Immunofixation electrophoresis (IFE) is the method of choice to identify monoclonal components.” David F. Keren, MD, Arch. Pathol. Lab. Med. - Vol. 123, February 1999 - “Characterization of monoclonal gammopathies immunofixation”; Page 129, 1st paragraph. The Immunofixation test, as described, is the current trend in the field of immunofixation, in terms of the “Gold Standard”. In order to make “conscientious, judicious and explicit use of current best evidence”, standards are needed which have been defined by means of systematic, retrospective review, to search for and eliminate sources of bias and develop randomised, prospective trials, in order to achieve significant clinical results. Laboratory objectives are broad and ever-increasing, and the questions they raise involve all disciplines. Everything that has been achieved by means of EBM can be transferred to the laboratory. From EBM, one can learn that a certain bias influences laboratory results, but the possible effects in relation to the size of individual studies or studies of populations are not yet clearly understood. For diagnostic purposes, old and new tests need to be assessed by comparison with the “Gold Standard” and, where such a standard is not available, as is often the case, disturbance factors must be taken into account. The fact that tests with the same name (e.g. electrophoresis in dry acetate and in agarose) can yield different numerical results from method to method makes it very hard to define the principles of EBLM. Different kinds of evidence are accepted in laboratories: e.g. data on the analytical performance of an assay; data on internal and external quality control and data concerning the specificity and sensitivity of tests in particular clinical situations. However, there is scant evidence that the use of a laboratory test can change the clinical attitude to diagnosis or treatment of a given patient or group of patients. EBLM should include all these types of evidence. The standards for assessment of reviews and original articles for the purpose of evaluating the efficacy of laboratory tests are given below:
Standard 1: The composition of the population that is being studied The sensitivity and specificity of a test depend on the characteristics of the population that is being studied. The sensitivity and specificity values of studies carried out on populations with serious pathologies may not be applied to populations with less serious pathologies. Therefore reports should cover at least three of the following criteria: distribution by age and sex; summary of the clinical picture and/or stage of the disease and eligibility criteria for the subjects studied. Standard 2: Relevant groups The sensitivity and specificity of a test may be deduced from average values for a given population. Except for the condition for which the test is used, indices may vary in relation to different clinical groups. In order for a specific test to be used successfully, accuracy indices are required for each subgroup within the range of patients being tested. This standard has been achieved when results for accuracy indices refer to each clinical or demographic subgroup to which they belong. Standard 3: Confirmation distortion (the difference between actual and expected values) Patients for whom the diagnostic examination has yielded positive and negative results must not be used to confirm the reference test at a different percentage. All subjects should undergo both the reference test (Gold Standard), and the assessment test. Standard 4: Distortion in review Care must be taken to ensure that the “Gold Standard� test, and the test which is being evaluated, are analysed objectively, and the results of the two tests must be interpreted separately, irrespective of the results obtained from the other test.
Standard 5: The precision of results of the accuracy test The reliability of sensitivity and specificity tests depends on the number of patients assessed. In order to achieve this standard, the confidence intervals or standard deviation in relation to the numbers involved must be taken into account. Standard 6: Presentation of indeterminate results Indeterminate results are often obtained when assessing a test and their frequency can limit the applicability of such tests to a clinical environment; or it can increase costs, because more tests will be needed in order to confirm the results. Standard 7: The replicability of the test An estimate must always be done of the variability of a result and the cause thereof must always be estimated. Once the method to be applied in the laboratory has been chosen, the following must be verified: 1) Whether the test is available, accurate, replicable and accessible in the context in which it is used; 2) What the pre-test probability is; 3) Whether the post-test probability obtained is such that patient management needs to be modified; 4) Whether the medical consequences of a particular test are acceptable to the patient. Whatever the type and size of the laboratory, it must be suitably equipped, in order to participate in the process that leads to the answering of these questions. The rationale used by doctors in the diagnostic process has been and still is a matter of study. In order to explain the diagnostic process, four main reference models have been identified: The laboratory is responsible for: 1) Analytical identification of the profile The doctor is responsible for: 2) Recognition of the profile 3) Physiopathological reasoning 4) Probabilistic diagnosis
The diagnostic capacity of a laboratory is closely related to its ability to assemble knowledge obtained from different areas of competence, and integrate it and bring that knowledge to bear, in such a way that “the best methodological truth available” is ascertained. The “Gold Standard” concept is meant to be a fusion of the following sub-types: 1) the personal Gold Standard 2) the independent Gold Standard 3) the separate Gold Standard In the final analysis, what was stated at the beginning of this chapter will have to be analytically assessed, in order to understand the overall reliability of the electrophoretic system, which depends on the following: 1) the reliability of the serum protein and urinary profile (preparatory to M-protein research) 2) the reliability of the immunofixation profiles
3. The “good quality” electrophoretic profile The good quality electrophoresis, on agarose or cellulose acetate, is the method used to look for monoclonal components. In order to define the term “good quality electrophoresis”, we need to refer to the “Official Recommendations of the SIBioC 05 Committee” 3.1 Introduction to specific proteins Types of electrophoresis Characteristics of zonal electrophoresis, of an obsolete type: • short run • 5 zone • information about 2 - 3 specific proteins • densitometric reading • numerical expression • loss of detail of monoclonal components Characteristics of the good quality electrophoresis: • long run (resolutive power: buffer / support / stain) • separation of 8 - 11 specific proteins • information about all the specific proteins separated by electrophoresis • visual reading (molecular interpretation)
• comments on interpretation: - identification of qualitative and quantitative changes in specific proteins - knowledge of physiopathology - knowledge of clinical data • visual identification of roughly twice the number of monoclonal components, compared with the short run with 5 zones 3.2 Requirements of the “good quality” electrophoresis (see Fig. 1.1) 1. The pre-albumin band must be visible. 2. In the event of heterozygosis of alpha-1 antitrypsin, it must be possible to identify the protein in the two heterozygotic forms. 3. The alpha-2 macroglobulin specific proteins and haptoglobin phenotypes must represent the anodic and cathodic migration fronts, morphologically and respectively. 4. The specific proteins transferrin and complement 3 must be kept separate. 5. The gamma zone must be seen to be widely extended, in order to permit assessment of the heterogeneous polyclonal – immunoglobulin profile. 6. Because of what is stated in the previous point, it must be possible to identify – in the polyclonal field – small homogeneous components weighing less than 1 g/L. Fig. 1.1(3) Example of the “good quality” electrophoresis, by SIBioC 05 standards
(3) AAT Alpha-1 antitrypsin
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3.3 Proteic changes First group: qualitative and quantitative changes Heterozygotic or homozygotic conditions, with changes that adversely affect: 1. Albumin 2. Alpha-1 antitrypsin 3. Transferrin 4. Complement group 3 fraction 5. Haptoglobin Second group: qualitative changes (genetic or acquired changes) Conditions in which a specific protein, which is structurally normal, behaves differently, because of chemical and physical changes: 1. Activation of complement 3 (in vivo/vitro) 2. Haptoglobin - haemoglobin complex 3. Detection of an MC, which is a sign of anomalous activation of a B lymphocyte, with loss of the normal molecular heterogeneity of the immunoglobulins. 3.4 Specific Proteins most often involved in visual inspection On the anodic front: 1. Pre-albumin 2. Albumin 3. Alpha-1 antitrypsin 4. Alpha-2 macroglobulin 5. Haptoglobin 6. Transferrin 7. Complement group 3 fraction 8. Immunoglobulins 9. Oligoclonality 3.5 Data expression 1. Of a semi-quantitative type, using photodensitometers: a) percentage expression b) semi-quantitative expression 11
2. of a qualitative type, defined as a visual inspection, based on the following findings: a) an increase/decrease in the intensity of each specific protein, compared with “normal” results b) the absence of specific proteins usually present in “normal” results c) the appearance of supernumerary protein bands, compared with “normal” results d) an increase or decrease in the width of each band, compared with “normal” results; this is an indication of increased molecular heterogeneity or homogeneity e) fusion of one or more bands, which is indicative of an increase in molecular species, with intermediate electrophoretic mobility f) doubling of one or more bands, compared with “normal” results g) recognition of phenomena due to the transport of exogenous or endogenous substances Qualitative expression of data 1. Descriptive stage through the use of adjectives a) big decrease b) big increase c) moderate decrease d) moderate increase e) absence of ............ f) presence of ........... 2. Interpretative stage a) This can be worth consulting and, because of its possible complexity, exhaustive clinical information must be obtained. 3.6 Definition of a M-protein (MC) 1. Expression of anomalous protein production by a B-lymphocyte clone. 2. Homogeneous mobility in electrophoresis: determined by the uniform electric charge assumed by the constituent protein. 3. Immunologically consisting of just one type of: a) complete immunoglobulin 12
b) heavy immunoglobulin chain c) light immunoglobulin chain d) fragments of complete immunoglobulin e) fragments of light immunoglobulin chain 3.7 Research process for studying monoclonal components 1. Choice of investigative method for typing purposes 2. Identification of suspect anomalies 3. Search for Bence-Jones proteinuria 3.8 Monoclonal components in electrophoretic traces 1. The possible position[s] that can be assumed at the end of migration: a) in any zone, from the alpha-1 zone to the gamma cathodic zone and sometimes with retromigration 2. Position: a) in zones free of other visible proteins b) in zones overlapped by other proteins c) in the gamma zone, with a decrease in polyclonal immunoglobulins d) in the gamma zone, with an increase in polyclonal immunoglobulins e) in the retromigrated gamma zone 3. Morphology: a) monoclonal IgDs with bands blurred by post-synthetic degradation b) diseases due to heavy chains (IgA, IgM) with wide-based bands and diffuse margins due to a variable molecular mass and the formation of dimers with a high carbohydrate content c) Because of their polymeric structure, isotype-M MCs can form complexes with a low or non-existent migrability index compared with the seeding point; or they can induce deformations and distortions of the electrophoretic trace. d) A number of B-lymphocyte clones, with the production of a number of MCs. e) Polymerisation 13
f) Simultaneous presence of one complete immunoglobulin and of a light chain of the same kind as that of the complete immunoglobulin. 3.9 Qualitative anomalies not attributable to M-proteins, but simulating an MC. Anomalies due to heterozygosis: 1. Alpha-1 antitrypsin 2. Transferrin 3. Complement group 3 fraction Anomalies with a restricted band: 1. Alpha-fetoprotein at a high concentration 2. Protease complex Alpha-1 antitrypsin 3. Haptoglobin - haemoglobin complexes 4. Converted C 3 (which is found in unfresh serum) 5. Fibrinogen in the event of slowed-down coagulation 6. Lysozyme in myelomonocytic and monoblastic leukosis 7. C-reactive protein (which can appear with non-barbiturate buffers in particular) 3.10 List of some factors that can adversely affect the quality of electrophoretic results Choice of: 1. Electrophoretic wet chambers 2. Migration support 3. Buffer solution 4. Quantity of biological sample applied 5. Stain solution 6. Quality of stain 7. Transparency of support 8. Biological fluid concentrators Careful selection of all the stated parameters is crucial, in order to obtain the good quality electrophoretic protein profile. 14
3.11 Is the Microgel System capable of producing the good quality PE (protein electrophoresis) serum protein profiles, according to the requirements of the SIBioC 05 Committee? The advantages of the Microgel system are: 1. Standardisation of the whole process 2. Agarose support 3. Barbiturate buffer 4. Sensitive stains 5. Micro application Is the kind of “Micro” application used by Microgel compatible with the “Official Recommendations of the SIBioC 05 Committee”? It has already been shown that PE can be done in two ways: the first can provide qualitative and semi-quantitative information about 2/3 of specific proteins and a limited number of monoclonal components; the second can provide such information about 8/11 of specific proteins and a much higher number of monoclonal components. Furthermore, in 1977, direct proof was provided that, in the case of acetate and agarose, the individual PE bands were determined by individual specific proteins. Therefore, the claim that PE bands – which are caused by many specific proteins – cannot be used for precise symptomatology, only applies to the micro-zone technique. This technique is under attack for two basic reasons: 1. Poor resolution and sensitivity, related to the cellulose acetate support; 2. Low concentration of proteins per unit area, in relation to the low volume of biological sample used (poor sensitivity). A critical appraisal of the kind of biological sample application that uses the Microgel system. Use of the Microgel system has enabled us to do a critical appraisal of the above two claims. Microgel is designed to work under the following conditions: 1. With an agarose gel support with a controlled negative charge, which makes it possible to obtain broad retromigrations (high EEO = electroendoosmotic power). 15
2. During migration, the support is kept at a constant temperature, with the result that diffusion phenomena, which are caused by the Joule effect and which reduce resolution, are eliminated. 3. The biological sample application time is set to obtain the optimum levels of protein concentration in the support. That makes it possible to achieve a high degree of sensitivity, which is directly proportionate to the amount of biological sample used. 4. Electrophoresis times are short (because of the following parameters which have already been discussed, i.e.: high voltage; temperature control; high EEO value). That makes it possible to limit induced diffusion phenomena to a considerable extent, mainly on the proteic species with lower net charges and limited electrophoretic runs. 5. Use of stains with increasing sensitivity, e.g.: Amidoblack and Acid Violet. Results: The results obtained using the Microgel system have enabled us to re-examine the two issues raised by the SIBioC Protein Committee, which are repeated here: The Microzone technique is to be avoided for the following reasons: 1. Poor resolution and sensitivity, due to the cellulose acetate support. 2. Low concentration of proteins per unit area, in relation to the low volume of biological sample used (poor sensitivity). Let us tackle point 1 first: “Poor resolution and sensitivity....................” The article “Official Recommendations of the SIBioC 05 Committee” (Giornale Italiano di Chimica Clinica [Italian Journal of Clinical Chemistry], Vol. 15, No. 1, 1990) includes the following table on page 52: 16
Table 1.1 Number of M-proteins found in 100 serum specimens containing one or more M-proteins and analysed by means of three electrophoretic techniques Electrophoretic technique
Number of bands
Agarose gel
131
Cellulose acetate (7 bands and visual inspection)
123
Microzone
62
From a reading of this table, an immediate conclusion can be drawn: The technique using agarose is the best in terms of both resolution and sensitivity, because it identified all 131 MCs, which were differentiated by concentration and electrophoretic position. Microgel uses an agarose support; therefore its resolution and sensitivity are higher than those of the systems that use cellulose supports. Now let us tackle point 2: “Low concentration of proteins per unit............� Our aim is to demonstrate that, by using Microgel, the concentration of proteins applied per unit area, using the variable application time, is equal to or higher than the concentration in semi-micro applications on acetate. This would demonstrate that the micro technique limitation is groundless. The volume of biological sample applied, without the occurrence of any diffusion phenomena, which greatly restrict resolutive power, is the true limiting factor in terms of sensitivity. The volume of biological sample applied is, in turn, a direct function of the volume of the applicator, which is related to the thickness of the support. In fact, if the applicator volume were greater than what the support could absorb per unit area of the applicator, there would be widespread diffusion phenomena. Table 1.2 gives the dimensions of the applicators normally used, and the thicknesses of the supports. 17
Table 1.2 Applicators: dimensions and volumes that can be deposited Semi-micro applicators
Microgel micro applicators
Length
8.0 mm
4.2 mm
Width
0.5 mm
0.3 mm
Height
0.25 mm
0.8 mm
Area of application
4 mm2
1.26 mm2
Internal volume of the applicator
1 mm3
1 mm3
Applicator dimensions
Table 1.3 gives the average thicknesses of the supports Table 1.3 Average thicknesses of the supports Supports
Thickness
Dry acetate
0.13 mm
Wet acetate
0.20 mm
Agarose
0.5 mm
Table 1.4 shows the maximum applicable volumes, without the occurrence of diffusion phenomena, in relation to the thicknesses of the individual supports. Table 1.4 Maximum applicable volumes without diffusion phenomena Supports
18
Maximum applicable volume without diffusion
Dry acetate
0.52 mm3
Wet acetate
0.8 mm3
Agarose
1.25 mm3
One now needs to calculate the concentration of proteins deposited on the individual supports in Table 1.4, per unit area (1 mm2), assuming that one has a sample of human serum containing 80 g/l total proteins. Table 1.5 gives the values in grammes of proteins applied, using each individual method, in relation to the supports listed in Table 1.4. Table 1.5 Volumes that may be deposited Dry acetate
Wet acetate
Interlab agarose
8
8
8
Volume applied without diffusion
0.52 mm3
0.8 mm3
1 mm3
Concentration in grammes of total protein for the volumes applied
4.2 x 10-5
6.4 x 10-5
8 x 10-5
Concentration in grammes of total protein applied per 1 mm2
1.05 x 10-5
1.6 x 10-5
6.35 x 10-5
Support Human sample with 8 g/dl total protein
Just by observing the data, one can see how the Microgel system, with its particular mode of application, enables one to apply significantly higher amounts of protein per unit area than those obtainable using conventional methods. A higher protein concentration per unit area makes it easier to appreciate the assessment of the individual specific proteins, as well as being a more sensitive method. Greater sensitivity also makes it possible to observe proteins at low concentrations, which could otherwise be missed, using alternative methods. General Conclusions The Microgel system has a number of advantages that can be appreciated in terms of higher sensitivity and resolution. This work shows that the two points under discussion are invalid, because of the use of an agarose support and a particular mode of application, which permits a “micro� application in terms of size, but not 19
protein concentration per unit area. This situation is translated into serum protein electrophoretic migrations with a high degree of sensitivity and resolution. A further advantage is the use of more sensitive stains, such as Amidoblack or Acid Violet. At the end of the day, the Microgel system can produce electrophoretic migrations which show a high degree of condensation [sic] of individual specific proteins, in narrow isoelectric zones, which represent a state of equilibrium between electrical transport and contraendoosmosis, i.e.: the “right” sort of electrophoretic migrations, in compliance with the “Official Recommendations of the SIBioC 05 Committee”.
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