INTERLAB S.r.l. - Via Rina Monti, 26 - 00155 Rome (Italy) Tel. +39 - 06 - 227.54.350 - Fax +39 - 06 - 227.54.534 E-mail: info@interlab-srl.com - http: //www.interlab-srl.com

Dr. AnDreA CiApiniand the

interlAb SCientifiC teAm

protein electrophoresis and immunofixation: from the laboratory to the clinical practice

Protein electrophoresis and imm

unofixation: from the laboratory to the clinical practice

Dr. AnDreA CiApiniand the

interlAb SCientifiC teAm

protein electrophoresis and immunofixation: from the laboratory to the clinical practice

III

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 tech-niques 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.

�

Table of Contents

1. Introduction............................................................................ page 1

2. Researchonmonoclonalgammopathiesintheclinical laboratory........................................................................ page 3 2.1 EBMandEBLM..................................................... » 33. The“rightkindof”electrophoreticprofile.................. » 9 3.1 Introductiontospecificproteins............................ » 9 3.2 Therightapproachtoelectrophoresis.................. » 10 3.3 Proteinchanges....................................................... » 11 3.4 Thespecificproteinsmostoftensubjecttovisual inspection................................................................. » 11 3.5 Dataexpression....................................................... » 11 3.6 Definitionofamonoclonalcomponent(MC)....... » 12 3.7 Theprocessofresearchforstudyingmonoclonal components............................................................. » 13 3.8 Monoclonalcomponentsinelectrophoretictraces » 13 3.9 Qualitative anomalies not attributable toMCs, butwhichsimulateanMC..................................... » 14 3.10Listoffactorsthatcanadverselyaffectthequality ofelectrophoreticfindings...................................... » 14 3.11 IstheMicrogelsystemcapableofproducingthe good quality serum proteins electrophoretic profiles (EPs), according to the requirements oftheSIBioC(1)05committee?............................... » 15

(1)  SIBioC - Società italiana di biochimica clinica e biologia molecolare = Italian Clinical Biochemistry and Molecular Biology Society

�I

4. Specificproteinsidentifiable.......................................... page 21 4.1 Specific proteins: morphological appearance, positionandfunction.............................................. » 21 4.2 Serum protein profile of the most frequently occurringspecificproteins..................................... » 26 4.3 Serum protein profile of the least frequently occurringspecificproteins..................................... » 275. StandardisationoftheMicrogelsystem....................... page 29 5.1 TheMicrogelsystemasameansofstandardisation » 296. Immunoglobulins............................................................ page 49 6.1 Introduction............................................................ » 49 6.2 Cellbases................................................................. » 50 6.3 Structural,immunochemicalandfunctionalbases » 57 6.4 Summary of the main physicochemical, structuralandfunctionalcharacteristics of the 5 classesofhumanimmunoglobulins....................... » 747. How polyclonal and monoclonal Igs are distributed inelectrophoreticruns.................................................... page 87 7.1 PolyclonalIgs.......................................................... » 87 7.2 MonoclonalIgs........................................................ » 888. WhereMCscanmigrateinelectrophoreticprofiles.... page 91 8.1 Position.................................................................... » 91 8.2 Percentageintermsofpositionandfrequency.... » 92 8.3 Prevalence according to isotype, age and concentration........................................................... » 99. Monoclonalcomponentsinserumandurine............... page 97 9.1 Electrophoretic definition of a monoclonal component............................................................... » 97 9.2 Immunological definition of a monoclonal component............................................................... » 9910.Theconceptofelectrophoreticsemeiotics,asapplied toimmunoglobulins........................................................ » 103 10.1Introduction............................................................ » 103 10.2Serumproteinandurinaryproteinprofile,asa means of identification of immunoglobulin- relatedpathologies................................................. » 106 10.3Proteinurias:classificationaccordingtoBoylan » 108 10.4Immunoglobulinelectrophoreticprofiles............. » 111

�II

11.Homogeneousbandtypingmethods............................. page121 11.1Listofmethodsandcomments.............................. » 121

12.Bence-Jonesproteinuria................................................. page131 12.1Classification of proteinurias according to type........................................................................... » 131 12.2Persistentproteinurias........................................... » 131 12.3Pathogeneticclassificationofproteinurias........... » 132 12.4Qualitative methods used to identify the type ofproteinuria.......................................................... » 134 12.5Thechoiceofsupport............................................. » 135 12.6Urinaryelectrophoreticprofile:specificproteins » 139 12.7Technical andmethodological requirements for therightapproachtourinaryproteinprofiling... » 140 12.8Freelightchainsinelectrophoresis....................... » 140 12.9Visualandqualitativeinterpretation.................... » 143 12.10Visualinspectionastheonlypossiblemeansof interpretation.......................................................... » 144 12.11Methodologicaldetails.......................................... » 147 12.12Concentrationofbiologicalfluids....................... » 150 12.13Bence-Jonesproteinsize...................................... » 155 12.14Highlightingofmonoclonalcomponents............ » 156 12.15Bence-Jonesantiproteinantisera........................ » 159

13.The clinical significance of the presence ofMCs in serumandurine.............................................................. page167 13.1IdentificationofahomogeneousMCband.......... » 167

14.CriteriaforthedifferentiationofMGUSandMM(2)... page169 14.1Differentialcriteria................................................. » 169 14.2Classificationofmonoclonalgammopathies........ » 170

15.Which antisera should be used for Bence-Jones proteintyping?.......................................................... page173 15.1Thechoiceofantisera............................................. » 173

(2)  MGUS  =  Monoclonal  Gammopathy  of  Undetermined  Significance; MM = Multiple Myeloma

16.Primaryandsecondaryimmunodeficiencies............... page177

17.CAP(CollegeofAmericanPathologists)guidelinesand laboratory assessments concerning patients with monoclonalgammopathies............................................. » 183 17.1Differentialcriteriaandguidelines....................... » 183 17.2Follow-upofasymptomaticpatients..................... » 189 17.3Follow-up of patients with hyperviscosity syndrome.......................................................................... » 190 17.4Urinaryfollow-up................................................... » 191

18 Conclusions...................................................................... page193

19 Bibliography.................................................................... page195

1

1. Introduction

The  discovery  of  homogeneous  immunoglobulin  components,  in electrophoretic  profiles  of  serum  and  urinary  proteins,  is  an  increas-ingly 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 • �isual 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  (oligo-clonal profiles) was mainly described and taken into account in electro-phoresis 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 symptomato-logical – uncertainties was undoubtedly  the  fact  that  immunoelectro-phoresis 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  tech-niques,  clinicians  have  increasingly  focused  on  the  symptomatology of the possible diagnostic, evolutionary and predictive value of mono-clonal/oligoclonal components in serum, body fluids and urine.

Because we favour the immunofixation technique, whilst acknowl-edging 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 deter-mine the efficacy and efficiency of the system used – in other words, how to optimise efficiency and reliability (EBM and EBLM).

2.1.EBMandEBLMEvidenceBasedMedicine(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 contin-uous 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 medi-cine – 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;�)  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 effi-cacy 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 �9-�1, 1999 states :

5

“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  immunofixa-tion”; 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 ques-tions 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:

�

Standard1:ThecompositionofthepopulationthatisbeingstudiedThesensitivityandspecificityofatestdependonthecharacteris-

ticsofthepopulationthatisbeingstudied.The sensitivity and specificity values of studies carried out on

populationswithseriouspathologiesmaynotbeappliedtopopula-tionswithlessseriouspathologies.

Therefore reports should cover at least three of the followingcriteria:distributionbyageandsex;summaryoftheclinicalpictureand/or stage of the disease and eligibility criteria for the subjectsstudied.

Standard2:RelevantgroupsThe sensitivity and specificity of a test may be deduced from

averagevaluesforagivenpopulation.Exceptfortheconditionforwhichthetestisused,indicesmayvaryinrelationtodifferentclin-icalgroups.

Inorderforaspecifictesttobeusedsuccessfully,accuracyindicesarerequiredforeachsubgroupwithintherangeofpatientsbeingtested.

This standard has been achieved when results for accuracyindicesrefertoeachclinicalordemographicsubgrouptowhichtheybelong.

Standard3:Confirmationdistortion(thedifferencebetweenactualandexpectedvalues)

Patientsforwhomthediagnosticexaminationhasyieldedpositiveandnegativeresultsmustnotbeusedtoconfirmthereferencetestatadifferentpercentage.

All subjects should undergo both the reference test (GoldStandard),andtheassessmenttest.

Standard4:DistortioninreviewCaremustbetakentoensurethatthe“GoldStandard”test,and

thetestwhichisbeingevaluated,areanalysedobjectively,andtheresultsofthetwotestsmustbeinterpretedseparately,irrespectiveoftheresultsobtainedfromtheothertest.

7

Standard5:TheprecisionofresultsoftheaccuracytestThereliabilityofsensitivityandspecificitytestsdependsonthe

numberofpatientsassessed.In order to achieve this standard, the confidence intervals or

standarddeviationinrelationtothenumbersinvolvedmustbetakenintoaccount.

Standard6:PresentationofindeterminateresultsIndeterminate results are often obtained when assessing a test

andtheirfrequencycanlimittheapplicabilityofsuchteststoaclin-icalenvironment;oritcanincreasecosts,becausemoretestswillbeneededinordertoconfirmtheresults.

Standard7:ThereplicabilityofthetestAnestimatemustalwaysbedoneofthevariabilityofaresultand

thecausethereofmustalwaysbeestimated.

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;�)  Whether  the  post-test  probability  obtained  is  such  that  patient 

management needs to be modified;�)  Whether the medical consequences of a particular test are accept-

able 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 profileThe doctor is responsible for:2) Recognition of the profile  �)  Physiopathological reasoning�)  Probabilistic diagnosis

�

The  diagnostic  capacity  of  a  laboratory  is  closely  related  to  its ability to assemble knowledge obtained from different areas of compe-tence, 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 �)  the independent Gold Standard �)  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 (prepara-tory to M-protein research)

2)  the reliability of the immunofixation profiles

9

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 “OfficialRecommendations of the SIBioC 05Committee” 

3.1IntroductiontospecificproteinsTypesofelectrophoresisCharacteristics 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  electro-

phoresis • visual reading (molecular interpretation)

10

• 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 zones3.2Requirementsofthe“goodquality”electrophoresis(seeFig.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.

�. 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)Exampleofthe“goodquality”electrophoresis,

bySIBioC05standards

(3)  AAT Alpha-1 antitrypsin

11

3.3ProteicchangesFirst group: qualitative and quantitative changes   Heterozygotic  or  homozygotic  conditions,  with  changes  that 

adversely affect:1.  Albumin2.  Alpha-1 antitrypsin�.  Transferrin�.  Complement group � 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)�.  Haptoglobin - haemoglobin complex�.  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 SpecificProteinsmostofteninvolvedinvisualinspectionOn the anodic front:1.  Pre-albumin2.  Albumin3.  Alpha-1 antitrypsin4.  Alpha-2 macroglobulin5.  Haptoglobin�.  Transferrin7.  Complement group � fraction  �.  Immunoglobulins9.  Oligoclonality

3.5Dataexpression1.  Of a semi-quantitative type, using photodensitometers:a)  percentage expressionb)  semi-quantitative expression

1�

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” resultsg)  recognition of phenomena due to the transport of exogenous or 

endogenous substances

Qualitativeexpressionofdata1.  Descriptive stage through the use of adjectives a)  big decrease b)  big increasec)  moderate decreased)  moderate increasee)  absence of ............f)  presence of ...........

�.  Interpretative stagea)  This  can  be  worth  consulting  and,  because  of  its  possible 

complexity, exhaustive clinical information must be obtained.

3.6DefinitionofaM-protein(MC)1.  Expression of anomalous protein production by a B-lymphocyte 

clone.�.  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 

1�

b)  heavy immunoglobulin chain c)  light immunoglobulin chaind)  fragments of complete immunoglobulin e)  fragments of light immunoglobulin chain 

3.7Researchprocessforstudyingmonoclonalcomponents1. Choice of investigative method for typing purposes2.  Identification of suspect anomalies3.  Search for Bence-Jones proteinuria

3.8Monoclonalcomponentsinelectrophoretictraces1.  The possible position[s] that can be assumed at the end of migra-

tion:a)  in any zone, from the alpha-1 zone to the gamma cathodic zone 

and sometimes with retromigration �.  Position:a)  in zones free of other visible proteins b)  in zones overlapped by other proteinsc)  in the gamma zone, with a decrease in polyclonal immunoglobu-

lins d)  in the gamma zone, with an increase in polyclonal immunoglob-

ulins e)  in the retromigrated gamma zone �.  Morphology:a)  monoclonal IgDs with bands blurred by post-synthetic degrada-

tionb)  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

1�

f)  Simultaneous presence of one complete immunoglobulin and of a  light  chain  of  the  same  kind  as  that  of  the  complete  immu-noglobulin.

3.9 QualitativeanomaliesnotattributabletoM-proteins,butsimu-latinganMC.

Anomalies due to heterozygosis:1.  Alpha-1 antitrypsin�.  Transferrin�.  Complement group � fraction Anomalies with a restricted band:1.  Alpha-fetoprotein at a high concentration2.  Protease complex Alpha-1 antitrypsin�.  Haptoglobin - haemoglobin complexes�.  Converted C � (which is found in unfresh serum)5.  Fibrinogen in the event of slowed-down coagulation6.  Lysozyme in myelomonocytic and monoblastic leukosis7.  C-reactive protein (which can appear with non-barbiturate buffers 

in particular)

3.10List of some factors that can adversely affect thequality ofelectrophoreticresults

Choice of:1.  Electrophoretic wet chambers�.  Migration support �.  Buffer solution�.  Quantity of biological sample applied5.  Stain solution�.  Quality of stain7.  Transparency of support8.  Biological fluid concentrators

Careful selection of all the stated parameters is crucial, in order to obtain the good quality electrophoretic protein profile.

15

3.11 IstheMicrogelSystemcapableofproducingthegoodqualityPE(proteinelectrophoresis)serumproteinprofiles,accordingtotherequirementsoftheSIBioC05Committee?

The advantages of the Microgel system are:1.  Standardisation of the whole process2. Agarose support �.  Barbiturate buffer �.  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 �/� 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 indi-vidual 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).

AcriticalappraisalofthekindofbiologicalsampleapplicationthatusestheMicrogelsystem.

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).

1�

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.

�.  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.

�.  Electrophoresis times are short (because of the following param-eters  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).

Letustacklepoint1first:“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 5�:

17

Table1.1NumberofM-proteinsfoundin100serumspecimens

containingoneormoreM-proteinsandanalysedbymeansofthreeelectrophoretictechniques

Electrophoretictechnique Numberofbands

Agarose gel  1�1

Cellulose acetate (7 bands and visual inspection) 1��

Microzone ��

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 differen-tiated 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.

Nowletustacklepoint2:“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.� gives the dimensions of the applicators normally used, and the thicknesses of the supports.

1�

Table1.2Applicators:dimensionsandvolumesthatcanbedeposited

Applicatordimensions Semi-microapplicators

Microgelmicroapplicators

Length �.0 mm �.� mm

Width 0.5 mm 0.� mm

Height 0.�5 mm 0.� mm

Area of application � mm� 1.�� mm�

Internal volume of the applicator 1 mm� 1 mm�

Table 1.3 gives the average thicknesses of the supports

Table1.3Averagethicknessesofthesupports

Supports Thickness

Dry acetate 0.1� mm

Wet acetate 0.�0 mm

Agarose  0.5 mm

Table  1.�  shows  the  maximum  applicable  volumes,  without  the occurrence of diffusion phenomena, in relation to the thicknesses of the individual supports.

Table1.4Maximumapplicablevolumeswithoutdiffusionphenomena

Supports Maximumapplicablevolumewithoutdiffusion

Dry acetate 0.5� mm�

Wet acetate 0.� mm�

Agarose 1.�5 mm�

19

One now needs to calculate the concentration of proteins deposited on the individual supports in Table 1.�, per unit area (1 mm�), assuming that one has a sample of human serum containing �0 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.�.

Table1.5Volumesthatmaybedeposited

Support Dryacetate

Wetacetate

Interlabagarose

Human sample with � g/dl total protein � � �

�olume applied without diffusion 0.5� mm� 0.� mm� 1 mm�

Concentration in grammes of total protein for the volumes applied �.� x 10-5 �.� x 10-5 � x 10-5

Concentration in grammes of total protein applied per 1 mm� 1.05 x 10-5 1.� x 10-5 �.�5 x 10-5

Just by observing the data, one can see how the Microgel system, with  its  particular mode of  application,  enables  one  to  apply  signifi-cantly  higher  amounts  of  protein  per  unit  area  than  those  obtainable using conventional methods.

A higher protein concentration per unit area makes it easier to appre-ciate 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.

GeneralConclusionsThe Microgel system has a number of advantages that can be appre-

ciated 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 appli-cation, which permits a “micro” application  in  terms of size, but not 

�0

protein  concentration  per  unit  area.    This  situation  is  translated  into serum protein electrophoretic migrations with a high degree of sensi-tivity 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  electro-phoretic 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 contraendoos-mosis, i.e.:  the “right” sort of electrophoretic migrations, in compliance with the “Official Recommendations of the SIBioC 05 Committee”.