protein profiles derived by automated immune...

J. clin. Path., 28, Suppl. (Ass. Clin. Path.), 6, 27-32

Protein profiles derived by automated immuneprecipitationROBERT F. RITCHIE

From the Rheumatic Disease Laboratory (Department of Research), Maine Medical Centre, Portland,Maine, United States ofAmerica

This presentation will cover much the same groundas Professor Laurell's but from a slightly differentviewpoint. There is no great difference between us,though we use agarose gel electrophoresis only todetect paraproteins. Like others, we feel that scan-ning the electrophoretogram contributes nothing tothe interpretation of the test', as even with highresolution systems the various fractions are com-posed of mixtures. Instead, we prefer to estimatespecific proteins by the automated immunoprecipita-tion system, and we routinely estimate 12 plasmaproteins (albumin, ctl-antitrypsin, haptoglobin, oro-somucoid, ct2-macroglobulin, transferrin, comple-ment C3, complement C4, IgG, IgA, IgM, LDLprotein) and can estimate more than 30. The sameproteins could of course be estimated by the tech-niques outlined by Dr Laurell. Specific proteinanalysis is superior to electrophoresis in that inaddition to giving information on individual pro-teins it is quantitative. It is also reliable over a widerange of concentrations, it will differentiate proteinswhich occupy the same electrophoretic zone, and itis applicable to samples which may be unsatisfactoryfor electrophoresis due to haemolysis, etc. It can-not, however, replace electrophoresis in the identifi-cation of small paraproteins, particularly of freelight chains which cannot be detected with the pre-sent AIP system, nor in the study of genetic poly-morphism when the total amount of protein indifferent specimens may be the same even thoughthey contain different molecular variants.

Before discussing the interpretation of the profilesobtained by specific estimation of 12 proteins usingcurrent methods, it is pertinent to consider thequality of the analytical results obtained. About 18months ago, the Centre for Disease Control inAtlanta, Georgia, distributed to many laboratoriesin the United States three samples of serum con-taining known amounts of IgG, IgA, and IgM foranalysis. The great majority of the laboratories used

'The importance of scanning for the estimation of paraproteins isemphasized by Dr Carter and Dr Kohn.

radial immunodiffusion, and a few used the AIPsystem. The results for IgG (fig 1) were poor, to saythe least; whereas the actual value was about 11.5g/l, the observed values ranged from 7.5 to 18 g/l.Results from the five reference laboratories weresomewhat better than this. The results for IgA weresimilar; for one sample containing about 1-45 g/lIgM, some laboratories found less than 0 75 g/l andothers more than 3.0 g/l, although the referencelaboratories were all close to the true figure. Foranother specimen with an IgM content of about45 g/l a large number of laboratories found the 1gMcontent to be zero and only a few laboratoriesobtained values within an acceptable range (fig 1).Had the operator merely looked at the sample, itwould have been obvious that there was somethingseriously abnormal because the sample had theconsistency of syrup. Electrophoresis would haveshown that almost all of the plasma protein was IgM.There are probably several causes of these poor

results, including faulty technique, but in thisparticular circumstance the companies which manu-facture the plates containing antibody must take ashare of the blame.

Interpretation of the results of protein estimationis almost always based on a comparison of valuesexpressed in g/l or units, with a 'normal range'determined by measurements on a heterogeneouspopulation. However, a number of factors willinfluence the levels found in such a population andmust be taken into account when results are inter-preted. Most of us would accept that age, ethnicgroup and sex all influenice the serum levels of theimmunoglobulins. Other factors include upperrespiratory and gastrointestinal viral infection,pregnancy and oral contraceptives.When considering individual proteins the ques-

tion arises whether to correct for the total proteinconcentration which may be falsely lowered, eg, byparenteral administration of fluids, or falselyelevated, eg, by venous stasis during sampling orevaporation in the laboratory. The problem re-mains whether the observed value for total protein

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Robert F. Ritchie

Fig 1 Results obtained for IgG and IgM in sera distributed to many laboratories. Ig3-001, Ig3-002, and Ig3-003represent three different sera.

should be used or the value obtained after subtract-ing the amount of any paraprotein from the total.

Ideally, interpretation should be based on thenormal values for the particular individual, but suchinformation is rarely available, in contrast to clinicaldata such as the patient's normal height and weight,

PIStRIEIlITON OF RANDOMPOpLA7eN

etc. Well-person laboratory screening will benecessary to provide such base-line values.The limitations of a normal range determined in a

population is illustrated by the results of serumprotein estimation in 2200 randomly selected people(fig 2). The distribution is very wide for some pro-

Fig 2 Distribution ofserumprotein concentrations in 2200randomly selected people.

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Protein profiles derived by automated immune precipitation

teins, eg, IgA. An even wider range could result ifmeasurements happened to be made during aperiod when there were many 'normals' with sub-clinical upper respiratory or gastrointestinal infec-tions. Some proteins, such as 0£2-macroglobulin,have a bimodal distribution corresponding withsubjects over or under 21 years of age, but theranges in the two age groups overlap. Thus, apopulation norm is only of limited value for inter-preting data from an individual, but at the momentit is all that is available.A further difficulty in determining the normal

range is that of deciding whether all values obtained

FIG. 3

from an apparently normal population should beincluded, whether all values from an apparentlyabnormal population should be excluded, andwhether the criterion of 'normality' should beextended to the relatives of the individuals studied.For example, the range of values obtained for serumlow-density lipoproteins is very wide, but whenvalues from subjects who have relatives under 55years of age with cardiovascular disease are ex-cluded, the range is much narrower, although thereare still some outliers. Our data on low-densitylipoproteins show a distinct difference between theprepubertal individual, the middle-aged and theaged. We have a fairly large population aged over 80in northern New England and we found that thelow-density lipoprotein levels for those aged over 70approximated to those of adolescents, the levels inmiddle age being somewhat higher. This raised thepossibility that those with the higher values inmiddle age had less chance of reaching the 70s, andposed the question, Should they be treated prophylac-tically? We found outliers, even in children, sup-porting the notion that the latter may have bio-chemical disease for two or three decades before thefirst signs of coronary or cardiovascular disease be-come manifest; if so, low-density lipoproteinestimations in childhood may enable protectivemeasures to be taken.

It is well known that the concentrations of manyproteins alter dramatically in early life, so that inorder to interpret any given value properly it isnecessary to know the age of the individual. Thechanges in IgG and IgM are shown in figures 3 and

Fig 3 Variation ofserum IgGconcentration (ordinate) with tige(abscissa).

Fig 4 Variation ofserum IgM(ordinate) concentration with age(abscissa).

FIG. 4

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4. In addition to large changes with age, which makeinterpretation of IgG levels particularly difficult,there is also a very wide range of values at any givenage. In order to evaluate the benefit that would begained by following an individual's values from yearto year, we studied individuals at three monthlyintervals for many years. The variation was verysmall in the absence of intercurrent illness; forexample, for IgG we found variations of 1-0 to1.5 g/l as compared with the overall range of 8-0 g/lfor a large population. The fact that the physio-logical variation in an individual is so much lessthan the range of values found in a populationseriously compromises our ability to interpret asingle set of values.We have attempted to assist the physician to

overcome some of the difficulties of interpretinglaboratory data by displaying them in a mannerwhich is easy to read, and which does not requireknowledge of the numerical values or of the normalrange. This takes the form of a computer printout,with a bar representing the protein level as a percent-age of the normal mean (for the population) runningacross a colour-shaded background (fig 5); thecentral zone denotes approximately the normalmean ± 1 standard deviation, the intermediatezones ± 1 to 2 standard deviations, and the outerzones any deviation exceeding ± 2 standard devia-tions. The mean and standard deviations are ad-justed by the computer for various factors whereappropriate, such as age and sex. In the case ofhaptoglobin the distribution of values in a largepopulation is very skewed, so that the normal rangecannot be indicated by means of the standarddeviation. Our population mean for haptoglobin is

about 1.05 g/l, and 0-2 g/l is still within the normalrange. However, if the latter value were displayed interms of the mean and standard deviation, it wouldappear to be an abnormally low result. Therefore, inorder to display the result in the standard manner,we have to adjust the value for the skewed distribu-tion by means of an equation which brings the resultinto the low-normal range (fig 5).

Interpretation of the results by computer is anelaborate process (fig 6). The interpretative pro-gramme involving only 10 of the 12 proteins amountsto about 150 000 words and requires about 15seconds to process. The computer may print out upto two pages of information, which is of course toomuch for anyone to read. The machine goes throughabout 800 individual decisions, some of which areillustrated in figure 7. In the example shown thefirst question is, 'Is the IgG value less than orgreater than 40% of the "normal", taking account ofage etc?' If it is less than 40% the computer wouldprint out 'marked hypoimmunoglobulinaemia in-volving IgG'. If it is greater than 40% but less than65% a statement 'significant hypoimmunoglobulin-aemia involving IgG' would be printed. Thesevalues for 40 and 65% are, of course, only relevantto our particular population.The importance of correcting for total protein is

illustrated by the profile in figure 7. At first sight itlooks abnormal and was reported as such. However,we would now correct this for the raised total pro-tein, which would reduce all the values into thenormal zone. The new printout would now say thatthis study is normal, but would also state that thereis probably haemoconcentration. When to correctfor alterations in total protein concentration is very

. A

Fig 5 Illustration of the computer printout ofserum protein values. The central zone indicates the mean ± 1 SD fora normal population, the intermediate grey zone ± 1 SD to ± 2 SD. The lower display illustrates the effect ofacorrection by the computer for the skewed distribution of the normal range for haptoglobin.

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31Protein profiles derived by automated immune precipitation

Fig 6 Part of the interpretative program for the computer.

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Fig 7 Example ofcomputer printout'. Fig 8 Example ofcomputer printout'.

'The author has been unable to provide better photographs of the printouts illustrated in figs 7-10 but readers should be able to see thepattern in each.



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Fig 9 Example ofcomputer printout'.

difficult to assess and to program for but seems to bevery valuable.The situation illustrated in fig 8 is somewhat

different. Elevation of transferrin is the only ab-normality, and this is an unusual finding in a male.The commonest two conditions in which we haveseen this are chronic blood loss and lymphoma.Thepresence of hyperchylomicronaemia is also noted,probably indicating that the patient was not fasting;this was deduced from the blank value in the systemwhich gives a fairly good estimate of serum tri-glyceride levels.Sometimes the report has to be more detailed.

An example is given in fig 9 which shows o41-antitrypsin falling in the normal zone, haptoglobinfalling somewhere below it, and orosomucoidvirtually normal. The computer has stated that thereis a disparity between the level of haptoglobin andone or more of the other acute phase reactants.

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Fig 10 Example of computer printoutl.

In a case of very low transferrin accompanied byonly slight reduction in albumin (fig 10) the computerconcluded that the low transferrin could be as-sociated with bacterial infection, and suggested otherpossibilities. If the albumin were markedly reduced,a protein-losing syndrome would be more likely.

In addition to analysing such qualitative changesin the protein pattern, the computer can also beprogrammed to take account of the diagnosticsignificance of the degree of change in the levels ofindividual proteins.

If progress in this field is to continue, it is un-doubtedly necessary to stimulate wider interest andto accumulate more data which must be technicallyreliable. This requires proper training of technicians,suitable instruments and reliable reagents. Theclinician must be appraised of the clinical indicationsfor making these measurements and of the signifi-cance of the results.

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