Bulletin of the World Health Organization, 59 (5): 717-728 (1981)

Use and abuse of eight widely-used diagnostic procedures in clinical : a WHO Memorandum*

This Memorandum assesses eight widely-used diagnostic procedures with the aim of establishing their usefulness in patient care. For eachprocedure, the main methods that can be recommended at present are outlined and their pitfalls discussed. For each procedure, recommendations are made as to the clinical conditions for which the test is essentialfor diagnosis, the conditions for which the test will help in assessing and monitoring disease activity, and the conditionsfor which the test is usefulfor research purposes only.

The recent expansion of clinical immunology has application of some immunological techniques, with- been accompanied by the introduction of a variety of out detriment to patient care. The present Memor- immunological diagnostic tests in clinical labora- andum is an attempt to define indications for im- tories. Owing to increasing demands from clinicians munological tests. It has been restricted to the analysis for such procedures, their use has often been exag- of eight widely-used diagnostic procedures. gerated and there is a general feeling that a better For each procedure, two aspects have been con- definition of the indications for such tests, made in sidered. First, the main methods currently recom- relation to patients' needs, would be beneficial. mended are outlined and their pitfalls discussed. Obviously, immunological tests, like any other Technical details are not included since they are diagnostic tests, can be graded according to their use- readily available (J).a Second, particular attention has fulness in the care of patients. Some tests are essential been given to definition of the clinical conditions for for diagnosis, prognosis, or monitoring of disease; which the test is essential for diagnosis, those for many tests are useful but optional for routine investi- which the test is helpful in assessing or monitoring the gations; other tests are of interest only for research disease activity, and those conditions for which the purposes. In addition, a number of immunological test should be used only for clinical research purposes. tests are useless in some circumstances. The conclusions of this committee reflect the There is a consensus among immunologists that an present status of the art and do not preclude future effort should be made to reduce their share in the con- improvements. It was the feeling that the primary goal tinually increasing cost of medical laboratory investi- ofclinical immunology should be to help the patient in gations. This requires self-limitation in the routine the most cost-effective manner.

QUANTIFICATION OF IMMUNOGLOBULINS

The assessment of the three major immunoglobulin immunological methods is important in a limited classes in body fluids involves three laboratory tech- number of clinical conditions. This test is too often niques: serum electrophoresis, quantification of performed without good indication. major immunoglobulin classes, and immunoelectro- phoresis. The measurement of IgE requires more METHODOLOGICAL CONSIDERATIONS sensitive techniques (see pages 720-721). There is no clinical indication for the measurement of serum IgD. Quantification of the immunoglobulin classes by Many methods have been described for the quanti- tative assessment of immunoglobulins. Two of them * This Memorandum was prepared by the participants in a work- are currently of the most value and of comparable ing group organized jointly by the International Union of Immuno- logical Societies and the World Health Organization, in Geneva on accuracy: a) radial (RID) and b) 18-20 May 1981. The names of the participants are listed on pages . 727-728. Reprints should be requested from Chief, Immunology, World Health Organization, 1211 Geneva 27, Switzerland. A French a MACKAY, I.R. & RIrTS, R.E. WHO handbook of immuno- translation of this Memorandum will appear in a later issue of the logical techniques. Unpublished WHO document, WHO/IMM. Bulletin. TECH/79.1 (1979). 4107 717- 718 WHO MEMORANDUM

When the patient load is relatively low, RID will Normal values probably remain the method ofchoice. However, with Concentrations of immunoglobulins in sera vary a high patient load and if a nephelometer is already with age, geographical environment, and sex. Each available, nephelometry is useful. laboratory should measure serum immunoglobulin concentrations on a matched control group. Radial immunodiffusion has a constant coefficient of variation which under optimal conditions may be less than 10%, except at extremely low concentra- CLINICAL INDICATIONS tions. The limit of accurate protein measurements, using low concentrations of antisera, is about 10 mg/l (10 Mg/ml). Techniques using limited diffusion are Serum more accurate than those with timed diffusion. With Quantification of serum immunoglobulins is re- normal sera, results can be obtained after 24 h of dif- garded as essential in suspected primary or secondary fusion, but more time may be required for the assess- immunodeficiency (ID), even when no abnormality is ment of very high or very low levels. seen in electrophoresis. Concentrations of immuno- Pitfalls. RID is sensitive to differences in diffusion globulins cannot be used, however, as the sole constants; special precautions should be taken to criterion for diagnosis of primary ID. Selective IgA ensure that immunoglobulins in the standard and test deficiency may occur without any evidence of associ- sera are not split or aggregated and are in the same ated disease, and IgA is undetectable in approximately form. For instance, reliable measurements of such 0.03-0.2% of the normal population. On the other proteins as IgM of low relative molecular mass and hand, failure to respond to one or more can secretory IgA cannot be made unless a standard prep- sometimes be observed in patients with normal or high aration of these kinds of immunoglobulin is used. levels of all immunoglobulins. Thus, normal immuno- globulin concentrations do not exclude deficiency. Monitoring of serum immunoglobulin Nephelometric techniques levels is essential in patients with severe forms of These techniques are increasingly used for quantify- hypogammaglobulinaemia who receive immuno- ing serum immunoglobulin levels. Both turbidimetric globulin substitution therapy. procedures and the detection of -antibody Quantification of serum immunoglobulins is con- complexes by light scattering can be applied. The sidered helpful in distinguishing "benign" idiopathic advantages are that results can be obtained within a monoclonal gammapathies from paraproteinaemias very short time, they can be fully automated, and there caused by myeloma. In the latter case, the levels of are no problems with polymeric immunoglobulin. normal immunoglobulins are usually decreased, while they usually are unaltered in the "benign" form. In Pitfalls. Expensive instrumentation is required and this context, it should be stressed that monoclonal turbid serum samples may need to be clarified. immunoglobulins tend to give falsely high values in immunodiffusion assays. When large enough amounts of the monoclonal protein are present, it is Standards and antisera more accurate to measure the protein by the area Discrepancies in results have arisen from the use of under the spike on serum protein electrophoresis. different standards by different laboratories. WHO The value of quantifying serum immunoglobulins makes available International Reference Preparations for other clinical purposes has been established in only for the five classes of human serum few additional instances, such as the determination and it is recommended that working standards should of IgM levels in the cord blood of infants suspected of be related to these preparations. having congenital infections, and as an aid in the diag- Each antiserum, including those from commercial nosis of trypanosomiasis or tropical splenomegaly. sources, must be shown to be specific in the test in For research purposes, immunoglobulins may be which it is being used. Hybridoma-derived mono- quantified in diffuse hypergammaglobulinaemia and clonal may be useful in the future; how- in conditions such as some lymphoproliferative ever, many monoclonal antibodies do not precipitate diseases, liver cirrhosis, or systemic lupus erythema- antigen when used alone, and thus mixtures of such tosus. More promising might be immunoglobulin antibodies may be required. With these antibodies, it studies in the families of patients with immunodefi- may become easier to quantify subtypes and sub- ciency or homogeneous immunoglobulins, with the classes of the immunoglobulins. object of clarifying the role of genetic factors. DIAGNOSTIC PROCEDURES IN CLINICAL IMMUNOLOGY 719

Other bodyfluids ted CSF since concentration procedures will lead to the aggregation of immunoglobulins, especially IgG, Urine. Quantification of immunoglobulins in the and a falsely low value by RID. urine is possible but fraught with problems. For Quantification of immunoglobulins in CSF is of instance, immunoglobulin molecules in urine may be interest in diseases such as multiple sclerosis and sub- split or light-chains may exist in urine as monomers, acute sclerosing panencephalitis where the concentra- making standardization difficult. For the demonstra- tion of IgG relative to the total protein or albumin is tion of Bence Jones proteins, the combination of often, but not always, increased. In African trypano- protein electrophoresis and is somiasis, the increase in immunoglobulin levels in more useful. CSF is an indication of invasion of the central nervous Cerebrospinal fluid (CSF). Quantification of im- system by the parasite. munoglobulin should be performed on unconcentra-

IMMUNOELECTROPHORETIC ANALYSIS OF IMMUNOGLOBULINS IN BIOLOGICAL FLUIDS

Immunoelectrophoresis (IEL) permits ready identi- Bence Jones proteins and optional for myeloma pro- fication of the major immunoglobulin classes. It is the teins. The light-chain type may however have prog- method of choice for the identification of monoclonal nostic significance in myeloma. IEL with anti-K and immunoglobulins since it detects simultaneously their anti-A antisera allows the detection of small mono- electrophoretic and antigenic homogeneity. It is not a clonal components in the presence of diffuse hyper- good quantitative technique. It should not be used for immunoglobulinaemia, and sometimes the detection the systematic screening of serum proteins. of multiple monoclonal components. Pitfalls METHODOLOGICAL CONSIDERATIONS Because of poor availability of antigen determinants for cross-linking, many anti-light-chain IEL is a useful method for studying immunoglobu- antisera are unable to precipitate some whole mono- lins in other fluids in addition to serum, e.g., urine, clonal immunoglobulin molecules, especially IgA- cerebrospinal fluid, saliva, or intestinal juice. In these lambda, and /or some free light-chains (Bence Jones instances, it is usually necessary to concentrate the proteins). Thus for the diagnosis of heavy-chain proteins before performing IEL and to run a serum diseases (in particular alpha-chain disease), the use of sample from the same patient simultaneously. additional procedures is necessary- for example, IEL The medium of choice for IEL is either or with antisera containing precipitating antibodies to , using where possible the same type of gel for conformational determinants of the Fab region or the serum protein electrophoresis. immunoselection combined with IEL, using potent IEL requires the use of potent and specific antisera. antisera to light-chains or to Fab incorporated into the It is recommended to use, in the first step, polyvalent gel. As in all procedures, anti- antisera containing precipitating antibodies to the gen excess may preclude the visualization of a preci- various immunoglobulin classes and light-chain types. pitin line, especially when horse antisera are used. This In order to identify monoclonal immunoglobulins, is particularly the case when analysing Bence Jones monospecific antisera to the various Ig heavy- and proteins. light-chains are often required. These antisera are When a cryoglobulin is present in the serum, commercially available and should always be checked immunoelectrophoretic analysis of the whole serum for their content of precipitating antibodies and for should be performed after heating at 37 °C and their specificity. re-solution. The determination of the heavy-chain class of a In order to permit ready identification of some IgM monoclonal immunoglobulin sometimes, but not proteins and to ascertain their monoclonal nature by always, requires the use of class-specific antisera. light-chain typing, additional procedures may be Such antisera are necessary for the diagnosis of IgD or necessary, such as the addition of a reducing agent to IgE myeloma. The identification of the heavy-chain the fluid under study (in order to convert 19S IgM into subclass of monoclonal IgG or IgA components is 8S subunits) or preliminary separation of IgM from mainly of value in research. The identification of the K IgG by physico-chemical techniques. Immunofix- or A light-chain type is necessary for the diagnosis of ation, a technique more recently developed for the 720 WHO MEMORANDUM identification of monoclonal immunoglobulins, may munoglobulins) and cold-agglutinin disease, and in be particularly useful in such instances. diseases such as Gaucher's disease or mucinar In interpreting immunoelectrophoretic patterns one papulosis (monoclonal components) and trypano- should be aware of possible associations of mono- somiasis (elevated polyclonal IgM). clonal immunoglobulins with other proteins, such as IEL may be useful for research purposes in several serum albumin, a-l-antitrypsin, and lipoproteins. instances such as primary immunodeficiencies (in addition to measurement of Ig levels), bone marrow grafts in patients with leukaemias, marrow aplasia, or CLINICAL INDICATIONS severe combined immunodeficiency; some auto- immune diseases; some haematological conditions, Serum such as myelomonocytic leukaemias; various infec- tions, such as cytomegalovirus or congenital toxo- Analysis by IEL is essential: plasmosis; systematic survey of family members of (a) When the clinical, haematological, or patho- patients with monoclonal gammapathies. logical findings lead to the diagnosis or suspicion of the following diseases: myeloma, Waldenstrom's Urine macroglobulinaemia, heavy-chain diseases, amyloid- osis, or immunoglobulin deposition disease. IEL is essential in myeloma (with or without whole (b) In the presence of the following biological homogeneous serum Ig); amyloidosis, immunoglobu- abnormalities: lin deposition disease; in all cases in which a mono- clonal Ig has been found in serum, whatever the clini- (i) An abnormal narrow band on serum protein cal condition; in cases in which an abnormal narrow electrophoresis. However, it should be stressed that band has been found on the urinary protein electro- IEL allows the detection of monoclonal components phoresis pattern. in situations without a distinctive electrophoretic IEL is optional in malignant lymphoproliferative pattern. diseases other than myeloma (macroglobulinaemia, (ii) A cryoglobulin. In sueh cases IEL is necessary to chronic lymphatic leukaemia, lymphoma, heavy- identify the proteins of the cryoprecipitate and to chain diseases) and in primary immunodeficiencies. distinguish single-class homogeneous cryoprecipi- tating immunoglobulins from mixed cryoglobulins Otherfluids with or without a monoclonal component. IEL should also be done on whole serum. IEL of cerebrospinal fluid proteins is also useful in (iii) A Bence Jones type of proteinura. the search for oligoclonal components in patients with (iv) A pyroglobulin; serum hyperviscosity; or a dis- subacute sclerosing panencephalitis, or in myeloma or crepancy between immunoglobulin level as appreci- macroglobulinaemia with neurological involvement. ated by and In multiple sclerosis, the technique of isoelectric electrophoretic immunochemical pro- focusing is more productive. cedures. IEL of the intestinal juice is essential in "immuno- IEL may be useful in some immunoproliferative proliferative small-intestinal disease" with suspicion disorders such as chronic lymphocytic leukaemias of a-chain disease, when the abnormal protein is not (detection of M-chain disease and of monoclonal im- detected in the serum of the patient.

MEASUREMENT OF TOTAL AND SPECIFIC IgE

IgE is the most important mediator in atopic techniques and solid-phase . disease. Moreover it is highly increased in some para- The common principle ofthe two methods is the use sitic diseases. The clinical value of IgE determination, ofinsolubilized anti-IgE antibody. This reagent can be however, is limited. used either in a competitive-binding using radio- labelled IgE and IgE standard, or in a non-competitive TOTAL IgE assay using radiolabelled anti-IgE. Like other com- petitive assays, the first one is subject to non-specific Methodological considerations inhibition by other serum factors, is of limited sensi- tivity, and is not recommended. The advantages of The recommended methods of measuring serum non-competitive assays are: increased sensitivity and IgE (usually present in Mg/l quantities) are: ELISA- precision and the fact that they are usually free from DIAGNOSTIC PROCEDURES IN CLINICAL IMMUNOLOGY 721 interference by non-specific serum factors. RAST) and the other one, enzyme-labelled anti-IgE Although radioimmunoassays were used initially, (ELISA). Modifications of the standard RAST pro- ELISA has much potential and many advantages, cedures should be used cautiously. Frequently, in- especially for use in developing countries. creased sensitivity is achieved at the cost of decreased The main advantages of ELISA techniques are the specificity. The potential advantages of immuno- avoidance of isotope markers; long shelf-life of the enzyme assays for specific IgE are the same as those reagents; and evaluation by means of a photometer for total IgE. instead ofthey-counter. The only limitation is that the The interpretation of results is hampered by a ELISA techniques developed so far are not suffi- number of pitfalls: ciently sensitive to measure very low IgE levels. (a) The commercial kits that are commonly used Radioimmunoassay is therefore the method of serum. For choice in paediatric patients, in immunodeficiencies, yield results related to a single reference and for analysis of cord blood, supernatants of cell this reason, comparison with other results is almost cultures, etc. For the higher sensitivity and repro- impossible. ducibility required for research purposes, suitable (b) RAST classes for different are not double-antibody assays have been described. comparable. The values obtained must be compared with those (c) Most preparations are impure. of a control group matched according to age and (d) Antibodies of other immunoglobulin classes geographical location. An International Reference present in the same serum sample cause interference. Preparation is available from WHO. One important obstacle is the difficulty of trans- forming RAST results into levels of clinical sensitivity Indications that are meaningful for the practising physician. Determination of total IgE is not essential, except in the diagnosis of the rare hyper-IgE-syndrome associated with eosinophilia and recurrent infections Indications described by Buckley. The measurement of specific IgE is not essential in Determination of total IgE may be useful in differ- any clinical situation; it is no alternative to careful entiating IgE-mediated from non-IgE-mediated dis- history taking and skin tests. Usually skin tests are orders when this cannot be done by clinical means. more closely related to the manifestation than specific Such disorders include perennial , bronchial IgE assay. However, in vivo tests can be subject to asthma, , chronic urticaria, and food non-specific (irritant) influences. intolerance. However, IgE levels are of limited value Measurement of specific IgE is useful in the follow- since total IgE level can be in the normal range in IgE- ing situations: dermographism or severe dermatitis mediated diseases (e.g., hay fever) and it can be which preclude skin testing; when symptomatic treat- increased by non-atopic mechanisms such as infes- ment influencing skin reactions cannot be stopped tation with parasites. The result should therefore be (e.g., antihistamines); when during extremely high interpreted with caution taking into account all other levels of sensitization skin testing would be dangerous pertinent clinical information. Serial determinations for the patient; allergens that cannot be used for skin (e.g., during immunotherapy) are usually of limited testing (toxic, water insoluble, or highly sensitizing value, with the possible exception of allergic broncho- substances); food , for which skin tests are less pulmonary aspergillosis. reliable; and for the interpretation of doubtful skin In prospective investigations, IgE elevation in early tests. In this connection it should be stressed that the childhood may be a useful indicator of high risk for antigenic composition of skin-test solutions is not atopic diseases. necessarily the same as that used as a substrate in the in Determination of total IgE can be considered as a vitro test. tool for research in certain immunodeficiencies and in Properly used, measurement of specific IgE can atopic families. reduce the frequency of provocation tests. Measurements of specific IgE are used for research purposes in various IgE-mediated diseases and in SPECIFIC IgE some parasitic infections. Measurements of specific IgE should not be regarded as a "screen" for allergic diseases or be Methodological considerations requested for the evaluation of allergic conditions in Two main techniques are available. One using which IgE-mediated mechanisms are not involved radiolabelled anti-IgE (: (e.g., contact dermatitis). 722 WHO MEMORANDUM

COMPLEMENT MEASUREMENTS

Complement consists of a series of proteins that necessary in clinical practice unless a genetic comple- undergo sequential activation as a consequence of ment defect is suspected. Antisera to most comple- interaction with a variety of agents. Measurement of ment proteins are available, in particular to C3, C4, complement can be achieved either by functional Clq, Cl-esterase inhibitor, and Factor B. Estimation measurement of the whole system, by functional of individual components by immunochemical tech- measurement of individual components, or by immu- niques is adequate for the vast majority of clinical nochemical measurement of individual components purposes, and is particularly useful with poorly-stored using specific antisera. These measurements represent specimens. Although rare genetic defects occur that the balance between synthesis and consumption. result in the synthesis of abnormal molecules without Elevated complement levels occur as a result of functional activity, in general, component levels esti- increased synthesis, especially following acute mated immunochemically reflect in vivo functional inflammation and trauma, and low levels are found as levels. a result of increased consumption and/or decreased The immunochemical estimation of C3 and C4 and synthesis. The latter may be genetically determined. other complement proteins can be carried out either by the single-radial-diffusion test, or by some form of nephelometry. "Rocket" immunoelectrophoresis is METHODOLOGICAL CONSIDERATIONS not recommended because of the changes in electro- phoretic mobility of the molecules on storage. Inter- national Reference Preparations for C3, C4, Clq, and The total-complement haemolytic assay (CH5o) Factor B are available through WHO. The specificity This assay assesses the ability of serum to lyse a of the antiserum used in the analysis is important and, standard suspension of sheep erythrocytes optimally for C3, antisera specific for C3c only should be used. sensitized with anti-sheep-red-cell rabbit antibody. Estimation of C3 and C4 together form the most The test as usually performed assesses mainly the useful routine measurements of complement com- functional activity of the components that generate ponents. In some conditions, the level of C4 may be the classical pathway C3-convertase, and of C3 itself. abnormally low although the CHo may be normal. It is also a test of the presence of functionally-active Sometimes a low CH. is due principally to a low C2, terminal components C5-C9, although it is not sensi- but antisera to C2 are not widely available, and tive to variations in the levels of these components. functional tests for this protein are difficult to carry There are many ways ofperforming this test, but the out in routine laboratories. Cl-esterase inhibitor technique that is most reproducible and clinically levels are principally of value in the differential applicable is that described by Mayer. Variations in diagnosis of angio-oedema. this procedure use different concentrations of cells, and/or different volumes of reactants and incubation CLINICAL INDICATIONS times. It is also possible to measure CH.. by automated methods. The value of this assay is dependent on the condi- CH,, complement estimations are essential only in tions of the test, and the results may vary if the red those conditions in which a genetic defect in comple- cells are not fresh or not standardized properly, or are ment is suspected, e.g., in patients presenting with low in potassium, or not adequately sensitized. For recurrent infections, especially recurrent meningitis, this reason, a standard serum with a known value with hereditary angio-oedema, or with established should be included in all batches of estimations. Also, immune-complex diseases occurring in families. For inadequate collection and storage of test specimens the confirmation of angio-oedema, estimation of the may give falsely low values. Sera should be separated Cl-esterase inhibitor level is essential, and if a normal within one hour after collection of blood, and stored immunochemical level is obtained then a functional at -70 °C before testing. Where this is not possible, assay should be performed since 10-15e/. of blood the use of EDTA plasma has been recommended. In relatives may produce non-functional molecules. If sera containing cryoglobulin, falsely low functional the CH. is normal, functional assays of individual and immunochemical complement levels may be components are unnecessary except for the detection obtained. of heterozygous states. Complement estimations (CH50, C3, and C4) are helpful in the assessment and monitoring of patients Measurement ofindividual components with glomerulonephritis, in established immune- Functional measurement of components is seldom complexdiseases such as systemiclupus erythematosus DIAGNOSTIC PROCEDURES IN CLINICAL IMMUNOLOGY 723 and certain forms ofvasculitis, and in conditions such monitor treatment. as dengue haemorrhagic fever. In conditions in which Routine complement tests are of little value in most low levels are found, these frequently return to normal other acute and chronic inflammatory or infectious in remission, and complement levels can be used to diseases.

DETECTION OF IMMUNE-COMPLEXES IN HUMAN BIOLOGICAL FLUIDS

There is good evidence that immune-complexes The main pitfalls of these four methods are the (IC) are involved in the pathogenesis of tissue lesions following: in a variety of human diseases. (a) These methods will detect non-specifically Since 1972, more than 30 methods for the detection aggregated Ig as well as immunologically aggregated of circulating IC have been devised and used exten- Ig. Some of the methods require a pretreatment of the sively and it was expected that they would provide sample (heating at 56 IC) which may induce Ig ideal tools for the diagnosis of diseases due to IC. aggregation. However, these expectations have not been fully (b) The collection and storage of samples for IC realized. detection should be done with care, avoiding bacterial contamination and repeated freezing and thawing. METHODOLOGICAL CONSIDERATIONS Blood should be allowed to clot for 2 hours at 37 °C before separation of serum. The temperature of Most methods have been designed for the detection storage should be -70 'C. of immunologically aggregated immunoglobulins (c) Tests using Clq may be influenced by the without considering the nature of the antigen(s) presence of heparin, endotoxins, or free DNA in the involved in the IC. These methods are the most widely test sample. used in clinical practice. (d) Methods using rheumatoid factors (RF) are un- Some methods are based on physico-chemical suitable for IgM-containing IC and cannot be used differences between monomeric Ig and aggregated Ig. with sera containing RF or in the presence of elevated Precipitation in polyethylene-glycol (PEG) has been IgG levels. It has also proved difficult to standardize widely used as a routine method. Although it may be RF preparations. useful to concentrate complexes, it is not specific for (e) In the case of the RAJI cell assay, false positive IC since, even at low concentrations, a variety of large results may be obtained in the presence of anti- serum proteins are also precipitated. The quantifi- lymphocyte antibodies. The cells require particular cation of total protein or even individual proteins in care as regards culture conditions to avoid variations PEG precipitate is not recommended as a measure- in sensitivity. ment of IC level. (/) For the above reasons, the results of the Biological methods are based on the recognition of different tests for IC may not always be directly IC in humoral or cell-receptor systems. Although all comparable. of these methods detect IC, they do not allow direct (g) The quantification of IC has been done until quantification of IC proteins. Tests using Fc receptors now without appropriate reference preparations. on macrophages, K cells, or platelets have been largely Therefore published results expressed in "gig of abandoned for two reasons: (a) high sensitivity to complexes" or as "equivalent of Mg of heat-aggre- interfering factors, (b) difficulty in achieving repro- gated IgG" are not comparable from one laboratory ducibility. to another. Reference preparations of aggregated IgG Although interfering factors can lead to false- and of preformed IC (tetanus toxoid antigen-anti- positive results, it now appears that in most cases a body complexes) are now available on request.b positive result is likely to indicate the presence of IC when the following methods are used: Clq solid-phase Although antigen-specific detection of immune- or fluid-phase binding tests; conglutinin assays; complexes should be the main goal in this type of monoclonal RF inhibition; assay using RAJI cells. investigation, information regarding the nature of the These tests have been found the most acceptable in antigen(s) involved in the in vivo-formed IC has been recent WHO/IUIS collaborative studies. Some of obtained only in restricted clinical conditions, using them (e.g., solid-phase Clq or conglutinin) can be methods developed for that particular purpose (e.g., used to detect the class of antibody present in the b Requests should be addressed to Dr U. Nydegger, Service de complex by the use of appropriate specific antisera at Transfusion CRS, Laboratoire central, Wankdorfstrasse 10, 3000 the final stage. Berne 22, Switzerland. 724 WHO MEMORANDUM

microbial antigens, DNA, etc.). Information for an immune-complex disease. IC-induced lesions obtained through the analysis of IC purified from (e.g., glomerulonephritis) can exist without detectable serum indicates that IC may often result from specific circulating IC, and IC are often present in serum interactions between immunoglobulin molecules (RF, without evidence of typical IC-associated lesions. anti-idiotypes). Thus the presence of IC in serum The detection of IC may be helpful for assessment samples does not imply the presence of a particular and monitoring of disease activity in conditions such antigen of exogenous, microbial, or autologous as rheumatoid arthritis and systemic lupus erythe- origin. matosus. It is also ofvalue in monitoring the effects of plasma-exchange therapy. It may also have a prog- nostic value in some malignancies such as acute leukaemia. CLINICAL INDICATIONS In all conditions where an IC disease is suspected, a direct analysis of tissue samples (e.g., kidney, skin) The detection of IC is not essential in any clinical should be done, when possible. Such examinations condition. The presence of IC in serum is not specific cannot be replaced by the detection of circulating IC.

AUTOANTIBODIES BY INDIRECT

The most widely used method for detection ofauto- Anti-nuclear antibodies antibodies directed against tissue antigens is indirect immunofluorescence (IIF). However, many other To test for anti-nuclear antibodies (ANA), appro- in priate substrates are cryostat sections of rodent liver methods common use provide diagnostic infor- or kidney, but human leukocytes are used in special mation by employing defined antigens and, in the cases. Fixed tissue culture cells are available com- future, the use of purified antigens will become more mercially, but they are visually less satisfactory than common. tissue sections because they give more non-specific fluorescence. Different patients' sera may produce different METHODOLOGICAL CONSIDERATIONS patterns of nuclear staining. Antibodies producing a homogeneous pattern are mainly directed against The IIF procedure involves the application of a nucleohistones. Peripheral patterns are probably due patient's serum to a section of appropriate human or to antibodies against native DNA. The antibodies animal tissue, removal of unbound globulin by associated with speckled staining are directed against repeated washing, and the subsequent addition of soluble nuclear antigens such as the Sm or ribonucleo- antiserum to human immunoglobulin (prepared by protein (RNP) antigens. Nucleolar patterns are due to immunization of an experimental animal) which has reaction with RNA. been conjugated with a fluorescent tag. The site of antibody fixation can be visualized by means of fluoresence microscopy. Instead of a fluorescent dye, antibody can be labelled with an enzyme such as Thyroid autoantibodies peroxidase and appropriate cytochemical methods The substrate for demonstrating thyroid autoanti- used to trace antibody localization. The most bodies consists of frozen human or monkey thyroid important variables involved in a reproducible tech- tissue and the procedure is the same as described for nique are: (a) the type of substrate employed, ANA. At least two distinct autoantibodies can be dif- including source, method of fixation, storage, and ferentiated by IIF. They are directed against the preparation; (b) the duration ofincubation and wash- thyroid epithelial cells or colloid, respectively. A ing of the patient's serum; and (c) the specificity and positive test of a patient's serum on unfixed slides sensitivity of the anti-globulin conjugate. An essential appears as bright fluorescence of the epithelial cells. part ofeach test is the incorporation ofknown positive The autoantibody responsible for this reaction is and negative sera as controls. directed to a microsomal lipoprotein of the epithelial The four groups of autoantibodies that are most cell. Autoantibodies reacting to colloid can be seen requested are antibodies to nuclei, to thyroid, to only when using methanol fixed slides. These auto- mitochondria, and to smooth muscle. It is possible to antibodies can also be demonstrated effectively using prepare composite blocks of several tissues for haemagglutination tests with red blood cells coated processing at one time. with the respective antigens. DIAGNOSTIC PROCEDURES IN CLINICAL IMMUNOLGY 725

Mitochondrial antibodies Thyroid autoantibodies For the demonstration ofmitochondrial antibodies, Tests for thyroid autoantibodies are essential for rat kidney is usually employed as substrate and the diagnosis of chronic thyroiditis and spontaneous immunofluorescence is seen in the cytoplasm of adult myxoedema. Over 90% of thyroiditis patients epithelial cells lining the ducts. have autoantibodies directed against either cell micro- somal antigen, thyroglobulin, or both. A positive test, Smooth muscle antibodies however, does not eliminate the diagnosis of such Smooth muscle antibodies are generally tested with conditions as adenocarcinoma or Graves' disease, rat stomach sections as substrate. since 20%o of these patients have antibodies to thyroid antigen, although titres are generally lower than in those patients with thyroiditis.

CLINICAL INDICATIONS Mitochondrial antibodies Antibodies to mitochondria are characteristic but Requests for unspecified screening for autoanti- not specific for primary bodies should be discouraged. Clinicians should biliary cirrhosis. rather ask for precise autoantibody tests appropriate to the clinical context. Smooth muscle antibodies Antibodies to smooth muscle are frequently found Anti-nuclear antibodies in high titre in the sera of patients with chronic active Tests for ANA are essential for the diagnosis of hepatitis. These and mitochondrial antibodies are systemic lupus erythematosus (SLE). The occurrence found in many other conditions, but the tests may of ANA in low titres is relatively common and is become more useful when purified antigens become associated with a variety of disorders. Even sera from available. normal individuals show a low incidence of ANA, especially in aged persons. Therefore the greatest use of the ANA is to exclude the diagnosis of SLE, since Other autoantibodies the vast majority of active SLE cases are positive. Other autoantibodies of clinical interest are found Further confirmation of the diagnosis of active SLE in certain uncommon diseases. For instance, anti- requires the demonstration of antibodies to native bodies to the intercellular substance of stratified (double stranded) DNA, which can be achieved by IIF squamous epithelium are present in pemphigus, while (with Crithidia lucilleae kinetoplast) or by other a different fluorescent pattern involving the basement techniques; the demonstration of antibodies to Sm membrane of stratified epithelium is characteristic of antigen is also of great diagnostic value in this pemphigoid. condition. Antibodies to muscle striation are often detected in Tests for ANA are useful in the diagnosis of "mixed the sera of patients with myasthenia gravis. However, connective tissue disease" (speckled pattern associ- in this condition, a more useful test is the detection of ated with antibodies to RNP) and the autoimmune antibodies to the acetylcholine receptor by radio- form of chronic active hepatitis. They are also helpful . in many cases of drug-induced SLE and a character- Autoantibodies to adrenal cortex found in chronic istic pattern ofnucleolar staining occurs in progressive cases of idiopathic adrenal insufficiency or to pan- systemic sclerosis. ANA is sometimes of value in the creatic islets in some cases of insulin-dependent study of family members of patients with SLE, diabetes mellitus are not frequent enough to be of because it may lead to earlier detection of this disease. diagnostic value, but are useful for clinical research.

B AND T CELL DETERMINATION

A major advance in the study of the lymphoid these studies have been disappointing for most clinical populations was made when it was shown that they purposes, they have helped in the characterization of could be characterized by certain cell-surface markers. cellular markers and in our understanding of human Since then the T and B cells have been enumerated in lymphocyte physiology. health and disease in a number of studies. Although 726 WHO MEMORANDUM

METHODOLOGICAL CONSIDERATIONS B-cell markers Surface-membrane Ig is the most reliable B-cell Lymphoid cell separation marker if the test is properly carried out. Membrane Most of the studies of human T and B cells are immunoglobulin (SmIg) is most commonly identified performed on human peripheral blood and use the by fluorochrome-labelled anti-immunoglobulin anti- Ficoll-Isopaque method for mononuclear cell separ- sera. The recommended reagents for the enumeration ation. Such preparations contain a variable number of of B cells are antisera raised against the Fab portion monocytes which it is important to distinguish from and/or a mixture ofanti-kappa and anti-lambda light- lymphocytes. This can be most easily achieved by chain. They are commercially available but should be either latex particle ingestion or peroxidase staining. very carefully checked for specificity. Monospecific It is advisable to carry out studies ofcell markers on reagents to the various Ig chains are used for freshly-drawn samples of blood and to check the characterization of the heavy- and light-chains of the viability of the cells since this may influence cell cell membrane and in cytoplasm. The following pitfalls should be emphasized: (a) in- surface characteristics. volvement of Fc receptors that may bind autologous as well as the reagent's Ig-this is largely overcome through incubation and the use of labelled F(ab)2 T-cell markers reagents for immunofluorescence; (b) specificity and At the present time, two types of method are recom- potency of reagents needs careful checking; (c) inad- mended for the detection of all peripheral T cells; they equacy of monocyte identification; (d) potential inter- are the formation of sheep-red-cell rosettes (E ference by autologously reacting antilymphocyte anti- rosettes) and the use of T cell-specific monoclonal bodies, conferring positive surface staining to an antibodies. otherwise SmIg-negative cell. The formation of E rosettes is the most commonly Anti-immunoglobulins may also be labelled by employed and recommended assay for enumerating T enzymes, isotopes, or red cells for the determination cells. Different laboratories have reported great vari- of SmIg. ability in the percentage of E rosettes in the normal Using a similar approach to that employed for T population, and these variations are still frequent cells, antisera, and/or monoclonal antibodies reacting although improvement has resulted from better stan- specifically with all B cells have been recently de- dardization of techniques. The source of sheep red scribed. Antibodies to B-cell subsets have also been blood cells, their conservation, the presence of small reported and await further characterization. amounts of serum (fetal calf serum or human AB A group of other markers present on B-cell mem- serum), as well as the careful handling of the rosette branes have been described. Some, such as the preparations, are important factors. Serum factors complement receptor and the receptor for Fc, are not (like antibodies to cell-surface components, or lipo- specific for B cells. Therefore, procedures such as the proteins) may interfere with rosette formation under formation of EAC rosettes are not recommended at certain conditions by coating the cells and competing present for routine enumeration of B cells. However, with the sheep red blood cells for their binding sites. In studies ofthese receptors as well as that ofthe Epstein- such situations, short-term culture (1-18 h) of the Barr virus and mouse red-blood-cell receptors to dif- cells is usually very helpful in removing or shedding ferentiate B cells and B-cell subsets could be used for these substances. research purposes. Specific anti-T cell antisera are now used increas- In summary, the recommended basic methods for T ingly for detection of all T cells among peripheral and B cell determination are at present the SmIg, blood lymphocytes and in the lymphoid organs. Such formation of E rosettes and, where available, the use reagents are directed against the E receptor or against of suitable monoclonal antibodies. other common T-cell membrane determinants. The most promising and reliable reagents are monoclonal antibodies. The preferred method for the use of such CLINICAL INDICATIONS antibodies is by indirect fluorescent labelling rather than by cytotoxicity, which is less accurate. Enumeration of T and B cells is essential in the T-cell subsets were defined initially by the presence assessment and monitoring of primary immuno- of receptors for the Fc of IgM or IgG. However, deficiencies and useful in the diagnosis of secondary recently defined monoclonal antibodies are more immunodeficiency and for the classification of reliable and accurate reagents for defining T-cell lymphoproliferative disorders. It should include subsets. where possible more than one reagent, for example, DIAGNOSTIC PROCEDURES IN CLINICAL IMMUNOLOGY 727 monospecific anti-Ig and/or monoclonal antibodies purposes since so far enumeration of B and T cells has to lymphoid populations. not proved to be of clinical value in infectious, auto- In addition, study of T- and B-cell subsets may be immune, or non-lymphoid malignant diseases. useful in selected patients and mainly for research

LYMPHOCYTE RESPONSE TO MITOGENS IN THE EVALUATION OF CELL-MEDIATED IMMUNITY

The investigation of cell-mediated immunity (CMI) some immunodeficiencies. is important in the evaluation of the immunological In the evaluation of the proliferative response, the competence of the host. For this purpose, a group of level of background should be taken into account in vivo and in vitro procedures is commonly used. since it may clearly affect the final results. The use of It is essential that these assays should be employed overnight culture prior to the addition of the mitogen in an orderly fashion in order to obtain pertinent may help to explain depressed proliferative responses information, minimize abuse, and overcome pitfalls. secondary to inhibitory factors. Delayed hypersensitivity skin testing, using two or The most commonly used mitogens are phyto- more common recall antigens (streptokinase-strepto- haemagglutinin, concanavalin A, and pokeweed dornase, PPD, Candida, Tricophyton, mumps), extract; the first two are mainly T-cell mitogens should be the first assay performed.c It is only follow- whereas the latter is a T- and B-cell stimulator. It is ing this initial stage, if the results obtained suggest however likely that these, as well as some other mito- possible alterations of CMI, that cell function should gens, stimulate poorly defined subpopulations of T be explored in vitro. In addition to mitogen responses, and B cells. lymphocyte responses to foreign antigens and allo- antigens should also be investigated. The following remarks deal exclusively with pro- CLINICAL INDICATIONS liferative responses to mitogens. The assessment ofthe lymphocyte response to mito- METHODOLOGICAL CONSIDERATIONS gens is not indicated for routine use and should be used only selectively. Abnormal results from single isolated CMI assays are clinically meaningless and will A proliferative response of lymphocytes to several not necessarily indicate abnormalities of CMI in the mitogens is best measured by radioactive thymidine patient. uptake. Mononuclear cells separated by Ficoll- Evaluation of cell-mediated immunity is essential in Hipaque from peripheral blood should be used in the assessing a suspected or proven primary immuno- micro method. Results are commonly expressed as deficiency. Evaluation of CMI is useful in (a) total uptake of radioactivity. assessment of secondary immunodeficiencies, includ- In order to optimize the assay, it is essential to ing those associated with chronic infections; and (b) define the culture conditions, standardize the biologi- monitoring and evaluating theapplication ofimmuno- cal and commercial reagents, and control the number stimulatory therapy. It may be helpful for research of cells in the culture (including concentrations of purposes in diseases with possible impairment of monocytes). In addition, owing to the great variability immune function, such as autoimmune processes and inherent in the systems, the use of normal controls is cancer, and for evaluation of the effect of immuno- essential. These should consist of controls matched suppressive drugs. for the patients as well as controls for computing the daily variation of the laboratory. Results should then be expressed as the relative proliferative response index, which takes into consideration the above- mentioned factors. Dose-response curves are also of importance in order to obtain the optimal response, Z. Bentwich, Kaplan Hospital, Rehovot, Israel while suboptimal concentrations of mitogens may be N. Bianco, Unidad de Immunologia Clinica, Instituto useful in the study of certain disease states such as Anatomo-Patologico, Facultad de Medicina U.C.V., Caracas, Venezuela c Sensitization with l-chloro-2,4-dinitrobenzene (DNCB) is at present the only way to explore the primary response in vivo, but this L. Jager, Friedrich Schiller Polyclinic, Jena Univer- should be done only in selected patients. sity, Jena, German Democratic Republic 728 WHO MEMORANDUM

V. Houba, Immunology, World Health Organization, M. Seligmann, Institut de Recherches sur les Maladies Geneva, Switzerland du Sang, Hopital Saint Louis, Paris, France (Chair- P.H. Lambert, WHO Immunology Research & Train- man) ing Centre, Centre de Transfusion, Hopital R. Thompson, Regional Department of Immunology, Cantonal, Geneva, Switzerland East Birmingham Hospital, Birmingham, England W. Knapp, Institute of Immunology, Vienna, Austria G. Torrigiani, Immunology, World Health Organiz- N. Rose, Wayne State University School of Medicine, ation, Geneva, Switzerland Department of Immunology & Microbiology, A. de Weck, Inselspital, Berne, Switzerland Detroit, Michigan, USA

REFERENCES

1. BLOOM, B.R. & DAVID, J.R., ed., In vitro methods in 4. THOMPSON, R.A. Techniques in clinical immunology, cell-mediated and tumour immunity. New York, 2nd ed., Oxford, Blackwell Scientific Publications, Academic Press, 1976. 1981. 2. KABAT, E.A. & MAYER, M.M. Experimental immuno- 5. WEIR, D.M. Handbook of experimental immunology, chemistry. Springfield, ILL, C.C. Thomas, 1961. Oxford, Blackwell Scientific Publications, 1978. 3. ROSE, N.R. & FRIEDMAN, H. Manual of clinical immu- nology. Washington, DC, American Society for Micro- biology, 1980.