Laboratory investigations in clinical immunology: Methods, pitfalls, and clinical indications

CLINICAL

IMMUNOLOGY

Laboratory

AND

49, 478-497 (1988)

IMMUNOPATHOLOGY

Investigations in Clinical immunology: Pitfalls, and Clinical Indications

Methods,

A Second IUIShVHO Report IUIS/WHO Working Group Genevu.

Switzerland.

Fehruqv

1988

LIST OF CONTRIBUTORS P.H. LAMBERT

Z.BENTWICH R. Ben Ari Institute of Clinicul Kaplan Hospital PO Box I 76100 Rehovot. Israel

Microbiology .Serlice.s World Health I211 Genes

Immunology

Supply

Orgunizntion 27, Switzerland

N. R. ROSE

P. C. L. BEVERLEY ICRF Human Tumour Immunology Group University College and Middlesex School Medicine Faculty of Clinical Sciemes 91 Riding House Street London WIP SBT, United Kingdom

L. HAMMARSTROM

(Chairman. Working Group)

of

Depurtment of Immunology und Infectious Diseases The Johns Hopkins Uni~~ersity School of Hygiene und Public Health 615 North Wolfe Street Brdtimorc. Marylund 21205

R. A. ‘THOMPSON (Chairman, IUIS Clinical Immunology Committee)

Department of Clinical Immunology Karolinska Institute Huddinge Hospital S-141 86 Huddinge, Sweden

J.R. KALDEN Institute for Clinical Immunology Rheumatology University of Erlangen-Nurnherg Krankenhausstrasse 12 D-8520 Erlangen, West Germuny

and Immunonology

und

Regional Depurtment of Immunology East Birmingham Hospital Bordesley Green East Birmingham B9 5ST. United Kingdom

Contents. Introduction. Assessment of immunoglobuiin abnormalities. Recent advances in autoantibody testing. Tests of immune effector systems. Leukocyte markers. Tests of peripheral blood leukocyte function. Use of immunodiagnostic tests of infectious diseases in lesser developed countries. Future directions of laboratory tests in clinical immunology Acknowledgments. References.

478 0090-1229188 $1.50 Copyright (r, 1988 hy Academic Press. Inc. All rights of reproduction in any form raewed

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INTRODUCTION The development of immunological concepts and ideas in medicine continues at a rapid rate, and new laboratory investigations for the management of disease are constantly being reported in the scientific literature. The widespread application of newer technologies in immunology requires that consideration be given to the benefits for patient management of newer tests. The previous WHO/IUIS report’ critically evaluating eight widely used laboratory tests in clinical immunology was well received and the IUIS Clinical Immunology Committee concluded that an update was necessary. The tests covered in the first report, with their indications and contraindications, are still relevant today, although in many cases improved methods are now available. The approach of this report is the same as in the previous report; that is, to outline the nature of the newer tests, the methodological pitfalls, and the indications for requesting them. These indications will again be divided into those in which the test is essential for diagnosis, useful in patient management or for research purposes, or of minimal clinical value. An additional aim of this report was to consider the special requirements for immunodiagnostic tests for infectious diseases in the lesser developed countries. ASSESSMENT

OF IMMUNOGOBULIN

ABNORMALITIES

Introduction The measurement of the main immunoglobulin classes in serum, i.e., IgG, IgA, and IgM, remains a useful test in the evaluation of immunoglobulin deficiencies and in certain other conditions as outlined in the previous report. The four subclasses of IgG and the two subclasses of IgA were identified during the sixties and antisera able to identify these subclasses have recently become available. The organization of the heavy chain constant region genes on chromosome 14 has been elucidated and appears to be reflected in the order in which these genes are used during the maturation of the B-cell system +,-Ay3-yl-(rl-y2-y4+-ol2). This pattern is also often reflected in the sera of patients where deficiencies of IgG2, IgG4, IgA, and IgE are frequently associated. Serum levels of immunoglobulin classes and subclasses appear to be independently regulated during ontogeny and adult levels of IgM, IgG 1, and IgG3 are reached at an early age, whereas adult levels of IgG2, IgG4, and IgA are not reached until later, even up to adolescence. IgGl usually contributes about 60-65% of total IgG and IgG2 around 20-25%, with IgG3 and IgG4 forming the remainder. Published data suggest an appreciable IgG subclass restriction of antibodies against both protein (mainly IgGl with a minor contribution of IgG3 or IgG4) and polysaccharide antigens (mainly IgGl in children with a gradual shift to IgG2 as the immune system matures). This is of considerable importance in patients who lack a particular subclass, since it may severely affect the antibody repertoire. ’ Use and abuse of laboratory tests in clinical immunology: Critical used diagnostic procedures. WHO Bull. 59(5), 717-728, 1981, and C/in. 122-138. 1982.

considerations fmmunol.

of eight widely Immunopathol.

24,

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Methods and Pitfalls a. Measurements of total levels of IggC; subclasses. Immunoglobulin subclass levels may be measured using either polyclonal or monoclonal antibodies. Polyclonal sera require considerable absorption to render them specific and are usually unsuitable for most tests with the exception of immunoprecipitation assays. Some of the available monoclonal antibodies display a high degree of assay restriction and care should be exercised when selecting reagents. Some are poorly precipitating and a mixture of antibodies may therefore occasionally be required. A moderate proportion of sera from patients with deficiencies affecting selective IgG subclasses contains antibodies against immunoglobulins of rabbits (which are often the source of polyclonal antisera) and may give erroneous values in a number of different assays. Radial immunodiffusion (RID) is at present the most widely used method for quantifying IgG subclass levels in serum. The method works well with both polyclonal and selected monoclonal antibodies, and has a sensitivity which is sufficient for most routine purposes. Both IUIS reference sera and suitable monoclonal anti-IgG subclass reagents are available.’ Rocket immunoelectrophoresis. radioimmunoassays, enzyme-linked immunosorbent assays, nephelometric, and turbidimetric assays have also recently been developed using either polyclonal or monoclonal reagents. b. Measurements of the subclass distributiorl of specijic antibodies. Attempts have been made to quantify the levels of specific antibodies in the different subclasses. Pitfalls may, however, be encountered since antibodies of different isotypes may display marked differences in affinity with subsequent quantitative underestimation of low-affinity antibodies. For these reasons, standardization is also difficult to achieve. The antigens used in these types of assays should include both polysaccharide antigens (such as pneumococcal capsular polysaccharide) and protein antigens (such as tetanus toxoid).

Clinical

Indications

Measurement of total levels of IgG subclasses or assessment of the subclass pattern of specific antibodies is essential in patients with increased susceptibility to bacterial infections when the total IgG level is within or only slightly below the normal range. It is also useful for research purposes. It is not essential to monitor class or subclass levels during replacement therapy. Although isolated or combined deficiencies of all IgG and IgA subclasses have been reported to date, only IgG2 deficiency can as yet be regarded as a distinct disease entity causing an increased susceptibility to infections with capsulated bacteria such as Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. IgG2 deficiency is often associated with a lack of IgA. A number of patients who lack antibodies of a given specificity (most often to polysaccharide antigens) in their serum despite normal total levels of IgG subclasses have also been described. Determination of the antibody repertoire may therefore be usefuf in the limited number of patients showing undue susceptibility ’ Available

from

CDC.

Atlanta,

GA

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to selected pathogens. A number of different, commercially available, intravenous immunoglobulin preparations show a relative or total lack of the desired subclass or specific antibody and it is therefore advisable to analyze the content of the product in certain circumstances. Measurement of IgA subclasses is not essential in any clinical condition. Quantitation of IgA subclasses may, however, be useful for research purposes and in the evaluation of patients with transfusion reactions. c’. Zmmunoelectrophoretic analysis of immunoglobulins. As discussed in the previous report, electrophoresis of serum samples (and other biological fluids) followed by immunofixation is a useful method for the identification of class, subclass, and light chain type of monoclonal immunoglobulins, and IUISlWHO has recently published an evaluation of suitable anti-subclass antibodies for this type of assay. Isoelectric focusing followed by immunofixation has recently been shown to be more sensitive for detecting monoclonal immunoglobulins, but the problems of determining the clinical significance of such proteins remain. The method is presently used both for the analysis of cerebrospinal fluid in cases of suspected multiple sclerosis and for determination of the clonality of a given immune response. d. Measurement of antibodies involved in allergic reactions. IgE, which mediates the release of potent, biologically active substances from mast cells and basophils, is the most clinically important antibody in atopic disease. In recent years, much interest has also been focused on the possible involvement of IgG4 in allergic reactions. Methods

and Pitfalls

The recommended methods to measure serum IgE are radioimmunoassay or enzyme-linked immunosorbent assay, as outlined in the previous report. Measurements of specific IgE and IgG4 antibodies are also mainly performed with these two techniques. Some of the previously identified pitfalls of these methods, such as lack of appropriate standards and impurity of the allergen preparations, are being addressed and many of these reagents are now more reliable. Problems such as the quantitative underestimation of antibodies of low affinity and difficulties in comparing antibody levels between different allergens remain. Indications Determination of the total level of IgE is not essential for diagnostic purposes in any clinical condition, with the exception of the hyper-IgE syndrome. It may, however, be helpful in differentiating IgE from non-IgE-mediated disorders, but the measurements are still of limited value, since the total level of IgE may be in the normal range even in IgE-mediated diseases. Serial determinations of IgE levels are of limited value, with the possible exception of allergic bronchopulmonary aspergillosis. High IgE levels in cord blood may be a useful indicator of high risk for atopic diseases. The measurement of specific IgE or IgG4 antibodies is not essential in any clinical condition and it is no alternative to careful history taking. In certain clinical conditions such as penicillin allergy, insect venom hypersensitivity, and food intolerance in children, relevant specific IgE antibody tests may be useful.

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It has been suggested that high levels of specific Ig4 antibodies are associated with treatment failure during immunotherapy. However, the data are conflicting and measurement of specific IgG4 cannot as yet be recommended as a routine test. Measurements of specific 1gE should, for obvious reasons, not be requested for evaluation of allergic conditions in which IgE-mediated mechanisms are not involved. RECENT

ADVANCES

IN AUTOANTIBODY

TESTING

Introduction Since 1981 the clinical and thus diagnostic relevance of serum autoantibodies has increased considerably, with special emphasis on antinuclear antibodies, liver antibodies, antibodies against cell receptor structures, and autoantibodies associated with endocrine disorders. Besides indirect immunofhrorescence (IIF) for the detection of autoantibodies directed against tissue antigens, methods such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA), immunodiffusion, counter immunoelectrophoresis, and, more recently, immunoblotting techniques are applied. Methods

and Pitfalls

The most important variables in IIF are (i) the type of substrate employed, including source, methods of fixation, storage, and preparation; (ii) duration of incubation and washing; (iii) specificity and sensitivity of the antiglobulin conjugate; and (iv) quality of fluorescence microscope equipment. Problems of interpretation can be caused by heterophil antibodies or nonspecifically bound immunoglobulin. In RIA that employ an ammonium sulfate precipitation, only high affinity antibodies are measured. The lack of purity of antigens still remains a probIem. Because of the species specificity of some autoantibodies, it may be necessary to use human tissue, but preparations of autoantigens from different human subjects may show variable reactivity with different patients sera. Also, radiolabeling of antigen may block autoantibody-specific epitopes. In interpreting test results, the frequency of autoantibodies in healthy subjects and in different immunologic diseases and disease subsets, as well as the standard error of the method, has to be taken into account. False negative results might be due to an inactive disease state, to immunosuppressive therapy, or to a loss of immunoglobulin. Antinuclear

Antibodies

(ANA)

Methods and pitfalls. The pitfalls of the different IIF assay systems for antinuclear antibodies have been discussed in the previous document. IIF identities most, but not all, of ANA present in patients sera. If a serum is positive in the IIF, further analysis should be considered to determine the tine specificity of the ANA, using other methods such as immunodiffusion, passive hemagglutination, and counter-current electrophoresis, as well as RIA and ELISA. In contrast to the FARR assay detecting only high affinity antibodies, both high and low affinity

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antibodies are demonstrated in the Crithidia lucilliae IIF assay and the recently developed ELISA test. The disease incidence of different antinuclear antibody specificities is given in Table 1. Standards of the WHO are available for the indirect immunofluorescence test (1) homogeneous pattern WHO 66/233, and (2) anti-native DNA WHO U/80. Recently, an international standardization effort has succeeded in preparing reference material for the demonstration of ANA, which is available from CDC in TABLE THE

DISEASE

INCIDENCE

OF DIFFERENT

1

ANTI-NUCLEAR

Immunological specificity -___ A. Nucleic acids 1. Double-stranded DNA (dsDNA) Antigenic determinant identical on ds- and ssDNA

ANTIBODY

SPECIFICITIES

Disease incidence Present in 60-70% of SLE. In high titers practically a diagnostic marker. Occasionally, low titers in other rheumatic diseases.” Present in up to 95% of SLE. Present in other diseases, including nonrheumatic diseases.b

2. Single-stranded DNA (ssDNA) Antigenic determinant related to purines and pyrimidines B. Histones Determinants on H,, H,A, H,B, H,, H, or complexes of H2A - H,B; H, - H,

Present in 30-70% of SLE, 15-20% of RA, and up to 95% in drug-induced SLE.

C. Nuclear proteins I. Sm-antigen Antigenic determinant complexed to five species of small RNA (smRNA) 2. Ul-RNP Antigenic determinant complexed to Ul-RNA

Present in 25-35% of SLE. Diagnostic marker. Present in high titers (and 95% of mixed connective tissue disease (MCTD); in 3545% of SLE; low titers in scleroderma, discoid SLE, and Sjogren’s syndrome. Present in 3@-40% of SLE and in 60-70% of Sjogren’s syndrome. Related to neonatal SLE. Present in 15% of SLE and in 40-60% of Sjogren’s syndrome.

3. SSAiRo Antigenic determinant related to 61 kDa protein. 4. SSBlLa Antigenic determinant related to 43 kDa protein complexed to RNAs. 5. PCA (proliferating cell nuclear antigen) Antigenic determinant related to a 33 kDa protein. 6. ScL 70, related to anti-toposomerase I. 7. Jo-l, related to anti-histidylt synthetase.

Present in 3% of SLE.

8. Centromere, related to kinetochore antigens.

-

Present in 30% of diffuse scleroderma. Present in 5% of dermatomyositis and 30% of polymyositis. Present in 70% of CREST syndrome (i.e., calcinosis, Reynaud’s phenomenon, oesophageal dysmotility, sclerodactyly, teleangiectasia). Highly diagnostic.

0 Transient elevated titers in certain infectious diseases. ’ Increased incidence by IIF in autoimmune diseases, infectious diseases, malignancies, and, in modest titers, with increased age.

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Atlanta, (i) AFKDC ANA No. 1 (homogeneous pattern), (ii) AF/CDC No. 3 (speckled pattern), (iii) AF/CDC ANA No. 6 (nucleolar pattern), (iv) AF/CDC ANA No. 8 (centromerelkinetochore), and (v) AFKDC ANA No. 4 (antinuclear ribonuclear protein [Ul-RNP]). (See Table I.) Clinical indications. Tests for ANA are essential in the diagnosis of systemic rheumatic diseases. They are most often used to exclude the diagnosis of SLE, since the vast majority of all active SLE cases is positive. Antibodies against dsDNA or Sm antigen are strongly indicative for SLE, in which IgG anti-dsDNA antibodies are used to assess disease activity and response to therapy. Newborn children of pregnant SLE patients with anti-SSA/Ro antibodies should be examined for the presence of congenital heart block. Autoantibodies

and Liver Diseases

The detection of serum autoantibodies is of value in the differential diagnosis of chronic aggressive liver disease (CALD), a term that encompasses a spectrum of disorders ranging from autoimmune (“lupoid”) chronic active hepatitis (CAH) to primary biliary cirrhosis (PBC) and includes CALD related to: chronic infection with hepatitis B (HBV) or the non-A, non-B (NANB) viruses, drug-induced liver disorders, Wilson’s disease, cu-1-antitrypsin deficiency, alcoholic liver disease (ALD), primary sclerosing cholangitis (PSC), and hemochromatosis. All of these disorders can be present as CAH. Classical autoimmune CAH (AI-CAH), originally described as a disease of young women, has been broadened to include idiopathic CAH in both sexes of any age, with either ANA or anti-smooth muscle antibody (SMA). Methods and pitfalls. Routine screening for ANA and SMA can be regarded only as a guide to a probable diagnosis of AI-CAH and should be taken in conjunction with other clinical and pathological parameters. Although ANA and SMA are rare in some disorders, such as Wilson’s disease, hemochromatosis, and NANB infections, they occur at significant frequencies in HB-CAH, PBC, and PSC. Anti-mitochondrial antibodies (AMA) occur in more than 95% of patients with PBC, but are found only rarely in other disorders. Of at least nine different AMA specificities (Ml-M9), the M, antibody is highly specific for PBC. Two antibodies to liver membrane antigens, the liver membrane antibody (LMA) and antibodies to a liver-specific membrane lipoprotein (LSP), may have overlapping specificities. These antibodies may help to differentiate various forms of CAH and may be useful in monitoring therapy in AI-CAH. Clinical indications. The MZmitochondrial antibody is essential for the diagnosis of PBC. The other liver autoantibodies are not essential, but may assist in the diagnosis and treatment of various forms of CAH. Receptor Antibodies Autoantibodies to the acetylcholine receptor (AChR) in myasthenia gravis (MG) and to the thyroid-stimulating hormone receptor (TSHR) in Graves’ disease (GD) are of clinical relevance.

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Autoantibodies

Methods and Pitfalls. Anti-AChR antibodies are routinely demonstrated by RIA using AChR preparations from human skeletal muscle, radiolabeled by snake toxins such as a-bungarotoxin. They are found in up to 70% of adult cases with generalized myasthenia gravis. They also occur in D-penicillamine-induced MG and neonatal MG, but not in congenital or restricted ocular MG. Few patients with ocular MG and high anti-AChR titers may have subclinical generalized MG. AChR preparations from the torpedo species should not be used because of limited cross-reactivity with human AChR. A recently described ELBA test system utilizes crude human muscle AChR preparations. However, false positive results occur with anti-striatal muscle antibodies. Work on the standardization of the methods is in progress. Clinical Indications. The demonstration of AChR antibodies is essential in the diagnosis of suspected myasthenia gravis. The antibody titer does not reflect the clinical severity of the disease. Thyroid-Stimulating

Autoantibodies

Methods and pitfalls. Thyroid-stimulating autoantibodies bind to TSHR, exert different biological activities, and represent a heterogeneous population of antibody specificities and functions. Assays to measure anti-TSHR antibodies include “‘1 uptake in vivo using a mouse assay or three different bioassays on human thyroid tissue: (i) the colloidal droplet formation, (ii) the stimulation of thyroid adenocyclase, and (iii) cytochemical alterations. Other assays measure the inhibition of binding of radiolabeled thyrotrophin to human thyroid membranes or lyophilized TSHR. Test systems available thus far are all poor in their precision, and the interassay variation is high. Clinical indications. Anti-TSHR antibodies are not essential for the diagnosis of autoimmune thyroid diseases. They may, however, be useful in excluding active GD or in follow-up studies, since patients maintaining a relatively high titer are more likely to relapse. Other Anti-Receptor

Autoantibodies

Other anti-receptor autoantibodies, such as insulin receptor antibodies in insulin resistance, the gastrin receptor antibodies in pernicious anemia, and antibodies to the p-2-adrenergic receptor in bronchial asthma, may probably prove useful in the future. Autoantibodies

and Endocrine

Diseases

Other thyroid autoantibodies. In the diagnosis of chronic thyroiditis and spontaneous adult myxedema thyroid autoantibodies are helpful. Over 90% of thyroiditis patients have autoantibodies to the thyroid microsomal antigen (ATM) or to human thyroglobulin (AHT) or to both. A positive test, however, does not exclude conditions such as adenocarcinoma or Graves’ disease, since up to 80% of patients with thyrotoxicosis and up to 60% of patients with thyroid adenocarcinema do have AHT antibodies, and up to 80% of patients with GD exhibit ATM

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antibodies as well. Both antibody healthy persons.

IUIdWHO

specificities

REPORT

may be present in up to 10% of

Anti-islet cell antibodies (ZCA).3 ICA are demonstrated by the indirect immunofluorescence assay, 50% of which fix complement. The complement-fixing antibodies appear to be more specific markers for ongoing insulitis. ICA are increasingly being used as serological markers for type I (insulin-dependent diabetes mellitus); although not essential for the diagnosis, they might be useful in classifying and monitoring diabetic patients, and in identifying susceptible individuals liable to develop the disease. Autoantibodies to adrenal cortex. These autoantibodies are found in a proportion of cases of chronic idiopathic adrenocortical insufficiency. They are not frequent enough to be of diagnostic value but are useful for clinical research.

Other Autoantibodies Antiphospholipid antibodies, such as anticardiolipin antibodies and the lupus anticoagulant (LAC), may be associated with thrombo-embolic disease or multiple abortions. The antigen specificity of these antibodies is different. Phospholipid antibodies are measured by either RlA or ELISA systems, which have not yet been standardized. Circulating antibodies to the intracellular substance of stratified squamous epithelium are present in the majority of patients with pemphigus, but are not specific for these conditions except when detected by direct immunofluorescence in biopsies from juxta-lesional skin. Some antibodies to the basement membrane of stratified epithelium are characteristic for various forms of pemphigoid but, here also, direct examination of biopsies may be more useful. Antibodies to neutrophil cytoplasmic granules (ANCG) can be demonstrated in up to 70% of patients with acute Wegner’s granulomatosis, and are useful markers for the disease activity in this condition. They have also been described in some patients with small vessel vasculitis or polyarteritis. Work is in progress at present to identify the target antigen and to distinguish different antibody specificities in the related conditions. Anti-basement membrane antibodies are of specific differential diagnostic \qa/ue for the Goodpasture syndrome. The demonstration of these antibodies in serum is difficult; they are best demonstrated by direct immunofluorescence on kidney biopsies. They are rarely demonstrable in other forms of nephritis. More recently, autoantibodies against the ADP/ATP carrier of heart muscle in virus-induced myocarditis and in dilative cardiomyopathy have been described. These autoantibodies as well as those against calcium-channel proteins in the Eaton-Lambert syndrome are of major interest as models of immunopathogenic effects. TESTS

OF IMMUNE

The effector systems of immunity

EFFECTOR

SYSTEMS

are those involved

in the mediation

of anti-

3 Reference sera for internal standardization of ICA tests are available from Dr. H. Gleichmann. Diabetes-Forschungsinstitut. D-4000 Dhsseldorf 1. FRG.

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body-directed effects, such as the complement system, and those which are cell associated, such as the leukotrienes, prostaglandins, and interleukins. Complement

and Immune

Complexes

Tests of complement depend on measuring functional activity of the whole system (lysis of antibody-coated erythrocytes or appropriately buffered erythrocytes in systems for measuring the alternative complement pathway) or of individual components, in fresh or frozen serum. The use of specific antisera enables immunochemical measurements of individual components to be made in less rigorously stored serum specimens. As discussed in the first report, the major pitfall of functional complement assays is inadequate storage of the specimen. More recently a number of sensitive methods for detecting the activation products of complement have been described. These tests enable the determination of in viva utilization of complement even if the serum levels are normal. Such tests include (i) the determination of the activation products of C3, namely C3a and/or C3d (or C3dg as it is more correctly known); (ii) the determination of the activation product of C4, namely C4a; (iii) activation product of CS, namely C5a; and finally (iv) the presence of the assembled terminal component complex “CSb-9.” Methods. The methods for the determination of split products (C4a, C3a, C3dg, and C5a) involve either radioimmunoassay procedures or ELISA-based systems. These detect the split products in the supernatant after first precipitating the native components by specific antisera or by acid or polyethylene glycol, which leaves the low molecular weight peptides in solution. There are also rocket electrophoresis systems for C3dg which include an anti-C3c containing intermediate gel to remove native C3. The detection and measurement of the terminal C5b-9 complex is usually by an ELISA-based system that “captures” the complex via a specific C9 antiserum and detects it by an antiserum to the C5b component. This test can be used to assess serum specimens for the complexes, although the specimens must be fresh, since, while the C5b-9 complexes are not generated to a significant extent during clotting, they do increase with aging of the specimen. Pitfalls. Because of the great sensitivity of the newer tests, blood must be drawn into EDTA and should be separated within 30 min, and care must be taken to handle plasma specimens in a uniform manner. Because of the reactivity of the anti-fragment antisera with the native molecules, there is a need to remove the latter from the plasma before their measurement can be done. Clinical indications. Functional complement tests (total lytic activity or functional Cl esterase inhibitor tests) are essential in patients suspected of inherited complement deficiency. Measurements of complement components are helpful in the management of a variety of immunological disorders, such as SLE. The newer tests for complement activation products are not essential for the diagnosis of any condition. They may be useful in monitoring the effects of treatment in patients whose disease process is accompanied by in vivo complement activation, particularly when this does not result in low serum levels, such as in certain cases of SLE and rheumatoid arthritis. They are also useful for research.

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Immune Complexes Methods and pitfalls. These were extensively discussed in the first report. No improved new methods have since emerged, and the recommended techniques are Clq-binding assays and the conglutinin assay. Clinical indications. The detection of immune complexes is not essential in any clinical condition. Their detection may be helpfkl for assessing and monitoring of disease activity in conditions such as rheumatoid arthritis and SLE. The detection of antigen-specific immune complexes may be helpful in circumstances where free antibody is not detectable in serum. Leukotrienes and Prostaglandins These groups of molecules play an important role in inflammatory processes. While their release by activated macrophages and granulocytes can be measured by bioassay procedures, this has recently been made more practical by the development of sensitive radioimmunoassays. They can be detected in blood and tissue fluids, but the main use of the tests has been to detect the in vitro production of these molecules by cells challenged in the test tube in various ways. There is as yet little clinical application of these tests although they are useful in research. Cytokines These embrace a number of regulatory molecules, including the interleukins, tumor necrosis factors, and interferons, synthesized by macrophages and lymphocytes during the development of an immune response. Some of them are also synthesized by other cells particularly during inflammation. They are measured in body fluids and in culture systems by sensitive radioimmunoassay techniques often involving the use of monoclonal antibodies, as well as by bioassays. The use of these tests is not as yet essential in any clinical condition. There have been reports of patients with immunodeficiency in whom the defect was an apparent inability of blood monocytes to produce IL-l, and there are other situations of chronic infection such as leprosy in which defects of IL-Z production in vitro can be demonstrated. Thus, while these tests are still only useful for research purposes, it is likely that in the future they will become more important for monitoring therapy and defining new disease entities. LEUKOCYTE

MARKERS

Introduction The use of monoclonal antibodies (mAbs) is now firmly established as the method of choice for identification of different lymphoid cells. Antibodies to monocytic and granulocytic cells are mainly useful in immunohistology. However, despite the identification of many leukocyte antigens with mAbs and some understanding of the function of a number of these molecules, enumeration of leukocyte subsets is essential in relatively few clinical conditions. An internationally recognized terminology for leukocyte antigens has been developed at three workshops on Leukocyte Differentiation Antigens. Antigens are

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defined by grouping together monoclonal antibodies with similar serological reactivity. Thus each antigen is defined by a cluster of antibodies and assigned a CD (cluster of differentiation) number. This terminology is strongly recommended, although some antigens have not yet been assigned a CD number. Cell Suspension Methods and pitfalls. Table 2 summarizes the most satisfactory identification of mononuclear cell subpopulations.

reagents for

T-Cell Markers The use of sheep red cell (E) rosette formation alone for enumeration of T cells is no longer recommended, since some natural killer (NK) cells are also E positive. At present, the most reliable marker for peripheral T cells is CD3, since this is associated with the T-cell receptor for antigen. CD4 and CD8 identify the helper/inducer and suppressor/cytotoxic subsets, respectively. B-Cell Markers Surface membrane Ig (SmIg) remains the hallmark of a B cell. Polyclonal antibodies to Fab, K, and A heavy chains are commercially available and can be used to detect surface and cytoplasmic Ig. Direct immunofluorescence is commonly used. Alternatively, indirect immunofluorescence may be carried out using mAbs against Ig or non-Ig B-cell surface antigens. CD19 and 20 mAbs are the most reliable reagents for B-lineage cells, but CD22 is also present on the membrane of mature B cells and in the cytoplasm of immature B cells (Table 2). Natural

Killer

Cell Markers

The origin of NK cells is still in doubt. Many, but not all, granular lymphocyte morphology. They are heterogeneous in notype but some reagents have emerged which identify the population. CD16, the low affinity Fc receptor, is present on some monocytes as well as NK cells while HNK-1 antigen large granular lymphocytes as is NKH-1. Monocytes

NK cells have large surface antigen phemajority of the NK granulocytes and on is present on some

and Granulocytes

Blood monocytes are heterogeneous. The majority of these monocytes are adherent, will phagocytose latex particles, will express nonspecific esterase, and may express peroxidase activity. Most monoclonal antibodies to monocytes also react with other blood cells, but CD14 mAbs react only with monocytes. Granulocytes can be most easily detected by their characteristic morphology. Some mAbs react strongly with granulocytes, but their variable reactivity with monocytes makes them less reliable for enumeration of granulocytes, which may contaminate mononuclear cell preparations. Indirect immunofluorescence remains the standard method for enumeration of mononuclear cell subsets. These are generally separated by density gradients. Monoclonal antibodies usually give low background staining, but measures should be taken to further minimize nonspecific staining, e.g., (i) mouse IgG2a mAbs

TOIs, RFB4 OKM I, Mac- I

T8

CALLA Bp95. B4 Bp31, Bl

Bp135

Surface Ig CR,. ~160

CDS”

CD10 CDl9” CD20”

CD22

CDllb

These are the most reliable markers for enumerating

MY9

Leu-?

HNKl NKH- I

CD33

Leu- I I

FcTR, FcR-II

CDl6”

2

T cells

Mature B cells, cytoplasm of immature B cells B lymphocytes Monocytes. macrophages. granulocytes Tissue macrophages, dendritic cells Monocytes

Immature B cells B lymphocytes B lymphocytes

Suppressoricytotoxic

Cortical thymocytes T-ALL Mature and immature T cells Mature T cells, cytoplasmic staining of immature T cells Mature and immature T cells Mature and immature T cells Helper/inducer T cells

Main cell type identified

NK cells or large granular lymphocytes NK cells NK cells Myeloid precursor cell> .~ ~~~~~~ mononuclear cell subsets in peripheral blood.

3.9. KB23 M02. UCHMI

pl50

CD1 lc CDl4”

J5 84, Len12 Bl. Leu-16

OKT8. Leu-2a

Tl FcpR T4

OKT6, NA134 OKTl I, Leu-5 OKT3, Leu-4. UCHTl OKTI 1 Leu- I WTI, 3Al OKT4. Leu-3a

CD5 CD7 CD4”

E receptor

Examples of mAbs

T6 Tll. T3

Other names

CD1 CD2 CD3”

International workshop name

TABLE REAGENTSFOR~MMUNOPHENOTYPING

Brain and some epithelial tissues Brain and nerve AML.

Granulocytes Some macrophages and endothelial cells. granulocytes very weak Granulocytes. some macrophages

Some T cells. NK cells

Small subset of B cells and B-CLL Myeloid progenitors (weak) Thymocytes, monocytes, and macrophages (weak) Thymocytes, spleen sinusoid lining cells CALL germinal center cells Renal parenchymal cells Renal parenchymai cells, follicular dendritic cells

Langerhans’ cells NK cells Cerebellar Purkinje cells (some mAbs)

Other reactivities

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should be avoided, if possible, as they bind avidly to human Fc receptors; (ii) mouse IgM mAbs do not bind to human Fc, but are less stable and should be stored at 4°C; (iii) antibodies should not be frozen and thawed repeatedly, and aggregates should be removed by centrifugation prior to use; (iv) Fc receptors may be blocked by prior incubation with heterologous serum; and (v) secondlayer anti-mouse Ig reagents may need to be preabsorbed to remove crossreacting anti-human Ig antibody. The use of F(ab), fragments is recommended. Stained cells may be enumerated by fluorescence microscopy or flow cytometry. Results are best expressed as absolute counts by relating the percentage of cells counted to the total lymphocyte count. Ratios of subsets, e.g., CD4/CD8, are often misleading because they do not indicate which subset is present in abnormal numbers. Monocytes are present in variable numbers in gradient preparations. They often carry Fc-bound human Ig and may therefore complicate enumeration of B lymphocytes. They may be identified with CD14 mAbs or by phagocytosis of latex particles. Direct fluorescence with mAbs is also possible, but it may be difficult to design appropriate controls and it is less sensitive and more expensive. Enumeration of subsets can also be carried out on whole blood using immunoenzyme methods. This method appears specific and sensitive, but has not yet been widely adopted. Clinical

Indications

and infectious disease. Enumeration of T and B cells is and secondary immunodeficiency in order to define the nature of the deficit. In a few rare primary immunodeficiency syndromes, it is now possible to make a prenatal diagnosis by immunophenotyping fetoscopy samples in affected families. Rare cases of immunodeficiency, lacking the leukocyte function antigen (CD1 1 a-c and CDl8), HLA, or CD3, have been identified by immunophenotyping. In AIDS, there is a progressive decline in the absolute number of CD4 cells, and serial CD4 cell counts provide useful prognostic information. There are few other indications for immunophenotyping in infectious disease, but a raised CD8 cells count may occur in acute Epstein-Barr, cytomegalovirus, and other virus infections. A persistent elevation of CD8 cell numbers may indicate chronic viral infection. Lymphoproliferative diseases. Immunophenotyping is useful in the classilication of childhood acute lymphoblastic leukemia (ALL) subtypes and may help to identify those patients (with common ALL) who respond well to standard chemotherapy. Immunophenotyping is also useful for identifying acute undifferentiated leukemias and malignant cell populations in peripheral blood before the total lymphocyte count is greatly raised (Table 3). The test is essential to identify the abnormal cells in poor prognosis patients in order to use the best reagents for bone marrow purging prior to autologous marrow replacement. In autoimmunity and in cancers other than hemopoietic malignancy, immunophenotyping is of research interest only and does not yet provide useful diagnostic or prognostic information. immunodeficiency essential in primary

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TABLE 3 REAGENTS FORLEUKEMIAILYMPHOMA Antigen

AML

CALL

CD45 MHC II CD33 CD14b CD10 CD20 CD22’ Anti-Ig’ CD2 CD3’ CD5 CD7

+ or 2 - or + + - or + ~ or t - or 5

+ or t + + + or -

T-ALL

DIAGNOSIS"

Peripheral T leukemia/lymphoma

+ + + or +

t ~~ 01 t -

B-NH1

t -t -- or -t

B-CLL i F

t

t

+ +

r

$-

-t + + or ~-

-~

1-

Note. Abbreviations: AML, acute myeloid leukemia; CALL, common acute lymphoblastic leukemia; T-ALL, T cell acute lymphoblastic leukemia: B-NHL, B ceil non-Hodgkins’ leukemia: B-CLL, B cell chronic lymphocytic leukemia. a The more important reagents for surface phenotyping of common leukemias. Myeloma expresses few detectable surface markers, but cytoplasmic IG is usually present. Terminal transferase is present in the nucleus of most ALLs. b Indicates monocytic differentiation in AML. ’ Surface membrane staining is indicated. Cytoplasmic CD22 is present in all and cytoplasmrc Ig in some CALLS. Cytoplasmic CD3 is present in most T-ALLs.

Tissue Sections Methods and pitfalls. The majority of mAbs used to stain cells in suspension can be equally well used to identify leukocytes in cryostat sections. Staining can be carried out by indirect immunofluorescence or immunoenzyme methods. Counterstaining is possible for both methods, and preparations are semipermanent if properly mounted. For routine staining the indirect immunoperoxidase method is simple and reliable. Sensitivity can be increased by using a biotinylated anti-mouse Ig followed by avidin-peroxidase complexes (ABC method) or by using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method. All the problems associated with staining cells in suspension apply to staining tissue sections. While the T and B markers listed in Table 2 are suitable for staining in tissue sections, tissue macrophages are not reliably identified by CD14 mAbs Unt, but antibodies to CDllc (the ~150 chain of the LFA complex) are broadly reactive with these cells. Currently the majority of mAbs to leukocyte surface antigens are not suitable for use on routine formalin-fixed and paraffin-embedded tissue sections. However, a number of reagents have recently become available for the identification of leukocytes in fixed material. Several of these mAbs are against components of the leukocyte common (CD43 antigen. It is likely that such reagents will make an increasing contribution to immunopathology in the near future.

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Indications MAbs are essential for the precise identification of non-Hodgkins’ lymphoma (NHL). They allow the distinction between B- and T-cell lymphoma to be made as well as the definition of the maturation stage of the tumor to enable the design of appropriate therapy. MAbs are also useful in the identification of undifferentiated tumors in lymph node biopsies or biopsies from other sites. Antibodies to CD45 and other lymphocyte antigens identify lymphoma (Table 3), while secondary carcinoma or other tumors can be identified positively by mAbs to cytoskeletal elements. TESTS

OF PERIPHERAL

BLOOD

LEUKOCYTE

FUNCTION

Lymphocytes, neutrophils, and monocytes are the main cell types that compose the cellular arm of the immune response. Recent years have witnessed an impressive growth in the study of their functions that are important in evaluating immunological competence in different clinical situations. Methods. It is essential that assays for cellular function be employed in an orderly fashion in order to obtain pertinent information, minimize abuse, and overcome pitfalls. The first step in the assessment of cell-mediated immunity consists of total white blood cell and differential counts. This should be followed by delayed hypersensitivity skin testing with four or more common recall antigens, such as streptokinase-streptodornase, PPD, Candida, trichophyton, and mumps, for which semiquantitative test systems are also commercially available. Most adults will show positive responses for two or more of these antigens. In children, these tests are less reliable because of lesser likelihood of prior exposure. Cell Separation Neutrophils may be obtained by dextran sedimentation or by density gradients, separating them from monocytes and lymphocytes. Purer preparations of monocytes and different lymphoid populations may be obtained by procedures such as countercurrent centrifugation, adherence to plastic plates, panning on antibodycoated plastic plates, fluorescence-activated cell sorting, additional density gradients, adherence to antibody-coated magnetic beads, and by free-flow cell electrophoresis. Phagocytic

Cell Function

Assays

Neutrophil adhesion can be measured by adherence to nylon fibers. Neutrophil aggregation is commonly measured by aggregometers or by an automatic cell counter. The measurement of phagocyte mobility in the presence or absence of a chemoattractant is generally carried out by modifications of the methods introduced by Boyden. Alternatively, phagocytic cell migration and chemotaxis can be measured under agarose. Ingestion by phagocytes is usually measured by particle uptake-polystyrene beads, microorganisms, or immunoglobulin-coated erythrocytes. Metabolic activity of the cell following particle ingestion is detected by the nitroblue tetrazolium

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test (NBT) or by chemiluminescence. The microbicidal activity of phagocytes is usually based on the measurement of viable bacteria remaining within the cells after a defined time period or, more recently, on release of bacterial DNA. It is now clear that some of the causes of familial phagocyte dysfunctions are specific enzyme deficiencies. Pitfalls. The main pitfalls of these methods are the following: (i) Granulocyte adherence is influenced by the type of fiber employed, the incubation period, and the anticoagulant used. Aggregation tests are usually better for assessing the effects of aggregating substances than for determining the tendency of cells to aggregate. (ii) Measurements of phagocyte mobility are highly variable and therefore require multiple replicates in each experiment and repeated testing of the same patient. Among the factors responsible for these variations are differences in cell handling, the nature of the filter, and the protein content of the medium. The agarose method is less sensitive and requires a stronger chemoattractant. (iii) In phagocytic ingestion assays, it is difftcult to distinguish between particle uptake and particle attachment to the cell surface. (iv) Heparin may interfere in the NBT test, as it forms a precipitate with NBT. (v) Chemiluminescence assays give variable light emission with different stimuli and lack quantitative correlation between respiratory burst activity and light emission. (vi) The majority of phagocyte cell function assays may be affected by a variety of drugs, such as corticosteroids, colchicine, and nonsteroidal anti-inflammatory drugs. Lymphocyte Function Assays a. Lymphocyte proliferation. Proliferative responses of lymphocytes to mitogens, antigens, and pathogens are best measured by radioactive thymidine uptake. For this purpose, either mononuclear cells separated by density gradient or whole blood is used. Results are best expressed as the total uptake of radioactivity after subtraction of background values, but might also be expressed as a relative proliferative response index. Dose-response curves are important particularly in the study of disease states such as some immunodeficiencies. The most commonly used T-cell mitogens are phytohemagglutinin, concanavalin A, pokeweed extracts, and CD3 monoclonal antibodies of the IgG2a subclass. Potentially useful human B-cell mitogens are formalin-fixed staphylococci of the Cowan 1 strain, crosslinked anti-Ig antibodies, and Epstein-Barr virus (EBV). Proliferation of peripheral blood lymphocytes induced by various antigens, such as PPD, tetanus toxoid, streptokinase-streptodornase, herpes simplex virus, or Candida, is also used to assess immune function and detect previous exposure. In some instances, this may be the only way to detect prior immunity. Proliferation induced by allogeneic lymphocytes (mixed lymphocyte reaction, MLR) is also commonly applied to determine lymphocyte function and immune regulation. 6. Mononuclear cell-mediated cytotoxicity. Three main types of mononuclear cell-mediated cytotoxicity can be studied: natural killer (NK) activity, antibodydependent cell-mediated cytotoxicity (ADCC), and cytotoxic T lymphocyte (CTL) activity, the latter being induced either by allogeneic stimulation or by altered cell surface components. Cytotoxicity is measured by the release of radiolabeled isotopes from target cells. It is important to apply varying effector-

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to-target cell ratios in these assays. NK cell activity does not require presensitization and is usually measured on standard target cell lines such as K562. c. Zmmunoglobulin synthesis. All B-cell activation and proliferation leading to immunoglobulin synthesis reflect B-cell function as well as regulatory T-cell function. The most widely used stimulant for Ig synthesis is pokeweed mitogen. It is studied most commonly by determining the immunoglobulin content of culture supernatant, immunofluorescent staining of intracytoplasmic Ig, and Igspecific direct or reverse plaque-forming cell (PFC) assays. d. Lymphokines. See section on tests of immune effector systems. Pitfalls. The main pitfalls of these methods are the following: (i) Lymphocyte proliferation assays are influenced considerably by culture conditions, source and type of biological and commercial reagents, and number of cells (including concentration of monocytes), all of which should be standardized as far as possible. (ii) Great variability is inherent in all lymphocyte function assays, which therefore require the regular use of matched controls for the patient’s cells as well as controls for computing the daily variation of the laboratory. (iii) The background thymidine uptake by nonstimulated cells may vary considerably in disease states and will have a considerable effect on interpretation of the results. (iv) In cytotoxicity assays and especially NK cell assays, cell sensitivity is an important variable. (v) In ADCC assays, the nature and target determinant specificities of the antibodies are important variables. Clinical

Indications

The assessment of phagocyte and lymphocyte function should be used selecBoth are essential in the assessment of patients with suspected primary immune deficiency. Phagocyte function tests may be useful in the assessment of secondary immune deficiencies. They may also be applied for research purposes in diseases with impairment of immune function, in cancer patients, and in evaluating immunosuppressive and/or immunomodulating treatments. Lymphocyte function tests are useful in the assessment of secondary immune deficiency. They may also be helpful for research purposes in diseases in which immune mechanisms are involved. tively.

USE OF IMMUNODIAGNOSTIC TESTS FOR INFECTIOUS LESSER DEVELOPED COUNTRIES

DISEASES

IN

In addition to difficulties commonly encountered in the interpretation of results of standardized immunologic tests used for clinical and epidemiologic diagnosis in industrialized countries, there is a host of other constraints that affects their results in developing countries. At any one time, about one billion people are affected by six tropical diseasesmalaria, schistosomiasis, filariasis, leishmaniasis, trypanosomiasis, and leprosy. In tropical Africa, almost every child has malaria by the age of 1 year and at least one million die from it each year. More than 200 million people, mainly in Southeast Asia, are infected with hepatitis B. The problem of HIV infection in Africa is

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becoming increasingly recognized. In some countries, individuals are likely to harbor four or more disease-producing organisms simultaneously. In addition to these biological constraints, there are those that stem from the serious shortages of medical personnel, equipment, equipment maintenance, and supplies. In many major hospitals, but even more in the peripheral health services, many essential laboratory services cannot be carried out due to their dependence on imported reagents and biologicals. Because of the complex health situation in lesser developed countries, there is a pressing need to improve the laboratory diagnosis for infectious diseases that constitute the major public health problems. To be of practical significance. the laboratory tests should be simple, reliable, and economic, and applicable to both curative medicine and public health. Their routine use should be as independent as possible of supplies of expensive biologicals from foreign manufacturers. as well as of costly repairs of laboratory equipment. The diagnostic approaches and needs of physicians and of epidemiologists are different. The physician needs a definite diagnosis to treat his patient promptly and specifically; the epidemiologist has to preselect the diagnostic criteria and laboratory tests that are best suited to identify all individuals who have the specific disease. A prerequisite for the use of any new immunodiagnostic laboratory method in both situations is the adequate validation of the new tests in the local population. The following represent areas of high priority: 1. Simple, reliable and inexpensive immunodiagnostic tests drawing on new technologies and innovative ideas for application: (a) to individuals in the field for diagnosis or for epidemiological screening; (b) in central laboratories for large populations. 2. Expanded training opportunities for laboratory personnel. 3. Improved monitoring, quality control, and standardization. 4. Partnerships between institutions in the more developed countries with their counterparts in less developed countries, to facilitate cooperation and collaboration. In addition, there are additional pitfalls in certain laboratory tests due to the high prevalence of parasitic and other infectious diseases in tropical areas. FUTURE

DIRECTIONS CLINICAL

OF LABORATORY IMMUNOLOGY

TESTS IN

The pace of new investigations in immunology and their applications in clinical medicine will undoubtedly be maintained, and the subject will continue to grow. Existing research procedures, which have as yet not yielded practical results, will probably be the base for new tests in clinical immunology. Some of the areas where progress is likely to be made are: 1. tests involving synthetic antigens and ligands, including recombinant DNA technology;

those produced

by

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2. anti-idiotypic reagents which recognize disease-specific idiotypes; 3. additional autoantibodies that may cause disease by interacting with specific cell receptors, or by interference with intracellular metabolism; 4. monoclonal antibodies identifying subsets of lymphocytes, monocytes, and polymorphonuclear leukocytes that correlate with functional and/or pathological changes in these cells; 5. better tests of neutrophil function; 6. tests for the production and functional activities of different interleukins and mediators of inflammation; 7. the use of DNA technology in tissue typing in identifying clinically significant immunogenetic disorders, and in recognizing pathological agents in tissue fluids and biopsy specimens; 8. tests for the adequate monitoring of immunotherapy. These and other similar advances are likely to be the subject matter of future joint IUISWHO reports on laboratory tests in clinical immunology. ACKNOWLEDGMENTS We are grateful to the following colleagues for helpful comments and suggestions: A. A. Buck, C. I. Civin, R. L. Dawkins, R. Djurup, M. Eible, J. L. Fahey, M. F. Greaves, L.-A. Hanson, E. Hedfors, G. Holm, R. L. Humphrey, G. Janossy, S. G. 0. Johansson, M. Kazatchkine, I. R. Mackay, D. Y. Mason, J. B. Margolick, E. Moller, J. Palmblad, E. Pick, B. R. Rabin, R. F. Ritchie, G. D. Ross, C. I. E. Smith, D. Stites, E. M. Tan, T. Totterman, N. L. Warner, R. Williams, J. Wybran, and B. J. M. Zegers.

REFERENCES Jefferis, R., Reimer, C. B., Skvaril, F., et al., Evaluation of monoclonal antibodies having specificity for human IgG subclasses: Results of an IUIS/WHO collaborative study. Immunol. Serf. 10, 223-252, 1985. Mason, D. Y.. Erber, B., Falini, H., Stein, K., and Gatter, K. C., Immuno-enzymatic labelling of haematological samples with monoclonal antibodies. In “Monoclonal Antibodies” (P. C. L. Beverley, Ed.), pp. 145-181, Churchill-Livingstone, Edinburgh, 1986. McMichael, A. J., ef al. (Eds.), “Leucocyte Typing III,” Oxford Univ. Press, 1987. Rose, N. R.. Friedman, H., and Fahey, J. L. (Eds.), “Manual of Clinical Laboratory Immunology,” 3rd ed., American Society for Microbiology, Washington, DC, 1986. Rose, N. R., and Mackay, I. R. (Eds.), “The Autoimmune Diseases,” Academic Press, Orlando, 1985. Thompson, R. A., “Techniques in Clinical Immunology,” 2nd ed., Blackwell, London, 1981.