Laboratory Tests in Rheumatic Diseases

Symposium on Rheumatic Diseases

Laboratory Tests in Rheumatic Diseases Wallace V. Epstein, M.D. *

The development of the modern clinical immunology laboratory is, to a significant degree, a by-product of the evolution of contemporary clinical rheumatology. No matter what the eventual utility of tests for rheumatoid factors, the discovery of such a circulating antibody with specificity for a human immunoprotein provided a powerful impetus for the growing concept of immunologically mediated chronic diseases. During the period of emphasis on humoral aberrations in rheumatic disorders, each new-found antibody associated with one or another rheumatic disorder furthered classification of disease on the basis of serologic characteristics. A polarity developed in which a nosology based on clinical and historical characteristics contrasted with rapidly evolving serologic profiles. The implications of the latter for etiology and pathogenesis were so powerful as to dominate evolving classifications of disease. Rheumatoid arthritis could be classified as either a seropositive or a seronegative disease and systemic lupus erythematosus became further defined by a wide variety of antibodies, immune complexes, and complement characteristics, each describing a disease subset with unique prognostic and therapeutic implications. The pattern of a new test showing extraordinary specificity when first described, and then gradually decreasing specificity with further clinical trial and modifications of test procedures, is well known. Major methodologic problems exist in the selection of the abnormal sera and the control sera used for evaluation of the test, and rarely have initial descriptions of the test adequately defined the accuracy and precision of the serologic technique. The population base used for estimation of the prevalence of the phenomenon is almost never broad enough to allow extrapolation of the test results from the research laboratory to the community. At present, we have the situation of changing clinical categories of rheumatic disease such as the emergence of mixed connective tissue diseases and the overlap syndromes simultaneously with the description of immunologic ';Professor of Medicine, University of California School of Medicine, San Francisco, California Preparation of this article was supported by a grant from the Robert Wood Johnson Foundation, Prince ton, New Jersey. The opinions, conclusions, and proposals in the text are those of the author and do not necessarily represent the views of the Robert Wood Johnson Foundation. Medical Clinics of North America - Vol. 61, No. 2, March 1977

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abnormalities which, in themselves, define subsets of disease. The demonstration of serum antibody to extractable nuclear antigens (ENA) strongly influenced the emergence of the entity, mixed connective tissue disease (MCTD). The laboratory de terminations used in the care of patients with rheumatic diseases share with other in vitro tests common problems related to the technique as well as to the motivation that led to ordering the test. The clinician must have well formulated reasons for ordering a single laboratory test or a group of tests and must decide, in advance, how the information will be used in diagnostic, therapeutic, and prognostic decision-making. Some relevant factors we will not discuss in this paper but which require consideration include simplicity, patient acceptability, and cost of laboratory tests. The precision of a test refers to the consistency of repeated measurements, an important characteristic if you plan to follow serum complement or DNA antibody levels. The accuracy of the test refers to the closeness of the test result to a true measure of the attribute being sought. The diagnostic sensitivity of a test expresses how frequently a test is positive when a particular disease is present, the disease, of course, not having the test itself as part of the diagnostic criteria. The diagnostic specificity of a test is an expression of how frequently a test is negative in the absence of a particular disease. We require such information concerning the tests and the laboratory performing the tests in order to determine how this information will be used, especially when tests are repeated frequently and changing results are used to guide management. As pointed out by Galen and Gambino,7 "accuracy and precision aren't good enough." It is the predictive value of a test which we frequently seek, but this depends on yet another factor-the prevalence of the disease under study. "The predictive value of a positive result defines the percentage of all positive results that are true positives. The predictive value of a test is determined by the complex interaction of three variables: sensitivity, specificity, and prevalence."7 For example, if we have a test which has a sensitivity of 95 per cent and a specificity of 95 per cent, the prevalence of the disease has a striking effect on the predictive value of our test (see Table 1). Beside the characteristics that cumulate in its predictive value we have the issue of the "normal range" of a test. The motivation for performing clinical laboratory tests ranges from screening to case finding to diagnosis, and each of these may have a normal test value. As shown in Figure 1 from Sackett,1s the value designated "A" would be an acceptable normal for someone screening for an illness, since no diseased patients would be missed. Someone doing case finding would select "B" as the limit of normal, since a positive test among disease-free persons is a false-positive for their purpose. Most interesting is the concept of "c" as a normal. This is a value selected as a guide to initiate therapy. In the usual clinical uses of laboratory test results there can therefore be at least three different "normal" limits. The applicability of this concept to a "normal" value of the serum total hemolytic complement while managing patients with systemic

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Table 1.

Effect of Prevalence of Disease on Predictive Value of Tests (Sensitivity 95%; Specificity 95%) PREDICTIVE VALUE OF DISEASE PREVALENCE

A POSITIVE TEST

O.IO/C

1.9%

16.1% 27.9% 50.0% 45.0%

1.0%

2.0% 5.0';7,· 50.0%

Note: The profound effect of prevalence on predictive value is independent of both the accuracy and precision of the test. Since most of the rheumatic diseases have a prevalence in the general community in the range 0.1 to 1.0 per cent, we see that even if we had tests of 95 per cent sensitivity and specificity, the tests would still have only 1.9 to 16.1 per cent predictive value when used for screening. From Galen, R. S., and Gambino, S. R.: Beyond Normality. New York, John Wiley and Sons, 1976. Reproduced with permission.

lupus erythematosus (SLE), or of the latex F II rheumatoid factor test to diagnose rheumatoid arthritis should be immediately apparent.

Ordering Specific Laboratory Tests At present no requirements exist that the laboratory test being ordered is cost effective. Few studies explain the wide variation of test-ordering behavior among physicians; further, few studies relate the manner and frequency of repetition of tests to outcome of disease. Bombardier et al,2 have reported an 81 per cent range of variation for laboratory tests ordered by university-based physicians caring for ambulatory patients with a similar form of rheumatoid arthritis without detectable differences in short term outcomes. The use of laboratory services in the patient care setting in contrast to the clinical research setting raises issues of cost effectiveness as well as the relation to the outcome of management. Almost no information concerning these issues is available at present. The following presentation deals with issues of test sensitivity and specificity but we must recognize that in a

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Figure 1. Theoretical range of test values in a population of disease-free and diseased individuals. (From Sackett, D. L.: Fed. Proc., 34(12): 2157-2161, 1975.)

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given clinical situation these qualities in themselves are insufficient justification for ordering a laboratory procedure. The decision to use the test result for screening, diagnostic, or therapeutic purposes should be made at the time the test is ordered. The Initial Panel . The similarity of the initial presentation of many rheumatic disorders leads to a clinical differential diagnostic list and to the use of a group of laboratory tests frequently referred to as a rheumatic disease panel. The normal value "A" of Figure 1 is frequently used in this situation since we wish to avoid missing the diseased individual, and we expect to detect the largest number of persons with the characteristic being tested for. It is assumed that clinical and other laboratory criteria will enable one to recognize the small number of positive reactors in the disease-free population. Lupus Erythematosus Preparation and Rheumatoid Factor In general, tests of low sensitivity are of high specificity and diagnostic import. Usually finding two typical LE cells makes a preparation positive. If one sets the positive LE cell preparation cut-off so that 8 to 10 typical LE cells must be seen per low power microscope field, other clinical conditions associated with a positive test, e.g., seropositive rheumatoid arthritis, or the procainamide associated lupus-like syndrome, usually will demonstrate a lower LE cell concentration, but even this is not absolute. The practical difference between these two "normal" values using this relatively insensitive biologic test is great. At present, the clinical laboratory is usually given no information as to which of these two purposes apply when an LE cell preparation is ordered. The methods for performing the test are well covered by Dubois,3 but beyond stating that the presence of two typical LE cells makes a positive preparation, there seems little correlation between the number of cells and clinical activity. Measurement of serum rheumatoid factor follows a somewhat similar pattern; however, it is believed that qualitatively dissimilar antibodies are measured by different techniques that use different immunoglobulins as the test reagent. The sensitized sheep cell test using rabbit IgG as the cell coating is said to be highly specific for rheumatoid arthritis, but this may be a factor of its relative insensitivity.9 Variations on the human IgG-coated latex particle test are performed in most clinical laboratories but even more sensitive tests are available. Here, again, it depends on whether we are calling for a diagnostic test for rheumatoid arthritis or a highly sensitive test for serum antiglobulin activity. A recent publication, "On Standardization of the Latex Fixation Test,"lI indicates that the test itself is still subject to considerable technical variation. The uses to which the report of a positive result will be applied help determine the selection of a positive/negative cut-off point. The commerciallatex-IgG coated particle is a satisfactory test if serum is first heated to 56° C for 1 hour and if serum dilutions in excess of 1 :40 are considered positive. Rarely, sera containing immunoglobulin com-

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plexes may clump uncoated latex particles but the control required to detect this phenomenon is not usually performed. This author prefers the more sensitive F II hemagglutination procedure lO (human IgG cell coating via tannic acid treatment of the cell), using a 1 :200 dilution of serum as the lowest positive dilution point. This test is from 10 to 40 times more sensitive than the latex procedure and allows precise quantitation of small amounts of antiglobulin. Rheumatoid arthritis remains a clinical diagnosis which is only rarely changed by the result of a serologic test.

Antinuclear Antibody (ANA) and Extractable Nuclear Antigens (ENA) ANA titers should be reported by titer endpoint and the pattern of staining. The pattern is best established at two or more doubling dilutions less than the endpoint. A difficulty arises when antibodies of two different specificities coexist in the patient's serum at different concentrations. It is difficult to identify an outline (synonyms: shaggy, peripheral, rim) pattern due to antibody to DNA when antibody to nucleoprotein is also present causing a diffuse pattern. The process of testing serial dilutions may result in more than one pattern being observed, so that a report of a diffuse pattern to a 1 :300 endpoint may be combined with a speckled pattern to a 1:1200 endpoint. The ANA pattern aids in classification of diseases. An ANA test of a speckled pattern, usually with a dilution endpoint over 1: 1000, warrants a more elaborate search, especially for scleroderma or mixed connective tissue disease. Serum from patients with scleroderma may occasionally cause a nucleolar pattern. An outline pattern is strongly associated with systemic lupus erythematosus. The titer endpoint aids in the diagnostic quality of the test; a 1: 1200 endpoint, outline pattern, is so high that it is unlikely to be found in anything other than systemic lupus erythematosus. ANA tests in which the pattern is predominantly diffuse have less diagnostic specificity than the other tests. Vague rheumatic-like symptoms combined with a positive ANA with end point in the 1:10 to 1:20 dilution, usually of a diffuse pattern, are not uncommon in otherwise healthy individuals. It is unfortunate that such low titer ANA results may become one of the major reasons for an extended clinical and laboratory search for a nonexistent collagen vascular disorder. Tests of Greater Sensitivity-Anti-DNA and Anti-ENA The development of assays for antibody to nuclear constituents using radiolabeled antigens has centered on antibody to double-stranded DNA. The two most commonly used techniques involve: (a) using ammonium sulfate to precipitate soluble complexes of radiolabeled antigen with serum antibody (Farr technique); and (b) the use of a millipore filter surface to adsorb complexes of radiolabeled antigen and serum antibody. These tests correlate generally with the outline pattern seen on the antinuclear antibody fluorescence assay. Sera of patients with most of the rheumatic diseases show normal or low levels of antibody to na-

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in tive, double -strand ed DNA. There is a chroni c technic al problem d strande singleinating contam is there not these tests as to whethe r or in al materi ed -strand double mostly of nts segme d strande DNA or singleshould most comme rcial prepar ations of double -strand ed DNA. One saferiate approp r whethe make periodi c inquiry of the laborat ory as to d strande singleto y antibod ring measu guards are being used to avoid ng changi and DNA native to y antibod serum of DNA. Chang ing levels pair of imlevels of serum comple ment (CH50 ) remain the most useful erythelupus ic system munolo gic tests in followi ng patient s with active 8 The occasio nal pasion. renalle matosu s, especia lly those with an active sts no tient with sustain ed high levels of such antibod y who manife assousually is this but , known is disease active of ce clinica l eviden obrecent A ment. ciated with a norma l concen tration of serum comple IgM or IgG be may DNA ed servati on18 that the antibod y to double -strand IgG than is of interes t, since greater clinica l activity is associa ted with , and tration concen ity, specific the report may tests IgM antibod y. Future y. antibod ting circula the of nature immun ologic ENA, Serum antibod y to non-hi stone nuclea r antigen s, design ated procetion glutina hemag a by red are of great clinica l interes t. Measu antibod y dure, the antigen contain ing ribonu cleopro tein (RNP) detects . This having a marked associa tion with mixed connec tive tissue disease . testing ANA on pattern antibod y is usually associa ted with a speckle d and disease the of course l clinica the Titers of this antibod y vary with erythem amay also be found in patient s with typical system ic lupus a difsing U tosus associa ted with antibod y to double -strand ed DNA. d12 claime is it sis), ferent assay techniq ue (count er immun oelectr ophore have s matosu erythe lupus ic that 46 per cent of patient s with system circula ting antibod y to ribonu cleopro tein antigen . leoThe other extract able nuclea r antigen does not contain ribonuc not and Smith Mr. protein and is design ated Srn antigen (named after a anSrn this to dy Antibo ). to be confus ed with smooth muscle antigen sysfor city specifi high has tion, glutina tigen, again measu red by hemag 4 is rarely, if temic lupus erythe matosu s and accord ing to Notma n et al.l ever, seen in closely allied rheum atic disease s. disease The rarity of renal disease in the mixed connec tive tissue serum high of on detecti by syndro me and the diagno stic aid provid ed s reason major the of one be may antibod y to ribonu cleopro tein antigen of tration concen serum the of s change Serial for orderin g an EN A test. (Fig. 2). these antibod ies may also provide a valuab le guide to therapy at tration concen A Since the same patient may have an elevate d anti-EN the with dealing re literatu t one time and not at anothe r, the presen based on a prevale nce of these antibod ies in the rheum atic disease s ined. reexam be to need may en single serum specim

Serum Compl ement as Just as a patient with rheum atoid arthriti s may be catego rized atoid rheum of tration concen the by gative serone or being seropo sitive be clasfactor in serum, so patient s with other rheum atic disorde rs may al (Norm ic. mentem sified as hypoco mplem entemi c or normo comple

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LABORATORY TESTS IN RHEUMATIC DISEASES 1973 ..u9/lT'l1 Se t Oct Nov Oec

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Figure 2. Serial de terminations in a patient with systemic lupus erythematosus. Normal values: Anti-ENA (Srn) < 1:200; Anti n DNA < 100 C.P.M.; urine light-chain < 20 ILg/ml; CH50 > 25 CH50 units.

serum CH50, 25 to 42. 50 percent hemolytic units per ml; Normal serum C3, 70 to 140 mg per dl.) By far the most frequent rheumatic clinical condition associated with serum hypocomplementemia is active systemic lupus erythematosus. There are occasional patients with systemic lupus who exhibit chronically low serum CH 50 values without other evidences of active disease but this is the exception. Occasionally, a patient may have a sufficiently high concentration of cryoimmunoglobulinemia (mixed type) that the low temperature used in the CH50 assay causes in vitro cryocomplex formation and decomplementation of serum. We have run the CH 50 assay with appropriate normal serum controls in this situation at room temperature and have been able to demonstrate a normal serum CH50 concentration which had been thought to be profoundly reduced. The association of hereditary complement deficiencies in patients with a lupus-like picture, especially C2 deficiency, is of great theoretical interest and should be sought when familial aggregation of this and related diseases is notedP It is the patient who demonstrates a low CH50 and/or C3 when the systemic lupus erythematosus is active and normal concentrations when inactive in whom serial measurements prove of greatest predictive value. Since low complement concentrations result from some mixture of diminished synthesis, increased catabolism, and tissue localization, it is usually difficult to easily relate changing concentrations to pathogenetic processes. Because of access to kits that measure C3 by a precipitin reaction in agar, this component of the complement system will continue

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to be the usual complement component measured. The total CH50 concentration has the theoretical advantage of constituting a measurement of the component present in the rate limiting quantity. The presence of less than "normal" concentrations of complement (CH50 and/or C3) in synovial fluid is characteristic of the active synovitis of rheumatoid arthritis. The synovial effusion of Reiter's syndrome has an appropriate or even elevated complement concentration. It is essential to measure both the serum and synovial fluid complement and protein concentrations so that the maximum possible synovial fluid complement concentration may be determined, since complement is found in synovial fluid as a result of a protein exudation from serum. 19

Urine Light Chain (L-Chain) Protein The presence of greater than normal concentrations of the light polypeptide chains of immunoglobulins in the urine of patients with systemic lupus erythematosus is a reflection of the tubular lesion which is a regular component of renal injury in systemic lupus erythematosus.4 The phenomenon of free L-chain proteinuria is due to failure of the proximal tubular cell to reabsorb and catabolize the L-chain protein normally filtered at the glomerulus. Unlike tests of renal creatinine excretory capability, the increased urine L-chain protein is an early (usually reversible) phenomenon. Normal, unconcentrated urine contains from 5 to 10 /-tg of free L-chain protein per ml, while the concentration found in active systemic lupus erythematosus is usually from 80 to 2560 /-tg per ml. It is interesting that the heat test for Bence Jones protein (monoclonal L-chain protein-B cell dysproteinemia), requires more than 2000 /-tg per ml of urine. Therefore, patients with the active renal lesion of systemic lupus erythematosus may have urine concentrations approaching that seen in multiple myeloma, although it is never monoclonal in nature. These urine concentrations may be seen in association with a normal serum creatinine concentration and with no clear relationship to the level of albuminuria. In a study of the sequence of changing laboratory values with therapy in systemic lupus erythematosus, the L-chain protein concentration in urine was found to return to normal after the serum complement concentration rose and the elevated level of anti-DNA decreased. 16 A rising L-chain concentration in urine occurred slightly in advance of a falling serum complement concentration, and a rising concentration of serum antibody to DNA frequently preceded a clinical exacerbation. Elevated urine concentrations of L-chain protein may be seen whenever there is diminished renal excretory capability. This therefore, is a test of low diagnostic specificity. It does, however, allow serial measurements of renal dysfunction that occurs before the more ominous rising serum creatinine. The frequent use of combined steroid and immunosuppressive drugs in the management of patients with severe renal systemic lupus erythematosus carries a high incidence of serious side effects. Laboratory tests that can indicate diminishing pathogenetic processes (e.g., rising CH50) or improving end organ function (e.g., fall-

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ing urine L-chain protein concentration) allow more rational adjustment of the types of medication and especially their dosage. Serum Protein Characterization and Immune Complexes As pervasive as any pathogenetic concept in the rheumatic diseases is the idea of circulating antigen-antibody complexes that localize and activate the complement system resulting in the local inflammatory process. Quantitation of the serum immunoglobulins is frequently performed as part of the work-up of a patient with symptoms suggesting a rheumatic condition, as a first step in seeking evidence of involvement of the immune system. The information returned from such quantitation is surprisingly small, whether one uses electrophoresis, immunoelectrophoresis, or actual quantitation of IgG, IgM, and IgA. IgA deficiency is associated with a wide range of "autoimmune" diseases/ or a profound hypogammaglobulinemia with a rheumatoid arthritis-like picture, but these are rare occurrences. Slight to moderate hyperglobulinemia, usually of IgG and IgM, is not uncommon but provides little assistance in diagnosis or management, and no specific conformational changes are found regularly by immunoelectrophoretic analysis. The cost/benefit ratio of these tests appears high, indeed, in these situations. A further, and at times more rewarding, technique for seeking the elusive circulating antigen-antibody complex is quantitation of cryoglobulins. Significantly increased quantities (over 0.12 mg per ml of serum) of mixed cryoglobulins may be found, especially in rheumatoid arthritis, systemic lupus erythematosus, and disseminated vasculitis. These cryoimmunoglobulins are mixtures of immunoglobulins, probably as part of an antigen-antibody complex, rather than the monoclonal variety more commonly seen in hematologic-oncologic disorders. Many cryoimmunoglobulin complexes have antiglobulin activity but the nature of the antigen is usually not clear. The cryoproteins form slowly (72 hours), and are present in relatively small quantities (less than 2 mg per ml of serum). The presence of cutaneous vasculitis in patients with any of the rheumatic diseases should raise the suspicion of a mixed cryoglobulinemia. The demonstration of immune complexes in glomeruli, perirenal tubular sites, and the dermal-epidermal junction of skin all may reflect tissue localization of such circulating complexes. Meltzer and Franklin have described a specific clinical picture of arthritis, renal disease, and anemia associated with such mixed cryoglobulinemia. 13 A further step in identification of circulating complexes is the C1 q precipitation reaction; a technically simple procedure in which the patient's serum reacts in a double diffusion arrangement in agarose with C1 q , the binding element of the first component of complement prepared from normal plasma. The presence of an antigen-antibody complex capable of complement fixation in the serum results in a precipitation line in the agarose and a crude quantitation can be made by finding the greatest dilution of the patient's serum that causes such a precipitation reaction. The relative insensitivity of these tests, i.e., cryoimmunoproteinemia and C1 q precipitation reactivity, make these valuable guides to disease activity when they are present, and rarely do we see

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persistence of these phenomena without some significant clinical activity. Their reappearance in a clinical remission may anticipate a serious clinical flare. 5 As more sensitive tests for minute amounts of immune complexes become available, we may find the phenomenon associated with a wide range of rheumatic as well as non-rheumatic disorders.

Tests of Cell Character and Function Progress in delineating the role of T-lymphocytes and Blymphocytes in a number of rheumatic diseases has been dramatic. Identification of T-helper and T-suppressor functions have partially clarified the ways in which hyperglobulinemia and depressed delayed skin test reactivity could coexist in systemic lupus erythematosus. At the present time, tests of T-cell enumeration and function have not reached a point of either general availability or clinical applicability. Cell functions possibly more related to pathogenetic processes, such as antibodydependent cellular cytotoxicity,6 offer considerable promise as a guide to the activity of conditions such as periarteritis; but again, the complexity of the procedures, at present, maintains these in the research laboratory - where the cost of further exploration is correctly borne by research funding and not by patient care resources. One test of unquestionable value based on cell characteristics in the diagnosis of rheumatic diseases, and now widely available, is the human lymphocyte antigen (HLA) typing of the patient's peripheral blood lymphocytes. Although still in its early stages, HLA typing has, for example, revealed the markedly enhanced susceptibility of HLA B27 individuals to develop ankylosing spondylitis. This subject is considered in detail elsewhere in this volume. Just as serologic profiles are redefining the clinical nosology of the rheumatic diseases, so the HLA phenotypes are about to re-categorize the serologic groups with the identification of even more homogeneous subsets. Only with the identification of homogeneous disease subsets can a more rational use be made of currently available laboratory tests in the rheumatic diseases.

REFERENCES 1. Ammann, A. J., and Hong, R.: Selective IgA Deficiency: Presentation of 30 cases and a review of the literature. Medicine, 50:223-236, 1971. 2. Bombardier, C., and Fries, J. F.: Use of laboratory tests and drugs: An analysis of the variation among physicians treating rheumatoid arthritis. Presented at the Fourth Western Regional Conference of Rheumatic Diseases, Berkeley, California, December 5-6, 1975. 3. Dubois, E. L.: Lupus erythematosus. Los Angeles, University of Southern California Press, 2nd ed., 1974. 4. Epstein, W. V., and Tan, M.: Increase of L-chain proteins in the sera of patients with systemic lupus erythematosus and the synovial fluids of patients with peripheral rheumatoid arthritis. Arthritis Rheum., 9:713,1966. 5. Epstein, W. V.: Immunologic events preceding clinical exacerbations of systemic lupus erythematosus. Amer. J. Med., 54:631,1973. 6. Feldman, J. L., et al.: Antibody-dependent cell-mediated cytotoxicity in selected autoimmune diseases. J. Clin. Invest., 58:173, 1976. 7. Galen, R. S., and Gambino, S. R.: Beyond Normality: The Predictive Value and Efficiency of Medical Diagnosis. New York, John WHey & Sons, 1976.

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8. Hecht, B., et al.: Prognostic indices in lupus nephritis. Medicine, 55: 163-181, 1976. 9. Honey, H. L., et al.: The application of the sensitized sheep cell test to sera of patients with nonrheumatic diseases and false positive serological reactions for rheumatoid arthritis. Amer. J. Med. Sci., 242:331-337, 1961. 10. Jacobson, A., et al.: The hemagglutination test for rheumatoid arthritis. Ill. A clinical correlation of the sheep erythrocyte agglutination test (SEA) and the gamma globulin (FII) test. Amer. J. Med., 20:489,1956. 11. Klein, et a!.: On standardization of the latex fixation test. Bull. Rheum. Dis., 26:866-869, 1975-76. 12. Kurata, N., and Tan, E. M.: Identification of antibodies to nuclear acidic antigens by counterimmunoelectrophoresis. Arthritis Rheum., 19:574-580, 1976. 13. Meltzer, M., et al.: Cryoglobulinemia, a clinical and laboratory study. 11. Cryoglobulins with rheumatoid factor activity. Amer. J. Med., 40:837-856, 1966. 14. Notman, D. D., et al.: Profiles of antinuclear antibodies in systemic rheumatic diseases. Ann. Intern. Med., 83 :464-469, 1975. 15. Sackett, D. L.: Laboratory screening: A critique. Fed. Proc., 34:2157-2161, 1975. 16. Spriggs, B., and Epstein, W. V.: Clinical and laboratory correlates of L-chain proteinuria in systemic lupus erythematosus. J. Rheum., 1 :287-292, 1974. 17. Stern, R., et al.: Hereditary C2 Deficiency: Association with skin lesions resembling the discoid lesion of systemic lupus erythematosus. Arthritis Rheum., 19:517-522, 1976. 18. Talal, N., et al.: Immunologic regulation of spontaneous antibodies to DNA and RNA. 1. Significance of IgM and IgG antibodies in SLE patients and asymptomatic relatives. Clin. Exper. Immunol., 25:377-382, 1976. 19. Townes, A. S., and Sowa, J. M.: Complement in synovial fluid. Johns Hopkins Med. J., 127:23-37,1970. U ni versity of California 350 Parnassus Avenue, No. 407 San Francisco, California 94117