Chapter 8 Laboratory tests

Laboratory

tests

CHAPTER 8

Laboratory Manuel

tests

Lopez, MD New Orleans, La.

LEUKOCYTE TYPING

Renal transplantation

The leukocyte surface antigens are currently defined by clusters of differentiation molecules (CDs) identified by monoclonal antibodies. Some of the CDs more commonly used in clinical practice are for T cells: CD1 = cortical T cells; CD2 = erythrocyte rosette; CD3 = T cell receptor; CD4 = helper/inducer T cells; and CD8 = suppressor/cytotoxic T cells. For B cells, the most commonly used clusters are: CD9 = pre-B cells; CD10 = common acute lymphoblastic leukemia antigen; CD14 = pre-B cells; CD20 = B cells; CD21 = B cell complement receptor; CD22 = activated B cell; and CD23 = low-affinity FC, receptors. Monoclonal antibodies and flow cytometry are valuable in defining lymphocyte cell surface phenotypes on T, B, and natural killer (NK) cells. The routine evaluation of lymphocyte subsets usually includes total T cells (CD3), helper/inducer T cells (CD4), suppressor/cytotoxic T cells (CD8), NK cells (Fc receptor CD16), and B cells (CD18 or CD20) (Table I).

Lymphocyte markers can be used to (1) monitor immunosuppressive therapy (to keep T cells below certain levels), (2) monitor the effect of rejection treatment with anti-CD3 or anti-CD4, and (3) evaluate rejection types. In the latter case, a CD4/CD8 ratio above 1 and interstitial changes in the kidney usually respond to rejection therapy; however, a ratio less than 1 with glomeruli and vascular changes do not respond to therapy and are associated with a high rate of infections .

Acquired

immunodeficiency

syndrome

Individuals in high-risk groups for acquired immunodeficiency syndrome (AIDS) but who are not infected may have elevated CD8 levels (low CD4/CD8 ratio). Patients with early AIDS may also have elevated CD8 levels, but the main abnormality, which gets progressively worse, is decreased CD4 levels (a low CD4/CD8 ratio). Infections Viral infections in patients with cytomegalovirus or Epstein-Barr virus cause elevated CD8 levels. The increase with cytomegalovirus is of short duration, whereas that with Epstein-Barr virus is long lasting. Tetanus immunization, measles, and malaria cause a transient decrease in CD4 levels. In sarcoidosis the circulating CD4 level decreases due to cell sequestration in granulomas.

From the Clinical Immunology School, New Orleans, La.

1040

Laboratories,

Tulane Medical

Bone marrow

transplantation

Phenotype studies are used to monitor the removal of donor T cells before transplant and to monitor recovery (initially NK cells, then CD3, CD4, and CD8 cells). MAJOR HISTOCOMPATIBILITY

COMPLEX

A region of the chromosome 6 contains several dozen genes that code for the human leukocyte antigens (HLAs). These genes were first recognized through the role of their products (HLAs) as targets and determinants of graft rejection. It is now clear that these gene products are critical in generating an immune response, particularly that involving T cells. The gene products of the major histocompatibility complex (MHC) are divided on the basis of their structure and function into two classes. Class I consists of HLA-A, -B, and -C molecules. Each loci have many alternative forms of the gene, called alleles. Class I antigens are expressed in all nucleated cells and platelets. They are detected by reacting the patient’s lymphocytes with a large panel of human antisera with known HLA specificity. Class II molecules make up the HLA-D region. The antigens are normally expressed on macrophages, B cells, and other antigenpresenting cells. These antigens are functionally recognized by the mixed lymphocyte reaction. The class II antigens of one patient will stimulate deoxyribonucleic acid synthesis by the lymphocytes of another. One way response can be elicited is by irradiating one set of cells. Other components of the MHC include several genes that direct complement production including C2, C4, factor B, and properdin.

VOLL ME 84 NUM3ER 6 FART

laboratory

TABLE 1. Reference

--

30% 25%-35%

41% 33%49%

7 1% 64%.78%

CD41CD8 ratio

Suppressorlcytotoxic T cells KD8)

Helper/inducer T cells (CD41

(CD31 -CI SD

1041

values for T cells Total T

Mean

tests

I .5 1.1-1.9

Data from the (‘linical Immunology Laboratories at Tulane University Medical School. New Orleans. La

TABLE

II.. Normal

values for immunoglobulins Range of normal

Group

hG

4dults

639-1349 345-1212

Age I yr

Dar;: from the Clinical Imrunology

Id

70-3 12 14-104

values

IgM

I@&

56-352 33-173

422-1252 236-660

(mgldl) I&,

W,

117-741 36-327

41-129 13-82

I&,

I-291 I-65

Laboratories at Tulane L’niversity Medical School, New Orleans, La.

HLA has clinical applications in transplantation, disease associations, ,2nd paternity testing. Its important disease associations include seronegative spondyloarthritis (HLA-B27), rheumatoid arthritis (HLADW4 to -DR4), narcolepsy (HLA-DR2), juvenile insulin-dependent disbetes (HLA-B8, -BW5, and -DW3), and systemic lupus erythematosus (SLE) (HI-A-DR3 and -B8)

IgE ANTIBODIES Total IglE Total 1gE antibodies are usually measured in vitro by one of two methods. In the competitive binding technique, the patient’s IgE antibodies inhibit the reaction between defined amounts of labeled (radioactive or enzyme) IgE and anti-IgE antibodies. The more IgE antibodies in the serum, the less labeled IgE is bound. Nonspecific inhibition occurs at low IgE levels, overestimating their values. In contrast, the double antibody sandwich technique is minimally affected by nonspecific serum factors and is the more commonly used test Fortotal IgE. Levels of total IgE ancibodiN;sin cord blood are less than 2 IUlml. Levels increase steadily until age 12 years, then decline moderately to normal adult levels (70 IUI ml). High IgE levels in cord blood are associated with risk of developing atopy. High IgE levels are correlated with atopy and are also present in allergic bronchopulmonary aspergillosis, parasitic infections. hyper-IgE syndrome, and IgE myeloma. Patients with a total serum IgE level 520 IU/ml rarely have detectable specific IgE antibody levels.

Specific IgE antibody RAST is the standard in vitro method for detecting specific IgE antibodiEs. This test and its several com-

mercially available modifications (MAST cellulose solid-phase matrix, iontophoretic microelisa, fluorescent allergosorbent) are allergosorbent tests, which means that the allergen is bound to a solid support. The patient’s serum reacts with the allergen bound to the matrix, and the presence of IgE antibodies is demonstrated with a labeled (radioactive or enzyme) antiIgE antibody. These tests are only semiquantitative. In general, RAST tests correlate well with skin tests and are less sensitive but more specific (rarely giving false-positive results but giving a few false-negative results). There is no group of allergens for which RAST is better than skin tests. The main clinical indications for RAST include evaluation of a small patient, dermographism, severe eczema. patients taking drugs that interfere with skin tests, and those with a history of anaphylaxis to previous tests. A modified RAST scoring system has been promoted as increasing the sensitivity of the test. However, it appears that this modified system adds relatively little to the clinical value of the RAST. Few studies have compared the different commercial in vitro tests; in general, RAST remains the standard in vitro test for specific IgE antibodies.

IMMUNOGLOBULIN

QUANTITATION

The major immunoglobulin classes can be quantitated by gel single radial immunodiffusion, nephrelometry. or ELISA methods (Table II).

COMPLEMENT A number of serum proteins act sequentially and play an important role in the normal inflammatory response and host defense against microorganisms. The terminal components (C3 through C9) are responsible for most of the biologic activity of the sys-

1042 Lopez

J. ALLERGY

tern, but must first be activated through two pathways (classic and alternative). The classic pathway is activated by IgG and IgM immune complexes, IgM being more effective than IgG in triggering activation. Clq binds to the Fc portion of the antibody and initiates the sequential activation of the complement components to their enzymatically active form. The alternative pathway does not require antibodies for activation and can be activated by bacterial cell walls. However, the presence of sialic acid on the bacterial surface (i.e., Escherichia coli, Group B streptococcus meningococci) inhibits activation of the alternative pathway. Complement components are acute-phase reactants and their levels are increased in acute inflammation. Low complement levels can result from decreased synthesis, as in congenital complement deficiencies, acquired deficiencies in liver disease (the liver is the primary source of synthesis for C3, C4, C2, and factors B and D), and malnutrition. Low complement levels may also result from their increased utilization in certain autoimmune diseases such as SLE. The most useful screening test for complement is the measurement of total hemolytic complement activity (CH,,). Complement components can be evaluated by activity or quantitation. Congenital abnormalities of complement components are associated with three major groups of diseases: collagen vascular disease, frequent infections, and glomerulonephritis. Congenital

deficiencies

A lack of C2 is the most common congenital deficiency. This is associated with a high incidence of SLE and glomerulonephritis. C4 deficiency is associated with SLE. With C3 deficiency the incidence of infections by Pneumonoccus, Hemophilus injuenza and Neisseria increases. Deficiencies in C5 through C9 are associated with an increased incidence of Neisseriu infections. Hereditary angioedema is associated with C 1 esterase inhibitor deficiency. Approximately 15% of patients produce the Cl inhibitor without detectable activity. C3 nephritic factor is an autoantibody that binds to C3 covertase, leading to uncontrolled C3 activation.

CLIN. IMMUNOL. DECEMBER 1989

and cystic fibrosis, among other diseases. An increase in specific IgG., antibodies has been associated with food sensitivity and with immunotherapy. IgG, , IgG,, and IgG, fix complement. IgA deficiency and ataxia telangiectasia have been associated with IgG, and IgG, deficiencies.

Laboratory

tests

Match the immune complex method with its description: 1. Bovine conglua. Binds immune complexes tinin assay containing 1gG or IgM antibodies b. Receptor that binds im2. Raji cell mune complexes that have fixed C3 3. Clq binding C. Protein A binds the Fc assay region of IgG or IgM 4. Staphylococcus d. Binds immune complexes containing split products binding assay of c3 l-d, 2-b, 3-a, 4-c

Answer:

McDougal JS, Hubbard M, Stobel PL, McDuffie FC. Comparison of five assays for immune complexes in the rheumatic diseases. J Lab Clin Med 1982;100:705-19.

Reference:

The techniques of nitroblue tetrazolium reduction and chemiluminescence are helpful in the diagnosis of: a. ChCdiak-Higashi syndrome b. Chronic granulomatous disease c. Cyclic neutropenia d. Myeloperoxidase deficiency e. None of the above Chronic granulomatous disease is characterized by an increased incidence of recurrent infections of the skin, lungs, liver, and bones. These patients have no normal respiratory burst associated with neutrophil phagocytosis. The respiratory burst can be measured by one of the above two tests.

Explanation:

IgG subclasses

Answer:

IgGZ are antibodies against polysaccharides. An IgG, deficiency is associated with an inability to produce antibodies against bacterial capsular polysacchat-ides. Also, infections with H. in&enzae and Pneumococcus are frequent in this deficiency. IgG, deficiency is associated with chronic pyogenic infections such as sinusitis, otitis, and pneumonia. IgG, levels are elevated in atopic dermatitis, asthma,

Which of the following statements is/are true regarding the evaluation of suspected immunodeficiency in children? a. Defects in antibody production predominate b. Elevated total IgE levels can be seen in primary phagocytic disorders c. IgG subclass determinations should be included

b.

VOLU f/It 84 NUMBER 6, F’.hRT :

Laboratorv

in the initial screening of all children with suspected immunodeficiency d. Deficiencies of 1gD may exist in antibody deficiency states but have limited clinical significance e IgA deticiency is the most common single immunoglobulin deficiency Exphnution: Screenin tests to evaluate suspected immunodefi;iency in children include: B-cell deficiency: IgG, IgM, and IgA levels; isoagglutinin titer: Schick test; and preexisting antibod) titers (e.g., polio. ru3ella, tetanus, diphtheria antibodies) T cell deficiency: lymphocyte count and morphology. thymic size by roentgenogram, delayed skin tests (e.g., Tr?+zo@z~ton, mumps, Can&&z. tetanus toxoid:,, and lymphocyte transformation Phagocytic deficiency: leukocyte count and morphology, nitroblue tetrazolium dye test, and IgE level Complement deficiency: CH,,, activity AnaMw: a. b. d. and e are true. Quantitative immunoelectrophoretic used for: ;!. Characterizing allergens tx. Quantitating antigens c Comparing allergens (:. Isolating allergens c’. All of the above

methods can be

Expfunution: Quantitative immunoelectrophoresis describes a group of highly resolving immunoelectrophoretic methods. These methods include crossed immunoelectrophoresis (used to characterize and quantitate antigens using polyspecific antibodies), crossed radioimmunoelectrophoresis (used to identify allergens and to evaluate patients’ responses to allergens), crossed-line immunoelectrophoresis (used to compare antigens and to determine partial identity reactions): and rocket immunoelectrophoresis (used to quantitate antigens using monospecific antibodies).

Rqference: Lowenstein H. Quantitative immunoelectrophoresis methods as a tool for the analysis and isolation of allergens. Progr Allergy 1978;25: l-62.

Match the antibodies with the disease: muscle a. SLE 1. Antismooth antibodies

2. Anti SSA!Ro antibodies 3. Anti SM antibodies 4. Anti RNP antihod& 5. Anticentromere antihodie\ Atww:

tests

1043

b. Progrcssivc systemic sclerc~~is c. Newho-n \vlth heart bloc!\ d. Mivrd connective tissue iliye~the ., L e. Chroruc active hepatitib

a-3. b-5, c-2, d-4, e-l

R&rewe: McCarty G. Autoantibodies and their relation to rheumatic diseases. Med Clin North Am 1986;70:23?-61. Which of the following statements is/ are true‘? a. Infections with Neisserin species are typical of early complement component deficiency b. C3 deficiency is associated with autoimmunity and pneumococcal and neisserial infections c. Nephritic factor is associated with low C3 levels d. The liver is the primary source for the synthesis of C2, C3, and C4 e. Low C4 levels help in the diagnosis of hereditary angioedema Answer: b. c. d, and e are true Referrrzce: Ross SC, Densen P. Compiement deficiency states and infections: epidemiology, pathogenesis and consequence of neisseriai and other infections in an immune deficiency. Medicine 1984:63:243-73. In which of the following conditions is there a complete lack of T-lymphocyte function’? a. X-linked agammaglobulinemia b. Chronic granulomatous disease of childhood c. Severe combined immunodeficienck d. Pre-AIDS e. DiGeorge syndrome Expluntrtion: Normal lymphocytes in culture medium after stimulation with mitogens such as phytohemagglutinin or antigens will respond by increasing deoxyribonucleic acid synthesis and cell division. The cell increases in size and resembles blast cells (blast transformation). This in vitro response correlates with the existence of delayed hypersensitivity. The test has broad applications, such as the evaluation of congenital immunologic defects. Patients with severe combined immunodeficiency and severe defects in T cell function demonstrate a complete lack of response. Atwwer: c and e