CLINICAL IMMUNOLOGY

MLS 4506C

Instructor: Office Hours:

Henry O. Ogedegbe, Ph.D. MW 9:00 AM – 12:00 Noon

hogedegb@fgcu.edu Additional office hours are available by appointment.

941-590-7486

Textbook:

Stevens, C. D. Clinical Immunology and Serology A Laboratory Perspective. FA Davis Co., Philadelphia, 1996.

Course Description:

Course integrates basic and clinical immunology featuring clinical presentation, immunopathological features, diagnosis and treatment of immunologically related diseases. Laboratories include procedures and diagnostic techniques utilized in a clinical immunology laboratory.

Course Goals:

This course introduces the student to the theories and techniques of clinical immunology. In addition, the course exposes the student to the knowledge, skills and abilities required to build confidence in the practice of clinical immunology. Achievement of these goals will lead to the production of accomplished and well-rounded clinical immunology professionals.

Course Expectations:

This course is designed in such a way as to provide you the opportunity to understand and learn the art and science of clinical immunology. Attendance in class will facilitate learning and understanding of materials taught. Following directions and instructions, asking questions and participating actively in the learning process will augment this. In order to complete the course requirements and pass the course successfully, you will have to be fully committed to the learning process. To this end, you must adhere to course policies and requirements.

Course Policies:

All reports for laboratory exercises are due the class meeting following that laboratory to receive full credit. Each class meeting time the laboratory report is missing, 5 points will be deducted. If you are absent from class, you will be able to make-up quizzes and laboratories if you have contacted the faculty of your absence and it is excused by the faculty. These policies will be strictly enforced.

Grading Policies:

Total Points

6 Quizzes 25 points each 150

10 Laboratory Exercises 20 points each 200

Final 100 points 100

450

A 405-450 points

B 360-404 points

C 315-359 points

D 270-314 points

F 0-269 points

Course Objectives:

These objectives indicate the abilities that the students are expected to acquire upon completion of lectures, readings and laboratory experiences in each content area.

A. Immunology Review:

1. Define immunology, antigen, hapten, antibody, immunoglobulin, and immune complex.

2. Classify an example of immunity as innate or adaptive immunity, cellular or humoral, and active, passive or adoptive immunity.

3. Discuss the function of each cell in the immune system.

4. Discuss the functions of the lymphatic system and spleen.

5. Define specificity and immunological memory.

6. Describe the steps involved in B-cell and T-cell antigen recognition.

7. Define the cells involved in the nonspecific immune response.

8. Describe the functions of the leukocytes.

9. Define and describe complement and the functions of each complement component.

10. Describe the phases or events that constitute inflammation.

B. Hypersensitivity:

1. Describe the pathogenesis of the four types of hypersensitivity.

2. List examples of each type of hypersensitivity.

3. Distinguish between the preformed and newly formed mediators of type I hypersensitivity.

C. Tumor Immunology:

1. Define the antigens associated with tumor immunity.

2. Describe natural immunity to tumors, including the functions of macrophages, natural killer cells, and nonimmunologic changes to the environment associated with tumor invasiveness.

3. Describe T-cell-mediated immunity to tumors, including cytokines and cytotoxic T-cell immune mechanisms.

4. Recall the tumor markers that are useful in the diagnosis, prognosis, and monitoring of the effects of therapy for various forms of cancer.

5. Describe the therapeutic approaches to eliminating tumor burden.

D. Transplantation and Major Histocompatibility Complex:

1. Define heterodimer, MHC, HLA, gene, allele, gene product, polymorphism, haplotype, genotype, linkage disequilibrium, and relative risk.

2. Compare and contrast the MHC class I, II, and III products with respect to structure, cellular or humoral distribution, function, and method of detection.

3. Given an HLA designation, identify the specificity.

4. Explain the principles of complement-mediated cytotoxicity, one-way mixed-lymphocyte culture, and the primed lymphocyte test.

5. Given the HLA phenotypes of parents and offspring, determine the genotype and haplotype of each.

6. Given the HLA phenotypes of an alleged father, mother, and offspring, determine if the alleged father is excluded as the biologic father, if possible.

7. Describe how relative risk can be related to a disease.

8. Classify a graft as autologous, syngeneic, allogeneic, or xenogeneic.

9. Compare and contrast hyperacute rejection, first-set rejection, and second-set rejection.

10. Explain why ABO and HLA compatibility are important in clinical transplantation.

11. Describe the cellular and humoral events that lead to graft rejection.

12. Describe the tests used to phenotype the ABO blood group and HLA-A, HLA-B, HLA-D, and HLA-DR.

13. State the general immunosuppressive effect of azathioprine, cyclophosphamide, corticosteroids, cyclosporin A, FK506, anti-lymphocyte serum, irradiation, and monoclonal antibodies.

14. Explain the difference between allogeneic bone marrow, autologous bone marrow, and peripheral blood stem cells.

15. State in what general conditions bone marrow or peripheral blood stem cells are transplanted.

16. List and explain the complications of transplantation.

17. Compare and contrast acute and chronic graft-versus-host disease.

E. Overview of Infectious Disease and Serology:

1. Differentiate between infection and disease.

2. Define colonize, pathogenicity, and virulence in relation to infectious diseases.

3. List and describe the stages of an infectious disease.

4. Define primary and secondary antibody response.

5. Discuss significant titers of IgG and IgM in determining recent and past infections.

6. Discuss the significance of a fourfold rise in antibody titer.

7. List several characteristics used to classify viruses.

8. Define capsid, envelope, capsomere, and peplomer.

9. Name and describe the steps in viral replication.

10. Discuss cytotoxic effects of viruses on host cells.

11. Describe the properties of transformed cells.

12. Discuss the role of interferon in response to viral infection.

F. Streptococcal Infection and Serology:

1. Explain the role of Streptococcus pyogenes and its components in the development of rheumatic fever (RF), rheumatic heart disease (RHD), and acute glomerulonephritis (AGN).

2. Describe the importance of immunologic procedures in the diagnosis of RF, RHD, and AGN.

3. Discuss the relationship of an increased anti-streptolysisn ) (ASO) titer with the antecedent streptococcal infection.

4. Differentiate between positive and negative ASO results.

5. Explain the relationship between a positive ASO and a negative anti-hyaluronidase test (AHT).

6. Outline the procedure for the ASO, AHT, anti-Dnase B, and the Streptozyme tests.

7. Describe the basic methods employed for diagnosis of antecedent streptococcal infections.

8. Identify significant test results.

9. Explain reasons for false positive results.

G. Syphilis:

1. State the treponemes that infect humans, the syndromes and infections they cause, and the epidemiological differences.

2. Describe the four stages of syphilis and congenital syphilis.

3. Identify appropriate therapy for syphilis and congenital syphilis.

4. Describe three types of direct detection tests for syphilis and in what stage of syphilis they are useful for diagnosis.

5. Describe the serological diagnosis of syphilis, indicating the differences between nontreponemal and treponemal tests, and name the stages of syphilis for which serological tests are most useful.

H. Borrelia burgdorferi Infection and Serology:

1. Describe the causative agent of Lyme disease.

2. List the major antigenic components of Borrelia burgdorferi.

3. Describe the stages of Lyme disease and the major symptoms in each stage.

4. Discuss the appearance of IgG and IgM in the course of Lyme disease.

5. Discuss the principles of the following tests used in the diagnosis of Lyme disease: Immunofluorescent assay, Enzyme-linked immunosorbent assay, Western immunoblot.

6. Discuss the methods of treatment, prevention, and control of Lyme disease

I. Rubella Virus Infection and Serology:

1. Describe the size and composition of the rubella virus.

2. Describe the symptoms and complications of acute rubella virus infection.

3. List the major abnormalities associated with congenital rubella infection.

4. Discuss the appearance of IgG and IgM antibodies in the course of acute disease and congenital infection.

5. Discuss the principles of the following tests used in rubella antibody testing: Hemagglutination inhibition, Passive hemagglutination, Solid- phase immunoassays, Sucrose density gradient ultacentrifugation.

6. List appropriate tests for diagnosing congenital infection and determining recent rubella infection and immune status.

7. Discuss the rubella vaccine in terms of composition, recommended administration, and contraindications for use.

J. Epstein-Barr Virus Infection and Serology:

1. Describe the Epstein-Barr virus

2. Discuss the diseases caused by Epstein-Barr virus.

3. Discuss the tests used to detect heterophile antibody.

4. Discuss specific tests used to detect antibodies to the Epstein-Barr virus.

5. Given laboratory results, suggest the most likely disease and whether the infection was recent, remote, or reactivated.

K. Viral Hepatitis:

1. Describe the pertinent characteristics of the five hepatitis viruses.

2. Compare and contrast the epidemiology and clinical manifestations of the five hepatitis diseases.

3. Describe the antigen and antibody responses for diagnosing acute and chronic stages of the five viral hepatitis diseases.

L. Human Immunodeficiency Virus Infection and Serology:

1. Describe the HIV virus, its structure, and its life cycle

2. Compare and contrast the epidemiology of HIV-1 and Hiv-2 infections.

3. Describe the clinical manifestations of AIDS and current therapy.

4. Describe the laboratory diagnosis of HIV infections, including the appropriate use of screening and supplemental antibody tests to include basic principles of PCR.

5. Describe several tests for directly detecting HIV viremia

M. Serology of Miscellaneous Infectious Diseases:

1. List two reasons why serology evaluation may be preferred over culture for the diagnosis of infectious disease.

2. List the four major rickettsia groups.

3. Name the organisms that are the causative agents of the following: epidemic typhus, endemic typhus, scrub typhus, and Q fever.

4. Describe the cold agglutinin test for mycoplasma.

5. Describe the method of detection of fungal precipitin antibodies.

6. List the two primary risk groups for toxoplasma infection.

7. Describe the use of cytomegalovirus serology in blood banking.

8. List the two clinical manifestations of HTLV-1 infection.

N. Immunologic Tolerance:

1. Define tolerance, anergy, and apoptosis.

2. Discuss the relationship between immune activation and tolerance.

3. Compare and contrast clonal deletion and anergy.

4. Describe general mechanisms by which tolerance is broken.

5. Explain the beneficial and deleterious effects of tolerance and broken tolerance.

O. Non-Organ-Specific Autoimmune Disease:

1. Define autoimmunity.

2. Discuss the general mechanism of autoimmunity.

3. Explain the theories that cause autoantibody production.

4. Describe the patterns seen when antinuclear antibody is detected by indirect imunofluorescence.

5. List the marker antibodies and explain the significance of each.

6. State the immunofluorescent pattern associated with the major nuclear antigens discussed in this chapter.

7. Define rheumatoid factor.

8. For each of the following diseases, state the general abnormality and major serological changes: SLE, RA, Sjogren’s Syndrome, Progressive systemic sclerosis, Polymyositis-dermatomyositis, Autoimmune hemolytic anemia.

P. Organ-Specific Autoimmune Disease:

1. Discuss the differences between non-organ-specific and organ-specific autoimmune diseases.

2. List the laboratory test procedures used to diagnose thyroid autoimmune diseases.

3. Describe the differences between Graves’ disease and Hashimoto’s thyroiditis.

4. List the laboratory test procedures used to diagnose liver autoimmune diseases.

5. Describe the differences between autoimmune chronic active hepatitis and primary biliary cirrhosis.

6. Discuss spinal fluid immunoglobulin quantitation in multiple sclerosis.

7. Identify autoimmune antibodies and immunologic changes associated with myasthenia gravis, diabetes mellitus, idiopathic adrenal failure, autoimmune bullous skin diseases, Good-pasture’s syndrome, and spermatozoa antibody-mediated infertility.

Q. Immune Deficiency:

1. Differentiate between primary and secondary immune deficiencies.

2. Describe the location of defects in the immune system causing the ID and in the pathways of normal lymphocyte differentiation.

3. Classify selected immune deficiencies as humoral, cellular, or combined.

4. Discuss the information generated from each of the laboratory tests described in the chapter and how they are used to evaluate immunocompetence.

5. Suggest the most likely type of immune deficiency when provided with laboratory and clinical data.

R. Hypergammaglobulinemia:

1. Characterize the laboratory findings of hypergammaglobulinemia as monoclonal or polyclonal based on serum protein electrophoresis, immunoelectrophoresis, immunofixation electrophoresis, and quantitation of immunoglobulin classes.

2. Identify abnormal laboratory results.

3. Compare and contrast polyclonal and monoclonal gammopathies with respect to cellular processes, the concentration of immunoglobulin, and the appearance on immunofixation electrophoresis.

4. Classify each of the following diseases or conditions as a monoclonal gammopathy and describe the expected changes on serum protein electrophoresis, immunoelectrophoresis, immunofixation electrophoresis and quantitation of immunoglobulin: chronic infection, Multiple myeloma, connective tissue disease, Primary amyloidosis, Waldenstroms macroglobulinemia, Heavy-chain disease, Liver disease, HIV infection.

S. Laboratories:

1. Describe, perform, and interpret laboratory methods for selected immunological and serological tests and report results in written and oral form as required and judge reasonableness of the result.

2. Discuss the clinical significance of the performed immunological and serological tests performed.

3. Correlate laboratory data with physiological conditions for the purpose of assessing the validity of the results.

4. Ask appropriate scientific questions and recognize what is involved in experimental approaches to the solutions of such questions and demonstrate ability to evaluate and draw conclusions.

5. Utilize correct instrumentation, statistical and mathematical applications, reagent preparation and storage, specimen collection and handling, quality assurance and quality control.

6. Apply laboratory safety as described in the OSHA of 1970, 29 USCA section 655 and all subsequent regulations including instructions in universal precautions which is incorporated by reference in the safe use of laboratory equipment and procedures.

7. Demonstrate effective interpersonal relationships in working with others in the laboratory.

In order to succeed in the laboratory portion of the course, the student will be required to produce written reports of all lab exercises. In addition the student will be expected to:

1. Correctly perform and interpret lab tests on at least two unknown specimens for each lab exercise.

2. Correctly provide a tentative diagnoses on the unknown samples

3. Correctly answer all pertinent questions indicated.

Each laboratory exercise will be worth 20 points,

Laboratory 1

Streptozyme Rapid Slide Test

Principle:

Streptozyme Rapid Slide Test is an agglutination test that detects antibodies to five group A streptococcal exoantigens, including streptolysin, streptokinase, hyaluronidase, Dnase, and NADase. These antigens are attached to aldehyde-fixed sheep red blood cells, and if a patient has antibodies to one or more of these antigens, agglutination results.

Anti-Streptolysin O Titer

Principle:

The anti-streptolysin O titer is performed to detect antibodies to streptolysin O, a hemolytic exotoxin produced by group A streptococci. It s a neutralization test based on incubation of dilutions of patient serum with a standardized preparation of streptolysin O. If specific antibody is present, when human type O red blood cells are added, no hemolysis takes place. The ASO titer is used in the diagnosis of nonsuppurative sequelae of streptococcal infection, which include glomerulonephritis and rheumatic fever.

Laboratory 2

RPR Card Test for Serological Detection of Syphilis

Principle:

The RPR (rapid plasma reagin) 18-mm Circle card Test is a nontreponemal serological test for syphilis that detects reagin, an antibody formed against cardiolipin during the progress of the disease. The antigen consists of cardiolipin mixed with carbon particles, cholesterol, and lecithin. If antibody is present in the patient specimen, flocculation occurs with co-agglutination of the carbon particles, resulting in black clumps against the white background of the plastic-coated card. Nonreactive specimens appear to have an even, light-gray color.

Laboratory 3

Infectious Mononucleosis

Principle:

Forssman or heterophile antigens are similar antigens found on tissue cells of many different species. The antibodies formed in response to these antigens are called Forssman or heterophile antibodies. Heterophile antibodies can be found in normal individuals, inpatients with serum sickness, and in patients with infectious mononucleosis (about 98% of all normal individuals have heterophile titers of about 1:56 or less). Al heterophile antibodies (including those produced by IM) will agglutinate sheep RBCs. IM antibodies (non-Forssman) can be distinguished from other heterophile antibodies (Forssman) by the fact that Forssman antibody is neutralized (or adsorbed) by guinea pig serum. IM antibodies are not neutralized by guinea pig serum. Forssman antibody, after neutralization by guinea pig serum, prevents the agglutination of horse RBCs; IM antibody is not neutralized and, therefore, agglutinates horse RBCs. Rapid agglutination testing is based on the findings of Paul and Bunnell that sera from patients with infectious mononucleosis contain heterophile antibodies that will agglutinate sheep and horse erythrocytes. Prior adsorption of serum with guinea pig or horse kidney will remove Forssman antibodies, and adsorption with beef erythrocytes will remove serum sickness and infectious mononucleosis antibodies. Agglutination patterns are then determined by reaction with horse erythrocytes.

Rubella Titer

Principle:

Laboratory testing for rubella is performed most often for diagnosis of acquired infections, primarily in children and young adults, and for rubella immunity screening for pregnant patents. Rubella titers offer a quantitative test for immunity screening. These may be performed by HI, PHA, CF, RIA, ELISA, and latex agglutination. Latex agglutination tests for the detection and quantitation of rubella antibodies in serial dilutions of human serum.

Laboratory 4

HIV Rapid Slide AgglutinationTesting

Principle:

Several agglutination tests have been developed using latex particles, gelatin particles, or microbeads. HIV antigen is adsorbed onto these carrier particles, and if antibody is present in the patient sample, clumping occurs. These tests are rapid, simple to perform, and can be read visually. They are especially useful for testing done in laboratories without more sophisticated equipment.

Laboratory 5

Cold Agglutinins

Principle:

Cold autoagglutinins are found in persons following Mycoplasma pneumonia, infectious mononucleosis, cytomegaloviris infection, and the mumps or neoplasms such as lymphoma or histiocytic lymphoma. These cold-reacting antibodies belong to the IgM class, and most are specific for the Ii blood groups on red cells. Testing evaluates the ability of antibodies to agglutinate group O erythrocytes at 4 degrees Centigrade. The presence of an elevated titer of cold-reacting antibodies can cause acrocyanosis or hemolysis. These antibodies can be demonstrated in patients with primary (chronic) or viral disease.

Febrile Agglutinins

Principle:

Febrile agglutinin testing is a rapid screening method for determining the cause of a fever of unknown origin that could possibly be attributed to the presence of a gram-negative microorganism. These particular gram-negative organisms are difficult to isolate in the laboratory, so their presence is detected indirectly by testing for antibody produced in response to them. the symptoms of most of the diseases being tested for are very similar and include malaise, headache, chills, and persistent fever. Chemically inactivated dyed suspensions of antigen are used to determine the presence of antibodies that might indicate any of several diseases. Dilutions of antibody are made by adding progressively smaller amounts of patient serum to standardized concentrations of antigen. A positive reaction is indicated by agglutination of the dyed antigen.

C-Reactive Protein

Principle:

Latex particles coated with antibody to CRP are reacted with patient serum. The CRP is acting as the antigen. If CRP is present above normal threshold levels, antigen-antibody combination will result in a visible agglutination reaction. An elevated CRP level is a sensitive, although nonspecific, indicator of inflammation.

Ouchterlony Immunodiffusion

Principle:

Ouchterlony immunondiffusion is a double-diffusion method that is used for comparison of antigens. Wells are cut in an agarose gel in a pattern that utilizes a central well surrounded by several equidistant outer wells. Antibody or a combination of antibodies are placed in the central well, and a combination of standards and unknown antigens are measured into the surrounding wells. Reactants diffuse from the wells, and if the antigen is capable of reacting with the reagent antibody, a band of precipitate forms between the wells. Patterns of precipitation between adjacent wells indicate whether or not the antigens are related.

Laboratory 6

Quantitative Enzyme-Immuno-Assay

Principle:

A quantitation of testing of both antigens and antibodies will be used. The method employs color-changed products of enzyme-substrate interaction or inhibition to measure the antigen-antibody reactions. EIA microwell format for the detection of Borrelia burgdorferi antibodies (IgG and IgM) is used.

Laboratory 7

Rheumatoid Factor

Principle:

Rheumatoid factor is an IgM antibody directed against IgG. It is found in 70-80 percent of patients with rheumatoid arthritis. Passive agglutination can be employed to test for the presence of the antibody, using a carrier particle such as sheep erythrocytes sensitized with IgG. When patient serum containing RF is mixed with the sensitized reagent cells, visible agglutination occurs.

Laboratory 8

Anti-Nuclear Antibodies

Principle:

The measurement of antibody to nuclear antigens will be performed. Antinuclear antibodies are found in 99% of patients with untreated systemic lupus erythematosus and in mixed connective tissue disease and rheumatoid arthritis. ANA can be used as a diagnostic indicator, a prognostic indicator, or as a means to monitor the effectiveness of therapy. Immunnofluorescence techniques will be employed.

Laboratory 9

IEP

Principle:

Immunoelectrophoresis is a gel electrophoretic techniques commonly performed in the clinical immunology laboratory. The procedure uses electrophoresis and double diffusion. Patient serum is placed in a well and electrophoresed. The parameters of IEP include the migration of serum protein with albumin migrating toward the anode and immunoglobulins migrating to the alpha, beta, and gamma globulin regions. Antihuman serum is placed into the trough and the antiserum and the separated patient proteins diffuse toward each other. Precipitin arcs form at the zone of equivalence between the antigen and specific antisera.

Laboratory 10

Immunofixation

Principle:

Immunofixation electrophoresis is a gel electrophoretic technique used for serum, urine or CSF. Patient samples are electrophoresed and followed by the application of antisera. A cellulose acetate strip impregnated with the antiserum is placed on the separated proteins. The antiserum diffuses into the gel rapidly, resulting in the precipitation of antigen-antibody complexes.

LECTURE/LABORATORY SCHEDULE

January 6 & 11

Immunology Review (Chapters 1-4)

Hypersensitivity (Chapter 11)

January 13 & 18

Tumor Immunology (Chapter 13)

Transplantation and Major Histocompatibility Complex (Chapters 4, 13)

January 20 & 25

Quiz 1 Chapters 1-4, 11 & 13

Overview of Infectious Disease and Serology (Chapter 16)

Streptococcal Infection and Serology (Chapter 16)

Laboratory 1 Laboratory Safety, ASO and Direct Streptococcus testing (Chapter 16)

January 27 & Febuary 1

Syphilis (Chapter 15)

Borrelia burgdorferi Infection and Serology (Chapter 15)

Laboratory 2 RPR (Chapter 15)

February 3 & 8

Quiz 2 Chapters 15, and 16

Rubella Virus Infection and Serology (Chapter 17)

Epstein-Barr Virus Infection and Serology (Chapter 17)

Laboratory 3 Monospot, Rubella Titer (Chapter 17)

February 10 & 15

Viral Hepatitis (Chapter 17)

February 17 & 22

Quiz 3 Chapter 17

Human Immunodeficiency Virus Infection and Serology (Chapter 18)

Laboratory 4 HIV Rapid Slide Agglutination (Chapter 18)

February 24 & 29

Serology of Miscellaneous Infectious Diseases (Chapter 9)

Laboratory 5 Cold Agglutinins, Febrile Agglutinins, CRP (Chapter 9)

March 2

Quiz 4 Chapter 9

Laboratory 6 Quantitative EIA (Chapter 10)

March 7 & 9

Spring Break

March 14

Serology of Miscellaneous Infectious Diseases (Chapter 9)

March 16 & 21

Immunologic Tolerance (Chapter 12)

Non-Organ-Specific Autoimmune Disease (Chapter 12)

Organ-Specific Autoimmune Disease (Chapter 12)

March 23 & 28

Laboratory 7 Rheumatoid Factor, (Chapter 12), Anti-Nuclear Antibodies (Chapter 12)

March 30 & April 4

Laboratory 8 Anti-Nuclear Antibodies (Chapter 12)

April 6 & 11

Quiz 5 Chapter 12

Immune Deficiency (Chapter 14)

Hypergammaglobulinemia (Chapter 14)

April 13 & 18

Laboratory 9 IEP (Chapter 14)

April 20 & 25

Quiz 6 Chapter 14

Laboratory 10 Immunofixation (Chapter 14)

April 27

Comprehensive Final

References:

1. Sheehan, C., Clinical Immunology. Second edition. Lippincott, Philadelphia, 1997.

2. Turgeon, M. L., Immunology and Serology in Laboratory Medicine. Second edition, C.V. Mosby Co., St. Louis, 1996.

3. Miller, L. E., Ludke, H. R., Peacock, J. E., Tomar, R. H., Manual of Laboratory Immunology. Second edition, Lea and Febiger, Philadelphia, 1991.

4. Janeway, C. J., Travers, P., Hunt, S., Walport, M., Immunobiology, the immune system in health and disease. Third edition, Current Biology Limited, London, 1997.

5. Bryant, N. J., Laboratory Immunology and Serology. 2^nd edition, W. B. Saunders Company, Philadelphia, 1986.

6. Sharon, J., Basic Immunology. Williams and Wilkins, Baltimore, 1998

7. Roittt, I., Roitt’s Essential Immunology, 9^th edition, Backwell Science Ltd. London, 1997.

8. Roitt, I., Brostoff, J., Male, D., Immunology, 5^th edition, Mosby, London, 1998

9. Kuby, J., Immunology, 3^rd edition, W. H Freeman and Company, New York ,1997

10. Playfair, J. H. L., Immunology at a Glance, Blackwell Science Malden1996

Web sites:

http://www.niaid.nih.gov/publications/allergens/title.htm

http://www.niaid.nih.gov/

http://www.cdc.gov/nip/vaccine/influenz.htm

http://familydoctor.org/

http://www.ama-assn.org/insight/gen_hlth/blood/blood.htm