Clinical Pathology: Haematology

1. Clinical Pathology: Haematology Group D

2. Scenario A 25 year old female presented with: • 2 week history of progressive tiredness and dyspnoea on exertion • 3 week history of intermittent migratory arthralgia • 2 years ago pleurisy • no meds • mucosal pallor with mild scleral icterus • palpable spleen (3cm) • normal joints • urine negative for bilirubin and blood

3. Scenario cont. moderate anisocytosis numerous spherocytes prominent polychromasia mild red cell agglutination nucleated red cells neutrophilia with occasional band forms

4. Scenario cont.

5. Scenario cont. Other results: • positive direct antiglobulin test (Coomb’s Test) • IgG and complement demonstrated on the red cells Increased polychromasia i.e. elevated reticulocyte count, is strong evidence for haemorrhage or haemolysis, and either may be associated with neutrophilia and or thrombocytosis. Warm type autoimmune haemolytic anaemia is suspected. Investigation for other autoimmune disorders is carried out, with the history of arthralgia and pleurisy suggesting systemic lupus erythematous.

6. Questions: • Explain the significance of the presence of spherocytes and mild red cell agglutination • Explain the result of the Direct Antiglobulin (Coomb’s) test • What medications and conditions are associated with warm type AIHA? • What place does testing for antinuclear antibodies (ANA) and antidoublestranded DNA play in informing a diagnosis of SLE?

7. Warm type AIHA is associated with a number of conditions and medications – explain ?

8. Classification of autoimmune haemolytic anaemia I. On the basis of serologic characteristics of involved autoimmune process A. Warm autoantibody type-autoantibody maximally active at body temperature, 37ºC B. Cold autoantibody type-autoantibody active at temperatures below 37ºC C. Mixed cold and warm autoantibodies II. On basis of presence or absence of underlying or significantly associated disorder A) Primary or idiopathic AIHA B) Secondary AIHA 1) Associated with lymphoproliferative disorders 2) Associated with rheumatic disorders, particularly systemic lupus erythematosus 3) Associated with certain infections 4) Associated with certain non-lymphoid neoplasms 5) Associated with certain chronic inflammatory diseases 6) Associated with ingestion of certain drugs

9. Diseases associated with warm type AIHA

10. Etiology and pathogenesis • Malignancy associated AIHA the origin of pathologic antibodies is unknown Theories 1) Malignancies disrupt the normal immune function surveillance, that allows both autoantibodies to form and neoplasia to proliferate 2) malignant cells are the source of the anti erythrocyte antibody • non-malignant disease often of immunologic origin, general immune system disturbance is the etiology • Erythrocyte autoantibodies produced in these diseases cover RBCs → complement system responds to these antibody coated cells causing RBC phagocytosis • In general there is an inverse relationship between the quantity of red cell bound auto antibodies and red cell survival.

11. Clinical Features and Management • In secondary AIHA the haemolytic anaemia and associated features (weakness, fatigue, fever, bleeding and other symptoms and signs of anaemia) may be overshadowed by the more serious symptoms and signs of the underlying disease. • Treatment of the secondary disease may also bring the AIHA under control, however in some situations the disease may need to be treated separately – glucocorticoids or transfusion.

12. Drug induced warm-type AIHA

13. Autoantibodies In SLE, B cells spontaneously produce a large amount of immunoglobulins, including three types of autoantibodies: • nucleic acid binding proteins (histones and ribonucleoproteins) • nucleic acids (DNA) • cell membrane antigens (phospholipids) The different antibodies vary widely in their diagnostic and pathological significance

14. Autoantibodies cont.

15. Antinuclear autoantibodies • Heterogeneous group • Their presence is associated with a variety of diseases and disease manifestations, thus they are a non-specific marker of disease. • Antinuclear antibodies are also detectable in individuals with no symptoms of autoimmune disease • Many diseases presumed to be autoimmune in nature have no known association with antinuclear autoantibodies

16. Anti-Ro autoantibodies • The Ro antigen is a small intracellular complex between RNA and a membrane protein • High anti-Ro titres in patients with SLE has been associated with lymphopenia and leukopenia, myocarditis and cardiac conduction defects Anti-La autoantibodies • The La protein has a molecular weight of about 50 kDa and is involved in RNA polymerase III transcription • The significance of its presence in patients with SLE is not fully understood

17. Antiphospholipid autoantibodies • Directed against negatively charged phospholipids including β2 glycoprotein, phosphatidic acid, phosphatidylserine, cardiolipin and phosphatidylinositol • The presence of antibodies against these phospholipids is significantly associated with arterial and/or venous thrombosis • Anti-cardiolipin antibodies have been associated with abnormal intracardiac anatomy

18. Anti-Sm autoantibodies • The Smith antigen is a small uridine rich RNA molecule bound to a common group of core proteins, forming part of the complex that splices pre-mRNA • Titres are usually constant Anti-RNP autoantibodies • RNP = ribonucleoprotein • High levels of anti-RNP may indicate potentially fatal features e.g. fibrosing alveolitis or polymyositis

19. Anti-dsDNA autoantibodies • In patients without SLE, binding is in a sequence-specific manner to linear epitopes • In SLE, binding depends on ionic interactions and requires DNA flexibility • Anti-dsDNA antibodies are 75-95% sensitive and 95-99% specific for SLE • A rising titre of anti-dsDNA often heralds the development of nephritis or vasculitis

20. Anti-dsDNA autoantibodies cont. • Autoantibodies bind with DNA or DNA-histone conjugates to form circulating immune complexes • These complexes deposit in different tissues to elicit inflammation • They may react with plasma membrane-associated antigens on different cells and stimulate release of mediators which amplify the inflammatory response

21. References American College of Rheumatology ad hoc Committee on SLE Guidelines: Guidelines for referral and management of systemic lupus erythematosus in adults. Arth & Rheum 42(9):1785-96 1999 Arbuckle MR. McClain MT. Rubertone MV. Scofield RH. Dennis GJ. James JA. Harley JB. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. New Eng J Med 349(16):1526-33, 2003 Esdaile JM. Abrahamowicz M. Joseph L. MacKenzie T. Li Y. Danoff D. Laboratory tests as predictors of disease exacerbations in systemic lupus erythematosus. Arth & Rheum 39(3):370-8, 1996 Mar Mok CC, Lau S. Pathogenesis of systemic lupus erythematosus. J Clin Path 56:481-90 2003 Ulvestad E, Kanestrem A, Madland TM, Thomassen E, Haga HJ, Vollset SE. Evaluation of Diagnostic Tests for Antinuclear Antibodies in Rheumatological Practice. Scand. J. Immunol. 52:309-15 2000 Venables PJW. Fortnightly Review: Diagnosis and Treatment of SLE. BMJ 307(6905):663-6 1993