Laboratory Tests

Marisa Fernandes das Neves¹, Rajendra Vara Prasad Irlapati² and David Isenberg³

¹ Medicine IV Department, Fernando Fonseca Hospital, Amadora; CEDOC ‐ Chronic Diseases, Faculty of Medical Sciences, New University of Lisbon, Lisbon, Portugal

² Nizams Institute of Medical Sciences, Hyderabad, India

³ Centre For Rheumatology, Department of Medicine, University College London, London, UK

This chapter describes investigations that may be performed in a patient with a suspected or established rheumatological disorder. Abnormal haematology tests, particularly anaemia and platelet abnormalities, are commonly found. Biochemical abnormalities include raised protein and globulin levels and reflect a non‐specific inflammatory response. Haematological and biochemical investigations are useful for both diagnostic and monitoring purposes, while most immunological investigations are mainly used to facilitate diagnosis.

Haematology investigations

A full blood count and erythrocyte sedimentation rate (ESR), although non‐specific, are used to monitor disease activity, to assess the effects of drug treatment, to exclude factors such as dietary deficiency or haemolysis that may be contributing to the morbidity of a rheumatological disease, and (rarely) to exclude a primary haematological malignancy that can mimic various forms of arthritis (Table 25.1).

images — **Table 25.1** Anaemia and rheumatological disease

Type	Indices	Causes
Iron‐deficient	↓Serum Fe ↑Serum TIBC Microcytosis, hypochromasia	Disease per se: e.g. oesophagitis in scleroderma
Megaloblastic	Macrocytosis ↓Folate ↓B₁₂ ↓TFTs	Azathioprine Methotrexate (↓folate) Pernicious anaemia
Haemolytic	Reticulocytes Haptoglobins Positive direct Coombs’ test	SLE Drugs e.g. dapsone
Chronic disease	Normochromic, normocytic ↓serum iron, ↓TIBC, ↓ferritin	Multifactorial, ↓EPO, abnormal erythrocyte development, ↑cytokines e.g. IL‐1, TNF‐α

Anaemia

Anaemia of chronic disease (ACD) is the most common abnormality observed in rheumatic diseases. The cause for this is not clear, but likely causes include shortened red cell survival or impaired bone marrow response to erythropoietin. Another major contribution is from inflammation‐induced activation of interleukin (IL) 6 and the hepcidin axis, which hampers iron absorption from gut and release from storage sites. It is sometimes difficult to distinguish iron deficiency anaemia from ACD as the smear in ACD can be normocytic or microcytic. Serum ferritin, which is an acute‐phase reactant, may also be elevated due to the underlying rheumatic condition. Soluble transferrin receptor levels may help in such scenarios.

Autoimmune haemolytic anaemia (AIHA) is a rare complication in rheumatic diseases seen in SLE and related conditions such as antiphospholipid syndrome (APS) and Sjögren’s syndrome. Evans’ syndrome is AIHA with thrombocytopenia. A positive direct Coombs’ test helps to identify AIHA. Non‐immune haemolytic anaemias may be due to microangiopathic haemolytic anaemia in conditions like thrombotic thrombocytopaenic purpura (TTP) and catastrophic antiphospholipid syndrome (CAPS) or disseminated intravascular coagulation (DIC).

Macrophage activation syndrome (MAS) is a unique haematological complication seen in systemic‐onset juvenile idiopathic arthritis (JIA) and SLE, characterized by pancytopenia, very high serum ferritin levels, rapid fall in ESR and elevated triglyceride levels.

Platelet abnormalities

Platelet abnormalities are common in rheumatic disorders. Mild to moderate thrombocytosis is one of the markers for acute or chronic inflammation. Platelet count is regulated by thrombopoietin, the synthesis of which is elevated in states of inflammation due to elevated IL‐6. Platelets are often elevated in disease like rheumatoid arthritis (RA) and Still’s disease when active. In the paediatric age group, thrombocytosis is often seen in systemic‐onset JIA and Kawasaki’s disease. A normal or low platelet count in a child with suspected active systemic‐onset JIA should raise the suspicion of underlying haematological malignancy or macrophage activation syndrome.

Thrombocytopenia, on the other hand, is often a manifestation of autoimmune rheumatic disease (ARD) and related diseases. An autoimmune thrombocytopenia (usually chronic but occasionally acute) occurs in up to 20% of patients with lupus and in patients with primary antiphospholipid antibody syndrome. Thrombocytopenia in APS is often mild, whereas it can be severe in SLE. In some of these patients, it has been possible to demonstrate the presence of antiplatelet antibodies. Approximately 15% of patients with ‘idiopathic’ thrombocytopenia later develop lupus, although identifying those patients prospectively remains a problem. Thrombocytopenia may also be seen in the subset of rheumatoid arthritis with Felty’s syndrome (see below). Infections associated with arthralgia such as cytomegalovirus, hepatitis C and human immunodeficiency virus (HIV) can also be associated with thrombocytopenia.

Critically ill patients with multiorgan dysfunction and thrombocytopenia, with or without vascular manifestations, may be due to TTP, catastrophic APS and DIC. Thrombocytopenia in patients with cutaneous vasculitis excludes primary systemic vasculitis and secondary causes like lupus should be considered.

In patients under long‐term follow‐up, thrombocytopenia is an early marker for marrow suppression due to cytotoxic therapies like cyclophosphamide, methotrexate and mycophenolate mofetil.

White blood cell abnormalities

Felty’s syndrome, the association of rheumatoid arthritis with leucopenia (predominantly neutropenia) and splenomegaly (and often leg ulcers), is rare. Leucopenia, particularly lymphopenia, is common in lupus. Most patients are asymptomatic but may be predisposed to infections. There are several causes for lymphopenia in SLE. Antilymphocyte antibodies are frequently found in patients with SLE; lymphocyte apoptosis is increased in active SLE and lymphocytes may be sequestrated at sites of inflammation or lymphoid tissues. Lymphopenia may be associated with flares of disease activity.

Bone marrow suppression is a well‐recognized complication of immunosuppressive drugs used to treat rheumatic disease such as azathioprine, methotrexate, leflunomide, sulfasalazine, cyclophosphamide and mycophenolate mofetil. Patients taking these drugs require regular haematological assessments to allow early detection of bone marrow suppression.

Systemic JIA characteristically has a severe neutrophilic leucocytosis. Mild to moderate neutrophilia is seen in RA and primary vasculitis. Leucocytosis is occasionally found in flares of lupus, but is more often a reflection of underlying infection or corticosteroid‐induced demargination of neutrophils. Infections should be ruled out in all cases of leucocytosis, though it may be a marker for severe inflammation.

Eosinophilia is commonly seen in parasitic infections or secondary to drugs, but it is also characteristic of some rheumatic and inflammatory diseases such as eosinophilic granulomatous polyangiitis (formerly known as Churg–Strauss syndrome). A range of blood test abnormalities in rheumatological diseases is shown in Table 25.2.

Table 25.2 Blood test abnormalities in some rheumatological diseases

		RA	SLE	PMR	Crystals	Myositis	SSc	Osteoporosis	Osteomalacia
Anaemia	Chronic disease	++	++	+	−	−	++	−	−
	Microcytic/hypochromic	++	+	++	−	−	++	−	−
	Megaloblastic	++	−	−	−	−	−	−	−
Acute − phase response	ESR	++	++	+++	+/−	+	+	−	−
Acute − phase response	CRP	+	−	+	+/−	−	−	−	−
Abnormal renal function		+	++	+/−	+	+/−	+	−	−
Abnormal liver function		+	+	−	−	−	−	−	−
Uric acid		−	−	−	+	−	−	−	−
Bone biochemistry	Alk phos	−	−	+	−	−	−	−	+/−
	Ca²⁺	−	−	−	−	−	−	−	↓/−
	PO⁴⁻	−	−	−	−	−	−	−	↓/−

Aik phos, alkaline phosphatase; CRP, C‐reactive protein; ESR, erythrocyte sedimentation rate; PMR, polymyalgia rheumatica; RA, rheumatoid arthritis; SIE ‐ systemic lupus erythematosus; SSc, systemic sclerosis

Acute‐phase response

This response defines a co‐ordinated set of systemic and local events associated with inflammation, triggered by tissue damage. The term is misleading, as changes may occur in both acute and chronic inflammation. About 30 acute‐phase proteins are known. Elevated serum concentrations of these proteins often last for several days after the initiating event, and their synthesis in the liver is triggered by cytokines, particularly IL‐1, IL‐6 and tumour necrosis factor alpha (TNF‐alpha). These cytokines derive from activated macrophages that have been demonstrated at the site of the injury. Other types of cells such as fibroblasts and endothelial cells are also sources of cytokines. There is some specificity in the cytokine/acute‐phase reactant interactions; for example, the synthesis of C‐reactive protein (CRP) is dependent on IL‐6, while haptoglobin production is influenced by the three cytokines mentioned above.

Measurement of the acute‐phase response is helpful to ascertain inflammatory disease, as well as for the assessment of disease activity, monitoring of therapy and the detection of intercurrent infection. It is impractical and unnecessary to measure all aspects of the acute‐phase response; the most widely used measurements are the ESR, CRP and complement components, especially complement C3 (Table 25.3). ESR correlates with fibrinogen levels and CRP is linked to IL‐6 activity. The ESR is the measure of the height of the layer of red blood cells that settle in a tube of anticoagulated blood in 1 hour (measured in millimetres per hour (mm/h)). Less common measurements include plasma viscosity, serum amyloid A (SAA) protein, haptoglobin and fibrinogen.

Parameter	Measurement	Pathophysiology	Affected by
ESR	Distance in mm that RBC column falls in 1 hour	Dependant upon rouleaux formation (aggregation of red cells) and PCV Indirect reflection of acute‐phase proteins and immunoglobulins	Plasma proteins (i.e. fibrinogen. β2 microglobulin and immunoglobulins) Anaemia, ↓ in sickle cell anaemia
CRP	Immunoassay (mg/L)	Pentamenc protein released from liver under influence of IL‐6 within 4 hours of tissue injury	↑↑ in infection, often normal in SLE