Immunoglobulins-high
Quick tip: it is important to distinguish whether a result returning high immunoglobulins are polyclonal (infective/inflammatory) or monoclonal (almost always malignant e.g myeloma) to help determine cause and significance.
Immunoglobulin Testing
- Immunoglobulins, commonly called antibodies, are proteins produced by plasma cells which are fully mature B lymphocytes.
- IgG represents 85% of all antibodies.
- IgM is the immunoglobulin produced early in humoral immune response (1-3 days) and subsequently IgG production ensues (5-7 days) which peaks at 14 days.
- IgA is mainly secreted by mucosae and is responsible for mucosal immunity.
- IgE mediates type 1 or immediate hypersensitivity reactions.
- Immunoglobulins are measured by automated analysers and any monoclonal proteins are detected by serum protein electrophoresis.
Causes of high Immunoglobulins (Hypergammaglobulinemia)
It is clinically important to distinguish if the increased production is monoclonal or polyclonal.
Immunoglobulins produced by a single clone of plasma cells are called monoclonal proteins or Paraproteins. This is confirmed by performing ‘Immunofixation’ - this is usually done automatically by the laboratory when a paraprotein is found on serum electrophoresis.
A. Monoclonal Hypergammaglobulinemia or Paraproteinemia:
- This is more commonly IgG but can be IgM, IgA or IgE rarely. Causes include
- Monoclonal Gammopathy of Undetermined Significance
- Plasma cell neoplasms like Multiple Myeloma
- B cell lymphomas
- Chronic Lymphocytic Leukaemia
- Lymphoplasmacytic Lymphoma
- Systemic Amyloidosis
- POEMS (Peripheral neuropathy Organomegaly Endocrinopathy M protein Skin changes)
B. Polyclonal Hypergammaglobulinemia:
These are produced by plasma cells belonging to multiple lineages and do not show a single band on protein electrophoresis. This is commonly IgG with IgM, IgA or IgE rarely.
This is almost always secondary to ongoing inflammation or infection.
All immunoglobulins can be elevated rarely in Hyper IL-6 syndromes.
Causes of polyclonal/'non-paraprotein' raised immunoglobulins include
| High IgG | Liver disease (Autoimmune Hepatitis, Alcoholic liver disease, viral hepatitis, Drug induced Hepatitis) Autoimmune disorders (Sjogren’s syndrome, SLE, Vasculitis, RA etc) Haematological conditions (Castleman’s disease, Angioimmunoblastic Lymphoma, CLL, Histiocytic diseases) IgG4 related Disease Chronic Infections (HIV, Chronic CMV, EBV, BK virus, Tuberculosis, Malaria) Solid organ tumours Iatrogenic (Exogenous administration of Immunoglobulins) |
| High IgA | Liver diseases including Autoimmune Hepatitis, Alcoholic liver disease Autoimmune diseases (SLE, RA etc ) HIV IgA vasculitis Chronic infections – HIV, TB, Osteomyelitis |
| High IgM | Primary Biliary Cirrhosis Autoimmune diseases (SLE, RA etc) Acute infection Hyper IgM syndrome (very rare) |
| High IgE | Atopy including allergic Asthma Hodgkin’s disease IgG4 related disease Hyper IgE syndrome (very rare) |
Immunoglobulin - low
Quick tip: when considering causes, secondary deficiency is more common than primary.
Causes of Low Immunoglobulins (Hypogammaglobulinemia):
A reduction in the level of immunoglobulins may be primary or secondary.
- Primary deficiency is due to reduced production and causes include the spectrum of primary immunodeficiency disorders involving B cells.
- Secondary deficiency is either due to loss or reduced production. These are more common than primary deficiency.
|
| Primary | Secondary |
| Low IgG | Agammaglobulinemia with absent B cells (XLA, ARA) Common Variable Immunodeficiency Severe Combined Immunodeficiency (mainly children involving T cells also) Good’s Syndrome Hyper IgM syndrome
| Protein losing enteropathy Intestinal Telangectasia Chylothorax Nephrotic syndrome Lymphoma CLL Multiple Myeloma Severe burns Medications – Rituximab, CART cell therapy, immunosuppressants including steroids, Azathioprine, Cyclophosphamide etc, antiepileptics (phenytoin, Lamotrigine), antipsychotics, Chemotherapeutic drugs Infections – HIV (rare), Herpes, acute bacterial infections, Measles, Mumps
|
| Low IgM | Agammaglobulinemia with absent B cells (XLA, ARA) Common Variable Immunodeficiency Severe Combined Immunodeficiency (mainly children involving T cells also) Good’s Syndrome Selective IgM deficiency
| Same as above IgM may be normal in Gastrointestinal and renal loss |
| Low IgA | Selective IgA deficiency (most common primary immunodeficiency) Agammaglobulinemia with absent B cells (X-linked or autosomal recessive) Common Variable Immunodeficiency Severe Combined Immunodeficiency (mainly children involving T cells also) Good’s Syndrome | Same as above
|
| Low IgE | Uncommon and may not be clinically significant Agammaglobulinemia with absent B cells (X-linked or autosomal recessive) Common Variable Immunodeficiency Severe Combined Immunodeficiency (mainly children involving T cells also) Good’s Syndrome Selective IgE deficiency
| Same as above |
Low levels should be discussed with immunology if identified without an obvious cause or if there are clinical sequelae or clinical concern.
Note: IgM levels are higher in adult females than males (check that the range quoted by the lab is different for males and females)
Serum electrophoresis
Quick tip: Immunofixation is useful in the typing of found paraproteins. See the haematology module on paraproteins in the ‘highs and lows’ section for interpretation in the context of a suspicion of plasma cell malignancy like myeloma or MGUS
Serum electrophoresis
Serum electrophoresis is a method used to separate proteins based on their charge and size. Once the proteins have been separated, a densitometer is used to measure the absorbance of each fraction and produced into a curve as below. The total protein level (a biochemistry) test is used to work out the amounts of each “zone”.
O’Connell TX, Horita TJ and Kasravi B (2005) Understanding and Interpreting Serum Protein Electrophoresis. American Family Physician 71:105-112
The following table summarises the proteins found in each section:
| Albumin | The first peak only contains albumin. |
| Alpha 1 | Alpha1-antitrypsin, thyroid-binding globulin, and corticosteroid binding globulin |
| Alpha 2 | Ceruloplasmin, alpha2-macroglobulin, and haptoglobin |
| Beta 1 | Transferrin, plasminogen , fibronectin |
| Beta 2 | Beta-lipoprotein, CRP, occasionally immunoglobulins and complement C3 |
| Gamma | Mainly immunoglobulins |
Serum electrophoresis is useful to investigate if there is a paraprotein present (a marker of plasma cell disorders such as multiple myeloma). A paraprotein is the immunoglobulin product of a single cell clone. It will have only one heavy chain type (e.g. IgG, IgM, IgA or IgD) and/or one light chain type (kappa or lambda). On serum electrophoresis it has the same size and charge and so migrates as a “band” that can be quantified.
Immunofixation
This is useful in typing the paraprotein when found, and also to find small paraproteins that may be hidden under the beta and gamma curves.
Lymphocyte subsets
This test measures the percentage and absolute numbers of different lymphocyte subsets.
Indications
- Suspected B cell deficiency/antibody deficiency
- Suspected combined Immunodeficiency
- Patients with HIV
- Genetic diseases like DiGeorge syndrome
It is performed using flow cytometry. This test identifies T cells (CD4 and CD8), B cells (CD19) and Natural Killer cells (CD56) based on the cell surface markers.
These tests need to be interpreted by age-appropriate reference ranges.
Sample needed – Fresh EDTA sample, 5 – 10ml.
Types of allergy test
Quick tip: patients need to avoid antihistamines for at least 7 days before skin prick testing.
Type 1 hypersensitivity reactions are immediate and can lead to anaphylaxis. These are usually in response to allergens that have been ingested, inhaled or injected. Investigations that will be offered by the allergy clinic include:
1) Specific IgE Tests (formerly RAST)
- Specific serum IgE tests can be done irrespective of age, medications, skin condition, disease activity, pregnancy. Things to consider:
- No risk of anaphylaxis.
- However false positives do occur both to foods and inhalant allergens. I.e. the patient can have positive results but no symptoms on contact with aeroallegen e.g. pet or consumption of food.
- A specific allergen must be requested – screening tests are not available.
- Expensive ~ £7-11 per test.
- Patient serum is added to antigen in lab test. The greater then amount of antibody present, the greater the colour change and higher result.
- Negative <0.35 KUA/L
- Positive > 0.35 kUA/L (measured up to 100)
Symptoms do not always correlate with degree of positivity in lab, e.g someone who has anaphylaxis to nuts may have a relatively low result of ~1kAU/L whereas some people may have ‘more positive’ result yet tolerate the allergen.
2) Skin Prick tests
- Useful in diagnosis of allergies such as aeroallergens causing hayfever, food allergy, drug allergy and venom allergy
- Not useful for contact dermatitis, chronic urticaria and angioedema, food intolerance and non-specific rashes
- Considerations:
- Low risk (although not no risk – some patients have had systemic reactions to testing if very sensitive)
- Cheap
- Need to avoid antihistamines for at least 7 days before
- Tricky if the patient has eczematous skin.
- Intradermal testing can be done by injecting a small amount of potential allergen under the skin. This is usually done for drug allergy testing as the allergen in questions needs to be safe for injection, i.e. not irritant!
How is it done?
- Usually on forearm – up to max of about 25 allergens.
- Drop of commercial reagent of allergen applied to arm followed by small prick with sterile lancet. Reactions occur in 15 minutes.
- A histamine control is also used which should become itchy and form a weal. A negative control with saline should show no response.
- Very different to patch tests for contact sensitivities.
3) Challenges
Challenges to food and medications are offered to patients who have negative test results. This is done in hospital setting and patients are typically monitored for two hours after a challenge.
4) Patch Testing
Type 4 hypersensitivity reactions present with delayed reactions such as with contact dermatitis. This are usually in response to topical agents but can also be through other methods, particularly in drug allergy. For common household chemicals and substances, the dermatology department can offer patch testing. For drug reactions, or if unsure of the mechanism, please contact your local Immunology department for advice.
N.B: DermNETNZ.org has excellent sections on patch testing with information sheets on each allergen
Cryoglobulins
Cryoglobulins are immunoglobulins that precipitate at temperatures below 37 degrees and redissolve on re-warming. If blood circulates through part of the body where the temperature is below the critical temperature, then the protein will precipitate in the capillaries causing obstruction, vascular damage and eventually necrosis. Symptoms are generated by immune complex activity and complement activation.
The reason for the insolubility is unknown, but may be due to abnormal amino acid composition such as paraproteins and abnormal protein glycosylation such as in connective tissue diseases.
Indications for testing
- All patients with new onset Raynaud’s phenomenon
- Winter onset of purpuric or vasculitic lesions on the extremities
- Ischaemia in the absence of peripheral vascular disease eg. abdominal pain associated with vasculitis of the GI tract
- Cutaneous vasculitis
- Chronic hepatitis C infection or Hepatitis B infection
- Autoimmune phenomena: Sjogren’s syndrome, membrano-proliferative glomerulonephritis, SLE, RA
- Patients with suspected symptoms with lymphoproliferative diseases eg. Myeloma
Cryoglobulin collection: ESSENTIAL TO BE PERFORMED ACCURATELY
This is a test that can only practically be performed in hospital because of the need to keep the sample warmed from collection to analysis.
- Phone local laboratory to inform that a cryoglobulin sample is being collected.
- Obtain 37 degrees thermos flask with pre-warmed blood bottles from the laboratory which are transported in the flask.
- Take blood using a warmed blood taking equipment (37 degrees). Do NOT use a syringe. Use a butterfly to take directly into tubes to avoid losing cryoprotein during collection. Keep sample in the 37 degrees flask and arrange for the sample to be transported immediately [ideally within 20 minutes of taking the sample] to the laboratory for processing.
- The laboratory will allow the sample to clot at 37 degrees and then cool the serum to 4 degrees. The sample is split with one aliquot incubated at 37 degrees and the other at 4 degrees for 3-7 days. If a cryoglobulin is present a precipitate will form in the 4 degrees sample, and no precipitate will be detected on the 37 degree sample.
- The precipitate is then washed and redissolved for analysis by electrophoresis and immunofixation. The redissolved precipitate is tested for rheumatoid factor.
Treatment
The underlying disease processes typically determines the treatment and prognosis of cryoglobulinaemia. Treatment can prevent the progression of cryoprotein related symptoms however reversal of significant tissue damage may not be possible.
- Mild symptoms (e.g. occasional purpura) can be controlled with NSAIDs or low doses of steroids.
- Symptomatic patients may get immediate relief from plasmapheresis to reduce the paraprotein concentration and viscosity. Rituximab may also be used.
N.B: Cryofibrinogen can be found in patients with malignancy.
Complement levels
Quick tip; There can be false ‘normal’ C3/C4 results in patients with e.g SLE, if there is C3/4 consumption due to an infective/inflammatory insult
Measurement of C3 and C4 components of complement allows analysis of both the classical and alternative complement pathways. C3 and C4 proteins are acute phase proteins and may therefore be normal at times of rapid complement consumption; assessment of C3 breakdown products in this scenario is advised.
Indications for testing:
- Complement deficiency [tested in combination with the CH50/AP50 function test]
- SLE
- Anaphylaxis [anaphylotoxins C4a and C5a]
- Hereditary angioedema [tested in combination with C1-esterase inhibitor and functional C1-esterase inhibitor]
- Acquired angioedema [with C1q level]
- Monitoring SLE and immune complex disease (infective endocarditis, serum sickness)
- Atypical haemolytic uraemic syndrome
- Cryoglobulinaemia
Interpretation
*Complement levels need to be interpreted in the context of the clinical setting, as pre-disease levels are often unknown*
Low C4, normal C3:
- Genetic deficiency [C4 allele copy number for C4 null alleles]*
- SLE (active) -check C3 breakdown products
- Hereditary angioedema
- Type II cryoglobulinaemia
- Eclampsia
Low C3, low C4:
Consistent with classical pathway activation and active inflammation
- Sepsis
- SLE (active)
- Infective endocarditis
- Rheumatoid arthritis
Normal C4, low C3:
Consistent with alternative pathway activation
- Post-streptococcal glomerulonephritis
- C3 nephritic factor
- Gram negative sepsis
High C3, high C4:
- Acute phase response
*C4 null alleles are common and affect the baseline level of C4; it is not possible to use C4 levels as a marker of SLE activity without knowing how many null alleles are present.