Immunology Flashcards
What are the main APC’s?
APCs are cells that express MHC class II the main APCs that link the innate and adaptive immune systems are:
Dendritic cell
antigen presentation is the main role of these cells
critical in initiation of most adaptive immune response by presenting antigen to lymphocytes
Macrophage
important phagocytes, particularly involved in control of intracellular pathogens
also present antigen on MHC II, helping to activate adaptive immune responses
What are the functions of cd4+ and cd8+ T cells?
CD4+
also known as “T helper cells”
after activation multiply and form numerous daughter “effector cells” migrate to sites of inflammation/infection via the bloodstream
produce cytokines that direct the immune response
their role is to “help” other cells carry out their functions
e.g. B cells to produce antibody
macrophages to fight intracellular pathogens
CD8+
kill infected cells (“cytotoxic T cells”) recognise antigen expressed on the cell surface in conjunction with MHC class I (expressed by all cells)
What are important tissues of the immune system?
consist of central (primary) lymphoid tissues
where immune cells are produced
consist of bone marrow and thymus
bone marrow is the site of haematopoesis
B cells (along with myeloid cells) produced and develop in the bone marrow
T cells produced in the bone marrow and travel to the thymus for ‘education
peripheral (secondary) lymphoid tissues
where adaptive immune responses are initiated
lymph nodes
spleen
mucosal associated lymphoid tissue
facilitates antigen and lymphocytes to come together, allowing adaptive immune responses
dendritic cells expressing antigen migrate from tissues to secondary lymphoid organs (e.g. lymph nodes) in lymph via lymphatic vessels
naïve lymphocytes circulate through lymph nodes via the bloodstream
What is the function of Lymph?
lymph is essentially extracellular fluid from all tissue, but contains APCs bringing antigen from tissues
drains in a series of (afferent) lymphatic vessels into secondary lymphoid tissues (e.g. lymph nodes)
fluid (lymph) then leaves lymph nodes via efferent lymphatics vessels, which drain into a collecting lymphatic vessel called the thoracic duct
this then drains into the bloodstream via the heart
naïve lymphocytes enter the lymph nodes from the bloodstream
there they sample the environment for antigen presented by APCs
if they encounter their antigen and are activated they divide to form ‘effector’ cells
effector lymphocytes (or naïve cells that have not encountered their antigen) leave lymph nodes via efferent lymphatics
drain into thoracic duct and back into bloodstream
activated ‘effector’ cells then home to inflamed tissues (e.g. sites of infection)
naïve cells home back to lymph nodes again and continue to circulate between lymph nodes and blood until antigen encountered
What are the warning signs for immunodeficiency?
- The European Society for Immunodeficiencies warning signs for ADULT primary immunodeficiency diseases:
- Four or more infections requiring antibiotics within one year (otitis, bronchitis, sinusitis, pneumonia)
- Recurring infections or infection requiring prolonged antibiotic therapy
- Two or more severe bacterial infections (osteomyelitis, meningitis, septicaemia, cellulitis)
- Two or more radiologically proven pneumonia within 3 years
- Infection with unusual localization or unusual pathogen
- PID in the family
- Also remember Failure to Thrive in paediatrics
What are the important questions to cover in an immunological history?
Frequency, site and type of pathogen often determines response
Always think of the full differential diagnosis
Details of all previous infections Site and frequency Pathogens Severity Need for antibiotics Hospital admissions Autoimmunity Malignancy Immunisation History Operations (grommets, lobectomies) Family History Serious infections Immunodeficiencies Autoimmune diseases Unexplained sudden deaths Medication history (secondary immunodeficiency) Lamotrigine can cause a combined immunodeficiency, with viral, fungal and bacterial infections Phenytoin can cause hypogammaglobulinaemia (reduced immunoglobulins) and enlarged lymph nodes that can look like lymphoma (pseudolymphoma)
What are the potential features associated with immunodeficiency?
Atypical eczema Chronic diarrhoea Failure to thrive Telangiectasia Hepatosplenomegaly Endocrinopathy Chronic osteomyelitis/deep-seated abscesses Mouth ulceration Autoimmunity Family history
What are the features associated with secondary antibody deficiency?
Possible associated features to consider… Extremes of age Uraemia Toxins Acute & chronic infections Burns Myotonic dystrophy Protein-losing states Lymphangiectasia
What are some important examination findings in immunological diseases?
Weight and height (FTT)
Structural damage from infections (ears, lungs, sinuses)
Autoimmune features (vitiligo, alopecia, goitre)
Absent tonsils (XLA)
Lymphadenopathy
Hepatosplenomegaly
Other potential diagnostic features (telangectasia, eczema)
How can Antibody deficiencies present?
Recurrent bacterial infection
Main sites are chest, sinuses (i.e. respiratory tract)
Ear and eye infections also common
Recurrent infection eventually causes end organ damage e.g. bronchiectasis
Primarily encapsulated bacteria
E.g Streptococcus pneumoniae (Pneumococcus), Haemophilus, Klebsiella, Pseudomonas etc.
These have polysaccharide capsule that impairs phagocytosis by phagocytes (e.g. neutrophils and macrophages)
Antibodies are produced against polysaccharide antigens in capsule
Binding of antibodies results in ‘opsonisation’, greatly enhancing phagocytosis by phagocytes
Viruses can usually be cleared, but difficulty forming protective immunity can lead to recurrence (e.g. recurrent shingles)
Age at presentation varies by disease
There can also be a number of non-infectious complications
Increased rates of
Autoimmunity (e.g. autoimmune cytopenias / anaemia, endocrine, rheumatological e.g. SLE-like etc.)
Enteropathy (Coeliac-like condition not gluten sensitive, IBD)
Granulomatous inflammation
Sarcoid-like granulomatous lung disease (GLILD)
Lymphoproliferation, hepatosplenomegaly
Malignancy, particularly lymphoma
What are the causes of antibody deficiency
Primary antibody deficiency Common variable immunodeficiency (CVID) X-linked agammaglobulinaemia IgG subclass deficiency Specific antibody deficiency with normal immunoglobulins Hyper IgM syndrome (HIGM) – most are really a combined immunodeficiency Hyper IgE syndrome (HIGE) (Selective IgA deficiency)
Combined immunodeficieny
Secondary antibody deficiency More common than primary Drugs [cytotoxics, anti-convulsants, anti-rheumatics, Rituximab] Radiation Malignancy [CLL, myeloma] Loss [gut, kidney] Nutrition [B12] Metabolic Infections (HIV, CMV, EBV, Toxoplasma) Extremes of age / immunosenescence
What Investigations can be done for Antibody
Full blood count Haemoglobin & indices Malabsorption, anaemia of chronic disease Evidence for marrow failure Thrombocytopenia White cell count Lymphopenia or lymphocytosis Neutropenia Blood film Abnormal cells [smear cells, blasts
Biochemistry Liver function tests Albumin for renal or GI loss Thyroid function Creatinine & urea [renal disease] Blood glucose [HbA1c] Urinary protein excretion [Stick test first]
“Immunoglobulins” and “antibodies” are essentially interchangeable terms
Routine testing involves testing IgG, IgA and IgM (together constitute almost all serum immunogobulin)
Normal range (adults)
IgG 5.8 - 15.4 g/L
IgA 0.64 - 2.97 g/L
IgM 0.24 – 1.9 g/L (male), 0.71 – 1.9 (female)
Age-specific ranges in children
Normal levels do not exclude significant immunodeficiency
If considering hyper-IgE syndrome also need to check total IgE [typically very raised often >50,000 kU/l]
Always have a least two separate measurements of serum immunoglobulins if low.
Low serum immunoglobulins do not always lead to significant infections
Secondary causes of antibody deficiency particularly are often suprisingly well tolerated
Low serum immunoglobulins do not automatically mean that immunoglobulin replacement therapy is required!!
Lymphocyte analysis Basic panel CD19 or CD20 [B cells] CD16/56 [NK Cells] CD3 [pan-T] CD4 [Th] CD8 [Tc] For CVID, consider markers to identify memory and class-switched memory B cells (low in more severe disease) CD27, CD19 IgM, IgD
Secondary panel CD5 vs CD19 [CLL] Kappa & lambda [clonality] BTK expression (XLA) CD40, CD4OL
Other markers absent in rare immunodefiencies, including:
ICOS
Iga, Igb
HLA Class I & II
For CVID, consider following to identify memory B cells and type CVID
CD27, CD19
IgM, IgD
How does X-linked Gammaglobulinemia occur and present?What treatment is given?
The prototypic primary antibody deficiency
First described in 1952 by Ogden Bruton (sometimes called Bruton’s agammaglobulinaemia)
Defect is loss of function mutation in BTK (Bruton’s tyrosine kinase)
This is involved in signalling of pre B cell receptor and B cell receptor
Absence causes block in maturation at pro-B cell stage
No mature B cells form
Classically leads to:
Total absence of circulating B cells
Absent immunoglobulins (IgG, IgA, IgM)
clinical manifestation
Presents in male children, with infections usually starting in first year of life (when maternal IgG dwindles, so not at birth)
Recurrent upper and lower respiratory tract infections
Encapsulated bacteria, e.g. Strep pneumoniae, Haemophilus influenzae, Staph aureus, Pseudomonas
Invariably develop bronchiectasis, especially if delayed diagnosis
Other bacterial infections e.g. meningitis, osteomyelitis, septic arthritis
Most viral infections can be cleared, but sensitive to enteroviruses, including polio live vaccine strain
Treatment
Immunoglobulin replacement (subcutaneous or intravenous) is essential life-long
Sometimes prophylactic antibiotics are also required
Goal is to prevent end organ damage particularly bronchiectasis
Severe refractory cases can treated curatively with haematopoietic stem cell transplant (HSCT)
No live vaccines
How Does CVID present and how is it treated?
Relatively common as PIDs go, but still rare (approx. 1 in 25000)
Variable, as in reality it is probably many different genetic defects, with broadly similar phenotype (many identified, many likely yet to be)
Primarily antibody deficiency, particularly low IgG (and IgA)
Can present from childhood (not infancy) until well into adulthood
Diagnosis often delayed many years after first manifestations
Manifestation
Bacterial infections, as with XLA
Autoimmune disease
Enteropathy, Coeliac-like, Malabsorption
Lymphoproliferation
Sarcoid-like granulomatous disease, can affect any organ
Granulomatous lymphocytic interstitial lung disease (GLILD)
Liver disease (nodular regenerative hyperplasia)
Malignancy, especially lymphoma (40 fold increased risk)
Treatment
Immunoglobulin replacement as with XLA
Prophylactic antibiotics
Treatment of complications, e.g. autoimmune disease as appropriate
Some patients with active autoimmune disease or GLILD require immunosuppression, including steroids
Monitoring for malignancy, particularly lymphoma
Small (but increasing) numbers of complex patients treated with HSCT (curative but high mortality risk in adults, especially with comorbidity)
What are the diagnostic criteria for CVID?
At least one of:
Increased susceptibility to infection
Autoimmune manifestations
Granulomatous disease
Unexplained polyclonal lymphoproliferation
Affected family member with Ab deficiency
AND
Marked decreased in IgG and IgA (+/- IgM)
AND at least one of:
Poor vaccine response (or absent isohaemaglutinins)
Low class-switched memory B cells
AND
Secondary causes excluded
AND
Diagnosed after 4th year of life (although symptoms may have begun earlier)
AND
No evidence of a profound T cell deficiency(2 or more of):
CD4 numbers/microliter: 2-6y <300, 6-12y <250, >12y <200
% naive of CD4: 2-6y <25%, 6- 16y <20%, >16y <10%
T cell proliferation absent
What is SpAD and how is it treated?
It is possible to have normal total antibody levels, but fail to produce antibodies to certain types of antigens (particularly polysaccharide)
This can be clinically significant, resulting in recurrent bacterial infections and risk of bronchiectasis
By definition IgG, IgA and IgM levels are normal
Specific bacterial antibodies, particularly to Strep pneumoniae (Pneumococcus) and Tetanus should be checked.
If low, this does not necessarily mean a problem (may not have been vaccinated or significantly exposed
Test vaccination given (e.g. PPV23), and levels repeated at 4-6 weeks
Failure to adequately respond suggests SpAD
Diagnostic criteria also required no evidence of T cell defect (combined immunodeficiency)
Treatment
Prophylactic antibiotics in first instance
Immunoglobulin replacement if ongoing recurrent infection
How does selective IgA present?
Selective IgA deficiency (absent IgA, IgG and IgM normal) is common (approx. 1/800), usually incidental finding (Coeliac screening) and mostly not clinically relevant
In absence of recurrent infection no further investigation needed
Rarely it can be associated with other antibody deficiencies (e.g. specific antibody deficiency) so if recurrent infections warrants investigation
What are the Pathogens seen in combined T and B cell deficiency?
bacterial intracellular (mycobacteria) Salmonella spp fungal Candida spp Aspergillus spp Cryptococcus neoformans protozoal Pneumocystis carinii Toxoplasma gondii Cryptosporidia Viral Respiratory: RSV, Parainfluenzae GI: rotavirus (vaccine strain), norovirus Other: CMV, adenovirus
What can cause SCID?
Severe combined immunodeficiency(SCID,Glanzmann–Rinikersyndrome,alymphocytosis)
Definition: A rare genetic condition caused bynumerous genetic mutations that result in the defective development of functionalB cellsandT cells.
Etiology:various mutations, the most common of which are:
X-linked recessive: mutations in thegeneencoding the commongamma chain →defectiveIL-2Rgamma chain receptor linked to JAK3 (mostcommonSCIDmutation)
Autosomal recessive
Adenosine deaminase(ADA)deficiency→accumulation of toxic metabolites(deoxyadenosine anddATP) and disruptedpurinemetabolism →accumulation ofdATPinhibits the function of ribonucleotidereductase →impaired generation of deoxynucleotides
Janus-associatedkinase3 (JAK3) deficiency
RAGmutation results in faultyVDJ recombination(see “Immunoglobulinproperties”).
How does SCID present?and how is it diagnosed and managed?
Clinical features
Normal atbirth
Severe,recurrent infections: bacterialdiarrhea, chronic candidiasis(thrush), viral andprotozoalinfections
Failure to thrive
Chronic diarrhea
Lymph nodesandtonsilsmay be absent
Diagnosis
QuantitativePCR:↓T-cellreceptor excision circles (TRECs)
Flow cytometry:absentT cells
CXR: absentthymic shadow
Lymph nodebiopsy: absentgerminal centers
Treatment
IVimmunoglobulins0
PCP prophylaxis
Bone marrow transplantorstem cell transplantation
Avoidance oflive vaccines
Prognosis: often fatal in the first year of life if left untreated[ref]
What is Wiskott Aldrich syndrome and how does it present?
Definition:genetic condition characterized byimpaired function ofT cellsandthrombocytopenia
Epidemiology: occursprimarily in males
Etiology:mutatedWASp gene(X-linked recessiveinheritance) →impaired signaling toactincytoskeletonreorganization →defectiveantigenpresentation
Clinical features
Onset of symptoms: frombirth
Classic triad
Purpura(bleeding diathesis)
Eczema(high risk ofatopicdisorders)
Recurrentopportunisticinfectionswith encapsulated organismsin the first years of life(e.g.,otitis media)
Increased risk of autoimmune diseases and hematological malignancies(e.g.,lymphoma,leukemia)
How can wiskott Aldrich syndrome be diagnosed and managed?
Diagnosis
Normal or↓IgGandIgM
↑IgEandIgA
Thrombocytopeniawithsmallplatelets
Genetic analysis (confirmatory test): mutatedWASpgene
Treatment
IV immunoglobulintherapy
Prophylacticantibiotics
Platelet transfusions
Stem cell transplantationmay be curative.
Prognosis:shortened life expectancy
What is Omenn’s Syndrome?
Omenn syndrome is an autosomal recessive form of severe combined immunodeficiency (SCID) characterized by erythroderma (skin redness), desquamation (peeling skin), alopecia (hair loss), chronic diarrhea, failure to thrive, lymphadenopathy (enlarged lymph nodes), eosinophilia, hepatosplenomegaly, and elevated serum IgE levels.[1][2][3] Patients are highly susceptible to infection and develop fungal, bacterial, and viral infections typical of SCID. In this syndrome, the SCID is associated with low IgG, IgA, and IgM and the virtual absence of B cells. There is an elevated number of T cells, but their function is impaired.[1] Omenn syndrome has been found to be caused by mutations in the RAG1 or RAG2 genes.[1][3] Additional causative genes have been identified.[1] Early recognition of this condition is important for genetic counseling and early treatment. If left untreated, Omenn syndrome is fatal. The prognosis may be improved with early diagnosis and treatment with compatible bone marrow or cord blood stem cell transplantation
What are the deficiencies of the innate immunity?
Phagocyte disorders
Reduced numbers
Reduced function
NK cell defects
Cytokine deficiencies
Toll-like receptor defects
Complement deficiency
What are the common phagocyte deficiency?
Neutrophils are the chief phagocyte
Function to isolate, engulf and kill pathogens
Express adhesion receptors and complement receptors to facilitate uptake of opsonised dangerous elements.
Defects in neutrophils (function or number)
Account for nearly 20% PIDs
Include chronic granulomatous disease, Chediak-Higashi syndrome, Griscelli syndrome, leukocyte adhesion deficiency, neutrophil granule deficiency, myeloperoxidase deficiency, cyclic neutropenia, severe congenital neutropenia (Kostmann syndrome) & x-linked neutropenia.
Can be secondary too
Myeloperoxidase deficiency
G6PD deficiency
Both present with similar presentations to CGD but milder forms.
Diagnosis by measuring enzyme levels
Leukocyte adhesion deficiency
Rare
Inability of leukocytes to localise to sites of inflammation due to defective adherence mechanisms.
3 types
Clinical manifestations vary but all have defect in CD18 present on surface of leukocytes
CD18 is the beta chain of 3 integrin receptors – LFA-1, MAC-1 and P15-/95
Complete deficiency of CD18 results in death in early life but those with partial defects benefit from prophylactic and acute antibiotics
BMT may be required
Chediak-Higashi syndrome
Abnormal neutrophil granules and defective killing
Oculocutaneous albinism, neurological symptoms and increased pyogenic infections
Can develop unchecked inflammation
HSCT can cure immunological defects but not neurological symptoms
Griscelli syndrome
Global pigmentary dilution with sivery gray hair
Abnormal management of intracellular granules
Pyogenic infections
HSCT can cure immunologic abnormalities
How does Chronic granulomatous disease occur and present?
Defect in NADPH oxidase complex resulting in inability to produce respiratory burst.
Several components of NADPH complex encoded by different genes
One of these is on x chromosome so can get x-linked and autosomal recessive forms
Failure to kill micro-organism leads to granuloma formation
Key features
Early presentation – usually
Abscess of skin and deep seated infections of lungs, lymph nodes, liver and bones
Catalase positive bacteria – Staphylococcus, Kebsiella, Serratia & Burkholderia
Fungal infections – Aspergillus (historically leading fatal cause of death)
Inflammatory bowel disease
How can Chronic granulomatous disease be diagnosed and managed?
Neutrophil assay Dihydrorhodamine test (DHR): flow cytometry test showing abnormal NADPH oxidase activity (inability to metabolize dihydrorhodamine to fluorescent product, rhodamine → decreased green fluorescence) [21]
Nitroblue tetrazolium dye reduction test: negative, i.e., incubated leukocytes fail to turn blue when exposed to nitroblue tetrazolium
Hypergammaglobulinemia
Anemia
Genotyping is confirmatory
Culture for bacteria and fungi
Evidence of gut inflammation
Evidence of obstruction e.g. urethra secondary to granuloma
Total numbers of neutrophils are normal on FBC
Treatment Prophylactic antibiotics Prophylactic anti-fungals Interferon Gamma Bone marrow transplantation ? Gene therapy
What can cause Neutropenia?
Severe congenital, cyclic or x-linked
Septicaemia, bacterial respiratory infections, soft tissue infections, gingivstomatitis, periodontitis and oral, vaginal and rectal ulcerations
Severity parallels the deficiency
Diagnosis
Neutrophil count on FBC
Persistent or cyclical drop every 21 days
What are the causes of Natural killer defects and how does it present?
Function to control infection through Cytotoxicity Cytokine production Co-stimulation of other cells NK cell deficiencies may be found in association with other immunological conditions e.g. XLP (x-linked lymphoproliferative disorder), Wiskott-Aldrich syndrome, and NF-kB essential modulator (NEMO deficiency) Can be secondary Meds, malignancy or infection
Primary causes
Reduced numbers / absent
GATA2 ( a haematopoietic transcription factor) deficiency
Reduced function / normal number
Can again be secondary (meds, malignancy, infection)
Key features
Herpes virus infection – VZV, CMV, EBV and HSV
Unusual features of HPV – excess malignancy
What are cytokines defects?
Autosomal recessive
Increased susceptibility to mycobacterial disease
Patient with IL-12 deficiency also develop susceptibility to salmonellosis
Diagnosis
Measurement of cytokine levels (specialist tests)
IFN- gamma levels low in IL-12 deficiency
Raised in IFN-gamma receptor deficiency
Treatment
antimycobacterial regimens, prophylaxis, IFN-gamma, HSCT
What are Toll like receptor function defects?
TLRs
= pattern recognition receptors for lots of molecules (self and non-self) that contain danger signals.
Key initiators of innate immune response
Some defects affect a number of TLRs by impairing signalling pathways downstream of the TLR
E.g. IRAK-4 and MyD88 – patients susceptible to pyogenic organisms
Investigations (specialised)
Measure production of cytokines after exposure of PBMC (peripheral blood mononuclear cells) to TLR ligands
What are complement deficiency?
Increased susceptibility to infection
Increased susceptibility to autoimmunity
Hereditary angioedema
What is Secondary Immunodeficiency?
Immune system is compromised due to an extrinsic factor
Rather than an intrinsic genetic defect as seen with primary immunodeficiency (PID)
What is Secondary Immunodeficiency and what’s important to ask in the history?
Increased susceptibility to infections Unexpected severity Unusual infections Main difference from Primary Immunodeficiency (PID) Age of onset External cause More common than PID
Ask Type of infections Duration of symptoms Severity of symptoms and Medication Use Other medical conditions Identify any organisms cultured
What are 10 warning signs of immunodeficiency(both primary and secondary
Four or more infections requiring antibiotics within one year (eg, sinusitis, bronchitis, pneumonia, otitis media, especially with perforation).
Two or more serious sinus infections within 1 year
Two or more months on antibiotics with little effect
Two or more pneumonias within one year
Failure of an infant to gain weight or grow normally
Recurrent, deep skin or organ abscesses
Persistent thrush in mouth or fungal infection on skin
Need for intravenous antibiotics to clear infections
Two or more deep seated infections including septicemia
A family history of primary immunodeficiency.
- How do biological therapies cause immunodeficiency?
Likelihood of clinically significant infection depends
action of drug
treatment duration
Underlying disease process
Patient co morbidity
Concomitant use of other immunosuppresive medications.
Monoclonal Antibodies to B cells Monoclonal Antibodies to T cells Anti Cytokine therapy Agents disrupting T cell Costimulation Agents inhibiting leucocyte Movement Monoclonal Antibodies to Complement Proteins Small Molecule Kinase Inhibitors Checkpoint Inhibitors
What are the common causes of secondary immunodeficiency
Severe Malnutrition Affects 50% of population is some countries Age extremes (young and old) Infections HIV Drugs –most common Immunosuppressives medication Prednisolone Monoclonal Antibodies Anti convulsants
Anatomic Abnormality Diseases causing protein loss Nephrotic syndrome Protein losing enteropathy Haematological Malignancies Surgery and trauma Burns Disruption of epithelial barriers Splenectomy Metabolic Disorders Diabetes Uraemia
Both the underlying disease and the treatment contribute in different ways to SID
CLL
Multiple Myeloma
Non Hodgkins lymphoma
What Investigations can be done and how can secondary immunodeficiency be diagnosed?
FBC and film Immunoglobulins and Electrophoresis Serum Free Light Chains Specific Antibodies Pneumococcal and Tetanus Lymphocyte subsets Urine protein/creatinine Ratio
Treatment
Removal of the causative agent
Consider Antibiotic prophylaxis
In some cases consider replacement immunoglobulin therapy
If untreated
Infections and end-organ damage e.g bronchiectasis
Significant morbidity and mortality.
What is flow cytometry?
The Flow Cytometry laboratory uses the flow cytometer to detect different populations of blood cells in the patient samples. The laboratory also provides a host of functional immunological testing as well as panels for haematological malignancies. In some hospitals it helps to assess and prepare samples for bone marrow transplants. A few common tests are mentioned below. If you are interested, we would encourage a visit to the Flow laboratory to see the full range of testing available.
How are Lymphocytic subsets interpreted?
Lymphocyte subsets are a panel of cells counted using a flow cytometer using labelled fluorochromes that emit light at different wavelengths of light.
The cells are first separated by their size and granularity as above, Lymphocytes, seen on the plot above, are then separated based on their attachment to specific fluorochromes labelled antibodies that bind to varies clusters of differentiation belonging to each cell population (e.g. CD3 for T cells). Each population is expressed as a percentage of all cells in the sample. A set number of beads are also added which can be expressed as a % of all cells counted. As the number of beads added is known, this is used to count the actual number of each cell population in the sample.
The basic panel includes:
- Numbers of total lymphocytes
- T cells (CD3+)
- B cells (CD19/20+)
- NK cells (CD16/56)
- CD4+ (T helper cells) and
- CD 8+ (T cytotoxic cells).
Lymphopaenia of different patterns is seen in both primary and secondary immunodeficiency (discussed in other resources). It can also be due to losses from gut, kidneys or thoracic duct drainage. Severe combined immunodeficiency for example is almost always seen with low T cells, with or without the involvement of B cell and NK cell lymphopaenia. CD4+ cells can be low with infection, malignancy or due to drugs. Absent B cells may be seen in immunodeficiency such as secondary agammaglobulinaemia. Lymphocytosis can be seen after viral infections or due to leukaemia and lymphoma. It is important to note that lymphocyte subsets are very tricky to interpret in the middle of an acute infection as any high or low counts could be due to ongoing inflammation, rather than the true baseline for the patient.
What other functional tests can be used?
Neutrophil Oxidative Burst:
This is a functional test that looks at the neutrophils ability to phagocytose and kill microbes by testing the activity of NADPH oxidase. Defective neutrophil function should be investigated in patients suspected of having Chronic Granulomatous Disease. CGD is characterised by repeated and life-threatening infections caused by bacterial and fungal organisms. It may mimic inflammatory bowel disease and lead to malabsorption and obstruction of the bowel. Abscesses involving the liver, lungs or lymph nodes are often found.
The cells are stimulated to activation and then a dye is used to see the “oxidative burst”. If this is abnormal, further tests are then considered to look for the specific cause of the defect.
T cell proliferation Assays
Upon interaction with microbes, T cells rapidly proliferate to propagate the adaptive immune response. Defects in these pathways can cause immunodeficiency. These assays look for defects in T cell proliferation by subjecting cells to various stimuli, mimicking different parts of the activation pathways and then incubating them for 72 hours. The cells are then counted and compared with a normal control to ascertain where in the pathway a defect might be. This assay is used for diagnosis as well as monitoring of patients post bone marrow transplant.
NK Cell Granule Release Assay
Defects in the cellular toxicity of NK cells and cytotoxic T cells are a feature in Haemophagocytic Lymphohistiocytosis (HLH). HLH is a life threatening immune disorder of severe hyper-inflammation caused by uncontrolled proliferation of activated lymphocytes and histiocytes. This assay is used to analyse resting and activated NK cell degranulation, looking for the products of degranulation after artificially stimulating NK cells.
What are protein assays?
The proteins laboratory offers assays to look at immunoglobulin levels, serum electrophoresis and subsequent immunotyping/immunofixation of any paraproteins found. It also assays serum light chain, complement activity (classical and alternative pathway) and other tests for complement components. A few common tests are mentioned below. If you are interested, we would encourage a visit to the Proteins laboratory to see the full range of testing available
How can Immunoglobulin levels be interpreted?
An immunoglobulin panel consists of IgG, IgM and IgA. IgM, produced first, is often the first antibody to be lost in immunodeficiency. Isolated low IgM can be seen in lymphoproliferative diseases. High polyclonal IgM can be seen in liver diseases and other inflammatory conditions. Raised polyclonal IgA can often be seen in the elderly, but can also be raised due to chronic infection and inflammation. Polyclonally raised IgG can be seen in a reactive state as the other two, but very high levels of polyclonal IgG can be seen in Sjogren’s syndrome and HIV.
Immunoglobulin levels cannot reveal if the rise is due to monoclonal or polyclonal increase. Therefore it is always advised to interpret these with serum electrophoresis (+/- serum free light chains), which can detect the presence of a paraprotein.
In some cases, IgE is also measured. It can be >50,000 (reference range <100) in patients with Hyper IgE syndrome.
What is serum electrophoresis?
An immunoglobulin panel consists of IgG, IgM and IgA. IgM, produced first, is often the first antibody to be lost in immunodeficiency. Isolated low IgM can be seen in lymphoproliferative diseases. High polyclonal IgM can be seen in liver diseases and other inflammatory conditions. Raised polyclonal IgA can often be seen in the elderly, but can also be raised due to chronic infection and inflammation. Polyclonally raised IgG can be seen in a reactive state as the other two, but very high levels of polyclonal IgG can be seen in Sjogren’s syndrome and HIV.
Immunoglobulin levels cannot reveal if the rise is due to monoclonal or polyclonal increase. Therefore it is always advised to interpret these with serum electrophoresis (+/- serum free light chains), which can detect the presence of a paraprotein.
In some cases, IgE is also measured. It can be >50,000 (reference range <100) in patients with Hyper IgE syndrome.
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.
Check table in notion.
Immunofixation
Immunofixation is a process where multiple samples of the patient sample are run on gel electrophoresis, which each sample stained for a particular heavy or light chain. 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. In the examples below we see some paraproteins:
One band in the same location in IgG and lambda regions showing an IgG lambda paraprotein. Serum free light chains if measured in this case may show an increase in lambda chains.
Two bands seen in the same location in the IgG and kappa regions, showing two separate IgG kappa paraproteins. Serum free light chains if measured in this case may show an increase in kappa chains.
In the resources section below you will find The Newcastle upon Tyne Hospitals NHS Foundation Trust guidelines for the interpretation and management of immunoglobulins and paraproteins in adults.
