Cellular Investigations
Introduction
Cellular investigations include:
identification of cell-surface phenotype
identification of intracellular proteins
identification of cellular function, including activation
identification of secreted products (cytokines, chemokines)
identification of abnormal cellular constituents (leukaemia/lymphoma).
Techniques used include:
flow cytometry
tissue culture
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genetic techniques (see Chapter 21), mainly PCR-based techniques.
Few functional assays are standardized and gold standard assays have not been defined.
Flow cytometry
Flow cytometry provides the cornerstone of diagnostic cellular immunology and depends on the availability of monoclonal antibody reagents that react with human surface and intracellular antigens. Fluorescent intercalating dyes can be used to detect DNA semi-quantitatively (for cell cycle analysis).
The technique involves the flow of fluorescent-labelled cells past the exciting laser and subsequent detectors.
Method is only applicable to single-cell suspensions, e.g. blood-derived cells or cultured cells.
It is possible to use disaggregated solid tissues, such as tumours.
Modern flow cytometers use a single exciting laser (monochromatic light) and can detect multiple different wavelengths of light emitted by fluorescent dyes.
There are detectors for forward and 90° light scatter, which are related to cell size and cell granularity, respectively.
Software permits complex multiparameter gating and analysis, including real-time data collection and analysis.
Fluorescent-conjugated monoclonal antibodies are used against surface antigens.
Cell permeabilization techniques are available to enable staining of intracellular antigens.
Surface and intracellular stains can be combined.
Appropriate controls are required to detect non-specific staining.
The major advantage of flow cytometry for analysis is that it is semiautomated and can analyse very large numbers of cells very rapidly compared with fluorescence microscopy. It is much more accurate.
Accurate absolute counts are available on single-platform analysers using bead technology. This obviates the errors from using haematology counter total lymphocyte counts.
Regular calibration of the instrument is required, and it is essential that compensation between the fluorescence detectors is correctly set up. This is done with beads of known fluorescence.
Tissue culture
In vitro functional studies of cells may require purified cells (blood, other fluids).
This is done by density gradient centrifugation, using Ficoll, metrizamide, or dextran solutions.
The different buoyant densities of blood cells permit separation when blood is centrifuged through the dense medium.
Further purification of lymphocyte populations can be undertaken using:
rosetting with sheep red cells
magnetic separation using monoclonal antibodies coupled to magnetic microspheres.
The more cells are handled in vitro, the more their characteristics are altered.
This affects activation parameters in particular.
Cell culture is usually carried out in tissue-culture medium supplemented with:
antibiotics to prevent contamination with bacteria
fetal calf serum (FCS) or human AB serum (no isoagglutinins)
other ‘black-box’ factors that are required for optimal cell growth (glutamine is added as this is labile).
Where proliferation assays are being carried out, it is essential to screen the FCS first, as some batches are mitogenic in their own right.
Good sterile technique is essential.
Culture is carried out in a 37°C humidified incubator, with a controlled atmosphere (usually 5% CO2) to maintain pH.
Many different types of tissue culture media are available. Most contain pH buffers (bicarbonate) and pH indicators.
Proliferation assays
There are numerous mitogenic stimuli that can be used (see ‘T-cell function: in vitro assays’, p.570).
These are added at the initiation of the culture.
Cells are pulsed with tritiated thymidine, which is taken up into newly synthesized DNA in dividing cells: this remains the gold standard.
Cells are then harvested onto filter papers and exposed to scintillant fluid.
Counts per minute are determined using a beta-counter.
Alternative assays have been described using flow cytometers (no radio-isotopes):
CD69 expression
DNA analysis with intercalating dyes (cell cycle analysis).
These do not give comparable results to those from tritiated thymidine uptake and appear less sensitive.
Immunohistology
Immunoperoxidase and other enzymatic immunostains are used in the diagnosis of lymph-node disease.
Multiple monoclonal antibodies which recognize different stages of lymphoid development or particular subsets of cells are used.
Many of the antibodies used will also work on paraffin-embedded sections, but this depends on whether the target antigen is stable under the conditions of fixation. Frozen sections are better at present.
In situ hybridization is used to detect viral nucleic acid (EBV, other herpesviruses).
Cytokine, chemokine, soluble protein assays
Detection of specific cellular products, such as antibodies, cytokines, and shed surface molecules (soluble CD8 etc.) are usually undertaken using EIA or RIA techniques, as described in Chapter 18.
Cytokines may also be detected by bioassays using cell lines whose growth depends on a given cytokine.
Strictly, both types of assay should be used, as EIA techniques may give spurious results due to naturally occurring cytokine-binding proteins in serum (soluble receptors, binding factors).
Bioassays are notoriously difficult to standardize and reproduce, and are not suited to routine diagnostic use.
Detection of intracellular cytokines with fluorescently labelled monoclonal antibodies in permeabilized cells has been used in conjunction with surface staining.
This does not indicate that the cytokines are secreted and therefore does not equate to functional assays of cytokines.
Apoptosis assays
Principles of testing
Flow cytometric assays exist for identification of degraded DNA in apoptotic cells.
Preferred method is the TUNEL method, using fluorescent nucleotides enzyme-inserted into DNA strand breaks present in apoptotic cells. Commercial assays are available.
Fluorochrome-conjugated annexin V can be used to detect surface phosphatidylserine which is exposed on the cell surface in apoptotic cells, but not in normal cells.
Expression of fas and fas-ligand by flow cytometry.
A functional assay is available using PHA+IL-2 stimulated T cells. These express high levels of Fas and can be induced to apoptose by the addition of Fas-ligand. Co-staining with annexin V (apoptotic cells) and propidium iodide (identifies dead cells) allows the response of the patient’s cells to be compared with a normal control. To improve the reliability of the assay, multiple dilutions of fas ligand are used.
Protein and molecular follow-up tests are required to confirm defects: few PID centres have the capacity to run the necessary assays.
Interpretation
Careful use of controls is required.
Samples must be run fresh.
Microscopic confirmation of assay results is advised to exclude artefacts.
Adhesion markers
Principles of testing
Flow cytometry.
Analysis should be carried out with CD18, CD11a (LFA-1), CD11b (Mac-1, CR3), and CD11c (CR4) for LAD-1, and CD15 for LAD-2. Neutrophils and lymphocytes should be tested.
Stimulation studies for upregulation in the presence of PMA or γ-IFN may be required where there is partial expression of CD18.
Interpretation
LAD-1 is associated normally with deficiency of CD18, the common β-chain for the integrins, which leads to absence of CD11a, CD11b, and CD11c, as well as CD18.
Absence of β-chains has been reported but is extremely rare.
LAD-2 is exceptionally rare and is associated with deficiency of the hapten-X receptor on neutrophils (CD15).
Under certain circumstances it may be appropriate to look at the expression of the other complement receptors: CR1 (expressed on red cells, eosinophils, and B cells) and CR2 (CD21, EBV receptor expressed on B cells, NK cells, and follicular dendritic cells).
Reduction of red cell CR1 has been found in SLE.
Some patients with CVID may lack CD21 on some of their B cells.
Bronchoalveolar lavage (BAL) studies
Normal adult values for non-smokers:
total cells, 130-180×103/mL
macrophages, 80-95%
lymphocytes, <15%
neutrophils, <3%
eosinophils, <0.5%.
Normal adult values for smokers:
total cells, 300-500×103/mL
macrophages, 85-98%
lymphocytes, <10%
neutrophils, <5%
eosinophils, <3%.
Principles of testing
Cells recovered from bronchi by saline lavage during bronchoscopy can be stained and counted using neat BAL fluid. Total count and percentage differential counts are required.
Subsets of lymphocytes can be analysed by flow cytometry.
Indications for testing
Unexplained interstitial lung disease.
Sarcoidosis.
Hypersensitivity pneumonitis.
Idiopathic pulmonary fibrosis (IPF).
Eosinophilic granuloma.
Connective tissue diseases.
Interpretation
In sarcoidosis, there is a marked increase in lymphocytes (to about 30% of the total cells), predominantly CD4+ T cells, giving a CD4:CD8 ratio (which is normally 2:1) of between 4:1 and 10:1.
Values improve with treatment, but the levels and the ratio do not predict the severity of the disease.
Occasionally there is an increase in neutrophils and mast cells, which is said to indicate a poorer prognosis.
In hypersensitivity pneumonitis, the BAL lymphocytosis comprises mainly CD8+ cells, with the highest levels occurring in the acutely exposed.
In IPF, a neutrophilia in excess of 10%, particularly if there is an increase in eosinophils, is associated with a poor prognosis.
A lymphocytosis (a rare finding) is associated with a better prognosis and indicates a probable response to steroids.
In eosinophilic granuloma (histiocytosis X), there is an increase in OKT6-positive (S-100, CD1+) histiocytic cells, up to 20% of total cells, which is diagnostic.
CD40 ligand expression
Principles of testing
Flow cytometry is used to demonstrate upregulation of expression of CD40-ligand (CD154) upon stimulation of T cells in vitro with mitogens (PMA).
CD69 expression is used as an activation control.
Indications for testing
Suspected CD40-ligand deficiency.
Interpretation
Gating stimulated cells can be difficult because of clumping and size changes: this makes the activation control important.
Variants of CD40-ligand deficiency have been identified in which there is normal upregulation of non-functional ligand. Normal results do not exclude the diagnosis.
Failure of upregulation is highly suggestive of CD40-ligand deficiency.
Abnormal results should be followed up with genetic testing for mutations in the CD40-ligand gene.
Complement membrane regulatory factors
Principles of testing
Flow cytometry is now used exclusively.
Functional assays of cell lysis (Ham’s test) have been withdrawn.
Indications for testing
Suspected paroxysmal nocturnal haemoglobinuria.
Interpretation
Deficiencies of a group of surface proteins with an unusual glycosylphosphatidylinositol membrane binding are associated with paroxysmal nocturnal haemoglobinuria (PNH).
This is a clonal disorder leading to unusual susceptibility to homologous complement lysis, particularly of red cells.
The proteins in question are regulatory proteins which prevent destruction of cells by homologous complement and include:
decay accelerating factor (DAF, CD55)
homologous restriction factor-20 (HRF20, CD59)
C8-binding protein (HRF65)
acetylcholinesterase.
Cytokine and cytokine receptor measurement
Principles of testing
Enzyme immunoassay (serum, cell culture supernatant).
Bioassay.
Flow cytometry for intracellular cytokines; surface staining for receptors.
Immunoblotting.
In vitro stimulation assays with mycobacterial and salmonella antigens may be required to demonstrate defects.
Elispot assays can identify specific cytokine production in response to stimulation.
Indications for testing
Only absolute indication is suspected cytokine/cytokine receptor deficiency (e.g. IL-12, γ-IFN receptor deficiencies).
Interpretation
EIA assays are unreliable because of the presence of natural cytokinebinding proteins and soluble receptors.
Bioassays are difficult to standardize, time-consuming, and unsuitable for routine diagnostic use.
IL-6 rises very early in acute-phase responses, before a rise in the CRP can be detected. However:
CRP is readily available and an is acceptable surrogate for IL-6
CRP levels are raised in myeloma, reflecting elevated IL-6
CRP levels are also raised in Castleman’s syndrome, reflecting raised IL-6.
Cytokine and cytokine receptor deficiencies are exceptionally rare (see Chapter 1).
Flow cytometric tests for intracellular cytokine detection are available.
Technique works well for IL-2 and γ-IFN but poorly for IL-4.
It has the significant advantage that specific T-cell subpopulations can be studied using multicolour flow cytometry.
Cytotoxic T cells
Cytotoxic T cells can be generated during a one-way mixed lymphocyte reaction (sMLR), stimulating the responding cells with irradiated or mitomycin-treated allogeneic target cells and then assessing the ability of the responders to kill Cr51-labelled targets, in a similar assay to the NK-cell assay (see ‘NK-cell function’, p.575).
This is a complex and fiddly assay, and has been used mainly as part of the cross-matching procedure (see Chapter 21).
FOXP3 (regulatory T cells—IPEX syndrome)
Principles of testing
Flow cytometric test to detect the presence of regulatory T cells (Treg) by intracellular detection of FOXP3.
This requires a permeabilization step on separated lymphocytes.
Treg are FOXP3+ CD4+ CD25bright and CD127weak (CD127 is IL-7 receptor-α).
Interpretation
Assays that involve permeabilization of separated lymphocytes are intrinsically more prone to technical problems.
The assay must be run on fresh samples with a normal control.
This is a screening, not a quantitative, assay and will also pick up non-functional FOXP3—essential to do follow-up genetic analysis.
Genetic and protein studies
Protein and genetic studies are essential for the identification of gene defects in primary immunodeficiencies.
Surface proteins and some intracellular proteins, relevant to the diagnosis of primary immune deficiencies, can be identified by flow cytometry.
Protein studies, including surface and intracellular protein detection, are frequently used as a screening test prior to genetic testing, e.g. for:
Abnormal protein expression should be followed up by molecular mutation analysis.
Molecular analysis is also required where there is a high degree of clinical suspicion but apparently normal protein expression (expression of non-functional protein).
Family studies are valuable to identify asymptomatic carriers, who can then receive appropriate counselling.
Leukaemia phenotyping
Leukaemia phenotyping is undertaken to identify the origin of the malignant cell and the presence or absence of markers that are known to be of prognostic significance. This will always be undertaken in conjunction with other studies, including examinations of blood films, bone marrow smears, and trephines stained for enzymatic cytoplasmic and membrane markers.
Principles of testing
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