20 Cellular investigations

Flow cytometry

Tissue culture

Proliferation assays

Immunohistology

Cytokine, chemokine, soluble protein assays

Apoptosis assays

Adhesion markers

Bronchoalveolar lavage (BAL) studies

CD40 ligand expression

Complement membrane regulatory factors

Cytokine and cytokine receptor measurement

Cytotoxic T cells

FOXP3 (regulatory T cells—IPEX syndrome)

Genetic and protein studies

Leukaemia phenotyping

Lymphocyte subsets

B-cell function: in vivo assays

B-cell function: in vitro assays

T-cell function: in vivo assays

T-cell function: in vitro assays

Lymphoma diagnosis

Neutrophil function testing

NK-cell function

NK granule release

Perforin expression

Toll-like receptors (TLRs)

TRECs

T-cell receptor and immunoglobulin heavy-chain gene rearrangements

Techniques used include:

Few functional assays are standardized and gold standard assays have not been defined.

EQA exists only for basic lymphocyte phenotyping and then only in the context of testing for HIV.

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 semi-automated 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.

This is done by density gradient centrifugation, using Ficoll, metrizamide, or dextran solutions.

Further purification of lymphocyte populations can be undertaken using:

The more cells are handled in vitro, the more their characteristics are altered.

Cell culture is usually carried out in tissue-culture medium supplemented with:

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.

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):

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).

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.

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.

Samples must be run fresh.

Microscopic confirmation of assay results is advised to exclude artefacts.

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.

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.

Normal adult values for smokers:

Subsets of lymphocytes can be analysed by flow cytometry.

Sarcoidosis.

Hypersensitivity pneumonitis.

Idiopathic pulmonary fibrosis (IPF).

Eosinophilic granuloma.

Connective tissue diseases.

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.

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.

CD69 expression is used as an activation control.

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.

Functional assays of cell lysis (Ham’s test) have been withdrawn.

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:

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.

Intracellular cytokines have been used to identify functional Th1/Th2 balance.

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:

Cytokine and cytokine receptor deficiencies are exceptionally rare (see Chapter 1).

Flow cytometric tests for intracellular cytokine detection are available.

This is a complex and fiddly assay, and has been used mainly as part of the cross-matching procedure (see Chapter 21).

This requires a permeabilization step on separated lymphocytes.

Treg are FOXP3⁺ CD4⁺ CD25^bright and CD127^weak (CD127 is IL-7 receptor-A).

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.

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.

Morphology on peripheral blood and bone marrow.

Enzymatic studies.

Molecular studies:

Follow-up panels may be required (see next section).

Leukaemic cells often correspond to particular stages of cellular differentiation, which can be matched to normal cell ontogeny.

Aberrant antigens, expressed out of sequence, may occur.

This may give rise to biphenotypic leukaemias.

Bone marrow studies are complex because of the very different light-scattering properties of the cellular constituents.

Familiarity with the patterns of antigen expression at each stage of differentiation for each lineage is required.

Re-examination of bone marrow after treatment is important to detect the presence of minimal residual disease. This can be done using the following techniques.

Flow cytometric karyotyping is now possible as an alternative to molecular techniques.

T lineage:

Lymphoblast:

AML lineage:

Erythroid:

Megakaryocyte:

T lineage:

AML:

B lineage, secondary:

T lineage, primary:

T lineage, secondary:

Direct conjugation of the fluorochrome to the antibody is preferred.

Beads are used to calibrated absolute counts.

Results should be reported as absolute counts: percentages and ratios are not helpful for the basic markers but may be useful for extended panels (see next section).

Robust EQA schemes exist for common markers.

Monitoring immunotherapeutic agents (anti-T-cell antibodies, cytotoxic drugs).

Some of the fluorochromes are large molecules, and multiple staining of markers on cells may give rise to steric hindrance and reduced binding.

Correct set-up of compensation for the flow cytometer is essential. This must be checked regularly and especially after servicing.

Regular quality control checks should be carried out with commercial fluorochrome coupled beads.

A basic panel for primary immunodeficiency work should include the following.

Additional markers may include the following.

Lymphocyte numbers have a marked circadian rhythm. In serial monitoring, samples must be taken at the same time of day.

In a baby with suspected SCID, the presence of mainly activated CD8⁺ T cells raises suspicion of materno-fetal engraftment, while the presence of activated CD4⁺ T cells, in the presence of large numbers of eosinophils, is suggestive of Omenn’s syndrome.

Absence of CD8⁺ T cells is a feature of ZAP-70 kinase deficiency.

Abnormalities of T- and B-cell populations are also seen in CVID, with CD4⁺ T-cell lymphopenia, affecting particularly CD45RA⁺ T cells, and absence of class switch memory B cells.

After HSCT, high levels of activated T cells often indicate GvHD.

Very low CD4⁺ T-cell counts are not a diagnostic feature of HIV disease.

Persistent CD4⁺ T-cell lymphopenia has also been reported as a cause of opportunistic infections in the absence of any evidence for infection with either HIV-1 or HIV-2 (idiopathic CD4⁺ T-cell lymphopenia).

Rare deficiency of the binding site for the anti-CD4 mAb OKT3 is recognized, giving spuriously low CD4 counts. This variant CD4 appears functionally normal.

Abnormal lymphocyte profiles are also seen in:

Generalized proportionate reductions in lymphocyte counts are seen with long-term immunosuppressive therapy.

CD4/CD8 double-negative Tcr γδ T cells are increased in ALPS (see Chapter 1).

Antibody levels should be measured to exposure and immunization antigens.

Testing should include protein and polysaccharide antigens (see Table 20.1).

Subclass-specific responses can be measured to some antigens.

Serotype-specific responses can be measured to pneumococcal polysaccharides (not all serotypes are equally immunogenic).

Isohaemagglutinins, in appropriate blood group patients allow detection of IgM responses.

In the USA, the bacteriophage Φ'174 is used as an immunogen. This neoantigen permits detection of primary and secondary antibody responses.

Antibodies will normally be detected by EIA on serum, or by complement-fixation assays (viral antibodies).

ELISPOT assays allow the detection of specific antibody-producing B cells.

EQA schemes exist for viral and bacterial antibodies.

Dynamic responses after immunization give a better view of B-cell function.

Target should be a rise into the normal/protective range with a minimum of a fourfold rise in titre.

Assays for bacterial antibodies are poor with CVs of 15–25%. Pre- and post-immunization samples should be run on the same assay.

Assays must be interpreted with caution.

Only killed or subunit vaccines should be given to patients with suspected immunodeficiency.

Role of subclass- and serotype-specific assays is uncertain at present. Multiplex assays may be valuable for rapid screening of responses to multiple serotypes.

Move to conjugated polysaccharide vaccines may lead to loss of pure polysaccharides for test immunization.

IgG, IgA, and IgM production can be measured at 7 days by sensitive ELISA of the supernatant.

Testing is time-consuming.

Flow cytometric detection of class-switch memory B cells is quicker and easier than using anti-IgM + IL-2 system to identify prognostically important subgroups of common variable immunodeficiency (Chapter 1).

Antigens are pricked through the skin (Merieux Multitest CMI^®) or injected intradermally.

Most useful antigens include PPD, Candida, mumps, tetanus, and streptokinase/streptodornase, which are available (some with difficulty) as single antigens, or are part of the battery in the Multitest.

At 72–96 hours, in a positive reaction, there will be a cellular infiltrate that is palpable, with overlying erythema.

Reactivity to the panel is low in early childhood and increases with age.

Poor responses are seen in:

Mononuclear cells are separated from neutrophils by density gradient centrifugation (Ficoll^®).

Proliferation of the T cells is measured by the incorporation into DNA of tritiated thymidine in replicating cells.

Other methods used to study T-cell function in vitro include flow cytometric tests for the following.

There is little value in the MLR as a test of T-cell function, although it forms a part of cross-matching bone marrow.

ELISPOT assays can be used to measure cytokine production in response to antigens and will also give a precursor frequency.

Functional assays are rarely required in secondary T-cell immunodeficiency.

Monitoring of the T-cell proliferative response after HSCT provides a useful marker of returning function that will determine safe release from laminar flow.

For PHA-stimulated cells the uptake should be >5000cpm and the increment over the unstimulated cells should be >4000cpm. The stimulation index should be >10.

For antigens such as Candida, the response is smaller and an increment of 2000cpm and a stimulation index of 3.0 are satisfactory.

The requesting clinician must also arrange a control sample from a healthy volunteer, where possible of the same age/sex as the patient.

Each laboratory should establish its own age- and sex-specific normal ranges for each mitogen.

The most useful stimuli are the following.

Single-cell suspensions produced by disaggregating the tissue can be used.

Most information is gained from looking at tissue sections.

Staining is usually done with monoclonal antibodies followed by anti-mouse antibody conjugated to a reagent for developing a colour reaction (peroxidase, alkaline phosphatase–anti-alkaline phosphatase, etc.).

Most studies can now be carried out on paraffin sections.

Immunophenotyping will normally be undertaken in parallel with morphological, virological, and enzymatic studies.

The primary panel usually includes:

The secondary panel for T-cell antigens includes:

The secondary panel for B-lineage antigens includes CD10, CD21, CD22, CD23, CD24, CD79a, and CD5 (also expressed on T cells).

Confirmation of the presence of Reed–Sternberg cells can be obtained by using CD30 and CD15.

Histiocytes are reactive with CD68.

LMP-1 is a surface marker of EBV⁺ cells and is expressed on Reed–Sternberg cells.

In situ hybridization can be used to detect oncogene expression and viral genes.

Initial and supplementary antibody panels will be used.

Extracted material can be used as a source of DNA for molecular studies of T-cell receptor and Ig heavy-chain gene rearrangements as markers of clonality.

EQA systems exist.

Lymphomas often express aberrant patterns of surface and cellular antigens:

Lymphoid tumours need to be distinguished from other (metastatic) malignancy:

Hodgkin’s disease is distinguished by the presence of characteristic Reed–Sternberg cells:

Evidence of clonality can now be obtained by studies of Ig and Tcr gene rearrangements by molecular techniques:

Abnormal expression of oncogenes and tumour suppressor genes can be detected by in situ hybridization. Oncogenes routinely screened for include:

Viral screens include EBV, CMV, HHV6, HHV8.

The classification of lymphomas is constantly being revised in the light of new findings. Readers are advised to consult an up-to-date detailed text to understand the process.

Screening test for defects of oxidative metabolism (CGD) is nitroblue tetrazolium reduction test, in which a colourless intracellular dye is reduced to an insoluble blue compound, formazan, when the neutrophil’s oxidative machinery is activated.

Flow cytometry using dye reduction (dihydrorhodamine (DHR)) allows more cells to be analysed more quickly.

Other tests of the oxidative machinery include chemiluminescence (amplified by luminol) and the iodination test, which relates to hydrogen peroxide production.

Phagocytosis can be measured by simply counting the number of latex beads or yeasts ingested by neutrophils or, more accurately, by flow cytometry using labelled bacteria.

Bacterial killing assays allow the whole process to be tested, including opsonization, phagocytosis, and oxidative metabolism.

Chemotaxis assays are usually carried out by measuring migration under agarose or by the Boyden chamber method, in which cells migrate into a microporous filter which is examined under a microscope with a vernier gauge on the focusing ring, allowing the distance travelled to the leading edge to be measured.

Monocytes can be studied on flow cytometers at the same time as neutrophils.

No EQA schemes exist. Therefore laboratories must establish their own normal ranges and set up normal controls in parallel with patient samples for all assays.

With sensitive flow cytometric assays, heterozygotes for CGD mutations may have half the normal activity.

Bacterial killing assays may be abnormal in healthy children under the age of 2 years.

Chemotaxis is an important part of the process and rare defects due to the lack of anaphylotoxin receptors have been reported.

Neutrophil function testing should always include testing for adhesion molecule deficiency (see ‘Adhesion markers’, p.559) and for neutrophil enzymes, especially myeloperoxidase (a common deficiency of doubtful significance), G6PD, and alkaline phosphatase (reduced in specific granule deficiency).

Neutrophil assays must be done with fresh samples.

Any intercurrent infection will cause abnormal function.

Follow-up protein studies and genetic investigations are required where defects of oxidative metabolism are identified in screening tests.

Erythroleukaemia cell line K562 is known to be susceptible to lysis by NK cells.

Assay is carried out by incubating mononuclear cells with labelled K562 cells at varying effector target cell ratios and then identifying the death of the targets.

The assays can be modified using different targets to look at antibody-dependent cell-mediated cytotoxicity (ADCC) and lymphokine-activated killer (LAK) activity.

No EQA exists. Laboratories must establish normal ranges and run normal controls with each assay.

The test depends on availability of high-quality K562 cells. Long-term cell culture must be meticulous.

NK-cell deficiency has been reported (rarely) causing severe infections with herpesviruses.

Routine screening of patients with simple cold sores is not justified.

NK-cell function may be relevant in graft rejection and assessment of rare NK-cell leukaemias.

Chromium-release assay depends on the complete disintegration of the cell.

Clinical diagnostic value of the NK assay remains to be fully evaluated. Routine evaluation of ADCC and LAK activity is not undertaken.

Excessive NK-cell activity has been associated with an increased risk of graft loss in mismatched bone marrow transplants (particularly host NK-cell activity).

Low/absent NK activity has been reported in rare patients with recurrent infections with herpes family viruses.

Very high activity may be found in NK-cell leukaemias.

Number of NK cells identified by flow cytometry does not necessarily correlate with the activity.

K562 cells are used as a target for NK cells to stimulate degranulation, as they do not express MHC Class I antigens.

Expression of CD107a on NK cells (CD3⁺ CD56⁺) is detected by flow cytometry after incubation of separated PBMNC with K562 cells.

PHA is also used as a non-specific activator of degranulation.

Patients with perforin deficiency will have normal granule release.

A normal control is required with each run.

Very few NK cells should express CD107a unstimulated; >7% should express it when stimulated.

Assay depends on ready access to high-quality K562 cells.

Perforin is expressed in the granules of NK cells, some CD8⁺ T cells, CD56⁺ T cells, and δ T cells.

XLPS should be excluded in males.

All 10 TLRs identified signal via MyD88 and IRAK-4 (clinical deficiencies of both are described (see Chapter 1)). Four TLRs signal via UNC-93B (TLR3, TLR7, TLR8, TLR9).

Activation of neutrophils via TLRs will lead to loss of CD62L.

Lipopolysaccharide (LPS) is the ligand for TLR4; CL097 (an imidazoquinoline) is a ligand for TLR7/8. PMA is used as a positive control (bypasses TLRs to activate neutrophils).

UNC-93B-deficient patients will shed CD62L normally with PMA and LPS but not with CL097.

As TRECs do not replicate during cell division, progressive dilution occurs post-emigration.

Assays are not yet widely available for routine use.

TRECs can be detected on magnetic-bead-separated T-cell subpopulations by quantitative PCR techniques.

Panels of Tcr Vβ-specific labelled monoclonal antibodies can be used to carry out testing by flow cytometry.

This enables monoclonal expansions in the T-cell repertoire (lymphoma, response to chronic infection) and selective clonal loss (e.g. Omenn’s syndrome, DiGeorge syndrome) to be identified.

Spectra type of Tcr Vβ and IgH gene usage can be constructed to demonstrate polyclonal, oligoclonal, and monoclonal gene usage, using fluorescent PCR products.