Laboratory methods for the detection of cellular immunity

Dr.M.MalathiFinal year postgraduate

Department of MicrobiologyChengalpattu Medical College

Chengalpattu

Introduction

• Immune system – Humoral immunity and Cellular immunity

• Cellular immunity – T cell function

• The most consistent and reproducible of the methods for evaluating cellular immunity employ immunochemical means for detecting cellular antigens or markers.

Methods employed

• Assays for leukocyte phenotyping

• Delayed type hypersensitivity skin testing

• Lymphocyte activation assays

• Assessment of monocyte – macrophage function • Determination of granulocyte function

LEUKOCYTE PHENOTYPING• Number and types of cell surface molecules

(markers)• Quantify B cells, T cells, monocytes and

granulocytes• Enumeration of subsets of these cells• By means of monoclonal antibodies (mAbs)• The mAbs are conjugated with either

fluorescent dyes or enzymes and then they are used to stain leukocytes in tissue sections or in fresh cell suspensions.

Flow cytometry

The detection of leukocyte antigens is by FLOW CYTOMETRY

• Most widely used method for detecting the binding of mAbs to leukocyte surfaces is flow cytometry.

• An instrument capable of analyzing single cells as they pass through an orifice at high velocity.

• Measures the properties of light scattering by the cells.

• Detection by the emission of light from flourescently labelled mAb bound to the surface of the cell

• Light scattering property – related to size and intracellular content or complexity

• Forward light scattering – Size

• Side light scattering – intracellular complexity

Sample preparation

• Samples accepted – Bonemarrow samples, lymph node biopsies, tissue from lymphoid malignancies, peripheral blood

• Density gradient centrifugation on Ficoll hypaque

• Whole blood lysis method

Data collection and analysis

• Four pieces of data for every cell that passes through the laser:

1. Forward light scatter2. Side light scatter3. Green fluorescence4. Red/Orange fluorescence

X – Y plot of data

Results

• Rapid, accurate and highly reproducible separation of cells based on their differential staining with mAbs.

• Eg: CD4 count estimation:% of CD4 lymphocytes × Total lymphocytic count

= Total no of CD4 count

Applications of Flow cytometry

• Leukocyte phenotyping1. Diagnosis of congenital immunodeficiency

diseases2. Assessment of prognosis of HIV positive patients3. Monitoring of immune reconstitution in bone

marrow transplant recipients4. Monitoring of immunotherapy or chemotherapy

in immunodeficiency diseases

• Tumor cell phenotyping:1. Diagnosis and classification of leukemias and

lymphomas2. Determination of clonality of

immunoglobulin bearing cells from lymphomas and leukemias

3. Differentiation of hematopoeitic from non haemotopoeitic tumors of cells

4. Assessment of prognosis of cancers

• DNA analysis:

1. Determination of aneuploidy2. Determination of cell cycle kinetics

• Neutrophil function assays

DELAYED TYPE HYPERSENSITIVITY TESTING

• To assess the immune response• To determine whether a patient has memory T

cells that recognize a particular pathogen• Procedure:

Intradermal injection of sterile prepared antigens into the forearm or other easily accessed skin site.

• The DTH skin response requires antigen specific memory T cells produces inflammation in 48hours

• Inflammation due to production of local cytokines and chemokines recruitment of large numbers of neutrophils and mononuclear cells

To assess the CMI fungal antigens are used

ANTIGEN COMMENTS

Candida albicans Common organism against which normal patients should respond

Trichophyton Used as control

Coccidioidin Antigen of coccidiomycosis

Histoplasmin Antigen of histoplasmosis

Tuberculin purified protein derivative

Antigen of M.tuberculosis

Mumps Used to validate prior vaccination

Tetanus toxoid As like mumps

• To assess CMI by DTH skin testing difficult in first year of life limited exposure to the various antigens control difficult to interpret

• Congenital immunodeficiency diagnosed by leukocyte phenotyping and invitro assays for T cell function

Tuberculin testing

• By intradermal injection of Purified Protein Derivative (PPD)

• Can assess past infections and latent infection• 0.1 mL volume containing 5 TU (tuberculin

units) PPD intradermally• Reading after 48 hours

Interpretation of Tuberculin testing

Tuberculin positive

• A tuberculin reaction is classified as positive based on the diameter of the induration.

• In a healthy person whose immune system is normal, induration greater than or equal to 15 mm is considered a positive skin test

10 mm is positive

• Recent immigrants from high-prevalence areas• Residents and employees of high-risk areas• IV drug abusers• Children under 4 years old• Pediatric patients exposed to high-risk adults• People who work with mycobacteria in

laboratories

5 mm is positive

• People whose immune system is suppressed• HIV infected people• People with changes seen on CXR that are

consistent with previous TB• Recent contacts of people with TB• People who have received organ transplants

Booster effect

• Repeated PPD testing in elderly people who have had prior infection with TB but whose CMI has waned over years initial test negative subsequent test is positive incorrect conclusion of patient diagnosed with TB now

• To avoid this two stage tests i.e twice a month done

Other DTH tests

• Contact hypersensitivity testing for allergic dermatitis

• Patch testing for allergies

LYMPHOCYTE ACTIVATION ASSAYS

Two primary types of assays are:1. Determination of changes in cell surface

phenotype2. Ability of lymphocytes to proliferate following

stimulation

• Measures the functional capability of lymphocytes to respond to antigenic or mitogenic stimulation

• More direct test of immunocompetence

• Not standardised between different lab and hence it is qualitative.

Lymphocyte activation

• Activated T cells undergo a series of morphologic and phenotypic changes.

1. Expansion in size2. Open chromatin by histologic staining3. Expression of surface proteins• Determination of the markers done by Flow

cytometry

Lymphocyte proliferation

• Done by polyclonal activators of lymphocytes or lymphocyte mitogens.

• T cell stimuli:1. Phytohemagglutinin2. Concanavalin A3. Superantigens4. Phorbol myristate acetate5. Calcium ionophores6. Cytokines

Measurement of proliferation:

• Proliferation is measured by purified lymphocytes cultured in vitro in small 96 well microtitre plates for 48 hours.

• DNA synthesis measured by pulse labelling the cultures with tritiated thymidine (Radioactive)

• Fluorescent dyes in DNA (Nonradioactive)• Bromodeoxyuridine assays

Cytokine production

• Activated T cells produce a large repertoire of cytokine products.

• Determined by ELISA or ELISPOT

MONOCYTE – MACROPHAGE ASSAYS

• Identification of monocytes in peripheral blood is performed by flow cytometry using CD14 mAb staining.

• Histochemical staining for nonspecific esterase is commonly performed on leukemic samples to define monocyte derived disease.

NEUTROPHIL FUNCTION ASSAYS

• Any inflammatory stimuli PMN cells first to enter degranulate release antimicrobial peptides and proteins limited numbers of proinflammatory cytokines.

• Tests done for1. Neutrophil adhesion2. Chemotaxis3. Phagocytosis4. Respiratory burst

Neutrophil adhesion

• PMN use cell surface receptors like selectins and integrins to attach to endothelial surfaces during inflammation.

• L – selectin• Leukocyte adhesion deficiency (LAD) –

assessed by flow cytometry• LAD I patients – lack beta-2 subunit (CD18) of

the major leukocyte integrins• LAD II patients – lack a fucosyl transferase

Chemotaxis

• Directional motility of PMNs• By modified boyden chamber assay

Neutrophil degranulation assays

• Slide Nitroblue tetrazolium test• Flow cytometric dichlorofluorescein (DCF) test

Granules

• Degranulation – initially it will release of secondary and tertiary granules

• Marker for primary granules – Myeloperoxidase and beta glucuronidase

• Marker for secondary granules – Lactoferrin• Marker for tertiary granules – albumin

Frustrated phagocytosis method

Bactericidal assay for granulocytes

• Bacteria growing in log phase are incubated with human serum and freshly isolated PMNs ratio of roughly five to ten bacteria per PMN.

• After 30 minutes, add gentamicin• Intracellular organisms survive• Lysis of neutrophils with sterile water and the

no. of viable organisms is determined by plating serial dilutions

Result:

• Normal PMNs show a two log reduction in viable intracellular S.aureus after 1 hour incubation, but killing is virtually absent in PMNs from Chronic granulomatous disease.

LABORATORY EVALUATION OF IMMUNE COMPETENCE

Indications for lab testing for immune competence

1. Clinical diagnosis, therapeutic monitoring or prognosis

2. Congenital and acquired immunodeficiency diseases

3. Immune reconstitution following bone marrow or other lymphoid tissue grafts

4. Immunosuppression induced by drugs, radiation or other means for transplant rejection, cancer treatment or autoimmune diseases

5. Autoimmune disorders6. Immunization to monitor efficacy or immune

status7. Clinical or basic research

Assessment of immunologic competence

• Initial evaluation: Patient age General clinical history History of infections Physical examination

Investigations

• Chest xray to rule out thymic agenesis• Complete blood count• Morphological examination

• Lymphopenia SCID , CVID and MHC class II deficiency and XLA

• Lymphocytosis Duncans` syndrome• Leukocytosis Leukocyte adhesion deficiency

(LAD)• Abnormal neutrophils Chronic

granulomatous disease and Chediak Higashi syndrome

• Abnormal Platelet morphology Wiskott – Aldrich syndrome

Suspected patients of immunodeficiency by peripheral smear

• Do assays for assessing humoral immunity• Do assays for assessing cell mediated

immunity

• Do individual marker analysis for final diagnosis

THANK YOU…..

Reference

• Medical Immunology , tenth edition, Daniel P Stites