flow cytometry, neutrophil assays, CD4

1. Laboratory methods for the
detection of cellular immunity
Dr.M.Malathi
Final year postgraduate
Department of Microbiology
Chengalpattu Medical College
Chengalpattu

2. 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.

3. Methods employed
• Assays for leukocyte phenotyping
• Delayed type hypersensitivity skin testing
• Lymphocyte activation assays
• Assessment of monocyte – macrophage function
• Determination of granulocyte function

4. 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.

5. Flow cytometry
The detection of leukocyte antigens is by
FLOW CYTOMETRY

6. • 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

7. • Light scattering property – related to size and
intracellular content or complexity
• Forward light scattering – Size
• Side light scattering – intracellular complexity

8. 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

12. Data collection and analysis
• Four pieces of data for every cell that passes
through the laser:
1. Forward light scatter
2. Side light scatter
3. Green fluorescence
4. Red/Orange fluorescence

13. X – Y plot of data

14. 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

15. Applications of Flow cytometry
• Leukocyte phenotyping
1. Diagnosis of congenital immunodeficiency
diseases
2. Assessment of prognosis of HIV positive
patients
3. Monitoring of immune reconstitution in bone
marrow transplant recipients
4. Monitoring of immunotherapy or
chemotherapy in immunodeficiency diseases

16. • Tumor cell phenotyping:
1. Diagnosis and classification of leukemias and
lymphomas
2. 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

17. • DNA analysis:
1. Determination of aneuploidy
2. Determination of cell cycle kinetics
• Neutrophil function assays

18. 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.

19. • 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

20. 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

21. • 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

22. 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

23. Interpretation of Tuberculin testing

24. 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

25. 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

26. 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

27. 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

28. Other DTH tests
• Contact hypersensitivity testing for allergic
dermatitis
• Patch testing for allergies

29. LYMPHOCYTE ACTIVATION ASSAYS
Two primary types of assays are:
1. Determination of changes in cell surface
phenotype
2. Ability of lymphocytes to proliferate
following stimulation

30. • 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.

31. Lymphocyte activation
• Activated T cells undergo a series of
morphologic and phenotypic changes.
1. Expansion in size
2. Open chromatin by histologic staining
3. Expression of surface proteins
• Determination of the markers done by Flow
cytometry

32. Lymphocyte proliferation
• Done by polyclonal activators of lymphocytes
or lymphocyte mitogens.
• T cell stimuli:
1. Phytohemagglutinin
2. Concanavalin A
3. Superantigens
4. Phorbol myristate acetate
5. Calcium ionophores
6. Cytokines

33. 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

34. Cytokine production
• Activated T cells produce a large repertoire of
cytokine products.
• Determined by ELISA or ELISPOT

35. 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.

36. NEUTROPHIL FUNCTION ASSAYS
• Any inflammatory stimuli  PMN cells first to
enter  degranulate  release antimicrobial
peptides and proteins  limited numbers of
proinflammatory cytokines.
• Tests done for
1. Neutrophil adhesion
2. Chemotaxis
3. Phagocytosis
4. Respiratory burst

37. 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

38. Chemotaxis
• Directional motility of PMNs
• By modified boyden chamber assay

39. Neutrophil degranulation assays
• Slide Nitroblue tetrazolium test
• Flow cytometric dichlorofluorescein (DCF) test

40. 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

41. Frustrated phagocytosis method

42. 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

43. 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.

44. LABORATORY EVALUATION OF
IMMUNE COMPETENCE

45. 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

46. 5. Autoimmune disorders
6. Immunization to monitor efficacy or immune
status
7. Clinical or basic research

47. Assessment of immunologic
competence
• Initial evaluation:
 Patient age
 General clinical history
 History of infections
 Physical examination

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

49. • 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

50. 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

51. THANK YOU…..
Reference
• Medical Immunology , tenth edition, Daniel P
Stites