CELLULAR IMMUNOLOGY

Cellular Immunology

The field concerning the interactions among cells and molecules of the immune system, how such interactions contribute to the recognition and elimination of pathogens

CD Markers

an abbreviation for “cluster of differentiation.”

CD molecules

are cell surface markers which are very useful for the identification and characterization of leukocytes and the different subpopulations of leukocytes

CD45

CD Markers

Backbone marker; hematopoietic marker expressed on all leukocytes

CD3

CD Markers

Pan T Lymphocytes

CD4

CD Markers

T-Helper Cells

CD8

CD Markers

T Suppressor cells / Cytotoxic T cells

CD16/56

CD Markers

Natural Killer Cells

CD19

CD Markers

Pre B Cells

CD20

CD Markers

Pan B Cells

Flow Cytometry

The passage of cells in single file in front of a laser so they can be detected, counted and sorted. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths. Detected by detectors

Light Scatter

Flow Cytometry

Collected by forward (FCS) and side scatter (SSC) creates patterns that are characteristic of the size and internal complexity of the cell. This indicates what type of cells (ex., lymphocyte, monocyte, granulocyte, and platelet) are present

Forward Scatter

Flow Cytometry

Cell size

Side Scatter

Flow Cytometry

Cell granularity / lobes

BD Facs Lyric

Flow Cytometry

Machines

8 color lasers

BD Facs Calibur

Flow Cytometry

Machines

3 color lasers

Tests with Flow Cytometry as Principle

FACS CD4 (Fluorescence Activated Cell Sorting)

Used to enumerate the absolute counts of CD4 T lymphocytes

1 EDTA, 1 Sodium Heparin (4mL whole blood)

Tests with Flow Cytometry as Principle

FACS CD4 (Fluorescence Activated Cell Sorting)

Sample requirements (2)

CD3, CD4, CD8

Tests with Flow Cytometry as Principle

FACS CD4 (Fluorescence Activated Cell Sorting)

CD Markers tested (3)

2:1

Tests with Flow Cytometry as Principle

FACS CD4 (Fluorescence Activated Cell Sorting)

CD4:CD8 ratio – Normal

1:2

Tests with Flow Cytometry as Principle

FACS CD4 (Fluorescence Activated Cell Sorting)

CD4:CD8 ratio – HIV+/Px with AIDS

Primary Immunodeficiency Panel 1 (PID 1)

Tests with Flow Cytometry as Principle

Also known as LYMPHOCYTE SUBSET ENUMERATION TEST

1 EDTA, 1 Sodium Heparin (4mL whole blood)

Tests with Flow Cytometry as Principle

Primary Immunodeficiency Panel 1 (PID 1)

Sample requirements (2)

CD3, CD4, CD8, CD16/56, CD19, CD20

Tests with Flow Cytometry as Principle

Primary Immunodeficiency Panel 1 (PID 1)

CD Markers tested (6)

Primary Immunodeficiency Panel 3 (PID 3)

Tests with Flow Cytometry as Principle

Also known as LYMPHOCYTE SUBSET ENUMERATION TEST

1 EDTA (plasma), 1 Sodium Heparin, 1 Red top (serum) (4mL whole blood)

Tests with Flow Cytometry as Principle

Primary Immunodeficiency Panel 3 (PID 3)

Sample requirements (3)

CD3, CD4, CD8, CD16/56, CD19, CD20

Tests with Flow Cytometry as Principle

Primary Immunodeficiency Panel 3 (PID 3)

CD Markers tested (6)

IgG, IgA, IgM, IgE, Complement 3 (GAME 3)

Tests with Flow Cytometry as Principle

Primary Immunodeficiency Panel 1 (PID 1)

With Serology tests (5)

Flow PRA (Panel Reactive Antibody)

Tests with Flow Cytometry as Principle

A routine screening measure to assess the degree of a potential kidney recipient’s sensitization, as a result of prior exposure to external HLA antigens during previous blood transfusions, pregnancies, or organ transplantations.

Flow PRA (Panel Reactive Antibody)

Tests with Flow Cytometry as Principle

Also known as LYMPHOCYTE ANTIBODY TEST

2x 4mL Red Top (serum)

Tests with Flow Cytometry as Principle

Flow PRA (Panel Reactive Antibody)

Sample requirement

Percentage

Tests with Flow Cytometry as Principle

Flow PRA (Panel Reactive Antibody)

Result via _ (%-Qualitative: Positive or Negative with percentage)

10% (0-10% - Negative; >10% Positive)

Tests with Flow Cytometry as Principle

Flow PRA (Panel Reactive Antibody)

Cut off

kidney transplant

Tests with Flow Cytometry as Principle

Flow PRA (Panel Reactive Antibody)

Commonly requested for _ _ patients

Screening

Tests with Flow Cytometry as Principle

Flow PRA (Panel Reactive Antibody)

_ test for kidney transplant (result must be negative

lymphocyte immunization therapy

Tests with Flow Cytometry as Principle

Flow PRA (Panel Reactive Antibody)

Usually requested by woman taking LIT (_ _ _) to be pregnant (result must be positive)

Flow Cytometric Crossmatching

Tests with Flow Cytometry as Principle

Also known as T and B cell crossmatching

same time

Tests with Flow Cytometry as Principle

Flow Cytometric Crossmatching

Both recipient and donor must be tested at the _ _

Time

Tests with Flow Cytometry as Principle

Flow Cytometric Crossmatching

_ sensitive

antibodies, antigen

Tests with Flow Cytometry as Principle

Flow Cytometric Crossmatching

Assesses performed _ in the recipient against donor’s _

2 ACD tubes, 1 Red Top each

Tests with Flow Cytometry as Principle

Flow Cytometric Crossmatching

Sample requirement (2)

Acid-Citrate-Dextrose

Tests with Flow Cytometry as Principle

Flow Cytometric Crossmatching

anticoagulant for HLA studies due to its longer cell viability

hemodialysis

24-48

Tests with Flow Cytometry as Principle

Flow Cytometric Crossmatching

Always ask if the recipient undergoes _? If YES, samples must be collected after _-_ hours post HD

CD3 (T Cell), CD22, IgG (B cell)

Tests with Flow Cytometry as Principle

Flow Cytometric Crossmatching

CD Markers (3)

Immunophenotyping

Detects the presence or absence of white blood cell (WBC) markers called antigens. These antigens are protein structures found on or within WBCs. It is for leukemia test.

CD34

Immunophenotyping

marker for blasts

2-4 mL Sodium Heparin

Immunophenotyping

Accepted samples except PNH:

Bone marrow aspirate

2x 4mL Sodium Heparin

Immunophenotyping

Accepted samples except PNH:

Peripheral blood

3-5 mL

Immunophenotyping

Accepted samples except PNH:

Cerebrospinal Fluid

Pleural

Immunophenotyping

Accepted samples except PNH:

Other bodily fluids (e.g., _ fluid)

Comprehensive Leukemia Panel

Comprehensive Lymphoma Panel

Minimal Residual Disease Panel (ALL or AML)

Myeloma Panel

Acute Myelogenous Leukemia

Immunophenotyping

Tests (5)

Paroxysmal Nocturnal Hemoglobinuria

Immunophenotyping

Characterized by destruction of red blood cells, blood clots, and impaired bone marrow function

CD55

CD59

Immunophenotyping

PNH Panel

Decreased or absent expression of _ and/or _

2x 4mL EDTA

Immunophenotyping

PNH Panel

Sample requirement

CD45, CD55 (DAF-Decay Accelerating Factor), CD59 (MIRL – Membrane Inhibitor of Reactive Lysis), FLAER (Fluorescent Aerolysin)

Immunophenotyping

PNH Panel

CD Markers tested (4)

HAM

Immunophenotyping

PNH Panel

Also known as _ Test

Complement Dependent Cytotoxicity

Isolating donor cells usually lymphocytes from peripheral blood and exposing donor cells to recipient serum with the addition of exogenous complement

kidney

tissue crossmatch, HLA B27

Complement Dependent Cytotoxicity

Used for _ transplant.

Principle of _ _ and _ _

Tissue Crossmatching

Complement Dependent Cytotoxicity

Also known as T CELL CROSSMATCHING

recipient, donor

Complement Dependent Cytotoxicity

Tissue Crossmatching

Both _ and _ must be tested

antibodies

Complement Dependent Cytotoxicity

Tissue Crossmatching

Assesses performed _ in the recipient against donor’s antigen

2 ACD Tubes, 1 Red Top each (for blood typing; forward and reverse) (20 mins @ 2200 rpm)

Complement Dependent Cytotoxicity

Tissue Crossmatching

Sample requirements, mins and rpm

Hemodialysis

Complement Dependent Cytotoxicity

Tissue Crossmatching

Always ask if the recipient undergoes _? If YES, samples must be collected after 24-48 hours post HD

inverted

Complement Dependent Cytotoxicity

Tissue Crossmatching

Read in _ microscope

Human Leukocyte Antigen (HLA B27)

Complement Dependent Cytotoxicity

Disease associated: Ankylosing Spondylitis, Reactive Arthritis, Uveitis (inflammation of the eyes)

2 ACD

Complement Dependent Cytotoxicity

Human Leukocyte Antigen (HLA B27)

Sample requirements

inverted

Complement Dependent Cytotoxicity

Human Leukocyte Antigen (HLA B27)

Read in _ microscope

NSS, Lymphoprep

lymphocyte ring

Tris lysing

Complement Dependent Cytotoxicity

Human Leukocyte Antigen (HLA B27)

i. _ & _ is used to create a lymphocyte ring

ii. Harvest the _ _

iii. _ _ reagent

1:2 tryphan blue

nylon wool

Mccoy's medium

Terasaki

Complement Dependent Cytotoxicity

Human Leukocyte Antigen (HLA B27)

iv. _ _ _ is used to check the viability and concentration

v. To remove B cells, use _ _ to trap the B cells

vi. _ _ preserves the cells and used for washing

vii. _ plate is used

HLA Typing

A procedure in which the tissues of a prospective donor and recipient are tested for compatibility prior to transplantation. Mismatched donor and recipient tissues can lead to rejection of the tissues; test used to match patients and donors for bone marrow, cord blood, or organ transplants

Serological Method, Micro SSP

RSSO (Reverse Sequence Specific Oligonucleotide)

HLA TISSUE TYPING CLASS I AND II	HLA DNA TYPING WITH C AND D
Principle: HLA Class I done by _ _. HLA Class II DNA typing by _ _	Principle: _ (_ _ _ _)

kidney

bone marrow

HLA TISSUE TYPING CLASS I AND II	HLA DNA TYPING WITH C AND D
Commonly for _ transplant	For _ _ transplant / kidney transplant (rarely)

Serological

Molecular

HLA TISSUE TYPING CLASS I AND II	HLA DNA TYPING WITH C AND D
_ method	_ method

A, B, C, DR, DQ

A, B, C, DR, DQ, DP

HLA TISSUE TYPING CLASS I AND II	HLA DNA TYPING WITH C AND D
Locus: HLA Class I – (3) HLA Class II – (2)	Locus: HLA Class I – (3) HLA Class II – (3)

2x ACD 10cc, 1x Red top 10cc

2x ACD 10cc

HLA TISSUE TYPING CLASS I AND II	HLA DNA TYPING WITH C AND D
Sample Requirements	Sample Requirements
_ _ _ and _ _ _ _	_ _ _ ONLY

With

Without

HLA TISSUE TYPING CLASS I AND II	HLA DNA TYPING WITH C AND D
Sample Requirements	Sample Requirements
_ BLOOD TYPING	_ BLOOD TYPING

HAPLOIDENTICAL, COMPLETE

HLA TISSUE TYPING CLASS I AND II	HLA DNA TYPING WITH C AND D
Sample Requirements	Sample Requirements
NO MATCH: _ MATCH; _ MATCH	NO MATCH: _ MATCH; _ MATCH

RECIPIENT, DONOR

HLA TISSUE TYPING CLASS I AND II	HLA DNA TYPING WITH C AND D
Sample Requirements	Sample Requirements
ALWAYS ASK IF THE PATIENT IS A _ OR A _	ALWAYS ASK IF THE PATIENT IS A _ OR A _

ABI 7500 FAST REAL TIME PCR

LUMINEX LABScan 100

HLA TISSUE TYPING CLASS I AND II	HLA DNA TYPING WITH C AND D
Sample Requirements	Sample Requirements
Machine: _ _ _ _ _ _	MACHINE: _ _ _

Compatible

Not compatible

Negative Crossmatch	Positive Crossmatch
_	Not _

1

8

Negative Crossmatch	Positive Crossmatch
Reading: _	Reading: _

Ag-Ab complex

Negative Crossmatch	Positive Crossmatch
No _-_ _	With _-_ _

HBV/HCV/HIV (Viral Load)

Other Tests Performed in Cellular Immunology

Quantitative monitoring test for HBV/HCV/HIV

2 EDTA; 2200 rpm; 20 mins

Other Tests Performed in Cellular Immunology

HBV/HCV/HIV (Viral Load)

Sample requirement, rpm, and mins

Cepheid GeneXpert Instrument Systems

Other Tests Performed in Cellular Immunology

HBV/HCV/HIV (Viral Load)

Assay is performed on _ _ _ _

real-time reverse transcriptase PCR

Other Tests Performed in Cellular Immunology

HBV/HCV/HIV (Viral Load)

GeneXpert Instrument Systems automate and integrate sample purification, nucleic acid amplification, and detection of the target sequence using _-_ _ _ (RT-PCR) and real-time PCR assays

Cervical sample (most common; female)

Other Tests Performed in Cellular Immunology

Human Papilloma VIrus (HPV) High Risk with 16, 18/45 Genotyping

Sample requirement

Tongue mass; Laryngeal mass (male)

Other Tests Performed in Cellular Immunology

Human Papilloma VIrus (HPV) High Risk with 16, 18/45 Genotyping

Other samples accepted (2)

Cepheid GeneXpert Instrument Systems

Other Tests Performed in Cellular Immunology

Human Papilloma VIrus (HPV) High Risk with 16, 18/45 Genotyping

Assay is performed on _ _ _ _

Cartridge

Other Tests Performed in Cellular Immunology

Human Papilloma VIrus (HPV) High Risk with 16, 18/45 Genotyping

_ based assay

TAT

Other Tests Performed in Cellular Immunology

Human Papilloma VIrus (HPV) High Risk with 16, 18/45 Genotyping

Advantage: Faster _

BK Virus

Other Tests Performed in Cellular Immunology

Also known as Human polyomavirus 1 that may cause trivial symptoms in the immunocompetent, although it may cause significant pathology in the immunosuppressed transplant recipient (kidney transplant)

CMV Viral load

Other Tests Performed in Cellular Immunology

BK Virus

Monitoring test together with _ _ _

BK Virus

Other Tests Performed in Cellular Immunology

BK Virus

an abbreviation of the name of the first patient whom the virus was isolated from. Usually, primary infection is occurred during childhood then the virus could be latent through life, especially in the kidneys and urinary system

Real time PCR

Other Tests Performed in Cellular Immunology

BK Virus

Principle: _ _ _

Kidneys

Other Tests Performed in Cellular Immunology

BK Virus

Organ of interest

Plasma / Serum / Urine

Other Tests Performed in Cellular Immunology

BK Virus

Specimen (3)

3M Integrated Cycler

Other Tests Performed in Cellular Immunology

BK Virus

Machine

IGRA (Interferon Gamma Release Assay)

Other Tests Performed in Cellular Immunology

T-Spot TB Test

Principle

IGRA

Other Tests Performed in Cellular Immunology

T-Spot TB Test

a blood test used to see whether a person has been infected with Mycobacterium tuberculosis (the bacteria causing TB)