Immunodiagnostics 2

Question 1

Flo Cytometry Outline

Answer 1

Qualitive and quantitive cellular analysis of biological and physical properties. High quantity of data quickly processed. Lasers shine light on 1 cell of a sample at a time; cell count, phenotype (cell size and complexity)

Question 2

Flo Cytometry 3 Components

Answer 2

Fluidics, optical and electronics

Question 3

Fluidics System Functions

Answer 3

Transfers sample from sample tube to flow cell

Question 4

Optical System Functions

Answer 4

Excitation light sources, lenses and filters used to collect light

Question 5

Electronics Systems Function

Answer 5

Detector signals are digitised, processed and saved for subsequent analysis

Question 6

What light scatter indicates cell size

Answer 6

Forward scatter

Question 7

What light scatter indicates cell complexity and granularity

Answer 7

Side scatter

Question 8

2D light scatter histogram

Answer 8

x axis = forward scatter (size), y axis = side scatter (complexity): 4 quadrants (top left = small + complex, bottom right = large + simple)

Question 9

Small, simple cells

Answer 9

Neutrophils

Question 10

Large, simple cells

Answer 10

Monocytes

Question 11

Small, complex cells

Answer 11

neutrophils, eosinophils and basophils

Question 12

Fluorescent Detectors Outline

Answer 12

Specific to cell receptors counts cells. Antibodies are conjugated to flurophores

Question 13

Fluorescence Tag 2D Histogram

Answer 13

y-axis = FITC (Green fluorescence) and x-axis = PE (Red Fluorescence)

Question 14

4 stages of cell death

Answer 14

Healthy/normal (no fluorescence), early apoptotic (green), late stage apoptotic (green + red) and necrotic (red). Occurs as gradual breaking down of cell membrane

Question 15

What does green fluorescent (Annexin V) bind to

Answer 15

Exposed Phosphatidylserine in early + late stage apoptotic

Question 16

What does red fluorescent (PI) bind to

Answer 16

Exposed nuclear chromatin in late stage apoptotic and necrotic cells

Question 17

Functions of Flo Cytometry

Answer 17

Protein expression (+ post-translational modifications), RNA, cell status + sorting, immunophenotyping and multiplex bead array

Question 18

Cell Cycle Status

Answer 18

Analysis of cell proliferation and activation

Question 19

Immunophenotyping Outline

Answer 19

ID and characteristics of heterogenous cell types

Question 20

Multiplex Bead Array Outline

Answer 20

Quantative measurement of analysis of samples multiple analytes

Question 21

How to know if sample control is working

Answer 21

Top right is blank

Question 22

Fluorescence Activated Cell Sorter (FACS) Outline

Answer 22

Fluidics and fluorescence components similar to Flo Cytometer. Divert specific population from heterogenous sample based on fluorescence characteristics

Question 23

Fluorescence Activated Cell Sorter Method

Answer 23

Fluorescent antibodies bind to specific cell types in samples, cells pass through laser beam + electrode (FSC + SCC detectors and electrodes give charge) and charged plate attracts oppositely charged particles to tubes

Question 24

FACS Function

Answer 24

Cancer Research, Immunology Research, Stem Cell Reasearch and Development Biology Research (embryonic cells)

Question 25

Which cytokines have higher affinity for green fluorescence

Answer 25

Higher green fluorescence = higher selective antibodies. Most to least: IL-8, IL-6 and IL-1Beta

Question 26

Red Fluorescence Affinity Most to Least

Answer 26

IL-6, IL-1 beta and IL-8

Question 27

Polymerase Chain Receptor Outline

Answer 27

Method to amplify DNA from small template. Qualitive (only quantitive when specified)

Question 28

RT-PCR Outline

Answer 28

Reverse transcription produces a DNA sequence from an RNA source to be amplified

Question 29

qPCR and qRT-PCR Outline

Answer 29

Quantitive analysis of DNA amplification

Question 30

PCR Mechanism

Answer 30

Double stranded DNA is heat denatured, primers align DNA strands, DNA primers are extended by DNA polymerases, 2 copies of DNA strands produced

Question 31

RT-PCR Mechanism

Answer 31

RNA transcribed to cDNA using reverse transcriptase. Amplification of original RNA species. Used to detect: gene expression, transcript variance and cDNA cloning

Question 32

qPCR/ qRT-PCR

Answer 32

Normal mechanism with fluroescent tags to measure progression of RNA quantity. eg dsDNA binding dye

Question 33

3 fluorescence tag

Answer 33

ground state fluorophore (binds to primer), excited state fluorophore (binds dsDNA template + primer extension) and fluroescense quencher

Question 34

Amplification Plot Outline

Answer 34

x-axis = copies produced per reaction, y-axis = fluroescence. 2 pahses: exponential and plateu. Larger quantity at start = fewer PCR cycles needed to go over threshold

Question 35

qRT-PCR in HIV Detection

Answer 35

Measures baseline plasma HIV-1 RNA level (eg Alinity m HIV kit). Targets 2 highly conserved sequences in HIV1 genome