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Immunology final > Practice #4 > Flashcards

Practice #4 Flashcards

1
Q

Flow cytometry/ FACS

A

Fluorescence activated cell sorting

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2
Q

Advantages of flow cytometry as oppose to microscope

A
  1. Objective 2. Large num of cells can be examined in 1 measurment 3. Automatic 4. Fast
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3
Q

The basic structure of flow cytometer

A
  1. Fluidics: cell flow and hydrodynamic focusing to the point of detection 2. Optics: Laser beam is concentrated and passes through one cell at the time on the other side the emitted light is detected by FSC (front scatter detector) and a series of SSC (side scatter detectors) 3. Electronics: transforming light signals to electric ones for FSC its photodiode and for SSC its PMT 4. Analysator unit: collection, storage and processing
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4
Q

Hydrodynamic focusing is done by

A
  1. The shape of the flow cell (funnel shape) 2. Sheath fluid (isitonic buffer) : flowing in laminar flow to the opposite direction of the sample stream
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5
Q

What are the functions of FSC and SSC?

A

FSC - Indicator of the cell size with direct proportionality SSC - Indicator for the complexity of the cell (granular or not)

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6
Q

What are the possible fluorescent markers used to tag the cell

A

Direct, indirect, biotin-avidin, intracellular molecules

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7
Q

Possible data imaging methods

A
  1. Histogram - one single parameter depending on cell count 2. Dot plot - 2 or more parameters
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8
Q

Applications of flow cytometry

A
  1. cell sorting 2. total cell count 3. detecting different cell populations in the sapmle 4. detection of the functional state of cells in the sample
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9
Q

Preparation of the sample for flow cytometry

A
  1. Fresh suspension (not coagulated blood) 2. Erythrolysis 3. Staining if necessary
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10
Q

Dot plot complexity vs. size

A
  1. Eosinophils
  2. Neutrophils
  3. Monocytes
  4. Lymphocytes and Basophils
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11
Q

Dot plot diagnosis

A

Left: Normal bone marrow smear

Right: Bone marrow leukemia

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12
Q

What happened between A and B?

A

In A there is more fluorescence proteins attached to each cell

Left shift

(intensity shift)

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13
Q

What happened between A and B?

A

In B the number of cells expressing CD3 is increased

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14
Q

What are the different quality check possibilities in flow cytometry?

A
  1. Technical:

setting the machine

Fluorescence compensation for molecules with lower intensity fluorescence

  1. Biological:

Unstained preparation

or

Unspecific binding to control isotope

Detection of autofluorescence

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15
Q

Markers for NKC

A

CD3-

CD56+

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16
Q

Markers for B cells

A

CD19+

17
Q

Markers for B1 cells

A

CD5+

18
Q

Markers for plasma cells

A

ic light chain +

CD19-

19
Q

Markers for Th cells

A

CD3+

CD4+

20
Q

Markers for T cells

A

CD3+

21
Q

Markers for NKT cells

A

CD3+

CD56+

22
Q

Markers for Tc cells

A

CD3+

CD8+

23
Q

Markers for gamma/delta T cells

A

CD3+

gamma/delta TCR +

CD4-

CD8-

24
Q

Markers for Treg cells

A

CD4+

CD25++

Foxp3+

25
Q

Markers for Th1 cells

A

IFNgamma+

IL12+

TNFalpha+

TNFbeta+

26
Q

Markers for Th2 cells

A

IL4+

IL5+

IL10+

IL13+

27
Q

Markers for Th17 cells

A

IL17+

TGFbeta+

28
Q

Markers for lymphoid progenitor cells

A

CD34+

cKit+

HLA-DR+

TdT+

29
Q

Soluble cytokine detection by flow cytometry

A

A microbead with specific Ab against the cytokine is mixed with the sample

then a secondary Ab (with fluorescence dye) against the cytokine is added

30
Q

Cell cycle analysis by flow cytometry

A
  1. membrane lysis
  2. DNA staininng by propium iodide

* elevation of the S is an indicatorfor malignancy

31
Q

Phagocyte function analysis by flow cytometry

A

Phagosytosis of fluorescnece labled latex beads will release the dye and the intensity of fluorescence will be higher

(the x axis on the right the y axis on the left)

32
Q

Cell sorting by flow cytometry

A

Two possibilities:

  1. Mechanic separation by laser interrogation
  2. Electrostatic separation
33
Q

Acute myeloid leukemia markers

A

CD14+

HLD-DR+

CD13+

CD33+

icMPO+

34
Q

Acute lymphoid leukemia markers

A

HLA-DR+

icTdT+

Non T all

CD19+ CD20+

(CALLA)

CD10+

T all

CD2+ CD3+ CD5+ CD7+

35
Q

CD45

A

Present on all WBC except plasma cells

36
Q

CD38

A

Typical activation marker of Th, Tc, B, NKC

Prognostic marker for leukemia

37
Q

CD138

A

Highly expressed on all tumor cells

38
Q

FCM in pharmacology

A

Pgp detection by immunophenotyping

Measure the time it takesto the cell to pump out the fluorescent labled drug

39
Q
A

Immunology final (24 decks)

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