L8 - Flow Cytometry

Blood Contents

• White blood cells (leukocytes)
• Red blood cells (erythrocytes)
• Platelets (thrombocytes)

Blood cells are made in the bone marrow, mammalian stem cells differentiate into several kinds of blood cell within the bone marrow.

They take around 3 months to renew

Complete Blood Count (CBC)

• The most common blood test
• Provides information for Blood Contents
• Find the cause of symptoms (e.g. fatigue, weakness, fever, bruising, weight loss)
• Diagnose conditions (e.g. anaemia, infection, other disorders)

White Blood Cell (WBC) Count

• Protect our body from infection
• Attack and destroy bacteria and viruses
• Number rises very quickly in case of infection
• See how the bodying is dealing with cancer treatment

White Blood Cell Types (WBC differentiation)

• For finding infections, allergic/toxic reaction, other conditions (e.g. leukaemia)

Red Blood Cell (RBC) Count

• Low $\to$ anaemia
• High $\to$ polycythemia

Flow Cytometer

Characterises

• ~ 10,000 - 100,000 cells/second
• Extract scattered and fluorescent light signal from the cell
• Integrated optics electronics, fluidics technologies

Importance

• Separate “live” from “dead” particles
• High throughput ($10^5$ to $10^6$ particles in 1 minute)
• Measure particle-scatter, auto-fluorescence or exogenous fluorescence
• Sort single particles for analysis

Steps

1. Inject cell sample into a stream of fluid
2. The cell are accelerated and individually pass through a laser beam for interrogation
3. Th optics (lenses, optical filters) gather and direct the light to photodetectors
4. The photodetectors (mostly photomultiplier tubes (PMTs)) detect the scatted and fluorescence light signal from each cell
5. Peripheral electronics and computer systems convert the data into digital format and perform data analyses

The laser is the light source for light scattering and fluorescence measurements

Fluidic System

Hydrodynamic Focusing

1. Inject sample into a flow stream of sheath fluid or saline solution
2. Under laminar flow, the core fluid will not mix with the sheath fluid

• Differential pressure significantly influence the sheath flow rate
• Sample pressure $\propto$ width of sample core $\to$ change sample flow rate

Core pressure > sheath pressure, otherwise the sample cannot flow (the opposite of Palpatory (Riva-Rocci))

Reynolds Number ($R_e$)

• $R_e<2300\to$ always laminar
• $R_e>2300\to$ can be turbulent

$$R_e=\frac{\rho vD}{\mu }$$

$D$	Tube diameter
$v$	mean velocity of fluid
$\rho$	density of fluid
$\mu$	viscosity of fluid