Saliterman, Fundamentals of BioMEMS and Medical Microdevices, Ch.

Clinical Laboratory
Medicine
Introduction to BioMEMS
MN-BIO4600 Lecture 8
2015
 Sample collection

Needle &
sample
collection kit

Black: Red: Violet: Red/yellow:

Whole blood Serum EDTA Serum
sedimentation cloth whole cloth
activator blood activator w/gel

Fig 1: Blood collection tubes for clinical laboratory analysis.

Transport by postal service
Clinical Laboratory
 Processing steps
Sample collection
Venipuncture

Sample dispatched to laboratory

Vacuum vials
Closed plastic containers

Automated specimen processor

Read specimen bar codes and file
specimen in the database.
Additional processing as required
(centrifuge separation/upcons).
Sorting and sent to department.

Results
Automatic recording in database, and
forwarded to approp. person.
Urgent matters given priority.

Fig 1: The robotic LAB InterLink automated specimen processor control station
Laboratory Studies by Category

Chemistries
Hematology
Immunology
Microbiology
Urinalysis
Anticoagulation
Blood gas
Chemistries
Enzyme-Linked Immunosorbent Assay (ELISA)

Diagnostic tool in medicine:

A suspected antigen (virus/
bacteria) is affixed to a surface
Serum (containing antibodies) from
a patient is applied over the surface
binds to antigen if match
A specific anti-human antibody is
applied over the surface so that it
can bind to the antibody
Linked to an enzyme (marker)

A substance containing the

enzyme's substrate is added
The subsequent reaction produces
a detectable signal
Color change in the substrate
Temperature
Fluorescence
Chemiluminescence
Fig 2: Steps of the ELISA process.
 Chemiluminescence

The emission of light energy

(luminescence) as the result of
a chemical reaction.
Firefly (Mg2+): Luciferin + O2 +
ATP oxyluciferin + AMP +
CO2 + PPi + light
Forensic (Fe2+): luminol + H2O2
3-APA[] 3-APA + light

Detection method:
Analysis of organic species
Product involved in
reaction
Environmental air-quality e.g.
NO (nitric oxide) detection
NO+O3 NO2 + O2 + light
Emergency lighting, glow
sticks (party decorations).
diphenyl oxalate + H2O2
1,2-dioxetanedione + light
Fig 3: The emission of chemiluminescent light from a
firefly (also known as bioluminescence in animals)
 Nephelometry
Nephelometry: The intensity of
the scattered light
Used to determine the levels of
IgM, IgG, and IgA
The consentration of (antibody/
antigen) measured as function
of the amount of light scatter
Kinetic nephelometry
The rate of scatter is measured
right after the reagent is added.
the rate of change can be seen
as directly related to the
amount of antigen present.
Turbidimetry: the intensity of
transmitted light
Fig 4: The level of antigen/antibody is determined by the amount of turbidity from the scattered light.
 Lipoprotein Analysis
Measures the total lipid content
in blood.
Cholesterol & Triglycerides
Lipoproteins
HDL
LDL
VLDL
Chylomicrons

Electrophoresis
Plasma applied to an agarose
gel
Detect separation bands of
lipoproteins

Ultrasentrifugation
Separation into lipoprotein
bands based on density.

Fig 5: Process of lipoprotein analysis.

 Lipoprotein Analysis

Fig 5: Lipoprotein profiles showing the separation differences in NaBiEDTA.

 Spectrophotometric Assays
Measures a change in light
absorption (spes. wavelength)
Determine the concentration
of a substance of interest
Various biological samples
(urine, blood, plasma, tissue)
Validate a wide range of
assays (EIA / ELISA)

Fig 6: Quantification of endpoint products by Spectrophotometry.

 Protein Electrophoresis

Method for analysing the

proteins in a fluid or an extract.
Separation of proteins into
bands by gel electrophoresis.
Identification of disease
Nutritional deficiencies
Protein disorders
Inflammation
Cancer

Abnormal bands
Abnormal expressions

Fig 7: Protein separation by gel electrophoresis.

Hematology
 Hematology

The study of blood, the

blood-forming organs, and
blood diseases.

Blood diseases affect the

production of blood and its
components:
Blood cells
Hemoglobin
Blood proteins
Coagulation cascade

Hemopoiesis: the formation

of blood cellular components
from mesenchymal stem
cells.

Fig 8: White and red blood cells.

 Hemopoiesis

Fig 9: The formation of blood cells from bone marrow (mesenchymal) stem cells.
 Automated blood cell counter

Perform automated complete blood

counts.
White blood cell differentials
Reticulocyte counts

A small aperture separating two

electrodes
Each particle displaces its own
volume of conducting liquid
Momentary increase in impedance
Change in current flow translated
into voltage pulses
Pulse amplitude proportional to
particle size.

Size distribution and numbers/

sample volume.

Fig 10: Automated blood cell counting.

 Flow cytometry
Cells flow in a single cell stream past a
laser beam (coherent light source) and
detector
Detection of forward scatter
Diffraction (cell volume)
Detection of side scatter
Refraction (cellular granularity)

Conductivity
RF-arc: info cell size, internal structure,
chem. composition, nuclear volume
Opacity
Correction of conductivity signal so its
no longer influenced by cell size
Relation to internal structure (nuclear:
cytoplasm) differentiate lymphocytes
Rotated Light scatter
Sepatation of white blood cells based on:
granularity, nuclear lobularity, and cell
Fig 11: Basic flow cytometry. surface structure
 Flow cytometry with cell markers

Method to perform precise

identification of cells

Cell suspensions incubated with two

or more monoclonal antibodies

Each monoclonal antibody labeled

with a specific fluorochrome

Cells bound with antibodies pass

through the detector cuvette, -
excitation of fluorochrome
Excitation at different wavelenghts
Cells identified based on intensity,
forward and side scatter

Fig 12: Flow cytometry with cell markers.

 Peripheral Blood Smear

A drop of whole blood

is applied across a
glass slide using a
pusher slide

Stained with Wright stain

Visualise white blood cells under
microscope

Assess red blood cell morphology

and manual counting of white blood
cell types
Identification of leukemia
High and low blood cell count
Abnormal morphology

Fig 13: Peripheral blood smear.

 Erytrocyte Sedimentation Rate (ESR)
Elevated ESR used as an index of
inflammatory states in the body:
Bacterial/virus infections
Arthritis
Neuromuscular disease

Anticoagulated blood
placed in upright tube
Allowed to settle for over 1 hour
Column of settled red blood cells
measured in mm

> 20 mm/h indicative of some disease

state.

The higher proportion of fibrinogen in

the blood during inflammation causes
red blood cells to stick to each other.
cell stacks rouleaux settle faster
Fig 14: Assessment of erythrocyte sedimentation rate.
Immunology
 Antibodies
Proteins synthesized and secreted
by B-cells in the body
5-classes: IgM, IgG, IgA, IgD, IgE
Variable region: antigen spesificity
Bind (noncovalent) to antigens:
Antibody: antigen combining site
Antigen: antigenic determinant
(epitope)
Antigens (invading pathogens):
Proteins
Polysaccharides
Nucleoproteins

Antigen labeled by antibody:

Visible for immune system cells

B- and T-lymphocytes specific for a

Fig 15: Structure of an antibody. particular antigen.
 Antibodies
Heavy chains in blue and
blue-green.
Light chains in green and
yellow.
Carbohydrate in red.

Fig 16: Modelled structure of an antibody.

 Antinuclear Antibodies (ANA)
Identification of autoantibodies
directed against host antigens in
the cell nucleus
Autoimmune diseases:
Rheumatoid arthritis
Mixed connective tissue disease
Systemic lupus erythematosus

Whole blood is sentrifuged and

serum diluted at a ratio 1:40
Dilute serum added to a monolayer
culture of Hep-2 cells
Anti-IgG fluorescent-labeled
antibodies - applied and incubated
Washing reveals positive bindings
(fluorescence pattern)

Fig 17: Test of antinuclear antibodies.

 Antinuclear Antibodies (ANA)

Fig 17: Immunofluorescence staining pattern of anti-centromere antibodies on Hep-20-10 cells.

 Immunofixation

Detection of monoclonal antibodies

or immunoglobulins in serum,
urine, cerebral-spinal fluid
Separation of fluid components on
high resolution gel:
Individual lanes.
Strips of filter paper soaked with
specific antisera.
Precipitation (fixation) when a
soluble antigen (Ag) is brought in
contact with the corresponding
antibody.
Wash, fixed & stained
Correlation with protein
electrophoresis plot:
Immunofixation reveals same
protein.

Fig 18: Immunofixation protocol.

 Immunofluorescence

Detection of a spesific antigen in

serum, cells or tissue
Sample preparation by smear
Direct Immunofluorescence:
fluorescent labeled antibody
with affinity for antigen
incubated with the sample slide
Indirect Immunofluorescence:
Incubation of unlabelled
antibody with affinity to antigen
Addition of labelled secondary
antibody with affinity to antigen-
antibody complex
Fluorescent readout (positive if
conjugation antigen/antibody)

Fig 19: Direct and indirect immunofluorescence protocol.

Microbiology
Bacteria
Viruses, Fungi and Parasites
 Gram Stain
Method of differentiating bacterial
species into two large groups:
Gram-positive & Gram-negative

Based on chemical and physical

properties of the cell wall
Peptidoglycan: thick layer in Gram
positive bacteria
Spesimen with infectious agent
dried on glass slide:
Stains applied sequentially (crystal
violet, iodine, Safranin dye)
Gram + (purple)
Gram (red)

Morphological assessment:
Cocci/rods, clusters/chains &
inside/outside cells

Fig 20: Gram staining of bacteria.

 Bacterial Culture

Suspected bacteria can be cultured

from body fluids, swabs and solid
samples:
Sterile platinum metal loop
Placed in contact with sample and
streaked out in a zig-zag pattern on
solid culture medium (agarose)
Rotated 90 and streaked again

Individual colonies growing on the

culture medium:
Selectively removed by loop
Sub-cultured for further
identification.

Fig 21: Bacteria culture on solid medium.

Antimicrobial Susceptibility Tests
Determine the concentration of
antibiotics required to eradicate a
microorganism

Dilution method:
Tubes with increasing concentration
of antimicrobial agent
Suspended microorganism added to
all tubes and assessed for growth
First tube with no growth: Minimal
inhibitory concentration (MIC)mg/L

Disc diffusion technique:

Suspension of organism distributed
over the surface of a culture plate
Disks with antimicrobial agents added
to culture plate
Size of (growth) inhibition zone
around disk indicate if organism
susceptible, intermediate, resistant
Fig 22: Antimicrobial susceptibility test by dilution and disk diffusion method.
Urinalysis
 Urinalysis

Processing of urine samples

Colour and turbidity

Multiple tests:
Specific gravity, pH, glucose, protein,
ketones, bilirubin, blood, esterase and
nitrite

Sediment
Centrifuge

Positive tests:
Esterase/nitrite: culture
Proteinuria: protein electrophoresis
Glucose: diabetes

Fig 23: Processing and analysis of urine samples.

Anticoagulation
 Anticoagulation

Anticoagulant drugs are used in patients with:

Heart disease (incl. coronary artery disease and mural
thrombi)
Atrial fibrillation (an arrhythmia)
Pulmonary embolism (clot in the lung vasculature)
Deep venous thrombophlebitis (DVT)
Artifical heart valves and other prosthetic cardiovascular
devices

Coagulopathies include genetic and acquired

deficiencies in coagulation factors, abnormal synthesis
performance of the liver in hepatic (liver) diseases.
Coagulation Pathways
The intrinsic and extrinsic
pathways of the coagulation
cascade
Prothrombine time (PT)
Evaluate extrinsic pathway
factor VII and common pathway
factors II, V and X
PT = number of seconds for
blood to cloth when mixed with
thromboplastin reagent

Fig 24: Coagulation analyzer.

 The International Normalized Ratio (INR)
Created by the World Health
Organization (WHO) because PT
results can vary depending on the
thromboplastin reagent used

The INR is a conversion unit that

takes into account the different
sensitivities of thromboplastins

The INR is widely accepted as the

standard unit for reporting PT
results

Fig 25: The INRatio (anti)coagulation reader.

Blood gas
 Arterial Blood Gases
Blood gas analyzers are based on a
subset of ion and gas selective
electrodes

pH
Internal AgAgCl electrode, external
ref. electrode, ion buffer solution

pCO2
pH electrode encased in a plastic
sleeve filled with a buffered solution
of sodium bicarbonate
CO2 from blood diffuse through
membrane causing a drop in pH

pO2
Clark electrode platinum cathode,
AgAgCl anode
O2 migrates through oxygen selective
membrane and is reduced at platinum
cathode
Fig 26: The 3 key sensors of the blood gas analyzer.
 Summary

Biomedical Micro Electro-Mechanical Systems

Topics of study (curriculum):
Introduction to BioMEMS
Principles of Biochemistry
Silicon and Soft Fabrication Techniques
Polymer Materials
Microfluidic Principles
Sensor Principles and Microsensors
Microactuators and Drug Delivery
Clinical Laboratory Medicine
Micro-Total-Analysis Systems
Detection and Measurement Methods
Genomics and DNA Microarrays
Proteomics and Protein Microarrays
Emerging BioMEMS technologies
Packaging, Power, Data, and RF Safety
Biocompatibility, FDA and ISO 10993
Thank you