Exhaled
breath condensation is a non-invasive method of gathering exhaled
breath samples useful for assessing a number of disease parameters.
The RTube™ is designed to provide maximum sample collection
in minimal time. Exhaled air is directed through a unique one-way
valve and into a cooled collection chamber where vapors, aerosols
and moisture in the breath condense along the protected inside
walls of the RTube. The one way valve is then used as a plunger
that collects droplets stuck to the inside wall and holds the
sample near the top of the RTube.
RTubeT EBC Collector
and Accessories Typical condensate fluid yield is 75-150 microliters/minute
for a child and 100-250 microliters/minute for an adult at
normal tidal breathing effort. Two minutes of normal breathing
will yield sufficient fluid quantity for most laboratory tests,
however a 7-10 minute collection time is commonly employed
so that extra sample might be frozen for other investigations.
These samples can be easily collected unsupervised in the
subjects' homes or worksites and naturally lend themselves
to occupational medicine and toxicology research. Also, due
to recent FDA requirements it is believed that objective measurements
of airway inflammation, such as those found in Exhaled Breath
Condensates, will soon become important for clinical studies
designed to measure the efficacy of anti-inflammatory medication.
The typical 5-step collection process is shown below with
some details left out for clarity. Please download our Customer
Information Sheet for supplemental information regarding
proper use, cautions, and general assay information. The instructions
below detail each of the five steps:
Note: Patients should not eat or drink anything
for 1 hour before EBC collection.
Step 1: Condense Exhaled Breath
using the RTube.
Step 2: Store/Transport Condensate
Step 3: Use the Plunger to Collect EBC into a
Pool at the Top of the RTube
Step 4 (Optional): Remove CO2 (Required for pH
measurement)
Step 5: Analyze the Sample
RTube Instructions
Step 1: Condense
Exhaled Breath Using the RTube |
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| 1-1 |
Prepare the cooling sleeve
before taking samples:
- Place the aluminum sleeve into a plastic bag to prevent
moisture from freezing to the inside of the sleeve
and contaminating the sample.
- Place the sleeve into a freezer of the appropriate
temperature, depending on the exhaled substance of
interest. Home freezers work well for stable compounds.
- Allow the sleeve to cool to the appropriate temperature.
While transporting the sleeve over long distances,
store in an ice chest filled with ice or frozen packets.
|
| 1-2 |
Prepare the RTube: The RTubes
are sent ready to use for most applications; however, when
studying compounds in EBC that are found diffusely in the
laboratory setting as contaminants, extra precautions should
be taken. These include, but are not limited to, nitrate
and nitrite. We recommend that you wash the condensing
chamber with ultrapure deionized water which meets or exceeds
specification ASTM D 1193 for Type I and type II Reagent
Grade Water and allow them to dry fully before use to eliminate
any possible contamination. |
| 1-3 |
Fill out label located on
the side of the collection Tube and note the location of
the red arrow located on the label as this should be pointing
up during the collection. |
| 1-4 |
Place the blue insulating cover over
the aluminum cooling sleeve to protect your hand and keep
the aluminum cold. |
 |
| 1-5 |
Place the aluminum sleeve
over the outside of the collection chamber.
 
|
| 1-6 |
The patient should immediately begin
breathing in and out through the mouthpiece with the red
arrow pointing upward. In some cases patients will have
a very shallow breathing pattern and should be encouraged
to exhale hard enough to "hear their breath flowing
through the top of the RTube." The one way valve will
direct the exhaled air through the cooling sleeve where
the sample will be collected. After extended periods of
storage, the RTube may present some resistance upon the
initial exhalation attempt. This is normal and easily corrected
using the pre-use verification procedure here. |
 |
| 1-7 |
Collection times should be standardized
for each application. We recommend a five to seven minute
collection time for most applications. |
|
| 1-8 |
a. After sample collection has been completed
and the sample stored properly, the patient should dispose
of the mouthpiece and place the cooling sleeve back into
the protective bag and refreeze. |
|
| |
Step
2: Store/Transport Condensate |
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| 2-1 |
Detach mouthpiece and discard.
Place the cap on the end of the RTube opposite the blue
duckbill valve and near the red arrow. Optionally, the
opposite end can be also capped in addition to the end
with the red arrow. Additional caps are available by request,
please call for more information.
 
Freeze the sample at the appropriate temperature or
leave at room temperature if appropriate for the compounds
of interest. When studying stable compounds a sample
can be stored at room temperature or in a home/laboratory
freezer until needed. If packaged properly and kept out
of high heat, the sample can be shipped by mail with
no contamination or loss of sample fluid. In fact, the
RTube is so simple to use that the unsupervised subject
should be able to perform all sampling from their hospital
bed, at their workplace, or from the comfort of their
own home. When studying common laboratory contaminates
the sample should be removed from the collector and stored
in the freezer as soon as possible.
Be sure to remove the cap on the lower end (opposite
the red arrow) if so equipped PRIOR to thawing. The RTube
incorporates a pressure relief to ensure sample protection
during the thawing process which will only function if
the lower cap is removed. Keep the upper cap in place
until sample is fully thawed and ready for analysis. |
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Step
3: Use the Plunger to Collect EBC into a Pool at the
Top of the RTube |
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| 3-1 |
In the laboratory, remove the cap and
pool the sample by plunging the one-way duckbill valve
toward the top of the Tube for easy collection. Simply
place the RTube over the top of Standard Plunger and push
down until the RTube touches the base of the Standard Plunger.
Ensure the red arrow marked "UP" is properly
oriented during this process. As the RTube is pushed down
over the Plunger the nose on the end of the Plunger engages
the duckbill valve and strokes it the entire length of
the condensation surface much like a syringe. The result
is a neatly presented .5 ml - 1.5 ml pool of pure EBC ready
for analysis. |
Syringe-style plunger pools condensate near the top of
the RTube for easy sample collection |
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Step
4 (Optional): Remove CO2 (Required for pH measurement) |
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| |
We are currently modifying
our pH deaeration plunger and we will soon be offering
a deaeration attachment for use with the RTube. As a temporary
solution for our customers wanting to deaerate their samples
please follow the simple protocol listed below (please contact
us if you need more information): |
| 4-1 |
You will need a source of
carbon dioxide-free gas. We prefer Argon, but oxygen and
nitrogen are suitable. You will also need a micro pH probe
available here suitable
for use in very dilute fluids, a regulator for the tank,
tubing and glass pasteur pipettes. |
| 4-2 |
Aliquot 150ul of EBC sample
in a 1.5 ml microcentrifuge tube. |
| 4-3 |
Attach one end of the tubing
to the tank regulator and the other onto the glass pipette. |
| 4-4 |
Turn on the Argon flow (very
gently). |
| 4-5 |
Slowly lower the pipette
into sample. Prevent the sample from bubbling out of the
microcentrifuge tube by adjusting the flow from the tank
until the sample is bubbling at a steady rate. |
| 4-6 |
Remove the Argon pipette
from the sample, but leave a gentle Argon flow over the
top of the sample. This very gentle flow will help keep
ambient carbon dioxide from reentering the sample. But
move along with celerity. Insert the micro pH probe into
the sample and wait for the sample pH to stabilize. It
is wise to not leave the pH electrode against the bottom
of the microcentrifuge tube, as the pH reading may be
inaccurate. We recommend lifting the tip of the electrode
into the middle of the EBC sample, and then record pH.
For more information about EBC Sample Deaeration please
visit our Online Library or
click the links below:
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Step
5: Analyze the Sample |
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| 5-1 |
This step is of course
dependent on the substance of interest. The substance
my be unstable, and require rapid analysis, or it may
be very stable (as is the case for pH) and be analyzed
anytime.
The RTube is designed as a disposable, single use item.
The cooling sleeve, the insulating cover and the plungers
are fully reusable.
A key feature is the ability to collect the condensate
sample, store the sample, transport the sample, and analyze
the sample all in the same RTube. No transfer of condensate
fluid into another device is generally required, minimizing
complexity in the lab and at the patient's home. Opportunities
for contamination are virtually eliminated and labor
required for sample processing is minimized. Every step
has been painstakingly thought out and this design created
to minimize the effort and complexity for the patient,
researcher, and lab technician. The system is designed
for ease of use even in very large clinical studies.
Processing samples in the laboratory is efficient, but
can potentially be enhanced by using customized designs
created specifically for your area of interest. If you
have special requests concerning any condensate collection
products or methods Respiratory Research is standing
by to assist with your requests.
For more information about the analytical services offered
by Respiratory Research please visit our Laboratory
Services page. |
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