Blood collection tubes
Hi there.
Welcome to another Chalk Talk episode where we’ll be introducing the six most commonly
used blood collection tubes.
These tubes are the serum tube, coagulation tube, erythrocyte sedimentation rate tube,
heparin tube, EDTA tube, and sodium fluoride tube.
You’ve probably noticed that we’ve shown two tubes for each type.
This is because the tube caps have a distinct color coding system either in accordance with
international or in accordance with European standards.
To clearly distinguish the difference between the tube caps of both standards, we’ll be
using vacutainers colored according to international standards and monovette tubes colored according
to European standards.
But don’t be misled!
The color of the tube cap isn’t determined by its shape, but by its content.
Let’s take a closer look.
EDTA tubes have a lavender cap in accordance with the international standard and a red
cap in accordance with the European standard.
They are usually used to measure blood counts.
To determine the different numbers of cells in the sample, coagulation in the blood sample
should be completely inhibited.
Therefore, the collection tube contains EDTA.
The underlying principle is actually quite simple:
Calcium, also termed factor four, is important in blood coagulation.
When calcium is removed, blood doesn’t clot.
So, let’s look into this in more detail: Calcium mediates the binding of coagulation
factors to negatively charged surfaces such as the cell surface, thereby facilitating
blood clotting.
You can find out more in part 3 of our series of Chalk Talk episodes on secondary hemostasis.
Let’s take a look at what EDTA does:
To ensure that there’s no calcium in the blood sample, a molecule that has a high binding
affinity to calcium is required: EDTA.
The EDTA molecule and calcium form a complex, which results in calcium unable to initiate
coagulation.
EDTA can bind to a maximum of six atoms; however, it also forms stable complexes with fewer
ions.
One molecule of EDTA can, therefore, bind to several calcium ions in blood, resulting
in an undefined ratio of both substances.
As a result, the inhibition of coagulation with EDTA is considered irreversible, as it
remains unknown how much calcium would be needed to restore original conditions in the
collection tube.
Blood cells usually preserve their morphology in the presence of EDTA; therefore, EDTA blood
is mainly used to analyze blood cells.
The most commonly performed hematology tests on EDTA blood include the complete blood count,
differential blood count, as well as blood group typing.
A larger collection tube is usually used for immunohematology tests.
EDTA blood is also suitable for determining hemoglobin A1c, several genetic tests, for
example, single point mutations such as factor five Leiden, and pathogen detection, for example,
HIV via polymerase chain reaction, in short PCR.
However, there are tests for which EDTA blood is not suitable, because EDTA can bind to
any divalent ion.
Therefore, calcium or magnesium levels can’t be determined in EDTA blood.
In addition, the activity of enzymes whose spatial structure is stabilised by such ions
also can’t be analyzed.
To determine the level of these ions and enzymes, blood should be collected in a serum tube.
A serum collection tube is used if serum is desired for analysis, which is obtained after
the blood is allowed to clot.
Clinical chemistry analysis of electrolytes, such as calcium, and various liver function
tests are usually performed in serum.
Serum tubes are also used in serology especially for the diagnosis and monitoring of rheumatic,
immunologic, and infectious diseases.
To promote blood clotting, silica particles are present in the tubes acting as a clot
activator.
You may have heard of glass initiating blood clotting.
This is due to silica, a major constituent of glass.
Silica has numerous negative charges on its surface, which help activate factor twelve,
also known as Hageman Factor.
Factor twelve initiates the intrinsic pathway of coagulation.
Nowadays, blood collection tubes are made of plastic and to compensate for this, they
are either coated with silica or contain silica-coated beads.
Serum tubes are characterized by a red cap according to the international standard and
a white cap according to the European standard.
These tubes don’t contain a serum gel separator; however, there are also tubes that contain
a serum gel separator.
These have a yellow cap according to the international standard and a brown cap according to the
European standard.
The separation gel acts as a diffusion barrier because it has a specific density between
that of serum and coagulated blood.
During centrifugation, the gel forms a stable barrier between these two layers.
This barrier significantly reduces serum contamination by cell components and increases serum yield.
However, if a variety of laboratory parameters are to be measured, several tubes should be
collected.
One advantage of serum samples is that they can be frozen and stored for future use.
To analyze coagulation parameters in blood, a sample is required in which coagulation
is not irreversibly inhibited, as accomplished in the EDTA tube.
But the blood sample must be reversibly stopped from further clotting before measurement.
This is achieved using coagulation tubes, which contain sodium citrate; therefore, they’re
also often referred to as citrate tubes.
Coagulation tubes have a light blue cap in accordance with the international standard
and a green cap in accordance with the European standard.
Temporary anticoagulation in the coagulation tube functions similar to that in the EDTA
tube.
The only difference is that calcium is removed using citrate, which binds less strongly than
EDTA.
The calcium citrate salt formed in blood in the collection tube, following the addition
of citrate, has a clearly defined stoichiometry.
Two citrate ions are able to remove three calcium ions.
This enables the required amount of calcium to be added to the blood sample to restore
original conditions in the collection tube.
Subsequently, coagulation parameters such as prothrombin time, or PT, and activated
partial thromboplastin time, in short aPTT, can be analyzed.
But take care!
To avoid false measurements, correct ratios are required.
The collection tube must be completely filled with blood and not contain any additional
air from the collection system.
The heparin tube has a green cap in accordance with the international standard and an orange
cap in accordance with the European standard.
It contains heparin.
Heparin is a polysaccharide that acts as an anticoagulant and forms a complex with antithrombin
three.
Antithrombin three inhibits blood clotting by binding to the activated forms of factors
ten and two.
However, as a complex, antithrombin three and heparin inactivate factors ten and two
more rapidly.
Heparin tubes are used, for example, to measure the immune function of white blood cells or
to perform chromosome analysis.
So, what is the difference between a heparin tube and an EDTA tube, if both tubes inhibit
the coagulation process?
Both tubes initially appear quite similar.
In fact, many clinical chemistry tests that can be analyzed in EDTA tubes can also be
analyzed in heparin tubes.
However, EDTA tubes have an advantage: EDTA also inhibits calcium-dependent enzymes, resulting
in a more stable sample.
Therefore, sensitive parameters can be detected over a longer period of time.
Consequently, EDTA tubes have become a standard in the measurement of numerous laboratory
parameters.
Yet, EDTA tubes also have a disadvantage.
EDTA affects cell function and is therefore unsuitable for functional analysis.
For this reason, heparin tubes are the preferred choice in analyzing cell function.
The sodium fluoride tube has a gray cap in accordance with the international standard
and a yellow cap in accordance with the European standard.
It’s used to determine glucose or lactate levels in blood.
To accurately measure blood glucose levels, glycolysis in red blood cells must be inhibited;
otherwise, measured glucose levels will be too low and lactate levels too high.
To prevent this, fluoride ions are used to inhibit important enzymes involved in energy
metabolism.
In addition to sodium fluoride, other agents are sometimes added to stop metabolic processes.
These include calcium binders such as citrate, oxalate, or EDTA, which are often added to
the sodium fluoride tubes.
Analysis of the erythrocyte sedimentation rate, in short ESR, can provide initial evidence
of the presence of an inflammatory process.
The collected blood is stored at room temperature for a certain period of time.
During this time, a sediment of blood cells is formed.
This occurs rapidly if acute phase proteins are increased in blood in response to inflammation.
One of the reasons is that acute phase proteins decrease the surface charge of red blood cells.
As a result, the red blood cells do not repel each other and therefore form large aggregates,
sinking to the bottom of the tube.
Because the ESR not only depends on serum protein composition but also other parameters,
results are only of significance if rates greatly differ from the normal reference values.
ESR measurement can only be performed in anticoagulated blood.
Therefore, the anticoagulant citrate is also used in ESR tubes.
The distance in millimeters at which red blood cells fall after an hour is read using a special
scale.
Although the ESR tube is easily recognizable by its narrower diameter, it can also be identified
by the distinct color of its cap.
ESR tubes have a black cap in accordance with the international standard and a purple cap
in accordance with the European standard.