A Brief Overview of the Important Functional Parameters of a PRP Kit

by Joel Suraci

A major consideration in determining the quality/efficacy of PRP treatments is to consider the

medicine itself - platelets, and their all-important growth factors - when assessing the choices available
to physicians at this time in kits “on the market” we need to know how much medicine is being
harvested and utilized for the patient’s benefit. The major question is, "is the biologic being injected
truly PRP, or platelet rich plasma, by definition?" It's been well established (originally in
maxillofacial modalities by such physicians as Marx) that a minimal level of platelets are needed to
achieve substantial tissue repair/regeneration; that number being 1,000,000 platelets/µL in a 5mL
sample (which is 3x baseline using average patient platelet counts), assuming delivery to one site. This
is a total of 5 billion platelets. Considering the average patient has 150,000 to 450,000 platelets per
µL, we can take the average of that to be 300,000 platelets per µL. So we need to consider how much
blood is being drawn to determine a major fact as our starting point - "How many platelets are there
in the initial blood volume?" Platelets can not be simply formed from thin air, so there is no getting
past this rule of thumb, no matter what marketing materials may say, or manufacturers may claim. Then
from there we can move on to the second important question - "How many of these platelets are we
recovering, without damaging/destroying/prematurely activating, before injecting into the site
of injury?" In other words, how much medicine is actually being given to the patient? Our third
question, based on recent evidence, is "What other beneficial and non-beneficial
cells/components are we leaving/taking out of our final injectable?"

Question One "How many platelets are actually there in the initial blood volume?" (The only truly
definitive way to know this answer for sure is to simply use a platelet counter or send the sample out for
testing. This is highly recommended for the baseline whole blood sample, and for the PRP final sample
after preparation by an existing system). Taking an average of 300,000 pl/µL, we can examine a few
example volumes:
If we were to draw 8mL of blood on the "average platelet number" patient, we would get 300,000 pl/µL
x 1000 = 300,000,000 pl/mL x 8 = 2,400,000,000 total platelets. This is already less than half of
what we need to meet the definition of PRP, assuming that all platelets would be recovered and
remain viable (not possible), and are being used in a single site injection: If we bring this to the 5mL
volume (assuming the system being used can adjust volume without losing platelets) we now have
2,400,000,000 platelets ÷ 5mL = 480,000,000 pl/mL, which then translates to 480,000,000 pl/mL ÷ 1000
= 480,000 pl/µL. Already, this does not come close to reaching the minimum level of necessary
platelets.

Using the same math with a: 10mL blood draw we would end up with 600,000 pl/µL in 5mL final
injection volume
16mL blood draw we would end up with 960,000 pl/µL in 5mL final
injection volume
25mL blood draw we would end up with 1,500,000 pl/µL in 5mL final
injection volume (we've now achieved the minimum level of definition
standard PRP)
50mL blood draw we would end up with 3,000,000 pl/µL in 5mL final
injection volume

* When considering total volume of whole blood it is important to take into account the amount of blood
drawn, as opposed to the volume size of the tube/container being used, due to the volume of
anticoagulant being used taking up a fraction of the total volume of the container. Determine your
calculations using the blood volume only, in order to be accurate in your assessment.
Now onto our second, very important question: "How many of these platelets are we recovering,
without damaging/destroying/prematurely activating, before injecting into the site of injury?"

There are many factors involved here:

• Platelets being lost due to adhesion to device surfaces (too much surface area, rough/porous
materials allowing for excessive platelet adhesion), or succumbing to accidental collection by
anticoagulant gels that poorly target separation of RBCs and plasma for lab analysis, instead of
viable recovery purposes.

• Platelets "missed" during extraction due to poor targeting methods that allow far too much
inconsistency and user error (e.g. sticking a needle down into the buffy coat and trying to
extract only the buffy coat while it is surrounded with less viscous plasma which the laws of
physics make easily susceptible to the vacuum draw

• Platelets being damaged due to centrifugation that is not properly calibrated/designed to keep
living components within blood preserved and undamaged:
• Fixed angle rotors cause "platelet slip phenomenon" due to g-force pressure "rolling"
platelets against surfaces that are rough (microscopically)
• Hard braking places extreme impact force on platelets and other living cells, possibly
damaging/destroying them on impact

• Some anticoagulants, such as EDTA, can harm platelet membranes, destroying them and
rendering their growth factors non-functional

• Any other device design or user error that may involve prematurely activating platelets, can
eliminate their benefit (some growth factors have half-lives of only minutes, while other hours)

With a number of varying systems on the market it is only possible to assess those who have
opted for independent testing (very few), as opposed to data stated in marketing materials that come
from internal studies, or even worse (though about as trustworthy as the prior), have no citation at all.
Examining multiple independent reviews of systems available shows that some systems are capable of
recovering up to 80% of the platelets from the whole blood sample, while other systems fall as low as a
30% recovery. On top of this, it should be considered that a percentage of these platelets were
activated prematurely due to processing, and their functionality impaired. The average loss in this
parameter appears to be around 5%.
When taking into consideration this additional loss, we can now analyze our actual platelets
being delivered, based on the maximum retrieval in whole blood minus the platelets lost in processing
of the whole blood.

Interestingly enough, the smaller test-tube systems using typical EDTA blood-analysis lab gel (though
stating it to be "proprietary" gel) have not only the lower volume of initial platelet potential due to lower
initial volume of blood, but the greatest amount of viable platelet loss due to significant quantities of
platelets getting trapped in the gel during separation, use of hard-braking and/or fixed angle
centrifugation (as opposed to swinging buckets), use of an anti-coagulant that damages the platelet
membrane (EDTA), and no ability to control the end volume without tossing away the baby with the
bathwater (platelets suspended in plasma). Some systems have come up with retroactive solutions to
controlling the end volume/platelet fraction, but do so with major breaches in sterility protocol, so they
won't be considered here.

With our initial 8mL blood draw analysis, we had a maximum potential of 480,000 pl/µL in the
5mL injectable end volume. However, if we consider that this system has only a 30% platelet recovery
rate and a 5% loss in viability (this is being very conservative), we can now approximate that 25% of the
platelets will be functional and available for tissue regeneration, in our final sample. This means
144,000 pl/µL, or 14.4% of the necessary requirement. Clearly this falls extremely short of the
established minimum for standard definition PRP, and most likely will not deliver the necessary level of
platelets/growth factors for substantial treatment efficacy.

Using the same math with the: 10mL blood draw, and considering the company's own stated 60%
recovery rate (no independent testing has been presented by the
company),and taking into account a loss in 5% viability (being very
conservative again), we end up with 330,000 pl/µL, or 33% of the
minimal requirement.
16mL blood draw (same platelet recovery numbers as the 8mL system)
we end up with 288,000 pl/µL (worse than the above, using more blood)
25mL blood draw (independently tested 80% recovery, minus an
illustrated 6.5% viability loss) we end up with 1,102,500 pl/µL, finally
achieving a level that meets the standard definition of PRP
50mL blood draw (same system as 25mL blood draw) we end up with
2,205,000 pl/µL

In order to give insight into the standard measurement many manufacturers use in their marketing
materials, we will now consider "platelet concentration over baseline". This will vary with some of
the existing systems due to their poor quality platelet recovery, but also based on the fact that they
have no way to control the final volume of the injectable, without eliminating large quantities of platelets,
and end volumes are essentially determined by the patients hematocrit level. Fluctuations in end
plasma volume compared to RBCs is the final determining factor in the size of the injectable volume,
and therefore "fixed" for each individual patient. This can be an extreme disadvantage, especially
when wanting to place a large number of platelets into a site that can only handle so much
volume (e.g. intradiscal, knee joint, specific tendon tear, etc) However, some systems that have
adapted to current protocols have the ability to remove plasma volume without sacrificing platelets,
permitting concentration levels to be controlled. With our above systems, and their platelet retrieval
analysis we can go a step further and estimate "platelet concentration over baseline". To determine
these systems' concentrations over baseline, we will use their actual end volume standards - the final
injectable volume that the systems actually "put out", though some systems (as will be noted) are
adjustable and this concentration can be changed. For systems that are hematocrit dependent we will
make our assessment based on the average human hematocrit ratio, though this has a significant
standard deviation (The normal hematocrit for men is 40 to 54% and for women it is 36 to 48%). We will
take our average then, to be 45%.
In our 8mL blood draw example, which is a "fixed system" (hematocrit dependent), we see our
initial whole blood sample had 300,000 pl/µL. That is our baseline. Our end viable platelet count was
720,000,000 total, and in a 4.4mL final injectable (based on average hematocrit levels) we end up with
163,636 pl/µL. This is a platelet concentration of .54 times baseline! The obvious idea is to achieve
higher concentrations of platelets than that which is found in the baseline whole blood, and yet this
system is coming in at half. One might guess that the patient in this situation would have been better off
left alone, than being given a rather expensive placebo.

Using the same math with the: 10mL blood draw, again a "fixed system", baseline 300,000 pl/µL,
evpc=1.65 billion, 4.5 mL final injectable, 366,667 pl/µL, translates to a
1.2x concentration over baseline.
16mL blood draw, "fixed system", baseline 300,000 plµL, evpc=1.44
billion, 4.5 mL final injectable, 320,000 pl/µL, translates to a
1.06x concentration over baseline.
25mL blood draw (completely adjustable from 1mL (much more
concentrated) to as high as the hematocrit permits (average of 13.2 mL)
(much less concentrated), however we will use the manufacturer's
 suggested 4mL end volume) baseline 300,000 pl/µL, evpc=5.5 billion,
4mL final injectable, 1,378,125 pl/µL, translates to a 4.6x concentration
over baseline.
50mL blood draw (adjustable as above from 1mL to 27.5mL, however
we will use the manufacturer's suggested 7mL end volume) baseline
300,000 pl/µL, evpc=11 billion, 7mL final injectable, 1,571,429 pl/µL,
translates to 5.2x concentration over baseline.

A physician would never prescribe a medicine without figuring out the proper dosage necessary to bring
about the positive physiological change/outcome desired. Regenerative medicine involving platelet rich
plasma and autologous stem cells has been studied for over three decades now and has been shown
to be highly effective, when done properly, following protocols backed by studies and research data.
Last year alone there were over 9000 such studies available on PubMed, and this year that number is
expected to almost double. It is important for patient outcome for doctors to understand this data, and
follow the protocols that point to the best change of efficacy and positive outcome for their patients.
Ignoring this data or dismissing real world scientific process is simply bad medicine.

The above mentioned factors should be taken into consideration when assessing a PRP system. If they
are not, then it is possible the physician will end up injecting biologics that fall far short of their
expectations and the treatment's potential. It is a simple concept - in a treatment involving a specific
delivery of biologics that are shown to provide significant tissue healing and regeneration, one
would want to make sure those biologics are actually being delivered to the patient, at the
appropriate site.

Our final question, “What other beneficial and non-beneficial cells/components are we
leaving/taking out of our final injectable?", is nearly as important as the first two we considered.
When administering PRP injections, pertaining to the components of the final injectable, if physicians do
not pay attention to what they are placing into a joint space, or near a tendon, they could be introducing
harmful biological components, unwittingly, into the area where they meant to only promote healing. As
well, if highly beneficial components (besides platelets) are being left behind, when they could be
instead delivered and benefit the treatment and overall efficacy, why would anyone want to not include
them? Recent research has brought about the importance of monocyte inclusion (tissue repair and
regeneration from Macrophage-2), as well as the importance of removal of red blood cells and
neutrophils (a growing body of research has illustrated they are both harmful to chondrocytes and
synovial cells found in the intra articular space, and neutrophils can cause an excess, over-
inflammatory response rendering inefficiency in areas such as fibroblast proliferation, and even inhibit
tenocyte proliferation and tendon progenitor cells from fully functioning and repairing damaged tendon
tissue). Many lab-gel systems completely remove monocytes from the equation, due to the functionality
of the gel. As well, quite a number of commercial systems leave no choice but to include red blood cells
and neutrophils in the final injectable, as their designs would not permit such removal without sacrificing
a great number of platelets. The misnomer of “leukocyte-rich” and “leukocyte-poor” PRP in the recent
studies has created a misunderstanding via generalization; neutrophils and monocytes are both
leukocytes, yet we would like to keep one (monocytes), and omit the other (neutrophils), in the majority
of treatment situations. The only treatment parameters that studies have pointed to showing the benefit
of neutrophil inclusion appears to be bone non-union, open surgery/closure, and wound care. Outside
of those instances, thus far, the studies have shown neutrophils in high concentration to be more-harm-
than-good, as far as PRP treatments are concerned. Further studies will hopefully illuminate this more
clearly, as well as the apparent benefits of monocytes in tissue regeneration.

As well, physicians should be considering if the system they are using is truly closed
(disallowing for breaches in sterility and possible contamination of the injectable). Having a closed
system that allows for an intrinsic level of caution, allows the user to focus more on the task at hand,
and also eliminates unnecessary risk for the patient and the physician or medical assistant. PRP has
been shown to be extremely safe and the adverse events, when searched for, are few and far between
– however, patient safety should be priority at all times, and utilizing a system that is as safe as
possible would never be a negative over use of a contamination-risk-laden system.
Another important factor is consistency and as much elimination of the possibility of user error
being designed into the system being utilized. Without this, treatments can have added variables that
can alter efficacy, along with so many of the other variables that cannot be controlled. Utilizing a proper
system, study backed protocols, and a physician’s medical experience and knowledge to assess and
administer the appropriate treatment, can all determine the efficacy and overall patient benefit. A
system designed to eliminate as many corrupting variables as possible will help add to the certainty that
everything within the treatment is being performed to the best of the physician’s ability, and with the
best tools available.
The best part of it all is that these are parameters than can easily be tested and judged
individually by physicians, instead of taking stated information on marketing materials to be
fact. Platelet counts on a few patient samples can truly give physicians some clarity on whether or not
they are giving the patient appropriate levels of growth factors, deemed vital to the treatment efficacy,
and overall end result. There are many other non-controllable variables at play, such as level of
damage to the intended area of treatment, patient's age, activity level, overall health, nutrition, and on
and on. The one thing that can be controlled and accounted for, in order to give the patient the best
opportunity for an increase in quality of life, and return to normal activities, or even possibly the ability to
avoid surgery, is physicians making sure their PRP systems are at least meeting the minimum
standard, and is not just delivering a high-priced placebo.
Physicians, when vetting various systems and attempting to determine which provide them the
necessary minimums and best overall efficacy, should demand a patient trial and send the sample
to their own lab for platelet counts (unless they have a cell counter readily available). If
companies have nothing to hide, and want your business, this shouldn’t be an issue, and they
will gladly satisfy the request. If they refuse to be tested individually, or against another
system, physicians should simply think twice about working with any company that will not
stand behind its products and illustrate the data they claim (or simply don’t have), while
possibly having a negative effect on numerous patients’ potential treatment outcomes.