Nuffield Report 2015

Darnell Conteh
Introduction

For my project this year, I was asked to assess the effect of umbilical cord bloods sample
size and collection process on CD34+ and CD45+ cell concentration. From this, I produced
two hypotheses to investigate. To investigate them I done statistical tests using the
programmes Microsoft Excel and Minitab. My first hypotheses were: samples of a greater
volume give higher cell concentrations, and ultimately significantly greater cell numbers. The
cells that I was counting were hematopoietic stem cells and leukocytes which were indicated
by using a specialised machine called the flow cytometer. I would investigate this in coalition
with SmartBank whom have already done their own research on this field and confirmed that
there is a relationship with baby weight, Caesarean section and cell count, however, have
not investigated the relationship between blood volume removed from the umbilical cord and
cell count. The second hypothesis that was retracted from this was to see if Caesarean
Section births give rise to larger volumes collections with higher cell concentrations
compared to natural births. I too, for this, used data collected from SmartBank which are a
company that take cord blood; collect data from the mothers and send it to Biovault. I was
asked to place all of the data into a database using a programme like excel, call back patient
files and populate it with data and then do the data analysis using statistical techniques. I
decided to collect around 450 samples of data to minimise the impacts of any anomalies.
My prior knowledge to the work at Biovault was limited. After looking through their website
and brochure, I familiarised myself with the product that they provided to their customers. I
also used various websites such as www.ncbi.nlm.nih.gov which provided me with many
reports such as Waller-Wise (2011). I used this to help me understand how the umbilical
cord blood is removed, analysed and frozen safely. After the initial meeting with my
supervisor, he too, gave me some links on where I can find research papers like
http://parentsguidecordblood.org/diseases.php and https://www.lls.org/support/other-helpfulorganizations/blood-cancer-treatment/transplantation-cord-blood.These papers included
Stem Cell Transplant (Peripheral Blood, Bone Marrow, and Cord Blood Transplants) from the
American Cancer Society which really helped me understand the importance of Stem Cells
in medical treatments of the future. Furthermore, these gave me a great in-sight into what
Biovault actually do to the incoming blood and the potential new areas that they could go into
such as the treatment of motor neurone disease and other autoimmune diseases like lupus.
Another promising field is the production of artificial blood by using stem cells to grow blood
outside of the body which, if successful, would solve to increasing pressures on blood banks
around the world. To evaluate, 1 in 3 people in the UK, alone, will be diagnosed with cancer
(Cancer Statistics Key Facts by CancerResearchUK- Sept 2014) so the importance of new
and better therapy is highly desired. Stem cell research, especially in mesenchymal stem
cells, have shown a potential of being this new therapy and as pharmaceutical companies
are failing to find a medically approved cure for cancer, there is an increasing pressure on
new research to help resolve this growing epidemic.
Abstract

Using completed questionnaires from women acquiring Biovaults service, I gathered

data and used it to see if there is any relationship between incoming blood bag volume,
CD34+/CD45+ cell count and their viability percentage. I am also seeing whether
caesarean section or natural birth gives arise to a higher blood volume collected from

the incoming blood bag and to see if this affects the cell count and viability. The data
gathered would be a mixture of questionnaires; data collected from processing the
blood into cassettes and data from the flow cytometer. I took this data and produced
various charts such as scatter graphs and pie charts. Due to my data not being
normally distributed, I converted the data to make it normally distributed using the
Johnson Transformation method so that I can statistically test it using parametric tests
such as the Pearsons test and the Student T-Test.
To do this I am taking around 450 sets of data to compare. This is to ensure that my
results are reliable and accurate as the large sample size would reduce the impact of
any anomalous result. I am using data collected from January 2012 to the end of
December 2012. Whist doing this I did use the same methods that was used to obtain
the previous data on samples that were coming in on the days that I was working at
Biovault and, therefore, got to use the specialised equipment such as the flow
cytometer. I also processed some of the incoming blood under a lamina flow hood, to
familiarise myself with the techniques that past scientists used. This allowed me to
understand the results that I was observing and better understand what I was
essentially reading. The methods that Biovault were using were originally used by
Pablo Rubenstein and published in his paper (Proc. Natl. Acad. Sci. USA, Vol. 92, pp.
10119-10122, October 1995, Medical Science).
Methodology

As the service Biovault was providing had to oblige to their JACIE and HPA
accreditation and, of course, the product had to be tightly controlled for medical use, we
had very strict rules and precautions to stick to. Before entering, all personal had to
wear protective clothing. This included a hair net; lab coat and steel capped lab boots.
The lab coats were replaced weekly with new, sterile ones and the hair nets were
discarded after use. This was to make sure that not only was the staff and technicians
protected from the potential hazards around the room but also the incoming samples
were protected from contamination. Moreover, every morning and evening daily
cleaning and maintenance tasks have to be completed. Tasks such as cleaning the inhatch required the use of chemicals such as IPA (Sterile Denatured Ethanol) and a
Sterile Biocide E. When doing these tasks all staff had to wear nitrile gloves to protect
their skin. Daily air samples were also taken to monitor atmospheric bacterial and
fungal levels. If these levels were too high, the whole lab would have to be cleaned.
Furthermore, located frequently around the lab was alcoholic hand sanitizer which was
used when leaving certain rooms. All of these measures were vital because if

atmospheric bacterial and fungal levels were too high there is a risk that when blood
gets sent in, it would get contaminated. If the blood gets contaminated it wouldnt be
able to be used for treatment as whoever receives this blood could potentially develop
diseases such as septicemia which, with a suppressed immune system, would certainly
cause serious health problems.
When a blood sample comes we immediately check the questionnaire to see if it is a
biohazard. Biohazards are samples that have a risk of containing foreign bacteria or
diseases such as HIV which would result in them not being able to be used on the
public for treatment. If a blood sample is a biohazard, it is immediately separated from
the other incoming blood samples to make sure that cross-contamination doesnt occur.
Once this is completed we create files for the samples. Once the files were completed
a process known as cleaning occurred. As I was dealing with blood that could harm
me, I had to wear a lot of protective clothing. This included a plastic apron; goggles;
gloves and a face mask. This protective clothing and equipment ensured that no blood
could touch my skin or enter my body. Once clothed, I took the blood sample and
removed any hypodermic needles that maybe left on it by using a heated clamp. The
needle was covered by a plastic tube ensuring that it would be impossible for it to
puncture my skin. Once removing the needles, I placed them in a sharps bin, so that
they would be removed and discarded accordingly. Soon after that I would use Biocide
E spray and wipe the blood bag so that any bacteria that maybe on the surface of the
blood bag are killed. This is to maintain the o% atmospheric bacteria concentration
levels that are found in the aseptic lab conditions. Whilst waiting for the Biocide E to
work, I then spray a metal tray (which the blood bag and corresponding file would be
placed into) with IPA. Before placing the blood bag into the in-hatch which leads to the
lab where it is processed, I wipe it with IPA. Along with the blood bag is maternal blood
which I place into a bag with a virology sheet. The bag is sent to Royal Devon and
Exeter Hospital to be tested for any blood diseases like Hepatitis B.
The next step for the blood is for it to be processed in the aseptic lab. This starts with
the blood collection bag being heat welded to a Marcopharma T&B set which is a type
of blood bag with separate compartments for different blood layers. Once clamped,
blood from the original blood collection bag is then transferred to the Marcopharma
T&B set by inverting the original blood bag. Moreover, some blood from the blood bag
is poured into two vials that would be used for the flow cytometer and for any further
testing in a future date that maybe required. The T&B set is then placed into a
centrifuge bucket carefully with two bags (containing de-ionised water) either side. The

two saline bags prevent the blood in the Marcopharma T&B set from being damaged in
the centrifugation process. The bucket is then placed into its place in the centrifuge and
the blood is then spun at 1500 revolutions per a minute for twenty minutes. After twenty
minutes it is then re-centrifuged for a further five minutes at 1500 RPM. Once the five
minutes is up, the blood should be separated into three layers. These layers are the
erythrocyte layer; buffy coat layer and the blood plasma layer. The Marcopharma T&B
set is carefully placed into a machine called the Optipress. It has to be carefully
removed from the bucket as I didnt want to remix the blood layers. Once the T&B set is
placed into its positions in the Optipress, a restricting clamp must be placed onto the
tube leading to the blood plasma bag and a Spencer-Wells clamp is placed onto the
tube that leads to the red blood cell bag so that you can control the blood flow to it.
Once this is done I would break the cannulas on the blood bags, so that blood can flow
from the central bag to the blood plasma bag and RBC bag.
Left: T&B set placed into the Optipress machine.
Note how the RBC layer is at the bottom layer
(due to it being heavier), the buffy coat is the
central layer and the blood plasma is the top
layer.

After that I would press start on the

machine and the Optipress would
put pressure on the bag so that the
different layers separate into their
corresponding blood bags. This
has to be done carefully (using the clamps to control the rate of blood flow) so that the
right volume of buffy coat layer is left in the bag which would increase the volume and
concentration of hematopoietic stem cells isolated, thus improving the quality of the
product. The Optipress would then heat clamp the tube leading to the red blood cell
bag (RBC) so that the Buffy coat bag and blood plasma bag becomes isolated from it.

Left: Different blood layers separated into their corresponding blood

bags. The blood plasma layer is on the far left, Buffy coat layer is in the
center and the RBC blood bag is on the far right.

After the blood has been separated into its different layers, it is ready to be placed into its
final product to be cryogenically frozen. The two blood bags separated from the
Marcopharma T&B set are placed under a laminar flow hood with 1 X 50ml syringe, 2 X 5ml
syringe, 2 X coupler spike, 1 X pair appropriately sized sterile gloves, 1 X DMSO/Dextran, 1
X 23G Needle, 1 X 2ml Syringe and a pair of gloves. The T&B set is placed between two
cool bags. An air sample, fungal and TSA plates are also placed under the laminar hood so
that the microbe levels can be checked to make sure that the staff are operating aseptically.
During the processing of the blood, a cryogenic agent known as DMSO is injected. This
protects the blood when frozen however, it is also toxic to the stem cells at room temperature
therefore, staff have to do this procedure quickly. Once it is done and the blood is in its final
cassette, it is placed into a controlled rate freezer. Meanwhile, the laminar flow hood is
cleaned using IPA spray for the next cord blood to be processed. The whole method of
reducing the whole blood into just a cassette is known as the volume reduction method. This
has many advantages over whole blood storage. For an example, when freezing larger
volumes of blood, it is harder to guarantee that all of the cells within it are frozen at a
controlled and safe rate as it can be difficult to control the temperature gradient with a larger
volume in a bigger bag and once volume reduced, the stem cells are frozen ready to use.
The cells only have to be thawed immediately before they are used. With whole cord blood
there is another step required between thawing and re-infusion which adds on unnecessary
time to potential life-saving treatment.
Left: Final product of the blood processing. In this cassette is the stem
cells that would be cryogenically frozen.

Next, the product needs to be dropped from room

temperature to around -190 degrees. This cant be
done instantaneously because the stem cells
would be destroyed during the freezing process as ice crystals can form between their cell
membranes in a process known as crystallization. So to store the stem cells safely, they
are placed into a machine known as the Controlled Rate Freezer (CRF). This machine is
connected to a tank of liquid nitrogen and would slowly drop the temperature of the stem
cells from room temperature to around -140 degrees over the course of 75 minutes. The
machine works by producing a laminar flow of air around the sample, cooling it to the
desired temperatures. After this the sample is then ready to be place
into the eterne to be frozen.

Left: a graph showing the

temperature drop throughout the 75
minutes the sample is in the CRF

During the cooling process in the CRF, two

vials containing the incoming cord blood are analyzed using a machine known as the flow
cytometer. This machine uses a laser beam to count how many cells there are in the blood
sample and also their viability. When the blood is passed into the machine, the cells are past
to a point known as the interrogation point one cell at a time via hydrodynamic focusing. It is
here that a cell has a laser past through it. The beam of photons causes side scatter and
forward scatter which is directly measured by detectors. The CD34 marker is a protein
cluster on the outside of a stem cell. Flourochromes are attached to antibodies specific for
the CD34+ markers. This is important as is there is a low amount of CD34+ cells (stem cells)
then there is a reduction in the likely hood that the stem cells would engraft to the patients
bone marrow meaning that theyll have to look for other donor-ship. One cord blood sample
has 20ul CD34/45 reagent; 20ul 7 amino actinomycin D (7AAD) and 100ul of blood. This
incubates for 20min in the dark box. Then 2ml of lyse is added and incubated for a further 10
minutes before being placed on the machine. All is forward pipetted apart from the blood
which is reverse pipette technique.

Above: the
flow machine BD
Facs Canto-2
and all of its
equipment.
Right: A flow
report. Similar to
that used to
obtain my data.

If the
sample has passed the flow check it, then goes into the eterne.
The eterne is a large storage vessel connected to large tanks of
liquid nitrogen. Each eterne can store over 1000 stem cell
cassettes and maintain internal temperature of under -190 degrees for 24 hours a day;
every day. Each eterne is fitted with an alarm which automatically sends off a signal to the

staff members if the temperature is dropping for some unknown reason. At Biovault, they
store each cassette through a method known as the steam vaporisation method. This is
using the evaporated liquid nitrogen steam rather than the actual liquid to store the
processed samples. This is beneficial as when submerged in the liquid, over time blood
could contaminate other samples, making them unusable.
For collecting the data, I used patient sheets from January 2012 to the end of December
2012. This provided me with a total of 450 sets of data. I decided to use a large sample
size like 450 data sets because it minimises the impacts of any anomalous results I may
get. I plotted all of this data on to Microsoft Excel. I used this program because I already
have training in this software and it is easy to use. However, all of this data was not
normally distributed. I calculated this by using an application called Minitab. Minitab
allowed me to plot the data into a graph which had a normal distribution line. I used Minitab
as it is a popular software used by many universities. There are many online tutorials on
how to use the software and it is very easy to use and find various statistical tests. In fact,
Minitab was a lot easier to use for the statistics than Excel. From using Minitab, I could see
that my data was slightly skewed and, by using the Johnsons Transformation, I converted
the data so that it is normally distributed. I done this so that I could use parametric
statistical tests rather than non-parametric tests such as Spearmans correlation coefficient
and Mann Whitney-U test. This is because with large sample sets such as mine,
Spearmans correlation coefficient becomes unwieldy and unreliable. So to make my
results as accurate as possible I decided to use parametric tests like the Pearsons test
and the Students T-Test.
Before, doing the statistical tests, I created graphs comparing volume against CD34+ and
CD45+ count; weight; CD34+ and CD45+ cell count against birth type. I used a scatter
graph to show the data for volume of blood against CD34+, CD45+ cell count because it
allows me to compare two variables. It also clearly shows trends between the two and it
leads on to allowing me do statistical tests such as the Pearsons test to scientifically prove
trends. I used a line graph to show type of birth against incoming blood weight; CD34+ and
CD45+ cell count because it is not just easy to do but it easily allows me to represent two
different data sets on the same graph and qualitatively analyse it. I also used box plots and
pie charts to display this data. Box plots handle large numbers of data easily which is
useful for my 450 sets of data. It also shows outliers which is why I decided to use this
technique. Pie charts are useful as they allowed me to easily compare two different data
sets together and it is really easy to see a difference between the two data sets.

After this I created null hypotheses for each hypothesis. For hypothesis one, my null
hypothesis was that there wasnt any significant relationship between incoming blood
volume and CD34+ or CD45+ cell count. For my second hypotheses, my null hypothesis
was that Caesarean section doesnt give arise to larger blood volumes with higher cell
counts than natural birth and thus any results showing this was by chance. After this I then
done the statistics and compared the results against a mixture of critical values from their
critical value tables and the p values which were calculated. I used Pearsons test for my
first hypotheses and the Student T-Test for my second hypothesis.

Results and discussion

For my first hypothesis, I created a scatter graph to see if there was any relationship
between incoming blood volume and CD34+ cell count.

Volume v CD34+
200
150

CD34+ cell count (ul) 100

50
0
20 40 60 80 100 120 140 160 180 200

Volume of blood of incoming bag (ml)

Immediately you can see that there is a slight positive correlation. The only problem is that
it isnt that strong. My Pearsons correlation coefficient test score of 0.209 was above the p
value of 0.164 thus, proving that there is a relationship at a significance of 0.001. Thus I am
95% certain that there is a relationship between incoming blood volume and CD34+ cell
count and therefore can reject my null hypothesis.
However, the relationship between incoming blood volume and CD45+ cell count is quite
different. From the scatter graph below, one might assume that there is definitely a
correlation between the two and even go as far as saying that this relationship is stronger
than that between incoming blood volume and CD34+ cell count.

CD45+ cells/ul
25000
20000
15000

CD45+ cells (ul) 10000

5000
0
20

40

60

80 100 120 140 160 180 200

Volume of blood of incoming sample (ml)

But when doing the Pearsons correlation coefficient, the Pearsons score of 0.115 was
below the critical value of 0.164 and therefore, I wasnt 99.9% sure that there was a
relationship between the two variables. However, the Pearsons value was above the
critical value of 0.098 at a significance level of 0.06 thus, resulting in a 94% certainty
that there is a relationship and that it is not due to chance. Therefore, I can reject my
null hypothesis. As I am below 95% certainty this result isnt scientifically accepted.
This is unusual as I believed that if there was an increase in blood volume there would
be an increase in both stem cells and leukocytes, however from this result there is only

an increase in leukocytes. This would most definitely need further research as it isnt
really logical.

For the significant difference between the means of the CD34+ numbers of women that
gave birth naturally and the CD34+ numbers of the women that had Caesarean births,
you can originally see that from the line graph and box plots, the two sets of data are
very similar.

CD34+ v Birth type

200
150
100
50
0
Natural birth
Natural Birth

This in general agrees with my null hypothesis. After doing the Students T-Test, my T-Test
result of 0.540 was equal to my p value of 0.540 and thus I had to accept my null

hypothesis at a significance of 0.05. So there was no significant difference between the

means of natural and Caesarean birth and so any relationship between the two is due
to chance. This is what I originally believed what would happen as even though the two
delivery methods are different, the placenta and umbilical cord arent being affected by
the way the baby is delivered thus giving there no reason for a difference in CD34+ cell
numbers.

CD45+ v Birth Type

25000
20000
15000
10000
5000
0
Natural birth
Natural Birth

Average

8464.8 7679.37

Here upon doing the T-test, I found

that my p-value was above my T value of 0.543 at a significance of 0.05 and therefore I can
accept my null hypothesis. All though disappointing, this was to be expected. You can see
from the pie chart on the right that there is a slight favor for natural births but there isnt a
significance favor so scientifically shouldnt be accepted.

Evaluation

If I was to continue the work, I would like to see what results I would get with the data that
wasnt transformed by using non-parametric tests such as the Spearmans rank and the Mann
Whitney-U test. These would give me more statistical data to reject or accept my hypotheses. I
would also like to gather more data to further eliminate any anomalous results that I have
obtained. I would also like to see there was any relationship between the mothers age and
CD34+ or CD45+ cell count. I would also recommend someone doing the same statistical test
on incoming blood samples that havent been treated with DMSO to understand the real
dangers of using it in the processing method done at Biovault. Different transformation method
rather than Johnsons. I would also like to see what might cause the lack of increase in
leukocyte concentrations when there is an increase in blood volume.
In the future I think my experiment would have been better if I had done statistics on data that I
had collected myself. However, because of the short period of time that I was at Biovault, the
sample size wouldnt have been large enough to produce accredited results.
Even though not very significant, these results would enable Biovault to really stress the
importance to Doctors (that are taking the Umbilical cord blood samples) of obtaining large
blood volumes, which could possibly reduce the number of rejected and unsuccessful samples
in the future and therefore potentially saving more lives. These results could also reduce the
numbers of unnecessary Caesarean sections which (like all types of operations) contain risks.
This can be done by proving that there isnt really any best way of producing a successful
sample and that really, your best chances of producing a good sample ironically rely on luck.

Appendix

http://parentsguidecordblood.org/diseases.php
https://www.lls.org/support/other-helpful-organizations/blood-cancertreatment/transplantation-cord-blood
http://www.cancer.org/cancer/leukemia-acutemyeloidaml/detailedguide/leukemia-acutemyeloid-myelogenous-treating-bone-marrow-stem-cell-transplant
http://parentsguidecordblood.org/diseases.php
https://www.lls.org/support/other-helpful-organizations/blood-cancertreatment/transplantation-cord-blood
These all contain interesting papers and published work on the latest information on
hematopoietic stem cells.

References
Umbilical Cord Blood: Information for Childbirth Educators by Renece Waller-Wise
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209739/
Pablo Rubenstein and published in his paper (Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 1011910122, October 1995, Medical Science)

Acknowledgements
I would like to say a big thank you to:
Michael Tindale For the help he has given me and for having the patience to look after me
for four weeks
Amy Stoke For supplying me with so much information that has helped me with this report
and for allowing me to get stuck in at the lab
Sean Mansfield For supplying me the specialised knowledge needed in this report and for
being so enthusiastic about the science he was doing everyday
And to everyone else at Biovault for their support; for allowing me to really understand how
science in industry works and for the key understanding that I have now gained in how
science can really impact and save so many lives.