University of Wolverhampton School of Applied Sciences

Division of Biomedical Science

In vitro assessment of platelets stored for
seven days in a platelet additive medium
A pilot study
1
Acknowledgements


I should like to express my appreciation of the kindness, courtesy and assistance I have
received from so many people on this research project and in particular, I should like to
thank Dr. James Vickers Ph.D., Award Leader (M.Sc. Transfusion Science) and Dr. Alex
Aquilina M.D., Head of the National Blood Transfusion Centre, St. Lukes Hospital Malta,
who most kindly accepted to act as my tutor and my main supervisor respectively.

I should also like to mention my gratitude to Dr. Liberato Camilleri B.A. (Educ.), M.Sc.,
PH.D. (Lancaster), Assistant Lecturer at the Department of Statistics and Operations
Research at the University of Malta and to my colleagues at the National Blood
Transfusion Laboratories for the co-operation extended to me in the course of my
research.

I should like to thanks Ms. Josette Zammit B.Sc. for dedicating much of her time to
correcting the text, and for her valuable suggestions.

I cannot let this opportunity pass without making special reference to my indebtedness to
Dr. James Vickers and other staff in various Departments at the University of
Wolverhampton for their courteousness and support during may stay in Wolverhampton.


Vanessa Zammit
Malta, 2006.
2
Abstract

Throughout the world most Blood Banks store platelet concentrates for a period of five
days. However some Banks have managed to validate a standard operating procedure
which enables them to extend such storage period to seven days.

The quality of platelet concentrates plays an important role in transfusion therapy.
Platelets are stored at a temperature of 22

C and are subject to storage lesions. These

lesions distort and disrupt platelet function that is their ability to stop bleeding. The
incidence of storage lesions increases with time, however it is possible to store platelets
for a maximum of seven days, as long as specific characteristics such as platelet counts,
bacterial contamination and pH are found to have retained acceptable parameters under
closely monitored conditions.

It was the aim of the current study to establish the bases for a more in depth
investigation that would ultimately validate a procedure to enable the National Blood
Transfusion Centre in Malta to store platelets for seven, instead of the current five days.
Apart from the above mentioned characteristics the study also included monitoring of
platelet activation, their metabolic activity, blood gases and platelet indices.

The study focused on the behavior of twenty recovered platelet concentrates suspended
in platelet additive solution, that were monitored over a period of seven days. In addition
to regular quality control checks, variations in Platelet Factor IV, glucose and lactate
dehydrogenase levels, platelet indices and blood gases were monitored.

Statistical analysis of the results showed that generally, changes in the characteristics of
the platelet concentrates under extend storage, were within acceptable parameters. In
one instance however invalid test results were obtained and consequently this test was
not conclusive.

This study confirms that the quality of locally produced recovered platelets permits their
storage for seven days and provides ground for further testing and eventual validation.
3
Contents


1. Introduction
1.1 General 6
1.2 Platelet concentrates 6
1.3 Bacterial contamination 7
1.4 Platelet additive solution 8
1.5 Investigations 10
1.6 Aims 13
1.7 Statistical analysis 13
1.8 New developments 13


2. Materials and Method
2.1 Overview 15
2.2 Platelet concentrate samples 15
2.3 Analysis of samples 16
2.3.1 Cell counts 16
2.3.2 pH and blood gases 17
2.3.3 Blood cultures 17
2.3.4 Measurement of Glucose and
Lactate dehyrdrogenase (LDH) 18
2.3.5 Platelet Factor IV 18


3. Results
3.1 Results 21

4
4. Discussion
4.1 Discussion 25
4.1.1 Filtration of samples having elevated counts of
Leucocytes and Erythrocytes 27
4.1.2 Blood Gases 28
4.1.3 Bacterial Cultures 29
4.1.4 Glucose and LDH levels 30
4.1.5 Platelet counts and indices 31
4.1.6 pH measurement 32
4.1.7 Platelet Factor IV (PF4) 33
4.2 Limitations 35
4.3 Conclusion 35


5. References 37


Appendix 1 45


Appendix 2 62


Appendix 3 70
5





Chapter 1
Introduction
6
1.1 General

Platelet transfusions have various uses, whether as part of treatments or as a
prophylaxes in cases of bleeding in patients with thrombocyte problems. Since platelets
are a blood-derived product, these carry the same risks associated with blood
transfusions such as the risk of infectious disease transmission and allo-immunization.

Platelets play an important role during the process of heamostatis, as they become
activated and stop the bleeding. However when dealing with recovered platelets this
function may be greatly compromised by platelet lesions which affect the platelets both
structurally and functionally, as well as in their metabolic activity.

These lesions are mainly caused by incorrect whole blood collection processes and poor
storage conditions, coupled with the duration of storage of the platelet concentrates
(PCs).

As discussed by Aleil et al. (2005), during storage the oxidation of long-chain fatty acids
is impaired, leading to an increase in glucose consumption, production of lactate and a
decrease in pH all leading to eventual platelet storage lesions i.e. the morphological and
functional changes mentioned previously.

1.2 Platelet concentrates

Recovered platelets are random donor platelet concentrates obtained from units of
whole blood that are pooled at the time of transfusion.

PCs may be stored in plasma or platelet additive solution (PAS), always at a
temperature of 22C. Bacterial growth flourishes at this temperature and this renders the
concentrates susceptible to bacterial contaminations with a consequent loss of platelet
function. The source of contamination is usually normal flora from the donor skin or
bacteria in the blood of donors who are asymptomatic carriers.

Availability of safe platelets is a constant challenge and can become accentuated at
times of emergencies. Outdating of platelets and their subsequent discard after five days
7
is an added problem and is essentially wasteful of resources of blood, equipment, and
personnel, that all add up to the expense of providing this blood product.

1.3 Bacterial contamination

Whiles, when considering the risks related to transfusions, the primary concern is that of
the transmission of viral agents causing hepatitis or AIDS, or other viral infections, such
risks being minimized by means of specified methods for donor selection and additional
sensitive analytical testing, bacterial contamination is sometimes misguidedly given less
importance. Septic platelet transfusion reactions (SPTRs) are a problem for platelet
recipients. The potential loss of platelet function due to the lowering of the pH as the
result of bacterial contamination is of major concern when transfusing PCs.

Since March 2004 the American Association of Blood Bankers (AABB), obliges blood
services to have strategies incorporated in their procedures which are meant to reduce
the risk of bacterial transmission. Such procedures cannot completely exclude the
possibility of bacterial contamination, and therefore the absence of bacteria prior to use
in effect is the only safe course for reducing infection risks.

As argued by Cardigan and Williamson (2003), the testing of platelets for bacterial
contamination requires a culture based test that takes a minimum of 48 hours to yield
results. Ultimately if the maximum storage life is not increased from five days to seven
days, PCs end up being unavailable for patients due to their decreased shelf-life.

While blood culturing remains the ideal method of detection for bacterial contamination
Lin et al. (1994) has suggested similar microbial safety may be achieved through
inactivation by use of psoralens and UV-irradiation.

Increasing PCs storage for more than 5 days is only permitted if bacterial contamination
can be excluded. With the improvements in techniques for the prevention of bacterial
contamination during blood collection, and the further sensitive testing for early bacterial
detection during storage, it is deemed possible to increase PC storage to 7 days and
longer, research into achieving an 11 day shelf-life is indeed underway. On the other
hand it must not be neglect that as remarked by Leytin et al. (2003) a inherent reduction
8
in platelet function as of the day of pooling, Day 0, may ultimately restrict their
effectiveness in transfusions.

1.4 Platelet additive solution

Research regarding appropriate PC storage conditions has been underway for the past
twenty-five years but despite such activity a standard procedure for the processing,
preparation and storage of PCs has not as yet been concluded. Currently focus is on the
development of PAS so that these may be a means of retaining platelet function whilst at
the same time extending their shelf-life.

Today, the use of these synthetic media for the storage of PCs is seen as having several
advantages. Foremost among these are a decrease in the number of transfusion
reactions to plasma allergens and the possibility of introducing pathogen inactivation
methods. As a consequence this results in a general increase of plasma stocks that is
thus available for other uses. The absence of plasma from PCs facilitates ABO
compatibility due to the absence of antibodies otherwise present in the plasma.

Hogman (1999) sees other advantage in the removal of plasma in view that this also
removes leukocytes from PCs. Their removal is beneficial because of their high
immunogenicity and capacity to produce cytokines during storage, that otherwise impair
usefulness of the concentrate the longer it is stored after Day 0.

Gulliksson (2000) highlighted some other characteristics in using an additive solution for
substitution of plasma as a storage medium for PCs. In brief these are:
1. An extended shelf-life while still retaining the haemostatic properties of platelets;
2. The presence of glucose in the platelet storage medium, necessary to retain
metabolic activity of the platelets, avoids platelet lesions;
3. Acetate used as an additional substrate for platelet metabolism, also reduces the
production of lactate by the platelets and, due to the formation of bicarbonate, it
maintains stable pH levels during storage;
4. The fall in pH can be rapid in PAS-containing media due to the very limited
buffering capacity of PAS compared with that of plasma;
9
5. Platelets stored in PAS at a citrate concentration of 8 mmol/L produce only half
the quantity of lactate as that of platelets at 14-26 mmol/L of citrate;
6. For PCs prepared by apheresis with ACD anticoagulant, presence of phosphate
in PAS seems to be a critical factor to avoid low adenine nucleotide levels during
storage.

Van Rhenen et al. (2004) mention that even though there is great potential for the use of
additive solutions as storage for platelets, only a few studies have evaluated the clinical
efficacy of platelets stored in these solutions.

Development of new PAS aims at assuring maintenance of good platelet quality
throughout storage. An optimal PAS media contains citrate, acetate, phosphate,
potassium and magnesium with the amount of glucose being determined by the amount
of plasma carryover. The less plasma carryover there is, the better the PAS. PAS-III and
ComposolPS, are two of the latest generation additive storage media that are available
on the market.

Other researchers in this field have reported as follows:
Fijnheer et al. (1991) - The in vitro quality of platelets stored in ComposolPS retained
the in vitro quality of platelets stored in plasma;
van der Maar et al. (2001) - Showed that platelets stored in ComposolPS had a more
constant pH throughout the storage period, favoring a capacity for a prolonged period of
storage and in this regard ComposolPS was deemed to have an improved buffering
capacity.
Aleil et al. (2005) Studies using 65% PAS showed that this was as effective as plasma
for the storage of PCs.

10
1.5 Investigations

Research in the storage of platelets suggests that there are three fundamental quality
standard parameters that must be considered for a proper evaluation of the effects of
prolonging the shelf-life of PCs namely: platelet counts, pH value and absence of
bacteria (Singh et al. 2003).

Platelet counts: Since platelet transfusions are given as prophylaxes or for therapeutic
purposes, the actual number of platelets transfused should meet pre-established
acceptable ranges. As a result of platelet storage lesions that invariably occur, as a
result of which the platelet count decreases during storage, monitoring of platelet levels
is important to ensure that an adequate amount of platelets is transfused.

pH: The pH value of the PAS greatly affects the metabolic activity of platelets. This fall in
pH level during storage time, is caused by an increase in the production of lactic acid
and carbon dioxide by the platelet metabolisms. As a consequence of this process
platelets become irreversibly damaged thus loosing their platelet functions.

Bacteria: Ensuring the absence of bacterial contamination of PCs shall benefit the
prolongation of the shelf-life of PCs. Platelets are particularly susceptible to bacterial
growth due to their having to be stored at 22C and therefore negative bacterial culturing
of platelets at Day 2 or Day 3 of storage would be advantageous for the extension of
their shelf-life.

This current study has therefore pursued these characteristics. In addition other tests
have been considered essential to monitor the quality of PCs being tested as to monitor
the quality and, more importantly, the safety of the product after seven day storage.
These additional investigations consist of:

1. Platelet Function IV (PF4): PF4 is a 30,000 Dalton high-affinity heparin-binding
protein which is produced in megakaryocytes and stored in platelet alpha
granules. It is a platelet-specific protein secreted when platelets are activated.
Levels of PF4 indicate the degree of platelet activation, the lower levels indicating
11
platelet inactivation required for good storage practice, as platelets should only
be activated in vivo to be effective.
In vivo measurements of levels of PF4 in plasma have been shown to be a
marker of platelet degranulation, and so can be used to detect activation of
platelets in vitro (Kaplan and Owen 1981).

2. Leukocyte counts: Leukocytes significantly affect the metabolic activity of platelet
concentrates. The quality of stored platelets can be improved by reducing the
number of contaminating leukocytes. Measurement of leukocytes may be
important in quality control of platelet concentrates. This is in accordance with
findings by Gottschall et al. (1984).

3. Gas analysis: Platelet storage lesions can be caused by a malfunction in the
process of gas exchange. Increase in pO
2
has been found to result in a decline in
oxygen utilization by platelets (Hunter et al. 2001). High oxygen permeability
induces anaerobic metabolism, reducing the production of lactate, and in turn the
carbon dioxide produced during glucose metabolism, that can thus acidify the
medium. This was the conclusion reached by Kostelijk et al. (2000). The quality
of platelets gradually deteriorates during storage due to fluctuations in pH levels
and the gas analysis test shall assist with pH monitoring.

4. Determination of glucose and lactate levels: Excessive metabolism of glucose to
lactic acid during platelet storage generally results in a fall in pH levels
(Gulliksson 2000). A sharp fall in pH levels is clearly undesired as it will lead to
substantial loss of platelet functions and consequently on storage viability.

As part of platelet count analysis the mean platelet volume (MPV), platelet distribution
width (PDW), and platelet large cell ratio (P-LCR) will also be recorded. MPV measures
platelet size and is a reliable measure of residual platelet function in stored PCs - an
increased MPV representing a deterioration of the product. PDW is a measure of platelet
volume heterogeneity, i.e. it measures platelet anisocytosis. A mixture of large and small
platelets may give a normal MPV but a high PDW, this being indicative of active platelet
release and consequent unsuitability of the product. Taken together MPV and PDW can
12
thus provide a more complete description of the platelet volume distribution than if
considering MPV alone (Singh et al. 2003).

This study therefore observed and recorded in vitro changes that occurred to platelet
concentrates suspended in ComposolPS over a period of seven days. Sampling on
days 0, 3, 5 and 7 was carried out aseptically by means of satellite bags which were
provided pre-attached to the original pool bag. When necessary, a transfer bag was
attached by means of a sterile connecting device (Compodock).
Transfer of the platelets from the satellite bag or transfer bag to the appropriate test
tubes was performed using sterile needle and syringes.

Investigations were performed on specific days as follows, where day 0 is the day of
pooling:

Day 0 Day 3 Day 5 Day 7
Blood gases
pH
Platelet indices
Residual leukocytes
Erythrocyte count
Glucose
LDH
Platelet Factor IV
Blood cultures
Table 1.1 Investigations schedule

13
1.6 Aims

The principal aim of this pilot study is to establish whether there is scope for further
initiatives to validate the introduction of additional quality control procedures at the
National Blood Transfusion Centre at St. Lukes Hospital, Malta that would lead to a
review of current policy regulating the storage shelf-life of platelets, which at present is
not allowed to exceed 5 days.

The Objectives were as follows:
1. Establish that the storage of currently produced platelets can be safely extended
from the current five to the proposed seven days;
2. Determine in vitro quality of seven-days stored platelets;
3. To map the way forward for further initiatives.

1.7 Statistical analysis

All results were entered into a personal computer, and the data analyzed using
Statistical Package for Social Sciences (SPSS) for windows (version 14) by SPSS Inc.

In accordance with standard statistical procedures a P value < 0.05 was considered to
indicate a statistically significant difference.

1.8 New developments

During the course of this study reference has been encountered to the discovery of new
techniques which could prolong the platelet shelf life.

Cryopreserved platelets, also referred to as frozen platelets, are being developed as an
alternative to recovered fresh platelets in clinical use (www.biomed.brown.edu) and the
introduction of these new procedures could mean that the shelf life of platelets would be
extended for much longer periods, possibly even ten years.
14





Chapter 2
Materials and Methods
15
2.1 Overview

For the purpose of this study the following main materials and equipment were used:

Whole blood from local donors
Platelet additive solution - ComposolPS
Helmer agitator
Sysmex cell counter
Negeotte chamber cell counter
Neubauer chamber cell counter
IL (instrumentation Laboratory System) blood gas analyzer
Bactec system for blood cultures
Roche Hitachi analyzer.
Platelet factor 4 reagent kit
Micro ELISA plate washing equipment and shaker
Micro ELISA plate reader

2.2 Platelet concentrate samples

Whole blood was procured from one hundred anonymous healthy volunteer blood
donations at the National Blood Transfusion Centre, Malta. This procurement had to be
spread over a period of six weeks, in view of the limited number of donors and heavy
demand for platelets at the time. On given days when five buffy coats were surplus to
requirements at the Centre these were utilized to pool a PC sample for use in this study.
Twenty pooled PCs were eventually obtained in this manner from standard processing of
the whole blood units donated. The procedure used for the production of the PCs is
described in Appendix 1 section 1.1.

ComposolPS was used as a PAS medium. Details of this mediums composition, as
provided by its manufacture, are included at Appendix 1 section 1.1.

The limitation of blood donors necessitated that, on each occasion that a PC pool could
be produced, the constituent buffy coats be kept in a Helmer agitator, care being taken
16
that twenty-four hours had not elapsed from the time of donation to pooling. Similarly
after pooling, and for the duration of the seven day test period for each PC, the pooled
platelets were also stored in the Helmer agitator. In all instances storage was carried
out at an electronically regulated monitored temperature of 22C with constant agitation.
This procedure was repeated for all twenty samples.

2.3 Analysis of samples

The following tests were carried out at the National Blood Transfusion Laboratories and
at the Pathology Laboratories at the same hospital:

Cell counts
pH and blood gases
Blood cultures
Measurement of glucose and lactate dehyrdrogenase
Platelet Factor IV

2.3.1 Cell counts

Platelet counts and PMV and PDW indices were measured using an automated cell
counter, Sysmex

. Although this instrument also provides leukocytes and erythrocytes

readings, quality directives given in the Guide to the preparation, use and quality
assurance of blood components 11
th
Edition, Council of Europe Publishing, require that
manual counts for leukocytes and erythrocytes be performed to compliment
measurements made by an automated counter.

Manual cell counts were therefore carried out utilizing the Negeotte chamber for
counting leukocytes and the improved Neubauer chamber to count erythrocytes.
Procedures followed for automated and manual countings are described in more detail
at Appendix 1 sections 1.3, 1.4 and 1.5.

The results of the platelet counts and of platelet indices determined for the samples were
appropriately converted to single unit equivalent (SUE) to confirm the satisfactory quality
of the finished PC.
17
2.3.2 pH and blood gases

Although in the course of routine PCs stock management at the Laboratory, pH testing
of the concentrate products is carried out on Day 1 of any PC in stock, for the purpose of
this study more frequent pH testing was performed.

The pH, pO
2
and pCO
2
values were determined immediately after sampling of each pool
using the IL - Instrumentation Laboratory System blood gas analyzer models 1306 and
1302 at a temperature of 22C, as described at Appendix 1 section 1.6. pH and blood
gases testing was conducted in conjunction with tests for the determination of glucose
and lactate dehydrogenase (LDH) levels in view that a decreased glucose level would
suggest a corresponding drop in pH.

2.3.3 Blood cultures

It is a requirement of the AABB that bacterial contamination in all platelet products is
detected and limited. To correlate the general results of this study with AABB
requirements, several bacterial contamination tests on each sample were performed at
various times during course of this investigation.

The Hospital is equipped with a Bactec continuous-monitoring blood culture system.
The machines two sterile culture bottles, where inoculated from PCs and incubated
aerobically and anaerobically at a temperature of 37C for 7 days. Even though an
automated system was used, the cultures were controlled visually for signs of growth,
cloudiness or a color change in the broth, and gas bubbles or clumps of bacteria. Details
of the procedures followed in this instance are included at Appendix 1 section 1.7. These
culture tests were carried out on days 5 and 7 of each PC.

18
2.3.4 Measurement of Glucose and Lactate dehyrdrogenase (LDH)

It is not normally the practice for this test to be carried out at the National Blood
Transfusion Laboratories. However for the sake of this investigation, and as an
additional monitoring and backup test for bacterial contamination monitoring, test were
carried out on Days 0, 3, 5 and 7.

A Roche Hitachi analyzer at the Hospital was used for this biochemical analysis,
Appendix 1 section 1.8 and 1.9, utilizing reagent kits supplied by the equipment
manufacturer. The data provided by this analyzer is quantitative in nature and was
obtained through enzymatic in vitro testing of appropriately prepared assays.

PC samples for measurement of glucose where injected in vaccutainers containing
sodium fluoride anticoagulant and in plain vaccutainers for the measurement of LDH.

2.3.5 Platelet Factor IV

It is necessary that stored platelets remain inactivated during storage. To monitor the
state of activation of platelets over the seven day test period the PF4 levels had to
remain to a minimum, ideally the level at Day 0.

Determination of PF4 had to be carried out in three stages. The first stage involved the
centrifuging of a vaccutainer containing the PC sample mixed with citrate anticoagulant,
and the extraction of the resulting supernatant in which any released PF4 would be
suspended if the platelets had been activated.

The second stage was introduced to reduce wastage of reagents. The supernatant
collected on the testing days of each PC pool was stored at a temperature of -20C so
that all supernatant specimens could be tested together.

The third stage of testing involved the introduction of quantities of the stored supernatant
into the pre-coated micro-wells of the test plate. Further addition of reagents was
subsequently performed simultaneously for all eighty supernatant specimens using
Enzyme-linked Immunosorbent Assays (ELISA) techniques. Appendix 1 section 1.10
19
provides further details regarding the measuring of PF4 by this sandwich enzyme
technique.

This technique combines the specificity of antibodies with the sensitivity of simple
enzyme assays, by using antibodies or antigens coupled to an easily-assayed enzyme.
20





Chapter 3
Results
21
3.1 Results

The results of the tests carried out on the platelet concentrate samples over the seven
day test period are shown at Appendix 2.

Appendix 2 Tables 2.1 to 2.4 show the results obtained when testing platelet counts,
PDW, MPV, P-LCR indices, leukocytes and erythrocytes counts, pH, pCO
2
, pO
2
, LDH
and glucose levels. Appendix 2 Tables 2.5 shows the results obtained from blood culture
tests and Appendix 2 Tables 2.6 shows the results obtained for PF4 tests when these
were carried out.

These tests, having been statistically analyzed as mentioned at paragraph 1.7 and
achieved a P value < 0.05, are deemed to have yielded normally distributed results as
shown in Appendix 3 Section 3.1, enabling a comparison of the means of the various
parameters as per Appendix 3 Section 3.2.


On Day 0, the weight and volume of each PC unit was noted and recorded. These
characteristics, when considered together with the platelet counts initially recorded for
each unit, were indicative of the quality of the PC unit.

The leukocytes and erythrocytes counts taken on Day 0 were reuired to confirm the
good quality of the PC units. Platelet concentrates which had elevated leucocytes and
erythrocyte counts where re-filtered and appropriately re-tested. In this regard fifteen PC
units were found to meet the required standards when their first leukocytes and
erythrocytes counts were taken. Five units had to be re-filtered and when their counts
were re-taken they were found to have achieved acceptability.

22
The following table summarizes the means and corresponding standard deviations of the
recorded tests data on the four established testing days throughout the seven day
testing period:

Day 0 Day 3 Day 5 Day 7
Platelet
count
1106.45
308.9707
1050.55
259.78705
1045.75
244.33301
1006.7
251.31195
PDW
10.625
1.54677
10.6850
2.08914
10.555
2.16831
10.94
2.8697
MPV
8.62
1.08511
8.64
1.37359
8.7050
1.3623
8.765
1.42433
P-LCR
17.045
5.8925
17.6950
9.30107
17.47
8.94869
18.125
9.83195
pH
7.4485
0.06964
7.4737
.08378
7.4846
.09527
7.492
0.13533
pCO
2

19.41
5.77097
18.55
5.70757
18.31
7.1781
17.455
7.11281
pO
2

110.44
31.91631
102.23
31.56155
95.25
29.21405
95.85
33.59711
LDH
132.65
24.69237
154.35
22.31184
170.85
24.66891
189.2
27.95410
Glucose
15.5175
2.98235
13.562
3.1756
11.8575
3.34315
9.6235
2.89177
Table 3.1: Summarized means and standard deviations of tests data

The results shown at Table 3.1 indicate that there was a minimal variation in the values
of the various parameters, which therefore indicated that the PCs were generally still
within acceptable limiting range at the end of the seven day storage period.

Variance was observed in platelet counts from Day 0 to Day 7 as shown in Table 3.1. It
has to be mentioned that although care was taken to ensure that PC units were suitably
whirled prior to sampling on each occasion, the recorded change in the means of the
23
platelet counts, may be attributed to the decrease in volume of the unit following
extraction of the sample and/or insufficient whirling of the PC prior to the sampling
process itself. This would have contributed to the observed variances in the platelet
count values between units. Another factor affecting platelet count variance on Day 0
may have been the use of PCs having an initially low platelet count, which would
otherwise have been discarded had there not been the limitation of donor availability.

Variance was also observed in glucose and LDH levels as also shown at Table 3.1.
Metabolism activity within the platelet cells gives rise to a significant decrease in the
glucose level and a rise in LDH readings of PC samples. In a study by van der Meer et
al. (2001) it was shown that low glucose, high lactate and high LDH levels indicate high
platelet metabolism and/or activation. Due to the fact that lactate testing is not performed
at the Hospital, but is farmed out to other overseas laboratories, it was not possible to
measure the lactate level during the current study.

No bacterial cultures resulted in a positive growth when tested on Day 5 and Day 7,
except for one PC which was intentionally inoculated and used as a positive control for
the investigation, Table 2.5 Appendix 2 Section 2.2 Table 2.5 refers.

Platelet Factor IV readings have mostly resulted in an out of range reading as shown at
Appendix 2 Section 2.3 Table 2.6. Given that the results from other tests indicated that
PCs were inactive during the entire testing period, the PF4 Out of Range readings can
only be attributed to samples having had a low dilution factor in the course of the tests.
Readings for standards and controls for the PF4 test were satisfactorily obtained
confirming that the procedure followed during the entire test procedure was properly
carried out. Therefore it can only be surmised that test results were not achieved due to
this being the first instance when PF4 tests were carried out at the Hospitals laboratory,
similar tests when needed, being also farmed out and undertaken at overseas labs. The
results of this test were therefore considered invalid and regretfully had to be discarded.





24





Chapter 4
Discussion
25
4.1 Discussion

The National Blood Transfusion Centre currently does not store platelets in excess of
five days. Validation of PCs over the five day storage period is performed by the Quality
Assurance (Q A) and Quality Control (QC) team at the Centre.

Following PC preparation, QC is performed on all units prepared using both visual and
electronic means. The visual investigations include:
1. The recording of the donation number,
2. Date of pooling and expiry date,
3. Lot and reference numbers of the storage bag,
4. The number of buffy coats used to prepare the unit,
5. Observation for red cell contamination,
6. Checking for any incidence of the swirling phenomenon.
The electronic investigations include:
1. Volume,
2. Platelet count and concentration,
3. Residual leukocyte content,
4. Residual erythrocyte count,
5. pH measurement.

The parameters that are required to be met are shown at Table 4.1.

Parameter to be checked Quality requirement
Volume >40mL per 55x10
9
of platelets
Platelet count >55x10
9
/SUE
Residual leukocytes:
1. Before leukocyte depletion
Prepared from buffy coat
2. After leukocyte depletion


>0.05x10
9
/SUE
>0.2x10
6
/SUE
Erythrocyte count >1.0x10
9
/SUE
pH measured at the end of the recommended shelf life. 6.4 to 7.4
Table 4.1: Defined quality requirements for PCs
26
Products which are found to fail these parameters are discarded.

pH readings are recorded for all products on the first day and on the last day of storage.

To ensure the sterility of products blood cultures are performed on random samples on
Day 2. When surplus products are available after Day 5, and which therefore have to be
discarded, some random PC units from these surplus products are kept to be tested on
Day 7 prior to their discarding.

Current facilities at St. Lukes Hospital only permit the assessment of cell counts, pH and
gas analysis, bacterial culturing and measurement of glucose and LDH levels. Other
tests which could have been needed to be performed to provide a wider view of the
quality of the PCs during this prolonged storage would have involved the determination
of aggregometric levels, screening for surface glucoproteins and flow cytometry.
Unfortunately the aggregometer at the Hospital was not operational at the time that the
tests were being carried out and a flow cytometer was on order for delivery in 2007. The
undertaking of Platelet Factor IV tests was possible through the provision of the PF4
testing kit by the School of Applied Sciences, University of Wolverhampton.

The results of the tests carried out, when analyzed using the SPSS statistics software,
generally showed that the in vitro data obtained in this study suggested that the quality
of PCs did not show signs of storage lesions and in fact retained an acceptable
parametric range during the entire seven day storage period. Statistical analysis for
glucose and LDH levels indicated substantial metabolic activity from Day 0 to Day 7,
which did not show corresponding pH variation in the samples. This is in all probability
due to the buffering effect of the ComposolPS platelet additive solution used.

27
4.1.1 Filtration of samples having elevated counts of leucocytes and erythrocytes

Beutler and Kuhl (1980) and Taylor et al. (1983), concur that low levels of erythrocytes
or low levels of leucocytes appear to have no effect on the glucose consumption, lactate
production or fall in pH. Therefore maintaining leucocytes at low levels ensures that the
viability of the platelets during in vitro storage is not compromised. Furthermore Mrowiec
et al. (1995), reported that levels of leukocyte contamination of platelet concentrates
correlate with the secretion of pro-inflammatory cytokines, IL-8, IL-1 and IL-6, the latter,
having been demonstrated to activate platelets in vitro and in vivo.

Ensuring low levels of leukocytes not only prevents platelet activation resulting from pro
inflammatory cytokine secretion but also reduces the risk of febrile transfusion reactions.

It is not possible to remove leucocytes from whole blood by filtration before the buffy
coat product is derived. Pooled platelet bags come equipped with special filters which
enable the filtration of suspended platelets prior to pooling of the final product. Due to
high levels of both leucocytes and erythrocytes having been noticed during the initial cell
counts, and in order to ensure that test samples complied with the required parameter
for leucocytes presence, another additional filtration of the pooled platelets was
performed Although every effort is made during platelet concentrate preparation to
minimize the number of contaminating blood cells, the preparations may still contain
variable amounts of erythrocytes and leucocytes. When counts were again taken for the
filtered PCs these were subsequently found to conform to acceptable parameters.

Heavy blood stained pooled platelets were discarded.


28
4.1.2 Blood Gases

It is a requirement that the level of blood gases should stay at a constant level, ideally as
on Day 0. This in practice does not occur but rather there are constant fluctuations in the
concentrate during its normal storage period of five days. For PCs to remain suitable for
transfusion on Day 7 it would be reasonable to require that the level of blood gases
should remain within the ranges found from Day 0 to Day 5.

pO
2
gas analyzer readings are shown at Table 3.1. On comparing this data using Paired
T-Tests for Day 0 and Day 3, Day 0 and Day 5, Day 0 and Day 7, and Day 5 and Day 7,
it is noted that there is no significant change in values. The results shown in Appendix 3
Section 3.1 Tables 3.3, 3.6, 3.9 and 3.12 confirm that the results of the Kolomongrov
Smirnov test were normal distributed, whilst the results shown in Appendix 3 Section 3.2
Tables 3.26, 3.44, 3.62 and 3.80 confirmed the hypothesis that there was no significant
difference in values for recorded pO
2
levels.

Similar statistical comparison of pCO
2
readings from tested samples and for the same
paired testing days showed no significant change in levels as Sig. values were invariably
greater than 0.05.

The fact that no significant changes in pO
2
and pCO
2
levels were recorded during this
study mitigates in favour of the assumption that PCs would retain a constant pH
throughout the seven day storage period.

The results obtained also confirmed the suitability of the storage procedure and the
quality of the material of the storage containers used, which allowed for free exchange of
oxygen and carbon dioxide between the outside air and the suspended platelets,
permitting a constant gas exchange of the PCs.

29
4.1.3 Bacterial Cultures

It is invariably essential that all blood products should be free of bacteria. Contaminated
products if transfused would lead to septicaemia and transfusion reactions which,
depending on the infecting organism, can be fatal.

As with all similar Institutions, bacteriological controls are quite extensive at the National
Blood Transfusion Centre. Care is taken during the blood donation process to ensure
that the site of puncture is well disinfected, bags are suitably inspected before use and
equipment employed in PC production has been inspected and validated for use. QA
procedures are in place at the Centre whereby random samples from the platelet stocks
are tested on Day 2. Should platelets remain in stock after Day 5 some units are
retained to be tested on Day 7 for bacterial growth. To date no bacterial contamination
has ever been detected at the Centre, and so no fatalities from bacterial contamination
have been recorded.

In the current study, no bacteria were cultivated when culturing on Days 5 and 7, except
for one PC which was intentionally inoculated to serve as a positive control throughout
the study.

Blood culturing being a very reliable means in the detection of bacterial contamination,
the procedure was utilized on all PCs used in the study, although as stated previously it
is not normal practice to test each concentrate. The volume of the sample taken from
each concentrate for this purpose is quite significant, 15mLs for each set of two culture
bottles. This naturally had an affect on the quality and quantity of the final product as it
decreased the volume and ultimately the number of platelets available in the pool. This
sampling procedure could therefore affect the volumetric and platelet concentration
characteristics of the respective units of concentrates, as a result of which the pH value
of the concentrates might, as a consequence, change.

30
4.1.4 Glucose and Lactate Dehydrogenase levels

Platelets are living cells, their metabolism requiring the take up of glucose as a nutrient
for the cells to survive. Glucose levels in PCs are subject to diminishing change as
glucose from the PAS is consumed. Glucose will also be consumed by bacteria if these
are present in the concentrate, however consumption is so high in this regard that
change in glucose levels in the PC is dramatic as this would fall suddenly and
appreciably. In the course of this study, as no bacteria was detected in the sampled
PCs, this incidence did not occur. Recorded drops in glucose levels could therefore only
be attributed to cell metabolism during storage.

Glucose has a dual role in cell metabolism first as a substrate for glycolysis and
subsequently, for the carboxylic acid cycle and oxidative processes resulting from the
cell metabolism.

In glycolysis the glucose is converted into lactic acid. High levels of lactic acid content in
the PCs lowers the pH values and as result the PCs would be unsuitable for transfusion.
Again, during the carboxylic acid cycle and the resulting oxidative process, carbon
dioxide and water are obtained. As a result of the concentration of carbon dioxide the pH
value is again lowered thus further rendering the PCs unsuitable for transfusion.

For PCs to be adequate for transfusion at Day 7 it has to be ascertained that the pH
levels of the PCs remain within the recommended range as shall be discussed in
subsequence instance. A change in pH would be caused by the by-products being
produced and released by the platelets as a result of the take up and break down of the
glucose during storage.

Verhoeven et al. (2005) in a study on mitochondrial membrane potential in human
platelets mentioned that deterioration of platelet quality induced by the consumption of
glucose during storage is accompanied by an increase in lactate production.

Normally high levels of LDH may indicate the presence of platelet lesions, particularly if
there is no swirling effect. This is also confirmed if there is an imbalance in the gas
31
exchange where the O
2
levels are increased and those of CO
2
are diminished which in
turn indicates that platelet metabolism has been terminated.

Paired T-Tests for the data collected in this instance having yielded p-values of 0.00, i.e.
less than 0.05, show that there is a substantial significant difference of LDH production
and glucose consumption between Days 0 and 7. This is shown in data at Appendix 3
Section 3.2 Tables 3.82 and 3.84. Such a change mitigates towards an expected
lowering of lowering of the pH which, if substantial, could result in platelet lesions.

4.1.5 Platelet counts and indices

The quantity of platelets in PCs has to be within standard defined range. Platelet lesions
will reduce the quantity of platelets and if this reduction is substantial the PC would not
be suitable for transfusion. As stated earlier platelet lesions may occur for several
reasons during storage and would result in a lowering of the platelet counts.

It was observed during the course of the study and as shown in Table 3.1 that reductions
in platelet counts were occurring. Results of Paired T-Tests are shown in Appendix 3
Section 3.2 Tables 3.14, 3.32, 3.50, and 3.68. Analysis of counts for Day 0 to Day 3, Day
0 to Day 5, Day 0 to Day 7 and between Day 5 and Day 7 gave Sig. values greater than
0.05 which indicated that the noted reductions were not significant and that for research
analysis purposes no significant change had occurred.

Slichter and Harker (1970), and Murphy (1985), showed that falls in platelet count are of
prime detriment to pH. On the other hand more recent study by Singh et al. (2003) could
observe no such correlation. The latter study however reportedly demonstrated that
consideration of platelet indices namely PDW, MPV and PLCR, in addition to platelet
counts constitute further parameters in the assessment of the quality of PCs when the
pH is above 6.8 at any time.

The indices measured in the current study are shown at Table 3.1 The Paired T-Tests
for this data indicated that these indices remained relatively constant, and without any
significant change, when compared from Day 0 to Day 3, Day 0 to Day 5, Day 0 to Day 7
and between Day 5 and Day 7. Sig. values were greater than 0.05 as shown in Appendix
32
3 Section 3.2 Tables 3.16, 3.18, 3.20, 3.34, 3.36, 3.38, 3.52, 3.54, 3.56, 3.70, 3.72 and
3.74.

The statistically no significant change results recorded for platelet counts therefore
meant that pH would not be affected and indices checks suggested that there were no
appreciable platelet lesions occurring during the seven day storage period.

4.1.6 pH measurement

It is essential that during storage the pH level of PCs remains within an acceptable range
of 6.4 - 7.4 for PCs to retain platelet function and thus be suitable for use. pH could be
affected by gas exchange, platelet metabolism, platelet concentration and bacterial
contamination.

From the results of the tests carried out for this study one can note that the average pH
levels of samples of the PCs tested on all due days of testing were slightly higher than
the maximum value of the accepted parameter range. These results are given at Table
3.1. Results of paired T-Tests for this test data, as shown at Appendix 3 Section 3.2
Tables 3.22, 3.40, 3.58 and 3.76 suggest that there was no significant change in pH
values over the testing period.

These observations correlate previous conclusions derived from the analysis of blood
gases, platelet concentration and the absence of bacterial contamination, although for
the latter instance Myhre et al (1985) had shown that pH may still remain unchanged.

These recorded measurements do not correlate with platelet metabolism results that had
suggested that a lowering in pH was expected. The fact that pH levels remained
constant indicate that the ComposolPS was performing well as a buffer and its
buffering capacity had not been compromised or exhausted at the end of the seventh
day. Another factor, as mentioned previously, that contributed to the maintenance of a
constant pH in the PCs over the seven day testing period may have been the quality of
the storage containers, which allowed for free exchange of oxygen and carbon dioxide
between the outside air and the suspended platelets, permitting a constant gas
exchange of the PCs.
33

It must be pointed out that at a pH value grater than 7.4 the tested PC units had pH
levels that where somewhat on the higher side of the pH scale. If these units had not
been used for the purpose of the study they would have been set aside for use only in
an extreme emergency when no platelets would have been available for transfusion.
The question arises as to whether significant change in pH values would be recorded if
the PC units sampled had lower pH values at Day 0.

4.1.7 Platelet Factor IV (PF4)

Various researchers have commented about the incidence of platelet lesions during PC
preparation and storage despite technological advances in platelet storage conditions.

Platelet activation can occur early in the process of platelet preparation. When platelets
become activated they release PF4, as a platelet specific protein, which would have
previously been stored in alpha granules, the level of activation being directly correlated
to the levels of PF4 released. This test therefore was intended to measure the quantity
of PF4 that might have been released in the PC samples.

As already mentioned difficulties were encountered in the procurement of whole blood in
sufficient quantities to obtain the twenty PC samples required in this study concurrently.
The sampling and testing procedure therefore had to be spread over a period of six
weeks. Consequently the supernatant that had to be extracted for testing on the due
days of the testing period had to be stored for the duration of the testing period at a
temperature of -20C and until all the required eighty supernatant samples had been
collected. It would not have been possible to programme this test otherwise given that,
due to financial limitations, only one Micro Elisa plate could be procured for this test.

The manufacturers procedure for the utilization and reading of the micro plate was duly
followed. The test results were completely inconclusive as shown at Appendix 2 Section
2.3 Table 2.6. Test results were out of order except for three random readings for Days
0, 5 and 7 for three different PC units. The fact that these three readings were obtained
suggests that these samples had a very low concentration of PF4 which in term could be
indicative of low platelet activation.
34
This result was duly analyzed and considered. The test inherently consists of measuring
the absorbance of light, using a spectrophotometer, by the treated substance lining the
walls of the wells of the micro plate. Concentration is proportional to absorption.
Consequently testing a high concentrate of a substance will result in a higher
absorbance than when testing a lower concentration of the substance.

The manufacturer of the PF4 kit recommended that the Low and High PF4 Control
substances provided in the kit be diluted two-fold. The manufacturer also suggested that
test samples could be diluted two - or five - fold, and if a high concentrate of PF4 was
suspected samples could be diluted ten - or twenty - fold.

During cell counts no platelet aggregates were observed at any time of the testing period
and it was assumed that during PF4 testing no platelet activation would have been
present. As a result, and in accordance with procedures at the Centre, samples were
diluted to the same proportion as the Controls provided.

When the colour of the test plate wells treated with the test samples was visually
compared to the colour of the wells treated with the Calibrators and Controls, it was
noted that the intensity of the colour of the test samples was very high compared to that
of the Calibrators and Controls. It is therefore highly probable, in the absence of any
other plausible reason, and seeing that practically eighty samples were involved, that
false PF4 results were obtained due to an inadequate dilution factor.

In any situation, it is necessary that tests on stored platelets should show that platelets
remain inactivated during storage as activation has to be in vivo after transfusion.

35
4.2 Limitations

Blood donations are generally sporadic and unallocated stocks of blood products
intended to be kept in reserve end up being allocated as well. The major difficulty
encountered during this study therefore was the unavailability of sufficient amounts of
surplus pooled platelets which could be made available for this study and which limited
the supply of samples.

The situation was further aggravated when in the course of the study the Centre
changed the supplier of blood collection bags in view that instances of bacteriological
contamination had arisen. The use of the new bags required the adoption of new pooling
procedures which were initially resulting in low platelet count pools being obtained. Units
intended for platelet production were therefore needed for validation by the QA thus
reducing further the availability of PC units for analysis in this study.

4.3 Conclusion

In 2005, a total of 14377 donations were available at the Centre. From these 628 pooled
platelets were produced, of which 74 were discarded due to their expiring of the five day
storage period. Although this amount of PCs may be considered somewhat on the low
side if spread over a twelve month period, on the other hand this amount can be deemed
appreciable when considering that on two or three occasions a month PCs are required
for the treatment of patients and stocks are not available.

Tests carried out in this study, with the exception of the PF4 which did not yield results,
in principle confirm that it is possible for PCs to be stored for seven days within the
storage parameters and capacities at the Centre, this especially in view that new
equipment has been installed recently for platelet monitoring.

Before such change in storage practice may be introduced however further study and
testing, especially in the field of platelet activation monitoring and bacterial screening of
PCs between the fifth and the seventh day of storage is required.

36
The sample size used in this pilot study is not sufficiently populated to provide a
complete picture of the resulting situation and validation tests have to continue for some
time until testing of a sufficient sample size has been achieved.

At the same time it shall also be appropriate to watch out for new developments in
alternative PC storage techniques, as progressing in recent years, that would enable
cryropreseved platelets to be stored for up to 10 years under appropriate storage and
clinical administration procedures.

It shall invariably remain of greater clinical benefit to patients undergoing this kind of
treatment, if PCs could be transfused after a storage time that is as short as possible.
Although increasing PC shelf-life is an important step towards helping to increase the
number of units readily available for increasing patient demands, the promotion of
educational campaigns for more donors to come forward shall invariably remain the best
means of ensuring that PCs availability is commensurate with demand.
37





Chapter 5
References
38
Journals

1. Adcock, D. M., Jensen, R., Johns, C. S. and Macy, P. A. (2002) Coagulation
Handbook. Esoterix Coagulation.
2. Aleil, B., Isola, H. and Cazenave, J. P. (2005) Effect of L-carnitine during storage
of buffycoat platelet concentrates in an additive solution. Vox Sanguinis, 89, 59-
60.
3. Beckers, E. A. M. (2005) Bacterial contamination of platelet components:
remaining risks after introduction of bacterial screening. Vox Sanguinis, 89, 116
4. Bergmeier, W., Burger, P., Piffath, C., Nieswandt, B. and Wagner, D. D. (2003)
Inhibition of metalloproteases during platelet storage leads to better platelet
recovery and improved platelet function. Journal of Thrombosis and
Haemostasis; 1 Supplement July: Abstract number: OC156.
5. Beutler, E. and Kuhl, W. (1980) Platelet glycolysis in platelet storage IV. Effect of
supplemental glucose and adenine. Transfusion, 20:97-100.
6. Bock, M., Rahrig, S., Kunz, D., Lutze, G. and Heim, M .U. (2002) Platelet
concentrates derived from buffy coat and apheresis. Biochemical and functional
differences. Transfusion Medicine, 12, 317324.
7. Bode, M., Glaser, A., Pfosser, A., Schleuning, M., Heim, M. U., Mempel, W.
(1993) Storage of single-donor platelet concentrates: metabolic and functional
changes. Transfusion, 33, 311-315.
8. Boekhorst, P. A. W., Beckers, E. A. M., Vos, M. C., Vermeij, H. and van Rhenen,
D. J. (2005) Clinical significance of bacteriologic screening in platelet
concentrates. Transfusion, 45, 514-519.
9. Boomgaard, M. N., Gouwerok, C. W., Homburg, C. H., de Groot, G., IJsseldijk,
M. J. and de Korte, D. (1994) The platelet adhesion capacity to subendothelial
matrix and collagen in a flow model during storage of platelet concentrates for 7
days. Thrombosis and Haemostasis, 72, 611616.
10. Brecher, M. E., Holland, P. V., Pineda, A. A., Tegtmeier, G. E. and Yomtovian, R
(2000) Growth of bacteria in inoculated platelets: implications for bacteria
detection and the extension of platelet storage. Transfusion, 40, 1308-1312
11. Cardigan, R. and Williamson, L. M. (2003) Quality of platelets after storage for 7
days. Transfusion Medicine, 13, 173187.
39
12. Castro, E., Bueno, J. L., Barea, L. and Gonzalez, R. (2005) Feasibility of
implementing an automated culture system for bacteria screening in platelets in
the blood bank routine. Transfusion Medicine, 15, 185195.
13. Currie, L. M., Lichtiger, B., Livesey S. A., Tansey, W., Yang, D. J. and Connor, J.
(1999) Enhanced circulatory parameters of human platelets cryopreserved with
second-messenger effectors: An in vivo study of 16 volunteer platelet donors. Br
J Haematol, 105, 82631
14. de Wildt-Eggen, J. and Gulliksson, H. (2003) In vivo and in vitro comparison of
platelets stored in either synthetic media or plasma. Vox Sanguinis 84, 256264
15. Dijkstra-Tiekstra, M. J., Pietersz, R. N. I. and Huijgens, P. C. (2004) Correlation
between the extent pf platelet activation in platelet concentrates and in vitro and
in vivo parameters. Vox Sanguinis 87, 257-263.
16. El Golli, N., Issertial, O., Rosa, J.P. and Briquet-Laugier, V. (2005) Evidence for a
granule targeting sequence within platelet factor 4. J Biol Chem. 280(34):30329-
35.
17. Fijnheer, R., Pietersz, R. N., de Korte, D., Gouwerok, C. W., Dekker, W. J.,
Reesink, H. W. and Roos, D. (1990) Platelet activation during preparation of
platelet concentrates: a comparison of the platelet-rich plasma and the buffy coat
methods. Transfusion, 30, 634638.
18. Fijnheer, R., Veldman, H.A., Van den Eertwegh, A. J. M., Gouwerok, C. W. n.,
Homburg, C. H. E., Boomgaard, M. N., De Korte, D. and Roots, D. (1991) In vitro
evaluation of buffy-coat derived platelet concentrated stored in synthetic medium.
Vox Sanguinis, 60, 16-22.
19. Fijnheer, R., Modderman,P. W., Veldman, H., Ouwehand, W. H., Nieuwenhuis,
H. K., Roos, D. and de Korte, D. (1990) Detection of platelet activation with
monoclonal antibodies and flow cytometry. Transfusion, 30, 2025.
20. Filip, D. J., Eckstein, J. D. and Sibley, C. A. (1975) The effect of platelet
concentrate storage temperature on adenine nucleotide metabolism. The
American Society of Hematology, 45(6), 749-756.
21. George, J. N., Pickett, E. B. and Heinz R. (1988) Platelet membrane changes
during the preparation and storage of platelet concentrates. Transfusion, 28, 123-
126.
40
22. Gottschall, J. L., Johnston, V. L., Rzad, L., Anderson, A. J. and Aster, R. H.
(1984) Importance of white blood cells in platelet storage. Vox Sanguinis, 47(2),
101-107.
23. Gulliksson, H. (2000) Additive solutions for the storage of platelets for transfusion
Transfusion Medicine, 10, 257-264.
24. Gulliksson, H. (2000) Storage of Platelets for Transfusion in Additive Solutions:
Effects of Different Factors and Compounds. Infusion Therapy and Transfusion
Medicine, 27, 90-93
25. Hagberg, I. A., Akkok, C. A., Lyberg, T. and Kjeldsen-Kragh, J. (2000) Apheresis-
induced platelet activation: comparison of three types of cell separators.
Transfusion, 40, 182192.
26. Hillyer, C. D., Josephson, C. D., Blajchman, M. A., Vostal, J. G., Epstein, J. S.
and Goodman, J. L. (2003) Bacterial Contamination of Blood Components:
Risks, Strategies, and Regulation. Hematology, 575-589.
27. Hogman, C (1999) Storage of blood components. Current Opinion in
Hematology, 6(6), 427.
28. Hunter, S., Nixon, J. and Murphy, S. (2001) The effect of the interruption of
agitation on platelet quality during storage for transfusion. Transfusion, 41(6),
809.
29. Jensen, R. and Ens, G. E. (1992) Platelet activation markers. Clinical
Hemostasis Review. 6(9), 1.
30. Kaplan, K. L. and Owen, J. (1981) Plasma levels of -thromboglobulin and
platelet factor 4 as indices of platelet activation in vivo. Blood, 57: 199-202.
31. Karnicki, K., Johnson, C., St Cyr, J., Ericson, D. and Rao, G (2003) Platelet
storage solution improves the in vitro function of preserved platelet concentrate.
Vox Sanguinis, 85, 262-266.
32. Kilkson, H., Holme, S. and Murphy, S. (1984) Platelet metabolism during storage
of platelet concentrates at 22
o
C. Blood, 64, 406-414.
33. Klinger, M. H., Josch, M. and Kluter, H. (1996) Platelets stored in a glucose-free
additive solution or in autologous plasma - an ultrastructural and morphometric
evaluation. Vox Sanguinis, 71, 1320.
34. Knutson, F., Alfonso, R., Dupuis, K., Mayaudon, V., Lin, L., Corash, L., Hgman,
C. F. (2000) Photochemical inactivation of bacteria and HIV in buffy-coat-derived
41
platelet concentrates under conditions that preserve in vitro platelet function. Vox
Sanguinis, 78, 209216.
35. Kostelijk, E. H., Gouwerok, C. W., Veldman, H. A. and de Korte, D. (2000)
Comparison between a new PVC platelet storage container (UPX800) and a
polyolefin container. Transfusion Medicine, 10(2), 131-139.
36. Kurt Yuksel, M., Arat, M., Arslan, O., Beksac, M. and Lhan, O. (2003) Autologous
Platelet Collection and Storage to Support Thrombocytopenia in a Leukemia
Patient with Platelet Alloimmunization Undergoing Chemotherapy. Turk J
Haematol, 20(4), 233-236
37. Lazarus, H. M., Herzig, R. H., Warm, S. E. and Fishman, D. J. (1986)
Transfusion experience with platelet concentrates stored for 24 to 72 hours at 22
C. Importance of storage time. Transfusion, 26, 131-135.
38. Lee, C. K., Ho, P. L., Chan, N. K., Mak, A., Hong, J. and Lin, C. K. (2002) Impact
of donor arm skin disinfection on the bacterial contamination rate of platelet
concentrates Vox Sanguinis, 83, 204208.
39. Leytin, V., Allen, D. J., Hannach, B. and Freedman, J. (2003) Extension of
human platelet storage up to 78 days does not reduce their in vivo viability in a
rabbit model. Journal of Thrombosis and Haemostasis; 1 Supplement 1 July:
Abstract number: P0070.
40. Lin, L., Londe, H., Janda, J. M., Hanson, C. V. and Corash, L. (1994)
Photochemical inactivation of pathogenic bacteria in human platelet
concentrates. Blood, 83, 26982706.
41. Liu, H., Yuen, K., Chen, T. S., Lee, K., Chua, E.K., Ho, P. and Lin, C. (1999)
Reduction of platelet transfusion-associated sepsis by short-term bacterial
culture. Vox Sanguinis, 77, 15.
42. Lopez-Plaza, I. (2001) Evaluation and Management of Platelet Refractoriness.
Transfusion medical updates. The institute for transfusion medicine, ww.itxm.org
43. McDonald, C. P., Colvin, J., Robbins S. and Barbara, J. A. J. (2005) Use of a
solid-phase fluorescent cytometric technique for the detection of bacteria in
platelet concentrates. Transfusion Medicine, 15, 175183.
44. Metcalfe, P., Williamson, L. M., Reutelingsperger, C. P., Swann, I., Ouwehand,
W. H. and Goodall, A. H. (1997) Activation during preparation of therapeutic
platelets affects deterioration during storage: a comparative flow cytometric study
of different production methods. British Journal of Haematology, 98, 8695.
42
45. Mohammadi, T., Pietersz, R., Vandenbroucke-Grauls C., Savelkoul, P. and
Reesink, H. (2005) Detection of bacteria in platelet concentrates: comparison of
broad-range real-time 16S rDNA polymerase chain reaction and automated
culturing. Transfusion, 45, 731-736.
46. Mrowiec, Z., Oleksowicz, L., Dutcher, J., De Leon-Fernandez, M., Lalezari, P and
Puszkin, E. (1995) A novel technique for preparing improved buffy coat platelet
concentrates. Blood Cells, Molecules, and Diseases, 21(3): 25-33.
47. Murphy S. (1985) Platelet storage for transfusion. Seminars in Hematology 22,
165177.
48. Murphy, S., Shimizu, T. and Miripol, J. (2000) Platelet storage for transfusion in
synthetic media: further optimization of ingredients and definition of their role.
Blood, 86(10), 3951-3960.
49. Myhre, B.A., Demianew, S. H., Yoshimori, R. N., Nelson, E. J. and Carmen, R. A.
(1985) pH changes caused by bacterial growth in contaminated platelet
concentrates. Annals of Clinical and Laboratory Science, 15(6), 509-514.
50. Ness, P., Braine, H.,King,K., Barrasso, C., Kickler,T., Fuller, A. and Blade, N.
(2001) Single-donor platelets reduce the risk of septic platelet transfusion
reactions. Transfusion, 41, 857-861.
51. Rinder, H. M., Murphy, M., Mitchell, J. G., Stocks, J., Ault, K. A. and Hillman, R.
S. (1991) Progressive platelet activation with storage: evidence for shortened
survival of activated platelets after transfusion. Transfusion, 31, 409414.
52. Ringwald, J., Haager, B., Krex, D., Zimmermann, R., Strasser, E., Antoon, M., De
Schrijver, E. and Eckstein, R. (2006) Impact of different hold time before addition
of platelet additive solution on the in vitro quality of apheresis platelets.
Transfusion, 46, 942-948.
53. Ringwald, J., Walz, S., Zimmermann, R., Zingsem, J., Strasser, E., Weisbach, V.
and Eckstein, E. (2005) Hyperconcentrated platelets stored in additive solution:
aspects on productivity and in vitro quality. Vox Sang, 89, 11-8.
54. Ringwald, J., Zimmermann, R. and Eckstein R. (2006) The New Generation of
Platelet Additive Solution for Storage at 22 degrees C: Development and Current
Experience. Transfusion Medicine Rev, 20,158-64.
55. Sacher, R. A., Kickler, T. S., Schiffer, C. A., Sherman, L. A (2003) Management
of patients refractory to platelet transfusion. Arch Pathol Lab Med. 127, 409-414.
43
56. Singh, H., Chaudhary, R. and Ray, V. (2003) Platelet indices as quality markers
of platelet concentrates during storage. Clinical Laboratory Haematology 25, 307-
310.
57. Slichter S. J. (1998) Optimizing platelet transfusions in chronically
thrombocytopenic patients. Semi hematol, 35, 269-78.
58. Slichter S. J. and Harker L. A. (1970) Preparation and storage of platelet
concentrates. British Journal of Haematology, 34, 403418.
59. Solberg, C., Holme, S. and Little, C. (1986) Morphological changes associated
with pH changes during storage of platelet concentrates. Beitr Infusionther Klin
Ernahr, 15, 107-17.
60. Taylor, M.A., Tandy, N.P. and Fraser, I.D. (1983) Effect of new plastics and
leukocyte contamination on in vitro storage ofplatelet concentrates. J Clin Pathol;
36, 1382-1386.
61. Tong, L. and Mendez, M. (2002) Therapeutic Considerations in the Management
of Patients with Heparin-Induced Thrombocytopenia. Prog Cardiovasc Nurs,
17(3), 142-147.
62. Triulzi, D. J., Kickler, T. S. and Braine, H.G. (1992) Detection and significance of
alpha granule membrane protein 140 expression on platelets collected by
apheresis. Transfusion, 32, 529533.
63. Vadhan-Raj, S., John, J., Kavanagh, J.J., Ralph, S. and Freedman, R. S. (2002)
Safety and efficacy of transfusions of autologous cryopreserved platelets derived
from recombinant human thrombopoietin to support chemotherapy-associated
severe thrombocytopenia: a randomised cross-over study. The Lancet, 359,
2145-2152.
64. Van der Meer, P. F., Pietersz, R. N. I. and Reesink, H. W. (2001) Comparison of
two platelet additive solutions. Transfusion Medicine, 11, 193-197.
65. van der Meer, P. F., Pietersz, R. N. I. and Reesink, H. (2001) Leucoreduced
platelet concentrates in additive solution: an evaluation of filters and storage
containers. Vox Sang, 81(2), 102-7.
66. van der Meer, P. F., Pietersz, R. N. I. and Reesink, H.(2004). Storage of platelets
in additive solution for up to 12 days with maintenance of good in-vitro quality.
Transfusion; 44:1204-1211.
67. Van Rhenen, D. J., Gulliksson, H., Cazenave, J. P., Pamphilon, D., Davis, K.,
Flament, J. and Corash, L. (2004) Therapeutic efficacy of pooled buffy-coat
44
platelet components prepared and stored with a platelet additive solution.
Transfus Med,14, 289-95.
68. Verhoeven, J., Verhaar, R., Gouwerok, E. and de Korte, D. (2005) The
mitochondrial membrane potential in human platelets: a sensitive parameter for
platelet quality. Transfusion, 45, 82-89.
69. Washitani, Y., Irita, Y., Yamamoto, K., Shiraki, H., Kiyokawa, H., Maeda, Y.,
Yoshinari, M. (1988) Prevention of acquired defects in platelet function during
blood processing. Transfusion, 28, 571- 575.
70. Yazer, M. H. and Triulzi, D. J. (2005) Use of a pH meter for bacterial screening of
whole blood platelets. Transfusion, 45, 1133-1137.
71. Zbigniew R. M., Oleksowicz, L., Dutcher. J. P., De Leon-Fernandez, M., Lalezari,
P. and Puszkin, E. G. (1995) A novel technique for preparing improved buffy coat
platelet concentrates. Blood Cells, Molecules, and Diseases, 21(3), 25-33.

Standard

Guide to the preparation, use and quality assurance of blood components 11
th
Edition,
Council of Europe Publishing, Chapter 13 pg 121-126.

Web sites

www.biomed.brown.edu
http://biomed.brown.edu/Courses/BI108/BI108_2005_Groups/10/webpages/platele
tslink.htm#platsub

45






Appendix 1

46
Note: All procedures, unless otherwise stated, have been carried out in accordance with
the standard operating procedures in use at the National Blood Transfusion Centre, St
Lukes Hospital Malta.

1.1 Preparation of pooled platelets using platelet additive solution (ComposolPS)

1.1.1 Composition of ComposolPS

ComposolPS 300mLmass340g

Composition of ComposolPS per litre:

Sodium chloride 5.26g
Sodium glucanate 5.02g
Sodium acetate anhydrous. 2.22g
Potassium chloride 0.373g
Magnesium chloride hexahydrate 0.305g
Sodium citrate 3.213g
pH 7.2 (7.0-7.4)
Osmolality Approx. 305mOsm/kg

1.1.2 Procedure

It is necessary to ensure that the temperature of the centrifuge is between
+20C to +24C. If the temperature is not within this range a pre- run is
performed.
It is essential that a maximum time of 24hours has not elapsed from the
preparation of the buffy coats being utilized for pooling into platelet units.
Five buffy coat units must be used to pool one unit of platelets, and preferably
these units would all be Rhesus negative.
A transfer bag is attached to the ComposolPS using a sterile tubing
connecting device. The transfer bag is placed on the balance and the tare
facility used. 100g of platelet additive solution is then added.
47
Five units of screened buffy coat units and ComposolPS transfer bag are
attached by the sterile docking device to the platelet transfer bag set with an
already attached leukocyte removal filter.
All docking connections of the buffy coat are opened, making sure that all
clamps of the buffy coats are open. All buffy coats are drained into the
platelet transfer bag.
Using ComposolPS, the buffy coat is washed several times, passing this
additive solution from one buffy coat to another, until it finally ends in the
platelet transfer bag (600mL).
The same process is repeated until all buffy coats and the entire quantity of
ComposolPS ends up in the platelet transfer bag.
Air is removed from the pooled platelet transfer bag by gently pressing with
both hands until all the air is expelled from the bag.
This pooled unit is sealed using the dielectric tube sealer.
The sealed pooled unit is placed in the centrifuge bucket. The filter is placed
horizontally between the final storage bag and the pooled unit transfer bag
and balance.
The balanced buckets are placed in the Hettiech centrifuge, the rotor shield
being securely attached and the lid latched. The required program is selected
and run. For platelet production, program 5, is used. This utilises a setting for
a light speed of 1240rpm for 9 minutes and 30 seconds. It has to be ensured
that the brakes are switched off.
After centrifuging the bucket is carefully removed. The plastic valve is broken
and by utilizing the manual separator, platelets are separated from the red
cells which are directly passing from the leukocyte removal filter to end up in
the storage bag.
It is good practice to allow the red cells to pass from both sides of the filter, so
that the procedure yields as much platelet concentrates as possible.
Once the platelet concentrates are in the final storage bag, the bag is sealed
using the dielectric tube sealer.
The pooled platelets are labelled with a pool number and placed in the
Helmer platelet agitator for storage.

48
1.1.2.1 Alternative method

The platelets are centrifuged at a speed of 1100rpm for 10 minutes and separated using
automated separators set to a specific program which allows a gentle compression.

1.1.3 Storage

It is necessary to store platelets under conditions that guarantee their
viability, thus ensuring that homeostatic activities are optimally preserved.
Plastic bags intended for platelet storage must be sufficiently permeable to
gases to ensure availability of oxygen to platelets. The amount of oxygen
required is dependent on the concentration of the platelets in the product.
The volume of the dilution fluid must be large enough so that the
concentration of the platelets is less than 1.5 X 10
9
/mL and that the pH of the
platelet product remains continuously between 6.4 and 7.4 at 22
0
C.
Agitation of platelets must be such as to permit a good availability of oxygen
to the platelets, but at the same time being as gentle as possible as to ensure
that the structure of the platelets is conserved.
Containers used to transport platelets should be kept open.

1.2 Platelet sampling

The platelet transfer bags come equipped with 3 satellite bags attached to them.
If more than one sample is taken the satellite bag is labelled accordingly.
To sample, the clip attached to the desired satellite bag is opened. This
allows the platelets trapped in the tubing during processing to mix with the
pool.
The clip is again closed and the platelet mixed gently so as to obtain a
representative sample.
The clip leading to the satellite bag is opened once again, the bag tilted
slightly and a few mLs allowed to enter the satellite bag. The clip is again
closed.
49
Using a seal generator device, a seal is obtained just above the position of
the clip, enabling the satellite bag to be detached by cutting the seal in the
middle.
A check for any leakages in the platelet pool is made before returning this to
the agitator.

If the satellite bags are not available, a connecting sealing device may be used to attach
a transfer bag to the pool. After breaking the connecting seal, the same procedure as
above is then followed.

1.3 CBC testing using Sysmexautomated cell counter

Whether an electronic cell counter or one of the non-automated manual methods is
used, the steps covered by the procedure include diluting the blood sample
quantitatively by using pipettes and diluents, determining the number of cells in the
diluted sample, and converting the number of cells in the diluted sample to the final
result.

1.3.1 Equipment

Borosilicate test tubes
Test tube caps
Mechanical rotor
Sysmex cell counter

1.3.2 Procedure

Daily controls must be run to ascertain that the Sysmex is working correctly,
the results being checked to verify that the readings are within the acceptable
range.
3-4mLs of platelet concentrate are gently transferred from a transfer or
satellite bag to the borosilicate test tube, borosilicate test tubes being used to
prevent platelet aggregation.
50
The test tube is capped and placed on the mechanical rotor which ensures a
continuous mixing of the contents.
When the pool number is entered the machine is ready to aspirate the
sample.
The cap is removed and the test tube fed to the aspirator syringe. Care is
taken to make sure that the syringe is half way down in the sample to ensure
a proper aspiration.
After aspiration the sample is removed. The Sysmex counter carries out the
count and the results are issued as a print out.

1.4 Manual Leucocyte Counts using the Nageotte Chamber

1.4.1 Principle

This counting method describes a procedure for visual counting of leukocytes present in
leucodepleted blood or packed red cell products.

The sensitivity of this method is 0.1 leukocytes/L.

1.4.2 Equipment

Nageotte counting chamber with large size cover slips as supplied with
chamber
Turks Solution
10-100 L pipettor
100-1000 L pipettor
10-1000 L pipette tips
12 x 75 mm plastic test tubes
Laboratory microscope
Covered petri dish containing moistened paper

51
1.4.3 Procedure

The test tube is labelled with the appropriate Donation or Pool number.
900 L of freshly filtered Turks Solution are added to the tube.
A 1:10 dilution of the sample is made by adding 100L of the sample to the
appropriately labelled test tube. The pipette tip must be flushed several times
to ensure transfer of sample into the diluent.
The diluted sample is well mixed by shaking the test-tube.
It must be allowed to stand for 10 minutes to lyse red cells.
Carefully the cover slip is placed on a clean, dry Nageotte chamber. The
cover slip must be centred exactly on the chamber.
The diluted sample is mixed once, more and about 600L withdrawn into a
pipettor or disposable pipette.
Without disturbing the cover slip, the Nageotte counting chamber is loaded
until it is full. Loading is from one side of the chamber only to prevent air from
being trapped inside. The cover slip must not be disturbed once the chamber
is loaded.
The chamber is allowed to stand in a damp petri dish for 15 minutes to allow
leukocytes to settle. The sample must be counted within 30 minutes of
charging.
The leukocytes are counted by scanning back and forth across the grided
area using the x10 magnification eyepiece. Further verification of the
leukocytes is carried out by viewing under the x40 magnification eyepiece.
All leukocytes in the area i.e. the 40 rectangles are counted, including the
cells touching the lines.
To calculate leukocytes per L the following formula is used:

cells counted X dilution
Leukocytes / L =

Volume counted (L)


The volume of grided area counted is 50 L
52

To obtain the residual leukocytes in the filtered unit leukocytes/L obtained must be
multiplied by 1,000 to convert the results to leukocytes/mL. This figure is then multiplied
by the volume (mL) of the initial filtered unit to obtain the total residual leukocytes.

1.4.4 Preparation of Turks Solution

Materials needed:
1% Gentian Violet
Glacial Acetic Acid
Distilled water
Measuring flask (100mLs)

Procdure:
1mL of 1% Gentian Violet stain is pipetted into a measuring cylinder.
3mLs of Glacial Acetic Acid are cautiously added, care being taken to avoid
inhaling any fumes of this acid.
The contents in the measuring cylinder are topped up with distilled water to
obtain a final volume of 100mLs.
The solution is mixed thoroughly and stored in an amber brown bottle in a
dark cupboard.

Each day before use it is necessary to sequentially filter part of the stain through a
0.45m sterile syringe filter, followed by a 0.2m sterile syringe filter using a 20 mL
syringe.

53
1.5 Manual Erythrocyte Counts using the Improved Neubauer Chamber

1.5.1 Principle

This counting method describes a procedure for visual counting of erythrocytes present
in pooled and single donor platelets.

1.5.2 Equipment

Improved Neubauer counting chamber with small size cover slips.
100-1000 L pipettor
10-1000 L pipette tips
12 x 75 mm plastic test tubes
Laboratory microscope
Covered petri dish containing damp paper

1.5.3 Procedure

The test tube is labelled with the appropriate Pool number.
The cover slip carefully placed on a clean dry Neubauer chamber. The cover
slip must be centred exactly on the chamber.
About 600L of the unstained and undiluted sample is withdrawn into a
pipettor or disposable pipette.
Without disturbing the cover slip, the Neubauer counting chamber, is loaded
until it is full. Loading is from one side of the chamber only to prevent air from
being trapped. Once the chamber is loaded the cover slip must not be
disturbed.
The chamber is allowed to sand in a damp petri dish for 15 minutes to allow
red cells to settle. Counting of the sample must take place within 30 minutes
of charging.
Using the microscope, the erythrocytes in the four large squares of each
corner and the large square in the middle of the grided area are counted. All
54
erythrocytes in the five squares are counted, including the cells touching all
the grid lines.


Red blood cells are counted under high dry magnification (x40 objective). The central
1mm
2
area of the counting chamber is used. It is best for the area to be located with the
low power objective and than to be viewed under the high power objective.

The cells in 80 of the 1/400 mm squares are counted. This is equivalent to counting the
cells of 1/5
th
of 1 mm
2
(80/400). The volume is determined by multiplying the depth
(0.1mm) by 1/5
th
and is equal to 0.02L.

The preferred method for counting the cells in the 1/5 mm
2
is to count the cells in five of
the 1/25 mm squares. Since each 1/25 mm square contains 16 smaller squares, eighty
1/400 mm squares will have been counted

The calculation of the erythrocyte counts is based on the same principles as those used
for the leucocytes count. The usual blood dilution is 1:200, the area counted is 1/5 mm
2
,
and the depth is 0.1 mm. Due to the fact that only a small amount of erythrocytes are
usually found, generally less than 50, the dilution factor has been omitted from the
formula. Thus the sample blood product to be counted should be not be diluted, in order
to obtain more accurate counts.

Thus, the calculation formula would be


Cells counted in 1/5 mm
2


volume

= Red Blood Cells / L

Where volume = area x depth of the chamber.

55
The National Blood Transfusion Laboratory is not equipped to carry out the remaining
tests in this section and consequently these tests were carried out the Pathology
Department Laboratories at St. Lukes Hospital.

1.6 Blood gases and pH testing

All platelet bags to be tested are selected and removed from the agitator.
To each of the platelet bags a sterile transfer bag is connected using a sterile
connecting device.
The platelet concentrate is mixed gently. The seal made with the sterile
connecting device is opened so as to transfer a sample of platelet
concentrate from the platelet bag to the sterile connected transfer bag. It is
important that the flow of platelet concentrate should move downward to
avoid any possible contamination taking place. While platelets are flowing,
checks for any possible leakage from the tube connection are made. If this
occurs, the platelet concentrate bag should be immediately discarded.
Once the tubing has been filled, the tubing is clamped and resealed by
means of a tube sealer. Whenever it is possible, the seal should be made on
top of the tubing connection previously performed. This will minimise any
damages or unnecessary puncture hole formations in the tubing connection
during handling. Before sealing the tube care must be exercised to leave a
length of tubing of at least 5-6cms protruding from the platelet bag. This
distance is necessary to be able to re-connect another sterile transfer bag for
any further testing.
A 2mLs syringe is labelled with the appropriate Pool number of the platelet
concentrate sample just taken.
The tubing from the platelet concentrate bag is carefully cut away using
scissors. Gentle pressure is applied on the platelet bag to once again
checking for any possible leakage from the remaining tube. If leakage occurs
the platelet bag is discarded.
The protective needle cover of the syringe is removed and the needle is
carefully inserted into the sample tubing. The sampled platelet concentrate is
aspirated into the syringe.
56
Once a volume of about 1mL has been aspirated into the syringe, the needle
is removed from the tubing and immediately covered by its protective cover.
Any empty tubing is discarded.
The syringe is primed, the covered needle removed and immediately a blind
cap is used to recap the syringe. The needle is discarded.
The platelet concentrate bag is returned to the agitator.
pH testing should take place by means of the Blood Gas Analyser as soon as
possible after sampling and at a temperature of 22
o
C.
The Quality Requirement range for the ph of Platelet Concentrates has to be
from 6.8 - 7.4.

1.7 Blood Cultures

1.7.1 Equipment

Sterile syringes
1 set of culture bottles per sample

1.7.2 Procedure

All work should be carried out in a safety cabinet to ensure sterility.
The sealed cap of the culture bottles is broken so as to expose the rubber
membrane.
From the original platelet concentrate bag a sample of 15mLs is collected
using a syringe.
9mLs of the sample are injected into the aerobic culture bottle and 5mLs are
injected into the anaerobic culture bottle.
The cultures are mixed by gently inverting the bottles to ensure complete
mixing of the sample with the medium.
Incubation is carried out at a temperature of 37C.
As a control, one set of culture bottles is inoculated with sterile saline.

57
A system known as the continuous-monitoring blood culture systems (CMBCS)
automatically monitors the bottles for evidence of microorganisms, usually at every 10
minute interval. All CMBCS systems comprise the detection system, incubator and
agitation unit in one apparatus. The data points are collected daily for each bottle and
fed into a computer for analysis.

1.8 Measurement of lactate dehydrogenase

1.8.1 Test principle

LDH catalyzes the conversion of L-Lactate to pyruvate; NAD is reduced to NADH in the
process. The initial rate of NAD formation is directly proportional to catalytic LDH activity,


LDH

Lactate + NAD
+
Pyruvate + NADH
+


and is determined by measuring the increase in absorbance at 340nm.

The process involves the kinetic determination of LDH activity according to the
recommendations of the International Federation of Clinical Chemistry and Laboratory
Medicine (IFCC):

1.8.2 Procedure

All platelet bags to be tested are selected and removed from the agitator.
The sampling procedure is repeated as in the case of sampling for pH
determination described at Section 1.6.
Transfer 4mLs to a plain bottle vaccutainer and centrifuge at 3500rpm for 10
minutes.
The vaccutainer is placed in the Roche Hitachi analyzer and print outs of the
results are issued.

58
1.9 Measurement of Glucose

1.9.1 Test principle

Glucose is oxidized by glucose oxidase (GOD) to gluconolactone in the presence of
atmospheric oxygen. The resultant hydrogen peroxide oxidizes 4-aminophenazone and
phenol to 4-(p-bensoquinone-monoimino)-phenazone in the presence of peroxidase
(POD) The colour intensity of the resulting red dye is directly proportional to the glucose
concentration and can be measured photometrically.


GOD

Glucose + O
2
+ H
2
O Gluconolactone + H
2
O
2


POD
2H
2
O
2
+ 4-aminophenazone + phenol
4-(p-bensoquinone-monoimino)-phenazone + 4H
2
O

The principle of this test is based on an enzymatic colorimetric assay technique.

1.9.2 Procedure

All platelet bags to be tested are selected and removed from the agitator.
The sampling procedure is repeated as in the case of sampling for pH
determination described at Section 1.6.
Transfer 3mLs to a vaccutainer containing sodium fluoride anticoagulant and
centrifuge at 3500rpm for 10 minutes.
The vaccutainer is placed in the Roche Hitachi analyzer and print outs of the
results are issued.

59
1.10 Platelet Factor IV (PF4)

1.10.1 Principle

ELISARA PF4 is a sandwich ELISA designed with affinity purified rabbit polyclonal
antibodies specific for human platelet factor 4. The diluted tested plasma or biological
fluid is introduced in a microwell, coated with affinity purified rabbit polyclonal antibodies
(F(ab')2 fragments) specific for human PF4. When present, this protein is captured onto
the solid phase. Following a washing step, the immunoconjugate, which is an affinity
purified rabbit polyclonal antibody coupled to horseradish peroxidase (HRP), is
introduced, and binds to the free epitopes of immobilized PF4. Following a new washing
step, the peroxidase substrate, Tetramethylbenzidine (TMB) in the presence of
Hydrogen Peroxide (H
2
O
2
), is introduced and a blue colour develops. The colour turns
yellow when the reaction is stopped with Sulphuric Acid. The amount of colour
developed is directly proportional to the concentration of human PF4 in the tested
sample.

1.10.2 Specimen Collection

The specimen is collected in 0.109M citrate anticoagulant containing theophylline,
adenosine and dipyridamole (CTAD tubes).
One third of supernatant is collected following a 30 minute centrifugation at 2,500g at 2 -
8C. The extracted supernatant may either be tested within 4 hours of collection or
stored frozen at 20C or below for up to a period of 6 months. In the event of freezing
extracted supernatant has to be thawed for 15 minutes at 37C just before use. A
thawed specimen must be tested within 4 hours.
ETP (EDTA, theophylline, prostaglandin E1) collected human plasma may also be used.

60
1.10.3 Preparation of sample and controls

The sample must be tested diluted two- (1:2) or five- (1:5) fold in the PF4-Sample
Diluent.

Controls I and II must be tested diluted two fold (1:2) as for plasmas.

1.10.4 Equipment

PF4 Elisa Kit
8-channel or repeating pipette allowing dispensing 50-300 L.
1-channel pipettes at variable volumes from 0 to 20 L, 20 to 200 L and 200
to 1000 L.
Micro ELISA plate washing equipment and shaker.
Micro ELISA plate reader with a wavelength set up at 450 nm.
Distilled water.


1.10.5 Calibration

Using the PF4 Standard (2 mL at 10 IU/mL) provided in the kit, the following standard
solutions are prepared:

PF4 concentration 10 IU/mL 5 IU/mL 2 IU/mL 1 IU/mL 0.5 IU/mL 0 IU/mL
Vol. of PF4 Std at
10 IU/mL
1 mL 0.5 mL 0.20 mL 0.1 mL 0.05 mL 0 mL
Vol. of PF4-Sample
Diluent
0 mL 0.5 Ml 0.80 mL 0.9 mL 0.95 mL 1 mL
Table 1.1: Preparation of standard solutions

The solutions must be mixed gently for complete homogenisation.
The standard dilutions are stable for at least 8 hours at room temperature.

61
1.10.6 Procedure

The lyophilized reagents are reconstituted according to the kit insert. From the
aluminium pouch, the required numbers of strips for the series of measurements to be
performed are removed. The strips are placed in the frame provided. The reagents are
introduced into the different wells of the micro ELISA plate. The various assay steps are
as follows:
200 L PF4 Standard solution, controls and the test sample are added to the
coated wells.
Incubation is 1 hour at room temperature (18 - 25C).
300 L Wash Solution (20 fold diluted in distilled water) are added, followed
by 5 successive washings using the washing instrument.
200 L Conjugate (anti PF4 polyclonal antibody coupled with peroxidase) are
added and restored with 7.5 mL of Conjugate Diluent.
Incubation is 1 hour at room temperature (18 - 25C)
300 L Wash Solution (20 fold diluted in distilled water) are added, followed
by 5 successive washings using the washing instrument.
200 L TMB/H
2
O
2
Substrate are added immediately after the washing.
Incubation is exactly 5 minutes at room temperature (18-25 C).
0.45M 50 L Sulphuric Acid are added following exactly the same time
intervals.
The plate is left to stand for 10 minutes in order to allow the colour to
stabilise.
Absorbance at 450nm is measured.

1.10.7 Construction of calibration curve

On a linear graph paper plot the PF4 concentrations on abscissae (IU/mL)
and the corresponding absorbances (A450) on ordinates.
From the resulting curve, the PF4 concentration for the tested dilution can be
extrapolated. This value then multiplied by the dilution factor (i.e. 2, 5, 10, or
20) to obtain the PF4 concentration in the test sample.

62





Appendix 2
63
2.1 Table of results

2.1.1 Day 0

Sample
No
Weight Volume
Platelet
count
PDW MPV P-LCR
RBC
count
WBC
count
pH pCO
2
pO
2
LDH Glucose
1 155 133 1344 9.1 7.7 11.5 0 0 7.554 13.6 91 91 16
2 197 175 938 8.8 7.6 11 12 0 7.502 9.9 98 134 9.5
3 293 271 1284 9.8 8 13.6 8 0 7.385 25 54 145 19.58
4 257 235 1116 11.1 9 19.9 10 0 7.41 21.3 58 164 17.25
5 238 216 557 9.4 7.9 12.5 0 0 7.29 25.6 157 129 14.33
6 271 247 1887 14 10.4 30 0 0 7.401 23.9 67 143 18.89
7 292 270 1125 11 9.1 18.9 3 2 7.409 19.3 105 154 19.1
8 164 142 993 8.7 7.3 13.4 6 0 7.446 24.1 129 119 12.9
9 250 228 1104 10.1 7.7 11.8 0 5 7.514 21.9 131 133 16.4
10 259 237 1244 9.4 7.4 10.9 0 1 7.398 10.2 96 159 19.7
11 280 258 899 8.9 8 12.9 2 2 7.491 15.6 107 99 17.5
12 276 254 954 12.1 10.3 24.9 1 0 7.431 26.3 169 167 14.7
13 282 260 1752 9.9 7.1 11.6 2 0 7.501 17.2 91 145 13.5
14 273 251 1321 9.8 8.1 13.3 5 3 7.532 9.8 110 168 11.9
15 269 247 1097 11.3 9.4 21.5 0 1 7.338 22.8 154 97 12.1
16 160 138 909 10.6 8.8 17.8 15 0 7.541 14.9 131 110 14.9
17 153 131 901 10.5 8.8 18.4 8 0 7.495 25.8 99 129 16.3
18 225 203 839 12.3 9.7 19.3 0 2 7.471 13.7 118 147 11.2
19 156 134 856 12.2 9.7 18.4 0 1 7.451 22.7 97 126 18.5
20 128 106 1009 13.5 10.4 29.3 1 0 7.409 24.6 147 94 16.1
Table 2.1: Results on Day 0




64
2.1.2 Day 3

Sample
No
Weight Volume
Platelet
count
PDW MPV P-LCR
RBC
count
WBC
count
pH pCO
2
pO
2
LDH Glucose
1 135 113 1210 8.8 7.6 10.5 0 0 7.611 12.3 106 105 14.89
2 147 125 942 9.3 8 13.7 0 0 7.422 11.3 99 169 7.32
3 289 267 1307 9.5 8 13.5 0 0 7.373 24.5 28 152 17.82
4 240 218 1074 11 9 20.3 6 0 7.456 18.9 53 198 15.92
5 180 158 563 9.2 7.8 11.5 0 1 7.513 10.9 87 132 13.2
6 258 236 1329 9.5 8.1 13.6 5 0 7.381 23.1 44 149 17.09
7 273 251 1103 10.3 7.4 11 0 0 7.501 21.3 97 163 18.2
8 152 130 891 10.4 8.3 15.4 9 0 7.498 25.9 107 142 10.3
9 250 228 1099 10.2 7.8 12.1 3 0 7.481 19.8 147 151 15.7
10 255 233 1235 9.3 7.2 10.4 0 0 7.493 12.9 94 170 18.3
11 268 246 890 9.1 8.3 13.4 0 0 7.581 17.2 99 132 14.3
12 259 237 947 12.3 10.3 25.1 0 2 7.526 28.3 141 176 12.4
13 266 244 1747 9.7 7.2 11.8 0 1 7.639 17.9 90 169 11.1
14 254 232 1318 10 8.3 13 5 0 7.428 9.5 114 180 9.8
15 254 229 1088 12.5 9.8 22.5 3 0 7.407 23.4 123 124 11.4
16 149 127 820 18 12.9 49.1 0 3 7.403 12.6 145 129 10.3
17 138 116 916 12.9 10.1 27.7 0 1 7.392 21.3 119 150 13.2
18 214 192 802 10.2 8.4 15.8 2 0 7.509 13.9 108 173 9.9
19 128 106 829 9.9 8.4 15.2 1 0 7.319 20.7 119 159 15.2
20 107 85 901 11.6 9.9 28.3 3 1 7.541 25.3 125 164 14.9
Table 2.2: Results on Day 3





65

2.1.3 Day 5

Sample
No
Weight Volume
Platelet
count
PDW MPV P-LCR
RBC
count
WBC
count
pH pCO
2
pO
2
LDH Glucose
1 124 102 1241 8.6 7.5 11 0 0 7.492 10.7 55 128 12.57
2 140 118 892 8.8 7.8 11.9 2 0 7.64 8.8 80 198 6.99
3 274 252 1272 9.3 8 13.3 3 0 7.458 18.7 62 160 16.84
4 211 189 1044 10.6 9.1 20.3 0 0 7.512 16 77 228 14.41
5 155 133 567 8.8 8 12.3 0 1 7.714 7.8 90 135 12
6 245 223 1270 9.5 8.2 14.2 0 2 7.509 20.1 86 153 15.94
7 269 247 1110 9.4 8.3 13.2 8 0 7.496 23.5 81 174 15.3
8 118 96 834 10 8.2 15.2 1 0 7.48 27.3 92 163 9.8
9 237 215 1087 10 7.7 11.8 0 3 7.503 17.3 164 161 13.7
10 241 219 1220 9.5 7.5 10.6 0 0 7.49 12.9 95 191 17.6
11 245 223 907 9.4 8.3 13.2 2 0 7.509 17.9 99 149 12.5
12 249 227 937 12.1 10.5 24.9 0 1 7.499 34.2 93 189 10.9
13 252 230 1684 9.7 7.3 11.8 0 0 7.569 21.3 74 173 10.3
14 238 216 1309 10.3 8.6 13.2 1 0 7.502 13.5 102 192 8.1
15 238 216 1089 12.1 9.6 22 1 0 7.399 19.4 99 131 9.4
16 141 119 937 18.2 13.1 48.9 0 6 7.302 9.3 168 172 5.3
17 127 105 987 12.9 10.3 27.8 0 0 7.421 24.3 84 169 11.9
18 203 181 773 9.6 8.1 13.5 3 5 7.487 11.2 136 179 7.9
19 113 91 828 10.4 8.7 17.5 9 0 7.298 25.1 82 181 14.1
20 103 81 927 11.9 9.3 22.8 2 0 7.412 26.9 86 191 11.6
Table 2.3: Results on Day 5




66

2.1.4 Day 7

Sample
No
Weight Volume
Platelet
count
PDW MPV P-LCR
RBC
count
WBC
count
pH pCO
2
pO
2
LDH Glucose
1 35 102 1220 8.6 7.7 11.7 4 0 7.503 9.4 50 160 8.99
2 45 118 864 9 7.8 12.5 0 0 7.712 8.1 78 225 5.06
3 146 252 1207 9.1 7.9 13.1 1 0 7.593 17.2 72 168 14.25
4 114 189 951 10.6 9 19.9 0 0 7.604 15.3 80 236 12.33
5 67 133 521 9.8 8.3 14 6 0 7.698 6.9 98 139 10.98
6 210 223 1341 10 8.5 16.1 3 2 7.619 18.6 92 161 12.56
7 222 247 1001 9.3 8.3 11.2 0 0 7.509 21.3 89 199 12.2
8 134 96 853 10.1 8.3 15.4 0 0 7.46 27.8 92 175 7.6
9 196 215 874 9.9 7.5 11.9 1 0 7.513 17.9 165 181 10.5
10 207 219 1042 9.6 7.4 10.5 0 1 7.381 10.9 79 209 13.5
11 210 223 1201 9.5 8.4 13.1 0 0 7.49 19.2 104 161 9.9
12 212 227 967 12.1 10.7 25.1 0 0 7.504 29.1 110 199 8.6
13 215 230 964 9.8 7.3 11.7 2 1 7.625 18.3 51 196 7.9
14 199 216 1647 10.5 8.4 13.5 0 0 7.493 19.1 109 213 6.9
15 203 216 1315 12.3 9.5 22.1 1 1 7.426 16.3 100 146 8.4
16 127 119 879 14.9 10.6 32.2 3 12 7.121 4.6 181 228 3.9
17 98 105 841 21.4 13.3 52.3 5 2 7.421 25.4 76 173 9.9
18 148 181 817 9.7 8.2 14.6 0 0 7.451 15.2 138 198 5.6
19 86 91 793 10.4 8.7 17.9 1 1 7.328 18.9 91 213 12.5
20 61 81 836 12.2 9.5 23.7 0 1 7.389 29.6 62 204 10.9
Table 2.4: Results on Day 7




67
2.2 Blood cultures

Sample
Number
Day 5 Day 7
1
No bacterial growth
detected
No bacterial growth
detected
2
No bacterial growth
detected
No bacterial growth
detected
3
No bacterial growth
detected
No bacterial growth
detected
4
No bacterial growth
detected
No bacterial growth
detected
5
No bacterial growth
detected
No bacterial growth
detected
6
No bacterial growth
detected
No bacterial growth
detected
7
No bacterial growth
detected
No bacterial growth
detected
8
No bacterial growth
detected
No bacterial growth
detected
9
No bacterial growth
detected
No bacterial growth
detected
10
No bacterial growth
detected
No bacterial growth
detected
11
No bacterial growth
detected
No bacterial growth
detected
12
No bacterial growth
detected
No bacterial growth
detected
13
No bacterial growth
detected
No bacterial growth
detected
14
No bacterial growth
detected
No bacterial growth
detected
15
No bacterial growth
detected
No bacterial growth
detected
16 Bacterial growth detected Bacterial growth detected
17
No bacterial growth
detected
No bacterial growth
detected
18
No bacterial growth
detected
No bacterial growth
detected
19
No bacterial growth
detected
No bacterial growth
detected
20
No bacterial growth
detected
No bacterial growth
detected
Table 2.5: Blood culture results
Note:
Sample 16 was intentionally inoculated so as to provide a positive control.
68
2.3 Platelet Factor IV readings


1 2 3 4 5 6 7 8 9 10 11 12
A 0.110 2.855 2.972 2.195 2.105 1.164 1.223 0.772 0.706 0.427 0.441 0.115
B 0.132 0.387 0.390 1.037 1.090
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
C
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
2.615
Out
of
range
Out
of
range
Out
of
range
Out
of
range
D
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
E
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
F
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
G
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
H
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
Out
of
range
2.987 2.770
Out
of
range
Out
of
range
Table 2.6: PF4 results

Legend:
A1: Blank
A2 B1: Standards in duplicates
B2-B3: Low controls in duplicates
B4-B5: High controls in duplicates
B6-H12: Samples.

Note:
Each sample has four readings due to repeated sampling on various days (i.e. Day 0, 3,
5 and 7)

69
2.3.1 PF4 Calibration curve

Calibration curve
0
0.5
1
1.5
2
2.5
3
3.5
0 0.5 1 2 5 10
PF4 Concentration (IU/ml)
A
b
s
o
r
b
a
n
c
e
s

(
A
4

Figure 2.1: PF4 calibration curve
70




Appendix 3
71
3.1 Normality of results

The Kolomongrov Smirnov test is used to test the normality assumption of the variables.

A Significance (Sig.) value is obtained. The smaller the Sig. value the greater is the
difference between the means. This value is said to be the p-value.

There are two hypotheses to be considered:

H
o
The variable has a normal distribution

H
1
The variable has a non normal distribution

If the p-value exceeds the level of significance of 0.05, H
o
is accepted. If the p-value is
less, H
o
is rejected and H
1
is accepted.

72
3.1.1 Day 0

Table 3.1

Table 3.2


Table 3.3

One-Sample Kolmogorov-Smirnov Test
1106.4500 10.6250 8.6200 17.0450
308.97070 1.54677 1.08511 5.89250
.176 .133 .184 .221
.176 .133 .184 .221
-.143 -.107 -.090 -.149
.787 .594 .823 .987
.565 .872 .507 .285
Mean
Std. Deviation
Normal Parameters
Absolute
Positive
Negative
Most Extreme
Differences
Kolmogorov-Smirnov Z
Asymp. Sig. (2-tailed)
Platelet count PDW MPV P-LCR
Descriptive Statistics
1106.4500 308.97070 557.00 1887.00
10.6250 1.54677 8.70 14.00
8.6200 1.08511 7.10 10.40
17.0450 5.89250 10.90 30.00
7.4485 .06964 7.29 7.55
19.4100 5.77097 9.80 26.30
110.4400 31.91631 54.00 169.00
132.6500 24.69237 91.00 168.00
15.5175 2.98235 9.50 19.70
Platelet count
PDW
MPV
P-LCR
pH
pCO
2
pO
2
LDH
Glucose
Mean Std. Deviation Minimum Maximum
7.4485 19.4100 110.4400 132.6500 15.5175
.06964 5.77097 31.91631 24.69237 2.98235
.129 .178 .121 .114 .114
.110 .116 .105 .114 .080
-.129 -.178 -.121 -.112 -.114
.579 .798 .542 .508 .511
.891 .548 .930 .959 .956
Mean
Std. Deviation
Normal Parameters
Absolute
Positive
Negative
Most Extreme
Differences
Kolmogorov-Smirnov Z
Asymp. Sig. (2-tailed)
pH pCO
2
pO
2
LDH Glucose
73
3.1.2 Day 3


Table 3.4


Table 3.5


Table 3.6

Descriptive Statistics
1050.5500 259.78705 563.00 1747.00
10.6850 2.08914 8.80 18.00
8.6400 1.37359 7.20 12.90
17.6950 9.30107 10.40 49.10
7.4737 .08378 7.32 7.64
18.5500 5.70757 9.50 28.30
102.2300 31.56155 28.00 147.00
154.3500 22.31184 105.00 198.00
13.5620 3.17560 7.32 18.30
Platelet count
PDW
MPV
P-LCR
pH
pCO
2

pO
2
LDH
Glucose
Mean Std. Deviation Minimum Maximum
7.4737 18.5500 102.2300 154.3500 13.5620
.08378 5.70757 31.56155 22.31184 3.17560
.107 .142 .165 .105 .112
.107 .142 .091 .092 .102
-.091 -.097 -.165 -.105 -.112
.480 .637 .737 .471 .501
.975 .812 .650 .980 .963
Mean
Std. Deviation
Normal Parameters
Absolute
Positive
Negative
Most Extreme
Differences
Kolmogorov-Smirnov Z
Asymp. Sig. (2-tailed)
pH pCO
2
pO
2
LDH Glucose
One-Sample Kolmogorov-Smirnov Test
1050.5500 10.6850 8.6400 17.6950
259.78705 2.08914 1.37359 9.30107
.155 .254 .269 .281
.155 .254 .269 .281
-.119 -.183 -.147 -.216
.693 1.137 1.205 1.255
.723 .151 .110 .086
Mean
Std. Deviation
Normal Parameters
Absolute
Positive
Negative
Most Extreme
Differences
Kolmogorov-Smirnov Z
Asymp. Sig. (2-tailed)
Platelet count PDW PMV P-LCR
74
3.1.3 Day 5


Table 3.7


Table 3.8


Table 3.9
Descriptive Statistics
1045.7500 244.33301 567.00 1684.00
10.5550 2.16831 8.60 18.20
8.7050 1.36323 7.30 13.10
17.4700 8.94869 10.60 48.90
7.4846 .09527 7.30 7.71
18.3100 7.17810 7.80 34.20
95.2500 29.21405 55.00 168.00
170.8500 24.66891 128.00 228.00
11.8575 3.34315 5.30 17.60
Platelet count
PDW
MPV
P-LCR
pH
pCO
2
pO
2
LDH
Glucose
Mean Std. Deviation Minimum Maximum
7.4846 18.3100 95.2500 170.8500 11.8575
.09527 7.17810 29.21405 24.66891 3.34315
.237 .099 .259 .096 .069
.237 .099 .259 .096 .069
-.181 -.072 -.133 -.080 -.069
1.059 .441 1.157 .428 .311
.212 .990 .138 .993 1.000
Mean
Std. Deviation
Normal Parameters
Absolute
Positive
Negative
Most Extreme
Differences
Kolmogorov-Smirnov Z
Asymp. Sig. (2-tailed)
pH pCO
2
pO
2
LDH Glucose
One-Sample Kolmogorov-Smirnov Test
1045.7500 10.5550 8.7050 17.4700
244.33301 2.16831 1.36323 8.94869
.122 .242 .217 .250
.122 .242 .217 .250
-.086 -.184 -.151 -.221
.545 1.081 .970 1.119
.928 .193 .304 .164
Mean
Std. Deviation
Normal Parameters
Absolute
Positive
Negative
Most Extreme
Differences
Kolmogorov-Smirnov Z
Asymp. Sig. (2-tailed)
Platelet count PDW MPV P-LCR
75
3.1.4 Day 7


Table 3.10

Table 3.11


Table 3.12

7.4920 17.4550 95.8500 189.2000 9.6235
.13533 7.11281 33.59711 27.95410 2.89177
.138 .153 .187 .146 .114
.138 .153 .187 .094 .068
-.106 -.126 -.089 -.146 -.114
.619 .685 .835 .653 .508
.839 .737 .488 .787 .959
Mean
Std. Deviation
Normal Parameters
Absolute
Positive
Negative
Most Extreme
Differences
Kolmogorov-Smirnov Z
Asymp. Sig. (2-tailed)
pH pCO
2
pO
2
LDH Glucose
Descriptive Statistics
1006.7000 251.31195 521.00 1647.00
10.9400 2.86970 8.60 21.40
8.7650 1.42433 7.30 13.30
18.1250 9.83195 10.50 52.30
7.4920 .13533 7.12 7.71
17.4550 7.11281 4.60 29.60
95.8500 33.59711 50.00 181.00
189.2000 27.95410 139.00 236.00
9.6235 2.89177 3.90 14.25
Platelet count
PDW
MPV
P-LCR
pH
pCO
2
pO
2
LDH
Glucose
Mean Std. Deviation Minimum Maximum
One-Sample Kolmogorov-Smirnov Test
1006.7000 10.9400 8.7650 18.1250
251.31195 2.86970 1.42433 9.83195
.163 .297 .224 .232
.163 .297 .224 .232
-.148 -.207 -.152 -.219
.728 1.329 1.001 1.036
.664 .058 .269 .234
Mean
Std. Deviation
Normal Parameters
Absolute
Positive
Negative
Most Extreme
Differences
Kolmogorov-Smirnov Z
Asymp. Sig. (2-tailed)
Platelet count PDW MPV P-LCR
76
3.2 Comparing of the means

The paired sample T-test is used to compare the mean score between two related
groups of data such that each value in one group is paired with a similar measure in the
other group. This is done by calculating the difference between the means and standard
deviations for the results being analyzed.

There are two hypotheses to be considered:

H
o
The mean score remains the same, i.e. there is no significant
change in the mean score

H
1
There is a significant change in the mean score.

If the p-value exceeds the level of significance of 0.05, H
o
is accepted. If the p-value is
less, H
o
is rejected and H
1
is accepted.

77
3.2.1 Comparing the means of Day 0 and Day 3

3.2.1.1 Platelet count

Paired Samples Statistics
1106.4500 308.97070 69.08795
1050.5500 259.78705 58.09015
Platelet count Day 0
Platelet count Day 3
Mean Std. Deviation Std. Error Mean

Table 3.13
Paired Samples Test
55.90000 126.14023 28.20581 1.982 19 .062
Platelet count Day 0 -
Platelet count Day 3
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.14

3.2.1.2 Mean platelet volume (MPV)

Paired Samples Statistics
8.6200 1.08511 .24264
8.6400 1.37359 .30714
MPV Day 0
MPV Day 3
Mean Std. Deviation Std. Error Mean

Table 3.15

Paired Samples Test
-.02000 1.29436 .28943 -.069 19 .946
MPV Day 0 -
MPV Day 3
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.16
78
3.2.1.3 Platelet distribution width (PDW)

Paired Samples Statistics
10.6250 1.54677 .34587
10.6850 2.08914 .46715
PDW Day 0
PDW Day 3
Mean Std. Deviation Std. Error Mean

Table 3.17

Paired Samples Test
-.06000 2.28828 .51167 -.117 19 .908
PDW Day 0 -
PDW Day 3
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.18

3.2.1.4 Platelet large cell ratio (P-LCR)

Paired Samples Statistics
17.0450 5.89250 1.31760
17.6950 9.30107 2.07978
P-LCR Day 0
P-LCR Day 3
Mean Std. Deviation Std. Error Mean

Table 3.19

Paired Samples Test
-.65000 8.66879 1.93840 -.335 19 .741
P-LCR Day 0 -
P-LCR Day 3
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)
Table 3.20

79
3.2.1.5 pH

Paired Samples Statistics
7.4485 .06964 .01557
7.4737 .08378 .01873
pH Day 0
pH Day 3
Mean Std. Deviation Std. Error Mean

Table 3.21

Paired Samples Test
-.02525 .09954 .02226 -1.134 19 .271
pH Day 0 -
pH Day 3
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)
Table 3.22

3.2.1.6 pCO
2



Table 3.23

Table 3.24

Paired Samples Statistics
19.4100 5.77097 1.29043
18.5500 5.70757 1.27625
pCO
2
Day 0
pCO
2
Day 3
Mean Std. Deviation Std. Error Mean
Paired Samples Test
.86000 3.75645 .83997 1.024 19 .319
pCO
2
Day 0 -
pCO
2
Day 3
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)
80
3.2.1.7 pO
2



Table 3.25


Table 3.26

3.2.1.8 Lactate dehyrdrogenase (LDH)
Paired Samples Statistics
132.6500 24.69237 5.52138
154.3500 22.31184 4.98908
LDH Day 0
LDH Day 3
Mean Std. Deviation Std. Error Mean

Table 3.27

Paired Samples Test
-21.70000 15.20769 3.40054 -6.381 19 .000
LDH Day 0 -
LDH Day 3
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.28

Paired Samples Statistics
110.4400 31.91631 7.13670
102.2300 31.56155 7.05738
pO
2
Day 0
pO
2
Day 3
Mean Std. Deviation Std. Error Mean
Paired Samples Test
8.21000 22.11991 4.94616 1.660 19 .113
pO
2
Day 0 -
pO
2
Day 3
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)
81
3.2.1.9 Glucose

Paired Samples Statistics
15.5175 2.98235 .66687
13.5620 3.17560 .71009
Glucose Day 0
Glucose Day 3
Mean Std. Deviation Std. Error Mean

Table 3.29

Paired Samples Test
1.95550 1.02311 .22877 8.548 19 .000
Glucose Day 0 -
Glucose Day 3
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.30

82
3.2.2 Comparing the means of Day 0 and Day 5

3.2.2.1 Platelet count

Paired Samples Statistics
1106.4500 308.97070 69.08795
1045.7500 244.33301 54.63452
Platelet count day 0
Platelet count day 5
Mean Std. Deviation Std. Error Mean

Table 3.31

Paired Samples Test
60.70000 140.81571 31.48735 1.928 19 .069
Platelet count Day 0 -
Platelet count Day 5
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.32

83
3.2.2.2 Mean platelet volume (MPV)

Paired Samples Statistics
8.6200 1.08511 .24264
8.7050 1.36323 .30483
MPV Day 0
MPV Day 5
Mean Std. Deviation
Std. Error
Mean

Table 3.33

Paired Samples Test
-.08500 1.30234 .29121 -.292 19 .774
MPV Day 0 -
MPV Day 5
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.34

3.2.2.3 Platelet distribution width (PDW)

Paired Samples Statistics
10.6250 1.54677 .34587
10.5550 2.16831 .48485
PDW Day 0
PDW Day 5
Mean Std. Deviation Std. Error Mean

Table 3.35

Paired Samples Test
.07000 2.33195 .52144 .134 19 .895
PDW Day 0 -
PDW Day 5
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.36

84
3.2.2.4 Platelet large cell ratio (P-LCR)

Paired Samples Statistics
17.0450 5.89250 1.31760
17.4700 8.94869 2.00099
P-LCR Day 0
P-LCR Day 5
Mean Std. Deviation Std. Error Mean

Table 3.37

Paired Samples Test
-.42500 8.63243 1.93027 -.220 19 .828
P-LCR Day 0 -
P-LCR Day 5
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.38

3.2.2.5 pH

Paired Samples Statistics
7.4485 .06964 .01557
7.4846 .09527 .02130
pH Day 0
pH Day 5
Mean Std. Deviation Std. Error Mean

Table 3.39

Paired Samples Test
-.03615 .13012 .02910 -1.242 19 .229
pH Day 0 -
pH Day 5
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.40

85
3.2.2.6 pCO
2



Table 3.41


Table 3.42

3.2.2.7 pO
2



Table 3.43

Table 3.44

Paired Samples Statistics
19.4100 5.77097 1.29043
18.3100 7.17810 1.60507
pCO
2
Day 0
pCO
2
Day 5
Mean Std. Deviation Std. Error Mean
Paired Samples Test
1.10000 5.62644 1.25811 .874 19 .393
pCO
2
Day 0 -
pCO
2
Day 5
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)
Paired Samples Statistics
110.4400 31.91631 7.13670
95.2500 29.21405 6.53246
pO
2
Day 0
pO
2
Day 5
Mean Std. Deviation Std. Error Mean
Paired Samples Test
15.19000 32.74343 7.32165 2.075 19 .052
pO
2
Day 0 -
pO
2
Day 5
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)
86
3.2.2.8 Lactate dehyrdrogenase (LDH)

Paired Samples Statistics
132.6500 24.69237 5.52138
170.8500 24.66891 5.51614
LDH Day 0
LDH Day 5
Mean Std. Deviation Std. Error Mean

Table 3.45

Paired Samples Test
-38.20000 22.10406 4.94262 -7.729 19 .000
LDH Day 0 -
LDH Day 5
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.46
3.2.2.9 Glucose

Paired Samples Statistics
15.5175 2.98235 .66687
11.8575 3.34315 .74755
Glucose Day 0
Glucose Day 5
Mean Std. Deviation Std. Error Mean

Table 3.47
Paired Samples Test
3.66000 1.60653 .35923 10.188 19 .000
Glucose Day 0 -
Glucose Day 5
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.48
87
3.3.3 Comparing the means of Day 0 and Day 7

3.2.3.1 Platelet count

Paired Samples Statistics
1106.4500 308.97070 69.08795
1006.7000 251.31195 56.19506
Platelet count Day 0
Platelet count Day 7
Mean Std. Deviation Std. Error Mean

Table 3.49

Paired Samples Test
99.75000 250.06270 55.91572 1.784 19 .090
Platelet count Day 0-
Platelet count Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.50

88
3.2.3.2 Mean platelet volume (MPV)

Paired Samples Statistics
8.6200 1.08511 .24264
8.7650 1.42433 .31849
MPV Day 0
MPV Day 7
Mean Std. Deviation Std. Error Mean

Table 3.51

Paired Samples Test
-.14500 1.31968 .29509 -.491 19 .629
MPV Day 0 -
MPV Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)
Table 3.52

3.2.3.3 Platelet distribution width (PDW)

Paired Samples Statistics
10.6250 1.54677 .34587
10.9400 2.86970 .64168
PDW Day 0
PDW Day 7
Mean Std. Deviation Std. Error Mean

Table 3.53

Paired Samples Test
-.31500 2.99987 .67079 -.470 19 .644
PDW Day 0 -
PDW Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)
Table 3.54

89
3.2.3.4 Platelet large cell ratio (P-LCR)

Paired Samples Statistics
17.0450 5.89250 1.31760
18.1250 9.83195 2.19849
P-LCR Day 0
P-LCR Day 7
Mean Std. Deviation Std. Error Mean

Table 3.55

Paired Samples Test
-1.08000 9.31578 2.08307 -.518 19 .610
P-LCR Day 0 -
P-LCR Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.56

3.2.2.5 pH

Paired Samples Statistics
7.4485 .06964 .01557
7.4920 .13533 .03026
pH Day 0
pH Day 7
Mean Std. Deviation Std. Error Mean

Table 3.57

Paired Samples Test
-.04355 .16888 .03776 -1.153 19 .263
pH Day 0 -
pH Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.58
90
3.2.3.6 pCO
2



Table 3.59


Table 3.60

3.2.3.7 pO
2



Table 3.61


Table 3.62
Paired Samples Statistics
19.4100 5.77097 1.29043
17.4550 7.11281 1.59047
pCO
2
Day 0
pCO
2
Day 7
Mean Std. Deviation Std. Error Mean
Paired Samples Test
1.95500 6.29089 1.40668 1.390 19 .181
pCO
2
Day 0 -
pCO
2
Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)
Paired Samples Statistics
110.4400 31.91631 7.13670
95.8500 33.59711 7.51254
pO
2
Day 0
pO
2
Day 7
Mean Std. Deviation Std. Error Mean
Paired Samples Test
14.59000 35.89537 8.02645 1.818 19 .085
pO
2
Day 0 -
pO
2
Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)
91
3.2.3.8 Lactate dehyrdrogenase (LDH)

Paired Samples Statistics
132.6500 24.69237 5.52138
189.2000 27.95410 6.25073
LDH Day 0
LDH Day 7
Mean Std. Deviation Std. Error Mean

Table 3.63

Paired Samples Test
-56.55000 28.29446 6.32683 -8.938 19 .000
LDH Day 0 -
LDH Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.64

3.2.3.9 Glucose

Paired Samples Statistics
15.5175 2.98235 .66687
9.6235 2.89177 .64662
Glucose Day 0
Glucose Day 7
Mean Std. Deviation Std. Error Mean

Table 3.65

Paired Samples Test
5.89400 1.59449 .35654 16.531 19 .000
Glucose Day 0 -
Glucose Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.66
92
3.2.4 Comparing the means of Day 5 and Day 7

3.2.4.1 Platelet count

Paired Samples Statistics
1045.7500 244.33301 54.63452
1006.7000 251.31195 56.19506
Platelet count Day 5
Platelet count Day 7
Mean Std. Deviation Std. Error Mean

Table 3.67

Paired Samples Test
39.05000 216.09342 48.31996 .808 19 .429
Platelet count Day 5 -
Platelet count Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.68

93
3.2.4.2 Mean platelet volume (MPV)

Paired Samples Statistics
8.7050 1.36323 .30483
8.7650 1.42433 .31849
MPV Day 5
MPV Day 7
Mean Std. Deviation Std. Error Mean

Table 3.69

Paired Samples Test
-.06000 .90693 .20280 -.296 19 .771
MPV Day 5 -
MPV Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.70

3.2.4.3 Platelet distribution width (PDW)

Paired Samples Statistics
10.5550 2.16831 .48485
10.9400 2.86970 .64168
PDW Day 5
PDW Day 7
Mean Std. Deviation Std. Error Mean

Table 3.71

Paired Samples Test
-.38500 2.07422 .46381 -.830 19 .417
PDW Day 5 -
PDW Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.72

94
3.2.4.4 Platelet large cell ratio (P-LCR)

Paired Samples Statistics
17.4700 8.94869 2.00099
18.1250 9.83195 2.19849
P-LCR Day 5
P-LCR Day 7
Mean Std. Deviation Std. Error Mean

Table 3.73

Paired Samples Test
-.65500 6.82299 1.52567 -.429 19 .673
P-LCR Day 5 -
P-LCR Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.74

3.2.4.5 pH

Paired Samples Statistics
7.4846 .09527 .02130
7.4920 .13533 .03026
pH Day 5
pH Day 7
Mean Std. Deviation Std. Error Mean

Table 3.75

Paired Samples Test
-.00740 .07094 .01586 -.466 19 .646
pH Day 5 -
pH Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.76

95
3.2.4.6 pCO
2



Table 3.77


Table 3.78

3.2.4.7 pO
2



Table 3.79


Table 3.80

Paired Samples Statistics
18.3100 7.17810 1.60507
17.4550 7.11281 1.59047
pCO
2
Day 5
pCO
2
Day 7
Mean Std. Deviation Std. Error Mean
Paired Samples Test
.85500 2.95144 .65996 1.296 19 .211
pCO
2
Day 5 -
pCO
2
Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)
Paired Samples Statistics
95.2500 29.21405 6.53246
95.8500 33.59711 7.51254
pO
2
Day 5
pO
2
Day 7
Mean Std. Deviation Std. Error Mean
Paired Samples Test
-.60000 11.09481 2.48087 -.242 19 .811
pO
2
Day 5 -
pO
2
Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)
96
3.2.4.8 Lactate dehyrdrogenase (LDH)

Paired Samples Statistics
170.8500 24.66891 5.51614
189.2000 27.95410 6.25073
LDH Day 5
LDH Day 7
Mean Std. Deviation Std. Error Mean

Table 3.81

Paired Samples Test
-18.35000 12.27867 2.74559 -6.683 19 .000
LDH Day 5 -
LDH Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.82
3.2.4.9 Glucose

Paired Samples Statistics
11.8575 3.34315 .74755
9.6235 2.89177 .64662
Glucose Day 5
Glucose Day 7
Mean Std. Deviation Std. Error Mean

Table 3.83

Paired Samples Test
2.23400 .92553 .20695 10.795 19 .000
Glucose Day 5 -
Glucose Day 7
Mean Std. Deviation
Std. Error
Mean
Paired Differences
t df Sig. (2-tailed)

Table 3.84