Platelet Storage

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University of Wolverhampton School of Applied SciencesDivision of Biomedical Science

In vitro assessment of platelets stored for

seven days in a platelet additive medium

A pilot study


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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.


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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 22C 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.


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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


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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


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Chapter 1

Introduction


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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 storagethe 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


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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


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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;


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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 vitroquality of platelets stored in ComposolPS retained

the in vitroquality 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.


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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


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platelet inactivation required for good storage practice, as platelets should only

be activated in vivoto 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 pO2has 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


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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


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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 vitroquality 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


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Chapter 2

Materials and Methods


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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

Helmeragitator

Sysmexcell counter

Negeotte chamber cell counter

Neubauer chamber cell counter

IL (instrumentation Laboratory System) blood gas analyzer

Bactecsystem 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 Helmeragitator, care being taken


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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 Helmeragitator. 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 11thEdition, 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.


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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, pO2and pCO2values were determined immediately after sampling of each pool

using the IL - Instrumentation Laboratory Systemblood 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.


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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 vitrotesting 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 -20 C 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


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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.


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Chapter 3

Results


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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, pCO2, pO2, 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


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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

PDW10.625

1.54677

10.6850

2.08914

10.555

2.16831

10.94

2.8697

MPV8.62

1.08511

8.64

1.37359

8.7050

1.3623

8.765

1.42433

P-LCR 17.0455.8925

17.69509.30107

17.478.94869

18.1259.83195

pH7.4485

0.06964

7.4737

.08378

7.4846

.09527

7.492

0.13533

pCO219.41

5.77097

18.55

5.70757

18.31

7.1781

17.455

7.11281

pO2110.44

31.91631

102.23

31.56155

95.25

29.21405

95.85

33.59711

LDH132.65

24.69237

154.35

22.31184

170.85

24.66891

189.2

27.95410

Glucose15.5175

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


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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.


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Chapter 4

Discussion


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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 55x109of platelets

Platelet count >55x109/SUE

Residual leukocytes:

1. Before leukocyte depletion

Prepared from buffy coat

2. After leukocyte depletion

>0.05x109/SUE

>0.2x106/SUE

Erythrocyte count >1.0x109/SUE

pH measured at the end of the recommended shelf life. 6.4 to 7.4

Table 4.1: Defined quality requirements for PCs


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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.


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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 vitrostorage 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-1and 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.


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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.

pO2gas 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 pO2levels.

Similar statistical comparison of pCO2 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 pO2and pCO2 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.


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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.


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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


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exchange where the O2 levels are increased and those of CO2are 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


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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.


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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.


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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 vivoafter transfusion.


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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.


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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.


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Chapter 5

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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


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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 11thEdition,

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


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Appendix 1


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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 Composol

PS

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.


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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 Hettiechcentrifuge, 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

Helmerplatelet agitator for storage.


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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 109 /mL and that the pH of the

platelet product remains continuously between 6.4 and 7.4 at 220C.

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.


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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 Sysmex

automated 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

Sysmexcell counter

1.3.2 Procedure

Daily controls must be run to ascertain that the Sysmexis 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.


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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 Sysmexcounter 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


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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


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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.


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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


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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

1mm2area 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 1/5th of 1 mm2 (80/400). The volume is determined by multiplying the depth

(0.1mm) by 1/5thand is equal to 0.02L.

The preferred method for counting the cells in the 1/5 mm2is 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 mm2,

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 mm2

volume= Red Blood Cells / L

Where volume = area x depth of the chamber.


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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 ster

Documents

Platelet Storage