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
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:
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
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(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.
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)
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.
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.
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.
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)