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Thrombocytopenia is a haematological condition marked by a decrease in platelet number in peripheral blood (5). To identify the source, it is important to understand the mechanism of platelet formation and its pathophysiology.
1.1.1 Role of platelets
Platelets were first described in the 19th century as “dust of blood”(44, 45). Over the last 100 years, there have been many advances in understanding the role of platelets. These cells have a diameter of 2-3mm and have been identified as playing a crucial role in repairing of damaged blood vessels, in response to injury; achieved via a clotting mechanism and the involvement of various clotting factors (1). It is a continuous cascade, where one factor activates the next with the end product being a fibrin clot. Formation of the clot prevents blood loss, maintaining cell concentration within vessels. Under normal conditions, 1×1011 platelets are released from the bone marrow into circulation, which helps provide the balance between formation and sequestration (43). Larger platelets are considered to be more reactive and efficient at performing their role than mature platelets (11). During vascular damage, there is increased activity from cytokines, transcription and growth factors to restore the balance, as more than 20-fold rise in cells are released into blood (45).
Thrombopoiesis is the process of platelet formation, occurring in the bone marrow. The mature cells have a life span of 10 days and make up 5litres of the total blood volume; one third of which is found in the spleen due to platelet senescence (43).
Thrombopoiesis consists of a negative feedback mechanism, regulated by the glycoprotein ‘thrombopoietin’ (TPO) (43). TPO was named after erythropoietin in 1950, after being shown to have inducing capabilities in a study performed on rats with bleeding disorders (43). In the last decade, a greater understanding of the growth factor has been achieved (41). Its association with the cellular oncogene; cellular myeloproliferative leukaemia (c-Mpl) found on the surface of megarkaryocytes and platelets has been identified (40). TPO has a high affinity for c-Mpl (43). Research carried out in mice in 19__ showed 15% of patients with thrombocytopenia had a defect in TPO or c-Mpl (41,[2,3]). It was suspected to have a role in the differentiation of megakaryocytes to platelets (41). Megakaryocytes are immature platelets derived from haematopoietic stem cells, via megakaryopoeisis, with the help of various cytokines (Figure 1) (45). They are larger than platelets, consisting of organelles, granules and soluble macromolecules bound within a cell membrane (45). As seen in the diagram, megarkaryocyte organelles are fragmented to form proplatelets (45). These are long and thin cells, with hallmark features, consisting of swollen tips, which constitute the cell organelles and granules (45). The shape is made up of overlapping microtubules (45). The cell content is further phagocytosed by macrophages prior to entering peripheral circulation (45). The formation of proplatelets are dependent on environmental factors, for example during inflammation, synthesis is increased due to the rapid consumption of platelets (41). TPO proliferate megakaryocytic activity by stimulating stem cells to enter the G0 phase of the cell cycle (1). Its involvement does not stop at platelets, as it also stimulates activity of other stem lines. This shows that TPO is of great importance in the formation of the blood cells.
1.1.3 Pathophysiology of thrombocytopenia
The cause of thrombocytopenia is of great importance to clinicians. To establish the correct treatment of patients, it is important to identify the source of the condition and its pathophysiology (17). There are many causes of thrombocytopenia; 2 of the main mechanisms include:
Hypoproduction of platelets
Hyperdestruction of platelets
Decrease in platelet production is associated with suppression of thrombopoiesis, resulting in megakaryocyte hypoplasia within the bone marrow (50). There are several factors, which may contribute e.g. exposure to radioactive substances, such as chemotherapy and radiotherapy (50). A defect in the stem line can be due to haematological malignancies, such as acute leukaemia, aplastic anaemia, myelodysplastic syndrome and multiple myeloma. In the hypoproduction of platelets, there is a decrease in all progenitor cells of platelets, thus an increase in plasma TPO and a decrease in immature platelets seen in blood
An increase in destruction of platelets in peripheral blood may be due to sequestration of platelets by the spleen, via reticuloendothelial system (22). To compensate for the loss of platelets, the bone marrow releases immature platelets into circulation, indicating normal megakaryocytic activity (5).
Hyperdestruction conditions can be further classified into immunological causes, such as idiopathic thrombocytopenia purpura (ITP) and non-immunological causes, e.g. disseminated intravascular coagulation (DIC) (23, 34). ITP continuing for periods longer than one year is known as chronic ITP (7). The condition is characterised by mucocataneous bleeding and a decrease in platelet count, often associated with the humoral cell-mediated mechanism (7). It is proposed that the complement pathway mediated by anti-platelet antibodies, may be a contributing factor (7).
1.1.4 Treatment of thrombocytopenia
The severity of the abnormality varies from chronic to acute and is commonly associated with bleeding (10). Therefore the treatment of thrombocytopenia is dependent on the progression of the symptoms. There are many management schemes available to patients with this condition, one being TPO drug therapy, such as Electrombopag and AM G531 (45). The molecules of TPO bind to c-Mpl receptors, stimulating megakaryopoiesis (45).
In many cases, there is a dramatic decrease in platelet count, resulting in excessive abnormal bleeding. The loss of whole blood is likely to be managed with red cell transfusion followed by other blood products. Due to the cause being thrombocytopenia, the patient is likely to need platelet transfusion. National blood transfusion guidelines state the platelet threshold as 20×109/l (___). This would be decided by clinicians and consultants.
1.2 Historical Review
1.2.1 History of Platelets
It wasn’t until 1962 that platelet function was truly understood (44). ‘Platelets’ named by Bizzozero was first detected in the mid 19th Century by Max Schulitz (44,45). They were noted as being the smallest blood cells, equivalent to 1/10 of erythrocytes (44). Following on from this theory, Bizzozero in 1882 studies these cells in vivo, using microscopy and detected platelets to consist of adhesion qualities, which was significant during vascular damage (45[2,3]). It was later suspected to play a role in thrombosis (44).
1.2.2 History of reticulated platelets
Reticulated platelets were first observed in 1969 in peripheral blood of dogs, following acute blood loss. Ingram and Cooper-Smith (1969) used methylene blue to stain the RNA of cells, based on which a count was achieved (13). They were microscopically shown to be more reactive than mature platelets and have resemblance with reticulocytes, thus was named ‘reticulated platelets’ (13, 18). Further investigation showed reticulated platelets to be RNA containing immature platelets derived from megakaryocytes, in the bone marrow (4,10). Similarly, Boayse and Rafelson observed the same platelet characteristics in humans, which then lead to opportunities for greater research into the area (35). By 1970, megakaryocytic progenitor cells could be detected, followed by the identification of growth factors responsible for differentiation of stem cells in 1980 and 1990 (45). To date, several discoveries have been concluded, which has lead to a better understanding of the mechanism. This has resulted in advances in the diagnosis of thrombocytopenia. Based on reticulocyte analysis, Kienast and Schmitz introduced a fluorescent dye; thiazole orange to stain the nucleic acid, using flow cytometry (24).
1.2.3 History of thrombocytopenia
The clinical symptoms of epitaxis and pupura were first classified with thrombocytopenia by Brohm, Kraus and Denys in 1883 (56). Later in the year, Kaznelson associated thrombocytopenia with a destruction of platelets (55). By 1946, increase numbers of megakaryocytes were seen in the bone marrow of patients with low platelets, forming the basis of idiopathic thrombocytopenia purpura (ITP) repectively (56).
In 1953, Brecher et al developed the first manual phase microscopy (55). Using counting chambers, platelets could easily be identified from red cells and counted (55). This method was soon replaced in 1950 by the Coulter principle, followed by automation in 1970 (55). Since then, there has been a vast improvement in the counting of platelets. The discovery of the light microscopy has also helped in achieving this and is still used in diagnosing thrombocytopenia to date.
However, in the last decade, Sysmex have designed new upgraded software, designed for Sysmex XE2100 and XE5000 analysers. It has adopted the flow cytometry technique discovered by Kienasr and Schmitz, where reticulated platelets can be measured, in the form of immature platelet fraction (IPF) (11).
1.3.1 Diagnosis of thrombocytopenia
Preliminary studies consisted of platelet counts to be achieved microscopically (44). It wasn’t until 1962 that platelet function was truly understood (44). A great deal of time and research has resulted in implementation of various specialised tests.
Current diagnostic methods
Thrombocytopenia can be an incidental laboratory finding or suspected from clinical feature presented by patients (40). Characteristics seen include bruising, purpura and bleeding. The bleeding condition may progress to severe forms (27). Therefore, the importance of diagnosis cannot be emphasised enough. Screening consists of full blood count marked with a decrease in platelet count followed by morphology examination for confirmation of results. On many occasions, larger megakaryocytes are released in peripheral, which may be visible by microscopy. Currently, a bone marrow examination is the gold standard method for detecting autoimmune thrombocytopenia (5). It investigates megakaryopoietic activity, where a hypoplastic image indicates decreased production (17). A bone marrow aspirate is not desired by patients, as it is an invasive and uncomfortable procedure. The method is vulnerable to sampling errors and examination can be subjective (22) There is limited specialised testing available to achieve this information, thus is crucial, especially in chronic thrombocytopenia, where 30% of cases are due to immune reaction or cytokine associated (45).
Another crucial test includes plasma TPO levels. This is carried out by ELISA (Enzyme-Linked ImmunoSorbent Assay), using anti-TPO monoclonal antibody against recombinant TPO (50). This technique is expensive and time-consuming (2). Plasma TPO concentration have are reported to increase in hypoproduction conditions and normal in ITP (22)
Plasma Glycocalicin (GC) levels can also be examined. It is a hydrophilic fragment made up of carbohydrates, which forms part of the Î±-subunit of the platelet glycoprotein membrane (22). Low levels have been suspected in thrombocytopenia due to decreased production, and elevated in ITP (22).
Thrombopoiesis is cytokine-mediated; therefore it would be useful to determine the expression of c-Mpl, as mutations in the gene have been identified in conditions such as congenital amegakaryocytic thrombocytopenia (50). This may be achieved by proteomic assays.
Immature platelet fraction (IPF): the future diagnostic tool
Over the past years, there have been several advances in detection of reticulated platelet. After years of research, Sysmex have introduced an automated flow cytometric method on the XE2100 and XE5000 analysers (11). It uses upgraded software to calculate an accurate measure of immature platelet fraction (IPF). The cells obtained from the peripheral sample is stained using fluorescence dyes and passed through a semi-conductor diode beam, where approximately 30,000 cells are counted and displayed on the basis of cell size and RNA content (12). The data is converted into a graph, showing the RNA content and cell size as forwards scatter and side scatter (figure 2)(12). Many authors have commented on the positivity of the technique in the diagnosis and management of patients (3).
IPF was shown to be increase in ITP patient of a study performed by (5). 46 AITP patients had a median IPF of 17.4%, signifying the probable increase in megakaryocytic activity occurring in the bone marrow (5). Abe et al. (2006) conducted a similar study comparing healthy volunteers with patients diagnosed with thrombocytopenia (17). It was proposed that patients with ITP have a significantly increased IPF (17). This was agreed by (3,), as ITP cases with platelet count >50 was observed to have an increase in numbers varying from 2.3-52.1%, and patients with <50 had a similar range of 9.2-48.3%, indicating the active thrombopoiesis (3).
A similar trend was observed in DIC patients by _____(16). Comparably, a positive correlation with D-Dimer was noted, whereas the data was inversely proportional to platelet count (16). DIC is commonly associated with a decrease in platelets, due to the intravascular formation of clots (16). In response, the bone marrow releases immature platelets into circulation, increasing the count (16).
A study which compared hypoproduction and hyperdestruction of platelets showed a significant deferent between the groups (34). The mean IPF in subjects with decreased production was 7.5%, whereas in-patient with ITP and DIC, the mean IPF was 30.0% (34). This is evident that IPF can be used as a marker to distinguish between hypoproduction and hyperdestruction of platelets (34).
Thrombocytopenia can be seen in patients with HIV, as they are most probably immune-compressed, thus are more vulnerable to infection. According to(8, [Briggs et al]), patients suffering from infections are more likely to have reduced platelet count, which explains why 40% of HIV patients in (8)’s investigation were observed to have a low platelet count and raised IPF (8).
IPF is a very quick and useful technique. The data obtained can be used for various clinical applications (2). It is relatively inexpensive and commercially available, therefore its use in monitoring drug therapy for bone marrow malignancies can be beneficial (2). (4) has shown the positive use of IPF in monitoring patients on chemotherapy, as well as (37), who reported IPF having 70% predictive value for detecting early platelet recovery (4, 37). The platelet count and IPF was monitored post exposure to chemotherapy. During the process, 3 pools of platelets were transfused. Completion of the second transfusion resulted in an IPF peak of 11.3% at day 11 (4). The increase in IPF indicates the existence of immature platelets in peripheral blood, predicting the increased release of cells from the bone marrow, which would eventually result in a normal platelet count. The improvement in platelet numbers was seen 3 days post transfusion (4). Within this period, another pool of platelet was given (4). Based on the results, the last transfusion was not required, thus could have been avoided (4). Briggs at al. (4, ) discovered similar findings to (4) and concluded IPF a better parameter. A similar scenario was presented by (10), who monitored patients undergone haematopoietic stem cell transplant (10). There was a rise in IPF, 3 days prior to platelet count. (4) and (5) also observed a decrease in IPF during the process, which was described by (4,) as being part of the feedback mechanism of TPO or due to dilution of patient blood with prophylactic platelets (4,). There was no significant difference observed between authors, suggesting good precision (5).
Paediatric patients are most likely to show increase levels of immature platelets, as there is increased megakaryocytic activity during foetal and neonatal periods (3, 9).
Previous reports obtained detected good sensitivity and specificity in IPF measurement using Sysmex XE2100 analysers. In majority of investigations, sensitivity was between 91-96%, with the specificity ranging from 67-100% for ITP (22). These figures suggest IPF to have a good degree of sensitivity. Specificity is widely distributed; however another study performed by (17) showed 86.8% sensitive and 92.6% specific. Therefore IPF (5) is an accurate parameter of reticulated platelets.
Precision measurement reviewed by (14, [Biggs et al. 2004]) showed the coefficient of variance (CV) between 10-78% (14, [Biggs et al. 2004]). The vast variation of intra-assays questions the precision of the assay, when using XE2100 analysers.
Other platelet indices
Other platelet parameter that may be useful in determining thrombocytopenia includes mean platelet volume (MPV) and platelet distribution width (PDW). A decreased MPV is detected in bone marrow malignancies, where thrombocytopenia is present, however levels are shown to increase in myeloid leukaemia (M1) (11). PDW is a good indicator in thrombocytopenia of autoimmune cause, as the variation of the size of platelets marks the types of platelets available in peripheral (11). These parameters are commonly used in conjunction with morphology, IPF and TPO as individually, they have minimal clinical significance due to its lack of standardisation and instability with samples embedded in EDTA anti-coagulant (11).
1.3.2 Clinical Management and utility of IPF
In severe cases with the need for rapid platelet increase, prophylactic platelet transfusions are given (18). National blood transfusion guidelines state the platelet threshold as 20×109/l (___). The platelet count may vary from one analyser to another and may be dependent on the method used to detect the cells, as (12) compared the impedance method to the reference immune count (12). It was showed a decrease in correlation with decrease in platelet count (12). A change in the threshold would have a direct affect on the sensitivity and specificity, as adopting a higher threshold would result in false negatives and adjusted the count to about 10×109/l would raise false positives (12). In comparison, the optical fluorescence method showed a good correlation (12). (10,[2,3]) studied the platelet transfusion guidelines and suggested a more accurate marker to detect early marrow activity to be used (10,[2,3]). (17) proposed the use of IPF (17)
Chemotherapy treatment due to haematological malignancies can result in myelosuppression of bone marrow (27). It is important that these patients are monitored regularly. Currently, the need to transfuse platelets is dependant on clinical observation and platelet count. An early report from the national external quality assessment scheme (NEQAS) has shown a varying coefficient of variation (CV) between 20-60% when measured using automation (27[Parlar-William 03(NEQAS__ 2003)). It has been reported that the accuracy of the measure of platelet count decreases as the rate of thrombocytopenia increases (27). Therefore, a more sensitive marker would be beneficial. IPF has been shown to have capabilities of a good diagnostic marker. Several authors have discussed its use in monitoring and management with platelet transfusion; however its use in the clinical field is yet to be established (17). It is believed that this preliminary study will enable further investigations into the field at King George Hospital, which will eventually result in the positive use of IPF, to aid in diagnosing and monitoring of thrombocytopenia and determining treatment, respectively.
1.4 Future Prospects
In the last decade, there have been many discussions on the diagnostic use of immature platelet fraction. Several studies have demonstrated the parameter to be beneficial in routine haematology laboratories for the diagnosis of thrombocytopenia. Regardless of the intensive studies performed, its use is yet to be established.
Investigations carried out have shown up to a 3 days increase in IPF prior to platelet count during recovery of patients undergone chemotherapy. This is evident that it is a more useful marker for predicting a rise in platelet count, thus may aid in preventing unnecessary platelet transfusions from occurring.
As stated by many authors, the lack of standardisation and questionable specificity has limited it from progressing. Therefore, a study confirming these findings would be useful. An audit can then be carried out, showing its effects follow the change, which can aid in interpreting the best possible technique for diagnosis of thrombocytopenia and monitoring chemotherapy patient. If successful, a larger secondary study can be performed, where a threshold for IPF can be calculated for clinical decisions regarding platelet transfusion. Hopefully, in the near future, IPF will be incorporated into routine laboratories for diagnosis of thrombocytopenia and management of patients with haematological malignancies. This would, in-time improve patient care and cost management of blood transfusion.
Based on the above information, a question is asked; is IPF a good indicator of bone marrow function, which can be used to diagnose and monitor thrombocytopenia and help make clinical decisions regarding platelet transfusion?
WORD COUNT: 3,140
Section 2: Design Study
Thrombocytopenia is a common haematological condition, which if not treated, can progress to bleeding abnormalities. It is diagnosed routinely at King George Hospital via a full blood count. Further action consists of morphology assessment and bone marrow examination. The bone marrow aspirate is the final step, only performed if diagnosis is not identified. It is an invasive technique, thus not favoured by patients.
A new parameter on the Sysmex XE-2100; known as immature reticulated platelet fraction (IPF) has been developed, which targets this problem. It is a measure of reticulated platelets; a form of immature platelets found in peripheral blood. It has been considered to be a true reflection of thrombopoietic activity of the bone marrow. IPF can be detected by a quick, simple and non-invasive technique, which can help in identifying the pathophysiology of the condition.
By investigating the parameter in peripheral blood, a prediction can be made on the status of the bone marrow recovery in chemotherapy patient. Therefore it may be a beneficial marker in monitoring of therapeutic patients and in aiding clinical decisions regarding treatment, i.e. platelet transfusions.
Based on the information above, the following aims, objectives and hypothesis are drawn, which form the foundation of the study.
To determine if IPF can be used as a diagnostic marker to identify the pathophysiology of thrombocytopenia.
To determine if IPF can be used as a predictive marker to assess bone marrow function in chemotherapy patients.
To determine whether IPF can be used to help clinical decisions regarding treatment with prophylactic platelet transfusion.
IPF is a good indicator of bone marrow function, which can be used to diagnose and monitor thrombocytopenia and help make clinical decisions regarding platelet transfusion.
Determine the reference range for IPF at King George Hospital.
Determine the stability of IPF measurement during storage
Determine the precision of the IPF assay.
Compare IPF values for thrombocytopenic patients with different clinical conditions in order to identify its use in determining the pathophysiology.
Determine whether IPF predicts a rise in total platelet count in-patient with bone marrow suppression.
Determine in how many cases monitoring of IPF could have prevented the need for platelet transfusion
2.2 Research Plan
2.2.1 Flow Chart
Figure 3: flow chart of sequential events to take place to accomplish the study
Table 1: timetable consisting of events and the time in which is aimed to be completed
Obtain 200 EDTA samples from patients with normal full blood counts and perform IPF analysis using Sysmex XE2100, to determine reference range
View patient’s clinical details provided. Samples should have a normal full blood count results.
Measure the stability and precision of IPF
Repeat analysis at 0hrs, 3 times to calculate the precision of the assay. Analyze 50 samples with normal full blood count over a 48hr at two different temperatures.
Identify thrombocytopenic patients and categorise into group 1 and 2
Measure the IPF
Monitor IPF and PLT count of chemotherapy patients.
Most samples will be taken from the haematology/oncology unit at King George and Queen’s hospital
For each patient, collect the full blood count results.
Perform statistical analysis on the information gathered
January -March 2011
Write up first draft
Meet up with supervisor and add any suggestions and improvements made by supervisor
April -May 2011
Complete project write up
Hand into campus office
Make sure two copies are submitted.
2.3 Experimental Approach
The study protocol is awaiting ethical approval from the Research and Development (R&D) department of Barking, Havering and Redbridge university trust (BHRUT). This must be awarded before the practical commences.
All analysis will be performed in haematology laboratory at King George hospital and is aimed to be completed by December 2010.
2.3.1 Sample collection and criteria
Samples will be collected from King George and Queen’s hospital with a minimum of 2-3 ml of blood, taken by venepuncture in anticoagulant ‘ethylene diamine tetracetic acid’ (EDTA). All data processed will be from adult samples, thus this study is not valid for paediatrics or neonatal.
In the study, ‘normal’ will be defined as a patient with a full blood count within the hospital reference range, excluding any haematological abnormalities or unexplained clinical symptoms. Samples selected will be from patients samples requested for a full blood count between 20-50 years of age, with no bias towards sex.
Thrombocytopenia is defined as a platelet count of <100x109/l. Samples from patients with known causes will be selected and separated into two groups. Further investigation regarding the cause of thrombocytopenia would have been taken, such as bone marrow examination for group 2 patients.
Group 1: Hyperdestruction
Group 2: Hypoproduction
Before executing the practical, performance of the analysers needs to be checked. The internal quality controls used are purchased from Sysmex and analysed twice a day to ensure quality of the machines. Other quality measures taken include participation in the national external quality assurance scheme (NEQAS) and the Addenbrookes program, run weekly.
2.3.2 Reference range
The initial step consists of generating a reference range of IPF (%). To accomplish this, 250 normal patient samples will be analysed on the Sysmex XE2100 analysers. The data will be tabulated and the reference range will be calculated as mean +/- 1.96SD of the normal distribution.
The precision of the test will be determined by repeating IPF analysis five times on 50 normal samples. The standard deviation (SD) and coefficient of variation (CV) will need to be calculated.
The stability of immature platelets in EDTA will be established by analysing 50 normal samples kept in different conditions. Each sample will be separated into 2 aliquots, one, which will be kept in the fridge between 2-8°C, and the other, which will be kept at room temperature at about 20-25°C. The room temperature and fridge temperature will be monitored daily. Each sample will be analysed at 24hrs and 48hrs. The IPF at 0hrs will also be recorded. The data will be tabulated and presented as a scatter diagram. The best condition for storage of samples will be identified along with the time frame of analysis.
2.3.4 IPF analysis
Immature platelet fraction will be analysed in the RET chamber of the Sysmex XE2100 analyser (Sysmex, Kobe, Japan), using upgraded softawre. 4.5ml of EDTA blood will be aspirated via the sample rotor valve. The blood will then be diluted in 1:200 using 0.8955ml RetSearch (II) diluent, before being stained with 18Î¼l RetSearch (II) dye for approximately 31 seconds. 2.8Î¼l of the stained and diluted sample will enter the optical detector block. With the use of flow cytometry, the sample will go through a semi-conductor laser beam. The samples are then counted and separated based on cell size and RNA content. This will appear as an image of forward scatter and side scatter.
Thrombocytopenia detection in group 1 and 2
30 samples for each group needs to be obtained to continue with the study. Patients of group 2 will be selected on the basis of bone marrow examination. The samples will be analysed for a full blood count and IPF, where the data will be recorded in a table. Further unpaired t-test analysis will be carried out to establish the relationship between platelet count and IPF, which can then be used to determine whether IPF is a better marker than platelet count in detecting thrombocytopenia and the underlying cause.
Monitoring thrombocytopenia in chemotherapy patients
The use of IPF in monitoring patients on chemotherapy will also be investigated. The hypothesis states IPF to be used to monitor thrombocytopenia and help make clinical decisions regarding platelet transfusion. To prove this, 50 patients with thrombocytopenia post chemotherapy will be monitored for 14 days. The platelet count and IPF will be recorded along with any transfusion that may be given.
2.4 Data and Statistical Analysis
To perform statistical analysis and ease interpretation of results all raw data will be tabulated and presented in the appendix of the final project. The design of the tables can be seen below.
2.4.1 Analysis for Reference Range
Table 2: shows raw data used to determine reference range of IPF (%)
The ‘normal’ samples selected will be from patients between the ages 20-50 years.
Table 3: shows the IPF (%) value obtained at each repeat interval,
The data will be used to determine the precision by calculating the standard deviation of the mean and CV (%).
Table 4: shows the IPF (%) data over 48% of samples stored at different temperatures
0 hrs (°C)
24 hrs (°C)
48 hrs (°C)
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