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?- Sitosterol with Co Analysis

COMPLEX OF ?- SITOSTEROL WITH Co AT DIFFERENT MOLE RATIO AND THEIR ANTIMICROBIAL ACTIVITIES
TALAT MAHMOOD, YASMEEN BIBI, IFFAT MAHMOOD, SYED NASEEM HUSSAIN SHAH, ANEELA WAHAB,SIKINDAR SHERWANI, HUMAIRA ANWAR

Abstract
The principle of present work is to build up a plant-based method to reduce toxicity in our body which is caused due to high level of some essential trace metals. This current effort is concerning to investigate complex of ?- Sitosterol with Co, at different mole ratio and their antimicrobial activities. Complexes of ?-sitosterol (?s-Co) were formed with Co, at mole ratio of (1:1) (1:2) and (1:3). By pH metric titrations complex formation has been determined. After the formation of complexes metals are unable to absorb in our body and are excreted out in the form of complex from our body. In vegetable most common plant phytosterol; ?-sitosterol is present through intake of vegetable toxic metal can be eliminate out from body. Antimicrobial results indicate that all the complexes of ?s-Co have potential to kill various types of fungi and bacteria therefore these complexes can be used as the therapeutic agent for the treatment of bacterial and fungal infection. These complexes might be an excellent therapeutic runner for sake of different treatment but when completely explore from all parameter.
Introduction
Chelation in Metal Intoxication
In our body metals are an integral part of many functional as well as structural components in pathological and physiological processes critical role of metals have always been of great interest for researcher. Metals are used to restore the normal healthy physiology of body; either by chelating out excess or toxic metals ,direct administration of essential metals or for tagging biomolecule for diagnostic and using them as carrier for targeted drug delivery. Exposure to heavy metals from various sources and essential metal overload are responsible for metal toxicity. Metal interfere with the function of several organ systems such as kidney, liver, lungs haematopoietic system, central nervous system [Swaran 2010]. Ions and molecule bind with metal ion by chelation, chelating agent are inorganic as well as organic compound by binding metal ions they form complex ring-like structure known as chelate [Andersen O, 1999]. This complex structure is easily excreted from body by removing them from intracellular or extracellular spaces. Organic compound ?-sitosterol is present in many vegetables as well as in many fruits. Many scientific researches proved that for maintaining health, from protection against many serious health disorders and diseases ?-sitosterol is safe and nontoxic plant nutrient. Patients on diets devoid of plant sterols quickly became free of ?-sitosterol [Oja 2009] generally which implies that nutrients should be taken daily for excellent health and better functioning of immune system [Matsuoka et al. 2008]. ?-sitosterol posse’s antidiabetic, antihyperglycemic, and antibacterial, anti inflammatory, antipyretic acridity also antimicrobial, anti cancers. It is also beneficial for uterus and used to improve blood parameter [Normen et al. 2001]. For physiological activities in mammal also in humans 23 elements are identified. From these elements eleven trace elements such as Cr, Mn, Fe, Co, Ni, Zn, Cu, V, Se, Mo and Sn are essential element. [Murray et al. 2009]. When the concentration of these essential metals exceeds which is necessary for their biological function they become toxic.
For example large doses of cobalt (Co) may stimulate thyroid and bone marrow fraction [Fraga 2005]. Metal toxicity is more severe than organic toxicity because organic compounds may decompose but metals retain their identity in body. Most commonly used technique to treat heavy metal toxicity is chelation therapy. It involves administration of chelating agents to eliminate heavy metals from body [Marsha 1996]. In present paper we explore complexation of ?-sitosterol with Co, at different mole ratio. pH metric titration is used to study the formation of complex [T Mahmood et al]. Antifungal as well as antibacterial activities of these complexes are also investigated.
Material and Method
Reagent and glassware: Analytical grade reagent were used, purchase from Bio Basic Inc and Merck. All glassware used was of standard quality. Glassware are properly cleaned and rinsed with distilled water and dried before used. For potentiometric study salt of cobalt nitrate is used.
Instrumentation
Electrical balance: For weighing, Denver Instrument, TP- 214 was used.
pH meter: For pH metric titration, Jenway, model 3510 was used.
Stirrer: for stirring, hot plate stirrer (lab Tech) with bead was used.
pH metric titration: pH metric titration was done at 25±50C. Before titration of sample solution Sodium hydroxide solution was standardized using standard oxalic acid every time. Conical flask cover with rubber stopper is sued for all pH metric titration. This rubber stopper has four holes, one for purging inert gas (Nitrogen), and another for removal of oxygen, third for glass electrode and fourth for burette for addition of standard base. By passing Nitrogen gas for 30 minutes inert atmosphere is obtained in solution.
pH metric titration of ligand (?-sitosterol): For this purpose, in a conical flask containing magnetic bead, 10mL of chloroform and 40 mL of ?-sitosterol solution (10-2) were taken. For half an hour purified nitrogen gas was purged in this solution. After that ?-sitosterol solution was titrated against 0.1 M standard NaOH solution. Sodium hydroxide solution was prepared in methanol. Sodium hydroxide solution was standardized using 0.05M oxalic acid solution prior to the pH metric titration of ?-sitosterol. Magnetic stirrer is used for continuous stirring during titration. With the help of burette standard NaOH was added in sufficiently small increments of 0.1 ml and after each increment pH of reaction mixture was recorded till pH was not affected by further addition of standard NaOH. pH values were plotted against the added volume of standard NaOH.
pH metric titration of Co (II) with ?-sitosterol: In order to obtain metal-ligand complex pH metric titration of Co with ?-sitosterol were performed. ?-sitosterol solution (10-2M) solution is prepared in methanol: chloroform (1:1) and metal solution (10-2M) is prepared in methanol. By taking 10mL of ?-sitosterol solution and 10mL of metal solution 1:1 metal ligand solutions is obtained then add 30mL solvent in this solution. The mixture obtained was subjected to titration by using (0.1M) NaOH solution as a standard and under the same condition as used for earlier mention titration. Change in color at different pH confirmed the formation of complex. The above procedure is also repeated for mole ratio i.e. (1:2) and (1:3). The above procedure is also performed for the formation of Nickel Sterol complex but no change in colour takes place at any mole ratio.
Biological Assay
Preparation of media for antimicrobial activity: Muller Hinton agar and Muller Hinton broth [10] was used as the media for culturing bacterial strains and Sabourd dextrose agar (SDA) [Smyth et al.] was used as the media for fungal strains.
Screening of antibacterial activity: Antibacterial activity of ?s-Co (1:1, 1:2, and 1:3) against the test organisms were determined by using agar-well method. The Autoclaved Muller Hinton broth was used to refresh the bacterial culture, later well were punched into Muller Hinton Agar and 10 micro liters of culture were poured into the wells [Perez and Bazerque 2009].For screening of antibacterial activity 10mg/mL of sample is used .All plates were incubated at 28±2oC for 24-48 h and after the incubation diameter of zone of inhibition was noted by vernier caliper. Gentamicin antibiotic was used as a standard.
Screening of antifungal activity: Antifungal activity of ?s-Co (1:1, 1:2, and 1:3) was determined by using the agar-well method. Autoclaved distilled water was used for the preparation of fungal spore suspension and transfer aseptically into each SDA plates [Wuthi-udomlert and Vallisuta 2011]. For screening of antifungal activity 10mg/mL of sample is used. All plates were incubated at 28±2oC for 24-48h and after the incubation diameter of zone of inhibition was measured by vernier caliper. Gresiofulvin antifungal agent was used as a standard.
Determination of Minimum inhibitory concentration (MIC): Minimum inhibitory Concentration (MIC) was determined by Micro broth dilution method using 96-well microlitre plate. Two fold serial dilutions of extracts was made in 100 µl broth and subsequently 10 µl of two hour refreshed culture matched with 0.5 Mac Farland index was added to each well. One well served as antifungal agent control while other served as culture control. Microtitre plate was incubated for 24 hours at 37 ºc. The MIC was read as the well showing no visible growth.
Result and discussion
Initially ?- sitosterol titration was performed as a reference. In a plot of pH against added volume of NaOH only one curve was observed near pH 12.2. Remarkable changes in titration curves of ?- sitosterol and its complexes were observed (Fig.1), which indicates complexation between metals and ?- sitosterol. Complexation of ?-sitosterol with cobalt was confirmed by a change in color from Pink to Peach in case of ?s-Co (1:1) at pH 7.68, ?s-Co (1:2) at pH 7.35, and ?s-Co (1:3) at pH 7.14. Complex of ?s-Co (1:1) (1:2) (1:3) are formed at relatively high pH has moderate stability. From the present learning it is exposed that ?- sitosterol is helpful for removing toxicity of essential element by forming metal sterol complex and this essential toxic metal are excreted out from the body.[Marsha 1996]. The antimicrobial activities of complexes of ?s-Co show that they are also helpful in minimizing bacterial as well as fungal infection. The results of antibacterial and antifungal activity are presented in Tables.

Fig 1: Plot b/w pH againt volume of NaOH added. ?s = ?- Sitosterol, ?s-Co (1:1), ?s-Co (1:2) and ?s-Co (1:3) = Complex of ?- Sitosterol with cobalt
Gram positive bacteria
Zone of inhibition in mm (mean S.D)
Gram negative bacteria
Zone of inhibition in mm
(mean S.D)
?s-Co (1:1)
?s-Co (1:2)
?s-Co (1:3)
?s-Co (1:1)
?s-Co (1:2)
?s-Co (1:3)
Bacillus cereus



Enterobacter aerogenes



Bacillus subtilis



Escherichia coli ATCC 8739



Bacillus thruingiensis



Escherichia coli



Corynebacterium diptheriae
22 2
23 0
26 0
E. coli multi drug resistance



Corynebacterium hofmanii
20 0
25 1
25 0
Klebsiella pneumoniae
14 2
15 1
18 1
Corynebacterium xerosis
19 1
29 0
27 2
Salmonella typhi
12 1
19 1
22 0
Staphylococcus epidermidis



Salmonella paratyphi A



Streptococcus saprophyticus



Salmonella paratyphi B



M. smegmatis



Shigella dysenteriae



Streptococcus fecalis



Serratia marcesens
17 2
20 1
24 2
Streptococcus pyogenes



Acinetobacter baumanii
19 0
16 1
17 2
Campylobacter jejuni



Campylobacter coli



Helicobacter pylori



Hemophilus influenzae


Vibrio cholerae


Aeromonas hydrophila


Table -1 Antibacterial potential of ?s-Co (1:1), ?s-Co (1:2) and ?s-Co (1:3)
Extract
Bacteria
Minimum Inhibititory Concentration MIC (mg/ml)
?s-Co (1:1)
?s-Co (1:2)
?s-Co (1:3)
Gram positive bacteria
Corynebacterium diptheriae
78
88
68
Corynebacterium hofmanii
42
22
10
Corynebacterium xerosis
44
24
18
Gram negative bacteria
Klebsiella pneumonia
42
56
46
Acinetobacter baumanii
44
58
50
Serratia marcesens
50
52
48
Salmonella typhi
92
94
72
Table 2– Minimum Inhibititory Concentration (MIC) of ?s-Co (1:1), ?s-Co (1:2) and ?s-Co (1:3) in mg/ ml were determined by Micro dilution method:
Yeasts
Zone of inhibition(mm)
Dermatophytes
Zone of inhibition ( mm)
Saprophytes
Zone of inhibition mm
?s-Co (1:1)
?s-Co (1:2)
?s-Co (1:3)
?s-Co (1:1)
?s-Co (1:2)
?s-Co (1:3)
?s-Co (1:1)
?s-Co (1:2)
?s-Co (1:3)
Candidaalbicans
18 3
20 2
24 2
Microsporumcanis
12 2
12 2
12 2
Aspergillusflavus
22 2
20 2
26 3
Candidaalbicans ATCC 0383
20 1
24 0
26 1
Microsporum gypseum



Aspergillusniger
16 0
15 1
16 2
Saccharomyces cerevisiae



Trichophyton rubrum



Fusariumspecie
10 2
12 1
13 1
Candidagalbrata
20 1
21 1
20 1
Trichophyton mentagrophytes



Penicilliumsp
15 2
16 2
18 3
Candidatropicalis
21 2
23 3
27 0
Trichophyton tonsurans



Rhizopus



Candida kruzei
18 2
19 2
17 1
Helminthosporum



Table 3 – Screening of antifungal activity of ?s-Co (1:1), ?s-Co (1:2) and ?s-Co (1:3) against pathogenic fungi.
Yeasts
MIC (mg/ml)
Dermatophytes
MIC (mg/ml)
Saprophytes
MIC (mg/ml)
?s-Co (1:1)
?s-Co (1:2)
?s-Co (1:3)
?s-Co (1:1)
?s-Co (1:2)
?s-Co (1:3)
?s-Co (1:1)
?s-Co (1:2)
?s-Co (1:3)
Candida albicans
90
92
72
Microsporum canis
12
12
12
Aspergillus flavus
66
60
64
Candida albicans ATCC 0383
94
92
22
Microsporum gypseum



Aspergillus niger
60
42
32
Saccharomyces cerevisiae



Trichophyton rubrum



Fusarium specie
70
40
48
Candida galbrata
88
90
80
Trichophyton mentagrophytes



Penicillium sp
76
56
86
Candida tropicalis
92
82
72
Trichophyton tonsurans



Rhizopus



Candida kruzei
86
78
58
Helminthosporum



Table 4– Screening of antifungal activity of ?s-Co (1:1), ?s-Co (1:2) and ?s-Co (1:3) against pathogenic fungi
Conclusion: In present research work complexation of beta sitosterol with Co, at different mole ratio along with their antimicrobial activities has been studied. Complexation of ?- sitosterol with Co i.e. ?s-Co showed that complex formation takes place at neutral pH and have moderate stability. From centuries chelation therapy has been used to treat metal toxicity (Marsha, 1996).Their target organ are Kidney, central nervous system and cardiovascular system (Howland, 2011). In present paper ?-sitosterol form complexes with Co successively, hence can be used as chelating agent in chelation therapy. From result it is revealed that in case of essential metal toxicity ?s-Co could be helpful in removing metal toxicity from the body. It could perform this by forming complexes with cobalt which exceed the toxic level. This metal after formation of complex will be unable to absorb in the body and then excreted from the body in the form of complex. It is concluded that these complexes are not only reduce cobalt toxicity also the antimicrobial activity of ?s-Co exposed that these complexes are effective against many bacterial and fungal infection.
References
Andersen O. (1999). Principles and recent developments in chelation treatment of metal intoxication.Chem. Rev 99:2683–2710
Fraga, Cesar G. (2005). Relevance, essentiality and toxicity of trace elements in human health. Molecular Aspects of Medicine 26: 235–244.
Howland, M.A. (2011). Deferoxamine. In: Nelson, L.S., Lewin, N.A., Howland, M.A., Hoffman, R.S., Goldfrank, L.R. And Flomenbaum, N.E. Goldfrank toxicology Emergencies. New York: Mc Graw-Hill.604-608.
Jones MM. (1994). Design of new chelating agents for removal of intracellular toxic metals. In: Kauffman GB, editor. Coordination Chemistry: A Century of Progress. The American Chemical Society; pp. 427–438.
Marsha, D.F. (1996). Heavy metals. In Emergency Medicine- A compressive review, 4th edition, edited by J. E. Tintinall, E. Ruiz, and R.L. Krome, 833-841.
Matsuoka, K., T. Nakazawa, A. Nakamura, C. Honda, K. Endo, and M.Tsukada. (2008). Study of Thermodynamic Parameters for Solubilization of Plant Sterol and Stanol in Bile Salt Micelles. Chem. Phys. Lipids 154: 87-93.
Murray, R. K., D. A. Bender, K. M. Botham, P. J. Kennelly, V. W. Rodwell, P. A. Weil, and P. A. Mayes. (2009). Chapter 44. Micronutrients: Vitamins

Fresh Frozen Plasma (FFP) Collection, Preparation and Uses

Samuel Good
Fresh Frozen Plasma
Introduction
Fresh Frozen Plasma (FFP) is the name for the liquid portion of human blood, which has been frozen and preserved. It is taken by blood donation and is stored until needed for blood transfusion.
FFP has been available since 1941 (Hoffman, et al, 1990), it was used initially as a volume expander (Erber, et al, 2006), but is now used for the “management and prevention of bleeding in coagulopathic patients” (Ho, et al, 2005).
The term FFP is confusing as the plasma cannot be frozen as well as fresh at the same time. What the term implies is that the plasma was frozen rapidly after it was taken and therefore can be considered fresh.
The plasma, from a transfusion aspect, contains essential components such as fibrinogen, albumin, globulin and coagulation factors. These allow for specific individual components to be transferred to a recipient who is in need.
The most efficient and effective way to make optimum use of blood which has been donated, is to separate it into its individual components. This process allows for a “wider availability of blood products” (Spence, et al, 2006) and also reduces the risk patients are exposed to “transfusion-related risks” (Erber, et al, 2006).
The use of FFP and its individual products has increased tenfold since its first introduction (Hoffman, et al, 1990). One reason for this may be the declining availability of whole blood because of the trend to use component therapy (Spence, et al, 2006).
Collection and Storage
When a donor gives a unit of whole blood, the blood is then separated into several components parts. These include; packed red blood cells (pRBC), platelets and FFP. If required the FFP can be further divided into cryoprecipitate and something called cryo-poor plasma. Cryo-poor plasma is rarely used as a therapeutic response (Lauzier, et al, 2007).
As mentioned previously, plasma is the non-cellular, liquid part of the blood. It is made up of; water, electrolytes and proteins. The proteins include the clotting factors and intrinsic coagulants (Murray, et al, 1995).
The plasma is separated from the blood after donation and then frozen. For the plasma to be considered ‘fresh’ it must be frozen “within eight hours of collection” (Murray, et al, 1995) and stored at a temperature of minus 18 degrees centigrade or lower. If this fails to happen, the product is known just as ‘frozen plasma’, which like cryo-poor plasma, is rarely used for therapeutic means. However, to maintain coagulation factors to optimum levels the plasma should be stored at minus 30 degrees centigrade (Lauzier, et al, 2007).
FFP can be prepared by separation from whole blood or via plasmapheresis. Plasmapheresis is the name given to a “broad range of procedures” where “extracorporeal separation of blood components” (Erber, et al, 2006) results in a plasma which is filtered.
Preparation
To summarise, FFP is collected in citrate-containing anticoagulant solution, frozen within 8 hours and stored at minus 30 degrees centigrade for up to a year.
Although every protection is taken to ensure sterility, it is quite possible for the donor to have an asymptomatic bacteraemia at the time of donation (Stanworth, et al, 2004). The bacteria will have its proliferation down-regulated by the plasma being frozen. However, FFP can still sometimes transmit infectious diseases. Therefore, screening and pathogen inactivation may be performed to reduce the risk.
FFP contains no RBC’s and also no WBC’s. As there are no WBC’s the plasma is referred to be as being leucodepleted. This is an indication as to why FFP can transmit said diseases. As mentioned pathogen inactivation can be performed and this is done by using either Methylene blue or a solvent/detergent process.
The Methylene Blue Technique
Methylene blue is a dye that has been shown to be very effective in the inactivation of pathogens. It binds to nucleic acids and, on illumination with white light, singlet oxygen is formed. This then destroys viral DNA and RNA, therefore viral replication cannot take place.
Solvent/Detergent Technique
This technique is used for the preparation of factors viii and ix as well as immunoglobulins. First, a solvent is added to the plasma which removes the lipid viral envelope. After this is complete, a detergent is added which inactivates the viral contents. The solvent and detergent are then removed by a physical separation technique, in which they are dissolved in oil. Column chromatography can then be used to isolate factors viii and ix.
Once any treatment that is required is complete, the FFP is ready for use. It is an accepted practice that FFP is thawed before use (Ho, et al, 2005). The required units of FFP are placed in a water bath set at 30 – 37 degrees centigrade for approximately 20 – 30 minutes.
Von Heyman, et al investigated the effects of 2 different thawing machines and running warm water of 43 degrees centigrade, on the activity of clotting factors, inhibitors and activation markers in FFP. They discovered no significant differences in the activity of coagulation markers over a 6 hour period post thawing. However, a major conclusion found was that, if FFP is immediately transfused after thawing, the product remained rich in clotting factors. Also, if the plasma is left, the activity of said clotting factors decline gradually and therefore FFP should only be maintained at room temperature for up to 4 hours.
If thawed FFP is not used within 24 hours it becomes a separate product known as ‘thawed plasma’ (Murray, et al, 1995). Most clotting factors are stable in thawed plasma, however some labile factors, such as v and viii are not. Their degradation actually accelerates whilst the plasma is in a liquid state (Lauzier, et al, 2007).
The only main advantage of having thawed plasma readily available, is that it can be transfused rapidly if a severely injured patient requires it.
FFP Blood Type Specific
It is widely accepted that O negative is the universal donor for pRBC’s, however for FFP this isn’t the case. A and B antigens of the blood are located on the red cells themselves. Type O individuals are devoid of these proteins on their red blood cells.
Plasma does not contain RBC’s, but it contains antibodies to the corresponding absent protein. An example of this is:
Type A individual has Anti-B antibodies in their blood.
Type O plasma has both Anti-A and Anti-B antibodies and is incompatible with about 55 percent of the population.
An individual with type AB blood has neither Anti-A nor Anti-B antibodies.
This makes the AB plasma ideal for universal use when the blood type of the patient is unknown.
The Rh status is irrelevant because any plasma with Anti-D is destroyed at the manufacturing stage.
Recipient blood
Acceptable blood groups of donor plasma
O
O,A,B,AB
A
A,AB
B
B,AB
AB
AB
The major problem with blood type AB is that the percentage of the population which has it is only 4 percent. Therefore it is better to use FFP which is blood type compatible, which will be determined at the blood bank.
Usage
There are very few actual specific needs for the use of FFP (Spence, et al, 2006). Usually FFP is used to treat “deficiencies of coagulation proteins where specific factor concentrates are unavailable” (Hoffman, et al, 1990).
Coagulation deficiencies can occur in a variety of different clinical situations. These include massive blood loss, surgery, and infection or acquired multiple coagulation factor deficiencies.
Examples of FFP usage:
Replacement of isolated factor deficiencies
Reversal of Warfarin effects
Massive blood transfusion
Antithrombin III deficiency
Treatment of immunodeficiency
Treatment of thrombotic thrombocytopenic purpura
Treatment of Disseminated intravascular coagulation
Replacement of isolated factor deficiency
FFP can be used to heat deficiencies of factors II, V, VII, IX, X and XI. It is only chosen as a treatment when no specific component therapy is available. Certain factors require a different haemostatic level, for example; severe factor X deficiency only requires a factor level of about 10 percent. Therefore FFP has a range of success when treating factor deficiencies.
Reversal of Warfarin effect
If a patient is being treated with Warfarin, they have been shown to be deficient in “functional vitamin K dependent coagulation factors II, VII, IX and X” (Spence, et al, 2006). Usually vitamin K will be administered, however anticoagulated patients will be actively bleeding, and therefore FFP can be used.
Massive blood transfusion
The use of FFP as a treatment on massive blood transfusion has increased over the decades. Massive bleeding is defined as “the loss of one blood volume within 24 hours” or as “50 percent blood loss within 3 hours” or a “bleeding rate of 150 ml/minute” (Lauzier, et al, 2007). It is indicated for use in patients who have documented blood clotting abnormalities after large blood loss and who are in need of urgent treatment. This is due to the fact that in most emergency situations it is unacceptable to wait hours for lab results to be returned.
Antithrombin III deficiency
FFP is sometimes used as a source of Antithrombin III in people who are deficient of this inhibitor. Especially if the patients are undergoing surgery or who use Heparin to treat thrombosis.
Treatment of Immunodeficiency
FFP has been used in children and adults with a humoral immunodeficiency as a source of immunoglobulin. It is also sometimes used for infants when parental nutrition is lacking, and they are suffering with severe protein losing enteropathy (Erber, et al, 2006).
Treatment of thrombotic thrombocytopenic purpura
The treatment recommended for this condition is a daily plasma exchange (Murray, et al, 1995). Prompt intervention is indicated if development of neurological abnormalities start to appear. This plasma exchange usually continues for at least 2 days after remission (Ho, et al, 2005).
Treatment of Disseminated intravascular coagulation
Disseminated intravascular coagulation (DIC) is a syndrome where the control of the coagulation system becomes disturbed and out of control. This is usually due to pro-coagulants being dispersed into circulation (Stanworth, et al, 2004). Most of the time this happens secondary to a disease or disorder, such as cancer. In the presence of DIC, fibrinogen, platelets and coagulation factors V and VIII become rapidly depleted. FFP is given as treatment to prevent further problems or progression. Treatment usually involves a patient being infused with a single line of FFP and then coagulation tests performed to assess the clinical benefit (Stanworth, et al, 2004).
There are also some conditional uses where FFP can be used but is not the first choice treatment, such as liver disease and Paediatric use. If patients have an abnormal coagulation profile and are suffering from liver disease, they can be treated with FFP. There is varying success and treatment must be monitored by regular transfusion coagulation tests.
Clotting times of infants have been shown to be longer than that of adults (Murray, et al, 1995), and even longer in premature babies (O’Shaughnessy, et al, 2004). Vitamin K deficiency is the most common cause of neonatal bleeding (Murray, et al, 1995). FFP can be used to counter the effects if required. In the case of babies suffering from haemorrhagic disease of the newborn, FFP can be used as treatment. But only if the “chance of bleeding is greater than the risk of harmful reactions” to the treatment with FFP (Lauzier, et al, 2007).
Risks
As with any transfusion there is a risk of infection, the main risks identified include:
Disease transmission
Excessive intravascular volume
Anaphylactoid reactions
Alloimmunisation
Transfusion related acute lung injury
The risks associated with viral infectivity of FFP are similar to that of whole blood and RBC’s. As mentioned earlier this risk can be countered by photochemically treating the plasma.
Allergic reactions that occur in response to FFP transfusion vary in severity from “hives to fatal non-cardiac pulmonary oedema” (Stanworth, et al, 2004). Transfusion relate acute lung injury (TRALI) is defined as a “new episode of acute lung injury within 6 hours of complicated therapy” (O’Shaughnessy, et al, 2004). It manifests as severe respiratory problems, including hypoxia and other symptoms linked to pulmonary oedema. Symptoms will usually subside 2 days after ceasing FFP treatment (Stanworth, et al, 2004).
Alloimmunisation can occur if Anti-Rh antibodies are formed after treatment with FFP. To counter this, plasma containing Anti-D antibodies should not be given to an RhD-positive recipient. There has also been reported incidences of post-transfusion Hepatitis, and depends on a number factors, including donor selection. Also with any intravenously transfused fluid, there is a chance of hypervolemia which could lead to cardiac failure, therefore administration of FFP should not be given in excessive doses.
Below is a suggested dosage breakdown:
Volume of 1 Unit Plasma: 200-250 mL 1 mL plasma contains 1 u coagulation factors 1 Unit contains 220 u coagulation factors Factor recovery with transfusion = 40% 1 Unit provides ~80 u coagulation factors 70 kg X .05 = plasma volume of 35 dL (3.5 L) 80 u = 2.3 u/dL = 2.3% (of normal 100 u/dL) 35 dL
In a 70 kg Patient: 1 Unit Plasma increases most factors ~2.5% 4 Units Plasma increase most factors ~10%
Figures taken from (http://reference.medscape.com/drug/ffp-octaplas-fresh-frozen-plasma-999499)
Conclusion
In conclusion, FFP can be used as an effective treatment for a number of different clinical issues. It also does not come without risk and therefore FFP should be collected, stored, prepared and used in an efficient and safe manner. Below I have summarised the administration of FFP.
FFP (Fresh Frozen Plasma) Volume: 240-300ml (mean 273ml)
Storage: designated temperature controlled freezer. Core temperature -30 o C
Shelf life: 24 months (frozen)
Must be ABO compatible, but Rh is not necessary to be considered for transfusion and no anti D prophylaxis is required if Rh-D negative patients receive Rh-D positive FFP.
Prior to the transfusion FFP must be thawed under controlled conditions using specifically designed equipment. Thawing usually takes approximately 15-30 minutes
Once thawed, FFP must not be re-frozen and should be transfused as quickly as possible. Post-thaw storage results in a decline in the quality of coagulation factors.
If stored at 4 degrees centigrade post thawing (in a designated temperature controlled refrigerator), the transfusion must be completed within 24 hours of thawing.
Pooled solvent-detergent treated plasma is also commercially available
Dose: typically 10-15ml/kg. This dose may need to be exceeded in massive haemorrhage depending on the clinical situation and its monitoring (BCSH 2004)
Typical infusion rate 10-20ml/kg/hr (approximately 30 minutes per unit)
Rapid infusion may be appropriate when given to replace coagulation factors during major haemorrhage. There is anecdotal evidence that acute reactions may be more common with faster administration rates.
(http://reference.medscape.com/drug/ffp-octaplas-fresh-frozen-plasma-999499)
REFERENCES
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http://en.wikipedia.org/wiki/Fresh_frozen_plasma
http://www.psbc.org/therapy/ffp.htm
http://reference.medscape.com/drug/ffp-octaplas-fresh-frozen-plasma-999499
http://ccforum.com/content/14/1/202

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