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Keywords:

  • plasma;
  • viral inactivation;
  • methylene blue;
  • solvent–detergent;
  • blood safety

The likelihood of transfusion of transmissible infection via blood components and fractionated blood products has declined dramatically in the last two decades. In the early 1970s, the risk of hepatitis transmission to recipients of blood or blood components was between 10% and 25% (Horowitz & Ben-Hur, 1995). Today, there is still a residual risk associated with labile blood components [fresh frozen plasma (FFP), platelets and red cells]. Recently, in the UK, this has been estimated for human immunodeficiency virus (HIV)-1 and -2 as 0·19/million donations, for hepatitis C virus (HCV) as 0·6/million donations and for hepatitis B virus (HBV) as 5–20/million donations (L. M. Williamson, personal communication). This risk reduction has been achieved largely by improved donor selection procedures and the screening of donated blood for agents that may cause transfusion-transmitted infections (TTI). Pooling of plasma donations to make fractionated blood products such as coagulation factor concentrates and intravenous immunoglobulin (IVIG) increases the chance of viral transmission. In the USA, between 1979 and 1985, 70% of severe haemophiliacs became infected with HIV-1 that contaminated factor VIII concentrates (Goedert et al, 1989). Since then, a number of viral inactivation strategies including dry heat, heating solution and solvent–detergent (SD) treatment have been developed for fractionated plasma products. These have resulted in an enormous reduction in risk such that the likelihood of viral transmission from such fractionated blood components can now only be estimated by calculations of probability. These are based on factors such as the risk per donor, quantity of virus that may be present in a unit that gives a negative test, the number of units pooled and estimations of virus killing and removal during the process. In 1990, this was determined for a factor VIII concentrate purified by a monoclonal antibody affinity method and SD treated. It was calculated that the risk for an HIV-1, HBV or HCV transmission was < 1 in 1016, in 1013 and in 106 vials administered respectively (Horowitz & Ben-Hur, 1995). This represented, at the time, a risk reduction of between 1000 and 10 billion compared with untreated single unit blood components. After licensing for the treatment of coagulation factor concentrates in 1985, it was shown at the New York Blood Center that SD treatment could be used for the viral inactivation of FFP (Horowitz et al, 1992). In 1991, it was estimated that > 3 million doses of SD-treated FFP prepared from pooled plasma had been administered in Europe (Horowitz et al, 1998). This process was licensed by the Food and Drug Administration (FDA) in 1999. During this time, it was also shown that the use of methylene blue (MB) in conjunction with visible light had significant antiviral activity (Lambrecht et al, 1991; Bachmann et al, 1995). In 1992, MB-FFP was introduced into clinical use by several Red Cross Transfusion Services in Germany and Switzerland (Mohr et al, 1997). In the 1990s, there were a number of reports describing the photodynamic treatment with psoralens and ultraviolet A (UVA) light to inactivate viruses in platelet concentrates (Dodd et al, 1991; Margolis-Nunno et al, 1997; Grass et al, 1998). Recently, viruses and other pathogens have been inactivated in FFP using a combination of the psoralen S-59 and UVA light (Grass et al, 1998).

It is important to balance the potential benefits of reduced viral transmission against the cost and logistics of implementing viral inactivation strategies, although in theory it is desirable that the risk of TTI is minimized wherever possible. Therefore, there is a reluctance to ignore new strategies that may help to achieve this and a need to assess their potential contribution to blood safety. In this article, the use of SD treatment and the use of MB plus white light and the psoralen S-59 plus UVA light are discussed and progress made towards their clinical implementation is described.

Avoidance of viral transmission

  1. Top of page
  2. Avoidance of viral transmission
  3. Methods used for virus inactivation in pooled plasma derivatives
  4. Quality assessment of virally inactivated plasma
  5. Solvent–detergent-treated ffp (sd-ffp)
  6. Methylene blue-treated ffp (mb-ffp)
  7. FFP TREATED WITH THE PSORALEN S-59 and ULTRAVIOLET A (UVA) LIGHT (S-59-UVA-FFP)
  8. Selecting the best option for clinical ffp
  9. Conclusions
  10. References

In the UK, this is based on rigorous donor selection criteria to eliminate those who may be at particular risk of transmitting infection. In addition, FFP is not made from first-time donors. This is coupled with testing of all donations for HIV-1 and -2 (HIV-1 and -2 antibodies), HCV (HCV antibodies) and HBV (HBsAg) (Guidelines for Blood Transfusion Services in the United Kingdom, 2000). Blood Services in the UK also introduced PCR testing for HCV-RNA in 1999 for FFP but not cellular products. In addition, in some countries, blood centres routinely use additional tests, including alanine aminotransferase, anti-HBc, PCR for HCV-RNA, p24 antigen (HIV-1) and HTLV-1 antibody. Plasma pools used to make solvent–detergent-treated FFP may also be tested by PCR for hepatitis A (HAV), HBV, HCV, HIV-1 and parvovirus B-19. However, in the UK, blood is not tested for HAV and parvovirus B-19. For fractionated plasma products, the risk is reduced further by partitioning of viruses during the fractionation process before viral inactivation. There is good evidence that HTLV-1 and cytomegalovirus (CMV) are not transmitted by FFP (Bowden & Sayers, 1990; Donegan et al, 1994), but there may be a number of other infectious agents, potentially transmissible via labile blood components, which may not be identified or known at the present time to be pathogenic.

It is important not to increase the chance of TTI by transfusing blood components unnecessarily and there is ample evidence that for FFP this is often the case. Guidelines produced by the National Institutes for Health (NIH) (Consensus Conference, 1985) and the British Committee for Standards in Haematology (BCSH) (Contreras et al, 1992) indicate where it is appropriate to transfuse FFP (Table I). The BCSH indications subdivide indications for transfusion of FFP into definite and conditional. Both guidelines indicate that FFP should not be used for nutritional support, volume expansion and as ‘formula replacement’ in patients with massive haemorrhage, e.g. administration of one unit of FFP for each 4–6 units of blood transfused. In addition, the BCSH guidelines do not recommend transfusion of FFP as a source of immunoglobulin in inherited immunodeficiency states (Contreras et al, 1992). Despite this guidance, there is evidence from audits that FFP is often transfused inappropriately (Snyder et al, 1986; Thomson et al, 1991; Schots & Steenssens, 1994). Lack of adherence to clinical guidelines was illustrated in the European Sanguis study, in which the use of FFP in total hip replacement, coronary artery bypass grafting and abdominal aortic aneurysm surgery varied from < 5% to > 95% of cases (Sirchia et al, 1994). In this study, the most common reason for receiving a transfusion of any sort was most specifically related to the hospital in which the operation was undertaken and, within that, to the hospital team performing surgery (Sirchia et al, 1994). Where clinicians completed a questionnaire specifying their triggers for prescribing FFP, the commonest indications were bleeding (43%), abnormal coagulation tests (26%) and signs/symptoms of hypovolaemia (16%) (Snyder et al, 1986).

Methods used for virus inactivation in pooled plasma derivatives

  1. Top of page
  2. Avoidance of viral transmission
  3. Methods used for virus inactivation in pooled plasma derivatives
  4. Quality assessment of virally inactivated plasma
  5. Solvent–detergent-treated ffp (sd-ffp)
  6. Methylene blue-treated ffp (mb-ffp)
  7. FFP TREATED WITH THE PSORALEN S-59 and ULTRAVIOLET A (UVA) LIGHT (S-59-UVA-FFP)
  8. Selecting the best option for clinical ffp
  9. Conclusions
  10. References

Our understanding of techniques for inactivating viruses in blood products derives in large part from the fractionation of plasma to make albumin, intravenous immunoglobulin (IVIG) and coagulation factor concentrates. The physical separation of plasma proteins is achieved primarily by adjustment of pH, temperature and ethanol concentration (Knowles, 1995). This process is known as Cohn cold ethanol extraction. Further purification steps include ion exchange, affinity and size exclusion chromatography and polyethyleneglycol precipitation. Subsequently, a number of techniques including dry heat, heating in solution, solvent–detergent treatment and β1 propiolactone/ultraviolet light treatment may be used. Most of these steps result in a significant reduction of infectious viruses. Cold ethanol fractionation itself removes 5 log of HIV (Burnouf-Radosevich et al, 1992) and other viruses, including pseudorabies, bovine viral diarrhoea virus (BVDV), Sindbis, vaccinia and vesicular stomatitis viruses (VSV) (Chandra et al, 1999) but not hepatitis viruses (Burnouf-Radosevich et al, 1992). Affinity chromatography is active in removing both enveloped (Sindbis) and non-enveloped (polio) viruses (Roberts et al, 1994). Pasteurization (60°C for 10 h) kills a wide range of both enveloped and non-enveloped viruses (Burnouf-Radosevich et al, 1992; Chandra et al, 1999). Combined treatment with β-propiolactone, detergent and ultraviolet (UV) C light was shown to inactivate 6·9 log of hepatitis B and non-A, non-B viruses in a chimp model and is used in the manufacture of some factor (F)IX concentrates (Prince et al, 1983). In other studies, UVB or UVC irradiation, laser-pulsed UVB irradiation and gamma irradiation (2·5–10 MRad) were found to inactivate between 4 and 6 log of polio, vaccinia, polio and HSV-1 and HIV-1 (Prodouz et al, 1987; Hiemstra et al, 1991; Hart et al, 1993). Solvent–detergent (SD) treatment is highly effective in killing a number of viruses but is relatively ineffective against non-enveloped viruses (Mitra et al, 1994). A number of chemicals damage viruses and have been evaluated for viral inactivation. These include sodium hypochlorite and dichloroisocyanurate (duck HBV) (Tsiquaye & Barnard, 1993) and members of the imine family such as N-acetylethyleneimine (polio and foot and mouth disease viruses). Methylene blue (MB) has good activity against enveloped viruses (Brown et al, 1998). Three principal technologies have emerged for the virus inactivation (VI) of unfractionated plasma. These are SD, MB plus white light treatment and the use of a novel psoralen (S-59) followed by UVA exposure.

Despite the use of viral inactivation procedures, there was concern after outbreaks of HAV infection occurred in patients with haemophilia A who were treated with SD-treated FVIII concentrates in Italy, Belgium, Germany and Ireland (Gerritzen et al, 1992; Mannucci, 1992; Temperley et al, 1992; Peerlinck & Vermylen, 1993). The cases that were described occurred between 1988 and 1993 and were characterized by jaundice and the presence of IgM anti-HAV. Causality has not been universally accepted; those who maintain that direct evidence is lacking point out that the risk factors for certain groups of haemophiliacs were not always clearly stated and that in some cases epidemiological definition of the control groups mentioned was lacking (Robinson et al, 1992). Moreover, in 330 patients with haemophilia A studied in Norway, 28 of 202 in whom testing was performed had evidence of infection with HAV. However, it was shown in 27 patients (one emigrated) using archival samples that antibody was present before the institution of treatment with SD-treated FVIII (Evensen & Rollag, 1993). Other investigations in Finland and the USA have not revealed conclusive evidence of HAV transmission by SD-treated FVIII (Prowse et al, 1994).

Quality assessment of virally inactivated plasma

  1. Top of page
  2. Avoidance of viral transmission
  3. Methods used for virus inactivation in pooled plasma derivatives
  4. Quality assessment of virally inactivated plasma
  5. Solvent–detergent-treated ffp (sd-ffp)
  6. Methylene blue-treated ffp (mb-ffp)
  7. FFP TREATED WITH THE PSORALEN S-59 and ULTRAVIOLET A (UVA) LIGHT (S-59-UVA-FFP)
  8. Selecting the best option for clinical ffp
  9. Conclusions
  10. References

This should include an initial extensive investigation of a range of parameters on a relatively small number of units (for example 20 units of virally inactivated FFP compared with control FFP). This will involve in vitro studies, including those in which units are thawed and tested at intervals, e.g. 3, 6 and 12 months after manufacture, and more reliable data may be obtained where each subject donates twice (paired study design). In vivo studies in healthy volunteers may be required. One donation is handled according to standard procedures and the other is subjected to the viral inactivation (or other) strategy being tested before reinfusion. Currently, the guidelines for evaluation of new FFP/cryoprecipitate components for transfusion in the UK (Guidelines for Blood Transfusion Services in the United Kingdom, 2000) suggest the following.

• Volume, platelet count, WBC (the last is particularly important where plasma filtration is included; in the UK, from November 1999, all FFP has been manufactured as leucodepleted).

• Prothrombin time, partial thromboplastin time.

• Specific coagulation factors assays for fibrinogen (FBG), factors II, V, VII, VIII, IX, X, XI, von Willebrand factor antigen (VWF:Ag), von Willebrand factor ristocetin cofactor activity (VWF:RiCof).

• Analysis of VWF multimers on a small number of units only.

• Inhibitors of coagulation – antithrombin III, protein C, protein S.

• Markers of unwanted activation of coagulation – such as prothrombin fragment1+2 or fibrinopeptide A.

• Markers of unwanted activation of kinin/complement – C3a, C5a, bradykinin, factor XIIa.

Other assays such as fibrin split products and thrombin antithrombin (TAT) complexes may be helpful. Testing for the presence of neoantigens should also be carried out.

It is also recommended that consideration is given to performing studies on representative units of FFP stored at −20°C as well as −30°C (to reflect differing hospital storage conditions). Sampling should be undertaken at 3, 6, 9 and 12 months. The minimum assays to be performed at each time point should include FBG and FV, VIII, IX, X and vWF:RiCof.

It is also important to demonstrate that the agents used in viral inactivation systems such as methylene blue, solvents, detergents and psoralens are neither toxic nor mutagenic. These studies are usually undertaken by the manufacturer. Ideally, the systems in place should include their removal from plasma (≥ 99%) before it is frozen. Finally, it is important in model systems to demonstrate that significant antiviral activity (usually ≥ 4 log) exists against both lipid enveloped and, if possible, also non-lipid enveloped viruses. Testing may also assess antiviral activity against extracellular as well as intracellular viruses.

Solvent–detergent-treated ffp (sd-ffp)

  1. Top of page
  2. Avoidance of viral transmission
  3. Methods used for virus inactivation in pooled plasma derivatives
  4. Quality assessment of virally inactivated plasma
  5. Solvent–detergent-treated ffp (sd-ffp)
  6. Methylene blue-treated ffp (mb-ffp)
  7. FFP TREATED WITH THE PSORALEN S-59 and ULTRAVIOLET A (UVA) LIGHT (S-59-UVA-FFP)
  8. Selecting the best option for clinical ffp
  9. Conclusions
  10. References

Activity and method of preparation

The solvent–detergent treatment process was first licensed in 1985 for the manufacture of factor VIII concentrates (Horowitz & Ben-Hur, 1995). The treatment process had also been validated in the manufacture of antiviral vaccines and damages lipid but not proteins (Horowitz, 1991). The combination frequently used is 1% tri(n-butyl) phosphate (TNBP) in combination with a detergent, usually 1% Triton X-100. Pools of between 380 and 2500 ABO identical units of FFP are made after thawing (Horowitz & Ben-Hur, 1995). Contributing units may be either Rh D positive or negative. The SD treatment is performed at 30°C, usually for 4 h (Horowitz & Ben-Hur, 1995). The starting plasma is filtered to remove residual cells and cell debris and after treatment the TNBP is removed (< 2 µg/ml) using oil extraction and phase separation and Triton X-100 (< 5 µg/ml) by means of hydrophobic chromatography on C18 resin (Horowitz et al, 1992; Horowitz & Ben-Hur, 1995). It is claimed that the chromatographic step used to remove solvent is effective in removing HAV (Evensen & Rollag, 1993). The plasma is then sterile filtered (0·2 µm) and frozen in aliquots of 200 ml (Horowitz & Ben-Hur, 1995).

Product quality

The levels of coagulation factors in SD-FFP are generally equivalent to those in the start pool (Horowitz & Ben-Hur, 1995) and, indeed, recovery is usually > 90% (Table II) (Piët et al, 1990; Hellstern et al, 1992; Horowitz et al, 1992, 1998). Although levels of VWF:Ag and VWF:RiCof are > 90%, there is loss of some high molecular weight (HMW) VWF multimers during processing (Piquet et al, 1992). Factor VIII has been shown to be stable during 3 months of storage (Piquet et al, 1992). There is no evidence of activation of coagulation or fibrinolysis (Horowitz & Ben-Hur, 1995). Levels of plasminogen, C1 esterase inhibitor, TAT complexes and fibrin split products are normal but levels of α-2-antiplasmin and protein S are significantly reduced during the SD treatment process. Levels of d dimers are normal (Hellstern et al, 1992). No evidence of activation of coagulation factors has been reported (Piquet et al, 1992). A lack of neoantigenicity has been demonstrated in experiments in New Zealand white rabbits using crossed immunoelectrophoresis (Horowitz et al, 1992).

Viral inactivation

A wide range of lipid-enveloped viruses are inactivated rapidly, usually within 15 min of addition of solvent and detergent (Horowitz et al, 1993; Biesert & Suhartono, 1998). A summary of viral inactivation studies is shown in Table III. In addition, it has been shown that an SD-treated immunoglobulin prepared from patients with HIV did not transmit infection to chimpanzees (Hellstern et al, 1992). Some viruses are studied because they cause clinical infection in man, whereas others act as models for viruses which cannot themselves be grown in culture, e.g. pseudorabies and duck HBV (DHBV) are model viruses for HBV and bovine viral diarrhoea virus (BVDV) is a model virus for hepatitis C. SD treatment does not inactivate non-lipid-encapsulated viruses such as HAV and parvovirus B-19. This has led to concerns that overall benefit might be limited as non-irradiated viruses in a batch of SD-FFP could be transmitted to a large number of susceptible recipients (Williamson & Allain, 1995).

Partitioning of the immunoglobulin fraction during preparation of coagulation factor concentrates leads to loss of protective IgG antibody, but in the case of FFP it has been estimated that pooling of donated plasma results in levels of IgG anti-HAV 30 times higher than that in intramuscular immunoglobulin preparations, which have not been shown to transmit hepatitis.

Levels of antiparvovirus B-19 IgG antibody are similar to those found in IVIG that can be used for treatment of persistent parvovirus infections (Horowitz & Ben-Hur, 1995; Horowitz et al, 1998) and, in data from Norway, showed uniform virus neutralization (Solheim et al 2000) (see below). However, in the USA, 19 batches of SD-FFP have been recalled because of seroconversions to parvovirus B-19 and detection of B-19 DNA by PCR in volunteers who had received SD-FFP (CBER Document no. 0694, 1999). The former might have been due to passive transfer of antibody. In addition, batch testing revealed higher than acceptable levels of parvovirus B-19 nucleic acid. It has been shown that by screening donor blood by genomic testing (PCR) for HAV and parvovirus B-19, SD-FFP can be prepared from PCR-negative plasma pools (Horowitz et al, 1999). The clinical sequelae of B-19 infection include marrow aplasia in patients with haemolytic anaemias or other erythropoietic abnormalities and hydrops fetalis if transfused during pregnancy. SD-FFP has not, to date, been associated with documented viral transmission in clinical practice. In a study of 343 adults undergoing cardiac surgery, 194 received transfusion support which included SD-FFP and 41 patients received only SD-FFP. Twenty-five batches of SD-FFP prepared from Norwegian plasma were used. All batches contained neutralizing antibodies to B-19 and 20 of 25 to HAV, whereas in the other five the anti-HAV reactivity was borderline. After a 6- to 12-month follow-up period to allow washout of transfused antibodies, testing revealed seroconversion to HAV in four patients, to HBV (anti-HBc) in one patient, to CMV in four patients and to parvovirus B-19 in nine patients. No seroconversions could be ascribed to transfusion of SD-FFP (Solheim et al 2000).

Secondary products

Cryoprecipitate has been prepared from SD-treated pooled FFP. In SD cryoprecipitate, levels of VWF activity and antigen were reduced to only 36% and 30% of control values respectively. The fibrinogen level was 72%. The highest molecular weight multimers of VWF were absent from cryoprecipitate, indicating that this product could be an alternative to standard cryoprecipitate for the treatment of hypofibrinogenaemic states but unsuitable for treating von Willebrand's disease (Keeling et al, 1997).

Toxicity

There has been no evidence of toxicity when SD-treated blood components or fractionated products are transfused. In a haemovigilance study in Belgium, no adverse events were reported during a year when 5064 units of SD-FFP were transfused to 894 patients (Baudoux et al, 1998).

Resuspension of red cells and platelets in SD-FFP

Occasionally, FFP from a different donor is used for the resuspension of either red cells or platelets before transfusion. Patients who require massive transfusion, for example after trauma, receive large volumes of blood components, including red cells, platelets and FFP. Studies over 5 d of storage using both red cells and platelets have shown no adverse effects of SD-FFP on parameters of red cell or platelet function (Tocci et al, 1993; Snyder et al, 1994).

Transfusion studies of SD-FFP

Studies using SD-FFP in volunteer subjects were not conducted before its clinical evaluation in patients. Subsequently, phase IV studies have been carried out in healthy volunteers (CBER Document no. 0694, 1999) but data concerning clinical efficacy, for example coagulation factor recovery, are not published at the present time.

Coagulopathy In 11 patients with hereditary deficiency of FVII, X or XI, administration of SD-FFP resulted in cessation or prevention of haemorrhage; the factor half-lives were as expected (Inbal et al, 1993). Similar findings were reported in patients with FXIII deficiency during 39 episodes of prophylaxis (Horowitz & Pehta, 1998). In the UK, haemophilia directors have recommended the use of virally inactivated FFP to treat single-factor deficiencies when no factor concentrate is available (United Kingdom Haemophilia Centre Directors Organization, 1997). A significant improvement in the prothrombin time and FBG level was demonstrated together with stabilization of blood pressure and cessation of clinical bleeding in 16 of 22 patients with disseminated intravascular coagulation (DIC) (Hellstern et al, 1993). Reports describe the use of SD-FFP in 75 patients with surgical bleeding and nine patients in whom warfarin reversal was required preoperatively (Horowitz & Ben-Hur, 1995). In addition, a group of 48 patients with coagulation factor deficiencies received a total of 788 units of FFP. These were given for surgical prophylaxis (47 episodes), active bleeding (51 episodes) and in FXIII deficiency (see above) (Horowitz & Pehta, 1998). In both reports, the expected levels of coagulation factors were achieved and bleeding was adequately controlled.

Heart surgery In 122 patients of whom 53 (43%) required plasma, SD-FFP was offered to 46 patients and given to 20. Twenty controls received standard FFP. There were no perioperative differences in cardiovascular or respiratory support given or transfusion requirements. Correction of the prothrombin time was equivalent and there was no laboratory evidence of complement activation (Solheim et al, 1993).

Thrombotic thrombocytopenic purpura (TTP) In some patients with this condition, abnormally large high molecular weight (HMW) VWF multimers are present. Cryosupernatant plasma lacks large VWF multimers and could be advantageous in treatment. A recent report suggests at least equivalent, and possibly superior, efficacy compared with standard FFP (Rock et al, 1996). As SD-FFP lacks some HMW VWF multimers, it may also be particularly useful in this group of patients. A number of reports of plasma infusion or exchange using SD-FFP show no adverse effects during treatment (Moake et al, 1994; Pehta, 1996; Evans et al, 1999). Two out of five patients with chronic relapsing TTP received alternate treatments with conventional vs. SD-FFP and 3/5 received only SD-FFP on study, with their responses compared with their historical control data; responses in platelet count and duration of remission were similar and no adverse events were reported. In a further two children with chronic relapsing TTP who received SD-FFP infusions, evidence of VWF-mediated, sheer stress-induced platelet aggregation together with an increase in HMW VWF multimers in platelet-poor plasma was observed. After plasma infusion, abnormal HMW VWF multimers disappeared within 1 h, reversal of abnormal sheer stress in 8/9 infusions studied occurred within 1–4 h and the platelet count usually normalized after a week. Therefore, an important plasma constituent responsible for the control of TTP is unaffected by the SD process (Moake et al, 1994). A further six patients with chronic relapsing TTP who received SD-FFP infusions showed a good response in the platelet count with reduction of lactate dehydrogenase (LDH) and stabilization of the haemoglobin (Horowitz & Pehta, 1998). In three patients with acute TTP all treated using plasma exchange against SD-FFP, the platelet count rose to > 50 × 109/l by day 3 in one patient, by day 7 in another patient and by day 10 in the third patient. All remain in remission > 1 year later (Evans et al, 1999).

Liver disease In a multicentre UK study, 24 liver disease and 25 liver transplant patients were randomized to received SD-treated or standard FFP. Equivalent correction of the international normalized ratio (INR), activated partial thromboplastin time (APTT) ratio and levels of FII, VII and protein C were noted. Blood component usage in each group for liver transplant patients was equivalent. In at-risk patients with sufficient follow-up, no seroconversions to HBV or HCV were seen and although seroconversion to HAV was suspected originally in four patients this was thought to be the result of transfused IgG antibody as three out of four had no detectable IgM anti-HAV and the IgG antibody eventually became undetectable. HAV-RNA testing was not carried out on these patients (Williamson et al, 1999a).

Children Few data on the use of SD-FFP are available in children. In two neonates and 18 children (1·5–17 years), no untoward effects followed SD-FFP transfusion and in Norway 75 transfusions of SD-FFP were given uneventfully to neonates and older children (Klein et al, 1998).

Other potential benefits

Front-end filtration of FFP before SD treatment removes cell and cellular/particulate debris. Leucocyte depletion reduces both the incidence of alloimmunization (AI) to HLA and the occurrence of non-haemolytic febrile transfusion reactions (NHFTR). Currently, in the UK, universal leucodepletion of all blood components has been introduced to reduce the theoretical risk of transfusion transmission of the pathogenic agent of new variant Creutzfeld-Jakob disease (nvCJD). However, studies using a time-resolved fluoroimmunoassay for the cellular isoform of prion protein (PrPc) in human blood has shown that 68·5% is associated with plasma and 26·5% with platelets (MacGregor et al, 1999). The significance of this regarding acquisition of the causative agent of nvCJD is unknown at the present time. Terminal filtration using a 0·2-µl filter effectively removes bacteria (Horowitz & Ben-Hur, 1995). Theoretically, the presence of neutralizing antibody to pathogenic non-enveloped viruses should prevent their transmission, although it is possible that this could occur if antibody in the donor population was present at a low frequency or was non-neutralizing. Because of the extreme dilution that occurs when pooling, SD-FFP may be beneficial in reducing the number of reactions that occur as a result of infusion of antibodies present in high titre in individual donations, for example transfusion-related acute lung injury (TRALI), and allergic reactions, due to dilution when pooling plasma. As red cells are removed, Rh sensitization does not occur and it is not necessary to state the Rh type of SD-FFP.

Cost benefit

A recent study reported a net benefit of 35 min of quality-adjusted life expectancy costing an extra $19 for the viral inactivation process. This would equate to $289 300 per quality-adjusted life year (QUALY) saved. The authors of this analysis concluded that the expenditure was unjustified (AuBuchon & Birkmeyer, 1994). A more recent study taking account of declining virus transmission rate and more recent knowledge of the costs of viral inactivation procedures concluded that transfusing virally inactivated FFP prolonged quality-adjusted survival by 1 h 11 min per patient, at a cost-effectiveness ratio of $2 156 398 ± $257 587 per QUALY (Periera, 1999). It is generally agreed that QUALY costs of more than $50 000 are not cost-effective.

Methylene blue-treated ffp (mb-ffp)

  1. Top of page
  2. Avoidance of viral transmission
  3. Methods used for virus inactivation in pooled plasma derivatives
  4. Quality assessment of virally inactivated plasma
  5. Solvent–detergent-treated ffp (sd-ffp)
  6. Methylene blue-treated ffp (mb-ffp)
  7. FFP TREATED WITH THE PSORALEN S-59 and ULTRAVIOLET A (UVA) LIGHT (S-59-UVA-FFP)
  8. Selecting the best option for clinical ffp
  9. Conclusions
  10. References

Activity and method of preparation

The use of methylene blue for viral inactivation of plasma was first described in 1991. It is a phenothiazine dye previously used in the treatment of methaemoglobinaemia and shown to inactivate viruses in media, plasma and red cells. It binds to DNA but also has an affinity for the surface structures of viruses (Lambrecht et al, 1991). Methylene blue in combination with visible light (red/white) in the presence of oxygen causes a formation of 8-hydroxyguanine and DNA–protein cross-links (Lambrecht et al, 1991; Wagner et al, 1998). The DNA affinity of phenothiazines varies. In a comparative study of dimethylmethylene blue (DMMB), methylene blue and two closely related analogues designated 6-136 and 4-140, DMMB had the highest DNA binding and was able to kill the non-encapsulated bacteriophage R17. In contrast, the least active compound 4-140, a hydrophobic molecule with high protein affinity, effectively killed a lipid-encapsulated bacteriophage, illustrating that whereas phenothiazines exert their effect primarily via DNA damage they also interact with the viral envelope, although in this case the virucidal effect was inhibited by red cells and plasma (Lambrecht et al, 1991).

Methylene blue is usually added to plasma to a final concentration of 1 µm. As methylene blue is inactive against intracellular viruses, plasma is frozen and thawed first to disrupt leucocytes (Mohr et al, 1997). Alternatively, leucocytes can be removed before freezing by filtration either of whole blood before FFP separation or of secondarily separated FFP. In a comparison, the mean residual viable leucocyte counts of freeze-thawed and filtered plasma were 0·06 × 106/l and < 0·002 × 106/l respectively (Rider et al, 1998). After leucocyte removal and addition of methylene blue, units of plasma are exposed to white light for approximately 30 min to 1 h. Until recently, the process did not involve MB removal. A filtration step using an adsorbent LeukoVir device has been reported to remove residual methylene blue to undetectable levels (AuBuchon et al, 1998). This may address concerns relating to the theoretical toxicity of methylene blue (see below). Unlike SD-FFP, the photoinactivation of viruses using the methylene blue process is performed as a single unit treatment with volumes in the range 235–310 ml.

Product quality

The recovery of coagulation factors after MB treatment is shown in Table II. Usually, a recovery of > 90% is seen, although recoveries of FBG and FVIII were 65–80% in some studies and FBG concentration was lower when measured using a functional assay than radial immunodiffusion (Lambrecht et al, 1991; Zeiler et al, 1994). The recovery of fibrinogen in MB-FFP in a two-centre study was 74% and 83% and FVIII recovery was 67% and 70% (Hornsey et al, 2000), so that only 40% of units met the current UK specification of > 0·7 IU/ml in 75% of units (Guidelines for Blood Transfusion Services in the United Kingdom, 2000). A revised specification of > 0·5 iu/ml in 75% of units has been proposed (L. M. Williamson, personal communication). In a study in which MB-treated and control FFP were compared, there were no significant differences between plasminogin, alpha-2 antiplasmin, TAT, proteins C and S and antithrombin III, all of which were in the normal range (Abe & Wagner, 1995) (Table II). Neoantigens have not been demonstrated in MB-FFP (Mohr et al, 1992).

Viral inactivation

Methylene blue has been shown to have significant activity against both retroviruses and herpesviruses (Lambrecht et al, 1991; Bachman et al, 1995). It has also been shown to kill the West Nile virus, which is closely related to HCV. Inactivation of > 5 logs of Varicella Zoster Virus (VZV), HSV-1 and influenza viruses were observed under standard conditions (Lambrecht et al, 1991). However, the non-enveloped adenovirus was inactivated by only 1 log under standard conditions and by 2 logs when the concentration of methylene blue was increased 10-fold (Lambrecht et al, 1991). In quantitative PCR studies for HCV and HIV-1-RNA, > 98·5% reduction was shown after 6 min in small-scale experiments under standard conditions and a similar rate of inactivation for HCV in production scale experiments. The presence of anti-HCV IgG did not influence antiviral activity (Müller-Breitkreutz & Mohr, 1998).

Secondary products

MB-FFP prepared after integral plasma filtration has been used to make cryoprecipitate. Mean recoveries in two blood centres of FBG and FVIII after photodynamic treatment were 79% and 69% respectively. Recovery of the VWF antigen and VWF:RiCof had a mean of 86% and 100% and 80% and 100% compared with control cryoprecipitate (S. V. Hornsey personal communication). These results were better than those seen with SD-FFP (Keeling et al, 1997). Recovery of fibrinogen in cryoprecipitate had a mean of 60% and no obvious change in the VWF multimeric pattern was seen. This suggests that this product would be suitable for the treatment of both von Willebrand's disease and hypofibrinogenaemic states.

Toxicity

Methylene blue is listed in the US Pharmacopaeia (USP) and, when prepared to the standards specified in the USP by reconstitution in sterile water for injection, is suitable for intravenous administration. In the European Pharmacopaeia, methylene blue is listed only as a reagent suitable for external use. It has been shown to cause DNA damage in combination with white light and to cause mutations in both bacterial and viral nucleic acid (Wagner et al, 1995). Investigation of genotoxicity in mice showed mutagenicity in lymphoma cells, cultured ex vivo, but intravenous administration did not result in demonstrable genotoxicity when this was assessed in a mouse micronucleus assay (Wagner et al, 1995). In adults, ≈ 70–140 mg of methylene blue is used to treat methaemoglobinaemia, whereas after light exposure the amount transfused in one unit of MB-FFP is < 1% of this.

Resuspension of red cells and platelets in MB-FFP

Single-donor apheresis platelets were resuspended in MB-FFP, and pH, pCO2, pO2, bicarbonate, lactate dehydrogenase (LDH), glucose, hypotonic shock response (HSR) and CD62 expression were measured during storage. Similar experiments were performed with red cells where the per cent haemolysis, supernatant potassium and osmotic fragility were studied. No differences between red cells or platelets resuspended in control or MB-FFP were observed (Perrotta et al, 1999). MB cannot be used to eradicate viruses in red cell components as the light energy is absorbed by the red cells.

Transfusion studies of MB-FFP

Autologous MB-FFP was well tolerated when transfused to beagle dogs. One patient with TTP received MB-FFP during plasma infusion and exchange and no adverse reactions were reported (Pohl & Mohr, 1994). In a crossover randomized study in which 12 volunteers received 540 ml of autologous apheresed plasma either cryopreserved unmodified or MB treated, no differences were seen in vital signs, full blood count, serum chemistries and coagulation parameters. There was no evidence of coagulation or complement activation (Simonsen & Sorensen, 1999).

One hundred and twenty-five patients with routine indications for administration of FFP, including those with liver disease and massive transfusion resulting in disordered coagulation, were treated with MB-FFP; 50 additional patients received standard FFP and 25 received both types of FFP. A mean of six units (range 1–59) of MB-FFP were given. One patient experienced hypotension and urticaria and there were two episodes of mild generalized erythema. Clinical efficacy was not directly assessed but no clinical differences between MB and standard FFP were recorded (Pohl & Mohr, 1994).

Two patients, one with FV and one with FXI deficiency, were treated with MB-FFP. After infusion, the factor levels rose from 0% to 30% in one patient and from 1% to 46% in the other. Bleeding was controlled in both and no adverse effects were recorded (Pohl et al, 1995).

Information is available on MB-FFP transfused to 28 neonates; in 18, this was during exchange transfusion and in 10 FFP transfusions were given for acquired coagulopathies. No complications were described. In seven instances in which intrauterine/amniotic injections of MB were carried out to assess membrane rupture no fetal/neonatal toxicity was recorded at doses of 2–70 mg apart from a rise in heart rate in one case, although there was blue staining of the skin and/or urine (C. Prowse, personal communication).

Other potential benefits

It is usual now to leucodeplete rather than freeze–thaw plasma before methylene blue treatment. As with SD-FFP, this should reduce the incidence of AI and NHFTR. Similarly, leucodepletion may reduce the theoretical risk of transmission of the pathogenic agent of nvCJD. One disadvantage might be that because single units are treated a donation from a patient with a window period infection might have a high viral titre that exceeds the capacity of the system for virus removal. Further, it may be that the antibody contained is not neutralizing. A pooled product such as SD-FFP is more likely to contain a significant amount of neutralizing antibody to viruses with a relatively high prevalence in the population, such as HAV, HBV and parvovirus B-19. However, complete neutralization of any contaminating viruses cannot be guaranteed.

FFP TREATED WITH THE PSORALEN S-59 and ULTRAVIOLET A (UVA) LIGHT (S-59-UVA-FFP)

  1. Top of page
  2. Avoidance of viral transmission
  3. Methods used for virus inactivation in pooled plasma derivatives
  4. Quality assessment of virally inactivated plasma
  5. Solvent–detergent-treated ffp (sd-ffp)
  6. Methylene blue-treated ffp (mb-ffp)
  7. FFP TREATED WITH THE PSORALEN S-59 and ULTRAVIOLET A (UVA) LIGHT (S-59-UVA-FFP)
  8. Selecting the best option for clinical ffp
  9. Conclusions
  10. References

Activity and method of preparation

Psoralens intercalate with nucleic acid and, after exposure to UVA, form stable photoadducts within and between nucleic acid strands to pyrimidine bases. Such psoralen–UVA processes were shown many years ago to be virucidal. Compounds studied include 8-methoxypsoralen (8-MOP), 4,5′,8-trimethylpsoralen (TMP) and 4′-aminomethyl-4,5′, 8-trimethylpsoralen (AMT). Studies show that a wide range of viruses, including VZV, DHBV, feline leukaemia virus (FeLV) and HCV, are inactivated to > 106 log (Morel et al, 1992) with good preservation of protein function. There are now several reports of the use of the psoralens 8-MOP and AMT for photoinactivation of viruses in platelet concentrates (PCs) (Dodd et al, 1991; Margolis-Nunno et al, 1997; Grass et al, 1998; Hei et al, 1999), but it is the psoralen S-59 which is under evaluation for the production of virus-inactivated, single-unit, clinical blood components including FFP. The preparation technique is straightforward: S-59 (150 µm) is added to plasma or PCs, mixed and then exposed to 3 J/cm2 UVA over ≈ 3–4 min, as described by Lin et al (1997). After this procedure, photochemically treated platelet concentrates or plasma are treated to remove residual S-59 and unbound photoproducts.

Product quality

The level of coagulation factors present in S-59-UVA-FFP was > 85% for all those measured with the exception of FVIII (73%) (Table II) (Alfonso et al, 1996). In preclinical volunteer studies, no changes in the levels of coagulation factors were observed after S-59-UVA-FFP infusion (see below) (Wages et al, 1997).

Viral inactivation

S-59-UVA treatment at the dosage described above was shown to inactivate cell-associated HIV-1 (from productively infected H9 cells), cell-free HIV-1, DHBV and BVDV; the log10 reductions were 6·4, > 5·9, 5·4 and 6·7 respectively (Alfonso et al, 1996). In further studies, inactivation of BVDV was compared with PCR of RNA sequences for HCV. The same photochemical treatment (PCT) resulted in > 106 log inactivation of BVDV and, at the same time, a 1000-fold inhibition of HCV RNA amplification, indicating that HCV was clearly sensitive to S-59-UVA treatment (Corten et al, 1996). The same process has also been shown to inactivate HIV-1, DHBV and BVDV in PCs. In addition, the S-59-UVA process achieves 2–5 log inactivation of non-enveloped viruses in both DNA and RNA species (L. Corash, personal communication).

Suspension of cellular blood products in S-59-UVA-FFP

Studies in which cellular components are resuspended in an alternative plasma have not been specifically performed. However, S-59-UVA PCT has been shown to inactivate viruses in PCs without detriment to the function or characteristics of the cells themselves. There is no reason therefore to assume that platelets prepared by another method could not be resuspended in S-59-UVA-FFP. There are no data available for red cells.

Toxicity

Some psoralens in combination with UVA light are known to be mutagenic. However, the combination of S-59 and UVA has not been shown to cause genotoxicity in the in vivo mouse micronucleus assay and rat unscheduled DNA synthesis. S-59 photoproducts formed after irradiation also are not mutagenic. No toxicities were seen when S-59-treated FFP was infused (see below).

Transfusion studies

A single-blind, crossover, ascending dose clinical trial was performed in 15 healthy subjects. The responses to autologous FFP and S-59-UVA-FFP were compared. Doses were escalated between 100, 200, 400 ml (three subjects each) and 1000 ml (six subjects). No changes in baseline haematological and biochemical parameters were observed. Similarly, there were no changes in the levels of coagulation factors I, II, V, VII, IX, X and XI (Wages et al, 1997). In a prospective randomized study of patients with acquired coagulopathy due to liver disease, either S-59-UVA-FFP or standard FFP was transfused before invasive procedures. Changes in the prothrombin time (PT) and APTT, normalized for FFP dose and recipient weight, were not significantly different in six and seven patients who received test and standard FFP respectively. No adverse effects were observed (Wages et al, 1999).

Other potential benefits

The S-59-UVA process has no requirement for pooling, freeze–thaw to disrupt leucocytes (as psoralen has good penetrance of intact cells) or prefiltration. Because psoralens effectively inactivate nucleic acid and prevent its transcription, the psoralen–UVA processes described above has other areas of clinical utility. Although these are of primary importance when preparing platelets, they would be relevant where FFP and platelets were produced in the same system.

Bacterial inactivation S-59-UVA treatment has been shown to kill > 6 logs of both Gram-positive and Gram-negative bacterial species such as Staphyloccus epidermidis and Klebsiella pneumoniae (Lin et al, 1997).

Inhibition of cytokine synthesis Damage to nucleic acid also prevents transcription of the DNA that encodes for the cytokine interleukin (IL)-8, which is associated with NHFTR (Hei et al, 1999). In a study comparing gamma irradiation (2500 or 5000 cGy) with 8-MOP, AMT and S-59, by day 5–7 of storage, although there was little reduction in IL-8 production from gamma-irradiated PCs, treatment with S-59-UVA reduced its levels to a mean 3% of control. Similar results were found at appropriate doses of 8-MOP and AMT (Grass et al, 1999).

T-cell inactivation and prevention of graft-versus-host disease (GVHD) Psoralens inactivate leucocytes and S-59-UVA treatment has been shown to inactivate > 5·4 log of T cells in plateletpheresis PCs. Reducing the doses of S-59 and UVA from 150 µm and 3·0 J/cm2, respectively, by a combined factor of 1000 still reduced T-cell proliferation to below the limit of detection. Gamma irradiation of 2500 cGy also inactivates > 5 log of T cells but it induces relatively infrequent DNA strand breaks. By comparison, treatment with S-59, AMT and 8-MOP induce a mean of 12·0, 6·0 and 0·7 photoadducts/1000 base pairs DNA respectively (Grass et al, 1998). In the case of S-59, this is sufficient to inhibit the PCR amplification of small DNA sequences, in this case a 242-base pair sequence in the HLA-DQ α locus (Grass et al, 1998). Moreover, in further in vivo studies in a murine model of GVHD, both S-59 and 2500 cGy of gamma-irradiation was shown to inhibit completely the development of GVHD (Grass et al, 1999).

Selecting the best option for clinical ffp

  1. Top of page
  2. Avoidance of viral transmission
  3. Methods used for virus inactivation in pooled plasma derivatives
  4. Quality assessment of virally inactivated plasma
  5. Solvent–detergent-treated ffp (sd-ffp)
  6. Methylene blue-treated ffp (mb-ffp)
  7. FFP TREATED WITH THE PSORALEN S-59 and ULTRAVIOLET A (UVA) LIGHT (S-59-UVA-FFP)
  8. Selecting the best option for clinical ffp
  9. Conclusions
  10. References

The characteristics of the different clinical products that are, or may soon be, available are shown in Table IV. In the UK, production of standard FFP is licensed by the Medicines Control Agency (MCA). SD-FFP also has an MCA licence when made from plasma sourced outside the UK. By contrast, MB-FFP is licensed as a biomedical device by the Medicines Device Agency (MDA) and S-59-UVA-FFP is not licensed because clinical trials are ongoing in the USA (Table IV). Selection criteria for clinical FFP must be informed by data regarding: (i) donor selection, e.g. the UK policy of exclusion of first-time donors; (ii) prevalence of TTI, including nvCJD, in the donor population and the effect that pooling donations to make virally inactivated FFP might have (see below); (iii) the microbiological tests for antiviral antibody and nucleic acid performed on each donation, plasma pool or finished product; (iv) risk assessment data relating to manipulations such as pooling; and (v) any data regarding suspected or proven viral transmission. It is also important to bear in mind actual, theoretical or predicted toxicities as well as any possible alteration to the incidence of immediate adverse events associated with transfusion. Specific considerations are as follows.

Use of virally inactivated or standard FFP As transfusion strategies usually aim to achieve minimal risk whenever possible, then it seems logical to change from standard to virally inactivated FFP. At the same time, it is important to remember that calculations based on the cost of treatment show that virally inactivated plasma does not meet the generally recognized cost-effectiveness standard of $50 000 per QUALY.

Use of UK vs. non-UK plasma In the UK, plasma must be imported for the manufacture of fractionated plasma products and SD-FFP. This is because of concerns that an increased risk of acquisition of the agent of nvCJD might be associated with products derived from large plasma pools. It is not felt at the present time that single-donor clinical FFP should also be sourced outside the UK. Because of the unknown prevalence of nvCJD, UK plasma cannot be exported for viral inactivation.

Use of pooled vs. non-pooled products A risk assessment recently carried out in the UK (Det Norske Veritas, 1999) indicated that pooling would have an adverse effect with regard to nvCJD transmission unless the estimated infectivity in its source was 200× less than in UK plasma. Pooling does have theoretical benefits, including a possible reduction in reactions to FFP such as urticarial and allergic episodes as well as TRALI. Further, although there is theoretical concern about increasing the infectivity of FFP prepared from plasma pools, the pooling process is likely to ensure adequate levels of antiviral antibody which may be protective. It is not certain at the present time whether advisory bodies in the UK will recommend the use of SD-FFP and in the USA there is evidence suggestive of transmission of parvovirus B-19 in volunteers (CBER Document no. 0694, 1999) (see above). It may be necessary to specify batch levels of antibodies against non-lipid-enveloped viruses and/or perform PCR testing for HAV and parvovirus B-19.

Which single-unit treatment protocol should be selected MB-FFP has been used extensively in Europe (> 1·5 million units transfused). There has been concern that one HCV transmission may have resulted from transfusion of MB-FFP in a seroconverting donor in Germany, but this is not certain. S-59-FFP may have some theoretical advantages in that there is better preservation of some coagulation factor levels and bacteria are also killed. However, it is at an early clinical development stage and clinical proof of efficacy compared with MB-FFP is unknown. At present, there is no available information on the relative cost of these products.

Conclusions

  1. Top of page
  2. Avoidance of viral transmission
  3. Methods used for virus inactivation in pooled plasma derivatives
  4. Quality assessment of virally inactivated plasma
  5. Solvent–detergent-treated ffp (sd-ffp)
  6. Methylene blue-treated ffp (mb-ffp)
  7. FFP TREATED WITH THE PSORALEN S-59 and ULTRAVIOLET A (UVA) LIGHT (S-59-UVA-FFP)
  8. Selecting the best option for clinical ffp
  9. Conclusions
  10. References

Three virally inactivated FFP products are now currently available or in clinical trial. In some European countries such as Norway and Belgium, use of virally inactivated FFP is mandatory, but requirements vary in other countries. There are no other definitive recommendations in the UK as to the best choice of product, with the exception of the current recommendation that patients with single coagulation factor deficiencies such as FV or XI should receive virally inactivated FFP. At the present time, the use of SD-FFP has only been licensed by the regulatory authority in the UK if sourced from non-UK plasma (see above). It is, however, used widely in continental Europe and the USA. MB-FFP is not used in the USA but is used widely in Europe. S-59-UVA-FFP is of potential interest but is not yet available for routine clinical use. The use of virally inactivated FFP may well increase, although it is an intervention without proven cost benefits. FFP products of any type should only be transfused in accordance with accepted guidelines. Currently in the UK, these are produced by different organizations. It has been suggested here that a unified body with overall responsibility for blood safety should be established (Williamson et al, 1999b). This would provide a sensible approach to the strategic implementation of new developments in blood safety.

References

  1. Top of page
  2. Avoidance of viral transmission
  3. Methods used for virus inactivation in pooled plasma derivatives
  4. Quality assessment of virally inactivated plasma
  5. Solvent–detergent-treated ffp (sd-ffp)
  6. Methylene blue-treated ffp (mb-ffp)
  7. FFP TREATED WITH THE PSORALEN S-59 and ULTRAVIOLET A (UVA) LIGHT (S-59-UVA-FFP)
  8. Selecting the best option for clinical ffp
  9. Conclusions
  10. References
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