Andrey Skripchenko

Factors affecting virus photoinactivation by a series of phenothiazine dyes.

A series of four phenothiazine dyes, including methylene blue (MB), were previously tested for their ability to photoinactivate viruses in red cell suspensions. One of the dyes, 1,9‐dimethyl‐3‐dimethylamino‐7‐dimethylaminophenothiazine (1,9‐dimethylmethylene blue), exhibited good intracellular and extracellular virucidal activity for several RNA and DNA viruses under conditions that minimally affected red cell properties. In order to understand why the virucidal specificity of 1, 9‐dhnethylmethylene blue was greater than other phenothiazines tested, the physical and chemical properties of the dye were compared to three other closely related analogues (MB, 1,9‐dimethyl‐3‐diethylamino‐7‐dlbutylaminophenothiazine [compound 4‐140], 1,9‐dimethyl‐3‐dimethylamino‐7‐diethylaminophenothiazine [compound 6‐136]). All compounds required light and oxygen for virucidal activity and had relatively high singlet oxygen yields (>0.5), but 1,9‐dimethylmethylene blue had a singlet oxygen yield approximately 50% greater than that of MB. In addition, the hydrophobicity/hydophilicity of the compounds varied, with the partition coefficients (2‐octanol : water) ranging from 0.11 for MB to 3560 for compound 4‐140. The dyes had the following affinities for DNA: 1,9‐dimethylmethylene blue > compound 6‐136 > MB ∼ compound 4‐140. This order was similar to the order of activities for photoinactivation of the nonenveloped bacteriophage, R17, by the four compounds. Results with the most hydrophobic compound, 4‐140, contrasted with those obtained with 1,9‐dimethylmethylene blue. Compound 4‐140 had a high affinity for protein and a low affinity for DNA. Although compound 4‐140 and light inactivated the nonenveloped bacteriophage R17 poorly, the dye readily photoinactivated enveloped viruses in buffer. However, unlike results with 1,9‐dimethylmethylene blue, viral inactivation of enveloped viruses by compound 4‐140 was completely inhibited by the presence of red cells and plasma. Thus, the high affinity of 1,9‐di‐methyymethylene blue for DNA and the dyes efficient singlet oxygen yield suggest viral nucleic acid as a potential target, which could explain the photosensitizers ability to inactivate viruses without adversely affecting anuclete red cells.

Preservation of red cell properties after virucidal phototreatment with dimethylmethylene blue

BACKGROUND: All published reports have described methods for virus photoinactivation which significantly alter red cell (RBC) properties during storage. In order to improve virucidal activity and reduce damage to RBCs, a series of phenothiazine derivatives were either synthesized or purified and screened for bacteriophage inactivation and red cell potassium efflux. One compound, 1,9‐dimethylmethylene blue (dimethyl‐methylene blue), had superior screening results and was chosen for further characterization.

Comparison of Methylene Blue and Methylene Violet for Photoinactivation of Intracellular and Extracellular Virus in Red Cell Suspensions

Previous studies with methylene blue (MB) in red cell suspensions have demonstrated that extracellular, but not intracellular, virus can be readily photoinactivated. To test if the resistance of intracellular virus to inactivation is related to the permanent positive charge of the phenothiazine, a series of uncharged phenothiazine dyes, methylene violet (MV), monodemethylated MV and didemethylated MV, were studied. Values of the sensitivity of intracellular relative to extracellular vesicular stomatitis virus (VSV) inactivation for the three dyes (D10 extracellular/D10 intracellular) in buffer were 1.0, 0.60 and 0.33, respectively. In contrast, intracellular virus was resistant to inactivation with MB, with a D10 extracellular/D10 intracellular of 0.05 in buffer. Because virucidal activity of MV was inhibited by the presence of plasma, the red cells (30% hematocrit) were repeatedly washed prior to photoinactivation and storage. Under conditions where MB and MV inactivated approximately 5 log10 of extracellular VSV, intracellular VSV was inactivated by more than 4 log10 with MV compared to 0.88 log10 with MB. These phototreatment conditions did not significantly affect red cell morphology, extracellular pH, ATP or 2,3-diphosphoglycerol levels during 42 days of 1-6 degrees C storage. There was enhanced potassium efflux and hemolysis over values obtained from untreated control; the extent of change from controls was comparable for each phototreatment. These results indicate that the uncharged phenothiazine dye, MV, can inactivate both intracellular and extracellular virus yet exhibit similar in vitro red cell storage properties as MB phototreatment.

Platelet products prepared by different methods of sedimentation undergo platelet activation differently during storage

BACKGROUND: The activation marker CD40 ligand (CD40L) has recently been demonstrated to be released from the cytoplasm of platelets (PLTs) during storage. CD40L may be associated with some adverse transfusion reactions including febrile responses and transfusion‐related acute lung injury. CD62P has been traditionally measured to assess PLT activation. This study compares the surface levels of CD40L and CD62P and accumulation of the soluble forms of these activation markers in the plasma of stored PLTs, prepared by PLT‐rich plasma (PRP) or buffy coat (BC) methods and with two apheresis instruments.

Periods without agitation diminish platelet mitochondrial function during storage

BACKGROUND: Prolonged periods without agitation produce platelet (PLT) storage lesions that result in reduced in vitro assay parameters and an increase of apoptotic markers during storage. The aim of this study was to evaluate the influence of periods without agitation on PLT mitochondrial function, blood gases, and activation.

In vitro variables of apheresis platelets are stably maintained for 7 days with 5% residual plasma in a glucose and bicarbonate salt solution, PAS-5.

BACKGROUND: Platelet additive solutions (PASs) facilitate improved recovery of plasma and may reduce the severity and/or frequency of plasma‐associated transfusion reactions. Current apheresis platelet (PLT) PAS products contain approximately 30 to 40% residual plasma. In an effort to further decrease the residual plasma, two in vitro studies were conducted with PLTs suspended in 5% plasma and a reformulated PAS‐3, named PAS‐5, that contains additional salts, glucose, and bicarbonate.

The influence of simulated shipping conditions (24- or 30-hr interruption of agitation) on the in vitro properties of apheresis platelets during 7-day storage.

BACKGROUND: Platelet (PLT) components undergo interruption of agitation during shipment. Studies have demonstrated maintenance of PLT quality of whole blood–derived PLT concentrates during a 24‐hour interruption of agitation, but data are not available for apheresis PLTs in 100 percent plasma.

Inactivation of WBCs in RBC suspensions by photoactive phenothiazine dyes : comparison of dimethylmethylene blue and MB

BACKGROUND: The transfusion of blood components containing WBCs can cause unwanted complications, which include virus transmission, transfusion‐associated GVHD, alloimmunization, febrile reactions, and immunomodulation. Phototreatment with 4 μM of dimethylmethylene blue (DMMB) and 13 J per cm2 of white light irradiation has previously been shown to be an effective way to inactivate different models of enveloped and nonenveloped viruses in RBC suspensions, with minimum damage to RBCs. The present study compares WBC photoinactivation in buffy coat after DMMB or MB phototreatment under virucidal conditions.

Maintenance of platelet in vitro properties during 7-day storage in M-sol with a 30-hour interruption of agitation

BACKGROUND: Extensive periods without agitation can occasionally occur during platelet (PLT) shipment and can affect PLT quality during 5‐ to 7‐day storage. The use of buffer‐containing PLT additive solutions (ASs) may better preserve PLT quality during storage by maintaining PLT pH and other in vitro variables. A newly described bicarbonate‐containing AS, M‐sol, was compared to plasma for preservation of whole blood–derived PLT concentrates in which a 30‐hour interruption of agitation was included.

Use of a flexible thiopyrylium photosensitizer and competitive inhibitor for pathogen reduction of viruses and bacteria with retention of red cell storage properties.

BACKGROUND: Progress in developing photochemical methods for pathogen reduction of red blood cells (RBCs) has been hampered by hemolysis. A flexible, nucleic acid‐intercalating thiopyrylium (TP) dye that is only photochemically active in the bound state and a competitive inhibitor of RBC membrane binding, dipyridamole (DP), was used to reduce photoinduced hemolysis stemming from free‐ and membrane‐bound dye.