CROSS EIGHT M^® is a monoclonal antibody-purified factor VIII concentrate produced by the Japanese Red Cross from donated blood plasma. During its manufacture, viruses are expected to be inactivated and eliminated by either solvent/detergent (S/D) treatment or both immunoaffinity and anion exchange chromatography.
To validate virus inactivation/elimination during the manufacture of CROSS EIGHT M^®, the amount of virus spiked in the manufacturing materials was measured before and after treatment, and the efficiency of virus clearance was calculated for each step. The viruses used were as follows: sindbis virus (SIN), vesicular stomatitis virus (VSV), human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV) and herpes simplex virus type 1(HSV-1) as enveloped viruses; and hepatitis A virus (HAV) and polio virus (Polio V) as non-enveloped viruses.
The result shows that enveloped viruses were effectively inactivated to below detection level after S/D treatment with logarithmic reduction values (LRVs) of 5.6 in SIN, >4.4 in VSV, >5.2 in HSV-1 and >4.9 in HIV-1. In contrast, non-enveloped viruses were not inactivated by S/D treatment. Using the immunoaffinity chromatography process, both enveloped and non-enveloped viruses were eliminated with the LRVs of >3.1 in polio V, >5.4 in VSV, >4.4 in SIN, 4.5 in HAV and 5.4 in HBV. LRVs with anion exchange chromatography varied among viruses, from 0 in HAV to 1.9 in Polio V, depending on the condition of the viral particles.

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We examined the effectiveness of PLANOVA 35 N filter with a mean pore size of 35nm in the elimination of viruses in monoclonal antibody-purified freeze-dried human coagulation factor VIII concentrate (CROSS EIGHT M^®) and intramuscular human immunoglobulin (Anti-HBs Human Immune Globulin “Nisseki”^®and Human Immune Globulin “Nisseki”^®). The filtration process was validated for removal of a variety of enveloped and non-enveloped viruses ranging in size from 200nm to 20nm including sindbis virus (SIN), vesicular stomatits virus (VSV), herpes simplex virus type 1 (HSV-1), hepatitis B virus (HBV), bovine viral diarrhea virus (BVDV), hepatitis A virus (HAV) and human parvovirus B 19 (B 19).
Results showed that logarithmic reduction values (LRVs) of viruses were >4.7 in SIN, >3.7 in VSV, >2.8 in HSV-1 and 5.0 in HBV.
Similarly, PLANOVA 35 N filtration removed SIN, VSV, BVDV and HBV in intramuscular immunoglobulin with LRVs of >4.5, >4.0, >3.7, 5.0, respectively.
While filtration did not effectively remove HAV and B 19 in CROSS EIGHT M^®, these viruses were partly reduced after filtration in intramuscular immunoglobulin solution.
From the above data, we conclude that the introduction of a PLANOVA 35 N filtration step to our manufacturing process will improve the viral safety of these plasma derivatives.

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We investigated virus inactivation by two albumin products (Sekijyuji Albumin^® and Sekijyuji Albumin 25^® derived from human plasma with heat treatment at 60°C in liquid-phase (pasteurization) using several enveloped and non-enveloped viruses as marker viruses. Enveloped viruses were sindbis virus (SIN), vesicular stomatitis virus (VSV), herpes simplex virus type-1 (HSV-1) and human immunodeficiency virus type-1 (HIV-1). Non-enveloped viruses were polio virus (Polio V) and hepatitis A virus (HAV). The infectivity of all viruses except HAV was lost after 1hr heating, with logarithmic reduction value (LRV) for SIN, VSV, HSV-1, HIV-1 and Polio V of >5.1, >5.7, >4.3, >3.8 and >5.9, respectively. No significant difference in LRVs were observed between the two products. The infectivity of HAV, however, remained at 10^4.2-5.0 TCID₅₀ even after 10hr heating and LRV stayed in the range of 1.9 to 2.7.

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We used a novel “real time” quantitative PCR method, TagMan PCR (T-PCR), in which the accumulation of amplified products is detected via the formation of dual fluorogenic probe complexes, to diagnose HTLV-I infection, and compared the results with the Nested PCR method (N-PCR). Currently, confirmation of HTLV-I infection requires the detection of anti-HTLV-I by a serological method and of HTLV-I genome by a polymerase chain reaction (PCR)—based methodology. Secondary PCR-based assay is required to screen false positives which may occur during the serological testing. N-PCR is normally employed to amplify specific sequences from a very few copies of HTLV-I genome, this, however, consists of two PCR (time consuming) steps and is not intended for quantitative analysis. In our testing model as few as 3 copies of a partial genome of HTLV-I (3′ end half of HTLV-I genome cloned in plasmid) were successfully amplified and detected with comparable accuracy to N-PCR. Moreover, the measured quantitative values (Th cycle) of T-PCR were highly correlated with copy numbers of integrated HTLV-I genome estimated by other routine methodologies. Lastly, less labor was required to perform T-PCR than N-PCR, and the use of closed tubes in T-PCR significantly reduced carryover contamination. We conclude that T-PCR is a rapid and accurate assay system for confirmation of the serological diagnosis of HTLV-I infection.

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We investigated the effect of irradiation and leukocyte filtration on red cells in MAP solution (RC-MAP) for premature infants.
RC-MAPs were stored for 3 or 7 days and pretreated with 15-Gy irradiation and a leukocyte depletion filter, with either the irradiation or filtration performed first. Infusion was performed using an infusion pump for 8hr at a speed of 2ml/hr through a 4-ml, 100-cm tube and a 24G needle passing into an infant incubator warmed to 34°C.
Free hemoglobin concentration in the supernatant of tested RC-MAP stored 7 days and irradiated after filtration was increased to the maximum level of 42.6mg/dl.
Potassium ion level in the supernatants and ATP and 2, 3-DPG concentration in red cells from tested RC-MAP were similar to pretreated values. Maximum potassium ion level was increased to 23.5mEq/l.
Our results showed that both irradiation and filtration against RC-MAP solutions stored for 3 or 7 days is safe for use with premature infants in warmed incubators. However, further investigation is necessary to clarify the risk of bacterial contamination in such transfusion situations.

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Life-threatening anaphylactic transfusion reaction, a rare side-effect of platelet transfusion, has been reported in patients who lack various plasma proteins such as IgA and the fourth complement component (C4). However, the cause of these reactions has in most cases not been determined.
We experienced two patients with hematological disorders who developed severe anaphylactic reactions after several platelet transfusions with a leukocyte removal filter. C3, C4 and immunoglobulins A, G and M levels in their sera were normal. Anti-platelet, plasma anti-IgA, and C4 and C9 antibodies were negative.
The COBE Spectra Leukoreduction System (LRS) produces platelet concentrates (PC) that contain less than 0.5×10⁶ residual white blood cells. Since anaphylactic reaction to plasma protein was unlikely in our cases, we transfused LRS-PC without a leukocyte removal filter. Both patients received frequent infusions of LRS-PC without further complications.
Although the cause of the anaphylactic reactions in these two cases remains unknown, LRS-PC transfusion was useful in avoiding anaphylactic reaction associated with platelet transfusions through leukocyte removal filters.

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