TT virus does not contaminate first-generation recombinant factor VIII concentrate

Recently, Azzi et al1 suggested TT virus (TTV) occurs in first-generation recombinant factor VIII concentrate (rFVIII) products with the stabilizer, human serum albumin (HSA) as its source. As these results differ from those of earlier studies2 3 and Baxter's internal findings, we reinvestigated 11 lots of rFVIII (Recombinate), and 13 lots of HSA. Lyophilized rFVIII was reconstituted with distilled water, or HSA used directly. One part of each sample was extracted directly and the other spiked with approximately 300 genome equivalents of TTV (derived from a TTV-positive plasma sample) before extraction, to control for inhibition of the polymerase chain reaction (PCR).

DNA was extracted from 200-μL samples (Blood Kit; Qiagen, Hilden, Germany) and the solution in the quantity described (6 μL)1 subjected to either nested PCR (primer sets untranslated region [UTR] A and B as described1,4), or single-stage PCR (primers T801 and T935, used previously for Baxter's internal investigations5), using a thermally activated DNA polymerase (HotStarTaq, Qiagen). All primer sets are specific for the non–coding region (NCR, also UTR) of the TTV genome.

Both the single-stage and nested PCR reactions were done in 50 μL containing 1 U polymerase, 200 μM dNTP, and 50 pmol each of forward and reverse primer. The samples were overlaid with mineral oil, incubated for 14 minutes at 94°C and amplified for 45 cycles (single-stage PCR) or 35 cycles followed by 25 cycles (nested PCR) in a TRIO-Thermoblock (Biometra, Göttingen, Germany) with the following cycle profile: 30 seconds at 94°C, 30 seconds at 55°C, 60 seconds at 72°C with a final elongation at 72°C for 1 minute. The PCR products were analyzed using a 3.5% low-melting agarose gel stained with ethidium bromide.

Amplification of a positive control sample (DNA extracted from human TTV-positive plasma) with primer pair T801/T935 resulted in a PCR product of 199 bp as expected, with the UTR A primers of 143 bp, and the UTR B primers of 141 bp.

All rFVIII samples were negative with each primer set evaluated. To control for false-negative results caused by inhibition of PCR by the sample matrix, all samples were extracted again and PCR-tested after spiking with a TTV control. All these samples showed positive PCR signals, verifying the results from the unspiked rFVIII samples. Positive samples (TTV-positive human plasma) and negative controls (buffer) also included with each PCR reaction again verified the experimental set-up used.

Additionally, 13 lots of HSA were tested as described above. All samples were negative, with positive results for the same samples after spiking with a low amount of TTV-positive human plasma.

When TTV was discovered in 1997⁶ and suggested to be associated with posttransfusion hepatitis, naturally patients, regulatory bodies, and the plasma products industry were concerned. Further investigations revealed a low viral load in plasma and high prevalence of TTV in up to 82% of plasma donations, and consequently in many plasma pools.2 More refined products (eg, albumin) were, however, consistently TTV-negative.2,3 These findings confirm longstanding experience, derived from numerous viral validation studies, of the Cohn HSA manufacturing process as one with a high capacity for removing viruses. The explanation for the recent unexpected findings1 thus remains enigmatic. Using the same 2 TTV primer sets and a third independent in-house primer system, we confirmed earlier reports2 3 from research groups at regulatory bodies who unanimously found HSA to be TTV-negative.