Blood, 15 September 2002, Vol. 100, No. 6, pp. 2271-2271
CORRESPONDENCE
To the editor:
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.
Thomas R. Kreil, Klaus Zimmermann, Sabrina Pable, Hans Peter Schwarz, and Friedrich Dorner
Correspondence: Thomas R. Kreil, Baxter BioScience,
Benatzkygasse 2-6, A-1221 Vienna, Austria; e-mail:
thomas_kreil{at}baxter.com
References
1.
Azzi A, De Santis R, Morfini M, et al.
TT virus contaminates first-generation recombinant factor VIII concentrates.
Blood.
2001;98:2571-2573[Abstract/Free Full Text].
2.
Pisani G, Cristiano K, Wirz G, et al.
Prevalence of TT virus in plasma pools and blood products.
Br J Haematol.
1999;106:431-435[CrossRef][Medline]
[Order article via Infotrieve].
3. Yu M-Y. Presented at: A meeting of the WHO International
Working Group on the Standardization of Genomic Amplification
Techniques for the Virological Safety Testing of Blood and Blood
Products; November 15, 1998; at the NIBSC, London, United Kingdom.
4.
Okamoto H, Takahashi M, Nishizawa T, et al.
Marked genomic heterogeneity and frequent mixed infection of TT virus demonstrated by PCR with primers from coding and noncoding regions.
Virology.
1999;259:428-436[CrossRef][Medline]
[Order article via Infotrieve].
5.
Simmonds P, Prescott LE, Logue C, et al.
TT virus
part of the normal human flora?
J Infect Dis.
1999;180:1748-1749[Medline]
[Order article via Infotrieve].
6.
Nishizawa T, Okamoto H, Konishi K, et al.
A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology.
Biochem Biophys Res Commun.
1997;241:92-97[CrossRef][Medline]
[Order article via Infotrieve].
Response:
First-generation recombinant factor VIII concentrates are free
from viral contaminations?
Kreil et al (from Baxter BioScience) failed to detect TT virus
(TTV) DNA in 11 lots of a Baxter first-generation recombinant factor
VIII concentrate (rFVIII) (Recombinate) as well as in 13 lots of human
serum albumin (HSA), a difference from our previously reported
results.1 As regards the rFVIII, the results of Kreil et
al are not significantly different from ours (3 out of 13 were positive
for TTV DNA). However, with regard to the contamination by TTV
in HSA lots, the difference between the results obtained by the 2 groups is more evident. Kreil et al cite, as we too have done, the
paper of Pisani et al,2 who failed to detect TTV DNA in
HSA, in order to strengthen their own conclusions. As we already
stressed, Pisani et al used only the N22 polymerase chain reaction
(PCR), which is unable to detect a high number of TTV variants.3 In this case the difference in the methods used may well justify the different results. In addition, it is well known
that, even using the same methods, the results obtained from different
laboratories are not fully comparable if international standards are
not available and used, as in the case of TTV so far. Furthermore, when
the size of the study is so small, a negative result can be affected by
a type II statistical error. According to the "rule of 3," the
one-sided 95% confidence intervals of the Kreil et al study are 0 and
27.3.4
Similar viral safety problems emerged concerning the possible
contamination of several blood products by parvovirus B19. As regards
the possibility of B19 contamination, we failed to detect B19
DNA in either rFVII or HSA, even using very sensitive nested PCR,
whereas other groups reported different results.5,6
It is likely that the residual amount of virus (or better, of viral
genome) (TTV or B19 virus or perhaps other viruses) in such products
after the manufacturing process is very low, near to the limit of
sensitivity of the analytical methods available at present.
Such a condition, in addition to the lack of standardization of
methods, makes a comparison of results obtained in different laboratories very hazardous.
We would like to emphasize again the need for continuous
drug-surveillance with prospective protocols of informative
hemophiliacs, treated for the first time even with rDNA-derived
clotting factor concentrates. In addition, there is also the need to
implement the standardization of the molecular methods for the
detection of viral contaminations by the development and use of
calibrated reference samples.
Alberta Azzi and Massimo Morfini
Correspondence: Alberta Azzi, Dipartimento di Sanita Pubblica,
(Sezione di Microbiologia e Virologia), Viale Morgagni 48, 50134 Florence, Italy
References
1.
Azzi A, De Santis R, Morfini M, et al.
TT virus contaminates first-generation recombinant factor VIII concentrates.
Blood.
2001;98:571-573.
2.
Pisani G, Cristiano K, Wirtz G, et al.
Prevalence of TT virus in plasma pools and blood products.
Br J Haematol.
1999;106:431-435[CrossRef][Medline]
[Order article via Infotrieve].
3.
Okamoto H, Takahashi M, Nishizawa T, et al.
Marked genomic heterogeneity and frequent mixed infections of TT virus demonstrated by PCR with primers from coding and non coding regions.
Virology.
1999;259:428-436[CrossRef][Medline]
[Order article via Infotrieve].
4.
Louis TA.
Confidence intervals for a binomial parameter after observing no successes.
Am Stat.
1981;35:154[CrossRef].
5.
Eis-Hubinger AM, Sasowski U, Brackmann HH, et al.
Parvovirus B19 DNA is frequently present in recombinant coagulation factor VIII products.
Thromb Haemost.
1996;76:1120[Medline]
[Order article via Infotrieve].
6.
Saldanha J, Minor P.
Detection of human parvovirus B19 DNA in plasma pools and blood products derived from these pools: implications for efficiency and consistency of removal of B19 DNA during manufacture.
Br J Haematol.
1996;93:714-719[CrossRef][Medline]
[Order article via Infotrieve].
