Submited on: 23 Dec 2010 02:34:12 PM GMT
Published on: 24 Dec 2010 10:51:49 AM GMT
 
Review
Posted by Dr. Trevor Barrowcliffe on 31 Mar 2011 04:56:08 PM GMT

1 Is the subject of the article within the scope of the subject category? Yes
2 Are the interpretations / conclusions sound and justified by the data? Yes
3 Is this a new and original contribution? Yes
4 Does this paper exemplify an awareness of other research on the topic? Yes
5 Are structure and length satisfactory? Yes
6 Can you suggest brief additions or amendments or an introductory statement that will increase the value of this paper for an international audience? No
7 Can you suggest any reductions in the paper, or deletions of parts? No
8 Is the quality of the diction satisfactory? Yes
9 Are the illustrations and tables necessary and acceptable? Yes
10 Are the references adequate and are they all necessary? Yes
11 Are the keywords and abstract or summary informative? Yes
  • Other Comments:

    This paper describes investigations on the effect of manufacturing processes, in particular pasteurisation, on clotting factors in one particular manufacturer’s IVIG preparations. The paper is important because concerns have been raised about increased thromboembolic events, probably caused by clotting factor contamination, in another manufacturer’s products.

     

    The experiments are well conceived and the results and conclusions clearly described. I have only a few minor comments as follows:

    1. Methods. Since FXI or XIa is a suspected contaminant in untreated IVIG preparations it is not clear why the NAPTT & TGT were performed with FXI deficient plasma. A positive result does not necessarily indicate the presence of FXI, since such a result could be given by Factors IXa, Xa, or VIIa in the case of the TGT. It would have been more logical to use FIX deficient plasma – the difference between this and normal plasma would then indicate the amount of activity due to FXI or XIa. However this limitation is recognised by the authors in the discussion and since the activity is in any case eliminated by pasteurisation this is not a major criticism.

    2. Results, Section 1. The activities of the clotting factors are given in %, and although it is implied that this represents recovery from the starting plasma, % is also often used to describe concentration (100% = 1 IU/ml). It might be helpful to insert the word “total” before “recovery” to emphasise the difference.

    3. Illustration 1. Some of the TGT results with PPP are given as negative values. Since the results are expressed as thrombin concentration this cannot be; results should be either zero or below the limit of detection.

    4. Illustration 3. The legend on the y-axis is not clear. The data are given as a ratio, presumably of sample to buffer blank, therefore seconds should not appear in the legend, and it should be more clearly explained in the description above.

    5. Illustration 5. The NAPTT tests show substantial reduction of activated clotting factors by acid pH treatment but hardly any reduction is found in the TGT in FXI deficient plasma. Would the authors care to comment on possible reasons for this?

  • Competing interests:
    I was previously a consultant for Instiuto Grifols ( and other Companies) but this activity has now ceased.
  • Invited by the author to review this article? :
    Yes
  • Have you previously published on this or a similar topic?:
    No
  • References:
    None
  • Experience and credentials in the specific area of science:

    Over 35 years working in haemostasis research with specific knowledge of manufacture and testing of blood products.

  • How to cite:  Barrowcliffe T .Review[Review of the article 'Pasteurization Inactivates Clotting Enzymes During Flebogamma® And Flebogamma® Dif Production' by Jorquera J].WebmedCentral 2011;2(3):WMCRW00633
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Answers to the reviewer

The experiments are well conceived and the results and conclusions clearly described. I have only a few minor comments as follows:

1. Methods. Since FXI or XIa is a suspected contaminant in untreated IVIG preparations it is not clear why the NAPTT & TGT were performed with FXI deficient plasma. A positive result does not necessarily indicate the presence of FXI, since such a result could be given by Factors IXa, Xa, or VIIa in the case of the TGT. It would have been more logical to use FIX deficient plasma – the difference between this and normal plasma would then indicate the amount of activity due to FXI or XIa. However this limitation is recognised by the authors in the discussion and since the activity is in any case eliminated by pasteurisation this is not a major criticism.

ANSWER: The approach suggested by the reviewer is well reasoned. However, NaPTT is not a quantitative assay, therefore we would not be able to subtract the results of NaPTT-FXI from the results of NaPTT-PPP. Concerning the TGT assay, obtaining the validated quantitative measures required for the suggested approach is something that can be considered in the future, maybe when additional reference materials are available. Our main initial goal was to find, as soon as possible, an analytical tool that could help distinguish products with higher thromboembolic risks. We focused on NaPTT and TGT with FXI deficient plasma because of unpublished observations from another laboratory and also because of the known fact that FXI may co-purify with IgG.

The TGT with other deficient plasmas (FVII, FIX and FX deficient) was also applied in the analysis of the final IVIG products (unpublished results). Using these deficient plasmas, no differences were found among all tested IVIG products. Some final products were also tested by TGT-PPP (normal plasma) and in this case (unpublished results), although the technique appears less sensitive than TGT-FXI, the differences observed with TGT-FXI remain (i.e.: Product A 5% 2010 vs. Flebogamma® and Flebogamma® DIF).

In the new version of the manuscript, more detail on the TGT and NaPTT methodologies has been added, such as the concentration of trigger (tissue factor), phospholipid and a statement that the read-out was peak thrombin, the  applicable concentration for NaPTT testing of IVIGs and dilution range.

2. Results, Section 1. The activities of the clotting factors are given in %, and although it is implied that this represents recovery from the starting plasma, % is also often used to describe concentration (100% = 1 IU/ml). It might be helpful to insert the word “total” before “recovery” to emphasise the difference.

ANSWER: Following the advice of the reviewer, the text has been improved as follows: “Assuming 1 IU/ml for the proenzymes in the normal human plasma pool used for the preparation of FrII+III, the total recovery of activities in the industrially re-suspended FrII+III (one-stage clotting assays on frozen samples with an average OD280 of 56.6±10.4, n=2) ranged from 15-33% for FX to 121-201% for FVII, with intermediate values for the remaining activities tested: 35-40% for FXII, 61-68% for FII, 67-70% for FIX and 76-92% for FXI.”

3. Illustration 1. Some of the TGT results with PPP are given as negative values. Since the results are expressed as thrombin concentration this cannot be; results should be either zero or below the limit of detection.

ANSWER: The reviewer is right. To clarify the meaning of negative results, the following sentence has been added to the table foot in illustrations 1 and 5: “Negative results indicate results below the value obtained for the vehicle.”

4. Illustration 3. The legend on the y-axis is not clear. The data are given as a ratio, presumably of sample to buffer blank, therefore seconds should not appear in the legend, and it should be more clearly explained in the description above.

ANSWER: The original figure legend was as follows: “Non-activated partial thromboplastin time (NaPTT) assays with FXI deficient plasma, in different lots (n= 1 to n= 4) from each of several products from different manufacturers and concentrations (A, B1, B2, C, D, E and F) (mean ± SD). The lot from Product A identified as ‘A 5% (2007)’ was manufactured in 2007 and was analyzed one year after its expiration date. The remaining lots were analyzed within their shelf life.”

Probably due to a limitation of characters that was unnoticed to us during the edition process, the figure legend was cut. In the new version of the manuscript the legend has been shortened, with further details being incorporated into the main text. Same approach has been followed in illustration 2.

5. Illustration 5. The NAPTT tests show substantial reduction of activated clotting factors by acid pH treatment but hardly any reduction is found in the TGT in FXI deficient plasma. Would the authors care to comment on possible reasons for this?

ANSWER: The FrII+III suspension used to spike the industrial materials before acid pH treatment or before pasteurization (illustrations 4 and 5, respectively), as mentioned in the first section of Results, contains, besides FXI, different amounts of other coagulation factors (FII, FVII, FIX, FX, FXII) and also showed very short clotting times in the regular NaPTT (NaPTT-PPP) assay, high PKA and “kallikrein” content, as well as high thrombin generation capacity. The NaPTT-PPP is a global test for different activated factors, the pH treatment could be effective for the reduction of some of these activated factors but, according to our purification process follow-up, is less effective for the reduction of others like FXI and consequently, this may be the reason for the absence of reduction found with the TGT performed with FXI deficient plasma. It is important to note that although after pH treatment the spiked samples show a substantial reduction in activated clotting factors, in all cases the clotting time of the samples was lower than the plasma control, indicating that total inactivation is not achieved, even at the lowest spike proportion (1/20 v/v).


Responded by Dr. Marta Jose on 10 May 2011 12:24:22 PM