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closeFlaws in the histopathology endpoint
Posted by Wolfpath1 on 14 Mar 2014 at 18:36 GMT
It should be recognized that the conduct and interpretation of the histopathology endpoint in this manuscript are seriously flawed. Consequently, the credibility of this study must be questioned. Examples of major problems include the following:
• Valid conclusions cannot be made with confidence because the sample size for the histopathological examination is too small (2 animals per group, for a total of 6 animals). Nine animals were sacrificed per group, therefore it is puzzling that only 2 per group were examined microscopically.
• The histopathological procedures are not described in sufficient detail to allow the reader to determine whether the sampling (animal selection, and selection of bone marrow areas for examination) was conducted in an unbiased manner, and that sufficient areas of bone marrow were examined.
• Although the authors state that “…the number of megakaryocytes (MKs) exhibited a positive correlation with the FA dose (0.5 and 3.0 mg/m3)…”, there is no description of any type of counting, scoring or ranking system that might have been used to quantitate megakaryocytes during the histopathologic evaluations.
• Myelofibrosis was one of the two reported histopathologic effects of formaldehyde treatment in this study. In Figure 3F, the authors attempt to illustrate this change using a black arrow and an inset image. However, the single indicated object, which is a thin-walled, tubular structure lined by endothelial cells, is clearly a normal arteriole (small artery) and not fibrosis. Furthermore, there does not appear to be evidence of myelofibrosis in other areas of Fig. 3F, or in any of the other five bone marrow images.
• In the Discussion section of their paper, the authors portray changes in peripheral blood hematologic values and bone marrow histopathologic findings as evidence of bone marrow toxicity. They state that “…changes in the numbers of circulating blood cells in several lineages may reflect toxicity to BM HSC [bone marrow hematopoietic stem cells].” They then further state that “In our study, significantly altered counts of WBC, RBC, LYM (decreased) and PLT (increased) in FA-exposed mice, the results are consistent with the clinical symptoms of some hematopoietic diseases such asaplastic [sic] anaemia (AA), myelofibrosis and megakaryocytic leukemia, indirectly suggesting that FA induces BM toxicity.” However, in their discussion, the authors neglect to acknowledge that alterations in numbers of peripheral erythrocytes and several lineages of leukocytes can also be induced by disturbances that have no obligate association with myelotoxicity, such as localized or systemic inflammatory disease. Additionally, it is patently incorrect for the authors to state that the results were consistent with such serious illnesses as aplastic anemia, myelofibrosis, and megakaryocytic leukemia, when in fact the particular hematological and histopathologic results of this study are not indicative or even suggestive of any of those potentially lethal conditions.
• Most importantly, the authors failed to recognize that a hallmark characteristic of bone marrow toxicity is the destruction or loss of one or more hematopoietic progenitor cell lines (Travlos, 2006). Such cell losses can occur via several mechanisms, such as direct cytotoxicity, chronic inflammation, bone marrow necrosis (e.g., infarction), stromal proliferation, or neoplastic invasion (Travlos, 2006). Loss of hematopoietic cell lines can be appreciated in bone marrow sections by observations such as hematopoietic cell necrosis, general hypocellularity, shifts in cell maturation, and/or by alterations in the relative proportions of myeloid and erythroid precursors (i.e., changes in the M:E ratio). The fact that none of these alterations were evident in the bone marrows of formaldehyde-exposed mice suggests that the hematologic changes (i.e., alterations in circulating blood cell counts) observed in this study, if accurate, were caused by mechanism(s) other than myelotoxicity.
Travlos GS (2006) Histopathology of bone marrow. Toxicol Pathol, 34:566-598.
RE: Flaws in the histopathology endpoint
dingshumao replied to Wolfpath1 on 20 Mar 2014 at 01:07 GMT
Thank you very much for your comments on our paper entitled as “Bone marrow injury induced via oxidative stress in mice by inhalation exposure to formaldehyde” (Ms. Ref. No.: PONE-D-13-28892).
The detail responses to the reader comments are as the following. We would like to express our deep thanks to the reader for their constructive comments and suggestions.
1. Sample size for the histopathological examination
Response:
Thanks for the kind advice. Because of the number of FA exposure equipment is limited, we could use 9 mice at most at the same time. In order to ensure all the experimental indexes are from the same group of mice, we cannot take every mouse to do histopathological examination. So we chose 2 mice from each group for histopathological examination, but we repeated the experiment three times to ensure the reliability of the data.
2. The histopathological procedures
Response:
Thanks for the careful observation. The slices were produced by biological company in an unbiased manner. The production procedures were normative.
3. Quantitating megakaryocytes (MKs)
Response:
Thanks for the kind advice. We found the number of MKs was increased after FA exposure through the microscope, and we didn't quantitate MKs by counting system during the histopathologic evaluations. Thanks for your constructive suggestion.
4. About Myelofibrosis
Response:
Myelofibrosis is characterized by fibrous tissue hyperplasia, the generation was accompanied by megakaryocytic hyperplasia. Besides, the femurs were ripped cutting and observated, so we think that it is not a normal arteriole.
5. About Myelofibrosis
Response:
The alterations in numbers of peripheral erythrocytes and several lineages of leukocytes could be induced by many factors including inflammation. We also detected the inflammation in bone marrow. The results of CBC, inflammatory markers and other indexes suggested that FA exposure could cause adverse effect to bone marrow. Some alterations were consistent with disease. We just stated that FA exposure caused adverse effect and increased the risk of some hematopoietic system disease.
6. Thanks for your constructive comments. Our group were dedicated to the research
of hematopoietic progenitor cell lines including the factors that induce the stem cells. The inadequacy will be improved in further study. Thank you very much.
RE: Flaws in the histopathology endpoint
dingshumao replied to Wolfpath1 on 20 Mar 2014 at 01:11 GMT
Thank you very much for your comments on our paper entitled as “Bone marrow injury induced via oxidative stress in mice by inhalation exposure to formaldehyde” (Ms. Ref. No.: PONE-D-13-28892).
The detail responses to the reader comments are as the following. We would like to express our deep thanks to the reader for their constructive comments and suggestions.
1. Sample size for the histopathological examination
Response:
Thanks for the kind advice. Because of the number of FA exposure equipment is limited, we could use 9 mice at most at the same time. In order to ensure all the experimental indexes are from the same group of mice, we cannot take every mouse to do histopathological examination. So we chose 2 mice from each group for histopathological examination, but we repeated the experiment three times to ensure the reliability of the data.
2. The histopathological procedures
Response:
Thanks for the careful observation. The slices were produced by biological company in an unbiased manner. The production procedures were normative.
3. Quantitating megakaryocytes (MKs)
Response:
Thanks for the kind advice. We found the number of MKs was increased after FA exposure through the microscope, and we didn't quantitate MKs by counting system during the histopathologic evaluations. Thanks for your constructive suggestion.
4. About Myelofibrosis
Response:
Myelofibrosis is characterized by fibrous tissue hyperplasia, the generation was accompanied by megakaryocytic hyperplasia. Besides, the femurs were ripped cutting and observated, so we think that it is not a normal arteriole.
5.
The alterations in numbers of peripheral erythrocytes and several lineages of leukocytes could be induced by many factors including inflammation. We also detected the inflammation in bone marrow. The results of CBC, inflammatory markers and other indexes suggested that FA exposure could cause adverse effect to bone marrow. Some alterations were consistent with disease. We just stated that FA exposure caused adverse effect and increased the risk of some hematopoietic system disease.
6. Thanks for your constructive comments. Our group were dedicated to the research
of hematopoietic progenitor cell lines including the factors that induce the stem cells. The inadequacy will be improved in further study. Thank you very much.