Lack of Association Between Complement Regulatory Proteins and Severe Malarial Anemia or Lack of Adequate Methodology?
Jose Stoute, Walter Reed Army Institute of Research
24 December 2007
Dear Professor Hommel,
We read with great interest the results of the study by Helegbe et al. entitled “Complement activation in Ghanaian children with severe Plasmodium falciparum Malaria.” The authors report a lack of association between levels of red cell complement regulatory proteins (CD35 and CD55) or C3b deposition and severe anemia due to P. falciparum malaria (SA).
Over the past seven years our group has reported that red cells of children with SA have reduced levels of complement regulatory proteins. The data came from three separate studies with a cumulative total of 174 cases of SA and a similar number of age and gender-matched controls with uncomplicated malaria [1-3], all with confirmed asexual parasitemia by Giemsa-stained smears. In each and every one of these studies, we observed reduced levels of red cell complement regulatory proteins in children with SA. These findings were very specific to SA as they are not found in patients with other categories of severe malaria such as cerebral malaria (CM). Furthermore, our more recent study revealed that red cells of children with SA have higher levels of C3b deposition than red cells of controls or patients with CM. Therefore, we are very surprised by the findings reported in the paper by Helegbe et al.
After carefully reading the paper, we find the flow cytometry methodology questionable, which may explain the discrepancy between our findings and theirs. The methodological description of the assays is very cryptic and does not provide enough details to allow reproduction. For instance, although the authors state that the antibodies used were FITC-conjugated, none of the primary antibodies listed (anti-CD35, anti-CD55 and anti-C3b) are FITC-conjugated. However, two of the three isotype controls are FITC-conjugated. The authors refer to the secondary antibodies (swine anti-rabbit and goat anti-mouse) as “controls”. Therefore, it is unclear whether they used direct or indirect fluorescent staining. If the latter technique was used, one wonders why FITC-conjugated isotype controls would be used. In addition, no mention is made of positive control cells for any of the assays, dilutions or antibody concentrations are not given, and there is no mention of whether the antibodies were used at saturation. These are essential requirements to insure that negative results are not simply due to technical failure.
The median fluorescent intensity (MFI) values provided also suggest that there are technical anomalies. In the words of the authors, a “cutoff” of 10 was chosen, meaning that values greater than 10 were taken as positive. However, looking at the results for the MFI in Table 5, one realizes that all the values are negative, i.e. < 10. While this is possible for C3b which is not constitutively present on red cells, it is not possible for CD35 and CD55 since both are constitutively present on red cells. CD55 is especially abundantly expressed on the red blood cells.
Taken together, the shortcomings in the flow cytometry methodology outlined above, not to mention the very small sample size used for the flow cytometry assays, cast serious doubts as to the validity of the results presented.
Lastly, we would like to take issue with the statement made to the effect that our studies “did not use strictly defined patient categories and, in some cases malaria diagnosis was not confirmed in the control groups.” In all our studies, our patient categories have been clearly defined with strict entry criteria for each. Most importantly, the diagnosis is always confirmed by the presence of asexual P. falciparum malaria parasites in Giemsa-stained thick or thin smears.
Sincerely,
José A. Stoute, M.D.
Walter Reed Army Institute of Research, Silver Spring, Maryland, USA
Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
E-mail: jose.stoute@us.army.mil
John N. Waitumbi, DVM, Ph.D.
US Army Medical Research Unit, Kenya
Kenya Medical Research Institute, Kenya
E-mail: Jwaitumbi@wrp-ksm.org
Reference List
1. Owuor BO, Odhiambo CO, Otieno WO, Adhiambo C, Makawiti DW, Stoute JA: Reduced immune complex binding capacity and increased complement susceptibility of red cells from children with severe malaria-associated anemia. Mol Med 2007. In Press.
2. Waitumbi JN, Donvito B, Kisserli A, Cohen JH, Stoute JA: Age-related changes in red blood cell complement regulatory proteins and susceptibility to severe malaria. J Infect Dis 2004, 190: 1183-1191.
3. Waitumbi JN, Opollo MO, Muga RO, Misore AO, Stoute JA: Red cell surface changes and erythrophagocytosis in children with severe Plasmodium falciparum anemia. Blood 2000, 95: 1481-1486.
Lack of Association Between Complement Regulatory Proteins and Severe Malarial Anemia or Lack of Adequate Methodology?
24 December 2007
Dear Professor Hommel,
We read with great interest the results of the study by Helegbe et al. entitled “Complement activation in Ghanaian children with severe Plasmodium falciparum Malaria.” The authors report a lack of association between levels of red cell complement regulatory proteins (CD35 and CD55) or C3b deposition and severe anemia due to P. falciparum malaria (SA).
Over the past seven years our group has reported that red cells of children with SA have reduced levels of complement regulatory proteins. The data came from three separate studies with a cumulative total of 174 cases of SA and a similar number of age and gender-matched controls with uncomplicated malaria [1-3], all with confirmed asexual parasitemia by Giemsa-stained smears. In each and every one of these studies, we observed reduced levels of red cell complement regulatory proteins in children with SA. These findings were very specific to SA as they are not found in patients with other categories of severe malaria such as cerebral malaria (CM). Furthermore, our more recent study revealed that red cells of children with SA have higher levels of C3b deposition than red cells of controls or patients with CM. Therefore, we are very surprised by the findings reported in the paper by Helegbe et al.
After carefully reading the paper, we find the flow cytometry methodology questionable, which may explain the discrepancy between our findings and theirs. The methodological description of the assays is very cryptic and does not provide enough details to allow reproduction. For instance, although the authors state that the antibodies used were FITC-conjugated, none of the primary antibodies listed (anti-CD35, anti-CD55 and anti-C3b) are FITC-conjugated. However, two of the three isotype controls are FITC-conjugated. The authors refer to the secondary antibodies (swine anti-rabbit and goat anti-mouse) as “controls”. Therefore, it is unclear whether they used direct or indirect fluorescent staining. If the latter technique was used, one wonders why FITC-conjugated isotype controls would be used. In addition, no mention is made of positive control cells for any of the assays, dilutions or antibody concentrations are not given, and there is no mention of whether the antibodies were used at saturation. These are essential requirements to insure that negative results are not simply due to technical failure.
The median fluorescent intensity (MFI) values provided also suggest that there are technical anomalies. In the words of the authors, a “cutoff” of 10 was chosen, meaning that values greater than 10 were taken as positive. However, looking at the results for the MFI in Table 5, one realizes that all the values are negative, i.e. < 10. While this is possible for C3b which is not constitutively present on red cells, it is not possible for CD35 and CD55 since both are constitutively present on red cells. CD55 is especially abundantly expressed on the red blood cells.
Taken together, the shortcomings in the flow cytometry methodology outlined above, not to mention the very small sample size used for the flow cytometry assays, cast serious doubts as to the validity of the results presented.
Lastly, we would like to take issue with the statement made to the effect that our studies “did not use strictly defined patient categories and, in some cases malaria diagnosis was not confirmed in the control groups.” In all our studies, our patient categories have been clearly defined with strict entry criteria for each. Most importantly, the diagnosis is always confirmed by the presence of asexual P. falciparum malaria parasites in Giemsa-stained thick or thin smears.
Sincerely,
José A. Stoute, M.D.
Walter Reed Army Institute of Research, Silver Spring, Maryland, USA
Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
E-mail: jose.stoute@us.army.mil
John N. Waitumbi, DVM, Ph.D.
US Army Medical Research Unit, Kenya
Kenya Medical Research Institute, Kenya
E-mail: Jwaitumbi@wrp-ksm.org
Reference List
1. Owuor BO, Odhiambo CO, Otieno WO, Adhiambo C, Makawiti DW, Stoute JA: Reduced immune complex binding capacity and increased complement susceptibility of red cells from children with severe malaria-associated anemia. Mol Med 2007. In Press.
2. Waitumbi JN, Donvito B, Kisserli A, Cohen JH, Stoute JA: Age-related changes in red blood cell complement regulatory proteins and susceptibility to severe malaria. J Infect Dis 2004, 190: 1183-1191.
3. Waitumbi JN, Opollo MO, Muga RO, Misore AO, Stoute JA: Red cell surface changes and erythrophagocytosis in children with severe Plasmodium falciparum anemia. Blood 2000, 95: 1481-1486.
Competing interests
The authors are competitors.