- Email: [email protected]
Testing for chronic granulomatous disease
3
Moher D, Fortin P, Jadad AR, et al. Completeness of reporting of trials published in languages other than English: implications for conduct and reporting of systematic reviews. Lancet 1996; 347:
4
363-66. Kessler DA, Rose JL, Temple RJ, Schapiro R, Griffin JP. class wars: drug promotion in a competitive marketplace.
5
D’Arcy Hart P. History of randomised control trials.
TherapeuticN Engl J Med
1994; 331: 1350-53. Lancet 1972; i:
965.
Testing for chronic granulomatous disease SiR-Liese
(Jan 27, p 220)’ present 11patients with granulomatous disease (CGD) who were remarkable unusually late diagnosis. Neutrophils from nine patients were said to test "weakly positive" in a nitrobluetetrazolium (NBT) screen. As suggested by Liese et al, the late diagnoses could be due to residual production of reactive oxygen metabolites by neutrophils. These cases et al
chronic for an
Figure: Numbers of randomised trials in six leading Germanlanguage general journals published in Germany (two), Switzerland (two), and Austria (two), 1985-94 Arrow indicates time when Klinische Wochenschrift became
an
English-
752-54.
suggest a limitation of the NBT assay where there is lowlevel residual oxidant production. In the ferricytochrome C quantitative spectral assay of superoxide production, normal neutrophils generally demonstrate a "burst" of oxidase activity, peaking at 10 min and complete by 20 min. With most CGD patients there is no detectable activity in this assay but some can produce very small amounts of superoxide, and in these cases there is no burst but steady activity for an hour or more. Because the NBT assay relies on the accumulation of the formazan precipitate, timing is important and when a CGD patient produces low levels of oxidants, it is possible for the NBT assay to become positive over time. We have described some improvements to a flow cytometric assay for granulocyte oxidase activity using dihydrorhodamine-123 (DHR) as indicator.2 DHR becomes fluorescent upon oxidation by reactive oxygen species. Because of the large number of CGD patients followed up at the US National Institutes of Health we are often asked to make or confirm a diagnosis of CGD from blood samples sent from patients at other centres. In the past year we have had three such referrals in which an NBT assay at the local hospital or commercial laboratory had been interpreted as positive (normal) whereas our DHR assay unequivocally indicated that the patient had CGD. The patients had been referred to us because of a clinical history suggestive of CGD. Like the NBT assay, the DHR assay is time-dependent but it is more accurate because it is quantitative and it unambiguously discriminates CGD granulocytes from normal cells. The NBT test is subjective, being based on visual inspection of a limited number of cells. The DHR assay has the added advantage of providing screening information suggestive of disease genotype.3 Now that flow cytometry is available in many centres the technique is beginning to be used for CGD diagnosis. The DHR assay for granulocyte oxidase activity is simple and can be done on as little as 0-2 mL of whole blood. The paper by Liese et al shows that a normal NBT test is not sufficient to rule out CGD and late-appearing cases of CGD mean that testing should not be limited to children. We would add that CGD testing should be done more frequently, even in cases involving single infection with selected opportunistic organisms (eg, aspergillus pneumonia or infections with Burkholdaria cepacia [formerly Pseudomonas cepacia] or Serratia marcescens). Finally, we suggest that the DHR assay provides the accuracy and ease of performance that suit this purpose and should replace the NBT assay for routine screening of CGD.
Bero L, Rennie D. The Cochrane Collaboration: preparing, maintaining, and disseminating systematic reviews of the effects of health care. JAMA 1995; 274: 1935-38.
*National Institutes of Health, Bethesda, MD 20892, USA
language journal.
Their results will reassure those making the effort to peruse the non-English literature. We are searching the six leading German-language general medicine journals (Dtsch Med Wochenschr, Klin Wochenschr, Schweiz Med Wochenschr, Schweiz Rundsch Med Prax, Wien Med Wochenschr, and Wien Klin Wochenschr). Using Cochrane criteria for trials, we identified 301 Germanlanguage reports of randomised trials for the years 1985-94, including 38 examined by Moher et al. In all three countries (figure), but most strikingly in Germany, the number of reports of randomised trials decreased over this 10-year period, from totals of 50 in 1985 to nine in 1994. In Germany, this trend accelerated after 1992 when Klinsche Wochenschrift became an Englishlanguage journal (Clinical Investigator). The proportion of placebo-controlled trials decreased whereas reports of comparisons between two active treatments tended to increase. Two drugs of the same therapeutic class were often compared (for example two "statins" or two H 2-receptor antagonists). This type of research, which is generally part of the drug promotion and marketing strategy of the manufacturers,4 is probably less relevant to systematic reviews and meta-analysis. Publication in German-language journals of randomised trials that are relevant to systematic reviews and metaanalyses has become uncommon in recent years. A likely explanation is that investigators in German-speaking Europe are now publishing controlled trials in English. Many more trials might be found in earlier volumes of these six joumals-indeed, preliminary results from a more extensive search of Dtsch Med Wochenschr indicate that randomised trials started to appear in large numbers in the mid-1970sbut not all will be relevant to current practice. The situation may be different for other languages (eg, French, Russian, Japanese, and Chinese) and more research into the importance of non-English publications is needed. We will continue to search the German-language general journals back to the 1940s, when the first trials featuring random allocation were published.5 *Matthias
Egger, Tanja Zellweger, Gerd Antes
*Department of Social and Preventive Medicine, University of Bern, 3012 Bern, Switzerland; and Department of Medical Informatics, University of Freiburg, Freiburg, Germany
1 2
Egger M, Davey Smith G. Misleading meta-analysis. BMJ 1995;
1048
310:
*Sarah J Vowells, Thomas A Fleisher,
Harry
L Malech
1 Liese J, Jendrossek V, Belohradsky B. Chronic granulomatous disease in adults. Lancet 1996; 347: 220-23. 2 Vowells S, Sekhsaria S, Malech H, Shalit M, Fleisher T. Flow cytometric analysis of the granulocyte respiratory burst: a comparison study of fluorescent probes. J Immunol Meth 1995; 178: 89-97. 3 Vowells S, Fleisher T, Sekhsaria S, Alling D, Maguire T, Malech H. Genotype dependent variability in flow cytometric evaluation of NADPH oxidase function in patients with chronic granulomatous 1996; 128: 104-07. disease. Pediatr J
CD reported by Rosenberg et al. According to our observation CD44v6 is expressed in colonic CD. Thus, we doubt that CD44v6 will be helpful as a diagnostic test in differentiating UC from CD lesions. We speculate that the expression of CD44v6 on colonic crypt epithelial cells reflect rather a cytokine-mediated epiphenomenon of inflammation than an UC-specific mechanism. *Walter Reinisch, Karl-Heinz Heider, Clemens Dejaco, Günther R Adolf
Expression of CD44v6 in ulcerative colitis and Crohn’s disease SiR-Rosenberg et al reported increased colonic crypt epithelial expression of the v6 variant of CD44 from biopsy samples of patients with ulcerative colitis (UC), whereas samples from individuals with Crohn’s disease (CD) and with normal colon lacked CD44v6 expression.’ CD44v6 isoform expression was detected by monoclonal antibody 2F10 in 23 of 25 UC samples compared with three of 18 colonic CD samples. The authors discussed the potential of CD44v6 analysis in differentiating UC from colonic CD. To test this hypothesis we examined the expression of CD44v6 in both diseases with the two different specific antibodies: BBA 13 (identical to clone 2FI0; R&D Systems, Abingdon, UK) and BIWA 1 (clone VFF18; Bender Med Systems, Vienna, Austria). Before using the antibodies for immunohistochemistry, we determined their epitope specificity and affinity. Competitive ELISAs and surface plasmon-resonance measurements revealed that both antibodies recognise a 14-aminoacid sequence encoded by CD44v6. BIWA 1, however, showed a three-to-four-fold stronger binding to its epitope than BBA 13. Therefore, we used BBA 13 in a higher concentration than BIWA 1 in our standard immunohistochemistry protocol (antigen retrieval by microwave, ABC-technique). Parallel staining of skin showed similar staining intensities of keratinocytes with both 2
antibodies. 20 biopsy samples from 12 UC patients and 17 samples from 12 CD patients were obtained by endoscopy. Samples were formalin-fixed and paraffin-embedded. Three UC and three CD patients were taking immunosuppressive therapy (steroids and/or azathioprine). Other patients were without treatment (four UC, four CD) or taking 5-aminosalicylates (five UC, five CD). Immunohistochemistry sections were read by two observers blinded to the diagnosis. Severity of inflammation in UC was graded histologically as inactive, mild, moderate, or severe. Samples from patients with CD were rated as normal or inflamed. For evaluation of staining we used Rosenberg and colleagues’ score: staining of fewer than 25% of crypt epithelial cells per sample was graded as negative, staining of 25-75% as +, and staining of more than 75% as ++. In our study eight of 16 UC samples (50%) with mild, moderate, or severe inflammation and five of 14 (35%) CD samples with inflamed mucosa showed crypt-epithelial The extent of staining with BBA 13 (X2 test, p<005). expression was not associated with histological grade of inflammation. Inactive lesions in UC samples and normal mucosa from CD patients remained negative. With BIWA 1, 88% of the active UC samples and 86% of the inflamed CD samples displayed a staining pattern that was not significantly different. With BIWA 1 even two of four inactive UC samples and two of three CD samples with normal mucosa were +. In colon from control patients no significant staining for CD44v6 was detected.2 Our data do not confirm the differing expression of CD44v6 on colonic crypt epithelial cells between UC and
Georg Oberhuber,
*Clinic of Internal Medicine IV, Department of Gastroenterology and Hepatology, of Vienna, A-1090 Vienna, Austria; Boehringer Ingelheim Research and Development, Department of Cell Biology, Vienna; and Clinic of Pathology, University of Vienna, Vienna
University
1
2
Prince C, Kaklamanis K, et al. Increased expression of CD44v6 and CD44v3 in ulcerative colitis but not colonic Crohn’s disease. Lancet 1995; 345: 1205-09. Heider K-H, Mulder J-WR, Ostermann E, et al. Splice variants for the surface glycoprotein CD44 associated with metastatic tumour cells are expressed in normal tissues of humans and cynomolgus monkeys. Eur J Cancer (in press).
Rosenberg WMC,
Leukaemia risk in Crohn’s disease SiR-In a report of with Crohn’s disease
long-term follow-up of patients treated with azathioprine or 6-mercaptopurine (Jan 27, p 215),’ Bouhnik and colleagues reviewed therapeutic outcomes in 157 patients. This is an extensive review, and it is of considerable interest that malignant disease developed in four patients who received azathioprine or 6-mercaptopurine. These workers report an association with malignant melanoma, cutaneous basal cell carcinoma, renal carcinoma, and brain lymphoma. We report a patient with Crohn’s disease who developed acute lymphocytic leukaemia after treatment with corticosteroids and immunosuppressant over 10 years. The 28-year-old man was admitted to our department with pancytopoenia. He had a history of Crohn’s disease since age 10. His treatments included sulfasalazin, and ascending colectomy, and had also received a short course of corticosteroid to control severe abdominal pain and diarrhoea. His pancytopoenia was apparently induced by sulfasalazin; however, after stopping this drug, cell counts did not recover. Bone marrow aspirates showed the presence of leukaemic lymphoblasts (LI on FAB classification) that had the phenotype of peroxidase negative, Sudan B negative, and terminal CD34, CD 19, CD 10, HLA-DR, He went into transferase. deoxynucleotidyl complete remission daunorubicin, vincristine, (chemotherapy: L-asparaginase, and prednisolone) for 18 months. His siblings did not match on HLA typing. He eventually died with acute respiratory failure and meningeal infiltration. Our Medline search uncovered 12 patients with Crohn’s disease who developed leukaemia 2-5 (table). The latency
1049
Moher D, Fortin P, Jadad AR, et al. Completeness of reporting of trials published in languages other than English: implications for conduct and reporting of systematic reviews. Lancet 1996; 347:
4
363-66. Kessler DA, Rose JL, Temple RJ, Schapiro R, Griffin JP. class wars: drug promotion in a competitive marketplace.
5
D’Arcy Hart P. History of randomised control trials.
TherapeuticN Engl J Med
1994; 331: 1350-53. Lancet 1972; i:
965.
Testing for chronic granulomatous disease SiR-Liese
(Jan 27, p 220)’ present 11patients with granulomatous disease (CGD) who were remarkable unusually late diagnosis. Neutrophils from nine patients were said to test "weakly positive" in a nitrobluetetrazolium (NBT) screen. As suggested by Liese et al, the late diagnoses could be due to residual production of reactive oxygen metabolites by neutrophils. These cases et al
chronic for an
Figure: Numbers of randomised trials in six leading Germanlanguage general journals published in Germany (two), Switzerland (two), and Austria (two), 1985-94 Arrow indicates time when Klinische Wochenschrift became
an
English-
752-54.
suggest a limitation of the NBT assay where there is lowlevel residual oxidant production. In the ferricytochrome C quantitative spectral assay of superoxide production, normal neutrophils generally demonstrate a "burst" of oxidase activity, peaking at 10 min and complete by 20 min. With most CGD patients there is no detectable activity in this assay but some can produce very small amounts of superoxide, and in these cases there is no burst but steady activity for an hour or more. Because the NBT assay relies on the accumulation of the formazan precipitate, timing is important and when a CGD patient produces low levels of oxidants, it is possible for the NBT assay to become positive over time. We have described some improvements to a flow cytometric assay for granulocyte oxidase activity using dihydrorhodamine-123 (DHR) as indicator.2 DHR becomes fluorescent upon oxidation by reactive oxygen species. Because of the large number of CGD patients followed up at the US National Institutes of Health we are often asked to make or confirm a diagnosis of CGD from blood samples sent from patients at other centres. In the past year we have had three such referrals in which an NBT assay at the local hospital or commercial laboratory had been interpreted as positive (normal) whereas our DHR assay unequivocally indicated that the patient had CGD. The patients had been referred to us because of a clinical history suggestive of CGD. Like the NBT assay, the DHR assay is time-dependent but it is more accurate because it is quantitative and it unambiguously discriminates CGD granulocytes from normal cells. The NBT test is subjective, being based on visual inspection of a limited number of cells. The DHR assay has the added advantage of providing screening information suggestive of disease genotype.3 Now that flow cytometry is available in many centres the technique is beginning to be used for CGD diagnosis. The DHR assay for granulocyte oxidase activity is simple and can be done on as little as 0-2 mL of whole blood. The paper by Liese et al shows that a normal NBT test is not sufficient to rule out CGD and late-appearing cases of CGD mean that testing should not be limited to children. We would add that CGD testing should be done more frequently, even in cases involving single infection with selected opportunistic organisms (eg, aspergillus pneumonia or infections with Burkholdaria cepacia [formerly Pseudomonas cepacia] or Serratia marcescens). Finally, we suggest that the DHR assay provides the accuracy and ease of performance that suit this purpose and should replace the NBT assay for routine screening of CGD.
Bero L, Rennie D. The Cochrane Collaboration: preparing, maintaining, and disseminating systematic reviews of the effects of health care. JAMA 1995; 274: 1935-38.
*National Institutes of Health, Bethesda, MD 20892, USA
language journal.
Their results will reassure those making the effort to peruse the non-English literature. We are searching the six leading German-language general medicine journals (Dtsch Med Wochenschr, Klin Wochenschr, Schweiz Med Wochenschr, Schweiz Rundsch Med Prax, Wien Med Wochenschr, and Wien Klin Wochenschr). Using Cochrane criteria for trials, we identified 301 Germanlanguage reports of randomised trials for the years 1985-94, including 38 examined by Moher et al. In all three countries (figure), but most strikingly in Germany, the number of reports of randomised trials decreased over this 10-year period, from totals of 50 in 1985 to nine in 1994. In Germany, this trend accelerated after 1992 when Klinsche Wochenschrift became an Englishlanguage journal (Clinical Investigator). The proportion of placebo-controlled trials decreased whereas reports of comparisons between two active treatments tended to increase. Two drugs of the same therapeutic class were often compared (for example two "statins" or two H 2-receptor antagonists). This type of research, which is generally part of the drug promotion and marketing strategy of the manufacturers,4 is probably less relevant to systematic reviews and meta-analysis. Publication in German-language journals of randomised trials that are relevant to systematic reviews and metaanalyses has become uncommon in recent years. A likely explanation is that investigators in German-speaking Europe are now publishing controlled trials in English. Many more trials might be found in earlier volumes of these six joumals-indeed, preliminary results from a more extensive search of Dtsch Med Wochenschr indicate that randomised trials started to appear in large numbers in the mid-1970sbut not all will be relevant to current practice. The situation may be different for other languages (eg, French, Russian, Japanese, and Chinese) and more research into the importance of non-English publications is needed. We will continue to search the German-language general journals back to the 1940s, when the first trials featuring random allocation were published.5 *Matthias
Egger, Tanja Zellweger, Gerd Antes
*Department of Social and Preventive Medicine, University of Bern, 3012 Bern, Switzerland; and Department of Medical Informatics, University of Freiburg, Freiburg, Germany
1 2
Egger M, Davey Smith G. Misleading meta-analysis. BMJ 1995;
1048
310:
*Sarah J Vowells, Thomas A Fleisher,
Harry
L Malech
1 Liese J, Jendrossek V, Belohradsky B. Chronic granulomatous disease in adults. Lancet 1996; 347: 220-23. 2 Vowells S, Sekhsaria S, Malech H, Shalit M, Fleisher T. Flow cytometric analysis of the granulocyte respiratory burst: a comparison study of fluorescent probes. J Immunol Meth 1995; 178: 89-97. 3 Vowells S, Fleisher T, Sekhsaria S, Alling D, Maguire T, Malech H. Genotype dependent variability in flow cytometric evaluation of NADPH oxidase function in patients with chronic granulomatous 1996; 128: 104-07. disease. Pediatr J
CD reported by Rosenberg et al. According to our observation CD44v6 is expressed in colonic CD. Thus, we doubt that CD44v6 will be helpful as a diagnostic test in differentiating UC from CD lesions. We speculate that the expression of CD44v6 on colonic crypt epithelial cells reflect rather a cytokine-mediated epiphenomenon of inflammation than an UC-specific mechanism. *Walter Reinisch, Karl-Heinz Heider, Clemens Dejaco, Günther R Adolf
Expression of CD44v6 in ulcerative colitis and Crohn’s disease SiR-Rosenberg et al reported increased colonic crypt epithelial expression of the v6 variant of CD44 from biopsy samples of patients with ulcerative colitis (UC), whereas samples from individuals with Crohn’s disease (CD) and with normal colon lacked CD44v6 expression.’ CD44v6 isoform expression was detected by monoclonal antibody 2F10 in 23 of 25 UC samples compared with three of 18 colonic CD samples. The authors discussed the potential of CD44v6 analysis in differentiating UC from colonic CD. To test this hypothesis we examined the expression of CD44v6 in both diseases with the two different specific antibodies: BBA 13 (identical to clone 2FI0; R&D Systems, Abingdon, UK) and BIWA 1 (clone VFF18; Bender Med Systems, Vienna, Austria). Before using the antibodies for immunohistochemistry, we determined their epitope specificity and affinity. Competitive ELISAs and surface plasmon-resonance measurements revealed that both antibodies recognise a 14-aminoacid sequence encoded by CD44v6. BIWA 1, however, showed a three-to-four-fold stronger binding to its epitope than BBA 13. Therefore, we used BBA 13 in a higher concentration than BIWA 1 in our standard immunohistochemistry protocol (antigen retrieval by microwave, ABC-technique). Parallel staining of skin showed similar staining intensities of keratinocytes with both 2
antibodies. 20 biopsy samples from 12 UC patients and 17 samples from 12 CD patients were obtained by endoscopy. Samples were formalin-fixed and paraffin-embedded. Three UC and three CD patients were taking immunosuppressive therapy (steroids and/or azathioprine). Other patients were without treatment (four UC, four CD) or taking 5-aminosalicylates (five UC, five CD). Immunohistochemistry sections were read by two observers blinded to the diagnosis. Severity of inflammation in UC was graded histologically as inactive, mild, moderate, or severe. Samples from patients with CD were rated as normal or inflamed. For evaluation of staining we used Rosenberg and colleagues’ score: staining of fewer than 25% of crypt epithelial cells per sample was graded as negative, staining of 25-75% as +, and staining of more than 75% as ++. In our study eight of 16 UC samples (50%) with mild, moderate, or severe inflammation and five of 14 (35%) CD samples with inflamed mucosa showed crypt-epithelial The extent of staining with BBA 13 (X2 test, p<005). expression was not associated with histological grade of inflammation. Inactive lesions in UC samples and normal mucosa from CD patients remained negative. With BIWA 1, 88% of the active UC samples and 86% of the inflamed CD samples displayed a staining pattern that was not significantly different. With BIWA 1 even two of four inactive UC samples and two of three CD samples with normal mucosa were +. In colon from control patients no significant staining for CD44v6 was detected.2 Our data do not confirm the differing expression of CD44v6 on colonic crypt epithelial cells between UC and
Georg Oberhuber,
*Clinic of Internal Medicine IV, Department of Gastroenterology and Hepatology, of Vienna, A-1090 Vienna, Austria; Boehringer Ingelheim Research and Development, Department of Cell Biology, Vienna; and Clinic of Pathology, University of Vienna, Vienna
University
1
2
Prince C, Kaklamanis K, et al. Increased expression of CD44v6 and CD44v3 in ulcerative colitis but not colonic Crohn’s disease. Lancet 1995; 345: 1205-09. Heider K-H, Mulder J-WR, Ostermann E, et al. Splice variants for the surface glycoprotein CD44 associated with metastatic tumour cells are expressed in normal tissues of humans and cynomolgus monkeys. Eur J Cancer (in press).
Rosenberg WMC,
Leukaemia risk in Crohn’s disease SiR-In a report of with Crohn’s disease
long-term follow-up of patients treated with azathioprine or 6-mercaptopurine (Jan 27, p 215),’ Bouhnik and colleagues reviewed therapeutic outcomes in 157 patients. This is an extensive review, and it is of considerable interest that malignant disease developed in four patients who received azathioprine or 6-mercaptopurine. These workers report an association with malignant melanoma, cutaneous basal cell carcinoma, renal carcinoma, and brain lymphoma. We report a patient with Crohn’s disease who developed acute lymphocytic leukaemia after treatment with corticosteroids and immunosuppressant over 10 years. The 28-year-old man was admitted to our department with pancytopoenia. He had a history of Crohn’s disease since age 10. His treatments included sulfasalazin, and ascending colectomy, and had also received a short course of corticosteroid to control severe abdominal pain and diarrhoea. His pancytopoenia was apparently induced by sulfasalazin; however, after stopping this drug, cell counts did not recover. Bone marrow aspirates showed the presence of leukaemic lymphoblasts (LI on FAB classification) that had the phenotype of peroxidase negative, Sudan B negative, and terminal CD34, CD 19, CD 10, HLA-DR, He went into transferase. deoxynucleotidyl complete remission daunorubicin, vincristine, (chemotherapy: L-asparaginase, and prednisolone) for 18 months. His siblings did not match on HLA typing. He eventually died with acute respiratory failure and meningeal infiltration. Our Medline search uncovered 12 patients with Crohn’s disease who developed leukaemia 2-5 (table). The latency
1049