- Email: [email protected]
In-vitro production of cytokines in serum
687
This strategy, combined with numerous negative controls in each PCR procedure, can reduce the rate of false-positive results. RICHARD W. CONE ANN C. HOBSON MEEI-LI W. HUANG MARILYNN R. FAIRFAX
Department of Laboratory Medicine, University of Washington Medical Center, Seattle, Washington 98195, USA 1.
Beyer-Finkler E, Pfister H, Girardi F. Anti-contamination primers to improve specificity of polymerase chain reaction in human papillomavirus screening. Lancet
1990; 335: 1289-90. Porter-Jordan K, Garrett CT. Source of contamination in polymerase chain reaction assay. Lancet 1990; 335: 1220. 3. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989; 339: 237-38. 4. Kitchin PA, Szotyori Z, Fromholc C, Almond N. Avoidance of false positives. Nature 1990; 344: 201. 2.
Phenobarbitone and
epilepsy
SiR,—Imuch enjoyed your Epilepsy Octet. Nevertheless, I am concerned that Dr Brodie (Aug 11, p 350) suggests an indication for phenobarbitone prophylaxis in febrile convulsions. There is little, if any, evidence of benefit from anticonvulsants in prophylaxis for febrile convulsions. In view of the very high frequency of important side-effects in young children treated with phenobarbitone, I am disappointed to see this recommendation in The Lancet. Dewsbury District Hospital, Dewsbury V1/F134HS, UK
*** We have shown this letter -ED. L.
P.EHRHARDT
to
Dr
Brodie, whose reply follows.
SIR,-I agree: "prophylaxis of febrile convulsions" has no place in a table entitled "prescribing anticonvulsants in adults". The item may not be entirely erroneous, however, since there is quite a lot of discussion about the prophylaxis of febrile convulsions. Although it is now becoming accepted practice in the UK not to give phenobarbitone, this is not so in other parts of the world, including the USA. Perhaps my error will provoke other comments. The question merits discussion in The Lancet’s correspondence columns. Department of Medicine and Therapeutics, University of Glasgow, Gardiner Institute, Western Infirmary, Glasgow G11 6NT, UK
In-vitro Autoradiograms of the 105 base pair HHV-6 PCR products. Upper autoradiogram = before decontamination; lower autoradiogram =after decontamination with HCI. Wipe test sites: lane 1= positive displacement pipette used for PCR set-up; lane 2=freezer door handle; lane 3= box containing PCR primers; lane 4= freezer shelf used for storing PCR reagents, lane 5= floor by laboratory bench; lane 6= PCR set-up hood, !ane7=f!oorby PCR set-up hood, lane 8=room door handle, lanes 9-13 = negative controls; lane 14 = positive control (500 molecules target DNA) The "shadow" band at about 130 base pairs is an artifact of liquid hybridisation, the method used to detect these PCR products, and does not interfere with interpretation of the specific band in our expenence.
handle) produced PCR products that were beyond the background level of contamination, suggesting that these locations harboured DNA which could contribute to the false-positive problem. Contaminated disposable materials were replaced. Other contaminated sites were vigorously cleaned with molar HCl (chosen for its deleterious effects on DNA). Retesting the suspect sites with the same procedure produced negative results (figure, lower part). We conclude that the PCR wipe test can make a substantial contribution
to
controlling environmental DNA contamination.
MARTIN J. BRODIE
production of cytokines
in
serum
SIR,-Dr Freeman and colleagues’ claim (August 4, p 312) that overnight storage of serum allows in-vitro production of tumour necrosis factor (TNF-a), thereby invalidating serum TNF-K measurements unless calcium-chelating agents are used, does not accord with our experience or much of the published data. We have measured serum TNF-of after overnight storage of whole blood at 4°C with a commercial ELISA (’Biokine’; T Cell Sciences), and have found measurable TNF (more than 10 pg/ml) in only 1 of 31 control children and 9 of 65 children in clinical remission of chronic inflammatory bowel disease. Similarly Balkwill et aP found only 1 of 31 controls to have detectable serum TNF. Freeman et al cite several references in support of significant in-vitro production of TNF-a in these circumstances. However, Maury and Teppoz found only low-level TNF-a (below 40 pg/ml) in 21 of 69 controls, Scuderi et aP found measurable TNF in only 7 of 88 controls and none of 14 cord-blood samples, and Michie and colleagues4 separated and froze serum without delay and found reproducible increments in multiple samples taken from the 3 positives among their 13 controls. Thus, the papers cited by Freeman et al do not support the contention that in-vitro production of TNF-x is a widespread problem. The serum TNF-K concentrations reported by Freeman and colleagues (above 1000 pg/ml, consistently found in specimens
688
taken before cardiac bypass surgery) have not been found before, suggesting that such patients cannot be regarded as normal controls or that there is a problem with the assay. Significant increases in serum TNF-ot have been reported in chronic heart failures and stimulated release of TNF from peripheral blood monocytes in vitro is increased by starvation before venesection.6 If the samples were taken just before bypass surgery, the patients would presumably have been starved, and may have had a cardiac cause for an increase in serum TNF. A more plausible explanation is some form of non-specific reaction in their assay. Abe et aF have reported artifactual increases in serum TNF-K in controls when using an ELISA with anti-TNF-ot polyclonal rabbit IgG for both solid and liquid phases: all sera tested negative on bioassay. A 250 kD protein cross-reacted in ELISAs for both TNF-a and murine epidermal growth factor. Freeman et al used a similar sandwich assay with rabbit polyclonal and mouse monoclonal antibodies. Overnight storage may have led to increased production of a cross-reactant, and the value of calcium-chelation may have been to inhibit its production, rather than directly affecting bioactive TNF. Departments of Child
Health
and Paediatric
Gastroenterology, St Bartholomew’s Hospital, London EC1A 7BE, UK
SIMON MURCH THOMAS MACDONALD
1. Balkwill F, Osborne R, Burke F, et al Evidence for tumour necrosis factor production m cancer. Lancet 1987, ii: 1229-32. 2 Maury CPJ, Teppo A-M. Tumour necrosis factor in the serum of patients with systemic lupus erythematosus. Arthritis Rheum 1989; 32: 146-50. 3. Scuderi P, Sterling KE, Lam KS, et al. Raised serum levels of tumour necrosis factor in parasitic infections. Lancet 1986, ii: 1364-65 4. Michie HR, Manogue KR, Spnggs DR, et al Detection of circulating tumour necrosis factor after endotoxin administration N Engl J Med 1988; 318: 1481-86 5. Levene B, Kalman J, Mayer L, et al Elevated circulating levels of tumour necrosis factor in severe chronic heart failure N Engl JMed 1990; 323: 236-41. 6. Vaisman N, Schattner A, Hahn T. Tumor necrosis factor production during starvation. Am J Med 1989; 87: 115 7. Abe Y, Miyake M, Miyazaki T, et al. Nonspecific reaction in the sandwich immunoassay for human tumor necrosis factor-&agr; (hTNF-&agr;) Clin Chim Acta 1989; 181: 223-30
on calcium ions. This would explain the differences between the samples treated with heparin or with chelating agents and would argue against a unique action of heparin itself.
depends
Department of Histopathology, St Mary’s Hospital Medical School, London W21PG, UK
SIR,-Peripheral blood mononuclear cells have been reported’ to produce interleukin 6 (IL-6) spontaneously. However, attention to the elimination of bacterial lipopolysaccharide (endotoxin) in the handling and preparation of monocytes has shown that such production can almost be completely removedDr Freeman and colleagues show that the method of blood collection affects the level ofTNF -ex in plasma. We have found that high levels of plasma IL-6 and It-1may result from collection of blood into endotoxincontaminated containers or anticoagulants; this endotoxin-induced production does not occur in blood collected into EDTA. Blood from healthy volunteers was collected into sterile ’Vacutainers’ containing lithium heparin or EDTA or into a sterile plastic syringe containing pyrogen-free sodium heparin for injection. Samples were distributed into sterile disposable plastic test tubes and incubated at room temperature (RT) or 37°C ; plasma was separated by centrifugation at time 0, every 20 min up to 1 h, and then at 2, 4, 6, and 24 h. Cytokine levels were measured by radioimmunoassay standardised against international reference standard 86/680 (IL-1) and interim standard 88/514 (IL-6). Significant cytokine production was detectable by 2 h of incubation at 37°C; levels at 24 h were: lL-6 (ng/ml) RT T 37°C Lithium heparin EDTA Sodium heparin
were
taken
at
all stages
to
avoid LPS
contamination, including baking of glassware and use of LPS-free media.
Monocytes treated in this way consistently produced a basal level of NO averaging 6-0 pmollllg protein. This increased more than ninefold to 55-5 pmollllg protein by the addition of LPS. Although a basal production of NO has been demonstrated in work on endothelial cells4 we had previously assumed that the production of such a potentially harmful substance by native monocytes reflected traces of LPS in the extraction and culture equipment and media. The work of Freeman et al suggests an alternative explanation-namely, that heparin can stimulate monocytes to produce NO as well as TNF. We have shown that basal NO production, like TNF production, is greatly increased in monocyte samples from patients with alcoholic hepatitis (unpublished). If heparin can induce monocytes to elaborate TNF and NO in vitro this would have important implications for the interpretation of research data and for diagnostic testing. However, the possibility remains that LPS-induced basal TNF production
30-0 04 0-4
IL-1 1
3.0 04 4 04
(nglml) (37’C) 23.3
ND 0-93
ND = not done
their
of nitrite ions.3 Precautions
N.C.A.HUNT
1. McClain CJ, Cohen DA. Increased tumour necrosis factor production by monocytes in alcoholic liver disease. Hepatology 1989; 9: 349-51. 2. Griffith T, Randall M. Nitric oxide comes of age. Lancet 1989 ii: 875-76 3. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR Analysis of nitrate, nitrite and (15N) nitrate in biological fluids Ann Biochem 1982, 126: 131-38. 4. Rees DD, Palmer RMJ, Moncada S. Role of endothelium-derived nitric-oxid inthe regulation of blood pressure. Proc Natl Acad Sci ( USA) 1989; 86: 3375-78
SIR,-The fact that monocytes can produce TNF-x has been demonstrated by several groups, as has the stimulatory effect of lipopolysaccharide (LPS).’ It is unfortunate that Dr Freeman and
colleagues did not try to reduce or measure LPS contamination of samples. Despite this they did find a clear difference in TNF production between monocytes isolated with heparin or with chelating agents. Nitric oxide (NO), besides its probable role as an endotheliumderived relaxing factor,z is also a mediator of macrophage cytotoxicity. Wehave studied NO production in monocytes by collecting whole venous blood in heparinised syringes followed by monocyte extraction on a density gradient and enrichment by adherence to a tissue culture vessel. Samples were then incubated for 24 h in medium with or without LPS (1 jg/ml). NO was measured by an indirect technique that is specific for the detection
R. D. GOLDIN
A lithium
heparin tube
was
washed
out
with 10 ml of
endotoxin-free water; this extract was found to contain high levels of
(greater than 250 units) when assayed by the limulus amoebocyte lysate assay standardised against international endotoxin
reference standard 84/650. The induced IL-6 proved to have bioactivity when assayed against a sensitive cell line (B9). The action of EDTA seemed to be a direct inhibition of endotoxin-induced IL-6 production because IL-6 production did not occur if blood collected into lithium heparin tubes was transferred into EDTA within 2 h of collection; beyond this time the induced response was irreversible. These results indicate that great care must be taken at all stages of blood collection and fractionation to eliminate endotoxin when studying production or presence of those cytokines that are readily induced by endotoxin. Plasma collected into endotoxincontaminated systems is unsuitable for use in cell culture
experiments. We thank Sue Selkirk, National Institute for Control, for the limulus assays.
Department of Immunology, Charing Cross and Westminster Medical School, Westminster Hospital, London SW1 P 2AP, UK
1. Aarden 2.
Biological
Standards and
PAMELA G. RICHES ROGER GOODING
LA, De Groot ER, Schaap OL, Landsdrop PM. Production of hybridoma growth factor by human monocytes. Eur J Immunol 1987; 17: 1411-16 Schindler R, Mancilla J, Endres S, Ghorbani R, Clark SC, Dinarello CA. Correlation and interactions in the production of interleukin-6 (IL-6), IL-1, and tumour necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood 1990; 75: 40-47.
This strategy, combined with numerous negative controls in each PCR procedure, can reduce the rate of false-positive results. RICHARD W. CONE ANN C. HOBSON MEEI-LI W. HUANG MARILYNN R. FAIRFAX
Department of Laboratory Medicine, University of Washington Medical Center, Seattle, Washington 98195, USA 1.
Beyer-Finkler E, Pfister H, Girardi F. Anti-contamination primers to improve specificity of polymerase chain reaction in human papillomavirus screening. Lancet
1990; 335: 1289-90. Porter-Jordan K, Garrett CT. Source of contamination in polymerase chain reaction assay. Lancet 1990; 335: 1220. 3. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989; 339: 237-38. 4. Kitchin PA, Szotyori Z, Fromholc C, Almond N. Avoidance of false positives. Nature 1990; 344: 201. 2.
Phenobarbitone and
epilepsy
SiR,—Imuch enjoyed your Epilepsy Octet. Nevertheless, I am concerned that Dr Brodie (Aug 11, p 350) suggests an indication for phenobarbitone prophylaxis in febrile convulsions. There is little, if any, evidence of benefit from anticonvulsants in prophylaxis for febrile convulsions. In view of the very high frequency of important side-effects in young children treated with phenobarbitone, I am disappointed to see this recommendation in The Lancet. Dewsbury District Hospital, Dewsbury V1/F134HS, UK
*** We have shown this letter -ED. L.
P.EHRHARDT
to
Dr
Brodie, whose reply follows.
SIR,-I agree: "prophylaxis of febrile convulsions" has no place in a table entitled "prescribing anticonvulsants in adults". The item may not be entirely erroneous, however, since there is quite a lot of discussion about the prophylaxis of febrile convulsions. Although it is now becoming accepted practice in the UK not to give phenobarbitone, this is not so in other parts of the world, including the USA. Perhaps my error will provoke other comments. The question merits discussion in The Lancet’s correspondence columns. Department of Medicine and Therapeutics, University of Glasgow, Gardiner Institute, Western Infirmary, Glasgow G11 6NT, UK
In-vitro Autoradiograms of the 105 base pair HHV-6 PCR products. Upper autoradiogram = before decontamination; lower autoradiogram =after decontamination with HCI. Wipe test sites: lane 1= positive displacement pipette used for PCR set-up; lane 2=freezer door handle; lane 3= box containing PCR primers; lane 4= freezer shelf used for storing PCR reagents, lane 5= floor by laboratory bench; lane 6= PCR set-up hood, !ane7=f!oorby PCR set-up hood, lane 8=room door handle, lanes 9-13 = negative controls; lane 14 = positive control (500 molecules target DNA) The "shadow" band at about 130 base pairs is an artifact of liquid hybridisation, the method used to detect these PCR products, and does not interfere with interpretation of the specific band in our expenence.
handle) produced PCR products that were beyond the background level of contamination, suggesting that these locations harboured DNA which could contribute to the false-positive problem. Contaminated disposable materials were replaced. Other contaminated sites were vigorously cleaned with molar HCl (chosen for its deleterious effects on DNA). Retesting the suspect sites with the same procedure produced negative results (figure, lower part). We conclude that the PCR wipe test can make a substantial contribution
to
controlling environmental DNA contamination.
MARTIN J. BRODIE
production of cytokines
in
serum
SIR,-Dr Freeman and colleagues’ claim (August 4, p 312) that overnight storage of serum allows in-vitro production of tumour necrosis factor (TNF-a), thereby invalidating serum TNF-K measurements unless calcium-chelating agents are used, does not accord with our experience or much of the published data. We have measured serum TNF-of after overnight storage of whole blood at 4°C with a commercial ELISA (’Biokine’; T Cell Sciences), and have found measurable TNF (more than 10 pg/ml) in only 1 of 31 control children and 9 of 65 children in clinical remission of chronic inflammatory bowel disease. Similarly Balkwill et aP found only 1 of 31 controls to have detectable serum TNF. Freeman et al cite several references in support of significant in-vitro production of TNF-a in these circumstances. However, Maury and Teppoz found only low-level TNF-a (below 40 pg/ml) in 21 of 69 controls, Scuderi et aP found measurable TNF in only 7 of 88 controls and none of 14 cord-blood samples, and Michie and colleagues4 separated and froze serum without delay and found reproducible increments in multiple samples taken from the 3 positives among their 13 controls. Thus, the papers cited by Freeman et al do not support the contention that in-vitro production of TNF-x is a widespread problem. The serum TNF-K concentrations reported by Freeman and colleagues (above 1000 pg/ml, consistently found in specimens
688
taken before cardiac bypass surgery) have not been found before, suggesting that such patients cannot be regarded as normal controls or that there is a problem with the assay. Significant increases in serum TNF-ot have been reported in chronic heart failures and stimulated release of TNF from peripheral blood monocytes in vitro is increased by starvation before venesection.6 If the samples were taken just before bypass surgery, the patients would presumably have been starved, and may have had a cardiac cause for an increase in serum TNF. A more plausible explanation is some form of non-specific reaction in their assay. Abe et aF have reported artifactual increases in serum TNF-K in controls when using an ELISA with anti-TNF-ot polyclonal rabbit IgG for both solid and liquid phases: all sera tested negative on bioassay. A 250 kD protein cross-reacted in ELISAs for both TNF-a and murine epidermal growth factor. Freeman et al used a similar sandwich assay with rabbit polyclonal and mouse monoclonal antibodies. Overnight storage may have led to increased production of a cross-reactant, and the value of calcium-chelation may have been to inhibit its production, rather than directly affecting bioactive TNF. Departments of Child
Health
and Paediatric
Gastroenterology, St Bartholomew’s Hospital, London EC1A 7BE, UK
SIMON MURCH THOMAS MACDONALD
1. Balkwill F, Osborne R, Burke F, et al Evidence for tumour necrosis factor production m cancer. Lancet 1987, ii: 1229-32. 2 Maury CPJ, Teppo A-M. Tumour necrosis factor in the serum of patients with systemic lupus erythematosus. Arthritis Rheum 1989; 32: 146-50. 3. Scuderi P, Sterling KE, Lam KS, et al. Raised serum levels of tumour necrosis factor in parasitic infections. Lancet 1986, ii: 1364-65 4. Michie HR, Manogue KR, Spnggs DR, et al Detection of circulating tumour necrosis factor after endotoxin administration N Engl J Med 1988; 318: 1481-86 5. Levene B, Kalman J, Mayer L, et al Elevated circulating levels of tumour necrosis factor in severe chronic heart failure N Engl JMed 1990; 323: 236-41. 6. Vaisman N, Schattner A, Hahn T. Tumor necrosis factor production during starvation. Am J Med 1989; 87: 115 7. Abe Y, Miyake M, Miyazaki T, et al. Nonspecific reaction in the sandwich immunoassay for human tumor necrosis factor-&agr; (hTNF-&agr;) Clin Chim Acta 1989; 181: 223-30
on calcium ions. This would explain the differences between the samples treated with heparin or with chelating agents and would argue against a unique action of heparin itself.
depends
Department of Histopathology, St Mary’s Hospital Medical School, London W21PG, UK
SIR,-Peripheral blood mononuclear cells have been reported’ to produce interleukin 6 (IL-6) spontaneously. However, attention to the elimination of bacterial lipopolysaccharide (endotoxin) in the handling and preparation of monocytes has shown that such production can almost be completely removedDr Freeman and colleagues show that the method of blood collection affects the level ofTNF -ex in plasma. We have found that high levels of plasma IL-6 and It-1may result from collection of blood into endotoxincontaminated containers or anticoagulants; this endotoxin-induced production does not occur in blood collected into EDTA. Blood from healthy volunteers was collected into sterile ’Vacutainers’ containing lithium heparin or EDTA or into a sterile plastic syringe containing pyrogen-free sodium heparin for injection. Samples were distributed into sterile disposable plastic test tubes and incubated at room temperature (RT) or 37°C ; plasma was separated by centrifugation at time 0, every 20 min up to 1 h, and then at 2, 4, 6, and 24 h. Cytokine levels were measured by radioimmunoassay standardised against international reference standard 86/680 (IL-1) and interim standard 88/514 (IL-6). Significant cytokine production was detectable by 2 h of incubation at 37°C; levels at 24 h were: lL-6 (ng/ml) RT T 37°C Lithium heparin EDTA Sodium heparin
were
taken
at
all stages
to
avoid LPS
contamination, including baking of glassware and use of LPS-free media.
Monocytes treated in this way consistently produced a basal level of NO averaging 6-0 pmollllg protein. This increased more than ninefold to 55-5 pmollllg protein by the addition of LPS. Although a basal production of NO has been demonstrated in work on endothelial cells4 we had previously assumed that the production of such a potentially harmful substance by native monocytes reflected traces of LPS in the extraction and culture equipment and media. The work of Freeman et al suggests an alternative explanation-namely, that heparin can stimulate monocytes to produce NO as well as TNF. We have shown that basal NO production, like TNF production, is greatly increased in monocyte samples from patients with alcoholic hepatitis (unpublished). If heparin can induce monocytes to elaborate TNF and NO in vitro this would have important implications for the interpretation of research data and for diagnostic testing. However, the possibility remains that LPS-induced basal TNF production
30-0 04 0-4
IL-1 1
3.0 04 4 04
(nglml) (37’C) 23.3
ND 0-93
ND = not done
their
of nitrite ions.3 Precautions
N.C.A.HUNT
1. McClain CJ, Cohen DA. Increased tumour necrosis factor production by monocytes in alcoholic liver disease. Hepatology 1989; 9: 349-51. 2. Griffith T, Randall M. Nitric oxide comes of age. Lancet 1989 ii: 875-76 3. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR Analysis of nitrate, nitrite and (15N) nitrate in biological fluids Ann Biochem 1982, 126: 131-38. 4. Rees DD, Palmer RMJ, Moncada S. Role of endothelium-derived nitric-oxid inthe regulation of blood pressure. Proc Natl Acad Sci ( USA) 1989; 86: 3375-78
SIR,-The fact that monocytes can produce TNF-x has been demonstrated by several groups, as has the stimulatory effect of lipopolysaccharide (LPS).’ It is unfortunate that Dr Freeman and
colleagues did not try to reduce or measure LPS contamination of samples. Despite this they did find a clear difference in TNF production between monocytes isolated with heparin or with chelating agents. Nitric oxide (NO), besides its probable role as an endotheliumderived relaxing factor,z is also a mediator of macrophage cytotoxicity. Wehave studied NO production in monocytes by collecting whole venous blood in heparinised syringes followed by monocyte extraction on a density gradient and enrichment by adherence to a tissue culture vessel. Samples were then incubated for 24 h in medium with or without LPS (1 jg/ml). NO was measured by an indirect technique that is specific for the detection
R. D. GOLDIN
A lithium
heparin tube
was
washed
out
with 10 ml of
endotoxin-free water; this extract was found to contain high levels of
(greater than 250 units) when assayed by the limulus amoebocyte lysate assay standardised against international endotoxin
reference standard 84/650. The induced IL-6 proved to have bioactivity when assayed against a sensitive cell line (B9). The action of EDTA seemed to be a direct inhibition of endotoxin-induced IL-6 production because IL-6 production did not occur if blood collected into lithium heparin tubes was transferred into EDTA within 2 h of collection; beyond this time the induced response was irreversible. These results indicate that great care must be taken at all stages of blood collection and fractionation to eliminate endotoxin when studying production or presence of those cytokines that are readily induced by endotoxin. Plasma collected into endotoxincontaminated systems is unsuitable for use in cell culture
experiments. We thank Sue Selkirk, National Institute for Control, for the limulus assays.
Department of Immunology, Charing Cross and Westminster Medical School, Westminster Hospital, London SW1 P 2AP, UK
1. Aarden 2.
Biological
Standards and
PAMELA G. RICHES ROGER GOODING
LA, De Groot ER, Schaap OL, Landsdrop PM. Production of hybridoma growth factor by human monocytes. Eur J Immunol 1987; 17: 1411-16 Schindler R, Mancilla J, Endres S, Ghorbani R, Clark SC, Dinarello CA. Correlation and interactions in the production of interleukin-6 (IL-6), IL-1, and tumour necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood 1990; 75: 40-47.