- Email: [email protected]
Fixing specimens
1397
obtained from patients with non-cardiac diseases complicated with bronchitis or pneumonia. A sample volume of 0-5 ml and 2 ml of 10 mmol/1 "tris" HCI buffer (pH 7-4) containing 1 mmol/1 edetic acid
was homogenised, diluted with 0-6% sodium dodecyl sulphate, and 2% ’Triton X-100’, and centrifuged. SP-A in the supernatant was measured by ELISA (Teijin, Tokyo).’ Sputum and aspirated secretion samples from patients with oedema showed 250 times higher SP-A concentrations than those from control patients-namely, 544 (SE 80) ng/ml (range 337-800, n=4) vs 2 (1) ug/ml (range 0-3-12, n = 10). With powerful diuretics and digitalis patients with chronic severe but stable heart failure can now be managed as outpatients or in hospitals where catheterisation facilities for estimation of cardiac function are limited. Such patients often have pulmonary oedema after a common cold, viral infection, bronchitis, or pneumonia, and it is sometimes difficult to distinguish this condition from pneumonia by chest X-ray alone. Airway secretion in pulmonary oedema is often contaminated with blood but serum SP-A concentration is 1 Ilg/ml or less.2 Further, measurement of SP-A concentration with this kit requires only 0-5 ml sample and a short time and is thus suitable for the clinical laboratory.’ Our preliminary findings suggest that in such cases measurement of SP-A concentration is useful. Further, if SP-A concentration in aspirated secretion were to be shown to correlate with cardiac function, including pulmonary artery wedge pressure (this work is in progress), it could also prove useful in the monitoring of patients with heart failure in intensive care, since airway secretion is routinely aspirated from intubated patients. First Department of Internal Medicine, Tohoku University School of Medicine, Sendai 980, Japan
TOHRU MASUDA SANAE SHIMURA TAMOTSU TAKISHIMA
results of immunohistochemistry, in-situ hybridisation and enzyme histochemistry are much better than those obtained with formaldehyde-fixed biopsies. These techniques are ideal for needle and endoscopic biopsies, where it is important to get as much diagnostic and prognostic information as possible from the limited amount of tissue available in a unique biopsy. Moreover, the blocks can be stored at room temperature for a long time and are thus available for retrospective study. Department of Pathology, University of Aberdeen,
GRAEME I. MURRAY STANLEY W. B. EWEN
Aberdeen AB9 2ZD, UK 1. Pearse AGE.
Histochemistry: Livingstone, 1980.
theoretical and
applied,
vol 1.
Edinburgh: Churchill
Murray GI, Ewen SWB. A new approach to enzyme histochemical analysis of biopsy specimens. J Clin Pathol 1989; 42: 767-71. 3. Murray GI, Ewen SWB. A novel method for optimum biopsy preservation for histochemical and immunohistochemical analysis. Am J Clin Pathol 1991; 95: 2.
131-36.
SiR,—Your editorial stresses that although most specimens can be treated with a standard fixative such as 4% formaldehyde at the time of surgical removal, some specimens require special attention and should be identified in advance. An example of this, not mentioned in the editorial, is the demonstration of crystals of sodium urate, which are water-soluble and can dissolve in a standard fixative. Samples of synovial membrane or other tissue in which the presence of urate crystals is considered possible should therefore be fixed in absolute alcohol. Crystals will then remain intact and can be seen with Gomori’s methenamine-silver stain or by polarising light microscopy. Department of Rheumatology, Charing Cross Hospital, London W6 8RF, UK
J. T. SCOTT
1. Masuda T, Shimura S, Sasaki H, Takishima T Surfactant aproprotein-A concentration in sputum for diagnosis of pulmonary alveolar proteinosis. Lancet
1991; 337: 580-82. S, Honda Y, Suzuki A, Kuroki Y, Akino T. Appearance of surfactant protein A m serum of patients with interstitial lung diseases Am Rev Respir Dis 1990; 141: A63.
2. Tsutahara
Fixing specimens SiR,—Your Oct 19 editorial (p 984) highlights the importance of specimen preservation for histopathological examination. The aim is to ensure structural preservation of tissue without loss of functional characteristics such as immunoreactivity or enzyme activity. The complete histopathological assessment of many biopsy specimens now requires specialised investigations with molecular and cell biological techniques such as immunohistochemistry or in-situ hybridisation, and for that purpose the preservation of immunoreactivity, enzyme activity, and DNA and RNA is essential. Fixation will always be a compromise between structural preservation and retention of tissue components in situ without destroying their functional groups.The search for the ideal fixative continues, but formaldehyde remains the most widely used agent. Unfortunately, fixation with formaldehyde is slow because its penetration varies with the type of tissue and on the rate at which formaldehyde reacts with tissue components. During formaldehyde fixation damaging effects on tissue include denaturation of antigens, extraction of mucosubstances, degradation of DNA and RNA, and loss of enzyme activity, whereas lipids remain unfixed. These adverse effects conflict with the aim of maintaining functional tissue and do not protect specimens from further damage during wax
embedding. Modern methods of tissue preservation do
not use
tissue
Plasma
tryptophan in postoperative delirium
SIR,-Dr van der Mast and colleagues (Oct 5, p 851) suggest that the mental symptoms of post-cardiotomy delirium are caused by a decrease in circulating tryptophan (Trp) availability to the brain, due to a catabolic state. They attribute this state to a rise in circulating cortisol and postulate two mechanisms by which this glucocorticoid could influence Trp availability to the brainnamely, accelerated hepatic Trp degradation through induction of Trp pyrrolase activity, and an increase in plasma concentrations of the five aminoacids known to compete with Trp for the same cerebral uptake mechanism. Although van der Mast et al did not report these aminoacid levels, their sums can be calculated from other data given, and seem not to be strikingly different (682, 788, and 623 umol/1 in the postoperative delirium, postoperative control, and healthy control groups, respectively). Increased competition with Trp can therefore be ruled out, leaving pyrrolase induction or induction of indoleamine dioxygenase, as Dr Weiss and colleagues suggest (Oct 26, p 1078). Indirect assessment of pyrrolase induction requires measurement of the plasma concentrations of free (ultrafiltrable) and total (free plus albumin-bound) Trp and of the Trp metabolite kynurenine. Furthermore, for cortisol to be the likely cause of any such induction in postcardiotomy delirium increased levels of this hormone would have to be demonstrated in this group of patients but not in their controls. Unless delirium per se is associated with hypercortisolaemia it is difficult to see why cortisol levels should differ between the two similarly stressed groups of surgical patients-and if cortisol levels are similar, alternative mechanisms (such as induction of indoleamine dioxygenase) will have to be
fIxation.2.3 Freeze-drying and freeze-substitution rely on the rapid
sought.
of fresh biopsy specimens to ensure an almost instantaneous cessation of cellular metabolism. Degradation or denaturation of labile cellular componenets such as antigens or mRNA is prevented and diffusion and redistribution of soluble substances cannot occur. Specimens are embedded directly in plastic at low temperature so that the damaging effects of wax are avoided. Labile antigens, enzyme activity, mucosubstances, and mRNA are all retained in specimens preserved in this way and the
The concentrations of circulating aminoacids (including Trp) and the albumin binding of Trp (a third peripheral determinant of Trp availability to the brain) are subject to dietary, hormonal, and pharmacological control and these factors and the metabolic ones mentioned above will have to be taken into account in future studies.
freezing
Biomedical Research Laboratory, Whitchurch Hospital, Cardiff CF4 7XB, UK
A. A.-B. BADAWY
obtained from patients with non-cardiac diseases complicated with bronchitis or pneumonia. A sample volume of 0-5 ml and 2 ml of 10 mmol/1 "tris" HCI buffer (pH 7-4) containing 1 mmol/1 edetic acid
was homogenised, diluted with 0-6% sodium dodecyl sulphate, and 2% ’Triton X-100’, and centrifuged. SP-A in the supernatant was measured by ELISA (Teijin, Tokyo).’ Sputum and aspirated secretion samples from patients with oedema showed 250 times higher SP-A concentrations than those from control patients-namely, 544 (SE 80) ng/ml (range 337-800, n=4) vs 2 (1) ug/ml (range 0-3-12, n = 10). With powerful diuretics and digitalis patients with chronic severe but stable heart failure can now be managed as outpatients or in hospitals where catheterisation facilities for estimation of cardiac function are limited. Such patients often have pulmonary oedema after a common cold, viral infection, bronchitis, or pneumonia, and it is sometimes difficult to distinguish this condition from pneumonia by chest X-ray alone. Airway secretion in pulmonary oedema is often contaminated with blood but serum SP-A concentration is 1 Ilg/ml or less.2 Further, measurement of SP-A concentration with this kit requires only 0-5 ml sample and a short time and is thus suitable for the clinical laboratory.’ Our preliminary findings suggest that in such cases measurement of SP-A concentration is useful. Further, if SP-A concentration in aspirated secretion were to be shown to correlate with cardiac function, including pulmonary artery wedge pressure (this work is in progress), it could also prove useful in the monitoring of patients with heart failure in intensive care, since airway secretion is routinely aspirated from intubated patients. First Department of Internal Medicine, Tohoku University School of Medicine, Sendai 980, Japan
TOHRU MASUDA SANAE SHIMURA TAMOTSU TAKISHIMA
results of immunohistochemistry, in-situ hybridisation and enzyme histochemistry are much better than those obtained with formaldehyde-fixed biopsies. These techniques are ideal for needle and endoscopic biopsies, where it is important to get as much diagnostic and prognostic information as possible from the limited amount of tissue available in a unique biopsy. Moreover, the blocks can be stored at room temperature for a long time and are thus available for retrospective study. Department of Pathology, University of Aberdeen,
GRAEME I. MURRAY STANLEY W. B. EWEN
Aberdeen AB9 2ZD, UK 1. Pearse AGE.
Histochemistry: Livingstone, 1980.
theoretical and
applied,
vol 1.
Edinburgh: Churchill
Murray GI, Ewen SWB. A new approach to enzyme histochemical analysis of biopsy specimens. J Clin Pathol 1989; 42: 767-71. 3. Murray GI, Ewen SWB. A novel method for optimum biopsy preservation for histochemical and immunohistochemical analysis. Am J Clin Pathol 1991; 95: 2.
131-36.
SiR,—Your editorial stresses that although most specimens can be treated with a standard fixative such as 4% formaldehyde at the time of surgical removal, some specimens require special attention and should be identified in advance. An example of this, not mentioned in the editorial, is the demonstration of crystals of sodium urate, which are water-soluble and can dissolve in a standard fixative. Samples of synovial membrane or other tissue in which the presence of urate crystals is considered possible should therefore be fixed in absolute alcohol. Crystals will then remain intact and can be seen with Gomori’s methenamine-silver stain or by polarising light microscopy. Department of Rheumatology, Charing Cross Hospital, London W6 8RF, UK
J. T. SCOTT
1. Masuda T, Shimura S, Sasaki H, Takishima T Surfactant aproprotein-A concentration in sputum for diagnosis of pulmonary alveolar proteinosis. Lancet
1991; 337: 580-82. S, Honda Y, Suzuki A, Kuroki Y, Akino T. Appearance of surfactant protein A m serum of patients with interstitial lung diseases Am Rev Respir Dis 1990; 141: A63.
2. Tsutahara
Fixing specimens SiR,—Your Oct 19 editorial (p 984) highlights the importance of specimen preservation for histopathological examination. The aim is to ensure structural preservation of tissue without loss of functional characteristics such as immunoreactivity or enzyme activity. The complete histopathological assessment of many biopsy specimens now requires specialised investigations with molecular and cell biological techniques such as immunohistochemistry or in-situ hybridisation, and for that purpose the preservation of immunoreactivity, enzyme activity, and DNA and RNA is essential. Fixation will always be a compromise between structural preservation and retention of tissue components in situ without destroying their functional groups.The search for the ideal fixative continues, but formaldehyde remains the most widely used agent. Unfortunately, fixation with formaldehyde is slow because its penetration varies with the type of tissue and on the rate at which formaldehyde reacts with tissue components. During formaldehyde fixation damaging effects on tissue include denaturation of antigens, extraction of mucosubstances, degradation of DNA and RNA, and loss of enzyme activity, whereas lipids remain unfixed. These adverse effects conflict with the aim of maintaining functional tissue and do not protect specimens from further damage during wax
embedding. Modern methods of tissue preservation do
not use
tissue
Plasma
tryptophan in postoperative delirium
SIR,-Dr van der Mast and colleagues (Oct 5, p 851) suggest that the mental symptoms of post-cardiotomy delirium are caused by a decrease in circulating tryptophan (Trp) availability to the brain, due to a catabolic state. They attribute this state to a rise in circulating cortisol and postulate two mechanisms by which this glucocorticoid could influence Trp availability to the brainnamely, accelerated hepatic Trp degradation through induction of Trp pyrrolase activity, and an increase in plasma concentrations of the five aminoacids known to compete with Trp for the same cerebral uptake mechanism. Although van der Mast et al did not report these aminoacid levels, their sums can be calculated from other data given, and seem not to be strikingly different (682, 788, and 623 umol/1 in the postoperative delirium, postoperative control, and healthy control groups, respectively). Increased competition with Trp can therefore be ruled out, leaving pyrrolase induction or induction of indoleamine dioxygenase, as Dr Weiss and colleagues suggest (Oct 26, p 1078). Indirect assessment of pyrrolase induction requires measurement of the plasma concentrations of free (ultrafiltrable) and total (free plus albumin-bound) Trp and of the Trp metabolite kynurenine. Furthermore, for cortisol to be the likely cause of any such induction in postcardiotomy delirium increased levels of this hormone would have to be demonstrated in this group of patients but not in their controls. Unless delirium per se is associated with hypercortisolaemia it is difficult to see why cortisol levels should differ between the two similarly stressed groups of surgical patients-and if cortisol levels are similar, alternative mechanisms (such as induction of indoleamine dioxygenase) will have to be
fIxation.2.3 Freeze-drying and freeze-substitution rely on the rapid
sought.
of fresh biopsy specimens to ensure an almost instantaneous cessation of cellular metabolism. Degradation or denaturation of labile cellular componenets such as antigens or mRNA is prevented and diffusion and redistribution of soluble substances cannot occur. Specimens are embedded directly in plastic at low temperature so that the damaging effects of wax are avoided. Labile antigens, enzyme activity, mucosubstances, and mRNA are all retained in specimens preserved in this way and the
The concentrations of circulating aminoacids (including Trp) and the albumin binding of Trp (a third peripheral determinant of Trp availability to the brain) are subject to dietary, hormonal, and pharmacological control and these factors and the metabolic ones mentioned above will have to be taken into account in future studies.
freezing
Biomedical Research Laboratory, Whitchurch Hospital, Cardiff CF4 7XB, UK
A. A.-B. BADAWY