CYTOLOGICAL EXAMINATION OF THE SPUTUM

1001

pension of the cells, and a drop is placed in a haemocytometer counting chamber. A simple stain (e.g., methylene-blue) is helpful. The cells are counted by standard methods applicable

CYTOLOGICAL EXAMINATION OF THE SPUTUM E. A. OPPENHEIMER

M. RIGATTO

M.D. Columbia

M.D. Rio Grande do Sul

to

DILYS SHORTRIDGE S. W. A. KUPER A.I.M.L.T. M.B. W’srand, F.C.Path. and the Medicine From Departments of Pathology, Royal PostMedical School of London, Hammersmith Hospital, London W.12, and the Brompton Hospital, London S.W.3

graduate

The importance of non-malignant sputum cytology is stressed. A method is described whereby sputum is fixed in formol/saline-solution, and liquefied in an ultrasonic disintegrator. Results using this technique are preferred to those obtained with chemical and enzymatic methods. Specimens in formalin can be

Summary

posted. Introduction

DURING the past twenty years the usefulness of sputum cytology in the early diagnosis of bronchial carcinoma has been increasingly recognised. There is, however, less awareness of the value of sputum cytology in the study of pathological processes other than cancer. The occurrence of eosinophils and bronchial casts, for example, is of considerable clinical importance, and cytological examination is the obvious way of determining the nature of the cellular response. Cells and organisms are irregularly distributed in sputum (May 1953, Rawlins 1955), and we considered that any reliable assessment must include examination of homogenised specimens. A new method for liquefying sputum and concentrating cancer cells (Kuper 1966, Kuper and Shortridge 1967) is here applied to the examination and enumeration of non-neoplastic cells. Materials and Methods Collection of Sputum We give the patient a standard bottle with printed instructions, asking him to spit into it all the phlegm coughed up from the chest in the first hour after getting up in the morning, and to return the bottle to the laboratory on the same day (Elmes et al. 1959).

Fixation of Sputum The technique is described by Kuper and Shortridge (1967). For total cell-counts, the initial formol/saline-solution volume and the total volume of the fixed specimen are carefully measured so that the dilution can be calculated. If examination of the structure of unliquefied sputum is required, the sputum is transferred to a petri dish; three to five very small cellular flakes are selected from the specimen and spread thinly on a clean microscope slide. This wet film is immediately placed in Schaudinn’s fluid (30 ml. absolute ethanol, and 60 ml. saturated aqueous solution of mercuric chloride; to which 5 ml. glacial acetic acid is added just before use) and left to fix for at least 30 minutes. An alternative to this technique for examining sputum structure is the paraffin-section method of Sanerkin and Evans (1965). "

"

"

"

Liquefaction of Sputum The fixed specimen is transferred to a glass cuvette and liquefied by high-frequency vibration (approximately 20 kilocycles per second, and an amplitude of 7fJ. peak to peak) with a 100W ultrasonic disintegrator (Measuring and Scientific Equipment Ltd.). Complete liquefaction is achieved in 20 to 60 seconds, the time varying with the nature of the specimen (mucopurulent sputum requires the longest time). The formalin-fixed cells are not disrupted. Total Cell-count The

liquefied sputum is well mixed

to ensure

uniform

sus-

cell-counts in other fluids.

Differential Count To achieve a satisfactory cell concentration in the smear, we centrifuge the specimen and resuspend the cells in a fraction of the supernatant. Using conventional haematological method (Dacie and Lewis 1963), a smear of the well-mixed cell concentrate is made on a clean microscope slide. It is stained with Harris’s hxmatoxylin and eosin, which gives the most consistently satisfactory results (Rawlins 1955). Slides are mounted in dibutylphthalate and xylene, including ’Distrene 80’. Preliminary microscopic scrutiny is made with x 10 and x 40 dry objectives; smears are only accepted if the cells are uniformly distributed and show little or no superimposition. The actual count is done with an oil-immersion x 50 objective, moving across the width of the smear, from upper to lower edge; the slide is then moved laterally, and counting is continued in this manner until 400 cells have been counted and classified. An eyepiece graticule and a simple counting register are helpful. We have been mainly interested in the eosinophil leucocytes. For this purpose, we found that slight understaining with eosin gave the best differentiation. We counted only leucocytes, and expressed the number of eosinophils as a percentage of the total leucocytes (Rawlins 1955). Better uniformity of cell distribution can be attained, with all the cells lying virtually in a single microscopic plane, by use of a membrane-filtration technique: we use type SM filter, pore size 5.0_1.2 (Millipore Filter Corporation). The ordinary smear technique is, however, quite satisfactory in routine

practice. Examination of Sputum " Structure " Slides prepared from unliquefied sputum (using either the smear method or paraffin sections) are stained with Harris’s haematoxylin and eosin; special stains may be indicated for demonstration of special structures. Intensive search may then be made for particular cell types and aggregations, Kurschmann spirals, plugs, crystals, fibrin, and mucus. Comment

The inadequacy of simple macroscopic visual examination of sputum has been well demonstrated (May 1954, Chodosh et al. 1962, Miller 1963). Organisms and cells are distributed irregularly in sputum, so that examination of samples without homogenisation does not provide a reliable assessment of the composition of the whole specimen. To overcome this difficulty, enzymatic digestion of sputum by incubation with pancreatin (Rawlins 1953) or trypsin (Miller 1963), has been recommended. Acetylcysteine has also been used. We have found the procedures reported here to be faster, more satisfactory, and easier to incorporate into laboratory routine than any of the enzymatic or chemical techniques. With ultrasonic liquefaction of formalinfixed sputum, morphology and staining characteristics of cells are unimpaired, and cellular detail is better preserved than after enzymatic digestion. Early formalin fixation of sputum greatly diminishes the risk to personnel of infection, and therefore specimens are easier to handle. Specimens in formalin may easily be posted to the laboratory, without the cells deteriorating. Other advantages of this method are the ease of obtaining quantitative rather than qualitative information, and the availability of fixed material which may be required for further examination. We thank Paul Hutchinson for technical assistance and Dr. Charles M. Fletcher for his advice and encouragement. E. A. 0. was supported by U.S. Public Health Service postdoctoral fellowship no. 1-F2-HE-32,211-01, from the National Heart Institute, National Institute of Health. M. R. was supported by a fellowship from the

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Ministry of Education of Brazil, with partial support from the Wellcome Trust. Request for reprints should be addressed to S. W. A. K., Department of Pathology, Brompton Hospital, London S.W.3. REFERENCES

Chodosh, S., Zaccheo, C. W., Segal, M. S. (1962) Am. Rev. resp. Dis. 85, 635. Dacie, J. V., Lewis, S. M. (1963) Practical Hæmatology. London. Elmes, P. C., Dutton, A. A. C., Fletcher, C. M. (1959) Lancet, i, 1241. Kuper, S. W. A. (1966) Int. Cancer Congr. published abstract no. SO867. Shortridge, D. (1967) Lancet, ii, 999. May, J. R. (1953) ibid. i, 534. (1954) ibid. ii, 839. Miller, D. L. (1963) Am. Rev. resp. Dis. 88, 473. Rawlins, G. A. (1953) Lancet, ii, 538. (1955) J. clin. Path. 8, 114. Sanerkin, N. G., Evans, D. M. D. (1965) J. Path. Bact. 89, 535. —

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(Deodhar 1960, Goodfriend et al. 1964, Haber et al. 1965, 1967), and a radioimmunoassay, based on such antibodies, has been used to determine angiotensin levels in human plasma (Vallotton et al. 1967). The results obtained in healthy individuals are high in comparison with bioassay data (Scomik and Paladini 1961, 1964, Boucher et al. 1964, Morris and Robinson 1964, Mulrow and Ganong 1964, Massani et al. 1966). We describe here a sensitive radioimmunoassay for angiotensin 11, based on a high titre antibody raised against the free hormone, and present preliminary data on circulation levels of angiotensin II in man.

Dietrich

Materials and Methods

RADIOIMMUNOASSAY FOR DETERMINING PLASMA-LEVELS OF ANGIOTENSIN II IN MAN

FELLOW, MEDICAL

UNIT

J. LANDON M.D. Lond. SENIOR

CHEMICAL PATHOLOGY AND MEDICINE, RESEARCH ASSOCIATE, IMPERIAL CANCER RESEARCH FUND LECTURER

IN

AND

W. S. PEART Lond., F.R.C.P.

M.D.

PROFESSOR OF MEDICINE

ST.

MARY’S

Summary

HOSPITAL MEDICAL

A

SCHOOL,

radioimmunoassay

used. Antibodies

were

were raised in rabbits by weekly intramuscular intraperitoneal injections of 1 mg. amounts of angiotensinamide adsorbed on to microparticles of carbon and suspended in Freund’s complete adjuvant. In some instances much smaller amounts of peptide were injected into popliteal and inguinal lymph-nodes and into the spleen. A detailed account of these techniques will be published elsewhere. Angiotensin n was radioiodinated by a modification of the chloramine-T method of Greenwood et al. (1963). Only

or

G. W. BOYD M.D., B.Sc. Melb., M.R.A.C.P. NUFFIELD RESEARCH

Synthetic asp(NH2)lva15 angiotensin n (angiotensinamide, Hypertensin’, Ciba), synthetic asplval5 angiotensin n, and natural asplisoleu5 angiotensin i and 11, both of equine origin,

LONDON

for

W.2

determining

plasma-levels of angiotensin II, in man, developed using antibodies raised against free angiotensinamide. The test can detect 30 pg. amounts of angiotensin II, and is not influenced significantly by angiotensin I. The plasma-levels found were, in general, lower than those reported by other workers for a similar test, and ranged from less than 8 to 56 pg. per ml. in twelve healthy volunteers. Evidence to support the validity of the assay when applied to circulating angiotensin II has been derived from data obtained during its

has been

intravenous infusion into controls. Plasma concentrations of the peptide fell rapidly after discontinuation of the infusion with a calculated half-life of less than 2 minutes. constant

Introduction

THE renin-angiotensin system has been reviewed by Peart (1965). Renin, probably secreted by the juxtaglomerular apparatus of the kidney, acts on a plasma substrate with the release of the decapeptide angiotensin i. This is then cleaved by a converting enzyme in plasma to give the physiologically active octapeptide angiotensin 11. Available evidence suggests that the renin-angiotensin system is concerned with the control of aldosterone secretion and with blood-pressure homoeostasis. Elucidation of its precise role, however, requires improved methods of assay, especially for angiotensin. Bioassay procedures lack specificity, and so attempts have been made to develop a valid radioimmunoassay for accurately determining circulating levels of angiotensin II. The small molecular weight of angiotensin has rendered antibody production difficult, although success has been achieved by chemically linking the hormone to larger molecules. Antibodies produced in this way have been shown to cross-react with native angiotensin II

preparations containing less than 5% of damaged labelled peptide and of unreacted iodide were used in the assay. Angiotensin levels were determined using 20 ml. blood samples drawn into plastic syringes containing 0-6 ml. of 0-2M 2,3-dimercaprol and 1-0 ml. of 0-3M edetic acid, which were used to inhibit " angiotensinase activity (Ryan 1967). The samples were transferred immediately to ice-cooled polyethylene tubes and centrifuged; the angiotensin n extracted from the plasma on to 10 mg. of finely sieved Fuller’s earth (a complex silicate). The peptide was subsequently "

eluted from the silicate with 0-88 ammonia in methanol and taken down to dryness. In six studies in which 10 ng. amounts of angiotensinamide were added to 20 ml. of whole blood, recoveries ranged from 50 to 68% with a mean value of 56%. When added to plasma the recovery exceeded 95%. Fuller’s earth has an extremely high avidity for angiotensin II, thus 10 mg. of this material rapidly removed 100% of 200 ng. amounts of angiotensinamide added to plasma, as determined

by bioassay. In the radioimmunoassay procedure, doubling dilutions of standard angiotensinamide or of plasma extract were set up in a final volume of 200 1. with buffer (0-05M barbitone, pH 8-6, containing human serum-albumin, 2-5 mg. per ml., 0’02M calcium chloride, and 0-02% neomycin sulphate). A constant amount of labelled peptide (usually 100 pg.) was then added to each tube, followed by an appropriate dilution of antibody such that some 60% of labelled angiotensin would be bound in the absence of unlabelled hormone. Control tubes in duplicate were set up with each standard curve, containing labelled peptide and buffer or labelled peptide, antibody, and buffer. After incubation at 4°C for 24 or 48 hours the antibody-bound and antibody-unbound fractions were separated by adsorbing the unbound functions on to an excess of dextran-coated charcoal using a minor modification of the procedure described for insulin by Herbert et al. (1965). Radioactivity was counted in a well-type crystal scintillation counter.

Three healthy volunteers were studied during constant 30-minute intravenous infusions of angiotensinamide at each of three dose levels (0-5, 1-0, and 2-0 !J.g. per minute). Bloodpressure and pulse-rate were monitored manually throughout.

Blood-samples

were

taken, using

an

indwelling

venous

catheter inserted into the other arm, immediately before each stepwise increase in the infusion-rate, and 0, 1, 2, and 4 minutes after stopping the infusion.