Current problems in clinical biochemistry

Pathology (1 979), 11, pp. 3 17-34

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

ABSTRACTSOFPAPERSPRESENTED AT THE 17th ANNUAL CONFERENCE OF THE AUSTRALIAN ASSOCIATION OF CLINICAL BIOCHEMISTS HELD IN ADELAIDE, SOUTH AUSTRALIA November 1978

THEME SESSION CURRENT PROBLEMS IN CLINICAL BIOCHEMISTRY CURRENT PROBLEMS IN CLINICAL BIOCHEMISTRY

D. H. CURNOW Department of Clinical Biochemistry, The Queen Elizabeth II Medical Centre, Perth Current problems to be faced by clinical biochemists in Australia include the following: ( I ) Laboratory standards especially in relation to accuracy and the role Australian laboratories will play in the world move towards definitive and reference methods. (2) The development and co-ordination of local, national and international quality control schemes and materials, lowering the cost and increasing the benefit. (3) To develop a better understanding of the distribution of reference values and patient results and the probabilities of normality and abnormality by clinical biochemists and clinicians. (4) Losses of service to emerging departments of toxicology, immunology, endocrinology, pulmonary physiology and consequent loss of expertise in clinical biochemistry departments. (5) The national co-ordination of highly specialized techniques and tests. (6) Australian production of antisera and their distribution to lower the cost of immunoassays and to develop local expertise. (7) The need to develop highly specialized meetings of clinical biochemists in this country. (8 j The development of a co-ordinated industrial policy. CLINICAL BIOCHEMISTRY SUPPORT FOR INTENSIVE CARE

G . D. PHILLIPSDepartment of Anaesthesia and Intensive Care, Flinders Medical Centre, Adelaide Patients are admitted to general intensive care units for monitoring, investigation, and management of actual or potential life threatening failure of an organ or system, particularly respiratory, cardiovascular, or metabolic. The range of investigations required by intensive care staff is not great, and includes blood gases, serum electrolytes, urea, creatinine, osmolality, blood glucose, liver function tests, serum calcium, phosphate, and magnesium. Response time varies from non-urgent, e.g. liver function tests, to urgent, e.g. blood gases in respiratory failure, to extremely urgent, e.g. serum potassium in a patient with hypokalaemia-induced ventricular tachycardia. Using brief case presentations, an attempt is made to illustrate the urgency ofcertain situations. The relative merits of a central laboratory and an in-unit laboratory are considered, as well as the current and future role of continuous on-line monitoring of selected biochemical parameters. REGIONAL STANDARDIZATION OF LABORATORY METHODS

A. N. BARKER& J. C . POWELL Department of Pathology, School of Medicine, University of Auckland, New Zealand There are numerous advantages in the clinical laboratories within a single region agreeing to use standardized methods of analysis: comparable clinical reference ranges can be used. The understanding of factors which produce

3 18

AUSTRALIAN ASSOCIATION OF CLINICAL BIOCHEMISTS

Pathology (1979), 11, April

analytical variability enables individual laboratories to improve precision and accuracy; the combined experience of a number of laboratories using the same method leads to more rapid solving of method problems; communication and co-operation between laboratories is improved with this form of continuing education in laboratory techniques. Regional standardization involves a group of laboratories initially agreeing to a free exchange of data and information, and then to the selection of appropriate methods, reagents, and calibration material. The progress of the 7 major Auckland laboratories in standardizing methods is described. The alkaline phosphatase method is used to illustrate the approach to the problems which arose, and the success in achieving the advantages outlined above.

THEME SESSION THE ROLE OF THE CLINICAL BIOCHEMIST IN HEALTH CARE

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

THE ROLE OF THE CLINICAL BIOCHEMIST IN HEALTH CARE

P. H. WISE Departmrnt of’ Medicine. Flinders Medical Centre, Adelaide Functions of a clinical biochemist are clearly area-dependent. In non-teaching hospitals, and in both state and private laboratories dealing with a wide range of sample origins (particularly from general practitioners). the clinical biochemist provides a uniquely valuable interface, providing guidelines on the performance and interpretation of laboratory investigations. In the teaching hospital setting however, the increasing laboratory orientation and subspecialization within clinical units allows clinical and scientific staff of such units to provide their own major areas of competence in clinical biochemistry: this renders the general clinical biochemist redundant in such specified areas, and significantly reduces job satisfaction. However, in this setting the appointment of a clinical biochemist with a formal, joint attachment to a clinical service (e.g., lipid, endocrinology, renal unit) can provide a significant input in clinical care, maximizejob satisfaction and provide significant opportunities for research and development at both clinical and laboratory level, whether he is scientifically or medically qualified. Cost-benefit analysis in laboratory investigation is a topical and infinitely valuable study: the monitoring and educational role of the clinical biochemist in this exercise is obvious, wherever the primary field of interest lies.

THE ROLE OF THE CLINICAL BIOCHEMIST IN HEALTH CARE

P. R. PANNALLDepartment of’ Clinical Chemistry, The Queen Elizabeth Hospital, Adelaide Clinical biochemistry evolved as a discipline dealing with the rational performance of biochemical tests of use in diagnosis. Preoccupation with performance rather than attention to clinical relevance has diminished the clinical biochemist’s role as advisor in the selection and interpretation of such tests. He must re-enter the clinical field, bringing with him a body of knowledge supplementary to that of the clinicians. His contribution should be partly in understanding the laboratory limitations of tests and the influence of non-specific factors, artefacts or drugs on measured values. He should also be able to interpret abnormal results in terms of disturbed biochemical and physiological processes rather than as indicators of specific diseases. By constantly evaluating current tests and assessing new ones in terms of their clinical value, the clinical biochemist can offer a comprehensive, but relevant. service.

DETECTING ABNORMAL LABORATORY RESULTS

D. W. THOMAS, R. W. PAIN,T. C. DURBRIDCE & M. GRAY Division ?I C’linIcal Chemistry, Instiiute of’ Medical and Veterinary Science. Adelaide In a laboratory handling many hundreds of patient samples each day, the identification of abnormal results poses a major problem. Usingcomputers, various abnormal listings are produced in our laboratory at regular intervals. For most tests the range of values observed in a hospital population has been divided into 7 zones: markedly, moderately and mildly low values, corresponding high values and values that approximate the reference range. A ‘Notify Medico Listing’ occurs instantly a markedly low or high value is detected of a life-threatening nature (i.e., with sodium, potassium, bicarbonate, calcium and glucose measurements). At 2-hourly intervals during the day an ‘Extreme Values List’ is produced of all markedly low and high values detected. At the end of each working day an ‘Abnormal List’ is produced of allmoderately and markedly low and high values. These listings permit transmission of vital information to clinical staff with the minimum of delay.

ABSTRACTS OF ANNUAL MEETING 1978

319

LEAD IN BLOOD. ITS MEASUREMENT AND USEFULNESS AS VIEWED BY THE LABORATORY AND THE CLINICIANS WHO SCREEN INDUSTRIAL WORKERS

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

T. F. HARTLEY*,P. COYLE*,J. S. ANDERSON,H. M. JOHN SON^, D. I. G U T H R I&~ I. D. STEVEN *Institute of Medical and Veterinary Science, Adelaide, ?Medical Officers at local South Australian organizations The measurement of blood lead concentration is an example of another laboratory investigation associated with occupational health care which is now becoming the responsibility of clinical chemists. This paper will report the experiences of laboratory and clinical staff involved in monitoring over 700 workers who use lead in four South Australian industries. The lead hazards encountered and the precautions taken to minimize personal exposure will be described. In every situation the clinicians use the workers’ blood lead concentrations to assess their exposure and determine whether or not their work task should be changed. This places considerable emphasis on the laboratory’s blood lead measurements, the accuracy and precision of which has been seriously undermined overseas by the results of interlaboratory surveys. Some mention therefore will be made of our method and its performance as well as the overall performance of Australian laboratories participating in the CAMBS scheme. QUALITY CONTROL PROGRAMMES: PRESENT AND FUTURE MONITORING LABORATORY PERFORMANCE BY MEANS OF EXTERNAL Q.C. PROGRAMMES

M . A. CRESSWELL Research Department, Wellcome Reagents, Wellcome Research Laboratories, Beckenham, Kent, England Interlaboratory Q.C. Programmes take many different forms with sample frequency varying from daily to quarterly depending on the primary purpose of the programme. Daily programmes are essentially a replacement for the internal Q.C. Programme but rarely have a large enough data base to provide useful comparisons of alternative methods. On the other hand programmes with less frequent samples are not immediately useful but provide a general picture of both individual and overall laboratory performance and an excellent opportunity to assess methods and instruments. The Wellcome Programme is of this latter type. The precautions necessary during the preparation of specimens for such programmes are described together with problems associated with the collation of results, particularly for enzymes. Data derived from the Wellcome Group Q.C. Programme will be presented to demonstrate that a general improvement in performance relative to the clinical requirements has occured during the past 7 years.

W(H)ITHER AUSTRALIAN QUALITY CONTROL?

R. C. BOWYER Department of Biochemistry. Royal Prrtli Hospiral Since 1974. due to the initiative of the R.C.P.A., Australian clinical biochemists have been exposed to several different external quality control programmes. All of them have had both good features and bad. Based on the lessons learned here and overseas, the following is proposed: that the A.A.C.B. takes a strong initiative in organizing an Australasian external quality control programme; that support staff be employed to run the programme under the direction of a representative advisory committee; that the matrix of the base material is demonstrably applicable to the methods and analyses to be tested; that performance indices are related to both accuracy and precision; that accuracy is related to the best available estimate of the true value. The time appears ripe to organize ourselves nationally: the alternative is to wither on the tenuous vine of large foreign schemes in which we represent a small regional minority. THE R.C.P.A./A.A.C.B. QUALITY ASSURANCE PROGRAMME

T. D. GEAKY* & D. W. THOMAS? *A.A.C.B. Rrpresenfutiw. Chiwrii~u/Pathology Committee, R . C .P . A . , t Chairman, Chemical Patholog,v Committee, R . C .P.A. Participation in the Comprehensive Clinical Chemistry Series of CAP Laboratory Surveys presented a number of difficulties. The current Burroughs Wellcome Programme is used and although the change has overcome some of the difficulties, the use of animal sera has presented new problems. The advanrages of a national programme are: large enough data base for reliable data analyses; number of participating laboratories large enough so that it can be run by a commercial organization; a national organization exists which can arrange and co-ordinate self-assessment

320

AUSTRALIAN ASSOCIATION OF CLINICAL BIOCHEMISTS

Pathology (1979), 11, April

programmes, mini-conferences and workshops to improve problem areas revealed by the programme; a committee from the professional organizations exists which can provide information and advice to accrediting bodies. The disadvantages include: in most cases there is no direct contact between the participants and the programme organizers; difficulty in revealing performance trends in the mass of data returned; the format used to present reports is restricted by requirements of other participating groups.

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

THE ROLE OF REGIONAL QUALITY CONTROL GROUPS IN IMPROVING LABORATORY PERFORMANCE

L. A. PENBERTHY Department of Clinical Biochemistry, Flinders Medical Centre, Adelaide Quality control procedures have been practised in clinical chemistry laboratories for 30 years. However, the objectives of these programmes have not always been clearly defined. Is their role only to monitor the performance achieved or to assist in improving the precision and accuracy of analyses until acceptable standards are reached? The South Australian Branch of the Australian Association of Clinical Biochemists has developed a regional programme which defines acceptable standards and directs participants’ attention to imprecise and inaccurate results. Unacceptable results, using defined limits, are determined immediately after assaying the unknown sera. Computer generated reports compare each participant’s results with the performance of all laboratories and the best laboratory. Unacceptable precision and accuracy are flagged and the percentage unacceptable results using defined limits is determined for each chemistry. The quality control sub-committee discusses and acts on these reports. A.A.C.B. SCIENTIFIC PAPER PRIZE COMPETITION EVALUATION OF FRACTIONATED URINARY STEROID MEASUREMENTS IN ENDOCRINE INVESTIGATION

G. PHILLIPOUEndocrine Laboratory, The Queen Elizabeth Hospital, Adelaide Recently we have published detailed methodology for the comprehensive analysis by capillary gas chromatography of all the major urinary androgen, progestin and corticoid metabolites (G. Phillipou etal., Aust. N.Z. J . Med., 1978, 8,63). Between assay variability (mean, pmo1/24h: C.V.) for several steroids as determined by this procedure in a control urine (N = 8; 10 ml, TV = 2260 ml) was: androsterone, 11.0, 12.5%; pregnanetriol. 2.4 12.5%; tetrahydrocortisone, 10.4, 10%. Patient samples (female 463, male 31) referred for analysis may be classified into 2 groups. Group A (9%) comprises disorders such as inborn errors of corticoid biosynthesis and adrenal hyperfunction which are uniquely characterized by the selective and marked elevation of certain urinary steroids. Group B (91 %) encompasses more common endocrine disorders represented by amenorrhoea, hirsutism, obesity and alopecia which cannot be defined by any one urinary steroid. Consequently, evaluation of these latter patients is complicated and contingent upon reference to a normal range.

IS THE L/S RATIO REALLY A LECITHIN/SPHINGOMYELIN RATIO?

C. G. DUCK-CHONG* & L. M. BROWN^ *Department 01 Histology and Embryology, University of Sydney and tBiochemistry Department, Royal Prince Alfred Hospital, Sydney Foetal lung maturity iscommonly assessed by determining the ratio of lecithin/sphingomyelin (L/S) in centrifuged amniotic fluid. In the routine determination of the L/S ratio by TLC, other bands were frequently noted between and overlapping the lecithin and sphingomyelin spots. Two-dimensional chromatography revealed that 2 additional phospholipids were present in the lecithin-sphingomyelin region. Treatment of dried extracts with cold acetone prior to TLC, as suggested in the original L/S ratio procedure, did not remove the interfering phospholipids. These phospholipids have been tentatively identified as phosphatidylserine and phosphatidylinositol, both normal components of amniotic fluid. A solvent system was developed to allow one-dimensional separation of these phospholipids from lecithin and sphingomyelin. Comparison of data obtained for individual cases using the new solvent and the conventional solvent indicates that the extra phospholipids contribute significantly to the routine ‘L/S’ ratio. It remains to be determined whether the true L/S ratio is a more reliable index of foetal lung maturity than theempirical ‘L/S’ ratio now in use.

ABSTRACTS OF ANNUAL MEETING 1978

321

APPLICATION OF GC-MS TO ESTIMATION AND CHARACTERIZATION OF METABOLIC PROFILES OF STEROlD EXTRACTS

G. E. JOANNOU, W. J. HENSLEY & J . R. TURTLE Blochemistry and Endocrinology Department, Royal Prince Alfred Hospital, Sydney

The urinary steroid metabolites of 2 male siblings with congenital adrenal hyperplasia of a suspected l l g hydroxylase deficiency have been studied in our laboratory by gas chromatography (GC) and gas chromatographymass spectrometry (GC-MS). Preliminary identification of steroids is based on the direct comparison of GC retention indices (RI) with those of reference compounds. The use of at least two oxime-trimethyl-silylderivatives not only enhances the reliability of the method when RI values are used for the sole criteria of identification but can afford some structural characterization of unknowns. Mass spectrometry is used for absolute confirmation. The profile pattern of this syndrome is distingulshed from that of normal subjects by greatly elevated secretion rates of atypical neutral steroids. The tentative identification of a pregnantetrol present in both these extracts has also been achieved.

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

THE ROLE OF ACANDROSTENEDIONE IN THE ASSESSMENT OF PATIENTS WITH DISORDERS OF SEXUAL DEVELOPMENT

J. MONTALTO, H. E. DAVIES, J. F. CONNELLY, G. WARNE & H. N. B. WETTENHALL Department of Clinical Biochemistry, Royal Children's Hospital, Melbourne The development of a radioimmunoassay for A4-androstenedione ( A 4-A.D.) in plasma has enabled the establishment of reference ranges In prepubertal boys and girls. Application ofthis assay in a variety of patients has shown the following: (1) An elevated plasma A 4-A.D. in untreated patients with congenital virilizing adrenal hyperplasia (C.V.A.H.) due to 21 hydroxylase deficiency. The A 4-A.D. levels were lower than the corresponding levels of 17 ahydroxyprogesterone (17 OHP). (2) In 2 cases of 1 la-hydroxylase deficiency A4-A.D. showed higher levels than 17 OHP, but lower than those of 11 deoxycortisol. (3) Elevated values were also seen in 2 infants with suspected testicular feminization and in 4 children with early pubic hair development. (4) In patients with C.V.A.H. on treatment, preliminary studies suggest that A4-A.D. in conjunction with 17 OHP may clearly define whether patients are receiving optimal steroid therapy. FRACTIONATION OF SERUM IgA IN PATIENTS WITH IgA NEPHROPATHY, ALCOHOLIC CIRRHOSIS AND SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)

A. M. WOOTTON*, P. E. M C K E N Z IA. ~ , A. G O R M L A. ~ , R. CLARKSON,M. E. COLES*& A. J. WOODROFT@ *Department of Clinical Chemistry, Institute of Medical and Veterinary Science, Adelaide and tRenal Unii. Royal Adelaide Hospital Deposits of IgA and C3 are found in renal biopsies from patients with IgA nephropathy, alcoholic cirrhosis and SLE. Many of these patients have raised serum IgA levels and it has been suggested that the glomerular deposits may be IgA aggregates rather than soluble immune complexes (IC). The molecular size of serum IgA was therefore determined in control subjects and in patients with these diseases using gel filtration (Sephadex (3-200 and Sepharose CL-6B) and sucrose density gradient (10-60%)ultracentrifugation. The fractions were assayed for IgA, IgG, IgM and C3 by laser nephelometry and for IC by'solid phase Clq RIA. Patients with alcoholic cirrhosis and SLE had increased amounts of > 7 s IgA and IC. There was no increase in polymeric IgA in the majority of patients with IgA nephropathy by 30% had IC. The data suggests that the serological and renal findings in these diseases result from IC rather than IgA aggregates. ASSAY OF SERUM FREE T, RADIOIMMUNOASSAY

FREE T, AND FREE rT, BY EQUILIBRATION DIALYSIS AND

R. G. SYMONS & M. L. WELLBY Department

of

Clinical Chemistry, The Queen Elizabeth Hospital,

Adelaide

The use of free (0 thyroid hormone assays to assess thyroid function is a controversial issue. We have therefore established assays for free T4, free T, and free rT, with a view to further investigating their role in the evaluation of thyroid disease. Serum was dialyzed against isotonic HEPES buffer. Aliquots ofdialysate were assayed for T4, T, or rT, by radioimmunoassay employing high specific activity tracer, high titre antisera andcharcoal as separant. Mean T,in euthyroid subjects were found to be similar, being 10.8 ? 4.4 pmol/l and 6.4 f 0.8 concentrations of ff4and t pmol/l respectively. Mean frT, concentration was 1.3 f 0.8 pmol/l. All subjects in an untreated clinically

322

AUSTRALIAN ASSOCIATION OF CLINICAL BIOCHEMISTS

Pathology (1979), 11, April

thyrotoxic group were characterized by elevated fT, and all hut one by an elevated fT4.Subjects in an untreated clinicallyhypothyroid group were found to have low to normal ff, and fT4.The results indicate that measurement of free thyroid hormone concentration is a valid approach to assessing thyroid status.

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

PROBLEMS ENCOUNTERED IN ESTABLISHING A KINETIC METHOD FOR SERUM CREATlNlNE BASED ON THE JAFFE REACTION

R. A. HAWKINS, H. E. DAVIES, A. YONG& J . F. CONNELLY Department qf'Clinica1 Biochemistry, Royul Children ',s Hospital, Melbourne Correlation studies between a kinetic approach to the estimation of serum creatinine and a conventional Lloyd's reagent system further highlighted the problems associated with the Jaffe reaction. The findings of these studies include the following: ( I ) The colour development of the creatinine-alkaline picrate complex is non-linear with respect to time. A first order or more complex system appears to exist. (2) Rate constants of the reaction for a given time interval are different for human serum, commercial quality control material and standard creatinine solutions. Differences in the reaction also occur between human and bovine albumin solutions. (3) Maximum colour development occurs at variable times (90-105). (4)Creatinine concentration determines the wavelength at which maximum absorption occurs. These findings, in conjunction with several recent publications, raise doubts as to the validity of any methody for measuring creatinine that employs the Jaffe reaction. ARE CARDIAC ISOENZYME STUDIES A BENEFIT TO THE PATIENT AND THE HOSPITAL?

K. FATCHES & M. ABDUL-KARIM Department of Clinicul Chemistry, The Prince of'Wales Hospital, Sydney

L.D.H. amd C.P.K. isoenzyme screens were performed on all patients with chest pain, seen through the Accident Centre and Coronary Care Unit ofThe Prince ofWales Hospital over a set period. This involved 3 blood collections: the first on admission, the second 6-13 h post admission and a third sample after 24-37 h. All electrophoresis was performed using a Corning cassette electrophoresis cell, and the isoenzymes were detected using Corning L.D.H. and C.P.K. substrate kits and scanned on the Corning 720 fluorimeterjdensitometer. The results suggest that besides confirming M.I., this profile is useful for differentiating between borderline M.I. and non M.1. patients. The L.D.H. and C.P.K. isoenzyme studies also provided further information on the clinical condition of the patient. Furthermore, it is suggested that the introduction of isoenzyme screens as a routine procedure in this hospital for all patients with chest pain could result in a saving of hospital bed time and eliminate prolonged enzyme studies currently in use. ECONOMICS AND THE CHOICE OF ANTICONVULSANT ASSAY METHODS

D. SAMPSON, J . B. WHITFIELD & W . J. HENSLEY Biochrmimy Departmenr, Royal Prince Alfred Hospital, Sydney Plasma levels of anticonvulsant drugs correlate well with the clinical status of epileptic patients and are assayed in many biochemistry laboratories. Five major techniques can be used for assay: manual gas chromatography, automated gas chromatography, liquid chromatography, radioimmunoassay and enzyme immunoassay. Technical considerations favour gas chromatography; precision is equal or superior to that of immunoassay methods and chromatographic methods allow more flexibility so that less common anticonvulsants such as clonazepam, sulthiame and methoin can be assayed. We have costed these 5 major techniques using experience gained in our laboratory and material published in current literature. In addition, we have used price lists and assay protocols distributed by the manufacturers of immunoassay kits. The major factors involved in costing are reagents, staff salaries and instrument depreciation. Automated gas chromatography was found to be the least costly method even for small numbers of anticonvulsants. PROBLEMS IN METHODOLOGY PROBLEMS WITH ISO-ENZYME DETERMINATIONS

A. ROFE,A. HUXTABLE, M. PHILCOX, R. B A ~& S J. B. EDWARDSDivision of Clinical Chemisrry, Institute of Medical and Veterinary Science. Adelaide Some of the interpretative errors in iso-enzyme determinations may be attributed to choice of support media, assay conditions, source of enzyme and the presence of contaminants. For instance: the separation of CK iso-enzymeson

ABSTRACTS OF ANNUAL MEETING 1978

323

agarose gel is much more revealing as t o the presence ofadenylate kinase than cellulose acetate; the electrophoresis of LD from human liver demonstrates an extra band; however this sixth band is due to alcohol dehydrogenase acting on 2-amino-propanol. the buffer in the assay system; rat liver lactate dehydrogenase is much more stable to heat than human liver; the correlation between the Beckman and Phadebas methods for measuring amylase activity is quite different for human and canine sera. This may be due to different iso-enzymes or the presence of activators or inhibitors in the sera. These factors are important in defining the conditions and the source of control materials for iso-enzyme determinations.

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

STANDARDIZATION OF COLORIMETRIC ANALYSIS-DOGMA

DISPUTED

M. J . PEAKF*, C . G . FRASER*& B. M . DUN CAN^ *Flinders Mrdicul Centre.Atieluidetrridtlnstitutr~f Medical arid Veterinary S(,ience,Adehide A favoured concept is that all analyses should be standardized with the purest materials available. This ideal is unattainable as a significant number of problems remain unsolved and, to eliminate certain of these problems, secondary standards are often used. An alternative approach is to relate concentration directly to absorbance by a calibration relationship dependent on the molar extinction coefficient of a reaction product or reactant. The present study examined the dogma that a standard must be analysed in each batch of tests by comparing the analytical performance achieved in 4 manual colorimetric tests- urea. glucose. phosphate and albumin-using daily primary or serum standards (the variable calibration mode) with that obtained using constant calibration relationships (the con.mni calibrarion mode). It was shown that the mode of standardization selected for each and every method must be chosen as an objective consequence of experimenial study. AN ANALYSIS OF THE BEHAVIOUR OF THE SOLID PHASE RADIOIMMUNOMETRIC ASSAY OF SERUM FERRlTlN

R. G. RYALL,D. R . TURNER & C. J. STORY Department of Huemarology. Flinders Medical Centre. Adelaide Two artefacts of solid phase radioimmunometric analysis of serum ferritin confuse the interpretation of results: ( I ) Samples with high ferritin content may give apparently normal results. (2) Progressive dilution of samples gives increasing final results. The behaviour of this analytical system was investigated using a theoretical model based on chemical equilibria. It was shown that artefactually low results are obtained if low affinity binding of ferritin to the solid phase antibody occurs in the first assay step. This loosely bound ferritin dissociates in the second assay step and complexes with radio-labelled antibody, preventing the latter binding to immobilized ferritin. Furthermore. competition between isoferritins for antibody binding sites results in different populations of ferritin molecules being bound to the solid phase antibody, depending on the dilution of the sample. This results in a non-linear dilution curve. If the serum ferritin is grossly heterogeneous, this methodology cannot give a valid result. KINETIC STUDIES IN THE TRICHLORACETIC ACID PRECIPITATION OF PROTEIN

A. MUIR& W. J. HENSLEYDepartment qf'Biochemistry. Royul Prince Atfred Hospital, Svdrzey Examination of the kinetics of protein precipitation by trichloracetic acid (T.C.A.) has led to the development of an automated urinary protein method using a centrifugal analyser. Attention to conditions 0fT.C.A. concentration, temperature and the time of reading of absorbance has eliminated the problem of the discrepancy between turbidity shown by albumin and globulins for which T.C.A. methods have always been criticized. The method requires 10 microlitres of urine sample and results have been shown to be linear to 2 g per litre. The coefficient of variation is 3.5% at a level of0.5 g per litre and 1.3% at 1.0 gper litre; recoveries are from 98.9 to 108",;. Estimation of 29 samples may be carried out in approximately 5 min. Pseudoproteinuria due to drugs in the urine, penicillins in particular, may be differentiated from true proteinuria by examination of their respective precipitation kinetics. EFFECT OF MEMBRANE POTENTIAL ON DIALYSIS OF CALCIUM IN A CONTINUOUS FLOW SYSTEM

M . J . STALEY& I. FARRANCEDepartnienr of C'linrcul Biochemistry. Flinders Medical Centre, Adelaide and Division of Biochemistry, The Geelong Hospitul. Geelong It has been found that the estimation of calcium by continuous flow analysis using a 12 inch dialyser leads to discrepant results if acidified aqueous calcium standards are used as the means of primary calibration. We have

324

AUSTRALIAN ASSOCIATION OF CLINICAL BIOCHEMISTS

Pathology (1979), 11, April

examined the effects of hydrogen ion concentration on the dialysis of 45Caand compared the rates of dialysis with dialyser membrane potentials. The amount ofcalcium dialysed increased from 5% to 12% when theconcentration of acid was increased on the recipient stream of the dialyser. This corresponded to a change in potential of 0 mV to + 24 mV of the donor stream with respect 10 the recipient stream. Further experiments including protein on either side of the dialyser show that the Donnan effect does not explain the differences in rate of dialysis between protein and aqueous samples and that the migration of H + does explain most of these differences. LIPID METHODOLOGY

THE MEASUREMENT OF APO-6 IN VLDL AND LDL

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

G. D. CALVERT & R. A. YEATES Department of Clinical Biochemistry, Flinders Medical Centre, Adelaide

Apolipoprotein-B (Apo-B) concentration may be measured by radioimmunoassay (RIA) or electroimmunoassay (EIA: ‘rockets’).If low density lipoprotein (LDL) is used as a standard, very low density lipoprotein (VLDL) Apo-B is of the order of 45-50X higher using EIA than RIA. Evidence suggests that the determination of Apo-B in VLDL by RIA is misleadingly low, perhaps because some immunoreactive sites are masked. The estimation of Apo-B by chemical means (it is insoluble in tetramethylurea) gives a value similar to that obtained by EIA. The height of rockets formed in the EIA system by LDLcan be increased by altering LDLcharge (maleylation). The approximate equivalence of the EIA and chemical assays for Apo-B may be because VLDL Apo-B is incompletely ‘recognized’ by the antibody, but at the same time it gives higher ‘rockets’ because VLDL particle charge is increased, compared with LDL. It may be better therefore to estimate Apo-B concentration in plasma containing VLDL by EIA rather than RIA. THE MEASUREMENT OF PLASMA-HDL-CHOLESTEROL BY PRECIPITATION TECHNIQUES

R. A. YEATES, T. MANNIK & G . D. CALVERTDepurtment ojClinica1 Biuchemisrry, Flinders Medical Centre , Adelaide Plasma high-density-lipoprotein cholesterol (HDLchol) is measured by precipitating low-density and very-lowdensity-lipoproteins (LDL and VLDL) with heparin-manganese, and measuring the supernatant cholesterol concentration. We have examined 8 heparin preparations in this assay, measuring supernatant apolipoprotein-B (Apo-B) by electroimmunoassay. None of the heparins precipitated plasma Apo-B completely. About 3-5% of total Apo-B remained in the supernatant. By inference, unprecipitated LDL-cholesterol may form 15-25% of supernatant cholesterol. Precipitation of LDL and VLDL by phosphotungstate-magnesium gives lower HDL-chol values, and probably more complete LDL and VLDL precipitation. Phosphotungstate-Mg’ precipitation would seem to be the method of choice for those who use a non-enzymatic method to measure cholesterol concentration. Phosphotungstate depressed an enzymatic estimation of HDL-chol (Calbiochem; Centrifichem) by a mean of 16%.Other precipitation techniques should be used with enzymatic analysis. Standardization of methodology is important if data from different laboratories are to be compared. +

A CENTRIFUGAL METHOD FOR THE MEASUREMENT OF HIGH DENSITY LIPOPROTEIN CHOLESTEROL J. K. ALLEN*,H. G. GALLAGHER?, W. J. HENSLEY*, A. V. NICHOLLS*, G. STELLIN@& J. B. WHITFIELD**Royal Prince A[fred Hospital, Sydney and t Medicheck Referral Clinic, Sydney

Interest in high density lipoprotein cholesterol (HDL-C), thought to be protective against coronary artery disease, has led to the need for a method capable of handling large workloads conveniently and economically. Estimation of HDL-C relies on the removal of LDL and VLDL from plasma. Commonly heparin-Mn++ is used to precipitate LDL and VLDL. However, Mn” interferes with enzymatic methods of measuring cholesterol. Our method circumvents this problem by precipitating LDL and VLDL with 10% polyethylene glycol 6000, followed by centrifugation and measurement of the supernatant HDL-C using a Centrifichem and an enzymatic method (Boehringer Mannheim CHOD-PAP kit). One person can analyse around 80 specimens in half a day with a reagent cost per test of approximately 12c. The coefficient of variation between batches is 3.6%.Accuracy has been assessed by immunoelectrophoresis of the supernatant. Ambulant subjects (990) attending Medicheck had HDL-C as follows: males 1.20 k 0.31 and females 1.51 f 0.38 mmol/litre. These results are similar to those reported by other methods for similar populations.

ABSTRACTS OF ANNUAL MEETING 1978

325

METABOLISM PORPHYRIA: A COMMON METABOLIC DISORDER

P. M. CARTER, K. SANDY, D. BLAKE& I. S. RATNAIKEThe Royal Melbourne Hospital, Melbourne Porphyrias represent a series of disorders due to specific enzyme abnormalities in haem synthesis. The abnormal porphyrin excretion patterns consequent upon these enzyme deficiencies can aid clinical diagnosis and patient management. The incidence and types of abnormal porphyrin excretion detected in our routine laboratory over an 18 mth period are presented. Patients (220) with suspected porphyria and relatives (30) were investigated. Abnormal porphyrin excretion was found in 52 patients and 7 relatives. Classification of these subjects according to clinical and biochemical data was as follows:

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

Diagnosis Porphyria cutanea tarda Porphyria variegata Hereditary coproporphyria Erythropoietic protoporphyria Uncertain Total

1

Number of patients

Number of relatives

29 5 1 2 15

6

52

7

1

The commonest presenting feature was photosensitivity. No cases were associated with acute abdominal pain and only one patient presented with acute neurological disturbance. DICARBOXYLIC ACIDURIA: A POSSIBLE DEFECT IN FATTY ACID METABOLISM

C . J. PULLIN, R. J . W. TRUSCOTT,B. HALPERNUniversity of Wollongong, N . S .W . B. WILCKENOliver Latham Laboratory, Sydney M. SILINK,H. GRIFFITHS, H. KILHAM Royal Alexandra Hospital. Sydney and F. GRUNSEITPrince of Wales Children’s Hospital. Sydney Investigations will be described on 3 unrelated males aged 6-14 mth who presented in a semi-comatose state with a history of 2 4 days vomiting and diarrhoea. The children were characterized by an easily correctable hypoglycaemia, mild metabolic acidosis and gross hepatomegaly with normal serum transaminases and amino acids. GC/MS of urinary acid extracts revealed large amounts of adipic, suberic and sebacic acids together with suberyl glycine. Only small amounts of lactic and fl-hydroxybutyric acid were present. The above diagnostic GC pattern was not present when the children were well but could be provoked by fasting. A genetic defect is suggested by the infantile death of 2 siblings of one patient following a similar pattern of illness. We suggest that these patients may have a defect in short chain acyl CoA decarboxylase which when fat is the major energy source, leads to increased o-oxidation of fatty acids and a consequent excretion of dicarboxylic acids. SCREENING FOR NEONATAL HYPOTHYROIDISM IN SOUTH AUSTRALIA

A. C . WILKINS,E. F. ROBERTSON & A. C. POLLARDDepartment of Chemical Pathology. Adelaide Children‘s Hospital, Adelaide Neonatal hypothyroidism is difficult to detect by clinical examination of the newborn baby. Undetected, it usually results in mental and physical retardation. The earlier the diagnosis is made, and treatment instituted, the better the outlook for normal development. Screening for low thyroxine levels was carried out using a modified Larsen radioimmunoassay method on a 3 mm disc of chromatography paper on which blood samples had been collected by heel stab at 5 days of age. This programme was commenced in February 1977. By April 1977, a T.S.H. method (Phadebas kit) was introduced using an 8 mm blood spot. The samples selected for T.S.H. measurement were those in the lowest 10th percentile of each thyroxine screen batch. The incorporation of the T.S.H. screen greatly reduced the number of patients recalled from 1% to 1 in 2500. By August 1978, approximately 30,000 newborns had been screened. In this sample, 5 cases of primary neonatal hypothyroidism and 3 cases of thyroxine binding globulin deficiency had been detected.

326

AUSTRALIAN ASSOCIATION OF CLINICAL BIOCHEMISTS

Pathology (1979). 11. April

A REVIEW OF THE FIRST 12 MONTHS OF THE S.A. PILOT SURVEY OF MATERIAL SERUM ALPHAFOETOPROTEIN SCREENING FOR FOETAL NEURAL TUBE DEFECTS

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

A. C. POLLARD,K. E. STROHM & R. K. OLDFIELD Department of'Cheniical Pathology. Adelaide Children's Hospital, Adelaide Neural tube defects represent the most common congenital nervous system malformation. In September 1977, a pilot survey of the measurement of material serum afoetoprotein (AFP) at 16-20 wk gestation, for the prenatal diagnosis of open neural tube defects was introduced. This survey was preceded by the accumulation of sufficient data for the calculation of reference percentile values for the relevant gestational ages. All assays were performed using the Behring Werke R.I.A. G-nost kit. The present test protocol will be discussed together with future modifications subsequent to the data generated from the first 12 months of the S.A. Pilot Survey. With approximately 3000 tests having been performed, 4 anencephalic foetuses and 1 spina bifida have been detected. Even though complete individual case records of all patients after delivery are not yet available. it does appear that maternal serum AFP screening is effective in reducing the number of severe birth defects. THE EFFECTS OF ORAL CHOLECYSTOGRAPHIC CONTRAST AGENTS ON THYROID FUNCTION TESTS

J. MARSHALL, R. G. SYMONS, M. L. WELLBY. C. G . BENG,R. G. REINER, M . LAWSON, G. T. DAVIES& W. G. TUCKER Departments of Clinical Chemistry, Gastroenterology and Radiolog).. The Queen Elizabeth Hospital, Adelaide Iopanoic acid (Telepaque) and sodium iopodate (Biloptin). two contrast agents commonly used for oral cholecystography, have been shown to reduce the serum concentration of triiodothyronine (T,) and increase that of reverse-triiodothyronine (rT,) by altering the peripheral metabolism of thyroxine (T4). This study reports the changes in serum TSH and thyroid hormones in over 30 patients undergoing routine oral cholecystography. Blood was sampled just before ingesting contrast and on 3 other occasions during the following 3 wk. Preliminary data show reduction of serum T, > 307; below basal levels (p = 0.01) on day 3,4 or 5, with a concomitant increase in rT, to > 2009( above basal levels = 0.01). Serum T4 showed slight but TSH marked increase. Concentrations of all hormones returned to basal levels by the third week. Consequently, thyroid function tests performed within 3 wk of ingesting these agents should be interpreted with caution.

INTERACTION BETWEEN PHENYTOIN AND SODIUM VALPROATE

T. D. O'LEARY*,L. N. SANS()*, B. J. CHRISTI@& R . G. EDWARDS* *Institute q/'Medical and Veterinary Science, Adelaide and ?School of Pharniucy, South Australian Institute of Technology. A deiaide Valproate, a short chain branched fatty acid. is increasingly used in Australia for treating epileptic disorders. The treatment of epilepsy often involves multiple drug therapy. This study has examined the interactions between sodium valproate and phenytoin. Using aniline hydroxylation as an indicator of phenytoin metabolism in rats, no inductive properties were demonstrated with valproate. Valproate caused neither activation nor inhibition of aniline hydroxylation. Valproate at concentrations of 300 pnol/l displaced phenytoin from rat serum protein causing a 50"/,increase in free phenytoin. Rat in vivo experiments demonstrated that valproate increased phenytoin clearance from 16.8 to 26.8 ml/kg/min, decreased the half life from 41.7 min to 34.3 min and increased the volume of distribution of phenytoin from 1.01 l/kg to 1.33 I/kg. The unconjugated p-hydroxylated metabolite of phenytoin (HPPH) increased twofold in the plasma compartment during phenytoin/valproate administration. This study demonstrated that valproate significantly interacts with phenytoin in the rat. Further work is therefore needed to determine the significance of these phenomena in man. METABOLISM AND METHODOLOGY COMPARISON OF SERUM FERRITIN, IRON AND TRANSFERRIN LEVELS IN A LARGE GROUP OF PATIENTS

Z. RUDZKI, M. COLES,G. CASEY,R. WALSH& R. BLUNDENDivisions of Huematology and Clinical Chemistry. Institute of Medical and Veterinary Science. Adelaide Serum levels of ferritin, iron and transferrin were measured in 1000 consecutive patients whose serum was sent for assessment of iron status. An attempt was made t o determine the relative usefulness of the different investigations.

ABSTRACTS OF ANNUAL MEETING 1978

327

Characteristic profiles were seen in normals, latent iron deficiency, established iron deficiency. increased erythryopoiesis, decreased erythropoiesis, iron overload and chronic and malignant disease. In general, ferritin was found to be the best indicator of iron stores but iron and transferrin often helped distinguish between the different groups. However. there are several cases where serum ferritin does not reflect iron stores and these will be discussed. In summary, a low or normal serum ferritin reflects body iron stores. However. a raised ferritin can be caused by disorders other than iron overload and in these cases iron and transferrin levels should be performed.

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

BIOCHEMICAL DISCRIMINATION OF HURLER AND SCHEIE SYNDROMES

J. J. HOPWOD & V. MULLER Department of Chemic.ul Puthologj,, Adelaide Childrrn iHo.rpiral. Adelaide The enzymatic delineation of the mucopolysaccharidoses has revealed that certain disorders. although phenotypically distinct, paradoxically appear to share the same enzymic defect. The clinically well defined Hurler and Scheie syndromes result from a deficiency in the same lysosomal enzyme, a-L-iduronidase. Using the 'synthetic' substrate, phenyl iduronide. it has not been possible to demonstrate residual a-L-iduronidase in cultured skin fibroblasts taken from patients with Hurler and Scheie syndromes. However, we have been able to detect residual aL-iduronidase activity in cultured skin fibroblasts taken from these patients using a 'natural' substrate (tritiated iduronosyl anhydromannitol sulphate). The apparent Km values of a-L-iduronidase in cultured skin fibroblast homogenates taken from Hurler. Scheie and normal controls were 212. 5 5 and 52 pmol/l respectively; the corresponding Vmax values were 11, 8 and 664 pmoljmin/mg protein respectively. These biochemical findings explain the marked clinical differences and provide evidence that Hurler and Scheie syndromes result from different allelic mutations at the a-L-iduronidase locus. ANTENATAL DIAGNOSIS OF CYSTINOSIS

P. V. NELSON& W. F. CAREY Department of Chenzical Pathology. Adelaide Childwn'.\ Ho,ypifal, Adelaide Cystinosis is a recessively inherited metabolic disorder characterized biochemically by a high intracellular content of free cystine; this results in cystine crystal deposition in many body organs. The primary genetic defect has not been identified. This disorder is diagnosable in cultured human skin fibroblasts and cultured amniotic fluid cells by radiochemically measuring the increased intracellular cystine levels. thus allowing antenatal detection of the disorder. Mrs P., who had had a previous child with cystinosis. presented at 16 wk gestation for antenatal diagnosis. Amniocentesis was performed. the amniotic fluid cells cultured for 6 wk, then 35S-cystinewas added to the culture medium of the test and control cells for 24 h. Mrs P.'s cultured amniotic fluid cells demonstrated elevated intracellular 35S-cystine levels compared with the controls. Subsequently, the pregnancy was terminated. The diagnosis of cystinosis was confirmed by histopathological examination of fetal tissues and by elevated "S-cystine accumulation in cultured fibroblasts derived from the umbilical cord and pericardium of the abortus. A SIMPLE, FAST ASSAY FOR HAEMOGLOBIN A1

A. CLAGUE & R. GRIFFITHRoyal Brishanr Hospital, Brishune

Methods for assay of haemoglobin A l are cumbersome and time consuming. We have modified the method of Kynoch and Lehmann (Lancet, 1977. 2, 16) so that 50 pl of blood is used, instead of 500 PI. and 12 assays can be performed in one h instead of 2 i h. Fluorideioxalate blood can be used. The method involves application of a haemolysate to a Bio-rex 70 column followed by elution of the haemoglobin A l , Haemoglobin concentrations in the haemolysate and the eluate are measured. The column may be reused for at least 3 mth. FAST MOVING HAEMOGLOBIN (FMH) AS A USEFUL NEW ROUTINE TEST OF DIABETIC CONTROL T. E. BOWEN, R. T. EDWARDS & R. G. MOSES 332 Crown Srreet. bollongong, N . S . W .

The measurement of minor components of haemoglobin ( H b ) has been hailed by diabetologists as a significant advance, furthering homeostasis of glucose in diabetic patients. We have adapted the method of Trivelli era/.for the measurement of Fast Moving Haemoglobins (FMH) in the routine laboratory. The major problem encountered was the deterioration of the specimens. Haemolysates prepared on the day ofcollection. dialysed overnight and then stored at 6 C for one day were found to have a small but significant drop in the FMH value (11 =0.05).This drop

328

AUSTRALIAN ASSOCIATION OF CLINICAL BIOCHEMISTS

Pathology (1979), 11, April

increased after one wk storage (p = ,025) but between one and two wk storage there was a significant and rapid rise in FMH values (p =0.005). Specimens stored at 6°C for one wk were found to show no significant change in value. The results indicate, however, a batch to batch variation greater than within batch differences. HbF is eluted with FMH, therefore diabetics with thalassaemia should have FMH values corrected for HbF. IMPROVING NEONATAL BILIRUBIN ANALYSES

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

A. ST.JOHN, L. R. WATK~NSON & L. A. PENBERTHY Departmeni of Clinical Biochemistry, Flinders Medical Centre, Adelaide In an effort to improve the poor interlaboratory performance of neonatal bilirubin analyses we have investigated various methodologies and forms of standardization. We found that a spectrophotometric method has significant advantages in terms of technical simplicity and gives improved precision. However the accuracy of this method depends on careful selection of the wavelengths used and on validated absorbance factors used to calculate results. We suggest that methyl orange is used as an artificial standard in conjunction with the spectrophotometric method for total bilirubin and that this standard is calibrated by a reference laboratory and subsequently distributed to laboratories in the same geographical area for use as a common standard. ENZYME IMMUNOASSAY SYMPOSIUM HOMOGENEOUS ENZYME IMMUNOASSAY IN PRINCIPLE AND PRACTICE

R. WEBSTERSyva, 3180 Porter Drive, Palo Alto, California Of the assays developed to measure drugs and hormones, homogeneous enzyme immunoassay has proved to be unique in that it is not only quick and easy, but also has high specificity and is thus effective in enhancing patient care. The technique utilizes an enzyme instead of a radioactive label and unlike radioimmunoassay, requires no separation of bound and free species. Specific antibodies can bind either the enzyme labelled conjugate or the unlabelled form. Binding of the enzyme conjugate causes either a diminution or increase in enzyme activity and this change depends on the amount of unlabelled drug or hormone from the patient sample which also competes for antibody sites. Observed enzyme activity can thus be correlated with drug or hormone level in the patient sample. An important feature in the development of such assays is the specialized chemistry needed to covalently link the drug or hormone or one of their congeners to the enzyme. Procedures are now available (EMIT@) for the assay of antiepileptic, antiasthmatic and cardioactive drugs as well as T4 and these can be performed using appropriate manual spectrophotometric equipment and certain automatic enzyme analysers. PERFORMANCE OF THE EMIT@ ANTI-EPILEPTIC DRUG ASSAYS ON THE ABBOTT ABA IOO

M. P. PRIOR Department oJ Clinical Chemistry,Institute of’Medical and Veterinary Science, Adelaide The Enzyme Immunoassay ( E M I m kit for Phenytoin, Phenobarbitone and Carbamazepine was evaluated against an H.P.L.C., G.C./M.S. and spectrophotometric method. Intrabatch and interbatch precision was acceptable for all kits. Accuracy was assessed by patient comparisons and the use of control materials with known or consensus values. The methods were linear within the stated ranges. The interference from lipaemia was negligible whereas gross haemolysis caused a negative interference. The speed, simplicity and performance of the kits makes them suitable for routine analysis of anti-epileptic drugs. SEMI-AUTOMATED ENZYME IMMUNOASSAY OF THYROXINE T,

N. D. H . BALAZS, S . TOBGUI& B. MCCLURE Department of Chemical Pathology. Prince Henry’s Hospital, Melbourne The EMI@ homogenous enzyme immunoassay T4 kit, developed for the Abbot ABA-100, was evaluated. Precision was good, especially between runs. Two separate kits (albeit from the same lot of antibody) gave statistically identical results. : 5.3% at 99 nmol/L CV (within run) 2.9% at 196 nmol/L (n = 7, CV (between run) : 6.5% at 104 nmol/L ](n = 24) 2.2% at 192 nmol/L Accuracy was acceptable when compared to RIA procedures carried out in 3 separate laboratories.

1

ABSTRACTS OF ANNUAL MEETING 1978

329

T,(EMIT) = 0.96 T.,(RIA) - 0.5 ( I = 0.929. n = 98) Agreement with quality control materials having extensive consensus values (Wellcome Survey, Victorian Q.C. Programme) was excellent, despite the use of non-human matrices. While occasional turbid sera having low levels of T4 gave negative ( < 0) results, few problems were encountered with this technique which provides a reliable alternative to RIA. Advantages are greater speed, stability and precision as well as eliminating the handling of radioisotopes. Costs compare favourahly with RIA kits. Performance data on a new T4 kit designed for centrifugal analysers currently under evaluation will also be presented.

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

EMIT@-MANUAL

OR AUTOMATED?

W. H. BETTS Department of Clinical Pharmacology. The Queen Eliiaheth Hospital. Adelaide The introduction of the homogeneous enzyme immunoassay into the clinical laboratory has been followed by a flood of automated improvements for the method. These procedures enable greater numbers of samples to be assayed in a given time with markedly reduced operator interaction. However, in some laboratories, the turn around time may be slow because the sample numbers may take several days to reach a sufficient level for an automated run. This may be convenient for the laboratory but such delay may not be in the interests of effective and efficient patient care. O n the other hand, with the manually operated Syva EMImsystem, assays of any type may be performed on arrival of the sample in the laboratory. In drug therapy, such a fast turn around results in prompt action in the case of the toxic o r overdose patient, and alternative management in the uncontrolled or noncompliant patient, and is essential for patients on lignocaine therapy. However, the manual method is not ideally suitable for large assay numbers as it is slower and very demanding o n operator concentration. When a laboratory is considering introducing an EMITQsystem, the question of sample numbers, turn around time, management problems of patients etc., need addressing before deciding between manual and automated systems. ABSTRACTS OF POSTER PRESENTATIONS AN IMPROVED METHOD FOR THE DETERMINATION OF HOMOVANILLIC ACID IN CEREBROSPINAL FLUID

J . EARL Division of Clinical Chemistry. The Prince Henrjp Hospiral, Sydney Homovanillic acid (HVA) in cerebrospinal fluid (CSF) is derived from dopaminergic neurons in the brain. Low C S F levels of HVA occur in neurological disorders associated with lowered dopamine production. Previous measurement of HVA in this laboratory required solvent extraction of 10 ml of CSF followed by fluorimetric measurement of the ferricyanide oxidation product of HVA against an unoxidized reagent blank. The major difficulty in the method was a broad fluorescence background which made measurement of HVA levels below the normal range (40-50 ng/ml) almost impossible. Modifications to the oxidation or further purification of reagents proved to be unsuccessful in removing this background fluorescence. The problem has now been partly overcome by recording fluorescence excitation spectra of the oxidized HVA and reagent blank (420 nm emission) and constructing a fluorescence difference spectrum. The sharp peak due t o HVA at about 330 nm is readily observed and allows measurement of 5 ng/ml of HVA using only 2 ml of CSF. ACCURATE MEASUREMENT OF HUMAN SERUM ALBUMIN BY BROMCRESOL PURPLE DYEBINDING

N . D. H. BALAZS& S. TOBGUI Department of Chemicd Pathology. Prince H e w j ' s Hospital, Melhournr The use of Bromcresol Green (BCG) to measure albumin is widespread despite the documented over-estimation by this technique in the clinically important low range. BCG binds to a & @globulinsin a time-related manner causing significant over-estimation even at 18 sec after mixing. Bromcresol Purple (BCP) shares the desirable features of dye-binding techniques, yields a stable chromogen, and gives excellent agreement with immuno-chemical measurements of albumin. Alb (BCP) = 0.95 Alb (RID) + 2.5 The recently published method of Pinnock et ul. (Clin. Chem., 1978,24,80) has been adapted to discrete analysers (Centrifichem 400, ABA-100). The optimized stable reagent and analytical conditions chosen ensure linearity to 60 g/L, and excellent precision (CV i25,, at 30g/L). However, heparin plasma is unsuitable (turbidity develops)

330

AUSTRALIAN ASSOCIATION OF CLINICAL BIOCHEMISTS

Pathology (1979), 11, April

and animal albumin (bovine, equine, porcine) is grossly under-estimated. With these limitations, BCP-binding appears to be the method ofchoice for the rapid, precise, accurate and economical measurement of human albumin suitable for automated equipment. ELEVATED MATERNAL xFP DUE TO FOETO-MATERNAL HAEMORRHAGE

D. L. HAY& I . HORACEKBi0chemisrr.y Department, Royal Women's Hospital. Melbourne

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

Elevated maternal serum levels of a-foetoprotein have been used as a screeningprocedure in the prenatal diagnosis of foetal neural tube defects where levels about the 98th percentile are indications for further testing. False positives due to multiple pregnancies, threatened abortions and incorrect gestations are well documented. however many false positives remain unexplained. In a survey of 150 women who gave birth to normal infants. 11/69 (16'7,) of women with anterior placentas revealed elevated maternal ctFP levels associated with increased numbers of foetal cells. This suggests that foeto-maternal hdemorrhage in an otherwise normal pregnancy can account for a percentage of raised aFP levels. The use of Kleihauer technique would eliminate elevated aFP levels due to foetomaternal haemorrhage that appears to be prevalent in women with anterior placentas. THE ELECTROLYTE CONTENT OF SOME AUSTRALIAN FOODS AND BEVERAGES

N. E. DALE. & J . F. ROGERS Department qfBiochemistry and Depurtmeni of'Diereiics,Royal Prince Alfred Hospital. Sydney Dietitians often need to know the electrolyte composition of foods and beverages in order to plan therapeutic diets. This study was undertaken because there are no recent analytical figures for many Australian foods. Solid foods were homogenized with distilled water and weighed aliquots analysed. For sodium and potassium estimations, the aliquots were pre-treated with hydrochloric acid, made up to volume and filtered. Estimations were performed on the filtrate using an IL flame photometer. For calcium, magnesium and phosphorus determinations, the aliquots were dried in an oven, ashed in a muffle furnace, and the ash dissolved in hydrochloric acid. Calcium and magnesium were measured by atomic absorption, and phosphorus by the Technicon autoanalyser. Canned drinks were analysed directly without prior treatment. Tea and coffee were treated with boiling water and the extract analysed. Examples: 100 g of canned baked beans contain 17.6 mmoles sodium, 9.3 mmoles potassium. 1.1 mmoles calcium, 1.4 mmoles magnesium and 3.2 mmoles phosphorus. 100 ml Coca Cola contains 0.2 mmoles sodium. no potassium or calcium, and has a pH of 2.6. Two g instant coffee contains 2.6 mmoles potassium, but no sodium or calcium. A CENTRIFUGAL ANALYSER TECHNIQUE FOR MEASURING ISOENZYMES OF ACID PHOSPHATASE IN COLUMN-CHROMATOGRAPHY FRACTIONS

A. V. NICHOLLS, A. MUIR& W. J . HENSLEYRoyal Prince Alfred Hospital, Sydnev A column-chromatographic technique for separation of acid phosphatase (ACP) isoenzymes has been combined with a rapid and sensitive automated method for detecting ACP using a Centrifichem 300. The substrate used is pnitrophenyl phosphate (PNPP), in acitrate buffer of pH 6.5. At this pH the ACP activity is not significantlyless than optimal, while the extinction coefficient of PNPP is high enough to detect changes in OD using a direct computer analysis of the Centrifichem OD readings. Three main peaks of isoenzyme activity are found; the second peak, containing isoenzyme 2. is the major fraction found in prostatic tissue and serum from patients with prostatic cancer. Serum from patients with normal values for total ACP was used to determine a normal range for isoenzyme 2. The value of this test is the sensitivity with which small amounts of isoenzyme 2 can be detected, increasing the possibility of detecting small elevations in prostatic ACP long before high total ACP indicates that prostatic cancer has metastasized. COMPARISON OF LIS RATIO, PALMITIC ACID CONCENTRATION AND P/S RATIO IN DIABETIC PREGNANCIES

A. G . ANDREWS & I. HORACEKBiochemistry Department, Royal Women's Hospital, Melbourne

The lecithin-sphingomyelin ratio (L/S ratio). palmitic acid concentration and palmitic to stearic acid ratio (Pis ratio) were estimated on samples of amniotic fluid obtained from 5 1 patients with diabetes. These were compared to the L/S ratio, palmitic acid concentration and PIS ratio obtained from 177 non-diabetic patients. Significant

ABSTRACTS OF ANNUAL MEETING 1978

331

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

differences were observed between the mean values of the L/S ratio and palmitic acid concentration in the gestational period 35 to 40 wk, when the t test between the two means was determined for the two populations ( P <0.5 whereas the mean P/S ratios for the two populations over the same gestational period show no significant difference (>0.5). The L/S ratio, palmitic acid concentration and P/S ratio were estimated on amniotic fluid obtained from 7 diabetic patients within 48 h of induction. In all 7 cases the PIS ratio predicted neonatal outcome correctly whereas palmitic acid was correct in 3 cases and the L/S ratio in only 2 cases. ASSESSMENT OF A SOLID PHASE RIA CORTISOL KIT L. S. Cox Deparlmenr 0/ Clinical Biochemistry, The Queen Elizuheth I1 Medical Centre. Pert11 The CORTCK-125 kit was evaluated and compared with the fluorimetric technique currently used in the laboratory. The radioimmunoassay is performed in antibody coated tubes using IiZ5-labelledcortisol as a tracer. There was good correlation between the assays (r = 0.97) on analysing patient sera but the radioimmunoassay results were higher than expected from a specific cortisol assay. The major interfering substances are prednisone, its metabolite prednisolone and progesterone (cross reactivities of3%, 537; and 1.43; respectively). The reproducibility of the assay was examined at three different concentrations of cortisol on 20-24 specimens. The intra-assay CV were 20:,.,, I I", and 139;, for concentrations of 110,370 and 920 nmol/l respectively. Inter-assay variation over 9 assays was 17",,,. 172, and l9Yc;respectively. In comparison. the within and between CV of the fluorimetric method varied between 2-7'4, and 6-87; respectively. The kit is convenient and simple to use; however, the precision achieved was disappointing.

PREPARATION OF HORMONE FREE SERUM FOR USE IN RADIOIMMUNOASSAY

M . C. STUART.S. ELLIS & L. GOWLLANDGarvan lnstirure a/' Medical Reseurcli. St Vincent'k Ho.vpital, Sydney The difficulties encountered in obtaining sufficient quantities of hormone free serum for addition to standards in radioimmunoassay have led to the development of a technique for stripping the pituitary glycoprotein hormones from normal serum. Normal human serum is eluted through columns containing wheatgerm lectin Sepharose 4 9 which specifically bindscarbohydrate containing N-acetyl-D-glucosamine residues. The eluted serum is freeze dried and rcconstituted to its original volume. The follicle stimulating hormone (FSH), luteinizing hormone (LH) and thyroid stimulating hormone (TSH) content of the serum measured before and after passage over the gel revealed that up to 200 pg/l of FSH. 190 pg/l LH and 3.5 mU/l of TSH were completely stripped from the serum. The displacement characteristics of the standards are identical in stripped serum t o those in untreated hormone free serum. This method thus provides a simple technique for the production of large quantities of hormone free serum. THE POSSIBLE RE-EMERGENCE OF LDH AS A DIAGNOSTIC TOOL

M. D. S. SHEPHARVDepartmerit of' Clinical Biochemisrry. Flinders Medical Centre. Adelaide Reactivity with a-ketobutyrate and the effect of heat inactivation were used to reassess the relative merits of H B D and LDH as diagnostic aids in liver and heart disease. Purified LD, and LD, both reacted with a-ketobutyrate over a wide range of temperature and substrate concentrations. N o one set of conditions adequately discriminated between these isoenzymes. Electrophoretic studies demonstrated that heat inactivation at 60 resulted in minimal loss ofcardiac isoenzyme (LD, activity after I h, while eliminating LD, activities within 30 min. Estimation of the percentage of the total LDH activity remaining after heat treatment revealed a clear demarcation between patients with liver and heart disease, whilst poorer discrimination was observed with HBD. The value of pre- and post-heat LDH estimations in the differential diagnosis of liver and heart disease was demonstrated in patients with minimally elevated liver and cardiac enzymes. A SIMPLE COLORIMETRIC METHOD FOR THE DETERMINATION OF URINARY IRON EXCRETION

R. TORMET & T. F. HARTLEY The Institute of' Medical and Veterinury S(.ience, Adelaide The method exploits the well-known reaction between thiocyanate and ferric ions. The latter are generated by reacting the urine with 5-molar nitric acid. MIBK (4-methyl-2-pentanone) is added before shaking the mixture with complex immediately it is formed. The 5-molar KSCN reagent. The MIBK is used to extract the red Fe (SCN)" 'L" colour is stable and has a maximum absorbance of 486 nanometers. The calibration graph is linear and is +

332

AUSTRALIAN ASSOCIATION OF CLINICAL BIOCHEMISTS

Pathology (l979), 11, April

constructed from the absorbances observed for a series of extracts of aqueous standards containing 14.8-74.0pmol Fe Cl,/l of 0.1 molar HCI. Blanks are necessary for each specimen and are prepared by substituting distilled water for the thiocyanate reagent. The method is sensitive, 0.0045 absorbance units/pmol Fe'+ / I , and accurate, mean recovery 97%.Precisions (CV%) are better than 5%. Data from patients with and without intravascular haemolysis will be presented to illustrate the usefulness of this investigation.

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

ASSESSMENT OF THE BECKMAN CREATlNlNE ANALYSER 2

C. G. FRASER & M. E. SONTROPDepartment of Clinical Biochemistry, Flinders Medical Centre, Adelaide An evaluation of the Beckman Creatinine Analyser 2 has been carried out. The assay is based upon a kinetic alkaline picrate method. Good day-to-day and within-day precision (CV = 3.4%; upper limit of reference range) was obtained. Analyses of quality control materials and materials with consensus values, and comparison of the results obtained for samples of plasma and urine from patients with the results obtained by an alkaline picrate continuousflow method, evidenced good accuracy. The method was linear up to 3.00 mmol/l. Glucose, lipaemia, haemolysis and icterus caused negligible interference in analysis of plasma creatinine. Albumin, ascorbate, glucose and acetoacetate caused negligible interference in analysis of urine creatinine. Acetoacetate in serum, however, caused significant positive interference. The desirable performance characteristics and practicability make this analyser attractive to the clinical chemist, particularly for paediatric and emergency laboratory use. AN EVALUATION OF THE CENTRIA@ SYSTEM FOR TESTS OF THYROID FUNCTION

N. KOSMADOPOULOS, T. READ,J . M. CROCKER & M. L. WELLBY Department qf'Clinical Chemistry,

The Queen Elizaheih Hospital, Adelaide The Centria is an automated radioassay system. Kits are supplied for the assay of serum T4, T, Uptake (T3U)and T, for the assessment of thyroid function. These kits were compared with the manual radioassay methods developed in our laboratory using the same patient samples. Laboratory results for T4were significantlyhigher than those by the Centria system @ <0.01, / I = 73, r = 0.87). T,U showed a good correlation (n = 96. I' = 0.81) even though calculated by different methods and the Free T4Index calculated from the same data also showed a goodcorrelation (n = 36, r = 0.87). T, was significantly higher by the Centria method but also showed a good correlation (p <0.01. n = 87,r = 0.74).Analysis of variance of low, medium and high Q.C. material showed significant differences between the two methods only at the medium level for T4 (CV Centria 18%, CV lab. 9%)and at low (Centria, 24"& lab. 350;) and medium (Centria 237". lab. 16%) levels for T,. THE EFFECT OF PRE-INCUBATION AND DIRECT PROCEDURE FOR THE ALANINE TRANSAMINASE ASSAY IN TWO KITS COMPARED

Department of Clinical Chemistr?. The Queen J . E. BUTTERY, C. R . MILNER & B. CHAMBERLAIN Elizaheih Hospital, Adelaide The kinetic measurement of serum alanine transaminase was carried out on 2 enzyme kits (Roche and Calbiochem), done as a direct method and a pre-incubation method. Variable results from commercial quality control sera and patient sera by these 4 procedures were obtained. Pre-incubation enzyme values were generally lower than the direct values (mean difference 10:4), but there were marked differences between the results from the 2 kits. This finding has important significanceespecially for laboratories participating in external Q.C. programmes. A frequent precision check of the method and a local reference range are therefore necessary. QUALITY OF COMMERCIAL OPTIMIZED REAGENTS FOR MEASUREMENT OF ALKALINE PHOSPHATASE ACTIVITY IN HUMAN SERUM

B. A. RUMBLE Deparimeni of' Clinical Biochemistry, Perth Medical Centre Three commercial kits for assaying alkaline phosphatase under optimized conditions were compared. The manufacturers stated that the optimized conditions conformed to the recommendations of the German Society for Clinical Chemistry. Measurement of pH, magnesium and p-nitrophenyl phosphate showed differences from the German recommended values, differences between kits and differences between vials within the same kit. These differences affected the results of alkaline phosphatase determinations on patients' sera and lyophilized control materials, depending on the source and type of isoenzyme present.

ABSTRACTS OF ANNUAL MEETING 1978

333

RESULTS OF AN ANTICONVULSANT ASSAY SURVEY

J . B. WHITFIELDBiochemistry Department, Royal Prince Alfred Hospital. Sydney Participants in the R.C.P.A./A.A.C.B. Chemical Pathology survey were asked if they wished to join in a drug survey and 50 respondents were sent 2 samples containing diphenylhydantoin, phenobarbitone, primidone, carbamazepine, ethosuximide, methylphenobarbitone, clonazepam and valproate. They were asked to return the results within the time they would take for a clinical sample. Twenty-seven replies were received within 4 wk and a further 12 after that time; if this is representative of the time taken to produce results on patients' samples then the performance of many laboratories must be considered unsatisfactory on these grounds alone. Histograms of the results will be displayed: the observed group coefficients of variation ranged from 14% to 36"" at the subtherapeutic level and from 13% to 23% at the elevated level. It is hoped that visitors to the poster display will take the opportunity to give their views on future directions for quality control for drug assays in Australia.

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

EVALUATION OF THE SPAC@-T, AND SPAC-T, UPTAKE RIA KITS

D. M. PENHALL, S. DEAM& G . H. WHITE Department qJ Clinical Biochemistrj,. Flinders Medical Centre, Adelaide The SPAC-T, assay utilizes antibody-coated tubes and requires 1 h incubation at 37 C. Analysis of Q.C. sera containing low, medium and high T4 levels yielded acceptable within-batch precisions (CV 5"b. 5.2'4, 4.9% respectively, n = 20). Day-to-day precisions for the same sera gave CV of 10.9%. 10.4% and 8.77, respectively (2 kits, n = 20). Recovery of different T4 levels added to stripped-serum was poor (80"$-108%, n = h), but linearity was good. Duplicate differences were high, 7.4% of pairs exceeding loo/, variation. The SPAC-T, uptake kit uses T, antibody-coated tubes and lZ5l-T3as tracer, and requires 45 min incubation at room temperature. Analyses of 2 Q.C. materials and a pooled pregnancy serum gave coefficients of variation for within-batch precision of 5.0%. 4.57/,, 4.3%, respectively (n = 20). For the same materials day-to-day precisions were4.7%, 5.47; and 6.5%, (n = 20). Both kits were technically simple, required care in their use, and provided rapid results suitable for thyroid function screening.

AN ASSESSMENT OF THE SUITABILITY OF DIFFERENT TYPES OF MICROFILTERS FOR USE WITH PROTEIN CONTAINING SOLUTIONS

R. L. WALSH& M. E. COLES Department qfClinica1 ChemiJtry. Institure of Mediculund Veterinary Science. Adelaide It was noted while clarifying diluted sera for protein estimations by laser nephelometry that certain types of microfilters absorbed immunoglobulin G (IgG). A range of 10 microfilters (0.45 p pore size) were evaluated for their ability t o filter diluted sera (1:lOO in saline) without removing IgG or albumin from the solutions. It was found that none of the filters removed albumin. However, most filters containing cellulose nitrate removed between 9-49?; of the IgG from diluted sera. The details of this study are presented. The results from this study suggest that microfilters should be selected not only on their ability to remove particles of a nominated size but also on the ability of the membrane t o be non-absorbent towards the material being filtered.

PRELIMINARY REPORT: ''C-TRIOLEIN BREATH TEST, A SCREENING TEST FOR FAT MALABSORPTION

R . N. BUTLER,N. J . GEHLING, E. BRAIOTTA & A. KERR GRANT Gasrroenrerologj Unit and Department of Clinical Chemistry, The Queen Elizabeth Hospital, Adelaide A simple reliable screening test for steatorrhoea is not yet available. We report here the use of a dynamic breath test to assess malabsorption of triglycerides. The results were compared to 3-day faecal fat estimations (3DFF). Fifteen normal volunteers and 10 patients with suspected steatorrhoea were studied. The subjects were fasted overnight and their ambulation and carbohydrate intake restricted during the test. Duplicate samples of 2 mmol of CO, were collected prior to a 5 ~c dose of "C-Triolein, and at hourly intervals up to 8 h after the dose. One abnormal and one borderline 3DFF result were obtained in the normal group. One normal and one borderline 3DFF result were obtained in the abnormal group. The 2 groups showed significant differences in the 2 4 h excretion rate (p < 0.001) and 4 h cumulative excretion 0, <0.001) of I4CO,. We conclude that this test could be a useful screening test for steatorrhoea.

334

AUSTRALIAN ASSOCIATION OF CI-INTCAL RIOCHFMTXTS

Patholoev

(197q1

I t April

LIVER FUNCTION TESTS AS AN INDICATION OF ALCOHOLIC PATHO-PHYSIOLOGY

B. C. SMITH Clinical Biochemistry Department, Flinders Medical Centre, Adeluide Liver function tests were performed on the following groups of males: (A) Normals (n = 130); (B) Two drinkdriving offences in the past 3 years, diagnosed non-alcoholic (n = 140); ( C ) As B, but diagnosed alcoholic (n = 149); (D) Known, long-term alcoholics (n = 46). Statistical comparison of the means of albumin. AP, bilirubin, ALT, GGT, AST and urate was applied to compare successive groups. Means ( f 1 SD) for GGT (IUjl, 37-C): A. 20.85 (f11.47); B. 38.66 ( k 39.19); C . 83.39 (k210.48); D. 155.70 (i229.22). A high GGT value in suspected alcoholics may be of significance provided no other enzyme-inducing factors, e.g., barbiturates, are present, and there is no underlying non-alcoholic liver disease. Statistically, there was significant difference between each group for AST. Fourteen per cent of group B. 34% of group C and 76% of group D were above 2 SD of group A. High values of AST may be useful in demonstrating patho-physiological damage due to alcohol. GLOBOID CELL LEUCODYSTROPHY (KRABBE'S DISEASE) IN SHEEP

Pathology Downloaded from informahealthcare.com by Monash University on 02/02/15 For personal use only.

A. J .

D. H. EMBURY*& D. PRlCHARDt *Veterinary Research Institute, Melbourne. Veterinary Laboratory, Hamilton, Victoriu

SINCLAIR*,

t Regional

Globoid cell leucodystrophy is a genetically inherited disease which occurs in man, dogs and cats. The clinical manifestations of the disease are almost exclusively neurological, and typical histological changes are apparent in brain white matter (presence of globoid cells). The primary defect in this disease is a deficiency of the enzyme galactocerebroside p-gdlactosidase. Recently we have examined sheep with neurological signs which had lesions in the brain similar to those changes described above. In order to confirm the diagnosis of globoid cell leucodystrophy we examined the activities of 3 enzymes in aqueous homogenates of control and affected sheep brains. The results showed that the activity of the galactocerebroside 8-galactosidase in the affected sheep brain was less than 52, of the activity in the controls whereas the activities of a- and /f-galactosidase in the control and affected sheep brains were the same. These results confirmed the initial diagnosis.