Optimal line fitting by computer

82

Pathology (1977). 9, January

R.C.P.A. AND A.A.C.B.

THE SEARCH FOR NEONATAL EVIDENCE OF FAMILIAL HYPERCHOLESTEROLAEMIA

MEANEY. R. F., BOULTON. T. J . C., CAREY.S. & POLLARD. A. C. Departnlent Pathology, Adelaide Children's HospituI. South Australia

OJ

Cllemiral

World opinion is divided as to the efficacy of cord serum cholesterol measurement for the detection of neonates at risk for familial hypercholesterolaemia. Investigations based on elevated cord serum total cholesterol have met with varying amounts of success. Making use of LDL-cholesterol measurements, others have been able to detect familial hypercholesterolaemia. when one parent is known t o have the disorder. By combining the convenience and rapidity of enzymatic cholesterol and triglyceride methods with the small sample volume requirement of the CentrifiChem centrifugal analyser, we have been able to begin a large scale analysis of cord sera. The coefficients of variation for the cholesterol and triglyceride assays are 2.57",, and 4.41",, respectively. The aim is to investigate the efficiencies of total and LDL-cholesterol in the detection of neonatal hypercholesterolaemia in a large unselected population of newborns. The final analysis must await a several year follow-up study. At this stage, we have found the results for sera stored at - 15 C (n = 500) for up to 7 yr prior to assay, agree very well with results from sera ( n = 1,000) assayed with a minimum delay after collection. However, results for LDLcholesterol in both our groups (95 percentile = 71 mg/100 ml) are much higher than values found in several literature reports (95 percentile = 42 mg/100 ml). This discrepancy may he explained in part by statistical aberrations caused by differences in sample numbers and by the possible overestimation of HDL-cholesterol when EDTA-plasma is used instead of serum for lipoprotein precipitations. CENTRIFICHEM EVALUATION: PART 3

HILL,G . N., LLOYD,D. & POLLARD. A . C. Department of Cheriiicul Patholugj,. Adelaide Childreri F.' Hospital. Soutll Australia At previous meetings of the A.A.C.B. we have reported on some of the problemsencountered in using CentrifiChem for routine work. The current report is concerned with our continuing experience, making particular reference to machine modifications initiated by the Electronics Department of this Hospital now that a significant part of the routine is being processed by CentrifiChem. Maintenance and adjustment of the system in-house over a 2-yr period has resulted in data accumulation which has been useful in predicting system malfunctions and failure modes, and the magnitude and bource of errors. As a result a maintenance quality control programme has been initiated to highlight problems before they become grossly manifest. Hardware and software development is proceeding locally (within the Hospital) to upgrade the system in terms of accuracy. precision and reliability. Tests have been divided into 2 groups: 1. Urea, Creatinine. Cholesterol: 2. Protein, Albumin, GOT, GPT, CK, AlkalinePhos., yGT. all tests in a particular group being done whenever 1 or more ofthat group is requested. Group 1 tests require a total volume of 65 pi; Group 2 120 ktl sample; these amounts are well within reason when dealing with infants and children. The results obtained by CentrifiChem have heen compared with those obtained by the standard manual techniques used in the laboratory. Now that CentrifiChem has been installed in many laboratories throughout Australia, there is a strong case for the publication or at least dissemination of comparable data. OPTIMAL LINE FITTING BY COMPUTER

DUNCAN, B. M. & LLOYD,J. V., A irstralia

Insfitute qf' Medico/ and C'eterinar.,, Science, Adelride. South

Current methods of line fitting used in laboratories are in many cases approximations made to simplify the arithmetic. Now that computing time is readily available to most laboratories, more accurate, though more complex, methods can he used. For instance, calibration curves for clotting factor assays are usually drawn on double logarithmic paper. This is not out of any theoretical considerations, hut simply because the points always seem to lie fairly close to a straight line on this sort of paper. There is no justification for employing logarithmic functions when using a computer for this sort of calibration; we have found that other methods can give better results. The problem of fitting straight lines where both variables s and y are subject to error is common in evaluations of new tests. The usually employed method for determining the coefficients a and h of the straight line y = a r + h is that of choosing values a and h which minimize the sum of the squares of the deviations of the y's. It is well known that treating?. as an independent variable and minimizing the sum of the squares of the deviations of the .YS gives a different straight line as best fit. If both variables are subject to error, there is no reason to prefer one of these regression lines to the other. In fact, neither equation gives the best structural relationship between s and J'. hut the

ABSTRACTS OF ANNUAL MEETINGS 1976 83 true line lies somewhere between. In the evaluation of new methods in our laboratory we use a computer to calculate the 'best' structural relationship, rather than fit a line by simple linear regression. THE USE OF AQUEOUS STANDARDS TO CHECK ROUTINELY THE LINEARITY OF SMA CHEMISTRIES

WHITNFY, G . & WARD.R. Victoria

Biocheinistrj' Department. Roj,al Melhoirrnr Hospitul. Mrlhourne.

This paper presents an investigation of the linearity of plasma chemistry results from the Technicon SMA 6/60 and 12/60instruments. We assay both plasma and urine electrolytes on the SMA 6/60. this investigation being prompted by the observation of non-linearity of urinary potassium for results that were greater than the reference material concentration. Current Technicon literature suggests no means of testing linearity beyond the values of their top serum standard. Scale 11, though a number of users have top scale values significantly greater than the stated value of this material. T o check the linearity from zero to the top scale value for each chemical analysis, we prepared a series of aqueous standards for sodium, potassium, bicarbonate. creatinine. urea, uric acid. calcium and inorganic phosphate. Bilirubin standards were prepared using lyophilized serum with a high bilirubin value. Albumin standards were assayed using Technicon SMA reference serum for calibration. As a result of these investigations non-rectifiable deviations from linearity were detected in two results. These results, viz. for urea and inorganic phosphate, have consequently had their top of scale values reduced. Linearity characteristics, particularly in the high scale regions of an SMA system, can alter significantly with changes in reagent batch. or mechanical or electrical deterioration of the instrument. We therefore recommend regular linearity checks encompassing the complete range of each chemical analysis even though there is no reason to suspect deviations from the assumed linear response. THE EFFECT OF SERUM TRIGLYCERIDE AND BILIRUBIN LEVELS ON THE ASSAY OF SERUM CALCIUM

HOGARTH, K.. HUNT.L.. O'HALLORAN. M . W. & WELLBY, M. L. Departrnrnt of'Clitiical Chrntistr:,.. The Queen Elixzbetli Hospital, 24delaide,South Australia The effect of serum triglyceride and bilirubin levels on the assay of serum calcium using 3 currently available methods, atomic absorption spectrophotometry (AAS),cresolphthalein complexone on the SMA 12160 (SMA)and the Corning calcium analyser (CCA) were studied. Two hundred and ninety sera were selected and grouped in such a way that a satisfactory working estimate ofthe amount of bilirubin or triglyceride could be made b! visual examination, so that a practical estimate of the likely effect of either of these substances could be readil! made. Grouping of bilirubin for visual examination at intervals of 34 pmol/l and triglyceride at intervals of 5 mmol I were found to be suitable. The effects of each of these interferents within each visually discernible group were studied by comparison of the slopes of the regression lines of CCA vs AAS and CCA 1's SMA. N o significant difference between these slopes was found for either interferent until levels of 175 pnoltl in the case of bilirubin and 15 mmolll for triglyceride had been achieved. The effects of both interferents at these levels were similar, calcium levels below 2.0 mmo1;l being overestimated by 8",, whilst calcium levels above 2.5 mmol,'l were underestimated by 6",,when the comparison of CCA and SMA were made against the AAS. RADIO IMMUNO-ASSAY OF THYROXINE-BINDING GLOBULIN

Iristitirte of' Metiicul and I,'ererinarj' GILLILAND, J . M , Department of' Clinical Cl~enzisrrj~, Science. Adelaide, Soirth Aiistraliu Thyroxine-binding globulin (TBG), the major extracellular thyroid hormone binding protein, is a glycoprotein with a single binding site for thyroxine. The serum concentration of TBG is known to vary over a wide range in normal subjects. to be raised during pregnancy and oestrogen therapy. and lowered in old age. malnutrition, cirrhosis, androgen therapy and nephrotic syndrome, and is also dependent on genetic factors. Thyroxine-binding globulin has been purified from pooled human sera by a three-step procedure consisting of affinity chromatography on thyroxine-Sepharose, followed by chromatography on Con A-Sepharose and finally ion exchange Chromatography on DEAE Sephadex A-50. The final product gives a single stained band on analytical disc gel electrophoresis. A sensitive radioimmunoassay for thyroxine-binding globulin has been developed. The assay is specific, being unaffected by human serum proteins other than TBG. TBG levels in normal adults were found to range between 10