167

Clinica Chimica Acta, 65 (1975) 167-173 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

CCA 7294

COMPARISON OF AN ALL-AQUEOUS AUTOMATED URINE PREGNANCY OESTROGEN METHOD WITH ONE USING ORGANIC SOLVENT EXTRACTION

A.J. LITTLE, Department

K. AULTON and R.B. PAYNE of Chemical

Pathology,

St. James’s

Hospital,

Leeds

LS9

7TF

(U.K.)

(Received August 10, 1975)

Summary A comparative study between an all-aqueous automated urine pregnancy oestrogen method and a well established one requiring organic solvent extraction showed good agreement (r = 0.992). Advantages offered by the all-aqueous method include a faster rate of sample analysis and elimination of the difficulties of phase separation. The storage of urine for 5 days did not affect the oestrogen concentration measured by the all-aqueous method. Glucose, which reduced the apparent oestrogen concentration, was readily removed by treatment of the urine with borohydride, and treated urines showed good recoveries. The recovery of oestrogen was inadequate when glucose was removed from urine by incubation with yeast, possibly due to the formation of acetaldehyde.

Introduction The measurement of the urinary excretion of total oestrogen in pregnancy is commonly undertaken in clinical chemistry laboratories serving maternity units. The oestrogen output, which is mainly oestriol, is an important means of monitoring the well-being of the foeto-placental unit [l] . Increasing workloads in recent years have led to the introduction of rapid and relatively simple methods, both manual and automated [ 2-41. In the automated fluorimetric technique of Hainsworth and Hall [4] untreated urine is diluted, heated with the sulphuric acid-quinol Kober reagent and the Kober oestrogen chromogen extracted into chloroform containing trichloroacetic acid. The fluorescent product so formed is measured using a fluorimeter with flow-through facilities. This method has been the subject of a further recent study [ 51. The principal difference between the method of Hainsworth and Hall [4] and the all-aqueous continuous flow method described by Lever et al. [6] is

168

that the chlor~fo~/trichloroacetic acid reagent is reptaced by an aqueous chloral hydrate/trichloroacetic acid solution. The all-aqueous method was reported to be capable of achieving a greater throughput of samples (40/h) than the method of ~~ainsworth and Hall (20/h) and it avoided the problem of phase separation. We have therefore undertaken a comparison of the two methods, and have also investigated the effects of urine storage and of glycosuria on the measurement of oestriol by the method of Lever et al. [6]. Methods Technicon AA1 modules were used in conjunction with a Locarte fluorimeter fitted with a thallium excitation lamp and a 560 nm interference filter on the emission side. The output signals were fed to a Servoscribe recorder. The two methods were set up alternately on the same modules. A manifold change could be achieved within fifteen minutes. The method of Hainsworth and Hall [4] was used as described except that the reagents were pumped directly using Acidflex pump tubing instead of by the displacement bottle technique. A Technicon B4 phase separator was used in place of the BO piece recommended by the authors. The method of Lever et al. [6] was used as described with the modification that the air segmentation of the chloral hydrate/trichloroacetic reagent was eliminated. To compensate for this, the air segmentation of the Kober reagent was increased to 0.06 ml/min. A prelimin~ study showed unaccep~b~e carryover at the recommended rate of 40/h, so a 60/h 1 : 1 sampling cam was used and water cups were placed between urine samples, giving a net sampling rate of 30/h. A stock standard (3 mmol/l) was prepared by dissolving a weighed amount of oestriol (Koch-Light Ltd.) in absolute ethanol, and stored in the deep freeze. A range of aqueous standards (30, 60, 90 and 120 pmol/l) were prepared freshly for each day’s run. Results

Pregnancy urines containing a range of oestriol levels were placed in sample cups in random order, and each plate was analysed twice by each method TABLE

I

WITHIN-BATCH

PRECISION

Range

BY THE METHOD ._~~_

Number

OF DUPLICATES

deviation . .-_-.

Standard ~~_.

of data

~_.

._-

- ~~.

Organic

solvent

All-a~euus

method

(4)

methud

I+.

(6)

___-.-

AI1 results

58

1.64

1.41

1.35*

>60

prnnlil

25

2.03

1.61

1.59*

=60

flmolll

30

1.15

1.18 _ -.

i_.

* Not significant.

__~

1.06”

~~ ~_

~_ _-.

__

_~_

169

TABLE

II

BETWEEN-BATCH TROLS

AT

PRECISION

TWO

USING

COMMERCIAL

UNASSAYED

URINE

OESTROGEN

Control value

Organic

solvent

Number

method

All-aqueous

E‘

method

S.D.

Number

S.D.

Approximately

30 pmol/I

36

2.24

36

2.32

1.08+

Approximately

70 ~.rmoI/I

22

4.11

38

4.21

1.05*

* Not

CON-

LEVELS

significant.

using the same aqueous oestriol standards. Analysis of a batch by the first method was immediately followed by analysis using the second method. Precision for each method was assessed from the standard deviation (S.D.) calculated by the method of duplicates: S.D. = &d*/2n where d is the difference between duplicates and n is the number of pairs. The F-test showed that the differences between the within-batch precision for the two methods were not significant (Table I). Between-batch

precision

Commercial unassayed urine oestrogen controls (IDA*) at two different levels, approximately 30 and 70 Irmol/l respectively, were analysed daily by each method over a period of several weeks. Precision was assessed from the standard deviation for each method. The F-test showed that the differences in precision between the two methods were not significant at either level (Table II). Correlation

of methods

The data obtained in the study of within-batch precision together with additional comparative data showed a good correlation between the two methods for 155 pairs over the range 15-120 pmol/l (Fig. 1). The correlation coefficient (r) was 0.992 and the regression equation was: Hainsworth

and Hall value = (1.008 X Lever et al. value) - 1.25 pmol/l

Thus, the methods

were of similar accuracy

with no significant

bias.

Carryover assessment Carryover in the cribed by Broughton 115-135 pmol/l of (b, , bz, b3) of urines

* International

Diagnostic

all-aqueous method was assessed using the technique deset al. [7]. Three aliquots ((I~, a2, ax) of urines containing oestriol were immediately followed by three aliquots with 20-40 pmol/l of oestriol. Carryover was expressed

Aids

Ltd.,

Gloucester,

U.K.

170

k 2 k 40

5 OESTRIOL

80

BY METHOD (vmol/

Fig. 1. Comparison of 155 oestriol and Hall 141 and by the all-aqueous

OF LEVER

c 120 et al

I measurements made by the solvent extraction method of Hainsworth method of Lever et al. [61 .‘The line is that of the regression equation.

as the mean percentage interaction calculated from 100%. The mean interaction for 13 pairs was 3.0%. Recovery

((b,

-

b,)/(a,

-

b3)) X

of added oestriol

Stock oestriol standard was added to a total of 25 urines containing no detectable oestriol to achieve a calculated concentration of 60 pmol/l. Single analyses were carried out during several routine batches. The mean recovery was 93.4% (range 80-105%). Effect

of urine ageing on measured oestriol

A small number of urines were selected from each of a number of analysed batches, stored at 4°C for a mininum of 5 days and then reassayed. A total of 100 urines were analysed. There was a good correlation (r = 0.983) between the fresh and the stored values (Fig. 2). The regression equation was : stored value = (1.018

X fresh value) + 0.34 pmol/l

Thus there was no evidence Effect

of glucose

of any significant

change due to ageing.

on measured oestriol

To investigate the effect of large amounts of glucose on the method of Lever et al. [6], concentrations up to 500 mmol/l were prepared in two urines

171

.

. . . .

a0

40 FRESH

URINE

.

120 GLUCOSE

OESTRIOL

(mmol/l)

(w-d/l 1 Fig. 2. Effect of urine storage for a minimum line is that of the regression equation. Fig. 3. Effect

of increasing

concentrations

of 5 days at 4OC on measured

of glucose

on measured

oestriol

oestriol

in 100 urines. The

in two urines.

with oestriol concentrations of 54 and 146 pmol/l. These solutions were analysed in triplicate for oestriol. There was a gradual reduction of measured oestriol as glucose concentration increased (Fig. 3). We found that added ethanol did not interfere with the recovery of oestriol, so the effect of incubating urines containing large amounts of glucose with yeast was investigated. A preliminary experiment showed that there were no differences in oestriol values before and after yeast treatment in 45 urines negative to Clinistix (Ames, Ltd.). Glucose was completely removed from urines containing 500 mmol/l of glucose by incubation of 3 ml aliquots with about 200 mg of dried baker’s yeast at 37°C for 2 hours, but the recovery of oestriol in five such urines averaged only 76% (range 69-U%). While this work was in progress, Ryan and Gray [El] demonstrated that aldehydes, including the aldehyde group in glucose, decreased the recovery of oestriol. They found that the interference due to 100 mmol/l of glucose was removed by treatment of 5 ml of urine for one hour at 37°C with 0.5 ml of 10% sodium borohydride in 0.1 N sodium hydroxide, with the addition of one drop of octan-2-01 to prevent frothing, followed by acidification with 0.5 ml glacial acetic acid. “Glucose negative” pregnancy urines treated in the same way showed an average increase in urinary oestrogen of about 5 pmol/l, an increase similar to that found when urines were diluted lo-fold. We added 500 mmol/l of glucose to eight pregnancy urines and measured oestriol before and after treatment with borohydride in the way described by Ryan and Gray [8]. The mean recovery of the borohydride-treated urines without added glucose was 105% (range lOO-108%), but the same urines containing 500 mmol/l of glucose gave a mean recovery of only 57% (range 3785%). It was suspected that the borohydride concentration recommended by

Ryan and Gray [S] was inadequate to deal with the high glucose concentrations which might be found in diabetic urines. Increasing concentrations of glucose ranging from 50 to 500 mmol/l were added to a urine and the recoveries of oestriol determined after treatment with borohydride, but using a concentration of 50% instead of 10%. For 12 observations at varying glucose concentrations the mean recovery was 102% (range 95-lo%%) and showed no change with increasing glucose. Thus, 50% sodium borohydride appears to deal adequately with the highest glucose concentrations likely to be encountered in clinical practice. Discussion The fundamental difference between the methods we have compared for the measurement of oestrogens in pregnancy urine is that the fluorescent product is measured in the organic phase following solvent extraction in the method of Hainsworth and Hall [4], while in the method of Lever et al. [6] there is no extraction. Despite this difference, the precision and accuracy of the two methods were very similar. We found a carryover of 3% at a net sampling rate of 30/h using the method of Lever et al. [6] . This compares favourably with the 4% carryover at a sampling rate of 20/h reported by Hainsworth and Hall [4] for their method. Moscrop et al. [5] have investigated several aspects of a modified version of the method of Hainsworth and Hall [4]. They found that dilution of the 24h urine to 20 litres improved the recovery of added oestriol, giving a mean of 94%, with a range of Sl-110%. Our recovery in undiluted urine by the method of Lever et al. [6] compared closely with these values, giving a mean of 93%, with a range of 80-105%. Moscrop et al. [ 51 also found that the apparent oestriol concentration in a “good number” of undiluted urines increased with storage, but this was eliminated by dilution of the 24h urine to 20 litres. This effect could mask a falling oestriol level in a urine collected and stored over a week-end. We found that urine ageing was no problem when measurements were made on undiluted urine by the method of Lever et al. [6]. The effect of glucose in lowering the apparent oestriol concentration is well recognised. Attempts to eliminate or reduce this have been made, for example, by high dilution of the urine [ 51. Our modification of the method of Ryan and Gray [8] for glucose removal using 50% borohydride gave good recoveries of oestriol from undiluted urine containing up to 500 mmol/l of glucose. We are not convinced by the argument of Ryan and Gray [S] that the average increase in oestriol of about 5 pmol/l which follows borohydride treatment of “glucose negative” pregnancy urines warrants the routine pretreatment of all samples. We suggest that urines should be screened with Clinistix, and only those urines giving a positive result should be treated with borohydride. However, once borohydride treatment has been used, it would probably be wise to treat any subsequent urines from the same patient whether or not glycosuria is present in these samples. Some laboratories remove glucose from pregnancy urine by incubation with yeast. We found this to be an efficient way of removing glucose. Ethanol

173

did not affect the measurement of oestriol by the method of Lever et al. [6] and yeast treatment did not affect oestriol measurement in “glucose negative” urines. Nevertheless, the recovery of oestriol after yeast treatment of urine containing 500 mmol/l of glucose averages only 57%. Acetaldehyde is known to affect oestriol measurement [S] . A possible explanation of the poor recovery is that acetaldehyde formed during the fermentation of glucose is incompletely metabolised to ethanol. Acknowledgement We are grateful

to Dr. R.E. Oakey

for his helpful

criticism

and advice.

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