6 Feb 1992 - beta subunitpresentin intacthCG. There is a clinically important hook effect in the Hybritech assay but not the. Stratus or lMx assays (to 1.2 x 16.
CLIN.CHEM.38/6, 887-897 (1992)
Immunoassays for Quantifying ChoriogonadotropinCompared for Assay Performance and Clinical Application L. R. Witherspoon,
S. E. Shuler, G. F. Joseph, E. F. Baird, H. R. Neety, and R. E. Sonnemaker
We examined calibration and accuracy, precision, sensitivity, specificity, and “hook” effects for recently revised
automatedchoriogonadotropin (hCG) immunoassaysystems (Baxter-DadeStratusII, Abbott lMx intact hCG and total beta hCG) and compared them witha widelyused immunoradiometricassay (Hybritech). We estimated hCG in pregnant women, women with trophoblasticdisease,
nonpregnantyoung and menopausalwomen, normal men, and men with testicular tumors. We found clinically unimportant differences in calibration (all calibrated to the
3rd InternationalStandard).Detectionof hCGby all four assays was limited by their responses in serum from nonpregnantwomenand men. Precisionwithin-runwas best for the automated instruments, but all four assays had similar between-run precision. The Hybntech, Stra-
tus,and lMxintactassaysare specificfor intacthCG.The lMxtotalbetaassayquantifiesbothfree betasubunitand beta subunitpresentin intacthCG. There is a clinically important hook effect in the Hybritech assay but not the Stratus or lMx assays (to 1.2 x 1 6 mt. units/L). Results for pregnant women were similar by all four assays. We
measured“hCG”to 8 mt. units/L in menopausal women, which weakly correlatedwith concentrationsof lutropin and follitropin and was, in part, explained by crossreactivity. There was no sample-probe carryover in either
instrument. We found the lMx diluting module as well as resultsat the extremes of the lMx calibration curves (1 x iO mt. unitsfL to ascertain if the sample-dispensing probe could be contaminated and carry over material from one sample to the next. We compared the results obtained when instrument and manual dilutions were used in the samples (>100) requiring it. We also measured with each method the time required to obtain an hCG estimate requested “stat” and assessed the relative utility of each assay in our laboratory. After completing an initial analysis of assay performance, we used first one, then the other instrument in parallel with the manual (Hybritech) assay, in an attempt to determine staff, time, and cost requirements for all assaysas they would be used clinically.
specific for intact hCG. The IMx total beta assay respends to both intact hCG and to beta subunit. Interference from subunits otherwise is negligible. Results of hCG estimation in menopausal women are shown in Table 4. All four assays detected an hCG-like material with similar affinity, estimatingapparentcon-
5 Table
7 4 7 475 Hybritect
90 26
90 2 20
3
3
121
115
2
lMx intact lMx total beta Stratus
2. WHO Callb ratlon-hCG Assays Concn, lid. unIts/I. lntact, 75/537 60000 66000
62 000 66000
Bats, 75/551
#{216}4t 26b
97000 36rI
Alpha, 75/588 123b 40b 65b
26b
#{149}1&t value, 70000 mt.units/I. b Apparent concentrationundiluted.
CLINICAL CHEMISTRY, Vol.38, No. 6, 1992
889
Table 3. PrecIsion Studies Samplea, no. of runsa
Table 5. DIscordant hCG Assay Results
Sam. run
MultIpl.runs
ApparenthCG, lid. unIts/I.
Mean (SD), Int. unIts/I.
CV, S
Mean (SD), lid. unIts/I.
CV,
23.7 (1.9)
7.8
25.0 (1.6)
6.3
B
96.9 (5.5)
5.7
100.2 (5.5)
5.5
4.2 3.0 2.9
9.2 (0.6)
6.1
23, 47
9.1 (0.4) 67.4 (2.0) 123.6 (3.6)
21, 50
24.6 (1.4)
C D Men A5 B C
C High
22,43 21, 43
102.6 (3.8) 592 (17)
5.6 3.0 2.9
lMx total beta C Low C Mid
21,62 21,55
23.1 (0.4)
1.6
Cen
Women A
HybritecM5
“25” “100” Stratus Cl
C2
20, 467 18, 469 23, 50 23,44
C3 lMx intact C Low C Mid
C High
20.54
68.2
(2.9) 4.3
128.0 (7.8) 25.4(2.1)
103.3 (5.5) 617 (45) 23.2 (0.8) 135(9.4)
130 (2.5) 2.0 3.5 665 (24)
679 (38)
IMx Intact
Mx beta
Stratus
0.4
0.0
6.9
0.6
3.1
2.4
9.4
2.0
0.0 1.6
1.5 0.5
3.7 0.1
5.2 5.3
5.0
1.0 2.9 3.3
5.0 1.5 1.8
1.4 0.3 3.3
Hybrlt.ch
S
6.1
8.2 5.4 7.4
22.0
3.6
Subject with benign testicular neoplasni.
assays. The distribution of results obtained for nonpregnant women is shown in Figure 1 and for men in Figure 2. Hook effect. Responses to hCG at high concentrations are shown in Figure 3. No anomalous high-dose hook effect was detected with the IMx or Stratus assays. The clinically important hook effect with the Hybritech
3.3 7.0 5.5
Samples in same run. b#{149} of assays.
is improved but not eliminated by reducing the calibration range to 5-200 mt. units/L. assay
Table 4. hCG Results in Menopausal Women y Slop. Intercept SE Correlation of hCG results(y) with Hybritech hCG concentrations
Comparison between Methods Table 6 compares IMx and Stratus results with Hybritech results (5-10 mt. units/L) for women with normal pregnancies and for women experiencing aborted pregnancies. hCG measurements in one patient with choriocarcinoma are shown in Figure 4. hCG was also detected in 4 of 5 women with molar pregnancies and in 3 of 20 men with testicular neoplasms. Results for these patients were similar by all four assays except for one man, who had discordant results (Table 5).
(x) (0-6.0 mt. unitsiL)
StratushCG lMxintacthCG IMxtotalbetahCG
1.07
-0.13
1.20 -0.76 1.13 -0.05 Correlation of hCG results (y) with LH concentrations (x) (7.8-165 mt. unitsiL) Hybritech hCG 0.031 0.70
StratushCG IMx Intact hCG IMxtotalbeta hCG Correlationof hCG results (y)
0.034 0.54 0.049 -0.56 0.039 0.55 with FSH concentrations
0.91 1.34 1.01
1.23 1.58 1.84 1.65
(x) (11-236 mt. unitsiL)
HybritechhCG StratushCG lMx intacthCG IMx total beta hCG
Instrument Evaluation 0.013 0.014 0.024 0.019
1.17
1.34
1.15
1.71
-0.08
0.92
1.95 1.74
centrations of hCG to be about 8 mt. unitsfL. This hCG correlated weakly with both LH and FSH results. No discernible interference from TSH was present in any
assay. Results in all but one hypothyroid but menopausal women were below the detection limits of the assays. Appropriate results were obtained in all dilutions of patients’ samples in all assays (dilutional parallelism was demonstrated). The results we considered discordant are summarized in Table 5. All results in these patients were confirmed to be replicable by reassay. In addition, the Hybritech assay yielded discrepant replicates for samples from two menopausal women (1.6, 21;
0, 16) and one normal man (0, 8.6); repeat measurement confirmed that the lower results were correct. Sensitivity. The analytical sensitivity, based on replicates of the zero calibrator, was 10 mt. units/L will appear to contain 5 x iO mt. units/I..will appear to contain
