Differential Diagnosis

With a careful history, physical examination, thyroid function testing, and pregnancy test, most hypothalamic-pituitary disease can be excluded. A single serum prolactin concentration is usually adequate to demonstrate hyperprolactinemia. Normal concentrations are generally <20 mg/mL. However, because stress can stimulate prolactin, serum concentrations between 25 and 40 ng/mL should be repeated. The diagnosis of prolactinoma can be confirmed with gadolinium-enhanced MRI. Large pituitary tumors in patients with serum prolactin concentrations <140 ng/mL are considered nonfunctioning macro-ademnomas and generally respond well to surgical removal, while those found with serum prolactin concentrations of > 375 ng/mL are associated with macroprolactinomas and should be treated with dopamine agonists.1,3

Serum prolactin is quantified by two-site "sandwich" immunoassays (Fig. 87.1a). In these tests, a capture antibody directed against one site on prolactin is immobilized to a tube, vial, well, or bead. Patient serum is added. The antibody binds, and immobilizes the pro-lactin in the serum sample (PRL + immobilized antibody). A second ("detection" or "tracer") antibody directed against a distant site on prolactin is linked to a tracer molecule (such as a radioisotope or an enzyme). This forms an immobilized "sandwich" complex (tracer-antibody + PRL + immobilized antibody. After washing, the amount of tracer is measured (in the case of enzyme assays, incubation with substrate is required). The amount of signal is directly proportional to the amount of prolactin in the sample. Quantitative prolactin tests are either "one-step" or "two-step." In a one-step assay, the serum is incubated with both immobilized and tracer antibodies simultaneously. In a two-step assay, the serum is first incubated with the immobilized antibody, and then washed before the addition of the

IIII

Yyyy Y

IIIlI

Wash

III I

Wash

Figure 87.1 Assay format for (a) one-step sandwich assay with optimal amount of ligand and (b) ligand excess leading to the high-dose "hook effect."

tracer antibody. The one-step protocol is by far the most common protocol used because it is the fastest and the least complicated. The slower two-step protocol, however, avoids problems with hook effect and limits false-positive results due to heterophilic antibodies (see the Case 86, "Where's My Baby?").

The High-Dose "Hook Effect"

The "hook effect" is a major limitation of all one-step immunoassays. It occurs when extremely high concentrations of an analyte, such as prolactin, occupy all the sites on both the capture and detection antibodies, and thus prevent the formation of a "sandwich" immunoassay (Fig. 87.1b). The end result is that few or no tracer antibody + PRL + immobilized antibody complexes will be formed, yielding a false-negative result. The "hook effect" does not occur with two-step quantitative assays because excess analyte is washed away before the tracer antibody is added. Manufacturers of one-step sandwich assays have become more aware of this problem and have designed assays with more capture antibody, hence decreasing the chances of the hook effect. However, the hook effect still occurs and should always be a concern with one-step assays. This problem has been documented for numerous immunoassays, including prolactin, hCG, myoglobin, and PSA.3-10 Physicians should be aware of these phenomena when evaluating large pituitary masses. As a rule, if a physician finds that the prolactin concentration is inconsistent with the clinical presentation or histological findings, the prolactin test should be repeated with 10- and 100-fold diluted samples.

Two reports, by St. Jean and Petakov, have described the clinical features of patients with pituitary macroadenomas who demonstrate the hook effect with serum prolactin.3,6 These two studies reported that the hook effect occurs in 6-14% of patients with macro-adenomas.3,6 Both papers were very similar in their findings. In general, the patients who exhibit a hook effect tend to be younger, be male, and have very large (> 20-mm) pituitary adenomas (Table 87.1).

Case 87 Elevated Concentrations, but Not Elevated Enough Table 87.1 Clinical Features of 69 Patients with Pituitary Macroadenomas

Macroprolactinomas Nonfunc- Hook effect tioning adenomas adenomas

Number

11 (16%)

54 (78%)

4 (6%)

Sex (M:F)

1:10

25 : 29

4:0

Age, median

29 (20-70)

51 (21-79)

38 (32-52)

(range)

Prolactin, median

428 ng/mL

72 ng/mL

100 ng/mL

(range)

(72-3937)

(81-151)

(69-211)

After dilution

18,062 ng/mL

(14,907-44,600)

Giant tumora

27%

30%

100%

Hypogonadism

100%

60%

50%

Galactorrhea

89%

28%

25%

Visual failure

10%

70%

100%

ACTH deficiency

50%

71%

25%

FSH/LH deficiency

18%

52%

25%

TSH deficiency

9%

24%

0%

aTumors with superior margin > 20 mm above jugum sphenoidal. Source: Adapted from St. Jean et al.3

aTumors with superior margin > 20 mm above jugum sphenoidal. Source: Adapted from St. Jean et al.3

It should be noted that this false-negative effect is also observed in qualitative serological agglutination assays used to detect serum antibodies.11 This phenomenon is referred to as the prozone effect. In agglutination assays the reaction will be positive when the optimal ratio of antigen to antibody results in an insoluble precipitate that is visible to the eye. This optimal ratio of antibody to antigen (two to three antibody molecules for each antigen molecule) is referred to as the zone of equivalence. In the zone of antibody excess, or prozone, false-negative test results occur because excess antibodies prevent antibody/antigen lattice formation and hence prevent precipitation (Fig. 87.2).

Figure 87.2 Assay format for agglutination assay and various ratios of antibody and ligand including (a) postzone due to antigen excess, (b) the optimal ratio resulting in a positive result, (zone of equivalence), and (c) prozone due to antibody excess.
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