Capillary Prolactin Measurement for Diagnosis of Seizures Robert S. Fisher, MD, PhD,"t Daniel W. Chan, PhD& Mary Bare, MSPH, RN,* and Ronald P. k s s e r , MD"T

Measurement of serum prolactin levels can be useful in the diagnosis of epilepsy, since prolactin levels often rise after seizures, but not after most imitators of epilepsy. Utility of the test is limited by the need to obtain blood 10 to 20 minutes after the episode. The present study documents the validity of prolactin measurements using capillary blood, which was obtained by the finger-stick method after a possible seizure and then applied to filter paper. Venous and capillary prolactin levels were determined 10 to 20 minutes after seizure-like episodes in 20 patients who were studied in an epilepsy monitoring unit. Venous and capillary prolactin values correlated, with a Pearson coefficient of 0.90. Using a criteria of any elevation above the laboratory upper limit of normal, capillary prolactin values correctly identified seizure versus pseudoseizure in 9 (1007o)of 9 patients with generalized tonic-clonic seizures, in 5 (71%) of 7 patients with complex partial seizures, and 4 (100%) of 4 patients with pseudoseizures. Prolactin values were unaffected by leaving filter paper samples at room temperature for up to 1 week. This study suggests the utility of diagnostic capillary blood collection kits to assist in the diagnosis of epilepsy in outpatients. Fisher RS, Chan DW, Bare M, Lesser RP. Capillary prolactin measurement for diagnosis of seizures. Ann Neurol 1991;29:187-190

iMaterials and Methods

The first step toward optimal treatment of epilepsy is accurate diagnosis. Alterations in consciousness, sensory-motor function, or behavior may be the consequence of epileptic or nonepileptic episodes. Imitators of epilepsy include psychogenic seizures, syncope, cardiac arrhythmias, transient ischemic attacks, transient global amnesia, hypoglycemia, migraine headaches, sleep disorders, intermittent tremors, and several rarer entities [l]. EEG is useful in diagnosis; however, findings on interictal EEG studies are often negative. A blood test able to aid in the diagnosis of epilepsy would be very valuable. Measurement of serum prolactin levels has been partially successful in addressing this need [2-131, but its utility has been limited by the need to draw blood samples within about 30 minutes after a seizure. In the current study we demonstrate that assay of prolactin with capillary blood applied to filter paper is as accurate in the discrimination of seizure versus pseudoseizure as is assay of prolactin with venous blood. Since the prolactin values are stable for days at room temperature, it should be practical to allow patients and families to obtain capillary blood samples at home, immediately after a possible seizure. Samples could then be delivered or mailed to a laboratory for testing. A preliminary account of this study has been presented 1141.

Twenty patients were selected from the epilepsy clinics at The Johns Hopkins Hospital, on the basis of a clinically secure diagnosis of epilepsy or pseudoseizures (psychogenic seizures).All patients were studied by video-EEG continuous monitoring. After a seizure or pseudoseizure, venous blood was drawn for prolactin assay and, simultaneously, capillary blood was obtained by lancet stick of a finger. Sampling was performed 10 to 20 minutes after the conclusion of the clinical episode. The capillary blood was applied directly to filter paper, similar to the paper used to assay newborns for phenylketonuria. This paper has several marked circles, 12 mm in diameter. Two circles were completely filled with blood by squeezing the finger against the filter paper. Filter paper and venous blood samples were then refrigerated at approximately 4°C for up to 1 day..Serum was prepared from venous blood by centrifugation and supplied to the laboratory conjointly with the filter paper. Samples were frozen in the clinical laboratory ( - 20°C) for up to several weeks prior to analysis. Measurement of prolactin {15] was performed with a solidphase, two-site immunoenzymetric assay (Tandem-E Prolactin, Hybritech, San Diego, CA). Samples were incubated at room temperature for 2 hours with plastic beads (solid-phase) coated with a mouse monoclonal IgG antibody directed against a unique antigenic site on the prolactin molecule, and with an enzyme-labeled (bovine alkaline phosphatase) monoclonal antibody directed against a different antigenic site on the prolactin molecule. Anti-prolactinantibody conju-

From the Departments of *Neurology, ?Neurosurgery, and $Pathology and Laboratory Medicine, The Johns Hoplclns University School of Medicine, Baltimore, MD

Addresb correspondence to Dr Fisher, Associate Professor of Neurology and Neurosurgery, The Johns Hopkins Hospital, Meyer 1-130, 600 North Wolfe Streec, Baltimore, MD 21205

Received Jun 21, 1990, and in revised form Aug 16 Accepced for publicauon Aug 17, 1990

Copyright 0 1991 by the American Neurological Association

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gate was mouse monoclonal IgG conjugated to bovine alkaline phosphatase in a protein matrix containing 0.1gj sodium azide. Beads were coated with anti-prolactin antibody comprised of mouse monoclonal IgG in buffer also with 0.1% sodium azide. The beads were then washed and incubated with p-nitrophenyl phosphate as a substrate for the enzyme for 45 minutes at room temperature. Colorimetric reaction, measured at 405 nm, gave an absorbance directly proportional to the prolactin concentration. Accuracy of the procedure was verified by calibration against commercially available standards with known concentrations of prolactin [ 161. These standards consisted of horse serum and 0.1o/c sodium azide containing either 20 o r 150 ng of prolactidml. Venous blood was tested in the conventional fashion. Capillary blood was tested by cutting two circles out of the filter paper by hand and placing them in an incubation mixture with an additional 200 p1 of the zero diluent, horse serum containing 0.1o/c sodium azide, but no prolactin. The calibrator and quality control samples were tested in the same manner. Calibration was performed with whole blood, as contrasted with the usual calibration done with serum, since the samples obtained from a finger were whole blood. Further methodological details are available from Hybritech package insert procedure summary for the Tandem-E Yrolactin kit, or from other commercially available prolactin assay kits. Each capillary prolactin value for the filter paper represents the average of the results from the blood on the two paper circles.

Results Data were obtained from 20 patients with presumed seizure disorders. Subjects (10 women and 10 men) were over 18 years of age. All patients had documentation of their seizure type by simultaneous video-EEG monitoring. In 18 patients, the seizure correlating t o the prolactin value was obtained during video-EEG monitoring. In the two remaining patients, for logistical reasons, prolactin was sampled after cessation of videoEEG monitoring; however, the seizure studied was in each case behaviorally identical to those analyzed previously in the epilepsy monitoring unit. By video-EEG criteria, 9 of the patients had generalized tonic-clonic seizures (GTCL), most of which were secondarily generalized from a temporal lobe focus. Complex partial seizures were recorded in 7 patients. Pseudoseizures, all with tonic-clonic behavior, occurred in 4 patients. Capillary prolactin values and venous prolactin values are summarized by seizure type in Figure 1. The prolactin values measured by both capillary and venous assays correlated highly among each of the patients, with a Pearson correlation coefficient of 0.90. This correlation is illustrated in Figure 1. Mean prolactin values, grouped by seizure types, are shown in Figure 2. For all 20 patients, the prolactin level was 41.9 2 9.1 nglml (mean standard error of mean) by capillary measure, and 38.8 2 6.2 by venous measure. These two outcomes do not differ Cp = 0.5 by two-tailed paired t test). For both capillary and venous prolactin assays, mean values were higher for GTCL seizures than for com-

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Fag 1. Cowelation of venous versus capillary prolactin values (ngl ml). Each poznt represents one paired sample drawn nearly simultaneously at 10 t o 20 minutes afer a seizure or pseudoseizure. Venous (brachtal vein serum) and capillavy (fingemtack whole blood applied t o Jilterpaper) vulues Correlated with a Pearson coeffinent of 0.90. Values on both axes tend t o be hzgher for tonicclonzc seizures than for complex partial seizures. and t o be lowest for vtdco-EE G documented pseudoseizures.

SEIZURE TYPE C CAPILLARY A VENOUS

Fig 2 . Mean capilkzty and venous prolactin values (nglml)f . r all seizures, generalzzed tonic-clonic (GTCL) seizures, complex partial seizureJ without generalization (CPS), and pseudoseizares (PSEUDO). See text and Table.

plex partial seizures. Statistical data are presented in the Table. The lowest mean prolactin values were seen in the patients with pseudoseizures. Correlation coefficients between capillary and venous measurements were not significant for the subgroups of only complex partial seizures and pseudoseizures, because of the small numbers in each group and relatively restricted range of values. The correlation for the 9 patients with GTCL seizures was 0.89, virtually identical to the correlation for the group as a whole. How useful the prolactin values are for diagnosis of seizures depends on the selection of a criterion for abnormality. Our present study was not designed to establish such criteria; however, we can assume a high (“strong”) and a low (“weak”) criterion and compare predictive value of capillary versus venous prolactin measurements. A low criterion is any elevation; in our laboratory this is a value above 18 ng/ml. A relatively high criterion is a requirement for elevation to 2 tunes the upper limit of normal; in our laboratory this is a

Summary of Prolactin Lmels (nglml) by Seizure Type All

Capillary Venous n

Correlation

41.9 k 9.1 38.8 k 6.2 20

+ 0.90

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Complex Partial Seizures

68.1 2 16.2

26.0

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value equal to or above 36 ng/ml. Using the low criterion, capillary prolactin values were correctly predictive of seizure versus pseudoseizure in 9 (100%) of 9 patients with GTCL seizures, in 5 (7191) of 7 patients with complex partial seizures, and 4 (100%) of 4 patients with pseudoseizures. Venous prolactin levels, under the low criterion, correctly identified seizures in 9 (100%) of 9 patients with GTCL seizures, 6 (865%) of 7 patients with complex partial seizures, and 3 (75%) of 4 with pseudoseizures. Diagnostic sensitivity deteriorated with use of the high criterion. Capillary prolactin values were correctly predictive in 6 (67%) of 9 patients with GTCL seizures, none (0%) of 7 with complex partial seizures, and 4 (100%) of 4 patients with pseudoseizures. Venous prolactin levels were correct with the high criterion in 5 (56%) of 9 patients with GTCL seizures, 1 (14%) of 7 with complex partial seizures, and 4 (100%) of 4 patients with pseudoseizures. In our study the selectivity of capillary prolactin measures of seizure versus nonseizure was excellent when we used the low criterion of any elevation above the upper limit of normal. Venous prolactin determinations were almost as good as capillary determinations, but did show one false-positive in a patient with pseudoseizures. Use of the high criterion (twice the upper limit of normal) in this data set obliterates the sensitivity of the test for detecting complex partial seizures, with either capillary or venous measurements. We recommend that further work be done on meaningful criteria for abnormality of serum prolactin levels; however, capillary assay gives comparable results to venous assay with both high and low criteria. Stability and replicability of capillary prolactin measurements are important issues in this test. We therefore performed a study of the stability of prolactin left on the filter paper for periods of 1 to 7 days. A pool of blood was prepared, “spiked” with prolactin, and spotted onto 7 pairs of filter paper. Pairs were used because the method averages values from two filter paper samples to reduce variance caused by uneven blood application to paper and circle cutting. O n consecutive days for ? days, matched pairs of paper were removed and left exposed to air and room temperature. Samples were run as a batch on the eighth day. Figure 3 plots the prolactin value as a function of the number of days exposed to air and room temperature.

Pseudoseizures

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Fig 3. Stability testing of prolactin values Ingiml) for a pool of blood ‘?piked”with prolactin, applied t o jlter paper, and ldt exposed t o room uir und room temperaturefor 1 t o 7 days. Each point is the azwage value of two identically prepured samples. Reasonable stability is observed.

Values were reasonably replicable and stable over time. This suggests valid prolactin measures can be done on filter paper samples mailed or delivered by patients.

Discussion Prolactin is a polypeptide hormone secreted by the anterior pituitary, first discovered in 1928 117). Prolactin serves co regulate lactogenesis and several related endocrine functions in mammals. Like other anterior pituitary hormones, prolactin secretion is regulated by the hypothalamic-hypophyseal axis. In 1978 Trimble first verified the hypothesis that seizures could raise serum prolactin levels {lo]. Subsequently, there have been several studies confirming that serum prolactin levels rise after GTCL or complex partial seizures 17, 9, 12, 13, 181. A small study of 7 children (age range, 3 months to 13 years) with various seizure types indicated a tendency for increased serum prolactin levels after complex partial and tonic-clonic seizures in children 1192. Prolactin is not significantly elevated after psychogenic seizures {lo, 11, 13, 20}, absence seizures 1111, simple partial seizures 141, or complex partial seizures originating from the frontal lobe {S}. Interictal spiking probably does not noticeably increase serum prolactin levels, but prolactin rises at night in patients with epilepsy, perhaps because of subclinical seizures {S]. After repetitive seizures, the rise in prolactin may habituate { 2 11. Pseudoseizures with muscular activity greater than that of many tonic-clonic seizures usually do not Fisher et

al:

Capillary Prolactin

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raise prolactin levels {20), indicating that increased levels after a seizure is not simply a consequence of physical exercise. Prolactin levels are usually stable in a given individual, except for a 50 to loo$%increase just prior to awakening from sleep [ZO}. However, prolactin increases are far from specific for epilepsy Ell, 13, 17, 22). Given this lack of specificity of prolactin rise, several investigators proposed criteria for a prolactin level or change that are diagnostic of a seizure Ell). Some proposed an upper limit of 23 &ml; others, two to three times the baseline prolactin level [13]. Yerby and associates [13} reviewed 12 studies of patients with GTCL seizures: 133 (89.9%) of 148 patients had increased prolactin levels. In seven studies of complex partial seizures, after 77 (69.4%) of 111 seizures there was a significant rise in serum prolactin levels. Among seven studies reporting prolactin values after psychogenic seizures, there were 2 findings of prolactin elevations (3.0%)in 66 patients. The predictive value of prolactin levels in the diagnosis of epilepsy depends on the population under study and the criteria chosen for abnormality of prolactin levels. In our study, prolactin measured on capillary blood was elevated above normal in 9 of 9 patients with GTCL seizures, in 4 of 7 patients with complex partial seizures, and in none of 4 patients with EEG-documented psychogenic seizures. In 1 patient with psychogenic seizures, the filter paper prolactin level was normal, while the venous prolactin level was elevated. These findings indicate that accuracy of diagnosis of epilepsy by the filter paper technique in a tertiary referral population is comparable to that reported for venous prolactin studies. In general, failure of prolactin levels to increase is not conclusive in ruling out complex partial seizures { 117. We did not perform serial prolactin studies, since the primary goal was correlation of venous and capillary prolactin levels. It is known that prolactin reaches a peak at 15 to 20 minutes after a generalized seizure, followed by a decline to baseline values at 60 minutes after the seizure [4].Paired capillary prolactin determinations at 15 minutes after a possible seizure, and again an hour later, might increase the sensitivity and specificity of the test. The prevalence of epilepsy in the United States is approximately 1%. The prevalence of individuals with episodes of altered sensorimotor function, behavior, or awareness, for whom epilepsy is an aspect of the differential diagnosis, is much larger. Capillary blood prolactin assay may be a safe and useful test to assist with the differential diagnosis. Because the sensitivity and specificity of increased prolactin levels for diagnosis of epilepsy are not yet well established, capillary prolactin determinations must be interpreted in context of the complete clinical picture.

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We acknowledge the helpful efforts of Dr William Gaillard in establishing the protocol, and of Barbara Cysyk, RN, in educating several of our patients about the protocol. Jane Lijewski performed the assays, for which we are grateful.

References 1. Wada JA. Differential diagnosis of epilepsy. Electroencephalogr Clin Neurophysiol 1985;37(suppl):285-311 2. Abbott RJ, Browning MCK, Davidson DLW. Serum prolactin and cortisol concentrations after grand mal seizures. J Neurol Neurosurg Psychiatry 1980;43: 163-1 67 3 . Aminoff MJ, Simon RP, Weidemann E. The hormonal responses to generalized tonic-chic seizures. Brain 1984;107: 569-578 4. Collins WCJ, Lanigan 0, Cdaghan N. Plasma prolactin concentrations following epileptic and pseudoseizures. J Neurol Neurosurg Psychiatry 1983;46:505-508 5. Dana-HaeriJ, Trimble MR, Oxley J. Prolactin and gonadotropin change followinggeneralized and partial seizures.J Neurol Neurosurg Psychiatry 1983;46:3 3 1-3 3 5 6. HBppener RJEA, Rentmeester TH, Arnoldussen W, et al. Changes in serum prolactin levels following partial and generalized seizures. Br J Clin Pract 1982;18(suppl):193-195 7. Laver KD, Mullooly JP, Howell B. Prolactin changes after seizures classified by EEG monitoring. Neurology 1985;35:31-35 8. Molaie M, Culebras A, Miller M. Nocturnal plasma prolactin and cortisol levels in epileptics with complex partial seizures and primary generalized seizures. Arch Neurol 1987;44:699-702 9. Sperling MR, Pritchard PB 111, Engel J Jr. Daniel C, Sagel J. Prolactin in partial epilepsy: an indicator of limbic seizures. Ann Neurol 1986;20:7 16-722 10. Trimhle MR. Serum prolactin in epilepsy and hysteria. Br Med J 197821682 11. Wroe ST. - . Henlv. R. -John R, Richens A. The clinical value of serum prolactin measurement in the differential diagnosis of complex partial seitures. Epilepsy Res 1989;3:248-252 12. Wyllie E, Liiders H, MacMillan JP, Gupta M. Serum prolactin levels after epileptic seizures. Neurology 1984;34: 1601-1604 13. Yerby MS, van Belle G, Friel PN. Wilensky AJ. Serum prolactins in the diagnosis of epilepsy: sensitivity, specificity, and predictive value. Neurology 1987 ;37:1224- 1226 14. Fisher RS, Gaillard W, Bare M, et al. Finger-stick blood for prolactin assay: use in seizure diagnosis. Neurology 1990; ~ O ( S U P1):442-443 P~ 15. Hwang P, Guyda H, Frisen H. A radioimmunoassay for human prolactin. Proc Natl Acad Sci USA 1971;68:1902-1906 16. Schuister D, Gaines Das RE, Jeffcoate SL. International standards for human prolactin: calibration by international collaborative study. J Endocrinol 1989;121:157-166 17. Frantz AG. Prolactin. N E n d J Med 1978;298:201-207 18. Pritchard PB 111, Wannamaker BB, Sagel J, et al. Endocrine function following complex partial seizures. Ann Neurol 1983; 14:27-32 19. Bye AME, Nunn KP, Wilson J. Prolactin and seizure activity. Arch Dis Child 1985;60:848-851 20. Pritchard PB 111, Wannamaker BB, Sagel J, Daniel CM. Serum prolactin and cortisol levels in evaluation of pseudoepileptic seizures. Ann Neurol 1985;18:87-89 21. Jackel RA, Malkowicz D, Trivedi R, et al. Reduction ofprolactin response with repetitive seizures. Epilepsia 1987;28:588 22. Franceschi M, Perego L, Cavagnini F, et al. Effects of long-term antiepileptic therapy on the hypothalamic-pituitary axis in man. Epilepsia 1984;25 :46- 52