Daylily S. Ooi Div. of Biochem. Dept. of Lab ... - Clinical Chemistry

696 Letters
2. Asplin JR, Favus MJ, Coe FL. Nephrolithiasis.
In: Brenner BM, ed. Brenner & Rector’s The
kidney, 5th ed. Philadelphia: WB Saunders,
1996:1893–935.
3. Smith LH. The medical aspects of urolithiasis:
an overview. J Urol 1989;141:707–10.
4. Mandel NS, Mandel GS. Urinary tract stone
disease in the United States veteran population.
II. Geographical analysis of variations in
composition. J Urol 1989;142:1516–21.
5. Coe FL, Parks JH. New insights into the pathophysiology
and treatment of nephrolithiasis:
new research venues. J Bone Miner Res 1997;
12:522–33.
6. Ruml LA, Pearle MS, Pak CYC. Medical therapy.
Calcium oxalate urolithiasis. Urol Clin North Am
1997;24:117–33.
7. Low RK, Stoller ML. Uric acid-related nephrolithiasis.
Urol Clin North Am 1997;24:135–48.
Daylily S. Ooi
Div. of Biochem.
Dept. of Lab. Med.
and Bone and Renal Stone Clin.
Ottawa Civic Hosp.
1053 Carling Ave.
Ottawa, ON K1Y 4E9, Canada
and Dept. of Pathol. and Med. Lab.
University of Ottawa
E-mail dsooi@civich.ottawa.on.ca
A spokesperson from Merck responds:
To the Editor:
We have not claimed that our semiquantitative
analysis is comparable
with a high-sensitivity infrared spectroscopic
analysis. Nevertheless,
Merck will evaluate the determination
of oxalate to check whether it is necessary
to modify the oxalate color scale
to prevent erroneous interpretations
(background). Blanking with water is
absolutely not allowed because all reactions
work correctly only under acid
conditions; the urate calculi must be
dissolved in concentrated sulfuric acid
and water, and all tests are optimized
for these conditions.
Ludwig Jakobi
Merck KGaA
Darmstadt, Germany
Cross-Reactivity of Fosphenytoin in
Four Phenytoin Immunoassays
To the Editor:
Fosphenytoin, 5,5-diphenyl-3-[(phosphonooxy)methyl]-2,4-imidazolidinedione
disodium salt (Cerebyx ® ; Parke-
Davis), is a phosphorylated form of the
anticonvulsant drug phenytoin. Fosphenytoin
itself has no pharmacological
activity but is dephosphorylated in
vivo by phosphorylases to the active
drug, phenytoin. The elimination halflife
of fosphenytoin in plasma is 8–15
min in healthy subjects [1, 2]. Fosphenytoin
can be used for parenteral or
intramuscular administration, where
its superior aqueous solubility results
in less severe side effects than does
phenytoin [2, 3]. Few data, however,
are available on the interference of
fosphenytoin in phenytoin immunoassays,
which are currently the most
common method used to monitor patients’
phenytoin concentrations. In an
abstract [4], Kugler et al. reported that
the TDx ® Phenytoin assay (Abbott
Labs.) was interfered with by fosphenytoin.
We report here the results of
detailed cross-reactivity studies for
fosphenytoin in four phenytoin immunoassays:
ACS:180 ® Automated
Chemiluminescence System (Chiron
Diagnostics), TDx Phenytoin and Phenytoin
II, and AxSym ® Phenytoin II
(also from Abbott Labs.).
A therapeutic total plasma phenytoin
concentration (10 mg/L) is
attained within 10–30 min after administration
of fosphenytoin. Conversion
of fosphenytoin to phenytoin
is reported to be complete in 2–4 h,
depending on mode and rate of administration
[5]. The plasma concentration
of fosphenytoin depends on
the route of administration and
length of time between administration
and patient sampling. The ordinary
half-life of fosphenytoin is reduced
by 50% in patients with
hepatic or renal diseases, apparently
because of less protein binding of the
prodrug [5].
A stock solution of fosphenytoin (a
gift from Parke-Davis) in methanol
was added to two separate serum
pools—one without phenytoin (pool
A), and the other containing 12.4
mg/L of phenytoin (pool B)—to give
final fosphenytoin concentrations of
52, 39, 26, and 13 mg/L. Apparent
phenytoin concentrations in both sets
of samples were measured by all
four assays according to the manufacturers’
directions.
The TDx and AxSym assays use
homogeneous fluorescence polarization
(FPIA) technology and were run
on the TDxFlx ® and AxSym automated
analyzers, respectively. The
TDx Phenytoin (TDx) assay uses a
polyclonal sheep anti-phenytoin antiserum,
whereas the TDx Phenytoin
II (TDx-II) and AxSym Phenytoin II
(AxSym-II) assays use murine monoclonal
antisera. The ACS:180 Phenytoin
(ACS:180) is a heterogeneous
chemiluminescent immunoassay that
uses murine monoclonal antibody; it
was run on the automated, randomaccess
ACS:180 chemiluminescent
system [6–8]. The analytical range
(0.5–40 mg/L) and detection limit (1
mg/L) for the TDx and ACS:180 assays
are similar [8], but the AxSym-II
assay has a lower detection limit (0.5
mg/L). Results of the ACS:180 assay
agree well with both TDx and TDx-II
assays [7, 8], whereas the AxSym-II
assay correlates with the TDx-II assay
(AxSym-II package insert).
The results for fosphenytoin crossreactivity
in pool A for the four phenytoin
assays (Table 1) show that the
TDx-II cross-reacted very strongly
(250%) with fosphenytoin; such
cross-reactivity would result in seriously
high TDx-II assay results, even
in samples containing very low concentrations
(5 mg/L) of fosphenytoin.
Fosphenytoin cross-reactivity in
the absence of phenytoin for the
other three assays exhibited the following
order: ACS:180 TDx
TDx-II AxSym-II. However, although
the ACS:180 cross-reactivity
was independent of fosphenytoin
concentration, that of TDx and Ax-
Sym-II decreased with increasing
concentrations of fosphenytoin.
Table 1 also presents our data on
the interference of fosphenytoin in
the four assays in serum pool B,
containing 12.4 mg/L phenytoin.
The fosphenytoin cross-reactivity in
TDx and AxSym-II assays was 2-
and 3-fold greater, respectively, in
the presence of phenytoin than in its
absence. Given that the rate of fosphenytoin
metabolism may differ
from person to person or in different
disease states, the higher cross-reactivity
in presence of phenytoin is of
concern. Cross-reactivity in the ACS:
180 assay, however, was indepen-

Clinical Chemistry 44, No. 3, 1998 697
Table 1. Cross-reactivity of fosphenytoin in TDx Phenytoin, TDx Phenytoin II, AxSym Phenytoin II, and ACS:180 Phenytoin
assays in serum pools A (no phenytoin) and B (containing phenytoin).
Phenytoin equivalent, mg/L (and cross-reactivity a,b ,%)
TDx TDx-II AxSym-II ACS:180
Fosphenytoin
concn., mg/L
A B A B A B A B
0.0 ND 12.4 ND 12.5 ND 12.1 ND 12.4
13.0 5.5 (42) 22.2 (75) 33.0 (254) 3.8 (29) 22.2 (78) 6.5 (50) 19.0 (51)
20.0 7.5 (38) 26.6 (71) 4.8 (24) 26.8 (74) 9.8 (49) 22.4 (50)
26.0 9.2 (35) 30.1 (68) 5.3 (20) 28.5 (63) 12.5 (48) 25.6 (51)
39.0 13.0 (33) 35.4 (59) NA NA 20.0 (51) 32.0 (50)
52.0 16.4 (32) 6.7 (17) 27.6 (30) 27.3 (52)
a Cross-reactivity (%) in absence of phenytoin (pool A) 100 (apparent phenytoin concentration observed)/(fosphenytoin concentration in sample).
b Cross-reactivity (%) in presence of phenytoin (pool B) 100 [(apparent phenytoin concentration observed in presence of fosphenytoin) (phenytoin
concentration in absence of fosphenytoin)]/(fosphenytoin concentration in sample).
ND, not detected; , greater than the range of the assay (40 mg/L); NA, not available.
dent of the presence of phenytoin.
Similar data have also been found in
interference studies of oxaprozin in
the TDx-II assay, in which cross-reactivity
to oxaprozin increased by 5%
in the presence of 10 mg/L phenytoin
[9].
In summary, different phenytoin
immunoassays may cross-react differently
with fosphenytoin, resulting
in discordant results for samples containing
fosphenytoin. Because of this
problem, serum specimens taken
from patients treated with fosphenytoin
before all of the prodrug is metabolized
(2–4 h after the drug administration
is complete) may yield
misleading phenytoin immunoassay
results. Finally, whereas the ACS:180
assay shows consistent cross-reactivity
to fosphenytoin in samples with
or without phenytoin, the TDx assay
cross-reactivity is dependent on both
fosphenytoin and phenytoin concentrations.
This underlines the importance
of cross-reactivity determination
in both the presence and the
absence of the primary analyte.
References
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[Letter]. Clin Chem 1997;43:
1468–9.
Pradip Datta*
Chiron Diagnostics
E. Walpole, MA 02032
Amitava Dasgupta
Dept. of Pathol.
Univ. of New Mexico
Albuquerque, NM 87106
*Author for correspondence. Fax 508-
660-4591; e-mail pradip.datta@chirondiag.
com.