We observed acute, asymptomatic elevations in measured serum Pi to as high as 4.75 mmol/l in 3 patients receiving high-dose L-AMB. This prompted us to consider the possibility that this medication was interfering with the LX20 Pi assay used in our laboratory. Our results indicated that serum specimens containing L-AMB yield abnormally high results only when analyzed with the LX20 instrument.
To prove that this discrepancy is due to interference of L-AMB with the LX20 analytical method, we performed spiking studies to achieve L-AMB concentrations similar to those seen in the sera of patients receiving high-dose L-AMB therapy [8
]. Spiking was done with normal clear serum pool in order to mimic normal physiologic conditions, with normal saline in order to remove the possibility of alternate interfering substances, and with lipemic serum pool to verify that endogenous hyperlipidemia does not interefere with the LX20 Pi method. There was a direct linear relation between L-AMB concentration and the Pi result for both serum pools and saline. In addition, the regression slope for the clear and lipemic specimens was comparable indicating that lipemia does not interefere with the Pi method. Furthemore, removal of L-AMB by ultrafiltration of the spiked and patient’s sera eliminated the overestimation of Pi. Based on these results a 100 mg/l increase in serum L-AMB concentration would cause a 0.9 mmol/l increase in serum Pi result when measured by LX20 analyzer.
Our index case, patient A, did have laboratory evidence of true hyperphosphatemia. This included increased parathyroid hormone and suppressed vitamin D. We suspect that she did indeed have mild hyperphosphatemia, due to her baseline chronic renal insufficiency, with superimposed pseudohyperphosphatemia due to the presence of L-AMB in her serum. This is supported by the mildly elevated Pis values we obtained when her sera were retested on the Vitros analyzer.
Pharmacokinetic studies have demonstrated that mean area under the concentration-time curve from 0 to 24 h (AUC24
) and maximum concentration of drug in plasma (Cmax
) increased with sequentially higher daily doses of L-AMB and reached the upper limit at 10 mg/kg/day [8
]. Furthermore, both AUC24
increased with the duration (days 1, 7, and up to 24 days) of drug therapy. The gradual increase in the LX20 Pi results that we observed for patient A receiving high dose L-AMB therapy is indicative of gradual increase in the serum L-AMB concentration. Thus, the increase in the estimated CL-AMB
from 193.3 (day 13) to 344.4 (day 16) mg/l while on 10 mg/kg/day dosage, and from 82.2 (day 18) to 215.6 (day 21) mg/l while on 7.5 mg/kg/day dosage, is consistent with the reported pharmacokinetics of the drug. In addition, on the first day of the 7.5 mg/kg/day dosage (day 18) the estimated CL-AMB
is in good agreement with the reported Cmax
(mean ± SD = 75.9 ± 58.4 mg/l) [8
The method for measuring serum Pi is based on the reaction of phosphate anions with acidified (sulfuric acid) ammonium molybdate to form a yellow molybdenum-phosphate complex that absorbs light at 340 nm. The majority of clinical analyzers, including those used in this study, determine the Pi concentration by directly measuring the absorbance of this complex with ultraviolet spectrophotometry. However, the reaction conditions of the assay, such as the concentration of molybdate and acidity of the reagent, vary among the different analyzers. If the pH of the reaction is extremely low, it may cause hydrolysis of organic phosphate compounds that are normally present in serum (i.e., lipids) or are released from red blood cells during hemolysis. Since the LX20 assay is carried out at a pH<1 we suspect the interference of L-AMB with the LX20 method is due to hydrolysis of the organic phosphate contained in the lipid bilayer of the liposomes. In comparison, the Vitros Pi assay is carried out at pH of 4.2. Furthermore, the Vitros analyzer employs dry slide technology where the isotropically porous spreading layer could act as a filter preventing the L-AMB from reaching the molybdate layer.
After we investigated this problem an occurrence of interference in the LX20 Pi method was reported [22
] for 2 patients that were treated with L-AMB. However, the possible cause of this interference was suggested to be due to a turbidity caused by biodegradation of the liposomal vehicle that could lead to light scattering or precipitation.
There are other drug products, mostly the anti-cancer drugs such as cytarabin, daunorubicin, doxorubicin and tretinoin, that are encapsulated in the liposomal bilayer. However, these drugs are given at a relatively much lower dosage and at longer time intervals (for example, the recommended dose for liposomal doxorubicin is 20 mg/m2 once in 3 weeks, or 45 mg/m2 once in 4 weeks, or 40– 80 mg/m2 once every 3–4 weeks) that their effect on the LX20 Pi result should be clinically insignificant.
Approximately 20% of clinical laboratories in the U.S. use a LX20 analyzer. In fact, a LX20 analyzer was used by the institution that previously reported severe hyperphosphatemia in a pediatric patient treated with high dose (25 mg/kg/day) of L-AMB [9
]. As that patient was not reported to have suffered any sequelae of hyperphosphatemia, this may also have been a case of pseudohyperphosphatemia related to erroneous overestimation of Pi by the LX20 analyzer. Clinicians and laboratory personnel should recognize this interference in order to avoid unnecessary diagnostic procedures and interventions.