Pseudohyperphosphataemia in Multiple Myeloma

Abstract

We read the article by Hawkins! with great interest and would like to report our findings on the subject.Serum phosphate concentrations were measured in 41 patients who have multiple myeloma with identified serum paraproteins by four methods, the first three methods being performed on a Cobas Mira analyser. (1) Direct single step method using ammonium heptamolybdate in sulphuric acid (absorbance: 340 nm); (2) a modification of the molybdate method with the addition of albumin and formate-(absorbance: 340 nm); and (3) an enzymatic method in which phosphate ions react with inosine in the presence of nucleoside phosphorylase to give ribose-l-phosphate and hypoxanthine. Hypoxanthine is oxidized to xanthine, then to uric acid and the hydrogen peroxide formed measured as the quinoneimine product when it reacts with 4-aminophenazone (absorbance: 550 nm). (4) Technicon SMAC 3 (absorbance: 340 nm) by courtesy of Mr Ian Gibb, Department of Clinical Biochemistry, Royal Victoria Infirmary, Newcastle upon Tyne, UK. Paraprotein concentration was determined by integration of the electrophoretic peak area using a Corning 720 Scanning Densitometer on 33 samples.Spuriously high phosphate concentrations were obtained in the direct acid/molybdate method in 11 out of 41 samples due to the occurrence of turbidity in the reaction cuvettes. Turbidity, and hence spuriously high phosphate concentrations, was noted in two out of 41 samples by the albumin/formate method which had previously been reported by Bakker et al? to be free of this problem. The enzymatic method and the Technicon SMAC which incorporates a dialysis step were not susceptible to turbidity problems.We did not find any association between paraprotein type and the occurrence of turbidity by chi-squared analysis. The mean of 10gIO paraprotein concentrations in samples where turbidity was noted by methods 1 and/or 2, n = 11, was significantly higher than the mean of Ann Clin Biochem 1992; 29: 237-238 10gIO paraprotein concentrations in samples where turbidity was not observed in both methods 1 and 2, n=22 (P<O'OI) by the unpaired t-test. The paraprotein concentrations in the 'turbid' group ranged between 2 giL and 64 giL with a median of 21 giL while the paraprotein concentrations in the 'non-turbid' group ranged between 2 giL and 37 g/L with a median of 10 giL.In conclusion, the direct acid/molybdate and albumin/formate methods are not suitable for the measurement of serum phosphate concentrations in patients with multiple myeloma. Serum phosphate in patients with paraproteinaemia should be determined by techniques which incorporate prior removal of protein, e.g. dialysis or by an enzymatic method.

Spuriously elevated inorganic phosphate level in a multiple myeloma patient

“…Pseudohyperphosphatemia in the presence of serum paraproteins has been documented on a number of occasions since the introduction of direct methods for the estimation of phosphate. Lai, McClure and Cornell (1992) found that not all paraproteins caused this problem, although the phenomenon is certainly more common than previously realized. The paucity of previous reports may in part reflect the scant attention clinicians routinely pay to phosphate measurements (Larner, 1992, 1995).…”

Pseudohyperphosphataemia and Monoclonal Gammopathy

“…The paucity of previous reports of spurious hyperphosphataemia suggests either that serum globulins do not interfere with phosphate determinations in general (hence the rare association of hyperglobulinaemia with pseudohyperphosphataemia) or that interference is common but of sufficiently minor degree to escape clinical detection in most instances. Lai et al , using a direct acid/moiybdate method, found previously unrecognised spuriously high phosphate concentrations in 11 of 41 patients with multiple myeloma, 14 demonstrating that the phenomenon is probably not uncommon.…”

“…The precise physicochemical mechanisms by which certain proteins interfere with the phosphomolybdate assay remain to be fully elucidated. As colorimetric and spectrophotometric methods are based on light absorbance, turbidity of the reaction mixture due to precipitation of protein may be relevant 5 , 9 , 10 , 11 , 13 , 14 . By analogy, 11 the increased light absorbance of lipaemic sera is known to interfere with colorimetric assays for chloride 15 and phosphate 5 , 16 .…”

“…Isoelectric focusing of globulins from some patients has revealed isoelectric points well above the value for normal globulins 11 . An association between paraprotein type and occurrence of turbidity has not been established 14 …”

Authors' Reply

“…Having read with interest the recent contributions from Dr Hawkins' and Dr Lai and colleagues/ concerning pseudohyperphosphataernia in multiple 2 find that not all paraproteins cause this problem, although the phenomenon is certainly commoner than previously realized; the paucity of previous reports may in part reflect the little attention clinicians routinely pay to phosphate measurements. 10 The important clinical message to emerge from these studies is that unexplained and persistent hyperphosphataemia should prompt the search for a monoclonal protein prior to potentially inappropriate treatment with phosphate binding agents.…”