Reference Range

Newly diagnosed nephropathic cystinosis patients are found to have WBC cystine levels in the range of 3 to 10 nmol half cystine/mg protein, while control individuals and heterozygous carriers generally have levels between 0.2 and 0.5 nmol half cystine/mg protein, respectively.^1,4,50,51 While on cystine-depleting treatment, patients targeting “good therapeutic control” should be maintained under a threshold level of 1 nmol half cystine/mg protein to delay disease progression.^1,3,50

Correlation with Clinical Outcomes and Minimally Important Clinical Difference

The WBC cystine levels at which progressive renal failure and extrarenal complications can be prevented is unknown. Therefore, the 90^th percentile of cystine levels in polymorphonuclear cells (less than 1 nmol half cystine/mg protein) found in asymptomatic heterozygotes has historically been used as the upper WBC cystine limit when monitoring therapy.³

Two retrospective studies have examined the relationship between depletion of WBC cystine levels and renal disease in nephropathic cystinosis patients at the point of diagnosis up until renal failure or transplantation.^24,52 Both studies examined peak WBC cystine levels recorded throughout treatment, within similar stratifications (less than 1.0, 1.0 to 2.0, and greater than 2.0 nmol half cysteine/mg protein) as well as the rate of deterioration in renal function. In one study,²⁴ a mean WBC cystine level was derived from all readings between the patient’s first visit to the time of renal failure, with an average of 35 ± 3 readings per patient. A composite score involving extent of WBC cystine depletion and duration was calculated for each patient.²⁴ Age at renal failure was found to vary inversely with mean WBC value, with a large scatter, and it was estimated that for every 1 nmol half cystine/mg protein increase in mean WBC cystine value, approximately nine months of renal function was lost.²⁴ There was a direct relationship found between consistently low levels of WBC cystine (less than 1 nmol half cystine/mg protein), preservation of remaining renal glomerular function, and increased age of end-stage renal disease (ESRD) (R² = 0.61).²⁴ Similar results were found with the second study,⁵² which also evaluated WBC cystine and renal function in cystinosis patients treated with cystine-depleting therapy. For this study, a parameter for rate of renal deterioration was used, based upon the linear relationship between reciprocal serum creatinine and age.⁵² In patients where median WBC cystine was less than 1 nmol half cystine/mg protein, a lower parameter value of renal deterioration was found than in those with a median WBC cystine value between 1 and 2 nmol half cystine/mg protein (P = 0.064), and those noncompliant with treatment (P = 0.006).⁵² Although in both studies renal damage associated with cystinosis was considered to be irreversible, consistently low WBC cystine levels were found to be inversely related to the degree of existing renal damage.^24,52

One prospective study⁵³ evaluated the white matter integrity in 48 children between the ages of three years and seven years with a mean age of 5.5 ± 1.3 years. Half of these children were diagnosed with infantile nephropathic cystinosis with no known history of pulmonary dysfunction or diabetes mellitus, and the remaining half were healthy children. A positive correlation was found between white matter alterations and elevated WBC cystine levels in children greater than five years of age, indicating that there may be an early delay in white matter maturation in children with consistently high WBC cystine levels. Furthermore, there was a signal that increased WBC cystine may have an influence on white matter organization and connectivity, resulting in persistent cognitive skill deficits.⁵³

Reliability

HPLC-MS/MS is known to have a low detection limit of WBC cystine concentrations.⁵⁴ The standard deviation range with this technique is small (less than 15% root mean square error), which reflects the reliability of this method for determining intracellular levels of cystine.⁵⁴

The main variability in the assay lies in the method of WBC separation, therefore separation methods should be carried out soon after blood draw, and techniques must be standardized for each laboratory. Shipping of whole blood samples is not advisable due to increases in intracellular cystine content when samples are left at room temperature for 24 hours.^3,55 The clinical expert involved in the CDR review noted that there are currently few laboratories in Canada set up to analyze WBC cystine levels; the laboratory at Montreal Children’s Hospital uses the assay to analyze samples and Calgary is setting up another reference laboratory. The expert stated that samples must remain on ice prior to testing, and that specific protocols must be followed; therefore, errors may occur leading to inaccurate analyses.

There is also documentation to show variability in the types of cells used in the assay, as there is a preferential accumulation of cystine found in polymorphonuclear leukocytes (PMN) and monocytes.⁵⁵ When using a traditional mixed WBC sample, there is an unpredictable risk of a falsely low reading of cystine levels if there is a high proportion of lymphocytes in the sample.^3,4,51 An assay using immunopurified PMN leukocytes has been demonstrated to be a more sensitive method of measuring WBC cystine levels. One study examined 26 blood samples of nephropathic cystinosis patients split into duplicates and prepared both by mixed WBC method and PMN leukocyte method.⁵¹ The values of cystine/protein measured in the PMN leukocyte sample were found to be higher than the values in the mixed WBC sample (1.9 versus 1.0 nmol half cystine/mg protein, P < 0.001).⁵¹ Due to the risk of a falsely low reading with a mixed WBC sample, a PMN leukocyte assay has been recommended to be used instead whenever possible.^3,4,51,55