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Diagnosis and monitoring of cystinosis using immunomagnetically purified granulocytes.

Cystinosis is an autosomal recessive lysosomal storage disease affecting an estimated 1 in 100 000-200 000 births (1). The disorder results from dysfunction or absence of the cystinosin protein, a lysosomal membrane transporter that serves to export cystine from the lumen of the lysosome (2, 3). Accumulation of cystine at high concentrations causes crystal deposition in many tissues, and the disease results in multisystem symptoms, including hypothyroidism, corneal damage, and most notably renal Fanconi syndrome and eventual renal failure (4-7). A variety of insertion, nonsense, and missense genetic mutations of cystinosin have been described in patients, though the most common is a 57-kb deletion that results in a complete loss of the cystinosin protein (3, 8, 9). The aminothiol cysteamine was shown to effectively reduce intracellular cystine concentrations both in vitro and in vivo (10) by forming a mixed disulfide with cysteine in the lysosome and allowing egress of the adduct through the PQLC2 transporter (11). Regular use of cysteamine has resulted in much improved patient outcomes, with greatly reduced or delayed renal failure, and overall improvement of the other aforementioned symptoms (12-14).

Quantification of intracellular cystine is critical to both diagnosis and therapeutic monitoring of cystinosis patients. Though recent gene sequencing technology has added an alternative tool for diagnosis (15), the biochemical assay remains the benchmark for evaluating cysteamine dosage levels and compliance. Circulating leukocytes are an accessible tissue to use as a surrogate marker of overall cystine burden, but there are special considerations. It is critical to isolate leukocytes from the larger amount of cystine in the plasma, which involves a density floatation centrifugation step for the preparation of mixed leukocytes (16). Granulocyte preparations may have advantages, including better discrimination of heterozygotes from controls (17, 18), but the preparation is somewhat more involved. Furthermore, mixed leukocyte preparations must be performed promptly to obtain reliable results (19), and so our laboratory has provided materials and instructions to outside clinics to permit cell isolation and lysis to be performed at the point of care. The most sensitive analytical technologies for cystine measurement currently involve LC-MS/MS (20, 21). The intracellular cystine content is then normalized to protein in the lysed cellular fraction. The greatest source of variation in the assay is in the cell isolation, and the requirement to carry it out at remote clinic sites has limited access to testing in many cases. An assay that allows for direct shipping of whole blood to cystine determination laboratories would therefore be very beneficial.

For years, the generally accepted therapeutic target for cystine in mixed leukocytes has been <1.0 nmol half-cystine/mg of protein, which was originally based on a retrospective study (22) that showed better preservation of renal function in the group with the lowest median mixed leukocyte concentration among 3 groups, stratified a posteriori with median values of <1.0, 1.0-2.0, or >2.0. Though this range has served as a useful guideline for cystinotic patients, there is associated uncertainty because the total mixed leukocyte population comprises an unpredictable proportion of several differential cell types, each adding unknown, varying amounts of cystine and protein. Lymphocytes from cystinotic patients contain much less cystine than do the granulocytes (23), and because the percentage of lymphocytes in mixed leukocyte preparations can vary significantly, a patient will have an artifactually lower cystine/protein at a time when he or she happens to have a high concentration of lymphocytes.

Here we describe a novel approach for measuring cystine in granulocytes from whole blood without requiring gradient centrifugation and fractionation, using a straightforward and reproducible kit that uses a tetrameric antibody complex to positively select granulocytes, which can be purified by magnetic nanoparticles. The fact that granulocytes contain higher cystine content necessitates reconsideration of the therapeutic target.

Materials and Methods

SAMPLE COLLECTION

All patients had blood drawn in anticoagulant collection tubes containing acid-citrate dextrose (ACD). [2] We have previously shown that cystine determination of blood stored in lithium or sodium heparin collection tubes displayed significant variation after 24 h at either 4 [degrees]C or room temperature (19). ACD type A collection tubes were used in all the analyses described, and chosen over type B because of the higher concentration of citrate, because the more acidic buffer should minimize thiol exchange. Single tubes were collected (the absolute minimum volume in our validation trials was 1.8 mL). The effects of shipping time and temperature on cystine determination were also evaluated, and we found that temperature at or below 10 [degrees]C was maintained only if samples, immediately stored in refrigerated conditions (4 [degrees]C), were shipped overnight in a foam box containing a minimum of three 6 inch X 6 inch frozen (-20 [degrees]C) ice packs (data not shown).

POSITIVE SELECTION OF CD66b-EXPRESSING CELLS

Granulocytes were isolated from whole blood using a commercial kit for positive selection (StemCell Technologies Inc.), containing a tetrameric antibody complex that recognizes both the CD66b antigen and the dextran of iron-coated nanoparticles. Aliquots of buffer and nanoparticles were prepared immediately after receiving each kit, with each aliquot containing volume for single use. An additional aliquot of wash buffer containing 1X PBS, 1 mmol/L EDTA, and 2% fetal bovine serum was prepared daily. We directly compared a single position, toroidal magnet (Stem Cell Technologies) and a 4-position cylindrical magnet assembly (Life Technologies, ThermoFisher), and observed no difference in results when using 12-min incubation times in the magnet. For purposes of cost and ability to perform up to 4 preparations simultaneously, we used the latter array for nearly all of the experiments described. The 10X lysis buffer was diluted to 1 X with water at least an hour before cell isolation. To 1.8 mL of blood, we added 1.8 mL of 1X lysis buffer. After mixing, 90 [micro]L of the antibody solution was added and mixed by gentle pipetting. The mix was left at room temperature for 15 min, followed by addition of 90 [micro]L of nanoparticle suspension, mixing, and then incubation for 10 min at room temperature. The sample was then brought up to 9 mL with the wash buffer, mixed by pipetting, and then placed in the magnet for 12 min. While holding the tube in the magnet, the liquid was poured off. The nanoparticles and bound cells were suspended in 9 mL of wash buffer by pipetting from the walls of the tube, and the tube was then placed in the magnet for 12 min, and the liquid decanted. These wash steps were repeated twice more. After discarding the liquid following the final wash step, we removed excess liquid while the tube was inverted and in the magnet. After removal from the magnet, water (300 [micro]L) was added to each tube and the particles were removed from the walls by repeated pipetting. The cell resuspension was added to cold sulfosalicylic acid (SSA) (3% final concentration), and vortex-mixed for 30 s. The sample was stored overnight at -20 [degrees]C, and then spun the next day at 13 000g in a refrigerated microcentrifuge for 10 min. The supernatant was removed for cystine determination, and the pellet was resuspended in 0.1N sodium hydroxide for protein determination by either a standard Lowry or bicinchoninic acid assay (both were used for many of the samples with no significant difference in concentration between methods; data not shown). Cystine measurement was performed by LC-MS/MS as previously described (24). Mixed leukocyte preparation was performed as previously described (16), except that SSA at 3% final concentration was added to 300 [micro]L of the resuspended mixed leukocyte pellet instead of NEM to quench thiol exchange.

STATISTICAL ANALYSIS

Histograms and log-gaussian distributions of the various donor groups were analyzed using Excel (Microsoft) and plotted with Sigma Plot (Systat Software). PassingBablok regression analysis, a nonparametric tool used for evaluating biases when comparing unique methods (25), was performed using the R-package, 'mcr' (26). Imprecision calculations were derived using an equation for root mean square error (RMSE) optimized for studies in which many independent sets of duplicates were acquired (27). Kolmogorov-Smirnov tests were also performed in R.

ETHICS APPROVAL AND INFORMED CONSENT

This study was approved by the UCSD Institutional Review Board, and the study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. All persons gave their informed consent prior to their inclusion in the study.

Results

ANALYSIS OF METHOD REPRODUCIBILITY AND STABILITY

Thirty-nine samples were prepared and analyzed in duplicate to determine reproducibility of the new method (Table 1). The imprecision calculations were separated into 3 different response levels (low, medium, and high), 1 for controls and obligate heterozygotes, another for patients compliant with medication, and a third for primarily noncompliant patients (1 of the samples was from an untreated patient at the time of initial diagnosis). A 17% RMSE was calculated for the control/heterozygote range, and the patient groups had RMSE's <15%. The RMSE calculations for these duplicates as well as for all comparisons below are derived from the final readout of cystine/protein, which also incorporates the error of both the LC-MS/MS analysis and the protein determination.

As shown in Fig. 1, the isolation method using CD66b positive selection effectively purified granulocytes from other leukocytes. Fixed slides of post-preparative cell resuspensions (Fig. 1C) showed an absence of other cell types, with homogeneous populations of purified granulocytes appearing intact, similar to their appearance in whole blood.

A subset of the samples drawn at our clinic were tested for stability over the course of 24-30 h to mimic the timeframe in which a sample that was shipped overnight would arrive at our laboratory. A total of 22 samples were included in this comparison, in which the granulocyte isolation was performed within 1 h after collection of the blood and the remaining aliquot of blood was stored at 4-8 [degrees]C, and then granulocytes were isolated 24-30 h later. As Fig. 2 shows, there was a strong linearity between the day 0 and day 1 comparisons using the method (Pearson's r, 0.99; P < 0.001). The RMSE between day 0 and day 1 was <15% at each range evaluated, similar to the error of measuring duplicates immediately after blood draw (Table 1). Thirteen day 1 blood samples were compared to replicate blood aliquots stored at 4 [degrees]C for 48 h as well, to test for the upper limit of storage/shipping times. There was a substantially increased RMSE in the day 2 values (48% at the compliant patient level and 39% at the noncompliant/diagnostic range, Table 1), resulting in a recommendation that samples processed by the current method be evaluated ideally within 30 h. A small cohort of 7 samples were tested at 36 h, and showed increased deviation (22% RMSE), but further testing is warranted to determine the exact cutoff for testing beyond 30 h.

To determine the role of shipping temperature in the cystine determination results, we arranged for 8 unique patient samples to be shipped overnight, both packed with ice, and at room temperature. Samples sent at room temperature had a 24% RMSE compared to identical samples that were shipped in ice.

DISTRIBUTION OF VALUES

We determined the distribution of results from 84 samples in 3 cohorts (47 cystinotic patient samples [30 female, age 20.2 (10.8) years, range 3.5-36.2years], 27 obligate heterozygote [14 female, age 41.8 (9.0) years, range 29.5-62.1 years], and 10 control samples [6 female, age 38.5 (16.6) years, range 21-62 years]). The histograms in Fig. 3 show the distribution of cystine/protein concentrations for the 3 cohorts. Log-gaussian curves were calculated for each distribution and are shown overlaid in Fig. 3D. There was little overlap between controls and obligate heterozygotes using the new method; the heterozygotes exhibited a wider range of variation in cystine content. The cystine concentrations in patients ranged from the lower heterozygote range to concentrations as high as 8 nmol/mg protein. The wide range in patients reflects a diverse patient population that includes those highly compliant with medication, those with low compliance or taking low cysteamine dosages, in addition to differences in genotype. The 3[sigma] point for the heterozygotes (which included >99.9% of the measurements) was approximately 1.90 nmol half-cystine/mg protein.

COMPARISON OF GRANULOCYTE METHOD WITH MIXED LEUKOCYTE METHOD

Twenty-six unique blood samples were split into duplicates and prepared both by the mixed leukocyte method as well as the granulocyte method. The values of cystine/protein measured in purified granulocytes were higher than those measured in mixed leukocytes, as previously reported in other granulocyte isolation methods (17, 18). To account for error in both axes (i.e., mixed leukocyte and granulocyte methods), and to determine the extent of proportional and constant bias, Passing-Bablok regression analysis was performed (Fig. 4). By interpolation, using the slope (2.03) and y-intercept (-0.1) of the regression, the mixed leukocyte target value of 1.0 nmol half-cystine/mg protein corresponded to a value of 1.9 by the granulocyte method, although there was substantial scatter. The Pearson correlation coefficient was calculated as r = 0.80 (P < 0.001). On the basis of the 95% CI for the intercept including zero, the analysis suggests there was no constant bias, though proportional bias for much of the observed range was evident, be cause a slope of 1 (the line of identity in the plot) lay outside the majority of the CI; this was expected because the cystine content of granulocytes is generally higher than that of mixed leukocytes (16-18, 23). Kolmogorov-Smirnov test results (P value = 0.0890; D = 0.346) suggest the 2 methods have similar distributions (i.e., meets criterion P > 0.05).

Discussion

Here we demonstrate an alternative method for measuring intracellular cystine, specifically in granulocytes, which reduces the unpredictable contribution of varying proportions of differential cell types in mixed leukocyte preparations. Another major advantage is that, unlike the previous method of measuring cystine in mixed leukocyte preparations, the current method does not require immediate isolation of the cells following blood draw (19) at the point of care, and samples can now be collected in standard tubes (ACD) and shipped overnight for analysis. The imprecision from duplicate and interday analyses met standard guidelines for bioanalytical methods (28, 29) using tandem mass spectrometry (<15% for diseased range and <20% for control samples, which are near the lower limit of quantification). We believe this is equal or superior to other methodologies currently used for clinical cystine determination, though prior published methodologies have never formally reported the imprecision and accuracy of the entire process, which includes cell separation, redox quenching, chemometric analysis, and normalization to protein. Though previous methodologies involving granulocytes have been presented (17, 18, 30), they have used density gradient centrifugation for cell fractionation, whereas the current method uses a simple positive selection of granulocytes with immunomagnetic nanoparticles. The new process may prove simpler and require simpler resources for performing cell fractionation, allowing greater accessibility for cystine determination laboratories, and therefore more frequent patient follow-up.

We believe the most important advantage of this method is cell specificity in measuring intracellular cystine from blood samples, which should lead to more consistent results, independent of differential counts. It has been previously shown that lymphocytes, for example, maintain a low concentration of cystine compared to granulocytes (23). The cystine content of mixed leukocyte preparations is a complex function of the differential cell count, and variations could potentially cause a misleading evaluation of drug efficacy.

We have determined that samples show tolerable error when measured within 30 h of blood draw and shipped cold overnight. Longer shipping times and different temperatures of shipping conditions were measured in smaller cohorts, with current data showing unacceptable stability for standard analytical precision requirements. We have tested the distribution of nearly 50 cystinotic patient samples and have compared these results to that of 27 obligate heterozygote samples to obtain a reference range for diagnosis. Nonparametric regression analysis showed the 2 methods as meeting basic requirements of overall comparability, but substantial deviations from the mean scale factor are seen for a considerable number of paired measurements, preventing confident direct translation between methods for any single measurement. We attribute this variability to the unique cell differentials for the different samples, a factor that varies only in the mixed leukocyte method. The mean scale factor obtained from regression analysis was effectively 1.9 X that of the previous cutoff of 1 nmol half-cystine/mg protein from mixed leukocytes. The 3ff of the heterozygote distribution, a logical starting point in establishing a therapeutic reference range, was also 1.9, enabling us to more confidently set the initial target range of the new method to 1.9 on the basis of this close agreement. As with any new clinical method, the range will be reevaluated periodically as increased numbers of patient samples are analyzed.

In summary, we demonstrate a new method that allows reliable determination of intracellular cystine, using samples of whole blood ([greater than or equal to]1.8 mL) collected at remote locations without special preparation steps, provided that they can be shipped to arrive promptly (<30 h) and at cool temperature to a laboratory for isolation of granulocytes and chemometric analysis. This allows for broader access to therapeutic monitoring for cystinosis patients. However, the values for granulocyte cystine content are higher than those for mixed leukocytes, and the relationship is not simply linear, so the therapeutic target range needs to be reconsidered. The most useful therapeutic target corresponded well to both the regression equation comparing the granulocyte and mixed leukocyte method as well as the upper limit of the heterozygote log-gaussian distribution (3[sigma]).

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: J.A. Gangoiti, UCSD Biochemical Genetics Laboratory.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: I. Gertsman, the Cystinosis Research Foundation; B.A. Barshop, the Cystinosis Research Foundation.

Expert Testimony: None declared.

Patents: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, and final approval of manuscript.

Acknowledgments: We would like to thank the Cystinosis Research Foundation for funding and support. We are also grateful to the Cystinosis Research Foundation and the Cystinosis Research Network for supporting our visits to their meetings, and to all the families who participated.

References

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Ilya Gertsman, [1] Wynonna S. Johnson, [1] Connor Nishikawa, [1] Jon A. Gangoiti, [1] Bonnie Holmes, [1] Bruce A. Barshop [1] *

[1] Biochemical Genetics and Metabolomics Laboratory, Department of Pediatrics, University of California, San Diego, La Jolla, CA.

* Address correspondence to this author at: Biochemical Genetics and Metabolomics Laboratory Department of Pediatrics University of California, San Diego, 9500 Gilman Dr., La Jolla, CA, 92093-0830. Fax 619-543-3565, e-mail bbarshop@ucsd.edu.

Received November 24, 2015; accepted February 12, 2016.

Previously published online at DOI: 10.1373/clinchem.2015.252494

[2] Nonstandard abbreviations: ACD, acid-citrate dextrose; RMSE, root mean square error.

Caption: Fig.1. Purification of granulocytes. Micrographs (600x magnification using oil immersion and Wright's stain) showing (A), an unpurified blood sample, containing primarily red blood cells, as well as 2 lymphocytes and a granulocyte in the field of view; (B), sample purified with mixed leukocyte method; and (C), sample purified using the new immunomagnetic isolation, in which only granulocytes are visible. L, lymphocyte: G, granulocyte; M, monocyte.

Caption: Fig. 2. Interday correlation. Pearson's correlation coefficient, r =0.99; P <0.001. Regression line equation was y = 1.06/+0.06.

Caption: Fig. 3. Distributions of cystine measurements. (A-C), Histograms of controls (10 samples), obligate heterozygotes (27 samples), and patients (47 samples). Log-gaussian curves [y = A/[sigma]x[square root of 2[pi]] x [r.sup.-]([(ln(x) - [mu]).sup.2]/2[[sigma].sup.2])] describing the histograms overlaid (A = 0.9, [mu] = -1.75, [sigma] = 0.35 for controls, A 0.9, [mu] = -0.4, [sigma] = 0.35 for heterozygotes, A = 35, [mu] = 0.7, [sigma] = 0.55 for patients. (D) The log- gaussian curves overlaid on a single plane, and the value at [mu] +3 [sigma] for the heterozygote distribution is depicted with a dashed vertical line, corresponding to 1.9 nmol half-cystine/mg protein.

Caption: Fig. 4. Passing-Bablock regression analysis comparing mixed leukocyte to granulocyte methods. The plot shows the slope, y Intercept, and 95% CIs of the regression line fitting paired comparisons from identical blood samples of cystine/protein evaluated by the mixed leukocyte and immunomagnetic granulocyte methods. The granulocyte value corresponding to a mixed leukocyte value of 1.0 was approximately 1.9 nmol half-cystine/mg protein.
Table 1. Imprecision of intracellular cystine determination in
granulocytes.

Patient classification   Mean (% RMSE     Mean (% RMSE   Mean (% RMSE
                         of replicates)   of day 0 to    of day 1 to
                                          day 1)         day 2)

Low range: controls      0.35(17.1%)      0.31 (14.4%)   NA (a)
and heterozygotes
Midrange: compliant      1.16(11.0%)      1.00 (14.9%)   1.44 (48.1%)
patients
High range: untreated    3.24(14.9%)      2.78(11.9%)    3.37 (38.6%)
or noncompliant
patients

(a) NA, not available.
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Title Annotation:Endocrinology and Metabolism
Author:Gertsman, Ilya; Johnson, Wynonna S.; Nishikawa, Connor; Gangoiti, Jon A.; Holmes, Bonnie; Barshop, B
Publication:Clinical Chemistry
Article Type:Report
Date:May 1, 2016
Words:4399
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