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Characterization of a Blood Spot Creatine Kinase Skeletal Muscle Isoform Immunoassay for High-Throughput Newborn Screening of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD[4]] is a progressive, lethal X-linked neuromuscular disorder with an estimated average world-wide incidence of 1:5000 (range 1:9337-1:3599) male live births (1-3). DMD is manifested by rapidly progressive proximal muscle wasting as early as 3 years of age, culminating in respiratory insufficiency and cardiac failure leading to premature death in the second decade of life (4). DMD is caused by gene mutations that result in the absence of dystropin, a protein required to maintain muscle infrastructure, which leads to necrosis and leakage of the enzyme creatine kinase (CK; EC (5).

It has long been recognized that CK enzyme activities are increased in asymptomatic boys with DMD and the development of the dried blood spot (DBS) CK enzyme assays (6, 7) resulted in several screening programs being implemented/piloted. Approximately 1.8 million newborns/infants have been screened worldwide over the last 40 years (8). However, widespread screening for DMD has not been adopted and currently no country nationally undertakes newborn screening for DMD.

Various CK enzyme activity tests have been used with different cutoffs ranging from 200 to 2000 U/L. The difference in cutoffs is a result of several factors: (a) lack of standardized assays (bovine, rabbit, and human CK sources used as calibrators) to quantify CK activity in DBS; (b) use of different assay reaction parameters, i.e., kinetic and endpoint reaction monitoring with different reaction temperatures; and (c) differing time points for sample collection (<48 h to >4 weeks of age) (1). Furthermore, the existing tests are nonspecific, in that they measure total enzyme activity. CK exists in blood in 3 isoenzyme forms (CK-MM, CK-MB, and CK-BB). It is the CK-MM isoform that is found predominantly in skeletal muscle and is significantly increased in the serum of patients with muscular dystrophies (9, 10). These factors, together with issues of reagent and analyte instability and resulting problems encountered with the automation of such tests on commercial analyzers, have prevented the widespread use of CK enzyme activity tests for newborn screening.

The Wales DMD newborn screening program was the most comprehensive screening program to date and was operational for 21 years (1990--2011) and screened a total of 343 170 boys. This program had a sensitivity of 81.6%, a false-positive rate of 0.023%, and positive predictive value of 38.6%. However, 13 false-negative cases were identified. This screening program was terminated following the withdrawal of the external quality assurance scheme in the US (a consequence of insufficient participants to support a viable scheme) and because of the lack of sustainability of the reagents used in the CK enzyme test (1).

In recent years the therapeutic landscape has changed significantly with the development of new molecular therapies for DMD that have been shown to be disease modifying (11). Furthermore, progress in the development of these therapies has led to biomarker discovery studies and testing strategies to assess response to therapy (12). Consequently, the premise of an early diagnosis to increase efficacy of therapy has resulted in renewed interest in newborn screening for DMD. However, there has been no focus on developing a standardized and automated high-throughput DBS screening test for DMD.

The ideal screening test needs to be sensitive, specific, and robust. Immunoassay is one of the main analytical technologies used in newborn screening laboratories and offers the potential for greater sensitivity, specificity, and stability than tests relying on the use of enzyme activity. Furthermore, immunoassay would provide improved standardization and traceability as opposed to an enzyme activity--based assay which is more dependent on assay conditions. Although both the CK-MM and CK-MB isoforms offer the potential to distinguish between DMD-affected and unaffected boys when used as biomarkers of DMD, the concentrations of CK-MM in the serum of individuals with DMD are 10-fold higher than those of CK-MB (9, 10). In a recent biomarker discovery study it was demonstrated that CK-MM was a good biomarker for DMD screening because CK-MM is significantly increased at an early age (13).

Accordingly, a high-throughput immunoassay to detect the CK-MM in DBS was developed. The aim of this study was to characterize the performance of this CK-MM immunoassay for the newborn screening of DMD.

Materials and Methods


Purified human CK-MM (>99%) and CK-MB (>99%) were obtained from Lee Biosolutions Inc. Whatman 903 filter paper (GE Healthcare Life Sciences) was used for all sample collections and preparations.

The GSP[R] CK-MM kit reagents (currently under development) used in this study were manufactured by PerkinElmer (cat. no. 4152- 0010, for research use only). The reagents included in the kit are calibrator paper cassettes with spots prepared from sheep blood enriched with human CK-MM, control paper cassettes with spots prepared from human blood enriched with human CK-MM, microtitration strips coated with mouse monoclonal CK-MM antibodies, tracer reagent containing europium chelate-labeled mouse monoclonal CK-MM antibodies in buffered salt solution with BSA, and a ready-for-use Tris-buffered salt solution with BSA, bovine globulin, Tween[R] 20, diethylenetriaminepenta-acetic acid, polyethyleneglycol 6000, blockers, and sodium azide.


The GSP CK-MM prototype assay is a solid phase, 2-site immunofluorometric assay. Calibrators and DBS disks (3.2 mm) are punched into the assay wells. CK-MM is eluted by the addition of 100 [micro]L of the assay buffer (Tris-buffered salt solution) and 5 [micro]L of the anti-CKMM-Europium tracer solution. CK-MM in the sample reacts with the immobilized mouse monoclonal antibodies and europium chelate-labeled mouse monoclonal antibodies, which recognize 2 separate antigenic sites on the molecular surface of CK-MM. After a 4-h incubation step at 25 [degrees]C with interval shaking for 15 s at 900 rpm, the paper disc and excess unbound label is washed away from the wells. Addition of the DELFIA[R] inducer solution to the wells (200 [micro]L and shaking for 5 min) dissociates the europium ions from the tracer antibody attached label chelates into solution where they form highly fluorescent chelates. The fluorescence of each sample is proportional to the concentration of CK-MM.


The effect of cross-reactivity to the various CK isoforms was assessed by spiking purified isoforms (CK-MB and CK-BB) at 3 different concentrations into adult human whole blood. Spiked samples were dispensed onto filter paper, dried, and measured (n = 6 per sample). The limit of blank (LoB) was assessed by the preparation of 3 samples from 3 different lots of nonhuman erythrocytes diluted in artificial serum (saline with 150 g/L sucrose). The limit of detection (LoD) and quantification (LoQ) was assessed by the preparation of 4 DBS samples from a single batch of nonhuman erythrocytes diluted in artificial serum and spiked with pure human CK-MM at concentrations < 20 ng/mL. The samples were dispensed onto filter paper and dried. All samples were analyzed using 2 different reagent lots, on 2 different GSP analyzers on 3 separate days (n = 60 per sample).

The recovery of CK-MM from human whole blood DBS samples was assessed by spiking human whole blood from 2 different donors at 3 different concentrations each with CK-MM. The samples were dispensed onto filter paper, dried, and analyzed in a single analytical run (n = 4 per sample). The expected concentrations after the spiking of the blood matrices were calculated from the concentrations of the spiking solutions. Linearity was assessed by preparing 2 series of DBS. Series 1 was prepared using human whole blood spiked with CK-MM and diluted with a sample pool of nonhuman erythrocytes diluted in artificial serum. Series 2 was prepared in human whole blood spiked with CK-MM and diluted with a sample pool of human whole blood with a low endogenous CK-MM concentration. The samples were assayed on 2 separate analytical runs using 2 different reagent lots (n = 4 per sample in each run).

Assessment of the high-dose hook effect was performed by the analysis of a serial dilution of a human whole blood sample spiked to 80 000 ng/mL of CK-MM. The dilutions were dispensed onto filter paper, dried, and measured in a single analytical run (n = 4 per sample). Assay imprecision was assessed with 3 control samples prepared by spiking human CK-MM into adult human whole blood, dispensing onto filter paper, and drying. Over a time period of 20 working days, 2 runs of 1 plate each were performed daily. On each plate, the samples were measured in duplicate.

The effect of nonhomogeneity in the DBS and DBS volume on the CK-MM results was assessed by preparing 4 separate samples at the concentrations 60, 89, 372, and 1650 ng/mL. Aliquots of 10, 25, 50, 75, and 100 [micro]L of each sample were dispensed onto filter paper and dried. Twenty spots of each CK-MM concentration and DBS size category were analyzed by punching once from the center and twice from the edge. Only central punches were taken from 10-[micro]L samples (area too small for >1 punch) and only edge punches were taken from the 25-[micro]L samples.


Newborn DBS (n = 277) used in the evaluation were residual anonymized DBS samples from a newborn screening laboratory in Italy, released for research use in accordance with the laws and regulations of Italy to provide anonymized material for research use without ethical committee approval. Adult samples (n = 8) were provided by healthy volunteers at the study site (6 female and 2 male, ages 25-53 years) collected into heparin tubes and then spotted onto filter paper. The 10 samples from known DMD patients were residual DBS samples used for the preparation of control materials in the DBS CK enzyme assay (1).


Four series of DBS samples were prepared by spiking whole blood with CK-MM to cover the analytical range (50, 256, 537, and 2710 ng/mL). These samples were then stored at low humidity (sealed in bag with a desiccant), ambient humidity (samples stored in a box with no lid), and high humidity (samples stored in a sealed plastic box with wet paper towels) at the following temperatures; -20 [degrees]C, 4 [degrees]C, and at 37 [degrees]C. Samples were analyzed in duplicate.


Intra- and interassay components of imprecision were calculated with fully nested ANOVA. Analysis of differences between groups was carried out using t-tests. The population cutoff was calculated using fitted log-normal distribution data. All analyses were performed using R-language and R-Studio software.



The assay run time was 4 h 40 min and 26 plates (2304 samples/standards and control samples) can be analyzed on the GSP analyzer in 12 h 50 min. The onboard stability of the CK-MM assay reagents was demonstrated for up to 14 days on the analyzer. The cross-reactivity of this DBS CK-MM immunoassay with pure CK-MB was <5% across the concentration range used. No cross-reactivity was observed with CK-BB. The LoB was <1 ng/mL, the LoD was 3.1 ng/mL and the LOQ was 8 ng/mL. The assay was shown to be linear throughout the tested range of4-8840 ng/mL (Fig. 1). The mean recovery of spiked CK-MM from human whole blood DBS at concentrations ranging from 126 to 4790 ng/mL was 101% (range 87%-111%). In the assessment of the high-dose hook effect, the measured CK-MM concentration started to lag behind the actual CK-MM concentration at >10000 ng/mL and the measured concentration decreased at actual CK-MM concentrations >40000 ng/ mL. The intraassay (n = 80) and interassay CVs (n = 40) for the CK-MM control samples, respectively, were as follows: low (123 ng/mL) 6.1% and 5.2%, intermediate (410 ng/mL) 5.1% and 6.5%, and high (1780 ng/mL) 6.4% and 5.1%.

Smaller blood spot volumes (10 [micro]L and 25 [micro]L) produced significantly lower results for CK-MM at all concentrations when compared to blood spots with volumes [greater than or equal to] 50 [micro]L (P < 0.05). CK-MM concentrations were 14%-27% lower in the 10-[micro]L volume DBS vs those from 50-[micro]L volume DBS across the concentration range used (60.4, 89.3, 372.3, 1648.4 ng/mL) (P < 0.001). The CK-MM concentrations obtained using a peripheral punch were 4% higher than those obtained from a central punch (P < 0.001).


The newborn DBS samples (n = 277) had a mean CK-MM concentration of 155 ng/mL and a 99th centile of 563 ng/mL (Fig. 2). The mean CK-MM concentration from the 10 DMD cases was 5458 ng/mL (range 1217-9917 ng/mL). The DBS CK-MM concentrations from adult volunteers (n = 8) gave a mean CK-MM concentration of43.3 ng/mL (range 13.9-87.3 ng/mL). There was a significant positive association between the CK-MM and total CK enzyme activity results (r = 0.96, P <0.01) obtained for the DBS from the 10 DMD cases (Fig. 3). The mean total CK enzyme activity was 3439 U/L (range 772-6084 U/L).


Both humidity and temperature affected sample stability (Table 1). At room temperature with low humidity, CK-MM was stable for the study duration. The same was observed for temperatures of 4 [degrees]C and -20 [degrees]C with both ambient and low humidity conditions. In the other tested storage environments CK-MM degraded with time. Degradation was faster at higher temperatures and in conditions of higher humidity. Samples stored desiccated at -20 [degrees]C are stable for up to 1 year (data not shown).


There is renewed interest in implementing newborn screening programs for DMD as an earlier diagnosis to allow timely intervention with steroids and physiotherapy, which improves outcomes (14). Furthermore, molecular therapies for the treatment of DMD, which have been shown to be disease modifying, are also on the horizon (11). Progress in the development of these therapies has led to biomarker discovery and testing strategies to assess response to therapy (12). These studies have shown that CK-MM is a more specific marker of skeletal muscle injury than the total CK enzyme assay that measures the activity of all CK isoforms (13, 15). CK-MM reflects muscle fiber leakage and is significantly increased in DMD patients at an early age and decreases as a function of age, reflecting the progressive loss of muscle mass with disease progression. A similar profile is seen for other biomarkers of muscle membrane leakage (12).

Both CK-MM and CK-MB are increased in serum from DMD patients, but the increase in CK-MM is 10fold higher than that observed for CK-MB (9, 10). Therefore, the observed cross-reactivity of <5% for CK-MB in our assay has no impact upon its clinical utility. No cross-reactivity to the CK-BB isoform was observed for the assay. The specificity of this assay to the CK-MM isoform is important as a former DMD screening program, which used a DBS total CK activity assay, identified numerous false positives (1:4000) that were due to a benign autosomal-dominant anomaly, which leads to the overexpression of the CK-BB isoform in erythrocytes (16). Such false positives would not be detected using the assay described here, resulting in an improved positive predictive value of a screening program based on this assay.

The DBS CK-MM concentrations obtained in our assay for the unaffected newborns and those DBS from DMD patients are comparable to serum CK-MM concentrations reported previously (9, 10, 17, 18). The CK-MM concentrations observed in the 10 DMD cases showed complete separation from the unaffected newborn DBS.

The lower concentrations of CK-MM observed in the adult vs newborn DBS samples suggest that appropriate CK-MM cutoffs may need to be derived based upon the age at which the screening samples are collected. Therefore, those countries where DBS are collected at 12-48 h of life would be expected to have a higher cutoff than those where samples are collected on day 5-8 of life. Furthermore, it should be recognized that both ethnic origin and gender may also have an impact on the screening cutoffs generated for CK-MM (18, 19).

Previous screening protocols for DMD used a single-tier screening protocol based upon the CK activity in DBS within the first week of life. Those infants with a CK above the screening cutoff were then referred for follow-up for venous blood CK testing to confirm/refute the finding (1). Using such a protocol resulted in a positive predictive value of <40% (1). False positives were due to birth trauma, congenital hypothyroidism, and other conditions (1). Following the advancement of molecular technologies for DNA mutation analysis, Mendell and colleagues (2) developed a 2-tiered screening protocol where those samples with a raised CK enzyme activity were followed up by analysis for DMD gene deletions and mutations on the initial newborn screening sample, thereby improving the positive predictive value of the screening program.

A linear correlation between the CK-MM concentrations and CK enzyme activities of the ten DMD DBS samples was observed. The highest CK activity observed was 6084 U/L with a corresponding CK-MM concentration of 9920 ng/mL. Therefore, the high-dose hook effect observed in our assay at >25000 ng/mL is unlikely to produce false-negative cases because concentrations > 100 000 ng/mL would be needed to have a significant effect and it is unlikely that such concentrations would be observed in the neonatal period, unless the infant was acutely unwell, e.g., severe rhabdomyolysis (9).

The imprecision and performance of this assay are similar to those of other immunoassays currently used for DBS (20). Smaller volumes of DBS produced significantly lower results for CK-MM, consistent with findings observed for other DBS metabolites (21). Humidity and temperature had a significant effect on the stability of CK-MM in DBS samples. It is reported that CK enzyme activity in DBS is relatively unstable and activities can decrease by 30% within a week of sample collection when stored at ambient temperature with normal air humidity (22). In contrast, DBS CK-MM concentrations measured in our immunoassay decreased by only 13% after 8 days and 21% after 34 days at room temperature with ambient humidity, reflecting greater stability of the molecular epitopes relative to functional enzyme activity. CK-MM DBS samples should be protected from moisture as much as possible and should be stored refrigerated for the short term. Acceptable long-term stability was achieved using desiccants and freezing DBS samples at -20 [degrees]C.

If screening for DMD does become universal, caution must be observed because CK is a marker of the disease process and is not a direct function of the gene defect per se. Therefore, both false negatives and false positives will occur (1). The utility of this standardized and automated test as part of a 2-tier screening protocol (CK-MM followed by DNA analysis) should prove to be a very effective protocol to screen for DMD, and further work is required to operationalize this test into the various healthcare infrastructures around the world.

This study demonstrates that the development and validation of the DBS CK-MM immunoassay on a high-throughput commercial analyzer provides a standardized approach for mass screening of DMD in newborns.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following3 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: T. Korpimaki, PerkinElmer; P. Furu, PerkinElmer; H. Hakala, PerkinElmer; H. Polari, PerkinElmer.

Consultant or Advisory Role: None declared.

Stock Ownership: P. Furu, PerkinElmer.

Honoraria: None declared.

Research Funding: None declared.

Expert Testimony: None declared.

Patents: None declared.

Other Remuneration: S.J. Moat, PerkinElmer, Wallac Oy. Role of Sponsor: No sponsor was declared.

Acknowledgments: The authors thank Anne-Mari Varjonen, Minna Alkio, Tiina Pihlaja, Alan Dodd, and Agathe Vatel for technical assistance.


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Stuart J. Moat, [1, 2] * Teemu Korpimaki, [3] Petra Furu, [3] Harri Hakala, [3] Hanna Polari, [3] Liisa Merio, [3] Pauliina Makinen, [3] and Ian Weeks [2]

[1] Wales Newborn Screening Laboratory, Department of Medical Biochemistry, Immunology and Toxicology, University Hospital of Wales, Cardiff, Wales, UK; [2] School of Medicine, Cardiff University, Cardiff, Wales, UK; [3] Perkin Elmer, Wallac Oy, Turku, Finland.

* Address correspondence to this author at: Wales Newborn Screening Laboratory, Department of Medical Biochemistry, Immunology and Toxicology, University Hospital of Wales, Cardiff, CF144XW, UK. Fax +44-29-20-744065; e-mail

Received October 18, 2016; accepted December 7, 2016.

Previously published online at DOI: 10.1373/clinchem.2016.268425

[4] Nonstandard abbreviations: DMD, Duchenne muscular dystrophy; CK, creatine kinase; DBS, dried blood spot; CK-MM, creatine kinase muscle isoform; LoB, limit of blank; LoD, limit of detection; LoQ, limit of quantification.

Caption: Fig. 1. Calibration curve for CK-MM obtained using the PerkinElmer GSP analyzer.

Caption: Fig. 2. CK-MM concentrations in DBS from healthy adult volunteers (n = 8), newborn infants (n = 277), and older boys with DMD (n = 10). Results are shown as mean, lowest, and highest results in the adult and DMD groups and the mean, 1st and 99th centile for the newborn group.

Caption: Fig. 3. Association between CK-MM concentrations and CK enzyme activity in blood spots from older boys with DMD.
Table 1. Stability of CK-MM in DBS measured by immunoassay under
various conditions of temperature and humidity (a).

Storage conditions                    Length of storage, days

                                     0     1     3     6     8    10

-20 [degrees]C; low humidity        100   105   101   108   108   104
-20 [degrees]C, ambient humidity    100   102   106   109   104   107
4 [degrees]C, low humidity          100   95    101   94    97    93
4 [degrees]C, ambient humidity      100   103   103   101   102   99
4 [degrees]C, high humidity         100   87    78    66    67    66
Room temperature, low humidity      100   106   97    101   99    96
Room temperature, ambient humidity  100   105   93    89    87    85
Room temperature, high humidity     100   87    78    66    67    66
37 [degrees]C, low humidity         100   91    91    85    80    76
37 [degrees]C, ambient humidity     100   90    83    80    79    74
37 [degrees]C, high humidity        100   85    69    56    53    45

Storage conditions

                                    13    17    20    24    27    34

-20 [degrees]C; low humidity        108   109   95    103   110   104
-20 [degrees]C, ambient humidity    106   94    109   102   106   104
4 [degrees]C, low humidity          95    87    89    98    96    95
4 [degrees]C, ambient humidity      104   98    102   102   96    99
4 [degrees]C, high humidity         51    46    48    41    35    30
Room temperature, low humidity      92    95    91    101   97    90
Room temperature, ambient humidity  86    77    82    83    83    79
Room temperature, high humidity     51    46    48    41    35    30
37 [degrees]C, low humidity         75    72    77    73    68    70
37 [degrees]C, ambient humidity     78    60    62    64    67    58
37 [degrees]C, high humidity        37    29    29    21    19    17

(a) Results are % residual concentration and are mean of duplicate
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Title Annotation:Pediatric Clinical Chemistry
Author:Moat, Stuart J.; Korpimaki, Teemu; Furu, Petra; Hakala, Harri; Polari, Hanna; Merio, Liisa; Makinen,
Publication:Clinical Chemistry
Article Type:Report
Date:Apr 1, 2017
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