Capillary Electro 20031

Clinica Chimica Acta 330 (2003) 1 30 www.elsevier.
com/locate/clinchim
Review
Capillary electrophoresis and its application in the clinical laboratory

John R. Petersen*, Anthony O. Okorodudu, Amin Mohammad, Deborah A. Payne
Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA Received 8 July 2002; received in revised form 6 December 2002; accepted 17 December 2002
Abstract Over the past 10 years, capillary electrophoresis (CE) is an analytical tool that has shown great promise in replacing many conventional clinical laboratory methods, especially electrophoresis and high performance liquid chromatography (HPLC). The main attraction of CE was that it was fast, used small amounts of sample and reagents, and was extremely versatile, being able to separate large and small analytes, both neutral and charged. Because of this versatility, numerous methods for clinically relevant analytes have been developed. However, with the exception of the molecular diagnostic and forensic laboratories CE has not had a major impact. A possible reason is that CE is still perceived as requiring above-average technical expertise, precluding its use in a laboratory workforce that is less technically adept. With the introduction of multicapillary instruments that are more automated, less technique-dependent, in addition to the availability of commercial and cost effective test kit methods, CE may yet be accepted as a instrument routinely used in the clinical laboratories. Thus, this review will focus on the areas where CE shows the most potential to have the greatest impact on the clinical laboratory. These include analysis of proteins found in serum, urine, CSF and body fluids, immunosubstraction electrophoresis, hemoglobin variants, lipoproteins, carbohydrate-deficient transferrin (CDT), forensic and therapeutic drug screening, and molecular diagnostics. D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Capillary electrophoresis; Clinical laboratory; Clinical applications; Molecular diagnostics
Abbreviations: AGE, agarose gel electrophoresis; CAE, cellulose acetate; CE, capillary electrophoresis; CZE, capillary zone electrophoresis; CGE, capillary gel electrophoresis; CIEF, capillary isoelectric focusing; MEKC, micellar electrokinetic chromatography; CITP, capillary isotachophoresis; CE-IS, capillary electrophoresis, immunosubstraction; CSF, cerebrospinal fluid, immunoelectrophoresis; CDT, carbohydrate-deficient transferring; HPCEC, high performance cation-exchange chromatography; IEP, immunoelectrophoresis; IFE, immunofixation electrophoresis; LOH, loss of heterozygosity; MC, monoclonal component; SNP, single nucleotide polymorphisms; STR, short tandem repeat; TDM, therapeutic drug monitoring. * Corresponding author. Tel.: +1-409-772-1350; fax: +1-409772-9231. E-mail address: jrpeters@utmb.edu (J.R. Petersen).
1. Introduction Clinical laboratories are under tremendous pressure to develop assays that are accurate, precise, fast, capable of being automated, and yet, inexpensive. For electrophoresis, this has been difficult. However, by using capillary electrophoresis (CE) many, if not all, of these goals can potentially be attained. This is because CE, even though based on the movement of molecules in an electric field, is not restricted to areas classically assigned to electrophoresis, namely separation of large molecules based on size or charge. It can also separate molecules that have low molecular weight, in addition
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J.R. Petersen et al. / Clinica Chimica Acta 330 (2003) 130
to neutral compounds, such as steroids. The commonly used modes of CE, which in many instances can be accessed by simply altering the buffer composition, are capillary zone electrophoresis (CZE), gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), micellar electrokinetic chromatography (MEKC), and capillary isotachophoresis (CITP). CZE, where a capillary is filled with a relatively low viscosity buffer, is the simplest form of CE. The analytes of interest migrate from one end of the capillary to the other with velocities determined by the charge to mass ratio to form discrete peaks (zones). CGE separates analytes, such as DNA or proteins, based on molecular weight. A variety of compounds, such as bis-polyacrylamide, agarose, or methylcellulose, have been used to act as molecular sieve to cause separation. MEKC takes advantage of a property of surfactants, such as sodium dodecyl sulfate (SDS), known as the critical micelle concentration where micelles (aggregates) are formed once the surfactant reaches a specific concentration. By taking advantage of the differential distribution of various analytes in the micelles separation of neutral and charged analytes is possible. CIEF is similar to classical isoelectric focusing where peptides and proteins are separated on the basis of isoelectric point (pI) by generating a pH gradient using ampholytes. The difference is that instead of creating a pH gradient in a gel it is created inside a capillary. CITP is similar to CZE, except that a discontinuous buffer (a combination of two buffers that have different mobilities) is used causing the analytes of interest to be concentrated in zones between the leading and terminating constituents. Enhanced separation can be achieved by using spacer compounds, such as various amino acids. The large number of methods and instruments, such as the traditional chromatographic techniques of gel electrophoresis, GC, and HPLC, that potentially can be replaced by CE along with the high separation efficiency (high number of theoretical plates) make CE an attractive technique in the clinical laboratories. Over the last 5 years, a number of review articles [1 12] and books [13,14] have been written on the use of CE in the clinical laboratory, however, with the exception of molecular diagnostics CE still has not had a major impact in the clinical laboratory. Although there are over 900 tests that can be performed in the clinical laboratory to help in the assessment of a patients health status it is unreasonable to think that
CE can be only used in more than a small fraction of these tests. Thus, this review will focus on the areas where CE shows the potential to have the greatest impact in the clinical laboratory and include analysis of various serum and body fluid proteins, forensic and therapeutic drug screening, and molecular diagnostics. Immunoassay, which shows future promise, especially with the advent of chip technology, will not be covered.
2. Applications of capillary electrophoresis in clinical settings: 2.1. Protein analysis 2.1.1. Serum protein Currently, the CE method that has had the most extensive clinical validation is in the analysis of serum proteins. The first indication that CE could be used in the separation of serum proteins into the classic six bands, e.g. albumin, a-1 globulin, a-2 globulin, h-1 and h-2 globulins, and g globulin, was reported by Jorgenson and Lukas [15]. Although variations in these protein fractions can be correlated with a patients health status it is usually the changes that occur in h and g fractions that are of the most clinical interest. It is in these fractions that monoclonal proteins are normally found and whose detection is an important part of the laboratory evaluation of patients with lymphoproliferative diseases. Large monoclonal bands are usually present in patients with multiple myeloma or Waldenstroms disease; however, lower concentrations may be seen in a variety of other diseases such as light chain disease, leukemia, lymphoma, amyloidosis, or monoclonal gammopathy of undetermined significance (MGUS). Patients that fall into the MGUS category should have annual follow-ups because the chance that these patients will develop myeloma or other lymphoproliferative diseases is increased [16]. Classically, screening serum has been done by either cellulose acetate (CAE) or agarose gel (AGE) electrophoresis. While the reagents for both of these methods are relatively inexpensive, these methods require a significant amount of labor. Considerable time is spent by technologists in applying the samples, fixing, staining, washing the gels to remove excess stain, and performing the densitometric scans. Although there has been
increased automation, classical electrophoresis methods still require a significant amount of labor and it is for this reason that various groups have evaluated CE with the hope that the labor component could be reduced. Although numerous research groups [17 19], including one from the industry sector [20], have looked at ways to improve on the initial method of Jorgenson and Lukas, none looked at large numbers of patients to fully evaluate the method as a replacement for AGE or CAE. It was not until Jenkins and Guerin [21 23], who used a single capillary CE instrument, was CE compared to AGE with large numbers of clinical samples. In these reports, a total of 6500 samples, of which 1357 contained a monoclonal component (MC), were compared. They were able to detect four IgA and one IgG MC that were not detected by AGE. Conversely, they reported that CE missed five IgM and three IgG MCs. However, by increasing the ionic strength and pH of the run buffer they were able to detect these MC indicating the importance of method validation with known samples. CE also missed a 1 g/l light chain band that was visible in AGE although in 11 other cases free light chain was detected by both CE and AGE. Although eight mini-MCs co-migrating with C3 were not detected by CE, they reported that most miniMCs in the range of 0.5 1 g/l were usually present as an irregularity in the gamma region. Due to potential interferences, such as elevated C-reactive protein or oligoclonal banding due to an acute inflammatory response identification of an irregularity as a miniMC required careful interpretation [24]. They also reported a good correlation (r = 0.96) between CE and AGE in estimating the monoclonal concentrations ranging from 1 to 71 g/l. In a prospective study, Katzmann et al. [25] used immunotyping to identify the presence or absence of an MC in 1518 clinical samples. They found that CE was more sensitive than AGE (94.9% vs. 90.7%) with similar specificity (98.6% vs. 98.9% for AGE). This was confirmed by Bossuyt et al. [26] using 58 specimens previously identified as containing an MC. Using immunofixation as the gold standard CE was able to detect an MC in 95% of the known samples. The same group found the same results in a prospective study of 1692 patients that were being screened for the presence of an MC or as follow-up of a known MC [27]. More recently, Jonsson et al. [28] developed computer algo-
rithms to detect MCs in the h and g regions. Using these algorithms, only one MC (concentration 1 g/l) out of 95 was not identified for a sensitivity of 98.9% while correctly identifying 604 of 607 samples that did not have an MC for a specificity of 99.5%. They were also able to correctly identify an MC due to circulating light chain in serum samples from five of nine patients with Bence Jones proteinuria even though light chain was not visible in two of these serum samples by AGE. In the initial evaluation of the Beckman Paragon CZE 2000 (Beckman Coulter, Fullerton, CA, USA), the first commercial CE developed exclusively for clinical purposes, Jolliff and Blessum [29] compared the precision of CE and AGE. They found that the within-capillary precision for CE was 0.6 3.3% (N = 10) and the between-capillary precision was 1.8 4.5% (N = 120), compared to an AGE precision of 3.8 8.0% (N = 120) for the classic five serum protein bands. This confirmed a previous study by Winen and van Dieijen-Vesser [32] who found a within- and between-capillary precision of < 3.1% for all protein bands. In addition, Jolliff and Blessum evaluated the ability of CE and AGE to discriminate between various disease states in 240 patient samples. They found that, although the concentrations of the various protein fractions were not identical, there was a 96% concordance between the two methods in the discrimination of various disease states involving a variety of dysproteinemias, such as hypogammaglobulinemia, acute and chronic inflammation, nephrosis, cirrhosis, poly- and monoclonal gammopathies. A normal serum protein electrophoregram using the Beckman Paragon CZE 2000 is shown in Fig. 1. Bossuyt et al. [30] reached similar conclusions when they studied 524 patient samples covering a similar population of dysproteinemias. They found a poor correlation between the concentrations of each protein fraction determined by CE and AGE (r < 0.61 for all protein fractions), although there was a reasonable correlation with CAE (r>0.85 for all fractions except the h globulin which was 0.67). These differences can be traced to the use of dye binding to detect the various proteins in AGE and CAE vs. CEs use of the amide bond absorbance at 200 214 nm. This will affect the quantification but will not change the clinical interpretation. Petrini et al. [31] established normal ranges for CE using 167 samples that were normal by CAE and
Fig. 1. Serum protein electrophoresis using the Beckman Paragon CZER 2000. Normal serum protein electrophoregram seen using the Beckman Paragon CZER. All parameters are preset by manufacture. Separation Conditions: capillary length is 20 cm (18 cm to detector) 25 Am (I.D.); detection was at 214 nm; temperature, 24 jC; a proprietary buffer is used at 10.5 kV for 4 min; injection by vacuum for 1 s. Superimposed on the electrophoregram is where the major components of the various protein bands would be expected to be found. The figure was kindly supplied by Beckman Coulter.
then verified the ranges using one thousand normal and abnormal serum samples. These studies have been confirmed by other groups in comparing CE with AGE [32 38]. Recently, Bossuyt et al. [39] expanded on these normal range studies by establishing pediatric serum protein reference ranges using a small number of children (151 males and 44 females, ages 1 16 years). Although there were differences between CE and AGE/ CAE, in particular the reference ranges (see Table 1); CE was found to be an efficient, reproducible method for the separation of serum proteins that is able to identify abnormalities that are not detected by either AGE or CAE. In addition, the technical skills required for the newer CE instruments, such as Beckman Coulters Paragon CZE 2000k or Sebias CAPILLARYSk, are less than those required for AGE/CAE making CE is a viable alternative to conventional electrophoresis, especially when large numbers of samples are submitted for serum protein analysis.
The ability to identify whether a pleural fluid is a transudate or exudate can affect patient care. Inaccurate classification can lead to unnecessary invasive procedures. To overcome this problem, a set of criteria was developed by Light et al. [56] to help identify exudates. While this criteria is sensitive (>95%), it is not specific (f 70%). Recently, CE has been used to analyze body fluids, pleural and ascetic, from a small number of patients (N = 47) using methods developed for serum protein separations [55]. By using CE to calculate the a2-macroglobulin/albumin ratio, the authors reported that CE could identify exudates with a sensitivity of 81% and a specificity of 91%. Combining the Light criteria with the a2-macroglobulin/albumin ratio obtained by CE, all exudates were correctly identified (sensitivity100%); however, a small number of transudates (2 out of 11) were incorrectly identified. Although evaluation of additional samples is needed to confirm the initial results of Claeys et al.,
Table 1 Reference ranges (% total protein content) for the five serum protein fraction by capillary electrophoresis. Comparison of representative literature results with agarose gel electrophoresis Author Beckman Coulter (Paragon CZE 2000) Sebia (Capillarys CE) Jolliff and Blessum (Paragon CZE 2000) Bossnyt et al. (Paragon CZE 2000) Petrini, et al. (Paragon CZE 2000) Bossnyt et al. (Pediatric) (Paragon CZE 2000) Reference Package Insert Package Insert [29] [30] [31] [39] Age (years) Adult Adult Adult Adult Adult Age 1 2 Age 3 4 Age 5 9 Age 10 14 Adult Adult Adult Albumin (%) 49.7 64.4 56 66 52.0 67.0 M 41.7 52.3 F 37.4 49.8 53.0 67.6 54.7 70.4 53.9 70.4 52.6 66.3 54.1 69.1 46.6 62.6 59.7 70.6 54.6 68.5 Alpha 1 (%) 4.8 10.1 3.6 6.0 3.8 8.3 2.6 4.5 2.6 5.1 3.3 8.4 4.2 8.5 4.8 8.1 4.2 7.6 4.4 8.0 1.7 4.1 1.4 2.7 3.6 7.8 Alpha 2 (%) 8.5 15.1 6 10 5.8 12.4 3.4 6.4 3.9 6.4 5.0 11.0 7.0 15.6 7.6 15.2 7.4 13.5 6.8 11.4 5.9 13.5 7.2 11.1 5.2 10.7 Beta (%) 7.8 13.1 9 14.5 9.8 13.0 5.8 9.5 5.5 8.7 8.1 13.3 7.5 11.6 7.4 11.5 7.9 11.3 8.5 12.9 10.9 18.9 8.0 14.7 8.5 13.7 Gamma (%) 10.5 19.5 11 19 9.3 20.4 5.3 13.2 9.6 20.5
Helena SPIFE 2000 Sebia Hydragel Beckman Paragon
Package Insert Package Insert Package Insert
4.7 16.0 7.1 17.8 8.5 18.7 8.8 17.6 11.6 24.4 8.4 16.3 9.7 19.2
the combination of Lights criteria in conjunction with the a2-macroglobulin/albumin ratio using CE should aid in the identification of transudates enhancing patient care. A possible exception to the usefulness of CE to identify serum proteins abnormalities is its apparent inability to detect an a-1 antitrypsin deficiency, specifically when the SS, SZ, and MS phenotypes are present [40], although the ZZ phenotype is detectable [30,40]. Although only one report, Gonzalez-Sagrado et al. indicated that if the concentration of the a-1 globulin fraction by CE was less than 4 g/l, then quantitation of a-1 antitrypsin is required. Perhaps, separate quantification of the a-1 acid glycoprotein and a-1 antitrypsin areas, which are distinguishable by CE (see Fig. 1), would eliminate this problem. However, until this issue can be resolved, AGEand not CEshould be used as a screening method if a deficiency in a-1 antitrypsin is suspected. Even though CE should not be used to screen for a-1 antitrypsin deficiency, it has been shown to be useful in the phenotyping of a-1 antitrypsin and thus could be used to identify a-1 antitrypsin variants associated with clinical disease [41].
2.1.2. Immunosubstraction electrophoresis In addition to detection, classification or typing of an MC is important in the long-term follow-up of patients [16]. For many of these patients, quantification and typing is used as a marker to help determine when malignancy has occurred or to monitor therapy. The most common methods to type an MC are by immunoelectrophoresis (IEP) or immunofixation electrophoresis (IFE). Although IFE is more sensitive than IEP [42 44], both are technically demanding and require a significant amount of labor. At present, neither of the classical forms of IFE nor IEP is possible by CE. However, a CE compatible method that can be used to type MC has been developed by modifying a procedure described by Aguzzi and Poggi [45]. This technique is called immunosubstraction electrophoresis or CE-IS. The method involves the removal of specific proteins from a sample using beads coated with anti-heavy chain (IgG, IgM, IgA) or anti-light chain (n or E) antibodies before separation by CE [46]. Identification is based upon which of the immunosorbants removes the MC (Fig. 2). Currently, the only commercial clinical instrument that uses CE-IS to type an MC is the Paragon CZE
Fig. 2. CE-IS of a monoclonal IgM-n using the Beckman Paragon CZER 2000. CE immunosubtraction using the Beckman Paragon CZER. All parameters are preset by manufacture. Separation Conditions: capillary length is 20 cm (18 cm to detector) 25 Am; temperature, 24 jC; a proprietary buffer was used at 9.5 kV for 5 min; injection by vacuum for 1 s. The serum protein electropherogram (SPE) shows a monoclonal peak in the beta region. After treatment of the serum is treated with beads coated with the various antibodies (IgG, IgM, IgA, n, E) the monoclonal peak is removed using beads coated with anti-IgM and anti-n. The monoclonal protein is thus an IgM-n. The figure was kindly supplied by Beckman Coulter.
2000. In the first published study using this instrument, Jolliff and Blessum evaluated the ability of CE-IS to correctly type an MC and found that if an MC was detectable (>0.5g/l for IgG and >0.75 g/l for IgA or IgM), the results of CE-IS were identical with IFE [29]. This was confirmed by Katzmann et al. [25], who also found that when an abnormality was visible in the electropherogram, CE-IS was able to correctly type the MC. The same group of researchers also found that CE-IS is easier to perform than IFE, and was extremely helpful in the interpretation of abnormalities that are difficult to analyze using AGE/IFE [47]. In a smaller study on 58 patients with an MC previously identified by IFE/IEP, Bossuyt et al. [48] found that the CE-IS is superior to CAE and equivalent to AGE in the identification of an MC. They indicated that increased automation and precision make CE-IS an acceptable replacement for the currently used IEF methods to
detect, quantify, and type MC. They did note, however, that CE-IS, like CAE and AGE, may miss a lowconcentration MC due to an MGUS, light chain disease, some lymphomas, etc., and suggested that if an MC is clinically suspected, a complimentary, more sensitive method, such as IFE, should be used. Similar conclusions were also reached by Bienvenu et al. [49] and Henskens et al. [50]. Litwin et al. [51], however, were not convinced of the utility of CE-IS. In their study, they first compared the ability of CE and AGE to detect the presence of an MC and found, like other researchers, that CE was more sensitive than AGE. However, the second part of the study, evaluating the ability to correctly interpret CE-IS results, found that four individuals were able to correctly identify and type only 60 75% of the MC identified by IFE. Although this is unacceptable, if the MC was prominent and discrete all MC were properly identified.
Interpretation of the subtle differences in the electrophoresis before and after CE-IS was difficult only when the MC was small and/or superimposed on a polyclonal background. As with any new technology, this problem may be resolved by appropriate, focused training on how to interpret the electropherograms or use of computer algorithms, such as the version developed by Jonsson et al. [28] to help interpret electrophorograms before and after IS. Overall, CEIS is an excellent alternative to IFE for the great majority of MC evaluations. However, if a question remains after CE-IS then IFE should be used to confirm the interpretation. It is also possible to use CE to detect cryoglobulins and, although typing of cryoglobulins using CE-IS has not been published, it would obviously be the next step in the evaluation. Like AGE, detection by CE is sometimes difficult due to precipitation of the cryoglobulin at temperatures lower than 37 jC. However, visual inspection of the electropherogram along with clinical information will usually indicate the presence of a serum abnormality that requires further investigation [33,52,53]. The advantage that CE has over AGE is that it is able to detect an MC in the cryoglobulin precipitate even when only low levels of protein are available. If quantification is desired precipitation of the cryoglobulin followed by redissolving, the precipitate before analysis is usually required. However, if the cryoglobulin concentration is < 4 g/l, underestimation can occur [54]. Thus, CE along with the future use of CE-IS, will give physicians additional information about a cryoglobulin faster and in a more costeffective manner. 2.1.3. Urine proteins Increased excretion of protein in urine (proteinuria) is one of the most common abnormalities seen in the clinical laboratory and can be caused by a number of pathologic conditions affecting the kidney and urinary tract [57,58]. Characterization of these proteins by electrophoresis can be helpful in determining the cause of increased urinary protein, whether tubular or glomerular. Classically, analysis has been by carried out AGE or CAE, although more recently CE has been successfully used to analyze urine proteins pattern. One of the problems with analyzing urine proteins by CE is that many compounds present in urine can absorb at the same wavelength (200 or 214 nm) that is
used to detect proteins, causing interpretation problems. To overcome this problem, Friedberg and Shahabe [59] used ethanol precipitation or low molecular weight cut-off filters (>15,000) along with washing, to concentrate urine proteins. They found good agreement between CE and AGE for selective and nonselective proteinuria and the detection of MC in urine. In a separate paper, Jenkins [60] successfully used CE to separate and detect Bence Jones proteinuria (range 0.04 9.7g/l), intact immunoglobulins, and Tamm Horsfall proteins in unconcentrated urine samples. More recently, Kolios et al. [61] used CE to evaluate unconcentrated urine from patients with glomerular, tubular, mixed tubular and glomerular, and overflow proteinuria. They found that as long as the protein concentration is slightly above normal (>150 200 mg/l), the analysis correlated very well with AGE. Both CE and AGE are able to distinguish tubular from glomerular proteinuria and detect the presence of free light chain. The advantage that CE holds over AGE is that concentration of the urine is not necessary, making CE faster and more cost-effective. In addition, although low molecular weight (< 10,000) substances have yet to be shown to have clinical significance, the ability to detect these substances in unconcentrated urine by CE may help give a more complete picture of the pathologic process occurring in the kidney. 2.1.4. Cerebrospinal fluid proteins The analysis of cerebrospinal fluid (CSF) proteins can be useful in the diagnosis and management of a variety of neurological diseases including conditions that cause immune responses, destructive brain diseases, or a breakdown in the blood brain barrier. The immune process may also produce a polyclonal or, perhaps more importantly, oligoclonal bands in the gamma region, which if combined with the IgG index can be helpful in making a diagnosis of multiple sclerosis in >95% of the cases [62]. Oligoclonal band may also be present in patients with a variety of other neurological conditions [63 65]. At present, the method routinely used in the clinical laboratory for the detection of protein profiles in CSF is AGE, which is labor-intensive and requires concentration of the CSF to visualize the protein bands. Crowdrey et al. [66] analyzed unconcentrated CSF from 30 random patients and were able to separate
between 20 and 25 peaks. Differences were noted in the electrophorograms; however, no attempt was made to correlate these differences with disease states nor was the method able to distinguish the fine features in the gamma region. Hiraoka et al. [67,68] used capillary gel electrophoresis to analyze proteins present in CSF. They were able to separate both high and low molecular weight proteins and found that an index based on the serum and CSF albumin and a2-macroglobulin was potentially useful in evaluating the blood brain barrier in patients with neurological disorders. Manabe et al. [69] used capillary isoelectric focusing (CIEF) to separate proteins in unconcentrated CSF and, although dialysis was required, they were able to identify proteins in the IgG region that were not obvious in the serum of the same patient. Sanders et al. [70] modified a method used to separate serum proteins for the analysis of CSF. This method gave a concordance of 87% when compared to high-resolution AGE in the detection of oligoclonal banding. When trying to use unconcentrated CSF, however, they found that a 3- to 6-fold increase in the injection volume was required to detect oligoclonal banding resulting in a loss of resolution. Sanders et al. speculated that by using extended path length cells (bubble or Z-cells) that give a 5- to 10-fold increase in sensitivity without a corresponding loss of resolution, detection of oligoclonal bands in unconcentrated CSF may be possible. One interesting feature of CE is its ability to calculate a CSF Ig index. The concentration of the albumin and immunoglobulin region can be estimated by using CE to separate both serum and CSF proteins. Substituting these values into the classic CSF IgG index equation [71] gives a CSF Ig Index. A good correlation (r = 0.934) was found when they were compared to the results obtained using nephelometry with only 2 discordant results out of 24 [70]. A side benefit of using CE to detect oligoclonal banding is this ability to determine the presence or absence of oligoclonal bands in addition to calculating a CSF Ig index using the same instrument. This reduces the number of instruments required to analyze CSF leading to a reduction in costs. 2.1.5. Hemoglobin and its variants Analysis of hemoglobinopathies and thalassemias is important in the diagnosis and management of the over 600 known congenital hemoglobin (Hb) defects [72]. Analysis requires identification and quantifica-
tion of the abnormal Hb along with the minor Hb components [73]. Historically, these variants have been characterized by electrophoretic separation using alkaline CAE or AGE combined with citrate agar at acid pH. However, these techniques are being replaced by high performance cation exchange chromatography (HPCEC) [74 76] and affinity methods. More recently, CZE [77 84] and CIEF [85 96] have been found to be helpful in making the differential diagnosis of a variety of hemoglobinopathies such as S/beta thalassemia, G-Philadelphia trait, S/C-Harlem disease, etc. Much of the pioneering work with CIEF identification of hemoglobinopathies was done by Hempe, Craver et al. [85,88,89,95]. In this initial work, they were able to identify the four most common variants (C, S, D, G), but could not separate Hb E, O-Harlem, and O-Arab due to the similar isoelectric points (pI). However, use of family history along with a sickle solubility test for O-Harlem could differentiate these variants. Using a standard sample containing known Hb variants and constructing a linear regression equation of pI vs. elution time, the pI of unknown variant hemoglobins can be calculated. The calculated pI is then used to help identify the unknown Hb. Hempe et al. [89] also used CIEF to analyze the isoelectric points of a number of common and uncommon variants using patient samples previously verified by a reference laboratory, commercial control samples, and proficiency testing samples. As with most CE methods, imprecision due to changes in the electroosmotic flow (EOF) can be a problem. By running a standard sample each day in addition to using the Hb A present in each sample as the reference peak, Hempe et al. was able to correct for changes in the EOF. In a separate set of experiments, Mohammad et al. added two pI markers, one at 6.6 and the other at 7.7, to samples before performing CIEF [91]. Using these markers, a migration index, which related the migration time of the Hb variant to the migration times of the two markers, was calculated. This reduced the migration CVof the Hb C, S, F, and A by approximately 4-fold from f 16% to f 4%. CIEF can also simultaneously detect the concentrations of minor Hb variants such as Hb A2 and Hb F, which are useful not only in the diagnosis of thalassemia syndromes but also in the long-term follow-up of sickle cell patients being treated with hydroxylurea. While it is possible to quantify Hb A2 and Hb F using
AGE or HPCEC alone, these methods are commonly supplemented by minicolumn ion exchange chromatography or alkali denaturation. In contrast, CIEF requires no other analytical procedure in the evaluation of patients with suspected hemoglobinopathies. Since minimal follow-up testing is required, use of a reference laboratory is a cost-effective way to identify the rarer Hb variants or, as necessary, confirm the identification made by CIEF [95]. CIEF can also be used to assess glycemic control through the measurement of Hb A1c, a chemically modified (glycated) form of normal adult Hb A. The Diabetes Control and Complications Trial [97] has shown that Hb A1c levels are a good long-term indicator of the average blood glucose and should be routinely monitored to check that diabetes is being properly controlled. Since Hb A1c results can vary with different analytical methods, national and international programs have been developed for standardization [98,99]. Although most laboratories use commercial HPLC or immunoassay to detect Hb A1c, problems with these assays have been noted [100]. Recently, CIEF has been used for separation and quantification of Hb A1c using the same conditions for separation and analysis of Hb variants. The method was shown to be highly correlated (r>0.98) with the gold standard HPLC method used by the Diabetes Control and Complications Trial [95,101]. A fixed negative bias of 1.4% however, was noted, indicating that other hemoglobin derivatives, such as carbamylated hemoglobins, may be co-eluting with the Hb A1c in the HPLC system [101]. Similar findings were also seen by Hempe et al. [95]. In addition, CIEF can also identify hemoglobin oxidation products formed during improper storage that can impose bias on Hb A1c results. Commercially available reagents used in CZE to separate and quantify Hb A1c have also been evaluated [101]. While the method is rapid ( < 4 min), relatively precise (CV < 4%), and unaffected by carbamylated hemoglobins and hemoglobin variants F, C and S, a separate proprietary buffer system is required to analyze the major hemoglobin variants. One benefit of using CIEF for Hb A1c is that the same instrument and reagents can also be used to separate hemoglobin variants. With the increased throughput available with multi-capillary instruments, CIEF should be able to compete with HPLC, since only one method and instrument is required for these analytes.
2.1.6. Carbohydrate-deficient transferrin Transferrin is an iron transport protein known to be microheterogeneous in carbohydrate and the terminating sialic residues [102]. The predominant sialioform in normal individuals is tetrasialotransferrin, although minor components are also present at lower concentrations [103]. The minor sialioforms, specifically those with two or less sialic acids, also known as carbohydrate-deficient transferrin or CDT, have been shown to be increased in chronic abusers of alcohol [104]. Studies have shown that CDT can detect excessive alcohol consumption (sensitivity of f 80%) when a person has consumed an average of >50 g alcohol per day over a time period of 1 week [105]. Later studies, however, have shown that diagnostic sensitivities of 30 50% for women and 50 70% for men are more realistic [106]. Even though CDT should not be used as a screening test for chronic alcohol abuse, it may be a way to monitor abstinence. An excellent review of the biology and use of CDT and chronic alcohol consumption has recently been published [106]. The reference method for the measurement of CDT is isoelectric focusing, followed by immunofixation, and quantification using densitometry [107]. Commercial methods suitable for the routine clinical laboratory using anion-exchange in conjunction with immunoassay are also available [108 110], although CDT recovery can be problematic [110]. Commercial methods combine all CDT sialoforms into one fraction and then quantify by immunoassay potentially obscuring information useful to the clinician [111]. CE which can examine the CDT profile via direct electrophoresis of serum may offer a solution to these problems [112 118]. The need for minimal sample preparation and the ability to directly detect the various sialoforms of CDT decreases analysis time compared to isoelectric focusing separations or commercial assays. As with other CE-based methods, protein capillary wall interactions can be a problem but this challenge can be reduced by using buffer additives, such as 1,4-diaminobutane [116,117], proprietary buffers [118], or coated capillaries [116 119]. Coated capillaries work well [112 115] but can give unpredictable results. Recently, Crivellente et al. [116] described a CE method to separate CDT, using 1,4-diaminobutane to dynamically coat the capillary surface. Although the separation of CDT was improved over their previous method [115], they were not able to detect the asialo isoform in
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a sample containing 61 U/l CDT. This is in contrast to IEF which can detect asialiotransferrin in samples containing CDT at much lower levels [119]. Giordano et al. [117] also used 1,4-diaminobutane and fused silica capillaries to separate the sialoforms of transferrin with similar results. These researchers were able to obtain a cleaner and more easily interpreted electropherogram by using protein A covalently attached to agarose to remove the IgG, but still were not able to see asialiotransferrin. It is possible that an even cleaner electropherogram may be obtained using the genetically engineered hybrid of Protein L and Protein A that binds all immunoglobulins subclasses including free kappa light chain [120]. This may allow better visualization of asialiotransferrin. While research methods were not able to detect asialiotransferrin, use of a commercial kit (CEofixk CDT kit, Analis, Belgium) and the Paragon CZE 2000 detected asialiotransferrin (Fig. 3). Although additional studies are necessary to
validate the use of CE in the detection and quantification of CDT, it appears to be an acceptable replacement for current methods in the evaluation of excessive alcohol consumption. 2.1.7. Lipoproteins Atherosclerosis, a chronic disease characterized by the localized accumulation of plaque, is the principal cause of coronary arterial disease leading to the obstruction of arteries [121]. Serum lipoprotein abnormalities, such as increased LDL, decreased HDL, increased Lp (a), etc., have been shown to be a factor in atherosclerotic development [122,123]. Because of this association, lipoprotein profiles consisting of serum cholesterol, triglycerides, HDL, and LDL (calculated using the Friedewald formula) are routinely run in the main clinical chemistry laboratory. It is possible to obtain the same information along with additional information on Lp (a), IDL, VLDL, LDL, and
Fig. 3. CEofixk CDT kit for determination of carbohydrate deficient transferrin (CDT) using the Beckman Paragon CZER 2000. Separation conditions: fused silica capillary with a capillary length is 57 cm (50 cm to detector) 50 Am; temperature, 40 jC, a proprietary buffer (CEofix CDT reagent set) was used at 28 kV for 7 min; injection by 0.5 psi for 1 s. The electrophoregram has been expanded to show the region where CDT is found. Results are shown for three social drinkers (electrophorograms labelled 1 2) and four heavy drinkers (electropherograms labelled 4 7). Peaks detected: (0) asialotransferrin, (2) disialotransferrin, (3) trisialotransferrin, (4) tetrasialotransferrin, (5) pentasialotransferrin, (6) hexasialotransferrin. Asialotransferrin is absent in the social drinkers but can be detected in the heavy drinkers. In addition, disialotransferrin can be seen to be increased in the heavy drinkers relative to the social drinkers. The figure was kindly supplied by Beckman Coulter.
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HDL by using AGE along with enzymatic staining of cholesterol to directly quantify the lipoprotein fractions [124]. Similar to other electrophoretic methods, CE can replace AGE in the separation and detection of all serum lipoproteins as discussed below. Using capillary isotachophoresis (CITP), it is possible to separate serum lipoproteins into 9 14 fractions [125 133]. Using Sudan Black as a lipoprotein stain, Schmitz and Mollers [125] were able to show that various hyperlipoproteinemias, classified according to the Frederickson classification, were clearly distinguishable by CITP. In addition, they were able to observe the effect of drug therapy on the various lipoprotein fractions. Recently, Schmitz et al. [126] used the lipophilic fluorescent dye, 7-nitrobenz-2-oxa1,3-diazole ceramide, to stain lipoproteins. Although the absolute values were not identical, reasonable correlations were found for HDL and LDL (r = 0.9 and 0.91, respectively) vs. routinely used methods for a small number of patients. Zorn et al. [127] used either an enzymatic specific staining of cholesterol or triglyceride to distinguish the various lipoprotein fractions. CITP has been shown to separate serum lipoproteins into multiple fractions, including at least five HDL and seven LDL subfractions. Once the clinical usefulness in monitoring these additional subfractions has been established, CITP should become the method of choice in monitoring disorders of lipoprotein metabolism. In addition, CITP can also be used to identify apolipoproteins directly from serum [129,134], however, significant sample preparation is required limiting it usefulness in the clinical laboratory. 2.2. Serum and urine analysis of drugs Studies have shown that CE has better resolution, reduced sample preparation, costs less, and is faster than high performance liquid chromatography (HPLC) [136 142], although the sensitivity and migration time precision for CE is not good as HPLC [135]. Sensitivity can be increased by using extended light pathlength capillaries, such as bubble cells or z-cells, where the inner diameter of the capillary is increased only at the detection window. Although extended light pathlength capillaries appear to work most researchers rely on field-amplified stacking where increases in sensitivity of up to a 1000 increase have been attained [143]. Stacking also improves the precision of analyte quan-
titation by increasing the amount of analyte on the capillary [144]. These and other ways to improve the sensitivity of CE has been recently reviewed by Hempel [145]. In addition to less-than-desirable sensitivity, reproducibility of analyte migration times in CE can also be poor, especially when MEKC or CZE are used to separate analytes. This is mainly due to the inability to control the EOF and is more pronounced when a capillary is first used. Although attempts to control EOF has met with limited success, the use of internal marker(s) to calculate an effective mobility (leff), which is independent of the EOF, has made it possible to account for these changes [146 149]. This has reduced the relative standard deviation (RSD) by over 3-fold from 3 5% to 0.7 1.5%. More recently, a new termcorrected leffwhich uses standards with known leff, has been introduced [150 152]. The leff of each standard is based on an average of multiple analyses. A standard curve is prepared and used to correct the leff obtained for analytes in each experiment. Using the corrected leff has reduced the intrainstrument RSD to < 3%, inter-instrument RSD to < 4%, and inter-laboratory to f 3%. Thus, by using the leff or the corrected leff, the RSD for CE should be able to favorability compete with HPLC. CE has been shown to be useful in the area of therapeutic drug monitoring (TDM). In addition to analyzing a drug and its metabolites, CE can also be used to determine the bound and free fraction of drugs, drug isomers, and a drugs physico-chemical properties [153]. In TDM, CE will have the most utility for those drugs that do not have an immunoassay or HPLC method already developed. CE is also useful in forensics [154,155] to screen urine for drugs of abuse, such as opiates [156], barbiturates [157], benzodiazepines [158], amphetamines [159], morphine [160], or impurities in heroin [161]. In the initial screening stages forensic toxicology laboratories are concerned only with identifying which drugs are present, if any, and are not concerned with quantification. Once detected, confirmation and quantification can be performed using a different method. The ultimate goal for screening samples is no false positive or false negative results [162]. However, this is not realistic. What forensic laboratories do to minimize false positives is to confirm all positive results by a definitive method, usually mass spectrometry (MS),
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which eliminates most false positive results. Although some false positive results are acceptable in the screening stage, false negatives should be kept to a minimum since a negative result will probably end further sample analysis. Currently immunoassay and HPLC are the methods of choice in forensic toxicology [163 167]. Immunoassays, however, often lack specificity and, at times, sensitivity, while HPLC methods usually require extensive sample pretreatment leading to modest sample throughput. CE has been used to separate and identify a wide variety of drugs; including drugs of abuse in body fluids [136 142,153 161,168,169] (see Fig. 4 for an example of the separation of the hallucinogenic amines, hydromorphone, morphine, and codeine). It has also been interfaced to MS [159,160,170], making it feasible to be used both as a screening and as a confirmatory method.
It is also possible to use CE for screening postmortem fluids for illicit drugs or elevated levels of legal drugs [155]. Hudson et al. has shown CE to be useful in analyzing whole blood specimens (whether fresh, hemolysed, or putrid) minimizing false negatives using a simple liquid liquid extraction procedure [155,171,172]. In addition, the disappearance of analytes due to thermal decomposition or irreversible absorption to columns is rarely, if ever, observed in CE. Use of a simple UV or diode array detector basic drugs allowed the detection of basic drugs at concentrations of 10 ng/ml (Fig. 5). They also characterized the relative mobilities of over 500 basic and neutral drugs of forensic interest [171,173]. Because of the ability to detect large number of drugs and a reduction in the screening cost the Royal Canadian Mounted Police forensic laboratory in Regina, Saskatchewan,
Fig. 4. Detection of the hallucinogenic amines, hydromorphone, morphine, and codeine, in whole blood by capillary electrophoresis. Whole-blood samples (1 ml) containing 10 ng/ml of hydromorphone (HM), morphine (M), and codeine (C) were extracted with 5 ml 1-chlorobutane plus 0.2 ml ammonia. The residue is redissolved in methanol (1% HCl), evaporated and dissolved in 30 AL water. Internal standard is 100 ng/ml nalorphine. Separation conditions: capillary 70 cm (60 cm to detector) 75 Am fused silica capillary containing 0.4% h-CD in 100 mM phosphate pH 2.38, at 21 kV; temperature, 25 jC; injection at 10 kV for 16 s; detection at 200 nm. The standard curve shows the linear response of hydromorphone over 3 logs. The figure was kindly supplied by J. Hudson, Royal Canadian Mounted Police, Regina, Saskatchewan, Canada.
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Fig. 5. Quality controls for basic drugs using the Beckman P/ACE MDQ. Whole-blood samples (1 ml) were extracted with 5 ml 1-chlorobutane plus 0.2 ml ammonia. The residue is redissolved in methanol (1% HCl), evaporated and dissolved in 30 Al water. Separation conditions: capillary, 60 cm (50 cm to detector) 75 Am fused silica capillary containing 100 mM phosphate, pH 2.38, at 18 kV; temperature, 25 jC; injection at 10 kV for 8 16 s; detection at 200 nm. (a) Quality control sample in water. (b) Quality control extract, 10 ng of each drug in porcine blood. (c) Quality control blank with only 50 ng/ml internal standard added. The figure was kindly supplied by J. Hudson, Royal Canadian Mounted Police, Regina, Saskatchewan, Canada.
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Canada is now using CE as the preferred method to screen forensic samples for drugs. 2.3. Molecular diagnostics Molecular pathology/molecular diagnostics is the newest and most rapidly growing area in laboratory medicine, in which the detection, characterization and/ or quantification of nucleic acids is used to assist in the diagnosis and management of disease. Molecular assays augment classical laboratory medicine by providing additional information that could not be obtained using standard methodologies. Currently, molecular pathology can be separated into six areas: (1) hematology/oncology; (2) solid tumors; (3) genetics; (4) pharmacogenetics; (5) infectious diseases; and (6) identity testing/forensics. Molecular pathology laboratories currently focus on infectious diseases. This focus probably will continue for the next several years; however, interest in other tests is increasing. CE-based molecular assays combine high sensitivity with high resolution that will allow it to become an important and integral part of the molecular pathology laboratory. CE has the following advantages over classical slab polyacrylamide gel electrophoresis (PAGE) in the detection of infectious agents, genetic polymorphisms, or quantifying gene expression [174]: (1) reduction in labor costs required cast and manually load slab gels; (2) small sample size capabilities and the ability to analyze non-ideal tissue samples such as archival paraffin embedded formalin fixed tissue; and (3) automated digital imaging capabilities that can use either peak height or peak area in a semi-quantitative or quantitative manner. The versatility of CE-based molecular platforms results from its ability to resolve single base changes for sequencing or larger base pair differences (greater than 5 bp) for fragment analysis applications. Sequence analysis is the gold standard for molecular testing. It yields the greatest amount of information since it identifies the order of each deoxynucleotide base of a particular target, usually amplified DNA or cDNA [175]. Following the initial amplification, a purification step removes residual deoxynucleotides and primers from the polymerase chain reaction (PCR) product. The products are then subjected to a second round of amplification similar to a typical PCR reaction. In this second reaction, however, deoxynucleo-
tides along with limiting amounts of chain terminating dideoxynucleotides labeled with a different flourophores on each base are present, resulting in a series of different colored, truncated DNA products due to a random incorporation of the dideoxynucleotides in competition with the normal deoxynucleotides. The single-base resolving capability of CE permits all four flourochrome products to be separated simultaneously (Fig. 6A and B) compared to classical sequencing with PAGE, where each chain terminator has to be loaded in separate lanes. The result is a multi-colored ladder where each color represents a different base. The order of the bases is then analyzed using secondary software that characterizes the sequence in terms of identity, and relatedness to prototypical sequences in a database. In contrast, fragment analysis incorporates one flourochrome-labeled PCR primer in a standard PCR reaction after which the product(s) are separated and visualized. Fragment analysis is more rapid and less expensive because the flourochrome is incorporated in the initial PCR reaction thus eliminating the need for sequencing reactions that incorporate the dye terminating bases. Interpretation of the fragment analysis data is also easier than sequence analysis because the fragments are readily identified by the imaging software and the need for secondary sequence software analysis is not required. While less expensive and more rapid, fragment analysis does not provide exact, detailed sequence data. In addition, PCR fragments can result from erroneous amplification and thus verification using sequencing techniques or independent probes is sometimes recommended for quality assurance purposes. In these cases, sequencing is often used to confirm the identity of the PCR product. 2.3.1. Infectious diseases An important aspect of molecular methods is that they do not require the presence of viable organisms permitting the identification of bacteria, viruses, and fungi that are difficult if not impossible to culture. Identification of infectious agents can also be used for both diagnostic and therapeutic purposes [176 180]. Although the simplest application of molecular techniques is to detect infectious entities in body fluids which are nearly sterile [181,182], more complex analysis is also done, such as detailed characterization of infectious agents via sequence analysis. For instance, detection of genetic polymorphisms in tuber-
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Fig. 6. A. Image of processed CE sequencing data. Raw sequence data was collected for BRCA mutation analysis. Note that different colors represent different base pair terminators. B. Analysis of BRCA gene mutation by comparison of patient and reference sequences. The patient sequence is on top with the reference sequence overlaid below. Peaks above and below the line denote changes from the reference sequence. (Data for Panel B was kindly provided by Tom Frank of Myriad Genetic Laboratories, Salt Lake City, UT).
culosis has been shown to be helpful in identifying rifampicin resistant isolates [178] while determination of the genotypes for Human Immunodeficiency Virus (HIV) or Hepatitis C Virus (HCV) can predict longevity and treatment modalities using antiviral therapies [183 185]. Also, if >2000 viral copies/ml of HIV are present in plasma, it is possible to determine the susceptibility of the virus to antiviral therapy. This is done by sequencing the reverse transcriptase and protease genes of the HIV that are isolated employing
RT-PCR and multiple sequencing primers. The sequencing results are aligned using a software program that overlaps sequences generated from forward and reverse sequencing primers (Fig. 7). This software identifies variations in the HIV sequence (lines in the top diagram) that may be associated with various antiviral drug resistance. PCR can also be used to target conserved stretches of DNA, such as ribosomal DNA (rDNA), resulting in new applications that can identify infectious agents
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Fig. 7. HIV Genotyping (ABI) shows alignment data of seven sequences using the Viroseq software permitting comprehensive data of both forward and reverse strands of the viral cDNA.
that cannot be cultured. The entire 16S rDNA sequence (1541 bp) for bacteria can be generated using a single pair of PCR primers and then sequenced using the Microseq Microbial Identification systems CE (Applied Biosystems, Foster City, CA). Normally, only 500 bases are required for routine identification, although sequencing of the entire rDNA is possible. Using this approach, various bacterial strains or biotypes have been identified for Streptococcus sp., Mycobacterium sp., coryneform bacterial isolates, as well as other bacterial species [175,186 190]. Identification of fungi using CE is usually done by amplifying and sequencing the large rDNA subunit because of the multiple gene copies normally present [191]. Although there are 12 domains present in the fungal rDNA, domain 2 is the largest (200 500 bp) and is routinely used for identification. Sequencing of domain 2 has allowed strain identification of the fungi Trichophyton rubrum and Schizophyllum communes associated with diseases of nail tissue and the sinus cavity, respectively [188,189]. Using this approach, Kurzai et al. [190] identified Candida dubliniensis as a new emerging pathogen. This fungi is often incorrectly identified as Candidia albicans using classical methods. Sequencing of rDNA by CE by Baele et al. [179] is also useful in the monitoring nosocomial infections. Typically, rDNA various isolates are amplified, sequenced, and then compared to determine if the strains are the same as, or in some manner related to, previously isolated strains. 2.3.2. Hematology/oncology CE is useful in the analysis of molecular hematology/oncology tests such as B- and T-cell clonality assays [174,192 198]. Using CE, monoclonal populations of B cells are detected are detected through analysis of changes in the immunoglobulin heavy
chain gene (Fig. 8). Because this region is polymorphic, a number of different primers (seven upstream VH region primers and three JH primers obtained from InVivoscribe LLC, CA) can be used to increase the ability to identify clonal heavy chain changes. It is also possible to identify changes in the Kappa chain using this approach with different primers. T-cell monoclonal populations can be detected using PCR, amplifying either the T-cell gamma and/or beta receptor [195,197]. Generally, the presence of one or two peaks with intensities greater than 2-fold over background is considered positive for a gene rearrangement. Prior to CE, distinguishing a monoclonal peak from a background of normal rearrangements was very subjective. Objective data could only be obtained using densitometry. In contrast, CE cannot only identify a peak but give numerical data using the peak height or area helping to facilitate interpretation. However, since several reports have demonstrated pseudo-spikes resulting from reactive rather than oncogenic processes [199,200], it is important to remember that identification of monoclonality does not always mean malignancy. Although CE facilitates interpretation of these difficult cases, it is critical that each clinical laboratory perform extensive validation to assure that interpretation of a rearrangement test is accurate. Detection of gene translocations by CE has been used to differentiate various types of lymphomas and leukemias aiding in the decision of appropriate therapy. In gene translocation assays, PCR uses primers from the two different chromosomes [194]. In this manner, mantle cell lymphomas have been found to be associated with the translocation of BCL1 t (11:14), follicular lymphomas with BCL2 t (14:18), and chronic myleogenous leukemia with Bcr/Abl t (9:22). The ability of CE to accurately determine the size of the monoclonal population and/or the translocation
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Fig. 8. Fragment analysis of gene changes for B- and T-cell monoclonality (Invivoscribe, LLC). Primers for the B- and T-cell monoclonality were obtained from Invivoscribe and are propritary. The B-cell primers are directed toward the heavy chain and framework III of the immunoglobulin gene. T-cell primers are directed toward conserved regions of the T cell gamma receptor. Each PCR product was generated using 35 cycles at 95 jC for 1 min, 55 jC for 30 s, 72 jC for 30 s. Blue spikes represent rearrangements corresponding to different size PCR products resulting from clonal populations. Monoclonal populations are represented by one or two prominent peaks (A and C) while normal clonal populations are seen as a single bell shaped curve for B-cell (JH) rearrangements using primers directed toward the immunoglobin heavy chain or as a bimodal curve using primers targeting the T-cell gamma receptor, respectively (B and D). The red spikes represent the standards.
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makes it useful for monitoring therapy by determining if treatment has failed or if reoccurrence is due to a new primary disease. Berg et al. [201] described a case involving a T-cell lymphoma resulting from allogenic bone marrow transplantation that clearly demonstrates the utility of CE in accurately sizing fragments. In this case, each of two sisters, one the donor and the other the recipient, developed an identical T-cell lymphoma as determined by CE, implicating transfer of the lymphoma cells from the donor to recipient during transplantation. In addition to accurately sizing PCR fragments, CE also has the ability to work with very small quantities of materials making it especially useful in cases where there is insufficient patient material or confounding results are found by using conventional methodologies. 2.3.3. Solid tumors Solid tumors can result from genetic polymorphisms including translocations, single nucleotide polymorphisms (SNP), loss of heterozygosity (LOH), and microsatellite instability. Translocations can also arise in various tumor types and are usually detected using fragment analysis as mentioned above for hematology/oncology samples. LOH, also known as
allelic loss, is useful in distinguishing between tumor recurrence vs. de novo cancer formation in addition to determining prognosis [202,203]. LOH is known to occur in various cancers, including head and neck and colon tumors [204 207] and can be identified using fragment analysis. These assays detect chromosomal differences between normal and tumor tissue. Thus, the presence in tumor tissue of normal tissue in the form of stromal tissue and blood vessels can be a source of contamination. As a result, identification of allelic loss requires calculating the ratio of band intensity of the two alleles in the tumor vs. normal tissue. A decrease of >30% is the threshold criteria to identify a specific allele loss. As shown in Fig. 9, CE provides improved resolution, sizing, and simplified quantification to determine if LOH has occurred. SNPs, on the other hand, are detected by sequencing and primer extension which can be used for prognostic purposes in solid tumor cases. For example, TP53, which is associated with a normal genotype, has been shown to predict enhanced patient survival in squamous cell carcinomas and rectal cancers [208,209]. Another example involves the recent success in the treatment of patients with a
Fig. 9. Use of STR PCR to detect LOH. This figure shows how the ratio of alleles can be used to help in distinguishing normal and tumor tissue using primers targeting the D1S2883 loci which is located near the gene associated with hereditary prostate cancer. In the normal tissue the ratio of the different alleles is 1.6. This ratio is greatly increased in the tumor with implicates a loss of the larger allele.
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specific tyrosine kinase inhibitor, STI571, in gastrointestinal stromal tumors expressing mutant c-kit [210,211]. In addition to being used as a solid tumor test, detection of SNPs can also be usefully in assessing risk in developing cancer. For example, in addition to being used to help identify the most appropriate treatment for these cancers, BRCA 1 and 2 variants can be used to help identify women at risk for developing breast and ovarian cancer. Genetic counseling is recommended for cancer patients to assist in understanding the relative risk for developing breast or ovarian cancer [213]. The presence of the BCRA 1 and 2 variants in a breast tumor can mean that certain interventions, such as prophylactic contralateral mastectomy, may be useful in preventing a second primary cancer [212]. 2.3.4. Identity testing/paternity testing/HLA testing Fragment analysis of short tandem repeat (STR) PCR products such as those used for LOH studies can also be used to identify specific individuals in a larger population [214 218]. Identity testing has applications that range from identifying suitable recipients for organ transplantation, paternity testing, bone marrow engraftment analysis, forensic testing, and quality assurance measures for determining the identity of mislabeled paraffin embedded tissue or foreign tissue embedded in the same cassette. Fig. 10 demonstrates the use of these tests for therapeutic follow-up of a bone marrow engraftment patient. In panel 3 of this figure, it can be seen from a 50% mixture of donor
and recipient DNA that treatment failure due to the recipients cells repopulation of their bone marrow has probably occurred. Identifying the cause of graft failure whether due to rejection, graft vs. host, or other mechanisms, is essential for deciding on the most appropriate treatment regimen. The figure also demonstrates the ease of identifying multiple alleles simultaneously using CE. This is the same basic technique that is used for forensic and paternity cases, although with forensic tests additional STRs are usually required to statistically assure the identity of the subject. 2.3.5. Genetics CE has simplified the methodology for performing both simple and complex genetic tests. Numerous inherited genetic diseases, such as Cystic Fibrosis (CF), fragile X, mitochondrial heteroplasmy, spinocerebellar ataxia, and genetic variants of cytochrome P450 that are involved with drug metabolism can be detected using CE [219 228]. Sequencing and fragment analysis of restriction fragment length polymorphisms (RFLP) and primer extension are commonly used to detect these variants. Fig. 11 demonstrates the results of DNA that is heterozygous for a splice variant of CYP3a5. This splice variant has decreased activity for some substrates such as protease inhibitors, which can potentially result in toxic drug levels. Using the same technology, it is also possible to detect the presence of certain genetic mutations that are associated with an increased risk having a child with
Fig. 10. Use of STR PCR to monitor bone marrow engraftment. Panels 1 and 2 represent the donor and the recipient, respectively. Panel 3 demonstrates a 50% chimera in the patient.
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Fig. 11. Single nucleotide polymorphism detection using primer extension (Snapshot, ABI) and fragment analysis. Wild-type base pair is red while the variant is black. (A) CYP3A5*3 Splice Variant 1, heterozygote; (B) CYP3A5*3 Splice Variant 2; (C) Simultaneous detection of heteroxygote compound for both CYP3A5*3 splice variants.
CF [229]. Currently, the American College of Gynecologists (ACOG) recommends screening for all Caucasian women and their partners for the presence of variants which over have been shown to have a causal association with CF. For routine screening purposes, however, screening of only the most common 25 specific variants has been recommended. Although the use of CE for complex inherited genetic tests has simplified testing, tremendous amounts of data are generated [227], necessitating the need for interpretative guidelines. This has led the College of American Pathologists (CAP) to recommend that laboratory reports include the following information to assist in test interpretation: (1) an estimate of the risk for false negatives and false positives arising from recombination between the probe and the disease associated gene; (2) an estimate of the residual risk of being a carrier for one of the variants not tested for; and (3) discussion of recessive or dominant inheritance, recurrence risk, penetrance, severity, and other genotype
phenotype correlatives. In addition to genetic counseling, the laboratory report is necessary to effectively relay residual risks and uncertainties, and the medical and reproductive options suggested by the test results [213]. 2.3.6. Other Currently, gene expression profiles using arrays permit pattern identification that may be useful for diagnostic and prognostic purposes. Classically, membrane and silicon arrays identified various patterns but required specialized equipment to perform and interpret the results. More recently, expression arrays formulated with internal expression controls have enabled analysis using standard CE equipment [230 234]. The advantages of automated quantification, as seen in previous CE-based clinical tests facilitate its application for high throughput analysis of gene expression. Other applications that hold promise for clinical applications involve microfluidic chambers [225,235
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237]. As shown in Fig. 12, these chambers can resolve DNA fragments with high resolution. Although these microfluidic devices could be applied to any point of care test, in the foreseeable future they will probably be used either for molecular analysis or identification of infectious agents.
3. Conclusion CE is a sensitive and versatile technique and represents an inexpensive and practical method for the determination of many clinically important analytes. Although CE may be maturing in the research,
Fig. 12. Microfluidic chamber (iChip) layout and an example of the resolution by chip and standard gel electrophoresis. The figure was kindly provided by Hitachi Chemical (Japan).
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genomic, and pharmaceutical arenas, in the clinical laboratory, it still appears to be in its early childhood. Initially, CE was heralded for its speed and low sample volume capabilities, and perhaps more importantly, its ability to be quantitative and to automate, as well as separating compounds that have been difficult to handle by traditional methods. As recently stated by one of the major contributors to the area of CE, Dr. James Jorgenson, CE is the best-kept secret in chemical analysis since it can be used to analyze complex mixtures and is responsible for virtually all microfluidics for lab-on-a-chip devices [173]. It is also possible to use one instrument for multiple purposes, such as serum, urine, and CSF protein; hemoglobin (both variant and A1c); lipoprotein; carbohydrate-deficient transferrin; and alpha-1 antitrypsin deficiency analysis. The main advantage of CE is its ability to screen large numbers of specimens with very little hands-on time at a relatively low cost per test. Although analysis of serum proteins by CE offers many advantages in terms of labor saving per test and reproducibility, it has not been generally accepted in the clinical laboratory. This is clearly seen by looking at the number of laboratories subscribing to CAP proficiency testing for serum proteins electrophoresis. In 200,1 the number of laboratories using CE for serum proteins was 13 out of a total of 739. So why is CE not being used in the routine clinical laboratory? Possibly because CE is still perceived as requiring above-average technical expertise precluding its use in a laboratory workforce that is less technically adept. Broader acceptance of CE in the main clinical laboratory will require the following: (1) the development of chemical reagent kits, instruments and software suitable for daily clinical laboratory use; (2) an increase of sensitivity and sample throughput; and (3) comparison and evaluation of various applications with current laboratory methods under daily laboratory conditions. Thus, a greater role for CE in the future clinical laboratory can be anticipated as the ease of use, number, variety of applications increase. Although CE has not been accepted in the main clinical laboratory, the technique is being used in the molecular pathology laboratory. Its use, however, is not apparent by simply looking at CAP proficiency
surveys, which indicate that only 12 of 70 molecular pathology laboratories use CE. From our experience, this did not appear to be correct. Thus, we initiated an informal survey of 33 molecular pathology laboratories, which showed that all but three use CE in some part of their testing. CE has simplified molecular tests by providing a platform that can address all areas of molecular testing. Because of its ease of use and robust nature, it is becoming a standard piece of equipment for every molecular pathology laboratory. In the future, CE will be the basis of additional molecular techniques, especially in the area of expression arrays.
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