Study of Cell Antigens and Intracellular DNA by Identification of

Mar 5, 2008 - Study of Cell Antigens and Intracellular DNA by Identification of Element-Containing Labels and Metallointercalators Using Inductively C...
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Anal. Chem. 2008, 80, 2539-2547

Study of Cell Antigens and Intracellular DNA by Identification of Element-Containing Labels and Metallointercalators Using Inductively Coupled Plasma Mass Spectrometry O. I. Ornatsky,*,† X. Lou,† M. Nitz,† S. Scha 1 fer,‡ W. S. Sheldrick,‡ V. I. Baranov,† D. R. Bandura,† and † S. D. Tanner

Department of Chemistry, University of Toronto, Toronto, Canada, and Lehrstuhl fu¨r Analytische Chemie, Ruhr-Universita¨t Bochum, D-44780 Bochum, Germany

The enumeration of absolute cell numbers and cell proliferation in clinical samples is important for diagnostic and research purposes. Detection of cellular DNA with fluorescent dyes is the most commonly used approach for cell enumeration in cytometry. Inductively coupled plasma mass spectrometry (ICPMS) has been recently introduced to the field of protein and cell surface antigen identification via ICPMS-linked immunoassays using element-labeled affinity reagents such as gold and lanthanide-conjugated antibodies. In the present work, we describe novel methods for using metallointercalators that irreversibly bind DNA and low concentrations of rare earth metals added to cell growth media for rapid and sensitive measurement of cell numbers by mass spectrometry. We show that Irand Rh-containing metallointercalators are useful reagents for labeling cells and normalizing signals when studying antigen expression on different types and numbers of cells. Results are presented for solution analysis performed by conventional ICPMS and compared to measurements obtained on the novel flow cytometer mass spectrometer (FC-MS) instrument, designed to analyze multiple antigens and DNA simultaneously in single cells.

It is an accepted assumption in cell biology that the cellular content of nucleic acids is a reasonable indicator of cell number owing to the tight regulation of DNA and RNA levels. DNA measurement is commonly used to estimate the number of cells in solid tumors as well as to characterize hematopoietic malignancies and monitor chemotherapy treatment.1,2 In combination with cell surface markers, DNA content reflects the ploidy or cell cycle phase for a subset of cells defined by the selected antigens in a heterogeneous cell population, or the distribution of a particular * Corresponding author. Phone: +1 416 946 8420. Fax: +1 416 978 4317. E-mail: [email protected]. † University of Toronto. ‡ Ruhr-Universita ¨t Bochum. (1) Labarca, C.; Paigen, K. Anal. Biochem. 1980, 102, 344-352. (2) Plander, M.; Brockhoff, G.; Barlage, S.; Schwarz, S.; Rothe, G.; Knuechel, R. Cytometry, Part A 2003, 54A, 66-74. 10.1021/ac702128m CCC: $40.75 Published on Web 03/05/2008

© 2008 American Chemical Society

antigen through the cell cycle, and may be used for disease diagnosis and prognosis.3,4 Conventional techniques for DNA identification include BrdU (5-bromo-2′-deoxyuridine) and 3H-thymidine incorporation into replicating live cells and the measurement of total nucleic acid content in cell lysates with colorimetric spectrophotometery or liquid scintillation. Fluorescent DNA intercalating dyes in conjunction with flow cytometry such as Hoechst 33258, propidium iodide, DAPI, and acridine orange have been shown to accurately estimate cell numbers. However, these reagents suffer from relatively low fluorescence enhancements upon binding to nucleic acids, interference from high cellular background fluorescence, and are difficult to use in multiparametric flow cytometry. Metallointercalators are a different type of DNA intercalating reagent that are extensively used as sensitive molecular probes and therapeutic agents owing to their site-selective targeting, reactivity, and stability.5,6 Most compounds consist of a transition metal complex core (Rh, Ru, Ir) bound by two nonintercalating ligands and one intercalating ligand which sits perpendicular to the DNA axis. Metallointercalators interact with double-stranded DNA through electrostatic forces, groove binding, and intercalationsinsertion of a planar aromatic ligand between two nucleotide base units of the nucleic acid.7 Substantial research has been devoted to the photoluminescent and photo-oxidizing properties of metallointercalators.8-13 The 9,10-phenanthrenequinone diimine (phi) complexes of rhodium(III) (Rh-intercala(3) Gong, J.; Li, X.; Traganos, F.; Darzynkiewicz, Z. Cell Proliferation 1994, 27 (7), 357-371. (4) Nowak, R.; Oelschlaegel, U.; Schuler, U.; Zengler, H.; Hofmann, R.; Ehninger, G.; Andreeff, M. Cytometry 1997, 30, 47-53. (5) Barton, J. K. Pure Appl. Chem. 1989, 61, 563-564. (6) Chow, C. S.; Barton, J. K. Methods Enzymol. 1992, 212, 219-242. (7) Long, E. C.; Barton, J. K. Acc. Chem. Res. 1990, 23, 271-273. (8) Chen, W.; Turro, C.; Friedman, L. A.; Barton, J. K.; Turro, N. J. J. Phys. Chem. B 1997, 101, 6995-7000. (9) Dandliker, P. J.; Holmlin, R. E.; Barton, J. K. Science 1997, 275, 14651468. (10) Erkkila, K. E.; Odom, D. T.; Barton, J. K. Chem. Rev. 1999, 99, 27772795. (11) Hill, M. G.; Jackson, N. M.; Barton, J. K.; Kelley, S. O. Abstr. Pap.sAm. Chem. Soc. 1997, 214, 144-INOR. (12) Sitlani, A.; Long, E. C.; Pyle, A. M.; Barton, J. K. J. Am. Chem. Soc. 1992, 114, 2303-2312. (13) Puckett, C. A.; Barton, J. K. J. Am. Chem. Soc. 2007, 129, 46-47.

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tors) have been shown to bind tightly (Kd < 10-8 M) to doublestranded DNA by intercalation in the major groove and to undergo a variety of photoinduced reactions with DNA.14,15 From photofootprinting experiments Barton and co-workers concluded that [Rh(phi)2(bpy)]3+ has a rigid rhodium complex that can occupy directly all sites not obstructed by DNA binding proteins, and at rhodium/base pair ratios of 2:1 binding to DNA is sequence neutral and random.16,17 Recent work from the same group has shown that metallointercalators which target major groove sites bind poorly to double-stranded RNA.18,19 However, other experiments indicate that the apposition of several noncanonical bases as well as stem-loop junctions and bulges in tRNA could result in intimately stacked structures with opened major grooves accessible for metal intercalator binding.19 Numerous studies have also focused on related compounds with a central Ir(III) atom such as the [Ir(bpy)(phen)(phi)]3+, a luminescent dipyridoquinoxaline complexes,20,21 and dipyridophenazine organometallic half-sandwich compounds that exhibit strong intercalative binding with DNA on the order of Kb ) 1.2 × 106 M-1.22,23 Recently, inductively coupled plasma mass spectrometry (ICPMS) has been introduced to the field of protein and cellular antigen identification via ICPMS-linked immunoassays using metal-containing immunoreagents such as lanthanide-conjugated antibodies24-28 and antibodies labeled with polymer tags bearing chelated metals.29-31 ICPMS offers absolute quantification that is largely independent of the analyte molecular form or sample matrix. Second, the abundance sensitivity of ICPMS, a measure of the overlap of signals of neighboring isotopes, is large (>106 for the quadrupole analyzer), and this ensures independence of the detection channels over a wide dynamic range. The third key property is the high sensitivity of ICPMS analysis; it was demonstrated that ICPMS-linked immunoassays can be at least as sensitive as radioimmunoassay.28 ICPMS is extensively used (14) Holmlin, R. E.; Dandliker, P. J.; Hall, D. B.; Barton, J. K. Abstr. Pap.sAm. Chem. Soc. 1997, 213, 335-INOR. (15) Sitlani, A.; Barton, J. K. Biochemistry 1994, 33, 12100-12108. (16) Uchida, K.; Pyle, A. M.; Morii, T.; Barton, J. K. Nucleic Acids Res. 1989, 17, 10259-10279. (17) Sitlani, A.; Barton, J. K. Abstr. Pap.sAm. Chem. Soc. 1992, 204, 479-INOR. (18) Chow, C. S.; Hartmann, K. M.; Rawlings, S. L.; Huber, P. W.; Barton, J. K. Biochemistry 1992, 31, 3534-3542. (19) Chow, C. S.; Behlen, L. S.; Uhlenbeck, O. C.; Barton, J. K. Biochemistry 1992, 31, 972-982. (20) Lo, K. K. W.; Chung, C. K.; Zhu, N. Y. Chem.sEur. J. 2006, 12, 15001512. (21) Stinner, C.; Wightman, M. D.; Kelley, S. O.; Hill, M. G.; Barton, J. K. Inorg. Chem. 2001, 40, 5245-5250. (22) Gencaslan, S.; Sheldrick, W. S. Eur. J. Inorg. Chem. 2005, 3840-3849. (23) Schafer, S.; Sheldrick, W. S. J. Organomet. Chem. 2007, 692, 1300-1309. (24) Baranov, V. I.; Quinn, Z.; Bandura, D. R.; Tanner, S. D. Anal. Chem. 2002, 74, 1629-1636. (25) Baranov, V. I.; Quinn, Z. A.; Bandura, D. R.; Tanner, S. D. J. Anal. At. Spectrom. 2002, 17, 1148-1152. (26) Ornatsky, O.; Baranov, V. I.; Bandura, D. R.; Tanner, S. D.; Dick, J. J. Immunol. Methods 2006, 308, 68-76. (27) Quinn, Z. A.; Baranov, V. I.; Tanner, S. D.; Wrana, J. L. J. Anal. At. Spectrom. 2002, 17, 892-896. (28) Zhang, C.; Wu, F. B.; Zhang, Y. Y.; Wang, X.; Zhang, X. R. J. Anal. At. Spectrom. 2001, 16, 1393-1396. (29) Lou, X.; Zhang, G.; Herrera, I.; Kinach, R.; Ornatsky, O.; Baranov, V.; Nitz, M.; Winnik, M. A. Angew. Chem., Int. Ed. 2007, 46, 6111-6114. (30) Tanner, S. D.; Ornatsky, O.; Bandura, D. R.; Baranov, V. I. Spectrochim. Acta, Part B 2007, 62, 188-195. (31) Ornatsky, O. I.; Kinach, R.; Bandura, D. R.; Lou, X.; Tanner, S. D.; Baranov, V. I.; Nitz, M.; Winnik, M. A. J. Anal. At. Spectrom. [Online early access]. DOI: 10.1039/b710510j.

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as a versatile analytical tool32 to study natural and induced metal incorporation into bacteria,33-36 plants,37 and in metalloproteomics research.38,39 A novel flow cytometer mass spectrometer (FC-MS) instrument was built recently in our lab and will be described in greater detail in a separate publication. Briefly, FC-MS is based on a custom-built research prototype of the inductively coupled plasma time-of-flight mass spectrometer for elemental analysis of single cells. The instrument comprises a three-aperture plasma-vacuum interface, a dc-turning ion optics for decoupling ions from the neutral component of the beam extracted from the inductively coupled plasma, dual rf-quadrupole ion guide discriminating against dominant plasma ions, a four-plate lens for matching the quadrupole output ion beam with the entrance geometry of the orthogonal acceleration reflectron time-of-flight mass analyzer, and an 8-bit 1 GHz digitizer. The cell suspension was introduced via a MicroFlow PFA-ST concentric nebulizer (Elemental Scientific, Inc.) and a low-volume spray chamber (Teflon, made in lab). High time-of-flight (TOF) spectrum generation frequency (i.e., number of TOF spectra per second registered) of up to 80 kHz provides the ability to collect multiple spectra for single-cell induced transient ion clouds, typically of 200-300 µs duration. The mass range of the instrument is limited to m/z ) 100-200. In this work, we investigated the use of rhodium and iridium compounds in the form of metallointercalators as labels for cellular nucleic acids. Additionally, rhodium and palladium salts at low nontoxic concentrations were added as supplements to cell growth medium for cell type identification by ICPMS and correlation to cell number in a heterogeneous cellular population. We then used the DNA binding properties of metallointercalators in the development of a rapid and sensitive assay for cell enumeration coincident with immunophenotyping using element-tagged antibodies in both solution format (applying a commercial ICPMS instrument) and in a single-cell detection format (applying the novel FC-MS instrument). METHODS AND INSTRUMENTATION Materials and Reagents. Rh(III) and Pd(II) ultrapure salts (chloride hexahydrates) were obtained from Sigma-Aldrich. Also used were phosphate-buffered saline with calcium and magnesium (PBS; 150 mM NaCl, 1.2 mM Ca2+, 0.8 mM Mg2+, 20 mM sodium phosphate, pH 7.4), concentrated 34% HCl (Seastar Chemicals Inc.), 37% formaldehyde (Sigma), Triton X-100 (Sigma), Ir (iridium) and In (indium) diluted from stock 1000 µg mL-1 solutions (PE) to 1 ng mL-1 in 10% HCl v/v. All solutions were prepared in deionized water (Elix/Gradient water purification system, Millipore). Antibodies. Primary mouse monoclonal antibodies (mAb) antiCD45, HLA-DR, anti-CD33 formulated at 12 µg/mL (Immunotech (32) Ammann, A. A. J. Mass Spectrom. 2007, 42, 419-427. (33) Li, F. M.; Armstrong, D. W.; Houk, R. S. Anal. Chem. 2005, 77, 14071413. (34) Zhang, B.; Li, F. M.; Houk, R. S.; Armstrong, D. W. Anal. Chem. 2003, 75, 6901-6905. (35) Binet, M. R. B.; Ma, R. L.; Mcleod, C. W.; Poole, R. K. Anal. Biochem. 2003, 318, 30-38. (36) Zhang, Y. S.; Zhang, Z. Y.; Suzuki, K.; Maekawa, T. Biomass Bioenergy 2003, 25, 427-433. (37) Sharma, N. C.; Sahi, S. V.; Jain, J. C. Microchem. J. 2005, 81, 163-169. (38) Prange, A.; Profrock, D. Anal. Bioanal. Chem. 2005, 383, 372-389. (39) Szpunar, J. Analyst 2005, 130, 442-465.

Figure 1. Chemical structure of the Ir-intercalator, (pentamethylcyclopentadienyl)-Ir(III)-dipyridophenazine.

Inc.) were used at 1:50 dilution; anti-CD34 mAb, 0.25 mg/mL (BD Biosciences), was used at 1:100 dilution; IgG1 κ, mouse, (BD Pharmingen) was used for negative controls; biotinylated rabbit antimouse IgG, 0.5 mg/mL (BD Pharmingen), was used at 1:50 dilution. Europium-labeled streptavidin (Eu-streptavidin; DELFIA, Perkin-Elmer), 0.7 mg/mL, was used at 1:200 dilution. Antibodies were labeled with the prototype MAXPAR reagents (DVS Sciences Inc., Richmond Hill, Ontario, Canada; www.DVSsciences.com), based on metal-labeled polymer tags described in detail by Lou et al.29 Oxides of the enriched lanthanides 144Nd, 147Sm, 151Eu, 176Yb (Trace Sciences International Corp., Richmond Hill, Ontario, Canada) were converted to chlorides for use in labeling protocols. Cell Lines. Human monocyte cell line MBA-1 was derived from Mo7e by retroviral induction of the p210 BCR/Abl cDNA.40 KG1a, a model human acute myelogenous leukemia cell line, with high CD34 antigen expression (approximately 100 000 copies per cell), as well as THP-1, human monocytic leukemia cell line, were obtained from the American Type Culture Collection (Manassas, VA). Cells were propagated in R-MEM, supplemented with 10% FBS (HyClone) and 2 mM L-glutamine (Invitrogen), in a humidified incubator at 37 °C and 5% CO2. Cells were split every 3-4 days, and viability was checked with trypan blue (>90% viable). Metallointercalators. The complex bis(phenanthrenequinonediimine)(bipyridyl) rhodium(III), [(Rh(phi)2(bpy)]3+, and referred to in the paper as Rh-intercalator, was synthesized as described previously.41,42 The compound is stable in water and is not inhibited by reducing agents, glycerol, divalent cations, or EDTA. Numerous studies have been devoted to the investigation of binding of transition metal polypyridyl complexes with DNA molecules. Increasing the surface area of the polypyridyl ligand is expected to result in a substantial increase in the intercalative binding strength. The novel Ir-containing metallointercalator (pentamethylcyclopentadienyl)-Ir(III)-dipyridophenazine, Ir-intercalator, (Figure 1) was prepared in accordance with the published procedure23 and is indefinitely stable in the solid state and in aqueous solution. This Ir-intercalator was found to have equilibrium constants Kb of 2.6 × 106 M-1. (40) Sirard, C.; Laneuville, P.; Dick, J. E. Blood 1994, 83, 1575-1585. (41) Pyle, A. M.; Chiang, M. Y.; Barton, J. K. Inorg. Chem. 1990, 29, 44874495. (42) Pyle, A. M.; Long, E. C.; Barton, J. K. J. Am. Chem. Soc. 1989, 111, 45204522.

Inductively Coupled Plasma Mass Spectrometry. Solution analysis was performed on a commercial ICPMS instrument ELAN DRCPlus (Perkin-Elmer SCIEX) described elsewhere43 and operated under normal plasma conditions (plasma power, 1400 W; nebulizer Ar flow 0.95 L/min; plasma gas Ar flow 17 L/min; auxiliary gas Ar flow 1.2 L/min; CeO+/Ce+ ratio in 10% HCl 50-fold excess of reagent over DNA binding sites for 1 × 106 cells. We assessed the binding of various amounts of Rh-intercalator to the nucleic acid of fixed cells by ICPMS. MBA-1 cells were fixed in 3.7% formaldehyde/ PBS for 15 min, washed once with PBS, and incubated for 45 min with 1 mL of increasing concentrations (2.5 nM to 250 µM) of Rh-intercalator. Each sample contained 1 × 106 cells, run in triplicate. Following washing the cell pellets were treated with concentrated HCl and analyzed by ICPMS. Figure 4A shows a linear response. Saturation of cellular DNA was not reached even at 250 µM concentration of Rh-intercalator. The normalized response obtained with Rh-containing metallointercalator is extremely high and stable. For typical Rh sensitivity, which the ICPMS can routinely provide (∼50 × 106 cps/(µg mL-1)), it is practical to keep the metallointercalator-DNA molar ratio well below equimolar, since even 10% intercalated DNA should produce a signal of >2 × 106 cps. The reagent is added to fixed cells for (44) Schafer, S.; Ott, I.; Gust, R.; Sheldrick, W. S. Eur. J. Inorg. Chem. 2007, 3034-3046.

Figure 4. Rh-intercalator binding to cellular DNA analyzed by solution ICPMS. (A) Linear concentration dependence between amounts of Rh-intercalator and signal obtained from treated cells (normalized response). (B) Normalized response for Rh obtained after treating live, fixed, or fixed/permeabilized (fix/perm) MBA-1 cells with Rh-intercalator.

less than 1 h and does not leach out during at least 24 h (data not shown), thus making it a preferable agent for cell enumeration with ICPMS. We then studied the reactivity of the Rh-intercalator with fixed and fixed/permeabilized versus live cells. MBA-1 cells were fixed in 3.7% formaldehyde as above, fixed and permeabilized with 0.1% Triton X-100, or left untreated (live cells). After two washes in PBS the cells were incubated with 200 µM Rhintercalator for 30 min, washed three more times, and dissolved in HCl. Figure 4B is a representative graph of two experiments. As evident from the figure, fixation and fixation with permeabilization gave similar results. Thus, it seems that the small size of the Rh-containing compound enables it to penetrate fixed cells without the need for additional permeabilization. In order to determine the saturation level and optimal amounts of metallointercalator, the following experiment was performed. THP-1 and KG1a cells were fixed in 3.7% formaldehyde, distributed into microtiter plates at 105 cells/well (0.45 µm Solinvert Filter MultiScreen plates, Millipore) and treated with 50 µL of 0.1 µM (5 10-12 mol per well), 1 µM (5 10-11 mol), 10 µM (5 10-10 mol), and 50 µM (2.5 10-9 mol) Ir-intercalator solutions for 30 min. Control wells had no cells. Washing was done four times with 200 µL of PBS in filter plates. Cells were resuspended in 100 µL of PBS and transferred to fresh filter wells to minimize Ir signal from background binding to filter. Cells were spun down and dissolved in 100 µL of HCL/well. In Figure 5, measured amount of Ir is related to this set of samples. For analysis, 10 µL of acidified sample was combined with 90 µL of H2O and 100 µL of 1 ng mL-1 Analytical Chemistry, Vol. 80, No. 7, April 1, 2008

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Figure 5. Ir-intercalator binding to cellular DNA analyzed by solution ICPMS. THP-1 and KG1a cells and empty wells (no cells) were fixed and treated with increasing amounts of Ir-intercalator. Results are presented as the average of four replicates per condition against the amount of Ir-intercalator in the original solution.

In internal standard. Results are presented as the average of four replicates per condition against the amount of Ir-intercalator in the original solution. Assuming that there are 3-23 × 108 DNA binding sites for Ir-intercalator per cell, the 105 cells sample should saturate at 5-38 × 10-11 mol of Ir-intercalator. Our results in principle support this rough estimate leading to the conclusion that only ∼1% of Ir-intercalator from solution was taken up by the cells. More concentrated solutions result in unacceptably high nonspecific absorbance of Ir-intercalator by surfaces and filters in the wells (see Figure 5, “no cells” plot). The intercalator amount of 10-11 to 10-10 mol per sample was considered optimal. Next, simultaneous cell surface antigen and DNA staining using the two intercalators, Ir- and Rh-containing, were carried out on cell lines KG1a and THP-1 which differ in the level of expression of several surface markers. Triplicate samples of 300 000 cells per tube were stained against surface antigens CD34, CD33, HLA-DR, and CD45, with a mixture of mAbs labeled with 169Tm, 141Pr, 147Sm (enriched), and 159Tb, respectively, using prototype MAXPAR reagents, or mouse immunoglobulins for controls (not shown). After washing, the cells were fixed in formaldehyde and incubated with 1 µM Ir- or Rh-intercalator. The data presented in Figure 6 show that similar results for antigen expression were obtained for KG1a stained with Ir-intercalator or Rh-intercalator: high CD45 and CD34, low CD33, and negligible HLA-DR. This pattern is characteristic for the KG1a line.45 Specificity of antibodies is evident from the staining of THP-1 cells, which do not express CD34, have high levels of CD45 and CD33, and moderate levels of HLA-DR.46 Furthermore, the dependence of the 193Ir+ signal measured by ICPMS on the concentration of Ir-intercalator solution was investigated. For this purpose, 96-multiwell filter plates were used. KG1a and THP-1 cells were distributed at 100 000/well in triplicate wells and stained for surface markers with the above-mentioned (45) Furley, A. J.; Reeves, B. R.; Mizutani, S.; Altass, L. J.; Watt, S. M.; Jacob, M. C.; Vandenelsen, P.; Terhorst, C.; Greaves, M. F. Blood 1986, 68, 11011107. (46) Tsuchiya, S.; Yamabe, M.; Yamaguchi, Y.; Kobayashi, Y.; Konno, T.; Tada, K. Int. J. Cancer 1980, 26, 171-176.

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Figure 6. Simultaneous cell surface antigen and DNA detection using Ir- and Rh-intercalators. Four antibodies against cell surface antigens (CD33, CD34, CD45, and HLA-DR) were labeled with element tags. Live cells were immunostained, fixed, and treated with Ir- or Rh-intercalators. Samples were analyzed in triplicate. Normalized response is presented as relative to 1 ng mL-1 In/10% HCl internal standard.

element-tagged antibodies. The plates were washed several times by low-speed centrifugation. The cells were then fixed in formaldehyde, washed once, and 100 µL of 1 µM Ir-intercalator was added to each well. Following several washes, the cells were digested with concentrated HCl (100 µL/well), the well contents were spun down into a collection plate, and 100 µL/well of 1 ng mL-1 In/10% HCl internal standard was added prior to the final centrifugation. Results are shown in Figure 7: a linear response for the measured Ir content (ng mL-1) with increasing intercalator concentrations (M) in staining solution is evident. Note that the antigen profiles measured with element-tagged antibodies for the two cell lines reflect data from Figure 6. To compare the dependence between responses from Rh- and Ir-intercalators and numbers of analyzed cells, we used samples of KG1a cells serially diluted to 104, 2 × 104, 4 × 104, 8 × 104, 1.6 × 105, and 3.2 × 105 and distributed in triplicate wells of 0.45 µm filter microplates. Following the procedure described above, cells were stained with 10 µM Rh-intercalator or 5 µM Ir-intercalator and analyzed after acidification. Results are presented in Figure 8A. The relationship between the uptake of Ir-and Rh-intercalators by KG1a cells was studied using Pearson’s correlation coefficient (Figure 8B). Owing to the difference in the chemical nature of the intercalators, as well as nucleic acid binding efficiency and stability, it is evident that Ir-intercalator produces a higher signal than the Rh-intercalator for a given number of cells. The use of Ir as the metal in the intercalator for detecting cells by FC-MS has several other advantages compared to Rh. First, Ir has two stable isotopes with abundance ratio of approximately 3:5; concurrence of both isotopes allows better correlated measurements of intracellular nucleic acid and surface antigens. Second, Ir mass is closer to the lanthanides mass range narrowing the mass range that is necessary for the TOF mass detector. Shorter mass range translates into improved duty cycle of TOF, thus allowing higher numbers of MS spectra to be acquired for every cell (transient signals generated in single-cell mode is around 200 µs). Third, Ir has higher ionization potential and can be used as a better reporter of ionization temperature changes during cell transport through the ICP indicating possible matrix

Figure 7. Quantification of the Ir signal in equal cell numbers with relation to the solution concentration of Ir-intercalator. Four antibodies against cell surface antigens (CD33, CD34, CD45, and HLA-DR) were labeled with element tags. Equal numbers of live cells were immunostained, fixed, and treated with increasing concentrations of Ir-intercalator. Samples were analyzed in triplicate. Normalized response is presented as relative to 1 ng mL-1 In/10% HCl internal standard.

effects. Therefore, subsequent experiments of multielement singlecell FC-MS analysis were performed using Ir-intercalator as the nucleic acid marker. Multielement Single-Cell FC-MS Analysis of DNA and Cell Surface Markers. The solution format of the assay linked to a quadrupole-based ICPMS does not allow analysis of an individual cell, since the whole cell population is digested in acid and the elemental composition of the resulting mixture is measured. To determine the elemental composition of a single cell, a novel class of analytical instrumentation, the FC-MS, was developed and employed. As a test sample for FC-MS analysis, the KG1a myeloid leukemia cells, which express large numbers of known markers on the cell surface (particularly the CD34 glycoprotein and CD45 biomarkers), were selected. The following isotopes of lanthanides chelated by the polymer tags were used to label specific antibodies against biomarkers characteristic of KG1a cells: CD7-139La, CD13-144Nd, CD44151Eu, CD45-159Tb, CD34-169Tm, and CD49d-176Yb. In separate samples, the same isotopes were employed to tag nonspecific IgGs. Immunostained cells were fixed in 3.7% formaldehyde and incubated with 1 µM Ir-intercalator solution. Cells were extensively washed with PBS, and 50% methanol/H2O was substituted for PBS directly prior to FC-MS analysis. The low-volume spray chamber

Figure 8. Comparison of Ir and Rh signals to the number of cells which were stained with either Ir- or Rh-containing intercalators. (A) Triplicate wells with increasing numbers of KG1a cells were set up in the 96-multiwell filter plates. Cells were fixed, incubated with 5 µM Ir-intercalator, or 10 µM Rh-intercalator, and treated for ICPMS analysis (normalized to 1 ng mL-1 In/10% HCl internal standard). (B) Pearson’s correlation coefficient for Rh- and Ir-intercalator binding to KG1a cells.

was used for sample nebulization employing the concentric nebulizer at 0.58 L/min Ar, 30 µL/min sample uptake (syringe pump), 1 mm alumina injector, and 1300 W of plasma forward power. The TOF sampling rate was fixed at 55 kHz. Such a simple sample introduction system is very harsh, and at least 50% of the cells may have been damaged during nebulization. However, cell fragments may still be registered as events (“cell-like” events). Half of the immunostained sample and control were simultaneously analyzed by solution ICPMS analysis (results are presented in Figure 9A). The signal-to-noise (S/N) values for CD13144Nd and CD44-151Eu and isotype-labeled IgGs are close to 1, indicating that either the specific antibodies have low affinity and/ or IgGs conjugated to these elements are particularly “sticky” and readily bind to cells. Lowering high background binding is one of the most important aspects of elemental immunophenotyping and requires additional protocol development. For the multielement FC-MS analysis at least two approaches to determine the S/N ratio are feasible: (1) for each analyte separately (one-dimensional S/N ratios) and (2) for all possible pairs of analytes (two-dimensional S/N ratios) averaged over all cells. The one-dimensional averaging, as presented in Figure 9B, is very similar to results of ICPMS assay (see Figure 9A), which Analytical Chemistry, Vol. 80, No. 7, April 1, 2008

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Figure 10. Example of two-dimensional projection of KG1a cells analyzed by single-cell FC-MS in an 8-plex experiment. The projection scale is based on the inverse hyperbolic sine of the registered signal, also called the modified logarithmic (mLn) scale, with nearly linear function on [0, 1] interval. Gray and black points represent the CD34- and CD45-positive events. The modified logarithmic scaling is similar to “logical” scaling introduced in ref 48 but is much simpler because it needs only one adjustment parameter a to define a specific

Figure 9. (A) Homogeneous solution ICPMS analysis of fixed KG1a cells stained with IgGs and Abs analyzed on a conventional ICPMS instrument. The intercalator concentrations was 1 µM Ir. The error bars represent the standard deviations of samples in triplicates. Signals are normalized on 1 ng mL-1 In signal (internal standard) to reduce effects of possible instrument sensitivity drift and mild matrix effects. (B) FC-MS single-cell analysis of fixed KG1a cells stained with IgGs and Abs The analyte signals are averaged over all cell-like events. The Ir-intercalator concentration was at 1 µM Ir.

does not fully represent the distribution of individual markers on a single cell, and corresponding S/N ratio can be artificially low. Other multidimensional projections are beyond the scope of this paper and will be discussed separately. Let us consider the two-dimensional projections of the multidimensional data set obtained as a result of the FC-MS experiment (see Figure 10 for the Tb/Tm projection). Some cell-like events are CD34- and CD45-negative and are positioned strictly on the axes of the projection. Single cells stained with labeled antibodies are positive in both directions with high probability. The same can be done for all possible two-dimensional projections. From results of the FC-MS experiment with KG1a cells stained with specific antibodies, we determined that the ratio of cells positive in both dimensions varies from 4% to 45% of total number of the cell-like events. On the contrary, the same ratio is small (1-7%) for KG1a cells stained with control IgGs. The twodimensional S/N ratios, presented in Table 1, are very different from the S/N ratios in one-dimensional projections (see Figure 9B) and provide more information about the distribution of individual cell biomarkers. Dramatic improvement in the S/N ratios can be achieved by normalizing signals from every cell on its intercalator signal, thus reducing the effect of cell size variability, including cell fragments. 2546 Analytical Chemistry, Vol. 80, No. 7, April 1, 2008

display: sinh-1(ax) ) ln(ax + x1+a2x2). The logical scaling is a very flexible approach designed to accommodate negative and zero signal values which routinely appear in conventional flow cytometry as a result of complicated compensation computations. The mLn scaling provides enough flexibility to meet the FC-MS display needs in the absence of compensation requirements. Table 1. Signal-to-Noise Ratios (S/N) for KG1a Cells Stained with Specific Antibodies (Signal) and Nonspecific IgG (Noise)a S/N La Nd Eu Tb Tm Yb

La 0.7 0.4 36 29 5.6

Nd

Eu

Tb

Tm

Yb

5.2

4.7 0.9

8.4 1.4 0.8

6.5 2.0 0.8 90

9.9 0.9 0.5 80 63

0.6 60 72 4.6

44 36 2.9

75 7.3

7.6

a The element-antibody combinations are CD7-139La, CD13-144Nd, CD44-151Eu, CD45-159Tb, CD34-169Tm, and CD49d-176Yb.

Table 2. Signal-to-Noise Ratios (S/N) for KG1a Cells Stained with Specific Antibodies and IgGa S/N

La

La Nd Eu Tb Tm Yb

2.6 1.6 220 200 28

Nd 8.2 3.4 450 500 31

Eu 6.6 4.3 330 290 20

Tb

Tm

Yb

15 8.3 5.4

11 11 5.1 720

11 5.1 3.5 470 270

620 33

46

a Signal intensities are normalized on Ir-intercalator for every cell individually. The element-antibody combinations are CD7-139La, CD13-144Nd,CD44-151Eu,CD45-159Tb,CD34-169Tm,andCD49d-176Yb.

Results are presented in Table 2. In this format, all biomarkers can be confidently measured. This presents an additional improvement in comparison with the ICPMS assay, where the intercalator normalization is successful in reducing the variability of the total

cell number per sample. Similar cumulative information can be also extracted from the FC-MS data set. For example, the twodimensional Ir-intercalator marker projections immediately highlight the fraction of marker-positive cells (all cells or cell-like events are selected to be intercalator-positive). CONCLUSIONS We describe a novel approach to nucleic acid detection and cell enumeration methodology using metal-containing reagents and elemental mass spectrometry. We show that the exclusive sensitivity, matrix tolerance, and absolute quantitation capability of ICPMS that have made it the method of choice in analytic chemistry may be successfully employed in biological studies. In combination with ICPMS, metallointercalators are a sensitive and practical reagent for cell enumeration and cellular DNA detection. They may also present convenient compounds for atomic mass spectrometry due to low detection limits in heavy organic matrixes. The addition of rare earth metals in nontoxic concentrations to cells in vitro conditions is a feasible alternative means of determining cell numbers in a multiplex setting. However, this method in comparison to metallointercalators is less reliable and reproducible due to analytical dependence on the sample preparation protocol (number of washes, volume of solutions, and so on). The choice of isotype IgGs tagged with the same elements as an indication of nonspecific binding of specific mAbs employed in this work relies heavily on the accuracy of concentration determination of tagged immunoglobulins and the assumption that all immunoglobulins have a similar chemical nature. These assumptions and experimental errors should be critically considered in future work. Gene expression and function may be disrupted by modifications in the cellular DNA copy number leading to various disease states. For example, human developmental defects are closely linked to gains and losses of chromosomes prior to or shortly after fertilization.47 Somatic cell alterations in DNA ploidy are (47) Pinkel, D.; Albertson, D. G. Annu. Rev. Genomics Hum. Genet. 2005, 6, 331-354. (48) Parks, D. R.; Roederer, M.; Moore, W. A. Cytometry, Part A 2006, 69A, 541-551.

thought to contribute to cancer. The proposed method based on ICPMS detection of metallointercalators that bind to DNA may provide a quantitative and sensitive method for detecting these aberrations, thus generating clinically relevant information. Due to the high efficiency of intercalation and the sensitivity of ICPMS as a detection system, attempts to achieve saturation levels with the Ir- and Rh-containing metallointercalators were unsuccessful. It was also difficult (statistically unreliable) to work with samples containing much smaller numbers of cells. Therefore, we will expand on this work using FC-MS single-cell analysis in conjunction with Ir-intercalators in future studies. Overall, the ability to detect and quantitate multiple element labels by plasma mass spectrometry in biologically relevant samples (solution or single cell) holds great promises for life sciences and clinical diagnostics. However, this new approach requires thorough method development aimed at quantifying a particular set of biomarkers. Because this method involves the use of novel elemental tags and intercalators their nonspecific binding to sample preparation containers and filters should be investigated. Multiparametric FC-MS technology is closely linked to data reduction, presentation, and classification in a multidimensional space, which are as yet in the early stages of research and development. Reliable quantitation of many biomarkers on every cell should open the possibility for cell state characterization unattainable by other bioanalytical methods. ACKNOWLEDGMENT This project was funded by Genome Canada through the Ontario Genomics Institute and Ontario Institute of Cancer Research. The authors thank Robert Kinach for his persistence in developing the lanthanide oxide conversion protocols, and Lavanya Nallapaneni, Alexei Antonov, and Sergey Vorobiev for their engineering efforts in developing the research prototype of the FC-MS.

Received for review October 16, 2007. Accepted January 29, 2008. AC702128M

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