as a small molecule probe , aptamers are composed of short , single - stranded oligonucleotides ( rna or ssdna ) ranging from 30 to 60 bases . similar to protein antibodies on the basis of their three - dimensional structures , aptamers specifically recognize and bind to their targets with high sensitivity and specificity . these targets can include small molecules , macromolecules , proteins , viruses , cells , and tissues . however , in contrast to antibodies , oligonucleotide aptamers are easily generated through chemical synthesis , and can be conveniently modified with a variety of functional molecules for different purposes . therefore , aptamers have been widely studied for potential biomedical applications as a  chemical antibody  and , more importantly , for developing new clinical applications that can not be performed with current protein antibody technology . metastasis or dissemination of primary tumor is the major cause of mortality in cancer patients . recent studies have demonstrated that circulating tumor cells ( ctcs ) in the bloodstream are key factors in the establishment of metastatic tumors . in addition , the number of ctcs present in whole blood is directly associated with cancer progression , recurrence , survival rate , and prognosis . therefore , accurate detection of ctcs will provide critical information for the proper management of cancer patients . antibody technology has been widely used for a variety of biomedical applications since it was first developed . however , the conjugated fluorochrome is constantly emitting a fluorescent signal , regardless of the status of whether the antibody is free or binding to cells , leading to a high level of background noise and potential off - target signals from normal blood cells . therefore , the current antibody - mediated assays require multiple steps to isolate ctcs from normal blood cells and eliminate potential off - target signals . repeated washes to remove excess free fluorochrome - conjugated antibody from the assays are also required . notably , these multi - step assays are time - consuming and , more importantly , lead to loss of the blood sample and damage to ctcs , which adversely impacts assay accuracy . to overcome these technical obstacles in antibody - mediated assays , a new reporter system that can selectively highlight only tumor cells without generation of background noise for point - of - care purposes , an  ideal  ctc assay should be ( i ) simple ( one - step reaction ) , ( ii ) high throughput ( complete in minutes ) , and ( iii ) accurate ( no loss of blood sample and no damage of tumor cells through the assay process ) . however , no such assay could be developed using current antibody - mediated technology as described above . to this end , we designed a unique aptamer - reporter , which is composed of an oligonucleotide sequence specific for a tumor cell biomarker conjugated with a reporter of paired fluorochrome - quencher molecules . the underlining hypothesis is that the intact aptamer - reporter is optically silent in the absence of tumor cells ( figure 1a ) , but can be activated by tumor cells to emit fluorescence . as showed in figure 1b , in assays containing patient blood the oligonucleotide sequence will selectively target ctcs through interaction with a specific surface biomarker , which triggers internalization of the aptamer - reporter through a natural cellular process and , subsequently , into cell lysosomes that are rich in different enzymes . this internalization mechanism has been successfully used for intracellular delivery of chemotherapeutic drugs for targeted therapy by a newly fda - approved antibody - drug - conjugate , brentuximab . rapid degradation of the aptamer - reporter by lysosomal nucleases results in separation of the paired fluorochrome - quencher molecules , the released fluorochrome becoming optically active and emitting bright fluorescent signals exclusively within the targeted tumor cells . notably , because it is controlled by a specific aptamer - biomarker interaction and an intracellular activation switch , the aptamer - reporter will generate no background noise or off - target signals from normal blood cells and thus , the assay can be carried out in one - step , load - then - read ( figure 1c ) . for the initial proof - of - concept studies , the aptamer - reporter was formulated by conjugating a 39-mer rna sequence specific for the cd30 biomarker with a fluorochrome cy3 compound and a bhq2 at the 5- and 3-ends , respectively ( figure 1a and figure 2a , left ) . due to covalent chemical conjugation , aptamer sequence is able to hold fluorochrome and quencher molecules close to each other and , thus , the bhq2 molecule will quench fluorescent signals emitted from the cy3 compound on the same aptamer . a control aptamer was also synthesized by conjugating the same sequence with the fluorochrome cy3 compound alone ( figure 2a , right ) . first , to validate the optical - activatable property , the aptamer - reporter was incubated with fresh lysates of cultured karpas 299 tumor cells of human alcl at room temperature as a cellular enzyme source . incubation with cell lysates activated the aptamer - reporter and triggered a rapid increase in florescent signals in a time - dependent fashion ( figure 2b ) . in contrast , cell lysates had no effect on the control aptamer which constantly emitted high levels of fluorescent signals . to rule out a potential effect due to conformation change , the aptamer - reporter was treated under different ph conditions , from ph 4.0 to physiological ph 7.4 , at room temperature for 30 minutes ( figure 2c ) . notably , cell lysosomes have a low ph environment ( ph 45 ) which can cause denaturing of the aptamer sequence . change in ph had no effect on fluorescent emission of the aptamer - reporter or the control aptamer . these findings indicated that the formed aptamer - reporter can be optically activated by cell lysates , but not affected by the low ph - induced structure change . to further strengthen these observations , the control and aptamer - reporter were also incubated in human serum alone or in serum containing fresh cell lysates under . kinetic analysis of fluorescence revealed that the aptamer - reporter was stable up to 90 minutes in human serum with minimal background , and was rapidly activated by the addition of cell lysates ( figure 2d ) . to study tumor cell - induced activation , the aptamer - reporter was simply added into a culture of karpas 299 cells , which express high levels of the cd30 biomarker on the cell surfaces . as shown in figure 2e ( upper panel ) , the aptamer - reporter was rapidly optically activated by the tumor cells and , thus , highlighted the tumor cells . the fluorescence was initially observed at 10 minutes postincubation and lasted up to 120 minutes , with no background noise . quantified fluorescent signals of tumor cells at different time points were calculated and summarized in graph in figure 2e . subsequently , to validate intracellular optical activation , cells were treated with the aptamer - reporter at room temperature for 30 minutes and cell membranes were then stained with the green fluorescent dye , alexa 488 . fluorescent signals derived from the aptamer - reporters ( red ) and prestained cell membranes ( green ) were recorded separately . merged images confirmed that the aptamer - reporter emitted fluorescent signals exclusively confined within the targeted cells . moreover , to detect intracellular distribution , the cells were treated with the aptamer - reporter and cell lysosomes were then stained with the fluorescent dye , lyso - id . merging of confocal microscopic images revealed the colocation of fluorescent signals from the aptamer - reporter ( red ) and prestained lysosomes ( green ) within the same cells ( figure 2 g ) . these findings confirmed that the aptamer - reporter could be optically activated and emit fluorescent signals exclusively within the targeted cells . to validate assay specificity , multiple cultured cells were incubated with the aptamer - reporter at room temperature for 30 minutes , and directly examined under fluorescent microscope . as shown in figure 3a , the aptamer - reporter was activated by the tumor cells that express the cd30 biomarker and , thus , highlighted the tumor cells of sudhl-1 , hdlm2 , and kmh2 cells , but remained optically silent in control cultures of maver-1 , u937 , and ca46 cells that do not express cd30 , with no background noise . additional studies with carcinoma tumor cells and normal peripheral cells are summarized in table 1 . to assess sensitivity , a cell mixture was made by diluting karpas 299 tumor cells at ratios from 1/10 to 1/10,000 in u937 control cells , which were prestained with green fluorescence ( figure 3b ) . the cell mixtures were incubated with the aptamer - reporter , and fluorescent signals derived from the aptamer - reporters ( red ) and prestained control cells ( green ) were directly recorded . merged images revealed that the aptamer - reporters were selectively activated and highlighted by cd30-expressing tumor cells , but did not react to control cells in the cell mixtures . taken together , these findings indicate that the aptamer - reporter was both specific and sensitive for detection of tumor cells . for preclinical evaluation , peripheral blood and marrow aspirate were collected from patients with alcl tumors under a protocol approved by the irb for the houston methodist research institute . diagnosis of alcl was made by tissue biopsy , and cd30 expression on lymphoma cells was confirmed by immunohistochemical staining of tumor tissues with anti - cd30 antibody ( figure 4a ) . to detect ctcs of alcl , whole blood samples were added into a 12-well plate ( 40 million total blood cells / well ) , which was preloaded with the aptamer - reporter ( 5 after incubation for 30 minutes at room temperature , plates were directly examined under a microscope . blood cells were well preserved for morphologic identification , including intact rbc , white blood cells , and possibly rare tumor cells that could not be distinguished ( figure 4b ) . however , the ctcs in whole blood samples were selectively highlighted by the aptamer - reporter with bright fluorescence and without background noise or off - target signals from normal blood ( figure 4b , right panels ) . similarly , patient marrow aspirate was also added into the assays , and ctcs were selectively highlighted by the aptamer - reporter as detected by fluorescent microscopy ( figure 4c ) . currently , antibodies have been used as the  gold standard  probe for tumor cell detection in the clinical setting . double - staining of ctcs were performed to confirm specificity of the aptamer - reporter . patient marrow cells were initially probed by the fitc - conjugated anti - cd30 antibody . after removal of free antibody from the reaction , the cells were then treated with the aptamer - reporter and directly examined under a fluorescent microscope . cell signals derived from antibody ( green ) and the aptamer - reporters ( red ) were recorded separately ( figure 4d ) . merged images revealed that the ctcs in patient marrow were double - stained by both antibody and the aptamer - reporters , confirming specificity of the one - step assay . notably , the aptamer and antibody did not compete for the same binding site(s ) on the same tumor cells . additional blood specimens from different patients with alcl tumor were also analyzed , and ctcs were specifically highlighted and detected with a high signal - background noise ratio ( figure 4e , upper panel ) . to validate assay capacity , a high - concentration blood sample was loaded into a 12-well plate ( 120 million blood cells / well ) to form multiple layers of cells ( figure 4e , lower panel ) . interestingly , cellular fluorescent signals were easily detected , although the highlighted ctcs were overlapped by normal blood cells and could not be observed under a light microscope . as a normal control , whole blood samples from healthy donors were tested under the same assay and showed no off - target signals ( figure 4f ) . for point - of - care purposes , an  ideal  ctc assay should be ( i ) simple ( one - step reaction ) , ( ii ) high throughput ( complete in minutes ) , and ( iii ) accurate ( no loss of blood sample and no damage of tumor cells through the assay process ) . however , no such assay could be developed using current antibody - mediated technology as described above . to this end , we designed a unique aptamer - reporter , which is composed of an oligonucleotide sequence specific for a tumor cell biomarker conjugated with a reporter of paired fluorochrome - quencher molecules . the underlining hypothesis is that the intact aptamer - reporter is optically silent in the absence of tumor cells ( figure 1a ) , but can be activated by tumor cells to emit fluorescence . as showed in figure 1b , in assays containing patient blood the oligonucleotide sequence will selectively target ctcs through interaction with a specific surface biomarker , which triggers internalization of the aptamer - reporter through a natural cellular process and , subsequently , into cell lysosomes that are rich in different enzymes . this internalization mechanism has been successfully used for intracellular delivery of chemotherapeutic drugs for targeted therapy by a newly fda - approved antibody - drug - conjugate , brentuximab . rapid degradation of the aptamer - reporter by lysosomal nucleases results in separation of the paired fluorochrome - quencher molecules , the released fluorochrome becoming optically active and emitting bright fluorescent signals exclusively within the targeted tumor cells . notably , because it is controlled by a specific aptamer - biomarker interaction and an intracellular activation switch , the aptamer - reporter will generate no background noise or off - target signals from normal blood cells and thus , the assay can be carried out in one - step , load - then - read ( figure 1c ) . for the initial proof - of - concept studies , the aptamer - reporter was formulated by conjugating a 39-mer rna sequence specific for the cd30 biomarker with a fluorochrome cy3 compound and a bhq2 at the 5- and 3-ends , respectively ( figure 1a and figure 2a , left ) . due to covalent chemical conjugation , aptamer sequence is able to hold fluorochrome and quencher molecules close to each other and , thus , the bhq2 molecule will quench fluorescent signals emitted from the cy3 compound on the same aptamer . a control aptamer was also synthesized by conjugating the same sequence with the fluorochrome cy3 compound alone ( figure 2a , right ) . first , to validate the optical - activatable property , the aptamer - reporter was incubated with fresh lysates of cultured karpas 299 tumor cells of human alcl at room temperature as a cellular enzyme source . incubation with cell lysates activated the aptamer - reporter and triggered a rapid increase in florescent signals in a time - dependent fashion ( figure 2b ) . in contrast , cell lysates had no effect on the control aptamer which constantly emitted high levels of fluorescent signals . to rule out a potential effect due to conformation change , the aptamer - reporter was treated under different ph conditions , from ph 4.0 to physiological ph 7.4 , at room temperature for 30 minutes ( figure 2c ) . notably , cell lysosomes have a low ph environment ( ph 45 ) which can cause denaturing of the aptamer sequence . change in ph had no effect on fluorescent emission of the aptamer - reporter or the control aptamer . these findings indicated that the formed aptamer - reporter can be optically activated by cell lysates , but not affected by the low ph - induced structure change . to further strengthen these observations , the control and aptamer - reporter were also incubated in human serum alone or in serum containing fresh cell lysates under . kinetic analysis of fluorescence revealed that the aptamer - reporter was stable up to 90 minutes in human serum with minimal background , and was rapidly activated by the addition of cell lysates ( figure 2d ) . to study tumor cell - induced activation , the aptamer - reporter was simply added into a culture of karpas 299 cells , which express high levels of the cd30 biomarker on the cell surfaces . cells were then directly examined under fluorescent microscope . as shown in figure 2e ( upper panel ) , the aptamer - reporter was rapidly optically activated by the tumor cells and , thus , highlighted the tumor cells . the fluorescence was initially observed at 10 minutes postincubation and lasted up to 120 minutes , with no background noise . quantified fluorescent signals of tumor cells at different time points were calculated and summarized in graph in figure 2e . subsequently , to validate intracellular optical activation , cells were treated with the aptamer - reporter at room temperature for 30 minutes and cell membranes were then stained with the green fluorescent dye , alexa 488 . fluorescent signals derived from the aptamer - reporters ( red ) and prestained cell membranes ( green ) were recorded separately . merged images confirmed that the aptamer - reporter emitted fluorescent signals exclusively confined within the targeted cells . moreover , to detect intracellular distribution , the cells were treated with the aptamer - reporter and cell lysosomes were then stained with the fluorescent dye , lyso - id . merging of confocal microscopic images revealed the colocation of fluorescent signals from the aptamer - reporter ( red ) and prestained lysosomes ( green ) within the same cells ( figure 2 g ) . these findings confirmed that the aptamer - reporter could be optically activated and emit fluorescent signals exclusively within the targeted cells . to validate assay specificity , multiple cultured cells were incubated with the aptamer - reporter at room temperature for 30 minutes , and directly examined under fluorescent microscope . as shown in figure 3a , the aptamer - reporter was activated by the tumor cells that express the cd30 biomarker and , thus , highlighted the tumor cells of sudhl-1 , hdlm2 , and kmh2 cells , but remained optically silent in control cultures of maver-1 , u937 , and ca46 cells that do not express cd30 , with no background noise . additional studies with carcinoma tumor cells and normal peripheral cells are summarized in table 1 . to assess sensitivity , a cell mixture was made by diluting karpas 299 tumor cells at ratios from 1/10 to 1/10,000 in u937 control cells , which were prestained with green fluorescence ( figure 3b ) . the cell mixtures were incubated with the aptamer - reporter , and fluorescent signals derived from the aptamer - reporters ( red ) and prestained control cells ( green ) were directly recorded . merged images revealed that the aptamer - reporters were selectively activated and highlighted by cd30-expressing tumor cells , but did not react to control cells in the cell mixtures . taken together , these findings indicate that the aptamer - reporter was both specific and sensitive for detection of tumor cells . for preclinical evaluation , peripheral blood and marrow aspirate were collected from patients with alcl tumors under a protocol approved by the irb for the houston methodist research institute . diagnosis of alcl was made by tissue biopsy , and cd30 expression on lymphoma cells was confirmed by immunohistochemical staining of tumor tissues with anti - cd30 antibody ( figure 4a ) . to detect ctcs of alcl , whole blood samples were added into a 12-well plate ( 40 million total blood cells / well ) , which was preloaded with the aptamer - reporter ( 5 after incubation for 30 minutes at room temperature , plates were directly examined under a microscope . blood cells were well preserved for morphologic identification , including intact rbc , white blood cells , and possibly rare tumor cells that could not be distinguished ( figure 4b ) . however , the ctcs in whole blood samples were selectively highlighted by the aptamer - reporter with bright fluorescence and without background noise or off - target signals from normal blood ( figure 4b , right panels ) . similarly , patient marrow aspirate was also added into the assays , and ctcs were selectively highlighted by the aptamer - reporter as detected by fluorescent microscopy ( figure 4c ) . currently , antibodies have been used as the  gold standard  probe for tumor cell detection in the clinical setting . double - staining of ctcs were performed to confirm specificity of the aptamer - reporter . patient marrow cells were initially probed by the fitc - conjugated anti - cd30 antibody . after removal of free antibody from the reaction , the cells were then treated with the aptamer - reporter and directly examined under a fluorescent microscope . cell signals derived from antibody ( green ) and the aptamer - reporters ( red ) were recorded separately ( figure 4d ) . merged images revealed that the ctcs in patient marrow were double - stained by both antibody and the aptamer - reporters , confirming specificity of the one - step assay . notably , the aptamer and antibody did not compete for the same binding site(s ) on the same tumor cells . additional blood specimens from different patients with alcl tumor were also analyzed , and ctcs were specifically highlighted and detected with a high signal - background noise ratio ( figure 4e , upper panel ) . to validate assay capacity , a high - concentration blood sample was loaded into a 12-well plate ( 120 million blood cells / well ) to form multiple layers of cells ( figure 4e , lower panel ) . interestingly , cellular fluorescent signals were easily detected , although the highlighted ctcs were overlapped by normal blood cells and could not be observed under a light microscope . as a normal control , whole blood samples from healthy donors were tested under the same assay and showed no off - target signals ( figure 4f ) . it is generally believed that there are only a scant number of detectable ctcs in patient blood , although the actual number ranges are not yet fully known . a recent study demonstrated that the average number of ctcs ranges from a mean of 79 to 196 cells per ml in whole blood specimens of patients with different types of cancers , which is much higher than the cut - off cell number ( 5 cells / ml ) detected by current commercial tests . in addition , employing the cell search profile kit could detect more than 30-fold of the median ctc counts that are identified by the clinically used cell search epithelial kit in the same blood samples of patients with breast or lung cancers . these findings indicate the presence of much more ctcs in patient blood than previously thought , suggesting the possibility that , with a sensitive detection approach , the current standard numeration of ctcs could be achieved using smaller blood samples . since our one - step assay will eliminate any potential loss of the blood sample and damage of tumor cells during the assay process , it will be able to detect the actual number of ctcs , and thus , require a much smaller blood sample . notably , although the number of ctcs present in blood may vary among different types of tumors and different stages of disease , our assay scale can simply be justified by employing different - sized plates as clinically indicated with 96- , 24- , 12- , 6- , and single - well(s ) to meet specific clinical needs . in addition , the load - then - read technology provides the possibility of performing high - throughput screening assays for rapid detection of ctcs . moreover , good preservation of intact ctcs in this one - step assay will allow us to collect viable ctcs for further analysis of cellular protein and/or gene expression profiles at a single - cell level . to eliminate background noise in whole - blood samples and enable intracellular highlighting of tumor cells , the one - step assay was designed by taking advantage of tumor cell internalization of the aptamer - reporter through a natural biological cell process , which is triggered by specific binding of aptamer sequences to surface cd30 receptors on tumor cells ( figure 1b ) . interestingly , a similar internalization mechanism is employed for intracellular delivery of chemotherapeutic drug by an antibody - drug - conjugate , brentuximab , a newly fda - approved , targeted therapeutic specific for cd30-expressing lymphomas . thus , it is considerable that , in addition to the potential for diagnostic applications , aptamers can also be used as a chemical antibody for targeted therapy . in summary , our study introduced a unique tumor cell - activatable aptamer - reporter technology and demonstrated a simple , one - step assay for detection of ctcs in patient whole blood . since this technology improves upon the existing antibody - driven methods , we believe it holds a high potential in early identification of patients who are at a higher risk for developing tumor metastases due to their elevated levels of systemic ctcs . reagents and cells . in the proof of principal studies , a 39-mer rna - based aptamer sequence , previously shown to specifically bind cd30-expressing tumor cells , was used . a novel tumor cell - activatable aptamer - reporter was then chemically synthesized ( bio - synthesis , lewisville , tx ) by conjugating a fluorochrome cy3 together with a black hole quencher 2 ( bhq2 ) molecule at the opposing ends of the aptamer sequence as follows : cy3 - 5-rgrarururcrarurarurgrgrgrurgrgrgrarurcrgrgrgrarargrgrgrcrurarurgrararurcrg-3-bhq2 . a control aptamer probe with the same sequence was conjugated with a fluorochrome cy3 compound at the 5 end . alexa fluor 488 conjugates for cell membrane staining were purchased from invitrogen ( grand island , ny ) and lyso - id green detection kit for lysosome staining was purchased from enzo life sciences ( farmingdale , ny ) . the fitc - conjugated anti - human cd30 antibody was purchased from bd biosciences ( franklin lakes , nj ) . cancer cells lines , including human anaplastic large cell lymphoma ( alcl ) cells ( karpas 299 and sudhl-1 cell lines from mark raffeld at nih ) , hodgkin lymphoma cells ( hdlm2 and kmh2 cell lines from barbara savoldo , baylor college of medicine , houston , tx ) , b cell lymphoma cells ( mino and maver-1 cell lines from atcc ) , and leukemia cells ( u937 cell line from atcc ) were cultured in rpmi 1640 medium supplemented with 10% fbs , 100 u / ml penicillin , and 100 g / ml streptomycin at 37 c under the atmosphere of 5% co2 and 95% humidity . fresh tumor cell lysates were prepared to confirm the cell - activatable property of the formed aptamer - reporter . briefly , cultured karpas 299 cells were harvested , washed twice , and suspended in pbs ( 5    10/ml ) . cells were frozen at 80 c , thawed at room temperature twice , and centrifuged at 12,000 g for 20 minutes at 4 c . finally , 100 l supernatants of cell lysates were collected and added into wells of a 96-well black wall plate , which was preloaded with the aptamer - reporter ( 5 after incubation at room temperature , fluorescence signals of the assays were kinetically quantified by a synergy h4 microplate reader ( biotek , winooski , vt ) at different time points , as described . each condition was tested in triplicate and experiments were repeated more than three times with similar results . similarly , the control aptamer , containing the same sequence and conjugated with the fluorochrome cy3 compound alone , was tested under the same treatment conditions and change in fluorescent signals of the control group was kinetically monitored as described above . to rule out the effects of low ph conditions , which is seen in cell lysosomes and might result in denature of the aptamer sequence 3d - structure , the aptamer - reporter ( 5 nmol / l final concentration ) was added into pbs with phs 4.0 , 5.0 , 6.0 , 7.0 , and 7.4 in a 96-well black wall plate . after incubation at room temperature for 30 minutes , fluorescence of the assays was quantified by a synergy h4 microplate reader . each condition was tested in triplicate and experiments were repeated more than three times with similar results . finally , the aptamer - reporter ( 5 nmol / l final concentration ) were incubated in 100 l human serum ( atlanta biological , lawrenceville , ga , usa ) in a 96-well black wall plate and changes in fluorescence were quantified as described above . in control group , 100 l of cell lysate supernatants each condition was tested in triplicate and experiments were repeated more than three times with similar results . one - step assay and tumor cell detection . to validate the tumor cell - induced activation , 100 l of cultured karpas 299 cells ( 5    10/ml ) were preseeded in a 96-well black wall plate in pbs , and 5 l of the aptamer - reporter was then added to each well at 5 nmol / l final concentration . without any additional preparation steps , the plates were directly examined under an olympus ix81 fluorescent microscope and cell fluorescent signals were kinetically imaged using a tritc filter under 547  nm of peak excitation wavelength and 572  nm of peak emission wavelength . for control purposes , cells were also treated with the control aptamer that was conjugated with fluorochrome cy3 compound alone under the same condition . fluorescent images were recorded using the same exposure time ( 300 microseconds ) at different time points post treatment as described in figures . each experiment with triple wells was repeated at least three times with similar results . for quantification , intensity of fluorescent signals from individual tumor cells a total of 90 cells from triplet wells ( 30 cells / well ) at each time point were randomly gated and recorded . to get the background value of fluorescent intensity , 10 random noncellular locations with an average area of cell sizes were gated and calculated similarly . the mean fluorescent intensity of tumor cells was derived after background signals were deducted , calculated with standard deviation , and shown in graph with  sd error bars . cultured karpas 299 cells were treated with the aptamer - reporter at room temperature for 30 minutes . after washing once with pbs , the cells were suspended in pbs at room temperature , stained with concanavalin a conjugated to alexa fluor 488 ( 1  mg / ml ) for 2 minutes , fixed with 4% formaldehyde for 10 minutes , washed with pbs , and suspended in pbs / glycerin ( weight / weight ratio of 1:1 ) . finally , 15 l of cell solution was loaded onto glass slides and examined by a confocal microscope to determine intracellular localization of the aptamer - reporter ( fitc filter for alexa for lysosomal detection , karpas 299 cells were treated with the aptamer - reporter as described above . after washing once with pbs , cells were stained with lyso - id green dye ( 1:1,000 dilution ) at room temperature for 1 hour , fixed with 4% formaldehyde for 10 minutes , washed with pbs , and then suspended in pbs / glycerin . finally , 15 l of cell solution was loaded onto glass slides and examined by a confocal microscope as described above to determined possible colocalization of aptamer - reporters with the lysosomes . specificity and sensitivity validation . cd30-expressing cells ( karpas 299 , sudhl-1 , hdlm2 , and kmh2 ) and cd30-negative control cells ( maver-1 , u937 , and ca46 ) were tested to confirm specificity of the aptamer - reporter . the cells were incubated with the aptamer - reporter for 30 minutes at room temperature and the fluorescence was detected as described above . additional cell lines ( breast , prostate , and colon carcinomas ) and peripheral blood collected from healthy donors were also tested and are listed in table 1 . to test sensitivity of the aptamer - reporter , cell mixtures were prepared from cd30-expressing karpas 299 cells and cd30-negative u937 control cells . for tracking purposes , u937 cells were stained with concanavalin a conjugated to alexa fluor 488 and karpas 299 cells were added in 1:10 to 1:10,000 ratios , as indicated . subsequently , cell mixtures were added into 96-well black wall plates ( 2    10 cells / well ) that were preloaded with the aptamer - reporting system ( 5 nmol / l final concentration ) . after 30 minutes incubation at room temperature , cells were examined by a fluorescent microscope ( fitc filter for alexa fluor 488 to detect u937 cells , tritc filter for cy3 derived from aptamer - reporters ) . detection of ctcs in patient whole blood and marrow aspirate samples in a single - step reaction . under an approved irb protocol , peripheral blood and bone marrow samples were collected from patients ( n = 6 ) with stage iv alcl tumors diagnosed by a histological evaluation of h&e - stained tumor tissue and immunohistochemical demonstration of cd30 expression on tumor cells . ten blood and marrow samples collected from healthy donors were used as a control group . following cell counting , whole blood and marrow aspirate specimens were added into wells of a 12-well plate that contained the aptamer - reporter ( 5 nmol / l final concentration ) for a total of 40 million blood or marrow cells / well . after incubation at room temperature for 30 minutes , single - step labeled ctcs were directly detected by a fluorescent microscope . for a morphological evaluation , ctc images were also obtained with a light microscope within the same view fields . to confirm specificity of the aptamer - reporter , marrow cells were double - stained with fitc - conjugated anti - human cd30 antibody and the aptamer - reporter . briefly , bone marrow cells were diluted in 1  ml pbs and incubated with fitc - conjugated anti - human cd30 antibody ( bd biosciences , san jose , ca ) at room temperature for 30 minutes . to eliminate unbound antibody and minimize damage to ctcs , the reaction supernatant containing some red blood cells ( rbcs ) was gently removed by a pipette . the remaining cells were then suspended in 200 l pbs and added into well plates that were preloaded with the aptamer - reporter at a 5 nmol / l final concentration . after 30 minutes incubation at room temperature , plates were directly examined by a fluorescent microscope and cell fluorescence ( green : antibody , red : aptamer - reporter ) recorded separately . merged images showed that ctcs in patient marrow were double - stained , confirming specificity of the aptamer - reporter . to test the effect of blood concentration , a total of 120 million blood cells from patients diagnosed with alcl were added into 12-well plates to form multiple cell layers . after 30 minutes incubation with the aptamer - reporter , plates were examined by light and fluorescent microscopes to detect ctc fluorescence as described above . merged images indicated that the assay was able to detect ctcs in high - concentration blood samples , even when ctcs overlapped with normal blood cells and could not be observed under a light microscope . to validate these results , whole blood samples from healthy donors were also tested in the single - step assay , which revealed no false - positive or off - target staining in the normal blood cells .