Background In the field of clinical mycobacteriology,  Mycobacterium tuberculosis  (MTB) can be a difficult organism to manipulate due to the restrictive environment of a containment level 3 (CL3) laboratory. Tests for rapid diagnostic work involving smears and molecular methods do not require CL3 practices after the organism has been rendered non-viable. While it has been assumed that after organism deactivation these techniques can be performed outside of a CL3, no conclusive study has consistently confirmed that the organisms are noninfectious after the theoretical 'deactivation' steps. Previous studies have shown that initial steps (such as heating /chemical fixation) may not consistently kill MTB organisms.
Background
In the field of clinical mycobacteriology,  Mycobacterium tuberculosis  (MTB) can be a difficult organism to manipulate due to the restrictive environment of a containment level 3 (CL3) laboratory. Tests for rapid diagnostic work involving smears and molecular methods do not require CL3 practices after the organism has been rendered non-viable. While it has been assumed that after organism deactivation these techniques can be performed outside of a CL3, no conclusive study has consistently confirmed that the organisms are noninfectious after the theoretical 'deactivation' steps. Previous studies have shown that initial steps (such as heating /chemical fixation) may not consistently kill MTB organisms.Mycobacterium tuberculosisMethods An inclusive viability study ( n  = 226) was undertaken to determine at which point handling of culture extraction materials does not necessitate a CL3 environment. Four different laboratory protocols tested for viability included: standard DNA extractions for IS6110 fingerprinting, crude DNA preparations for PCR by boiling and mechanical lysis, protein extractions, and smear preparations. For each protocol, laboratory staff planted a proportion of the resulting material to Bactec 12B medium that was observed for growth for 8 weeks.
Methods
An inclusive viability study ( n  = 226) was undertaken to determine at which point handling of culture extraction materials does not necessitate a CL3 environment. Four different laboratory protocols tested for viability included: standard DNA extractions for IS6110 fingerprinting, crude DNA preparations for PCR by boiling and mechanical lysis, protein extractions, and smear preparations. For each protocol, laboratory staff planted a proportion of the resulting material to Bactec 12B medium that was observed for growth for 8 weeks.nResults Of the 208 isolates initially tested, 21 samples grew within the 8-week period. Sixteen (7.7%) of these yielded positive results for MTB that included samples of: deactivated culture resuspensions exposed to 80°C for 20 minutes, smear preparations and protein extractions. Test procedures were consequently modified and tested again ( n  = 18), resulting in 0% viability.
Results
Of the 208 isolates initially tested, 21 samples grew within the 8-week period. Sixteen (7.7%) of these yielded positive results for MTB that included samples of: deactivated culture resuspensions exposed to 80°C for 20 minutes, smear preparations and protein extractions. Test procedures were consequently modified and tested again ( n  = 18), resulting in 0% viability.nConclusions This study demonstrates that it cannot be assumed that conventional practices (i.e. smear preparation) or extraction techniques render the organism non-viable. All methodologies, new and existing, should be examined by individual laboratories to validate the safe removal of material derived from MTB to the outside of a CL3 laboratory. This process is vital to establish in house biosafety-validated practices with the aim of protecting laboratory workers conducting these procedures.
Conclusions
This study demonstrates that it cannot be assumed that conventional practices (i.e. smear preparation) or extraction techniques render the organism non-viable. All methodologies, new and existing, should be examined by individual laboratories to validate the safe removal of material derived from MTB to the outside of a CL3 laboratory. This process is vital to establish in house biosafety-validated practices with the aim of protecting laboratory workers conducting these procedures.

Background Mycobacterium tuberculosis  (MTB), the causative organism of tuberculosis, has the distinction of repeatedly being ranked within the top five most commonly laboratory-acquired infections (LAIs) [ 1 - 3 ]. In 1976, Robert Pike prepared an extensive summary based on both published reports and surveys of 3921 LAIs that included both  M. tuberculosis  and other pathogens as the infectious agent [ 3 ]. He reported that laboratory and mortuary workers that are exposed to tubercle material have a TB incidence rate three times higher than that of the general population and indicated that only 18% of infections could be traced back to a known event. In 1987, a 25 year review at the National Animal Disease Center (NADC) described while only 35% of infections at the strict Biological Laboratory at Fort Detrick, MD, had a reportable, documented cause, the NADC could not account for 73% of LAIs occurring at its own facility [ 2 ]. With these reported statistics, it is negligent not to consider aerosol exposure in the absence of a known infecting episode, such as a needle prick [ 2 ]. A more recent report from 2003 demonstrated rates from 2 to 6.6 % of TB conversion among heath care workers (HCWs) in New York [ 4 ], in spite of current knowledge on precautions and safety measures in place. Furthermore, surveys suggest actual incidence of LAIs with MTB is greater than the amount of reported cases illustrate: these occurrences are likely underestimated due to the nature and length of the disease progression (i.e. workers move or retire before becoming symptomatic), and underreported to the social stigma attached [ 1 , 3 , 5 ]. Due to the nature of this organism, containment level three (CL3) laboratory operational and physical requirements have been recommended for manipulation of the live organism in North America [ 6 ]. Therefore, one would hypothesize that working in a CL3 with personal protective equipment including a respirator would be adequate to protect the worker. However, since conversions are still occurring, it is appropriate to consider the possibility that the procedure one is using to deactivate and extract material from the organism is not 100% efficient. Currently, the application of molecular methodologies for rapid diagnostics of MTB, such as nucleic -acid amplification based identification and subtyping schemes, in addition to extensive genomic and proteomic research in this area, necessitates the removal of material derived from this organism out of a CL3 laboratory to perform the work in a less restrictive containment level 2 (CL2) rated area. Commonly, due to both limited CL3 space, costs and preventative maintenance needs, high-tech equipment such as liquid handling robots and sequencers are shared and can be housed in a central "DNA core" CL2 laboratory. To consider the biosafety impact of removing organism material from a CL3 and manipulating it in a CL2, part of a risk assessment undertaken included the review of current literature on decontamination verification and viability testing of  M. tuberculosis . The existing literature is limited in regards to viability testing of material derived from MTB with respect to safe manipulation outside of a Biological Safety Cabinet (BSC). To date, no conclusive study has confirmed that this organism is noninfectious after theoretical 'deactivation' steps. A few reports concerning survival after different heating kill treatments for DNA extractions [ 7 - 9 ], heat fixing of smears [ 10 , 11 ] and chemical fixation [ 12 ] were found. Since, by definition, a risk assessment is based on, but not limited to, the properties of the agent used, personal risk factors, manipulation techniques, and the training and experience of staff, it is necessary to develop a method encompassing these factors to validate the safe removal of material derived from MTB from a CL3 laboratory for use in other laboratory areas. This approach was applied to all our methodologies and took into consideration variables such as interpersonal technique, culture load, temperature fluctuations and statistical significance.
Background
Mycobacterium tuberculosis  (MTB), the causative organism of tuberculosis, has the distinction of repeatedly being ranked within the top five most commonly laboratory-acquired infections (LAIs) [ 1 - 3 ]. In 1976, Robert Pike prepared an extensive summary based on both published reports and surveys of 3921 LAIs that included both  M. tuberculosis  and other pathogens as the infectious agent [ 3 ]. He reported that laboratory and mortuary workers that are exposed to tubercle material have a TB incidence rate three times higher than that of the general population and indicated that only 18% of infections could be traced back to a known event. In 1987, a 25 year review at the National Animal Disease Center (NADC) described while only 35% of infections at the strict Biological Laboratory at Fort Detrick, MD, had a reportable, documented cause, the NADC could not account for 73% of LAIs occurring at its own facility [ 2 ]. With these reported statistics, it is negligent not to consider aerosol exposure in the absence of a known infecting episode, such as a needle prick [ 2 ]. A more recent report from 2003 demonstrated rates from 2 to 6.6 % of TB conversion among heath care workers (HCWs) in New York [ 4 ], in spite of current knowledge on precautions and safety measures in place. Furthermore, surveys suggest actual incidence of LAIs with MTB is greater than the amount of reported cases illustrate: these occurrences are likely underestimated due to the nature and length of the disease progression (i.e. workers move or retire before becoming symptomatic), and underreported to the social stigma attached [ 1 , 3 , 5 ].Mycobacterium tuberculosisM. tuberculosisDue to the nature of this organism, containment level three (CL3) laboratory operational and physical requirements have been recommended for manipulation of the live organism in North America [ 6 ]. Therefore, one would hypothesize that working in a CL3 with personal protective equipment including a respirator would be adequate to protect the worker. However, since conversions are still occurring, it is appropriate to consider the possibility that the procedure one is using to deactivate and extract material from the organism is not 100% efficient.Currently, the application of molecular methodologies for rapid diagnostics of MTB, such as nucleic -acid amplification based identification and subtyping schemes, in addition to extensive genomic and proteomic research in this area, necessitates the removal of material derived from this organism out of a CL3 laboratory to perform the work in a less restrictive containment level 2 (CL2) rated area. Commonly, due to both limited CL3 space, costs and preventative maintenance needs, high-tech equipment such as liquid handling robots and sequencers are shared and can be housed in a central "DNA core" CL2 laboratory.To consider the biosafety impact of removing organism material from a CL3 and manipulating it in a CL2, part of a risk assessment undertaken included the review of current literature on decontamination verification and viability testing of  M. tuberculosis . The existing literature is limited in regards to viability testing of material derived from MTB with respect to safe manipulation outside of a Biological Safety Cabinet (BSC). To date, no conclusive study has confirmed that this organism is noninfectious after theoretical 'deactivation' steps. A few reports concerning survival after different heating kill treatments for DNA extractions [ 7 - 9 ], heat fixing of smears [ 10 , 11 ] and chemical fixation [ 12 ] were found.M. tuberculosisSince, by definition, a risk assessment is based on, but not limited to, the properties of the agent used, personal risk factors, manipulation techniques, and the training and experience of staff, it is necessary to develop a method encompassing these factors to validate the safe removal of material derived from MTB from a CL3 laboratory for use in other laboratory areas. This approach was applied to all our methodologies and took into consideration variables such as interpersonal technique, culture load, temperature fluctuations and statistical significance.Methods The current viability study consisted of the evaluation of a total of 226 material extracts, consisting of 208 initial extracts and 18 extracts tested after revision of faulty protocols (figure  1 ). For each test performed, laboratory staff members sub-cultured a proportion (100 uL) of the resulting material to Bactec 12B radiometric medium (Becton Dickinson, Oakville, ON) that was kept for eight weeks to observe for growth. The vials were incubated at 37°C and the growth index (GI) was read weekly on BACTEC 460 machines. All vials with positive GIs were sub-cultured to tryptic soy agar (TSA) with 5% sheep blood, Middlebrook 7H10 agar and stained with the Kinyoun method for the presence of acid-fast bacilli (AFB). Those positive for AFB were examined for the presence of MTB using a DNA probe specific for  M. tuberculosis  complex, Accuprobe (GenProbe, San Diego, CA). Methods that resulted in viable MTB were revised and retested. All work completed was performed in a CL3 environment, using Class II Type B2 biosafety cabinets (BSCs), and personal protective equipment (PPE) including N100 particulate respirators, double gloves, and protective gowns. The following describe the protocols used in our laboratory at the start of this study. Figure 1 Overall study flow chart with results after eight weeks incubation of material at 37°C DNA extraction for IS6110 fingerprinting This was performed according to the standard protocol [ 13 ]. A total of 125 Bactec 12Bs were inoculated with 0.1 mL of lysate materials. For initial heat deactivation steps, 1.5 mL screw-cap tubes were placed in a water-bath maintained at 80°C. The tubes were not submerged. Three technicians normally performing the procedure inoculated 12B media at different steps of the lysate protocol. Twenty-four samples were processed up to the point of lysozyme and proteinase K addition, prior to CTAB addition, and tested for viability. In 30 other samples the protocol was continued with the addition of choroform:isoamyl alcohol and subsequently centrifuged into organic and aqueous phases. For 10 samples, the organic (bottom) layer that is normally discarded was inoculated into 12B vials and the remaining 20 samples had their aqueous (top) layer planted. Sixty-one samples were processed to completion and inoculated into 12B vials. Finally, 10 extracts from frozen storage (-20°C) were retroactively tested by inoculation to 12B media. Crude lysate preparation A total of 45 lysates used for PCR testing were tested for viability. Thirty-six lysates were prepared from actively growing cultures by three technicians that normally perform the testing procedure. Briefly, a loopful of culture is placed in a pyrex glass bottle containing 1 mL distilled water and glass beads and vortexed in a BSC. Generally this suspension has a turbidity of > 1 McFarland. This suspension is transferred to a screw-capped vial and is placed in a boiling water bath for 10 minutes followed by transfer into a tube containing 0.5 mm silica beads and mechanically lysed for 2 minutes with a Mini-8-Beadbeater (BioSpec Products, Bartlesville, OK). The resulting lysate is spun down for removal of debris and the supernatant transferred to a new tube to be used for PCR. Lysates were processed in duplicate and the position in water bath was noted (i.e. periphery or centre). A proportion of lysates (100 uL) were planted pre and post bead-beating. An additional nine previously frozen lysates stored at -20°C were also inoculated into 12B media ( n =  45 lysates). Smear preparation Slides were prepared in duplicate according to our standard protocol: a loopful of organism is suspended in water with beads, vortexed and one drop added to a glass slide containing one drop of 0.5% phenol serum (made in-house). The slides were allowed to dry and were placed on a 95°C slide-warmer (Lab-Line Instruments, Melrose Park, IL) for 15, 30, 45 minutes, 1, 1.5 and 2 hours ( n =  12 slides). The heat fixed slide material was emulsified using a sterile swab and sterile distilled water. The suspension was transferred to a pyrex glass bottle containing 1 mL sterile water with beads, vortexed and 100 μl was inoculated into a 12B media vial. Protein extraction Twenty-six extractions of  M. tuberculosis  organism were processed as follows. A volume of 25 mL of Middlebrook 7H9 broth was inoculated and incubated at 37°C for 20 days. The culture was centrifuged at 1900 ×  g  for 15 minutes at room temperature and the pellet was resuspended with 2 ml of ice-cold phosphate buffer solution (PBS) pH 6.8 supplemented with 1% Tween-80. The sample was centrifuged as before at 4°C and the pellet washed twice in ice-cold 1% Tween-80 in PBS. Silicon beads (0.5 mm) and 500μl lysis buffer (2% CHAPS, 2% Triton X-100, 9.5 M urea and 1% DTT/TBP in water) were added to the pellet. The sample was mechanically lysed for 30 seconds and cooled on ice for 30 seconds, repeated eight times. The sample was then filter sterilized with a PES membrane, 0.22 μm Millipore filter unit (Millipore, Etobicoke, ON) into a 2 ml microcentrifuge tube on ice and stored at -80°C.
Methods
The current viability study consisted of the evaluation of a total of 226 material extracts, consisting of 208 initial extracts and 18 extracts tested after revision of faulty protocols (figure  1 ). For each test performed, laboratory staff members sub-cultured a proportion (100 uL) of the resulting material to Bactec 12B radiometric medium (Becton Dickinson, Oakville, ON) that was kept for eight weeks to observe for growth. The vials were incubated at 37°C and the growth index (GI) was read weekly on BACTEC 460 machines. All vials with positive GIs were sub-cultured to tryptic soy agar (TSA) with 5% sheep blood, Middlebrook 7H10 agar and stained with the Kinyoun method for the presence of acid-fast bacilli (AFB). Those positive for AFB were examined for the presence of MTB using a DNA probe specific for  M. tuberculosis  complex, Accuprobe (GenProbe, San Diego, CA). Methods that resulted in viable MTB were revised and retested. All work completed was performed in a CL3 environment, using Class II Type B2 biosafety cabinets (BSCs), and personal protective equipment (PPE) including N100 particulate respirators, double gloves, and protective gowns. The following describe the protocols used in our laboratory at the start of this study.M. tuberculosisDNA extraction for IS6110 fingerprinting This was performed according to the standard protocol [ 13 ]. A total of 125 Bactec 12Bs were inoculated with 0.1 mL of lysate materials. For initial heat deactivation steps, 1.5 mL screw-cap tubes were placed in a water-bath maintained at 80°C. The tubes were not submerged. Three technicians normally performing the procedure inoculated 12B media at different steps of the lysate protocol. Twenty-four samples were processed up to the point of lysozyme and proteinase K addition, prior to CTAB addition, and tested for viability. In 30 other samples the protocol was continued with the addition of choroform:isoamyl alcohol and subsequently centrifuged into organic and aqueous phases. For 10 samples, the organic (bottom) layer that is normally discarded was inoculated into 12B vials and the remaining 20 samples had their aqueous (top) layer planted. Sixty-one samples were processed to completion and inoculated into 12B vials. Finally, 10 extracts from frozen storage (-20°C) were retroactively tested by inoculation to 12B media.
DNA extraction for IS6110 fingerprinting
This was performed according to the standard protocol [ 13 ]. A total of 125 Bactec 12Bs were inoculated with 0.1 mL of lysate materials. For initial heat deactivation steps, 1.5 mL screw-cap tubes were placed in a water-bath maintained at 80°C. The tubes were not submerged. Three technicians normally performing the procedure inoculated 12B media at different steps of the lysate protocol. Twenty-four samples were processed up to the point of lysozyme and proteinase K addition, prior to CTAB addition, and tested for viability. In 30 other samples the protocol was continued with the addition of choroform:isoamyl alcohol and subsequently centrifuged into organic and aqueous phases. For 10 samples, the organic (bottom) layer that is normally discarded was inoculated into 12B vials and the remaining 20 samples had their aqueous (top) layer planted. Sixty-one samples were processed to completion and inoculated into 12B vials. Finally, 10 extracts from frozen storage (-20°C) were retroactively tested by inoculation to 12B media.Crude lysate preparation A total of 45 lysates used for PCR testing were tested for viability. Thirty-six lysates were prepared from actively growing cultures by three technicians that normally perform the testing procedure. Briefly, a loopful of culture is placed in a pyrex glass bottle containing 1 mL distilled water and glass beads and vortexed in a BSC. Generally this suspension has a turbidity of > 1 McFarland. This suspension is transferred to a screw-capped vial and is placed in a boiling water bath for 10 minutes followed by transfer into a tube containing 0.5 mm silica beads and mechanically lysed for 2 minutes with a Mini-8-Beadbeater (BioSpec Products, Bartlesville, OK). The resulting lysate is spun down for removal of debris and the supernatant transferred to a new tube to be used for PCR. Lysates were processed in duplicate and the position in water bath was noted (i.e. periphery or centre). A proportion of lysates (100 uL) were planted pre and post bead-beating. An additional nine previously frozen lysates stored at -20°C were also inoculated into 12B media ( n =  45 lysates).
Crude lysate preparation
A total of 45 lysates used for PCR testing were tested for viability. Thirty-six lysates were prepared from actively growing cultures by three technicians that normally perform the testing procedure. Briefly, a loopful of culture is placed in a pyrex glass bottle containing 1 mL distilled water and glass beads and vortexed in a BSC. Generally this suspension has a turbidity of > 1 McFarland. This suspension is transferred to a screw-capped vial and is placed in a boiling water bath for 10 minutes followed by transfer into a tube containing 0.5 mm silica beads and mechanically lysed for 2 minutes with a Mini-8-Beadbeater (BioSpec Products, Bartlesville, OK). The resulting lysate is spun down for removal of debris and the supernatant transferred to a new tube to be used for PCR. Lysates were processed in duplicate and the position in water bath was noted (i.e. periphery or centre). A proportion of lysates (100 uL) were planted pre and post bead-beating. An additional nine previously frozen lysates stored at -20°C were also inoculated into 12B media ( n =  45 lysates).n =Smear preparation Slides were prepared in duplicate according to our standard protocol: a loopful of organism is suspended in water with beads, vortexed and one drop added to a glass slide containing one drop of 0.5% phenol serum (made in-house). The slides were allowed to dry and were placed on a 95°C slide-warmer (Lab-Line Instruments, Melrose Park, IL) for 15, 30, 45 minutes, 1, 1.5 and 2 hours ( n =  12 slides). The heat fixed slide material was emulsified using a sterile swab and sterile distilled water. The suspension was transferred to a pyrex glass bottle containing 1 mL sterile water with beads, vortexed and 100 μl was inoculated into a 12B media vial.
Smear preparation
Slides were prepared in duplicate according to our standard protocol: a loopful of organism is suspended in water with beads, vortexed and one drop added to a glass slide containing one drop of 0.5% phenol serum (made in-house). The slides were allowed to dry and were placed on a 95°C slide-warmer (Lab-Line Instruments, Melrose Park, IL) for 15, 30, 45 minutes, 1, 1.5 and 2 hours ( n =  12 slides). The heat fixed slide material was emulsified using a sterile swab and sterile distilled water. The suspension was transferred to a pyrex glass bottle containing 1 mL sterile water with beads, vortexed and 100 μl was inoculated into a 12B media vial.n =Protein extraction Twenty-six extractions of  M. tuberculosis  organism were processed as follows. A volume of 25 mL of Middlebrook 7H9 broth was inoculated and incubated at 37°C for 20 days. The culture was centrifuged at 1900 ×  g  for 15 minutes at room temperature and the pellet was resuspended with 2 ml of ice-cold phosphate buffer solution (PBS) pH 6.8 supplemented with 1% Tween-80. The sample was centrifuged as before at 4°C and the pellet washed twice in ice-cold 1% Tween-80 in PBS. Silicon beads (0.5 mm) and 500μl lysis buffer (2% CHAPS, 2% Triton X-100, 9.5 M urea and 1% DTT/TBP in water) were added to the pellet. The sample was mechanically lysed for 30 seconds and cooled on ice for 30 seconds, repeated eight times. The sample was then filter sterilized with a PES membrane, 0.22 μm Millipore filter unit (Millipore, Etobicoke, ON) into a 2 ml microcentrifuge tube on ice and stored at -80°C.
Protein extraction
Twenty-six extractions of  M. tuberculosis  organism were processed as follows. A volume of 25 mL of Middlebrook 7H9 broth was inoculated and incubated at 37°C for 20 days. The culture was centrifuged at 1900 ×  g  for 15 minutes at room temperature and the pellet was resuspended with 2 ml of ice-cold phosphate buffer solution (PBS) pH 6.8 supplemented with 1% Tween-80. The sample was centrifuged as before at 4°C and the pellet washed twice in ice-cold 1% Tween-80 in PBS. Silicon beads (0.5 mm) and 500μl lysis buffer (2% CHAPS, 2% Triton X-100, 9.5 M urea and 1% DTT/TBP in water) were added to the pellet. The sample was mechanically lysed for 30 seconds and cooled on ice for 30 seconds, repeated eight times. The sample was then filter sterilized with a PES membrane, 0.22 μm Millipore filter unit (Millipore, Etobicoke, ON) into a 2 ml microcentrifuge tube on ice and stored at -80°C.M. tuberculosisgResults In total, 226 samples were tested for viability. As described below, 21 samples grew within the 8-week period, with 16 of those samples from three separate procedures yielding positive growth for  M. tuberculosis  (figure  1 ). Of the 125 RFLP lysates tested, 2 of 24 sample materials that were tested for viability before addition of CTAB remained viable for  M. tuberculosis . Following the complete extraction protocol as described [ 13 ], cultured samples yielded 0% viability for  M. tuberculosis . Four of ten 12B media vials planted with lysates from frozen storage did show growth, and upon further investigation were determined to be contaminants (Table  1 ). All other vials had negative GI readings after 8 weeks of incubation. Table 1 Results of positive Bactec 12B vials ( n  = 21). Description of material Growth on TSA + 5% SBA @ 48 hrs Growth on Middlebrook 7H11 Kinyoun Stain direct from positive 12B Probe Conclusion RFLP lysate – before CTAB addition (2 vials) - + , MTB morphology AFB+ (4+), serpentine cording ND Viable MTB 1 RFLP lysate from frozen storage #1 - + , KS of growth AFB - 4 AFB/slide, morph not consistent with TB MTB- contaminant present – ubiquitous mycobacteria RFLP lysate from frozen storage #2 - + , KS of growth AFB - 15 AFB/slide, morph not consistent with TB MTB- contamination present – ubiquitous mycobacteria RFLP lysate from frozen storage #3 & #4 - + , KS of growth AFB - No AFB contamination present Boiled lysate - + , Smooth colonies not consistent with MTB AFB+ 5–10/field, clumping, no obvious serpentine cording MTB- ubiquitous mycobacteria Smear, 1 h at 95°C & Smear, 2 h at 95°C 2 - + , MTB morphology AFB+ (4+), serpentine cording MTB+ Viable MTB Protein extracts (2) - +, MTB morphology AFB+ (4+), serpentine cording MTB+ Viable MTB 1  Accuprobe was not performed on these isolates, as MTB was expected due to morphology observed in addition to data from prior studies [7]. 2  All 12Bs from smears ( n  = 12) were positive, only 2 were chosen as representative for further testing Definitions: KS = Kinyoun stain, AFB = acid-fast bacilli, MTB =  M. tuberculosis Forty-five vials of TB PCR lysates were tested for viability. With the exception of one sample that was positive for growth and was later shown to be a contaminant (Table  1 ), all were negative for viable  M. tuberculosis . All vials containing suspensions from slide material that had been incubated for less than 1 hour on the slide-warmer exhibited identical growth rates, with a positive 12B vial in two weeks. Vials containing suspensions from slides that were incubated greater than 1 hour exhibited identical growth rates and had a positive 12B vials in 3–4 weeks. All 12 12B vials were positive for growth at 4 weeks incubation. Two representative vials were chosen for further analysis (1 hour and 2 hour slide incubations). These 12B culture vials were confirmed positive for  M. tuberculosis  complex using Accuprobe, in addition to displaying colony morphology consistent with TB on subculture to Middlebrook 7H10 agar and being AFB positive with the Kinyoun stain. Two of 25 vials tested for viability from protein extractions became positive. Confirmation of viable MTB in these test vials was confirmed as described above. Based on these results, the methods for slide fixation and protein extraction were modified. Slides were fixed prior to staining using 5% phenol in ethanol according to Chedore  et al . [ 10 ], and protein extractions samples were centrifuged at 4°C after lysis steps to rid the supernatant of cell debris before filter sterilization. These modified tests were planted again for growth of MTB, resulting in 0% organism viability for every test performed by each laboratory worker (Table  1 ). To date, ongoing viability testing of these tests have not shown any growth (data not shown).
Results
In total, 226 samples were tested for viability. As described below, 21 samples grew within the 8-week period, with 16 of those samples from three separate procedures yielding positive growth for  M. tuberculosis  (figure  1 ).M. tuberculosisOf the 125 RFLP lysates tested, 2 of 24 sample materials that were tested for viability before addition of CTAB remained viable for  M. tuberculosis . Following the complete extraction protocol as described [ 13 ], cultured samples yielded 0% viability for  M. tuberculosis . Four of ten 12B media vials planted with lysates from frozen storage did show growth, and upon further investigation were determined to be contaminants (Table  1 ). All other vials had negative GI readings after 8 weeks of incubation.M. tuberculosisM. tuberculosisResults of positive Bactec 12B vials ( n  = 21).nDescription of materialGrowth on TSA + 5% SBA @ 48 hrsGrowth on Middlebrook 7H11Kinyoun Stain direct from positive 12BProbeConclusion-+Viable MTB 1-+-+-+-+-+Viable MTB-Viable MTB1  Accuprobe was not performed on these isolates, as MTB was expected due to morphology observed in addition to data from prior studies [7].2  All 12Bs from smears ( n  = 12) were positive, only 2 were chosen as representative for further testing Definitions: KS = Kinyoun stain, AFB = acid-fast bacilli, MTB =  M. tuberculosisnM. tuberculosisForty-five vials of TB PCR lysates were tested for viability. With the exception of one sample that was positive for growth and was later shown to be a contaminant (Table  1 ), all were negative for viable  M. tuberculosis .M. tuberculosisAll vials containing suspensions from slide material that had been incubated for less than 1 hour on the slide-warmer exhibited identical growth rates, with a positive 12B vial in two weeks. Vials containing suspensions from slides that were incubated greater than 1 hour exhibited identical growth rates and had a positive 12B vials in 3–4 weeks. All 12 12B vials were positive for growth at 4 weeks incubation. Two representative vials were chosen for further analysis (1 hour and 2 hour slide incubations). These 12B culture vials were confirmed positive for  M. tuberculosis  complex using Accuprobe, in addition to displaying colony morphology consistent with TB on subculture to Middlebrook 7H10 agar and being AFB positive with the Kinyoun stain.M. tuberculosisTwo of 25 vials tested for viability from protein extractions became positive. Confirmation of viable MTB in these test vials was confirmed as described above. Based on these results, the methods for slide fixation and protein extraction were modified. Slides were fixed prior to staining using 5% phenol in ethanol according to Chedore  et al . [ 10 ], and protein extractions samples were centrifuged at 4°C after lysis steps to rid the supernatant of cell debris before filter sterilization. These modified tests were planted again for growth of MTB, resulting in 0% organism viability for every test performed by each laboratory worker (Table  1 ). To date, ongoing viability testing of these tests have not shown any growth (data not shown).et alDiscussion There are few publications that review the efficacy of methodologies that render material extracts from a  M. tuberculosis  culture non-viable [ 7 - 10 ]. One such study by Bemer-Melchior  et al . was prompted by a case of pulmonary tuberculosis acquired by a laboratory technician performing the standard method for IS6110-RFLP in a CL3 mycobacteriology [ 7 ]. In this study, placing the organisms in 80°C for 20 minutes gave breakthrough growth, which was also observed in our laboratory. Conflicting information exists which claims 100% loss of viability using this method, however, this report outlines potential reasons for the difference, such as the complete submersion of sample tubes, or volume and density of the suspension [ 9 ]. Bemer-Melchior  et al . also found that submerging the culture at 100°C for 5 minutes rendered the sample completely non-infectious; data that was supported by work previously done in 1994 by Zwadyk  et al.  [ 8 ]. Although these studies were the first of its kind in verifying the safety of laboratory protocols and addressed issues regarding temperature, timing, cell density and sample volume in deactivating MTB cultures, they clearly demonstrated that certain methods of heating may not be efficient in complete sterilization of a MTB culture. With the unique cell wall characteristics and ability of MTB to clump (or cord), variables to be considered include cell mass density, actual temperature reached and actual time exposed to the heat source. Zwadyk  et al.  showed that using a 95°C heat block failed to completely inactivate the culture, and in fact, using an internal temperature probe demonstrated that the tube did not reach the intended temperature even after 20 minutes [ 8 ]. Another study issued a caution in removing MTB fixed with 0.5 – 1% glutaraldehyde from a CL3 as the process used to sterilize the culture failed [ 12 ]. Again, both the efficacy of rendering MTB material non-viable as well as the effect of the clumping or cording factors not being known was questioned. Therefore, testing all deactivation methods was recommended for all preparations in this laboratory [ 12 ]. In preparing a risk assessment for our CL3, it became evident that this was a necessary course of action to reduce the risk of LAIs for both CL3 and CL2 mycobacteriology staff as well as other non-mycobacteriology laboratory staff in the shared CL2 environment. Genomic extractions, the most commonly performed test in our laboratory, are conducted using the standardized method outlined in 1997 by Van Embden  et al  for the purpose of RFLP typing [ 13 ]. Prior viability testing of this method in our lab showed that lysates contained viable MTB with the initial deactivation steps of this protocol, heating for 20 minutes at 80°C with addition of lysozyme and proteinase K, and thus could not be removed from containment until DNA extraction with CTAB. It is presumable that the small rate of viability observed (2 of 24), which is similar to what was seen by Bemer-Melchior  et al ., resulted from incompletely submerging sample tubes or cell density within the tube [ 9 ]. However, phenol-chloroform extraction such as the CTAB method and heating of specimens should be lethal to mycobacteria since phenolic-based disinfectants have been shown to be tuberculocidal [ 14 ], and to date our results reflect this fact. To both preserve the integrity of genomic DNA for the use of fingerprinting and allow the sample to be further processed safely outside a CL3 laboratory, it is recommended to complete DNA extractions with CTAB as per the standard protocol to confirm the complete inactivation of  M. tuberculosis . Further sampling of the DNA extractions performed in our laboratory (10% of each lysate batch) is ongoing for quality assurance purposes, i.e. to confirm integrity of reagents as well as to monitor staff performance and adherence to protocol. In addition, continuous sampling attaches statistical significance to the claim that this method ensures that the extracted material is 100% non-viable and guarantees staff safety. For procedures that do not require intact, high quality DNA such as PCR testing, our laboratory depends on the much more expedient lysate method of boiling culture at 100°C for 10 minutes, followed by mechanical lysis for 2 minutes to release DNA. Although crude, this procedure is adequate for our PCR testing needs, has been shown to completely destroy live organism in our laboratory, and is consistent with other studies [ 7 , 8 ]. The study by Zwadyk  et al.  concluded that inactivating mycobacteria by heat lysing at a temperature of 100°C for 30 minutes did not inhibit its ability to be amplified by PCR or strand displacement amplification [ 8 ]. Furthermore, this study has shown that inoculating the boiled lysate alone without mechanical lysis was adequate in rendering the sample non-viable. The viability testing of the two methods outlined above for DNA extractions were performed by various technicians who routinely follow these procedures, lending interpersonal variability to the study. It was demonstrated that the small nuances to procedure, such as the varying density of culture used, did not affect the method employed to deactivate the organism. The last routine test assessed for organism viability was the slide preparation. While phenolics are known to be tuberculocidal, the use of phenol serum alone or in conjunction with heat in slide preparation is not sufficient for killing of the live organism. Flaming of the slide was not an option due to facility requirements that prohibit open flames inside a BSC, despite this, flaming of smear material was found in prior studies to unsuccessfully inactivate smear material [ 10 ]. The primary method for slide fixation was a 2-hour incubation at 95°C on the slide-warmer, which was assumed adequate for staining purposes. Slides were then transported to an area where respirator usage was not mandatory. It was discovered that every slide preparation was positive for viable TB. As a consequence of the viability testing and subsequent risk analysis, the protocol was altered to include chemical fixation with a 5% fixative of phenol:ethanol [ 10 ]. This allows lab staff to safely remove slides from a CL3 area and examine slides under a microscope without interference by the need to wear a bulky respirator. Examining laboratory protocols for staff and building safety should be an integral part of any CL3 laboratory program. Implementation of a policy to test all procedures used routinely for viability in addition to new methodologies is highly recommended. As an example, applying this policy to a new procedure being utilized in the research arm of our laboratory proved valid: a protein extraction method being developed initially showed that of 2/26 tested samples yielded viable MTB. The researcher concluded that it was due to a clogging of the filter from large particles in the lysed culture used to isolate the proteins and revised the protocol to centrifuge the mixture after final lysis steps to remove cellular debris before filtration. This has proven successful so far, and continuous sampling of extracts is ongoing before removing the material from the CL3. To date, all have been negative.
Discussion
There are few publications that review the efficacy of methodologies that render material extracts from a  M. tuberculosis  culture non-viable [ 7 - 10 ]. One such study by Bemer-Melchior  et al . was prompted by a case of pulmonary tuberculosis acquired by a laboratory technician performing the standard method for IS6110-RFLP in a CL3 mycobacteriology [ 7 ]. In this study, placing the organisms in 80°C for 20 minutes gave breakthrough growth, which was also observed in our laboratory. Conflicting information exists which claims 100% loss of viability using this method, however, this report outlines potential reasons for the difference, such as the complete submersion of sample tubes, or volume and density of the suspension [ 9 ].M. tuberculosiset alBemer-Melchior  et al . also found that submerging the culture at 100°C for 5 minutes rendered the sample completely non-infectious; data that was supported by work previously done in 1994 by Zwadyk  et al.  [ 8 ]. Although these studies were the first of its kind in verifying the safety of laboratory protocols and addressed issues regarding temperature, timing, cell density and sample volume in deactivating MTB cultures, they clearly demonstrated that certain methods of heating may not be efficient in complete sterilization of a MTB culture. With the unique cell wall characteristics and ability of MTB to clump (or cord), variables to be considered include cell mass density, actual temperature reached and actual time exposed to the heat source. Zwadyk  et al.  showed that using a 95°C heat block failed to completely inactivate the culture, and in fact, using an internal temperature probe demonstrated that the tube did not reach the intended temperature even after 20 minutes [ 8 ]. Another study issued a caution in removing MTB fixed with 0.5 – 1% glutaraldehyde from a CL3 as the process used to sterilize the culture failed [ 12 ]. Again, both the efficacy of rendering MTB material non-viable as well as the effect of the clumping or cording factors not being known was questioned. Therefore, testing all deactivation methods was recommended for all preparations in this laboratory [ 12 ]. In preparing a risk assessment for our CL3, it became evident that this was a necessary course of action to reduce the risk of LAIs for both CL3 and CL2 mycobacteriology staff as well as other non-mycobacteriology laboratory staff in the shared CL2 environment.et alet al.et al.Genomic extractions, the most commonly performed test in our laboratory, are conducted using the standardized method outlined in 1997 by Van Embden  et al  for the purpose of RFLP typing [ 13 ]. Prior viability testing of this method in our lab showed that lysates contained viable MTB with the initial deactivation steps of this protocol, heating for 20 minutes at 80°C with addition of lysozyme and proteinase K, and thus could not be removed from containment until DNA extraction with CTAB. It is presumable that the small rate of viability observed (2 of 24), which is similar to what was seen by Bemer-Melchior  et al ., resulted from incompletely submerging sample tubes or cell density within the tube [ 9 ]. However, phenol-chloroform extraction such as the CTAB method and heating of specimens should be lethal to mycobacteria since phenolic-based disinfectants have been shown to be tuberculocidal [ 14 ], and to date our results reflect this fact.et alet alTo both preserve the integrity of genomic DNA for the use of fingerprinting and allow the sample to be further processed safely outside a CL3 laboratory, it is recommended to complete DNA extractions with CTAB as per the standard protocol to confirm the complete inactivation of  M. tuberculosis . Further sampling of the DNA extractions performed in our laboratory (10% of each lysate batch) is ongoing for quality assurance purposes, i.e. to confirm integrity of reagents as well as to monitor staff performance and adherence to protocol. In addition, continuous sampling attaches statistical significance to the claim that this method ensures that the extracted material is 100% non-viable and guarantees staff safety.M. tuberculosisFor procedures that do not require intact, high quality DNA such as PCR testing, our laboratory depends on the much more expedient lysate method of boiling culture at 100°C for 10 minutes, followed by mechanical lysis for 2 minutes to release DNA. Although crude, this procedure is adequate for our PCR testing needs, has been shown to completely destroy live organism in our laboratory, and is consistent with other studies [ 7 , 8 ]. The study by Zwadyk  et al.  concluded that inactivating mycobacteria by heat lysing at a temperature of 100°C for 30 minutes did not inhibit its ability to be amplified by PCR or strand displacement amplification [ 8 ]. Furthermore, this study has shown that inoculating the boiled lysate alone without mechanical lysis was adequate in rendering the sample non-viable.et al.The viability testing of the two methods outlined above for DNA extractions were performed by various technicians who routinely follow these procedures, lending interpersonal variability to the study. It was demonstrated that the small nuances to procedure, such as the varying density of culture used, did not affect the method employed to deactivate the organism.The last routine test assessed for organism viability was the slide preparation. While phenolics are known to be tuberculocidal, the use of phenol serum alone or in conjunction with heat in slide preparation is not sufficient for killing of the live organism. Flaming of the slide was not an option due to facility requirements that prohibit open flames inside a BSC, despite this, flaming of smear material was found in prior studies to unsuccessfully inactivate smear material [ 10 ]. The primary method for slide fixation was a 2-hour incubation at 95°C on the slide-warmer, which was assumed adequate for staining purposes. Slides were then transported to an area where respirator usage was not mandatory. It was discovered that every slide preparation was positive for viable TB. As a consequence of the viability testing and subsequent risk analysis, the protocol was altered to include chemical fixation with a 5% fixative of phenol:ethanol [ 10 ]. This allows lab staff to safely remove slides from a CL3 area and examine slides under a microscope without interference by the need to wear a bulky respirator.Examining laboratory protocols for staff and building safety should be an integral part of any CL3 laboratory program. Implementation of a policy to test all procedures used routinely for viability in addition to new methodologies is highly recommended. As an example, applying this policy to a new procedure being utilized in the research arm of our laboratory proved valid: a protein extraction method being developed initially showed that of 2/26 tested samples yielded viable MTB. The researcher concluded that it was due to a clogging of the filter from large particles in the lysed culture used to isolate the proteins and revised the protocol to centrifuge the mixture after final lysis steps to remove cellular debris before filtration. This has proven successful so far, and continuous sampling of extracts is ongoing before removing the material from the CL3. To date, all have been negative.Conclusions It is imperative to evaluate and record the actual rate of viability of DNA lysates from deactivated MTB cultures in individual laboratory settings [ 12 ]. This includes all material removed from the CL3 area. This process is vital to establish biosafety validated procedures and practices to protect laboratory workers conducting these procedures [ 2 ].
Conclusions
It is imperative to evaluate and record the actual rate of viability of DNA lysates from deactivated MTB cultures in individual laboratory settings [ 12 ]. This includes all material removed from the CL3 area. This process is vital to establish biosafety validated procedures and practices to protect laboratory workers conducting these procedures [ 2 ].Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions KB carried out the studies involving genomic DNA extractions and drafted original and final manuscripts. TB tested all smear preparations, compiled viability data and assisted with the writing of the manuscript. MS contributed by testing protein extractions and writing the method used. CT contributed by testing of crude lysates and editing of manuscript. MS contributed by testing protein extractions and writing the method used. AK and JW conceived of the study, and participated in its design and ongoing coordination. All authors read and approved the final manuscript
Authors' contributions
KB carried out the studies involving genomic DNA extractions and drafted original and final manuscripts. TB tested all smear preparations, compiled viability data and assisted with the writing of the manuscript. MS contributed by testing protein extractions and writing the method used. CT contributed by testing of crude lysates and editing of manuscript. MS contributed by testing protein extractions and writing the method used. AK and JW conceived of the study, and participated in its design and ongoing coordination. All authors read and approved the final manuscriptPre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Background Desmoplastic infantile ganglioglioma (DIG) is a rare WHO Grade I tumor of infancy that is characterized by large volume, superficial location, invariable supratentoriality, fronto-parietal lobe predilection and morphologically, by an admixture of astroglial and neuroepithelial elements in a desmoplastic milieu. With over 50 cases described, the histologic and radiographic spectrum of DIG has been well-characterized. The superficial location of DIGs may render them greatly amenable to preoperative assessment utilizing aspiration cytology; however, the cytologic features of this rare tumor have only been reported once previously.
Background
Desmoplastic infantile ganglioglioma (DIG) is a rare WHO Grade I tumor of infancy that is characterized by large volume, superficial location, invariable supratentoriality, fronto-parietal lobe predilection and morphologically, by an admixture of astroglial and neuroepithelial elements in a desmoplastic milieu. With over 50 cases described, the histologic and radiographic spectrum of DIG has been well-characterized. The superficial location of DIGs may render them greatly amenable to preoperative assessment utilizing aspiration cytology; however, the cytologic features of this rare tumor have only been reported once previously.Case Presentation We present herein cytomorphologic findings from the intraoperative aspiration of a typical case of DIG diagnosed in a 1-year-old male. As evaluated on a single liquid-based preparation, the specimen showed low cellularity and was comprised predominantly of a population of dispersed (occasionally clustered) large neuronal cells with eccentrically located hyperchromatic nuclei (which were occasionally binucleated) and abundant unipolar cytoplasm. Rare smaller astroglial cells were intermixed. Despite the tumor's characteristic desmoplastic histologic appearance, no stromal fragments were identified on the aspiration material.
Case Presentation
We present herein cytomorphologic findings from the intraoperative aspiration of a typical case of DIG diagnosed in a 1-year-old male. As evaluated on a single liquid-based preparation, the specimen showed low cellularity and was comprised predominantly of a population of dispersed (occasionally clustered) large neuronal cells with eccentrically located hyperchromatic nuclei (which were occasionally binucleated) and abundant unipolar cytoplasm. Rare smaller astroglial cells were intermixed. Despite the tumor's characteristic desmoplastic histologic appearance, no stromal fragments were identified on the aspiration material.Conclusions A differential diagnosis is presented and analyzed in detail and it is concluded that when these large neuronal cells are encountered in an aspirate of a brain mass in a child, a combination of clinical, radiologic and immunohistochemical parameters can eliminate most of the differential possibilities.
Conclusions
A differential diagnosis is presented and analyzed in detail and it is concluded that when these large neuronal cells are encountered in an aspirate of a brain mass in a child, a combination of clinical, radiologic and immunohistochemical parameters can eliminate most of the differential possibilities.

Background The clinicopathologic features of 11 examples of a distinctive pediatric tumor designated  desmoplastic supratentorial neuroepithelial tumors of infancy  (also known as desmoplastic infantile ganglioglioma, [DIG]) were originally described by Vandenberg et al in 1987 [ 1 ]. Since that seminal report, at least 40 additional cases have been described, such that the clinical, radiologic and histopathologic features of this tumor are now well-defined. An uncommon tumor that constituted less than 0.04% of all central nervous system (CNS) tumors in one series [ 2 ], DIG is classified as a Grade 1 tumor in the World Health Organization (WHO) classification of CNS tumors [ 3 ]. They most commonly occur in children less than 18 months of age [ 1 ] who typically present with symptoms related to an intracranial mass effect [ 3 ]. DIGs are generally of large size, are solid to cystic, show a predilection for the frontal and parietal cerebral lobes, and are typically superficially located with at least focal attachment to the overlying dura [ 1 - 3 ]. The superficial location of DIGs may render them greatly amenable to preoperative assessment utilizing aspiration cytology. However, there is a dearth of information on the cytomorphologic features of these tumors [ 4 ]. To contribute information of possible utility in their pre-operative or intra-operative assessment, we report herein cytomorphologic features associated with a typical case of DIG. Case Presentation A one-year-old boy was noted to have a striking increase in head circumference as compared to a previous measurement. Neurological examination and developmental status were normal at that point. Within 2 weeks, the patient deteriorated rapidly, with poor mobilization, feeding and verbalization. He was brought to the emergency room where an emergent computed tomographic scan showed a large left hemispheric cerebral mass (Figure  1-1 ) with an underlying cystic component and a more superficial area of bright enhancement; the rest of the brain showed massive edema. He was emergently admitted and within 24 hours, a gross resection of the tumor was carried out. At surgery, following a parietal craniotomy, 50–60 cc of straw colored fluid was aspirated from the cystic component through a taut dura. After the excision of the dura, the bright area of enhancement previously noted was an area of tumor attachment to the dura in the parietal region. Otherwise, the tumor showed a well-demarcated interface with the subjacent normal brain parenchyma and a complete gross resection was achieved. A follow-up magnetic resonance image at 12 months post-surgery showed no evidence of tumor recurrence. Functionally, the patient was felt to have a mild right hemiparesis and some probable language delay, but otherwise showed no neurological deficits. Figure 1 Radiologic, cytologic and morphologic appearance of the tumor.  1 : This computed-tomographic scan of the patient's cerebral mass shows a large cystic mass with peripheral enhancement at the solid portion which attached to the overlying dura;  2 : In addition to scattered individual cells, variably sized clusters of neuronal cells were identified, all composed of cells with eccentrically located, occasionally binucleated hyperchromatic nuclei and abundant unipolar cytoplasm [original magnifications ×400];  3 : Occasional neuronal cells were binucleated ( 3a ) while others showed bland nuclear features ( 3b ) [original magnifications ×400];  4 : Scattered astroglial cells with more convoluted nuclear contours and less cytoplasm were also present. [original magnifications ×400];  5 : Typical histologic appearance of desmoplastic infantile ganglioglioma, showing scattered ganglion cells in a desmoplastic and fibroblastic, vaguely storiform background (original magnification ×200, inset ×400) Materials and Methods For cytology, a slide was prepared from 50–60 cc of straw-colored fluid utilizing the ThinPrep ®  2000 Automated Slide Processor (Cytyc, Boxborough, MA) according to the manufacturer's instructions. For the tumor specimen, approximately 10 × 6.5 cm of fragmented gray and white cerebral tissue was received and entirely processed routinely: tissue sections were fixed in 10% neutral buffered formalin, processed, embedded in paraffin, sectioned to 4 μ-thick sections and stained with hematoxylin and eosin, Nissl stain and reticulin. The immunohistochemical profile of the tumor was evaluated on 4 μ thick, formalin-fixed, deparaffinized sections using a DAKO Autostainer (Carpinteria, CA, USA) based on the avidin-biotin-peroxidase complex with antibodies ki-67 (dilution 1:320, DakoCytomation Corp, Carpinteria, CA), synaptophysin (dilution 1:600, DakoCytomation) and glial fibrillary acid protein [GFAP] (dilution 1:10, DakoCytomation). Pathologic findings As evaluated on a single liquid-based preparation, the specimen showed low cellularity and was comprised predominantly of a population of dispersed (occasionally clustered) large neuronal cells (~70 μm diameter each) with round eccentrically placed uniform hyperchromatic nuclei, undulating and slightly convoluted nuclear membranes, and abundant unipolar granular cytoplasm (ganglion cells) (figures  1-2  and  1-3 ). Occasional cells were binucleated (figure  1-3a ). A spectrum in the degree of nuclear membrane irregularities was noted, with most cells displaying irregular features as described above, while other cell showed bland nuclear features (figure  1-3b ). However, all displayed nuclear polarity to the cytoplasm. Rare smaller cells interpreted as astroglial cells were interspersed between the larger cells. The latter cells showed nuclear hyperchromasia, more prominent irregularities in their nuclear membranes and a smaller cytoplasmic rim. (figure  1-4 ). Overall, significant proportions of both cellular populations showed varying degrees of degenerative changes manifested as lack of clear delineation of nuclear and cytoplasmic borders and a loss of nuclear detail. Several clusters were composed of large cells, and in these clusters, constituent large cells showed less cytoplasm but retained a unipolarity in relation to the nuclei and their nuclear features were identical to those of the more predominant population of large cells. Additionally, scattered foamy histiocytes were present. A finely granular background material consistent with necrosis was present, but there was no distinct neurofibrillary material. Vascular structures or stroma were not present. Histologically, the tumor was partially attached to the dura and was present in the subarachnoid space. The bulk of the specimen was a variably cellular desmoplastic component whose predominant constituent cells were elongated spindle cells arranged in a reticulin-rich, storiform pattern (figure  1-5 ). At higher magnification, ganglion-type cells (Nissl stain positive) with 1–4 round nuclei, prominent nucleoli, and abundant unipolar cytoplasm were present (Figure  1-5 , inset). Immature or abortive ganglion cells with enlarged single nuclei and markedly irregular nuclear membranes were rare but identifiable morphologically. Less "differentiated" aggregates of cells with hyperchromatic nuclei and minimal cytoplasm, as has been well-described in DIGs [ 1 - 3 ], were present. Mitotic figures were rare and small foci of necrosis were limited to the less differentiated component. Immunohistochemical stains for synaptophysin was positive in the ganglion cells only. GFAP was positive in astroglial elements within the desmoplastic regions; the latter was negative for synaptophysin. The ki-67 labelling index was 11.3% (evaluated in the area of greatest density of positive staining cells).
Background
The clinicopathologic features of 11 examples of a distinctive pediatric tumor designated  desmoplastic supratentorial neuroepithelial tumors of infancy  (also known as desmoplastic infantile ganglioglioma, [DIG]) were originally described by Vandenberg et al in 1987 [ 1 ]. Since that seminal report, at least 40 additional cases have been described, such that the clinical, radiologic and histopathologic features of this tumor are now well-defined. An uncommon tumor that constituted less than 0.04% of all central nervous system (CNS) tumors in one series [ 2 ], DIG is classified as a Grade 1 tumor in the World Health Organization (WHO) classification of CNS tumors [ 3 ]. They most commonly occur in children less than 18 months of age [ 1 ] who typically present with symptoms related to an intracranial mass effect [ 3 ]. DIGs are generally of large size, are solid to cystic, show a predilection for the frontal and parietal cerebral lobes, and are typically superficially located with at least focal attachment to the overlying dura [ 1 - 3 ]. The superficial location of DIGs may render them greatly amenable to preoperative assessment utilizing aspiration cytology. However, there is a dearth of information on the cytomorphologic features of these tumors [ 4 ]. To contribute information of possible utility in their pre-operative or intra-operative assessment, we report herein cytomorphologic features associated with a typical case of DIG.desmoplastic supratentorial neuroepithelial tumors of infancyCase Presentation A one-year-old boy was noted to have a striking increase in head circumference as compared to a previous measurement. Neurological examination and developmental status were normal at that point. Within 2 weeks, the patient deteriorated rapidly, with poor mobilization, feeding and verbalization. He was brought to the emergency room where an emergent computed tomographic scan showed a large left hemispheric cerebral mass (Figure  1-1 ) with an underlying cystic component and a more superficial area of bright enhancement; the rest of the brain showed massive edema. He was emergently admitted and within 24 hours, a gross resection of the tumor was carried out. At surgery, following a parietal craniotomy, 50–60 cc of straw colored fluid was aspirated from the cystic component through a taut dura. After the excision of the dura, the bright area of enhancement previously noted was an area of tumor attachment to the dura in the parietal region. Otherwise, the tumor showed a well-demarcated interface with the subjacent normal brain parenchyma and a complete gross resection was achieved. A follow-up magnetic resonance image at 12 months post-surgery showed no evidence of tumor recurrence. Functionally, the patient was felt to have a mild right hemiparesis and some probable language delay, but otherwise showed no neurological deficits. Figure 1 Radiologic, cytologic and morphologic appearance of the tumor.  1 : This computed-tomographic scan of the patient's cerebral mass shows a large cystic mass with peripheral enhancement at the solid portion which attached to the overlying dura;  2 : In addition to scattered individual cells, variably sized clusters of neuronal cells were identified, all composed of cells with eccentrically located, occasionally binucleated hyperchromatic nuclei and abundant unipolar cytoplasm [original magnifications ×400];  3 : Occasional neuronal cells were binucleated ( 3a ) while others showed bland nuclear features ( 3b ) [original magnifications ×400];  4 : Scattered astroglial cells with more convoluted nuclear contours and less cytoplasm were also present. [original magnifications ×400];  5 : Typical histologic appearance of desmoplastic infantile ganglioglioma, showing scattered ganglion cells in a desmoplastic and fibroblastic, vaguely storiform background (original magnification ×200, inset ×400)
Case Presentation
A one-year-old boy was noted to have a striking increase in head circumference as compared to a previous measurement. Neurological examination and developmental status were normal at that point. Within 2 weeks, the patient deteriorated rapidly, with poor mobilization, feeding and verbalization. He was brought to the emergency room where an emergent computed tomographic scan showed a large left hemispheric cerebral mass (Figure  1-1 ) with an underlying cystic component and a more superficial area of bright enhancement; the rest of the brain showed massive edema. He was emergently admitted and within 24 hours, a gross resection of the tumor was carried out. At surgery, following a parietal craniotomy, 50–60 cc of straw colored fluid was aspirated from the cystic component through a taut dura. After the excision of the dura, the bright area of enhancement previously noted was an area of tumor attachment to the dura in the parietal region. Otherwise, the tumor showed a well-demarcated interface with the subjacent normal brain parenchyma and a complete gross resection was achieved. A follow-up magnetic resonance image at 12 months post-surgery showed no evidence of tumor recurrence. Functionally, the patient was felt to have a mild right hemiparesis and some probable language delay, but otherwise showed no neurological deficits.Materials and Methods For cytology, a slide was prepared from 50–60 cc of straw-colored fluid utilizing the ThinPrep ®  2000 Automated Slide Processor (Cytyc, Boxborough, MA) according to the manufacturer's instructions. For the tumor specimen, approximately 10 × 6.5 cm of fragmented gray and white cerebral tissue was received and entirely processed routinely: tissue sections were fixed in 10% neutral buffered formalin, processed, embedded in paraffin, sectioned to 4 μ-thick sections and stained with hematoxylin and eosin, Nissl stain and reticulin. The immunohistochemical profile of the tumor was evaluated on 4 μ thick, formalin-fixed, deparaffinized sections using a DAKO Autostainer (Carpinteria, CA, USA) based on the avidin-biotin-peroxidase complex with antibodies ki-67 (dilution 1:320, DakoCytomation Corp, Carpinteria, CA), synaptophysin (dilution 1:600, DakoCytomation) and glial fibrillary acid protein [GFAP] (dilution 1:10, DakoCytomation).
Materials and Methods
For cytology, a slide was prepared from 50–60 cc of straw-colored fluid utilizing the ThinPrep ®  2000 Automated Slide Processor (Cytyc, Boxborough, MA) according to the manufacturer's instructions. For the tumor specimen, approximately 10 × 6.5 cm of fragmented gray and white cerebral tissue was received and entirely processed routinely: tissue sections were fixed in 10% neutral buffered formalin, processed, embedded in paraffin, sectioned to 4 μ-thick sections and stained with hematoxylin and eosin, Nissl stain and reticulin. The immunohistochemical profile of the tumor was evaluated on 4 μ thick, formalin-fixed, deparaffinized sections using a DAKO Autostainer (Carpinteria, CA, USA) based on the avidin-biotin-peroxidase complex with antibodies ki-67 (dilution 1:320, DakoCytomation Corp, Carpinteria, CA), synaptophysin (dilution 1:600, DakoCytomation) and glial fibrillary acid protein [GFAP] (dilution 1:10, DakoCytomation).Pathologic findings As evaluated on a single liquid-based preparation, the specimen showed low cellularity and was comprised predominantly of a population of dispersed (occasionally clustered) large neuronal cells (~70 μm diameter each) with round eccentrically placed uniform hyperchromatic nuclei, undulating and slightly convoluted nuclear membranes, and abundant unipolar granular cytoplasm (ganglion cells) (figures  1-2  and  1-3 ). Occasional cells were binucleated (figure  1-3a ). A spectrum in the degree of nuclear membrane irregularities was noted, with most cells displaying irregular features as described above, while other cell showed bland nuclear features (figure  1-3b ). However, all displayed nuclear polarity to the cytoplasm. Rare smaller cells interpreted as astroglial cells were interspersed between the larger cells. The latter cells showed nuclear hyperchromasia, more prominent irregularities in their nuclear membranes and a smaller cytoplasmic rim. (figure  1-4 ). Overall, significant proportions of both cellular populations showed varying degrees of degenerative changes manifested as lack of clear delineation of nuclear and cytoplasmic borders and a loss of nuclear detail. Several clusters were composed of large cells, and in these clusters, constituent large cells showed less cytoplasm but retained a unipolarity in relation to the nuclei and their nuclear features were identical to those of the more predominant population of large cells. Additionally, scattered foamy histiocytes were present. A finely granular background material consistent with necrosis was present, but there was no distinct neurofibrillary material. Vascular structures or stroma were not present. Histologically, the tumor was partially attached to the dura and was present in the subarachnoid space. The bulk of the specimen was a variably cellular desmoplastic component whose predominant constituent cells were elongated spindle cells arranged in a reticulin-rich, storiform pattern (figure  1-5 ). At higher magnification, ganglion-type cells (Nissl stain positive) with 1–4 round nuclei, prominent nucleoli, and abundant unipolar cytoplasm were present (Figure  1-5 , inset). Immature or abortive ganglion cells with enlarged single nuclei and markedly irregular nuclear membranes were rare but identifiable morphologically. Less "differentiated" aggregates of cells with hyperchromatic nuclei and minimal cytoplasm, as has been well-described in DIGs [ 1 - 3 ], were present. Mitotic figures were rare and small foci of necrosis were limited to the less differentiated component. Immunohistochemical stains for synaptophysin was positive in the ganglion cells only. GFAP was positive in astroglial elements within the desmoplastic regions; the latter was negative for synaptophysin. The ki-67 labelling index was 11.3% (evaluated in the area of greatest density of positive staining cells).
Pathologic findings
As evaluated on a single liquid-based preparation, the specimen showed low cellularity and was comprised predominantly of a population of dispersed (occasionally clustered) large neuronal cells (~70 μm diameter each) with round eccentrically placed uniform hyperchromatic nuclei, undulating and slightly convoluted nuclear membranes, and abundant unipolar granular cytoplasm (ganglion cells) (figures  1-2  and  1-3 ). Occasional cells were binucleated (figure  1-3a ). A spectrum in the degree of nuclear membrane irregularities was noted, with most cells displaying irregular features as described above, while other cell showed bland nuclear features (figure  1-3b ). However, all displayed nuclear polarity to the cytoplasm. Rare smaller cells interpreted as astroglial cells were interspersed between the larger cells. The latter cells showed nuclear hyperchromasia, more prominent irregularities in their nuclear membranes and a smaller cytoplasmic rim. (figure  1-4 ). Overall, significant proportions of both cellular populations showed varying degrees of degenerative changes manifested as lack of clear delineation of nuclear and cytoplasmic borders and a loss of nuclear detail. Several clusters were composed of large cells, and in these clusters, constituent large cells showed less cytoplasm but retained a unipolarity in relation to the nuclei and their nuclear features were identical to those of the more predominant population of large cells. Additionally, scattered foamy histiocytes were present. A finely granular background material consistent with necrosis was present, but there was no distinct neurofibrillary material.Vascular structures or stroma were not present. Histologically, the tumor was partially attached to the dura and was present in the subarachnoid space. The bulk of the specimen was a variably cellular desmoplastic component whose predominant constituent cells were elongated spindle cells arranged in a reticulin-rich, storiform pattern (figure  1-5 ). At higher magnification, ganglion-type cells (Nissl stain positive) with 1–4 round nuclei, prominent nucleoli, and abundant unipolar cytoplasm were present (Figure  1-5 , inset). Immature or abortive ganglion cells with enlarged single nuclei and markedly irregular nuclear membranes were rare but identifiable morphologically. Less "differentiated" aggregates of cells with hyperchromatic nuclei and minimal cytoplasm, as has been well-described in DIGs [ 1 - 3 ], were present. Mitotic figures were rare and small foci of necrosis were limited to the less differentiated component. Immunohistochemical stains for synaptophysin was positive in the ganglion cells only. GFAP was positive in astroglial elements within the desmoplastic regions; the latter was negative for synaptophysin. The ki-67 labelling index was 11.3% (evaluated in the area of greatest density of positive staining cells).Discussion The clinical presentation, radiographic appearance and histopathologic features of this case are entirely consistent with those described for desmoplastic infantile ganglioglioma [ 1 - 3 ]. There has been a significant evolution in the understanding of this rare tumor since its original description in 1987 [ 1 ]. DIGs are typically large supratentorial tumors that, at least as observed radiographically in one patient, are initially solid then become cystic [ 5 ]. Although this tumor is considered a grade 1 tumor based on the histopathologic features of cases described prior to the publication of the WHO monograph in 2000, at least one report has since documented anaplastic features in a case of DIG, which was ultimately fatal [ 6 ]. However, follow-up has generally been favorable following complete resection in the reported cases of DIG, with a median post-surgical interval of 8.7 years without metastases or recurrence in one series of 14 patients [ 2 ]. Additionally, in some cases, spontaneous regression of tumor following subtotal tumor resection has been documented [ 7 , 8 ]. The distinctive clinical features of DIG, being a typically superficially located tumor occurring in young children (with potentially unclosed fontanelles), may render them particularly amenable to pre-operative assessment using aspiration cytology. In addition, familiarization of practitioners with the cytopathologic features of DIG may be useful because 1) With the aforementioned cases of DIG regressing after subtotal resection [ 7 , 8 ], it might be unnecessary to aggressively resect these tumors to negative margins, and a preoperative aspiration diagnosis of DIG will be helpful in the neurosurgical planning and 2) In their intra-operative assessment, imprint cytopathology may potentially be more diagnostic than histopathology. However, to our knowledge, the cytologic features of DIG have been documented only once previously [ 4 ]. In that report, Hasegawa et al [ 4 ] reported aspiration and imprint cytology findings in two cases of DIG. Two distinct cellular populations were identified, a predominant population of small to intermediate sized astroglial cells and "a few" large cells with round nuclei, prominent nucleoli and profuse cytoplasm that was unipolar to the nuclei in all their illustrations. In the current case, the reverse was found, with the predominant cells being an identical population of large cells with round nuclei, prominent nucleoli and abundant unipolar cytoplasm, and only rare unequivocal astroglial cells. Most of the analysis of the aforementioned report [ 4 ] was on the imprint smears, and although a mixture of small and large cells were also identified on the aspiration smear, there was no stated assessment or low-power illustration of the relative ratio of small to large cells on the latter. In the current case, a cell-block for immunohistochemical confirmation of the nature of two-cell population was unavailable; however, the larger cells were positive for neurofilament (confirming their neuronal nature) while the smaller cells were positive for GFAP (confirming their astroglial nature) in the report of Hasegawa et al [ 4 ]. The finding of large cells with eccentrically located nuclei and abundant unipolar cytoplasm in an aspiration specimen of a cerebral mass occurring in a young person should generate a differential diagnosis that includes DIG, atypical teratoid/rhabdoid tumor (AT/RT), dysembroplastic neuroepithelial tumor (DNT), ganglioglioma, supratentorial primitive neuroectodermal tumour (PNET) with ganglionic differentiation (ganglioneuroblastoma), anaplastic large cell lymphoma and pleomorphic xanthoastrocytoma. In our opinion, clinical features as well as immunohistochemical analysis can significantly help reduce the likelihood for most of the aforementioned entities. The distinction of DIG from AT/RT is probably of the greatest prognostic significance, since in contrast to DIG, AT/RT is a highly malignant tumor that is almost uniformly fatal [ 9 ]. Although AT/RT occurs in infants or young children, most cases occur in the posterior fossa, in contrast to DIGs, which are invariably supratentorial [ 1 - 3 ]. Radiographically, AT/RT are typically not distinctly cystic, although necrosis may impart an irregularly cystic appearance. Immunohistochemically, the rhabdoid cells of AT/RT co-express vimentin and epithelial membrane antigen [ 10 ], in contrast to the ganglion cells of DIG. However, rhabdoid cells may rarely express neurofilament, an immunophenotypic overlap with DIG. Morphologically, the distinct cytoplasmic borders, "inclusion-like" cytoplasmic globule and overall dense eosinophilia of the cytoplasm of rhabdoid cells, in conjunction with the aforementioned clinicopathologic parameters, may help in their distinction from ganglion cells of DIG [ 9 , 10 ]. The separation of DIG from other tumors containing true ganglion cells based on cytomorphology alone would probably pose the greatest difficulty. These tumors include DNT, ganglioglioma, and supratentorial PNET with ganglionic differentiation; all have a predilection for, or at least may potentially occur in children. In addition to ganglion cells, cytomorphologic features of DNT include oligodendroglial-like cells arranged in lobules and neurons in abundant extracellular mucin or neurofibrillary material [ 11 , 12 ]; these findings were neither identified in the 2 cases of DIG reported by Hasegawa et al [ 4 ], nor the current case. The distinction of gangliogliomas form DIG based on cytomorphology alone may be impossible even in the presence of a significant stromal component on the aspirate. Gangliogliomas, like DIG may be solid to cystic and show desmoplasia, although in contrast to DIG, they have a predilection for the temporal lobe and most commonly occur in an age group slightly older than is typical for DIG [ 13 ]. However, it should be noted that conventional gangliogliomas and DIG may exist on a morphologic spectrum, and a case with morphologic features of both entities has been described [ 14 ]. Supratentorial PNET with ganglionic differentiation (ganglioneuroblastomas) also show significant clinicopathologic overlap with DIG, as they are supratentorial, occur in young children, may be cystic and may show desmoplasia [ 15 ]. Cerebral ganglioneuroblastomas are extremely rare, and in the absence of treatment-related cytodifferentiation, will show a significant neuronal component of smaller cells. However, the cytomorphologic features of pure ganglioneuroblastoma have not been well-characterized. Other less likely differential considerations include pleomorphic xanthoastrocytoma and anaplastic large cell lymphoma (ALCL), both of which may contain large cells with eccentrically located nuclei and abundant cytoplasm. The temporal lobe predilection, lack of neuronal differentiation, presence of xanthomatous cells, smaller tumor size and older age of patients with pleomorphic xanthoastrocytoma should permit an easy distinction of the large cells in this tumor from those of DIG. Primary brain ALCL is exceedingly rare and generally occurs in older individuals, with a mean age of 29 years in one series [ 16 ]. Immunoreactivity for CD30, ALK and CD45 in ALCL and absence of similar immunoreactivity in DIG should facilitate a distinction in rare cases that occur in very young children. When the cytomorphologic findings of DIG are described in more cases, it is likely that the cytologic spectrum will mirror the histologic heterogeneity of this tumor. For example, astroglial cells predominated in the two cases of Hasegawa et al [ 4 ] while the ganglion cells predominated in ours. In addition, it is conceivable that an aspirate would only capture the immature neuroepithelial cells which frequently characterizes DIG. Nonetheless, it is concluded that when the large neuronal cells are encountered in an aspirate of a brain mass in a child, a combination of clinical, radiologic and immunohistochemical parameters can eliminate most of the differential possibilities.
Discussion
The clinical presentation, radiographic appearance and histopathologic features of this case are entirely consistent with those described for desmoplastic infantile ganglioglioma [ 1 - 3 ]. There has been a significant evolution in the understanding of this rare tumor since its original description in 1987 [ 1 ]. DIGs are typically large supratentorial tumors that, at least as observed radiographically in one patient, are initially solid then become cystic [ 5 ]. Although this tumor is considered a grade 1 tumor based on the histopathologic features of cases described prior to the publication of the WHO monograph in 2000, at least one report has since documented anaplastic features in a case of DIG, which was ultimately fatal [ 6 ]. However, follow-up has generally been favorable following complete resection in the reported cases of DIG, with a median post-surgical interval of 8.7 years without metastases or recurrence in one series of 14 patients [ 2 ]. Additionally, in some cases, spontaneous regression of tumor following subtotal tumor resection has been documented [ 7 , 8 ].The distinctive clinical features of DIG, being a typically superficially located tumor occurring in young children (with potentially unclosed fontanelles), may render them particularly amenable to pre-operative assessment using aspiration cytology. In addition, familiarization of practitioners with the cytopathologic features of DIG may be useful because 1) With the aforementioned cases of DIG regressing after subtotal resection [ 7 , 8 ], it might be unnecessary to aggressively resect these tumors to negative margins, and a preoperative aspiration diagnosis of DIG will be helpful in the neurosurgical planning and 2) In their intra-operative assessment, imprint cytopathology may potentially be more diagnostic than histopathology. However, to our knowledge, the cytologic features of DIG have been documented only once previously [ 4 ]. In that report, Hasegawa et al [ 4 ] reported aspiration and imprint cytology findings in two cases of DIG. Two distinct cellular populations were identified, a predominant population of small to intermediate sized astroglial cells and "a few" large cells with round nuclei, prominent nucleoli and profuse cytoplasm that was unipolar to the nuclei in all their illustrations. In the current case, the reverse was found, with the predominant cells being an identical population of large cells with round nuclei, prominent nucleoli and abundant unipolar cytoplasm, and only rare unequivocal astroglial cells. Most of the analysis of the aforementioned report [ 4 ] was on the imprint smears, and although a mixture of small and large cells were also identified on the aspiration smear, there was no stated assessment or low-power illustration of the relative ratio of small to large cells on the latter. In the current case, a cell-block for immunohistochemical confirmation of the nature of two-cell population was unavailable; however, the larger cells were positive for neurofilament (confirming their neuronal nature) while the smaller cells were positive for GFAP (confirming their astroglial nature) in the report of Hasegawa et al [ 4 ].The finding of large cells with eccentrically located nuclei and abundant unipolar cytoplasm in an aspiration specimen of a cerebral mass occurring in a young person should generate a differential diagnosis that includes DIG, atypical teratoid/rhabdoid tumor (AT/RT), dysembroplastic neuroepithelial tumor (DNT), ganglioglioma, supratentorial primitive neuroectodermal tumour (PNET) with ganglionic differentiation (ganglioneuroblastoma), anaplastic large cell lymphoma and pleomorphic xanthoastrocytoma. In our opinion, clinical features as well as immunohistochemical analysis can significantly help reduce the likelihood for most of the aforementioned entities. The distinction of DIG from AT/RT is probably of the greatest prognostic significance, since in contrast to DIG, AT/RT is a highly malignant tumor that is almost uniformly fatal [ 9 ]. Although AT/RT occurs in infants or young children, most cases occur in the posterior fossa, in contrast to DIGs, which are invariably supratentorial [ 1 - 3 ]. Radiographically, AT/RT are typically not distinctly cystic, although necrosis may impart an irregularly cystic appearance.Immunohistochemically, the rhabdoid cells of AT/RT co-express vimentin and epithelial membrane antigen [ 10 ], in contrast to the ganglion cells of DIG. However, rhabdoid cells may rarely express neurofilament, an immunophenotypic overlap with DIG.Morphologically, the distinct cytoplasmic borders, "inclusion-like" cytoplasmic globule and overall dense eosinophilia of the cytoplasm of rhabdoid cells, in conjunction with the aforementioned clinicopathologic parameters, may help in their distinction from ganglion cells of DIG [ 9 , 10 ]. The separation of DIG from other tumors containing true ganglion cells based on cytomorphology alone would probably pose the greatest difficulty. These tumors include DNT, ganglioglioma, and supratentorial PNET with ganglionic differentiation; all have a predilection for, or at least may potentially occur in children. In addition to ganglion cells, cytomorphologic features of DNT include oligodendroglial-like cells arranged in lobules and neurons in abundant extracellular mucin or neurofibrillary material [ 11 , 12 ]; these findings were neither identified in the 2 cases of DIG reported by Hasegawa et al [ 4 ], nor the current case. The distinction of gangliogliomas form DIG based on cytomorphology alone may be impossible even in the presence of a significant stromal component on the aspirate. Gangliogliomas, like DIG may be solid to cystic and show desmoplasia, although in contrast to DIG, they have a predilection for the temporal lobe and most commonly occur in an age group slightly older than is typical for DIG [ 13 ]. However, it should be noted that conventional gangliogliomas and DIG may exist on a morphologic spectrum, and a case with morphologic features of both entities has been described [ 14 ]. Supratentorial PNET with ganglionic differentiation (ganglioneuroblastomas) also show significant clinicopathologic overlap with DIG, as they are supratentorial, occur in young children, may be cystic and may show desmoplasia [ 15 ]. Cerebral ganglioneuroblastomas are extremely rare, and in the absence of treatment-related cytodifferentiation, will show a significant neuronal component of smaller cells. However, the cytomorphologic features of pure ganglioneuroblastoma have not been well-characterized.Other less likely differential considerations include pleomorphic xanthoastrocytoma and anaplastic large cell lymphoma (ALCL), both of which may contain large cells with eccentrically located nuclei and abundant cytoplasm. The temporal lobe predilection, lack of neuronal differentiation, presence of xanthomatous cells, smaller tumor size and older age of patients with pleomorphic xanthoastrocytoma should permit an easy distinction of the large cells in this tumor from those of DIG. Primary brain ALCL is exceedingly rare and generally occurs in older individuals, with a mean age of 29 years in one series [ 16 ]. Immunoreactivity for CD30, ALK and CD45 in ALCL and absence of similar immunoreactivity in DIG should facilitate a distinction in rare cases that occur in very young children.When the cytomorphologic findings of DIG are described in more cases, it is likely that the cytologic spectrum will mirror the histologic heterogeneity of this tumor. For example, astroglial cells predominated in the two cases of Hasegawa et al [ 4 ] while the ganglion cells predominated in ours. In addition, it is conceivable that an aspirate would only capture the immature neuroepithelial cells which frequently characterizes DIG. Nonetheless, it is concluded that when the large neuronal cells are encountered in an aspirate of a brain mass in a child, a combination of clinical, radiologic and immunohistochemical parameters can eliminate most of the differential possibilities.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions All authors made substantial contributions to the intellectual content and/or presentation of the manuscript. ITO (cytologist), diagnosed the cytopathological aspects of the case and co-supervised the entire project. JHK (neuropathologist), diagnosed the histological aspects of the case and co-supervised the project. OF wrote the initial version of the manuscript. MRM, DH and AWZ collected pathological, clinical and/or photographical information and revised the manuscript.
Authors' contributions
All authors made substantial contributions to the intellectual content and/or presentation of the manuscript. ITO (cytologist), diagnosed the cytopathological aspects of the case and co-supervised the entire project. JHK (neuropathologist), diagnosed the histological aspects of the case and co-supervised the project. OF wrote the initial version of the manuscript. MRM, DH and AWZ collected pathological, clinical and/or photographical information and revised the manuscript.