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as clearly shown in previous articles within this special issue , dentin hypersensitivity ( dhs ) is a well - known patient complaint which is patent in middle - aged people and most probably will increase with aging . besides the importance of correct diagnosis [ 2 , 3 ] , appropriate treatment recommendations for patients are needed . it is important to emphasize once more that the basic requirements for having dhs is exposed cervical dentin ( ecd ) and open dentin tubules . in this respect , it is interesting to realize that dentists should be aware of the presence of ecd . most of the patients are not aware themselves of the present harm or the physiologic alteration of the dentition . moreover , patients who experience dhs wait to mention until the next recall visit , and most of them do not specifically seek treatment for this problem , most likely because they do not view it as a significant dental health problem . however , it is clearly shown that dhs can significantly be related to substantially impaired oral health - related quality of life [ 79 ] . as with all conditions or diseases , management strategies that include treatment are usually more successful than treatment alone ; therefore , the following approach was proposed by addy about 10 years ago .ensure the correct diagnosis of dhs , which is based on history and examination and compatible with the definition s clinical descriptor.consider a differential diagnosis , as suggested by the definition of dhs , which alone may explain the symptoms or identify the presence of other conditions contributing to the pain of dhs.treat any and all secondary conditions that induce symptoms similar to dhs ( differential diagnosis).identify etiological and predisposing factors , particularly with respect to erosion and abrasion . consider detailed , written , dietary histories and oral hygiene habits ( frequency , duration and timing of brushing , brushing frequency , estimation of brushing force , frequency of brush change , and appearance of brush at change ) . some of these aspects of tooth brushing behavior are best appraised by observing the patient brushing in the dental practice.remove or modify identified etiological or predisposing factors . offer dietary advice to minimize erosion and oral hygiene instruction to minimize abrasion and to distinguish abrasion from erosion.recommend or provide treatments appropriate to the individual needs of the sufferer . the number of teeth involved and the severity of the pain are important variables and should influence the treatment options . ensure the correct diagnosis of dhs , which is based on history and examination and compatible with the definition s clinical descriptor . consider a differential diagnosis , as suggested by the definition of dhs , which alone may explain the symptoms or identify the presence of other conditions contributing to the pain of dhs . treat any and all secondary conditions that induce symptoms similar to dhs ( differential diagnosis ) . consider detailed , written , dietary histories and oral hygiene habits ( frequency , duration and timing of brushing , brushing frequency , estimation of brushing force , frequency of brush change , and appearance of brush at change ) . some of these aspects of tooth brushing behavior are best appraised by observing the patient brushing in the dental practice . remove or modify identified etiological or predisposing factors . offer dietary advice to minimize erosion and oral hygiene instruction to minimize abrasion and to distinguish abrasion from erosion . the number of teeth involved and the severity of the pain are important variables and should influence the treatment options . the aim of this review was to design a decision tree which can be useful as soon as a patient present with ecd or suffers from dhs . the gaba forum group on dentin hypersensitivity was composed by:- bloch - zupan a. ( strasbourgh , france)- schmalz g. ( regensburg , germany)- gernhardt c. ( halle , germany)- schmidlin p. ( zurich , switzerland)- gillam d. ( london , uk)- sixou ( rennes , france)- hellwig e. ( freiburg , germany)- splieth c. ( greifswald , germany)- bekes k. ( halle , germany)- trp jc . ( basle , switserland)- lussi a. ( bern , switserland)- van loveren c. ( amsterdam , the netherlands)- martens l. ( gent , belgium)- west n. ( bristol , uk)- petersson lg . a pubmed literature search on dentin hypersensitivity was performed focussing on english written manuscripts using the mesh terms review ( 273 hits ) and management ( 12 hits ) . within the reviews , most manuscripts focussed on the etiology or summarized the current treatment strategies . only a few reports could be selected regarding treatment strategies including a decision tree . in addition , some websites of manufacturers were visited . finally , the adelaide university special topic nr 6 on dentin hypersensitivity was consulted . the best developed report came from the canadian advisory board on dentin hypersensitivity ( cabdhs ) who published in 2003 their consensus - based recommendations for the diagnosis and management of dentin hypersensitivity . since then , an interesting flowchart for the clinical management of dhs and only some reports dealing with the concept and methodology for its objective evaluation or reviewing diagnosis and treatment procedures of dentinal hypersensitivity were published . most recently , the academy of general dentistry published a dhs management document which was mainly based on the cabdhs report . from the cabdhs report , it became clear that there is a knowledge gap within the dentist and dental hygienist populations . to illustrate , the survey found that fewer than half of the 542 respondents ( 331 dentists and 211 dental hygienists ) considered a differential diagnosis for dhs , even though it is by definition a diagnosis of exclusion [ 2 , 3 ] . the survey also revealed that many respondents ( 64 % of dentists and 77 % of hygienists ) incorrectly cited bruxism and malocclusion as triggers for dhs , while only a small percentage of the respondents ( 7 % of dentists and 5 % of dental hygienists ) could correctly identify erosion as a primary cause . in this respect , it is clear that abrasion and erosion may be implicated here , but acid erosion seems to be predominant . furthermore , 17 % of dentists and 48 % of hygienists were unable to identify the accepted hydrodynamic theory of dhs . approximately half of the respondents reported that they lacked the confidence to manage a patient s pain caused by dhs . also , only half of the respondents reported that they would try to modify the patient s predisposing factors to control the pain . a total of 14 knowledge gaps were identified , and they were classified as related to the causes and diagnosis or the management of the condition ( see table 1).table 1summary of knowledge gaps regarding the etiology , diagnosis and management of dentin hypersensitivity as proven by the canadian advisory board on dentin hypersensitivity gaps related to etiology / diagnosisgaps related to management1 most of the respondents reported incorrectly that fluoride compounds are the most popular desensitizing ingredients4 . only 10% of respondents correctly thought that desensitizing toothpastes disrupt pain transmission by preventing repolarisation within the nerve.5 . a number of respondents did not believe that desensitizing toothpastes were effective in preventing caries even though most contain fluoride.6 . a number of respondents believed that desensitizing toothpastes were effective in preventing dentine hypersensitivity despite the lack of data 7 . most of the dentists and hygients incorrectly cited toothbrush abrasion as a reason for continued tubule exposure7 . although the latter gap , a number of respondents did not believe that desensitizing toothpastes relieved dentine hypersensitivity summary of knowledge gaps regarding the etiology , diagnosis and management of dentin hypersensitivity as proven by the canadian advisory board on dentin hypersensitivity the most striking gaps related to management were the lack in knowledge on the working mechanisms of the longest used and best known active ingredients in toothpastes : strontium chloride ( occluding the dentinal tubules ) and potassium nitrate ( decrease of excitability of nerves that transmit pain sensation ) . the following was not taken into account : having ecd without painhaving sensitive teeth without decrease of a patient s quality of lifean order of priority or hierarchy in treatment options having ecd without pain having sensitive teeth without decrease of a patient s quality of life an order of priority or hierarchy in treatment options furthermore , the recommendation to initiate for dhs management can be extended and should include also recommendations for the dental professionals and not for patients only as suggested by chu et al . based on drisko et al . . in the present review , an overall approach of the management of ecd and dhs will be given . illustrates how to start the thinking and managing process when ecd is occasionally found during routine check ups . as soon as ecd is seen , one should initiate a further screening asking some specific questions ( cfr fig . 2 ( aa)):fig . 1a treatment decision tree for patients with exposed cervical dentine ( ecd ) ( qol = quality of life , dhs = dentin hypersensitivity)fig . 2a treatment decision tree for patients with dentin hypersensitivity ( dhs ) and a decreased quality of life ( qol ) ( modified from the canadian advisory board on dentin hypersensitivity-2003)probing for lifestyle habits / practices , intrinsic and extrinsic acid ( citrus juices and fruits , carbonated drinks , wines , and ciders),obtaining detailed dietary information including the dietary intake relevant to medical problems that may have an impact on the oral cavity , probing for gastric acid reflux and excessive vomiting . a treatment decision tree for patients with exposed cervical dentine ( ecd ) ( qol = quality of life , dhs = dentin hypersensitivity ) a treatment decision tree for patients with dentin hypersensitivity ( dhs ) and a decreased quality of life ( qol ) ( modified from the canadian advisory board on dentin hypersensitivity-2003 ) probing for lifestyle habits / practices , intrinsic and extrinsic acid ( citrus juices and fruits , carbonated drinks , wines , and ciders ) , obtaining detailed dietary information including the dietary intake relevant to medical problems that may have an impact on the oral cavity , probing for gastric acid reflux and excessive vomiting . along with this specific screening , it is important to distinguish between a localized and a generalized problem which may be mild , moderate , or severe in nature . the latter will influence the future management which usually will involve a combination of at - home and in - office therapies . in practice , the regimen adopted will depend on the perceived severity of the condition and the number of teeth involved . the above - mentioned screening needs to be followed by the initiation of prevention of dhs in two directions : education of patients and self education of practitioners and care givers . in fig . 2 ( ba and bb ) if the patient presents with ecd combined with a complaint of dhs , one has to point out if this pain sensation affects the patient s quality of life ( qol ) . in this respect , the patient can be questioned on : pain and discomfortprobable limitations in dietary choices ( drinks?)the effectiveness of oral hygieneprobable negative esthetics probable limitations in dietary choices ( drinks ? ) the effectiveness of oral hygiene probable negative esthetics if qol is not affected , it is recommended to start the prevention program of dhs . if qol is decreased , it is strongly recommended to start a further management program of dhs as illustrated in fig . 2 . when a patient presents with dhs expressed by sharp pain sensations and with a negative influence on the patients qol , an additional screening obtaining a complete history of the patient especially focussed on nutritional habits ( fig . 2 ( aa ) ) and the promoted diagnosis by exclusion ( fig . 2 ( ab ) ) has to be performed . if there is no consistency between history and examination , other causes than dhs must be sought for . 2 ( ba ) ) as well as for dentists and caregivers ( fig . 2 ( bb ) ) . regarding the patients , dietary counseling and non - harmful oral hygiene habits are very important . therefore , a combination of individualized instructions on rather oral health behaviors , use of at - home products , and additional professional treatment may be required to manage the problem . while at - home treatment can be the first choice for generalized dhs , localized to one or two teeth practitioners may elect to use an in - office method as the first choice of treatment for dhs . regarding the dentists and caregivers , non - harmful professional dental care this must result in a well - considered choice and use of instruments and additional tools performing restorative dentistry . if , during follow up , symptoms are relieved or disappeared , improving the patient s qol , no further treatment is required . a well - maintained preventive management program must be followed . besides eliminating etiological factors , it is strongly recommended to advise a regimen of brushing with desensitizing toothpastes twice a day . regarding desensitizing toothpastes , two treatment approaches are well known : occluding dentinal tubules ( plugging ) or blocking the neural transmission to the pulp . these mechanisms will not further be discussed within this manuscript as other authors did this in the same issue of this journal . if necessary , a less traumatic brushing method may also be introduced . if symptoms are confirmed , no pain relief present , or a further decrease of the patients qol is present , treatment for dhs must be initiated . in this perspective , the non - invasive and invasive approach can be considered ( fig . 2 ( ca cb ) ) . it is recommended to start with minimal invasive in - office procedures such as the use of topical fluorides and dentin bondings , which were discussed in the corresponding reports within this issue or laser therapy . regarding the latter , apparently no significant reductions in sensitivity could be found . as a consequence when there is no long - term benefit of these minimal invasive procedures , more invasive procedures can be carried out after advanced diagnosis . mainly mucogingival surgery and/or pulpectomy will then be the most appropriate choices . if treatment is carried out successfully , one should maintain and review the therapy on a regularly basis . if treatment procedures did not result in pain relief , one should again start an advanced diagnosis based on exclusion ( fig . 2 ( e ) ) and especially focussing on : periodontal painreferred painneuropathic painchronic pain syndrome chronic pain syndrome depending on the results , a decision must be made to continue dhs management and treatment or the patient must be referred to an appropriate dental or medical specialist . a more handy flowchart [ 11 , 13 ] for daily use is also proposed ( fig . 3flowchart for the diagnosis and management of dentin hypersensitivity ( dhs ) in daily practice ( based on ochardsen and gillam 2006 and on the special topic nr 6 on sensitive teeth by colgate and the adelaide university ) flowchart for the diagnosis and management of dentin hypersensitivity ( dhs ) in daily practice ( based on ochardsen and gillam 2006 and on the special topic nr 6 on sensitive teeth by colgate and the adelaide university ) dhs is a problematic clinical entity that may become an increasing clinical problem for dentists to treat as a consequence of patients retaining their teeth throughout life and improved oral hygiene practices . for that , it is strongly recommended to screen routinely all dentate patients for ecd and dhs . in this respect , underdiagnosis of the condition will be avoided and the preventive management can be initiated early . active management of dhs usually will begin with at - home therapy of which brushing with desensitizing toothpastes is the most important . complete management will usually involve a combination of at - home and in - office therapies .

introductiondentin hypersensitivity ( dhs ) is a problematic clinical entity that may become an increasing clinical problem for dentists to treat as a consequence of patients retaining their teeth throughout life and improved oral hygiene practices.objectivesthe aim of this review was to develop a decision tree for the management of exposed cervical dentin ( ecd ) and dhs.material and methodsa brief pubmed literature search was performed on dentin hypersensitivity using mesh terms , review , and management . in addition , some websites and local guidelines were screened.resultsfrom this review , it became clear that all dentate patients should routinely be screened for ecd and dhs . in this respect , underdiagnosis of the condition will be avoided and the preventive management can be initiated early.conclusiona decision tree process and a flowchart for daily practice were designed which should be started up as soon as a patient present with ecd or suffers from dhs . this approach takes into account the possible improved quality of life of the patient and is further based on a hierarchy of treatment options . in this respect , active management of dhs will usually involve a combination of at - home and in - office therapies . starting with the use of desensitizing toothpastes is strongly recommended .

initiation and early growth of primary epithelial cancers , and acquisition of invasiveness is thought to herald the advanced stages of this multistep process that eventually leads to metastatic dissemination , with life - threatening consequences . the genetic control and biochemical mechanisms underlying the acquisition of the invasive phenotype are mediated by the activation of an epithelial mesenchymal transition ( emt ) program . emt is a biologic process that allows an epithelial cell to acquire the properties of a mesenchymal cell , thereby promoting invasion into the surrounding stroma and intravasation of tumor cells . a number of distinct molecular markers are engaged in order to initiate emt and enable its completion . one such marker is the s100a4 , a flament - associated calcium - binding protein , which is associated with the cells of mesenchymal origin , and deemed to play a significant role in emt . s100a4 expression levels strongly correlate with motility , which is a central element of the metastatic cascade . in oral squamous cell carcinoma ( oscc ) , metastasis results in 90% of cancer associated mortality . in this work , we studied the role of s100a4 in oral cancer as a biomarker of metastasis by assessing its expression in primary tumors from all the clinical stages of oral cancer and determined the association of s100a4 expression with aggressive clinico - pathological parameters including the pattern of invasion , distant metastasis and recurrence . assessment of s100a4 in oral cancer may be related to the phenotypic alteration and thereby predict its metastatic potential . the material for present study included a total of 57 formalin fixed paraffin embedded tissue sections retrieved from the departmental archives . out of these , 47 were patients with carcinoma of the bucco - alveolar mucosal complex and of the foor of the mouth . fhe remaining 10 were healthy mucosal tissues , which were obtained at the time of extraction of impacted teeth . only surgically treated cases of oral squamous cell carcinoma ( oscc ) with a minimum follow - up period of 5 years were included in the study . institutional ethics committee approval ( iec 36/2012 ) was obtained from kasturba hospital , manipal , prior to carrying out the study . the patient demographics and other details of the cases included in the study are presented in table 1 . a chart analysis was carried out to study the expression of s100a4 in all these cases by immunohistochemistry ( ihc ) . nt- no tobacco immunohistochemical staining was carried out using indirect streptavidin biotin immunoperoxidase technique . sections of 4 m thickness taken on amino propyl triethoxysilane ( apes ) coated slides were incubated overnight at 48c in a slide warmer to ensure complete adhesion of sections . following tissue sections deparaffinization and hydration through descending grades of alcohol , sections were placed in phosphate buffered saline ( pbs ) for 30 minutes . the slides were immersed in a microwave resistant plastic staining jar containing antigen retrieval solution ( 10 mm sodium citrate buffer , ph 6.0 ) and maintained at 700 w power for 10 minutes . subsequently , the solution was allowed to cool at room temperature for at least 20 minutes . the slides were incubated in 3% hydrogen peroxide for 5 minutes at room temperature , following which slides were washed with pbs . to decrease the background staining due to nonspecific binding of antibodies , the slides were incubated with 5% bovine serum albumin ( bsa ) for 10 minutes at room temperature and washed with pbs for 5 minutes . the researchers applied 100 l of the primary antibody ( rabbit polyclonal anti - s100a4 antibody , 1:250 ) over the section completely , which was incubated for 60 minutes at 37c in a humidified chamber . the slides were rinsed with pbs and placed in the pbs bath for 5 minutes . after draining and wiping of the slides , sections were incubated in 100 l of biotinylated secondary antibody , anti - rabbit igg ( whole molecule - peroxidase antibody produced in goat ) for 30 minutes at 37c . the slides were washed with pbs and placed in pbs bath for 5 minutes . for visualization , sections were incubated with freshly prepared substrate solution ( sigmafast 3 , 3'-diaminobenzidine tablets ) for 5 - 10 minutes . this was followed by washing the slides in running tap water and counter staining with mayer 's haematoxylin ( mhs1- haematoxylin solution , mayer 's 1 gil certified hematoxylin * ph 2.4 ) for 5 minutes , after which the slides were washed in running tap water for 1 minute . briefy , the sections were dehydrated through ascending grades of alcohol ( 70% , 95% and 100% ) for 2 minutes each , cleared in xylene for 1 minute , mounted in a resinous media and coverslipped . the neoplastic cell with cytoplasmic staining was viewed at 200 magnification , using a light microscope . the distribution of immunolabelling in neoplastic epithelial cells was determined from a minimum of five representative high - power fields at the invasive front . a positive cell demonstrated a diffuse brown signal in the cytoplasm , independent of its intensity . to eliminate any inter - observer bias , the degree of staining for s100a4 was evaluated using a semi - quantitative scale29 . tissues were graded as absent ( - ) when there was no staining , low ( 1 + ) when less than 25% of the tumor cells were positive , moderate ( 2 + ) when 25 to 75% of the tumor cells stained positively and diffuse ( 3 + ) when more than 75% of the tumor cells were positive for s100a4 . s100a4 expression was then correlated with clinicopathological parameters including primary tumor site , stage of tumor , degree of differentiation , lymph node metastasis and mode ( pattern ) of invasion . kendall 's tau - b statistics was applied to assess the measure of agreement between two observers . chi - square test was used to study the association between the s100a4 staining and clinicopathological parameters . the material for present study included a total of 57 formalin fixed paraffin embedded tissue sections retrieved from the departmental archives . out of these , 47 were patients with carcinoma of the bucco - alveolar mucosal complex and of the foor of the mouth . fhe remaining 10 were healthy mucosal tissues , which were obtained at the time of extraction of impacted teeth . only surgically treated cases of oral squamous cell carcinoma ( oscc ) with a minimum follow - up period of 5 years were included in the study . institutional ethics committee approval ( iec 36/2012 ) was obtained from kasturba hospital , manipal , prior to carrying out the study . the patient demographics and other details of the cases included in the study are presented in table 1 . a chart analysis was carried out to study the expression of s100a4 in all these cases by immunohistochemistry ( ihc ) . the antibodies and reagents were obtained from sigma aldrich co. ( st . louis , mo , usa ) . sections of 4 m thickness taken on amino propyl triethoxysilane ( apes ) coated slides were incubated overnight at 48c in a slide warmer to ensure complete adhesion of sections . following tissue sections deparaffinization and hydration through descending grades of alcohol , sections were placed in phosphate buffered saline ( pbs ) for 30 minutes . the slides were immersed in a microwave resistant plastic staining jar containing antigen retrieval solution ( 10 mm sodium citrate buffer , ph 6.0 ) and maintained at 700 w power for 10 minutes . subsequently , the solution was allowed to cool at room temperature for at least 20 minutes . the slides were incubated in 3% hydrogen peroxide for 5 minutes at room temperature , following which slides were washed with pbs . to decrease the background staining due to nonspecific binding of antibodies , the slides were incubated with 5% bovine serum albumin ( bsa ) for 10 minutes at room temperature and washed with pbs for 5 minutes . the researchers applied 100 l of the primary antibody ( rabbit polyclonal anti - s100a4 antibody , 1:250 ) over the section completely , which was incubated for 60 minutes at 37c in a humidified chamber . the slides were rinsed with pbs and placed in the pbs bath for 5 minutes . after draining and wiping of the slides , sections were incubated in 100 l of biotinylated secondary antibody , anti - rabbit igg ( whole molecule - peroxidase antibody produced in goat ) for 30 minutes at 37c . the slides were washed with pbs and placed in pbs bath for 5 minutes . for visualization , sections were incubated with freshly prepared substrate solution ( sigmafast 3 , 3'-diaminobenzidine tablets ) for 5 - 10 minutes . this was followed by washing the slides in running tap water and counter staining with mayer 's haematoxylin ( mhs1- haematoxylin solution , mayer 's 1 gil certified hematoxylin * ph 2.4 ) for 5 minutes , after which the slides were washed in running tap water for 1 minute . briefy , the sections were dehydrated through ascending grades of alcohol ( 70% , 95% and 100% ) for 2 minutes each , cleared in xylene for 1 minute , mounted in a resinous media and coverslipped . the neoplastic cell with cytoplasmic staining was viewed at 200 magnification , using a light microscope . the distribution of immunolabelling in neoplastic epithelial cells was determined from a minimum of five representative high - power fields at the invasive front . a positive cell demonstrated a diffuse brown signal in the cytoplasm , independent of its intensity . to eliminate any inter - observer bias , the degree of staining for s100a4 was evaluated using a semi - quantitative scale29 . tissues were graded as absent ( - ) when there was no staining , low ( 1 + ) when less than 25% of the tumor cells were positive , moderate ( 2 + ) when 25 to 75% of the tumor cells stained positively and diffuse ( 3 + ) when more than 75% of the tumor cells were positive for s100a4 . s100a4 expression was then correlated with clinicopathological parameters including primary tumor site , stage of tumor , degree of differentiation , lymph node metastasis and mode ( pattern ) of invasion . kendall 's tau - b statistics was applied to assess the measure of agreement between two observers . chi - square test was used to study the association between the s100a4 staining and clinicopathological parameters . positive s100a4 expression in the cytoplasm of the tumor cells was observed in 30 out of 47 cases ( 63.82% ) of oscc . kendall 's tau - b test between the two observers showed a very high level of agreement for the expression of s100a4 ( 0.8 , p<0.001 ) . in the control group , none of oral mucosal tissues were positive for s100a4 in the cytoplasm of the epithelium . out of 47 cases studied , 1/6 of stage i , 1/2 of stage ii , 13/17 of stage iii and 15/22 of stage iv cases showed positive s100a4 expression ( table 2 ) . a strong association was noted between the stage of the tumor and the expression score of s100a4 ( p=0.007 ) . s100a4 expression was positive in 12/14 ( 85.7% ) node positive stage iii cases , of which 7/12 were ( 3 + ) and 5/12 were ( 2 + ) for s100a4 expression . among the node negative cases , 1/3 cases showed ( 3 + ) for s100a4 expression . likewise , s100a4 expression was positive in 14/15 ( 93.3% ) stage iv cases , of which 13/14 were ( 3 + ) and 1/14 was ( 2 + ) . among the 7 node negative cases , 1/7 was ( 3 + ) and the remaining 6/7 cases were negative for s100a4 expression . although the cytoplasmic expression of s100a4 was strongly associated with lymph node involvement ( p<0.001 ) , there was no association between s100a4 expression in lymph node positive cases and primary size of the tumor ( p=0.15 ) . association of s100a4 expression with clinical stage , lymph node involvement , tumor size , metastasis , recurrence , grade of tumor and pattern of invasion wdscc - well differentiated squamous cell carcinoma ; mdscc - moderately differentiated squamous cell carcinoma ; pdscc - poorly differentiated squamous cell carcinoma with regard to distant metastasis ( m ) , 11/22 ( 50% ) cases in stage iv were positive for s100a4 expression , out of which 10/11 were ( 3 + ) and 1/11 was ( 2 + ) for s100a4 expression . a strong association could be established between the s100a4 expression in tumor cells and distant metastasis ( p=0.004 ) . likewise , 11/47 ( 23.4% ) cases that recurred after treatment were either in stage iii ( 2/11 ) or stage iv ( 9/11 ) . all these cases showed ( 3 + ) expression for s100a4 . among the stage iii cases that recurred , one was node positive and the other was negative , whereas all the 9 stage iv cases that recurred were node positive . there was a strong positive association between the s100a4 expression and rate of recurrence ( p=0.002 ) . with regard to the pattern of invasion among the positive cases , 1/6 cases of stage i showed a type 1 ( figure 1 ) pattern and 1/2 cases in stage ii showed type 2 ( figure 2 ) pattern . out of the 13 stage iii cases that showed positive s100a4 expression , 2 were type 1 , 2 others were type 2 , 3 were type 3 ( figure 3 ) , 3 were type 4c ( figure 4 ) and 3 were type 4d ( figure 5 ) . in the same manner , out of the 15 stage iv cases that showed positive s100a4 expression , 2 were type 3 , 4 were type 4c and 9 were type 4d . there was a strong association ( p<0.001 ) between s100a4 expression and pattern of invasion of oscc . however , there was no association seen between the s100a4 expression and histological grade of the oscc ( p=0.27 ) . oral squamous cell carcinoma ( oscc ) showing type 1 well defined borderline pattern of invasion ( s100a4 , 20x ) oral squamous cell carcinoma ( oscc ) showing type 2 cord like pattern of invasion ( s100 a4 , 20x ) oral squamous cell carcinoma ( oscc ) showing type 3 pattern of invasion with groups of cells with no distinct borderline ( s100a4 , 20x ) oral squamous cell carcinoma ( oscc ) showing type 4c diffuse cord like pattern of invasion ( s100a4 , 20x ) oral squamous cell carcinoma ( oscc ) showing type 4d diffuse widespread pattern of invasion ( s100a4 , 20x ) oral cancer is one of the major cancers worldwide , with a low five - year survival rate that has not significantly changed over the past few decades . metastasis , which is responsible for 90% of the cancer associated mortality , is one of the poorly understood factors in cancer progression . an important step for the invasion and metastasis is the epithelial mesenchymal transition ( emt ) , a complex process induced by modifications of multiple pathways leading to a spectrum of epithelial cellular changes , including loss of polarity and cohesion , increased motility , and the acquisition of mesenchymal phenotype . emt is critical for the invasion , progression , and metastasis in epithelial carcinogenesis . metastasis promoting s100a4 is a typical representative of s100 family of ca2 + -binding multifunctional proteins with dual extra- and intracellular function . the increased expression of the s100a4 in invasive front of the tumor suggests the pivotal role played by s100a4 in invasion of tumor cells into the surrounding matrix . the acquisition of invasive phenotype with altered pattern of adhesion is a result of e - cadherin down regulation by s100a4 , which the tumor cells acquire . further influences cell - matrix adhesions by interacting with liprin [ 1 and masking their protein kinase c - mediated phosphorylation sites . inhibition of the liprin [ 1-lar complex by s100a4 loosens cell adhesion and allows cell invasion . cell invasion is also facilitated by matrix metalloproteinases ( mmps ) , as s100a4 causes metastasis not only by affecting the motility of cells but also by affecting its invasive properties through influencing the expression of mmps and their endogenous inhibitors . a positive correlation of the s100a4 and mmp2 expression in oesophageal squamous cell carcinoma has been reported . s100a4 is located at the leading edge of migrating cells , where it induces the formation of fexible protrusions . the s100a4-myosin ii - a interaction does not only increase cell motility , but also enhances cell polarization and directed migration . s100a4 also binds to the septins 2 , 6 and 7 , which play a central role in cytokinesis , cell polarity determination , cytoskeletal reorganization , and membrane dynamics10 , . s100a4 binds to annexin ii on the endothelial surface , a complex that activates the tissue plasminogen activator , which in turn converts the plasminogen to plasmin , activating prommps . ca - dependent binding of s100a4 to metap2 modulates the metap2 activity , which could promote endothelial growth and angiogenesis . this process of angiogenesis favors the metastasis of the tumor to the regional lymph nodes . elevated s100a4 expression has shown to be the marker of metastasis and poor prognosis in patients with pancreatic ductal adenocarcinoma , prostate adenocarcinoma , colorectal cancer4 , and breast cancer . a higher level of s100a4 expression has shown to correlate with auxillary lymph node metastasis and overall patient survival . evidence suggesting the role for s100a4 in hnscc , especially in oscc , is scarce . elevated s100a4 as a prognostic factor in metastatic cases of oscc was demonstrated by moriyama - kita , et al . ( 2004).our findings showing increased expression of s100a4 in cases with regional lymphnode involvement and metastasis further emphasizes the role of s100a4 in oral cancer patients with poor prognosis . the final step in the metastatic cascade is the migration of the cells to a distant site and secondary colonization . in our study this finding has been elucidated earlier in papillary thyroid carcinoma and pancreatic cancer , in which poor outcome of patients was related to increased s100a4 expression coupled with decreased e cadherin expression . the increased expression of s100a4 in our study showed a significant correlation with the overall clinical stage of tumor , a finding that has been noted also in colorectal carcinoma and pancreatic cancer . however , no association could be drawn between the elevated s100a4 level and the primary tumor size or histological grading in our analysis . ( 2014 ) in lung cancer cells , in which s100a4 was frequently overexpressed , irrespective of histological subtype . in our study a strong association between s100a4 expression and pattern of invasion could be elicited , with an increased expression noted in type 4c and type 4d pattern of invasion . similar observations have been made in malignant melanomas where increased expression of s100a4 in the nodular variant was noted , linking this association to the pattern of invasion . significantly , our observation of high s100a4 expression with tumor recurrence is a novel finding . s100a4 has been identified as a candidate marker for maintaining the stemness in head and neck cancer initiating cells . s100a4 controls the notch and pten / pi3k / akt signaling pathways , which regulate the self - renewal and tumorigenicity of stem cells or cancer stem cells . s100a4 signaling pathways thus play a major role in the maintenance of cancer initiating cell population and contribute for the tumor recurrence . in summary , the data from our study adds weight to the growing body of evidence that s100a4 can be used to identify the subgroup of patients at high risk of metastasis and recurrence in oscc cases , and that targeting s100a4 signaling might be a potential therapeutic target for increasing the patient survival .

s100a4 , a biomarker of epithelial mesenchymal transition ( emt ) , plays an important role in invasion and metastasis by promoting cancer cell motility . in oral squamous cell carcinoma ( oscc ) , metastasis results in 90% of cancer associated mortality.objectiveto investigate the role of s100a4 expression as an important component of the epithelial mesenchymal transition ( emt ) program in oral squamous cell carcinoma ( oscc).material and methodss100a4 protein expression was assessed semi - quantitatively by immunohistochemistry in 47 histologically confirmed cases of oral squamous cell carcinoma ( oscc ) and 10 normal oral mucosal biopsies . the association between the s100a4 overexpression and the aggressive features of oscc were analyzed by x2 test.resultsmoderate to strong cytoplasmic expression of s100a4 was observed in 30 out of 47 specimens of oscc ( 64% ) . overexpression of s100a4 was significantly associated with the clinical stage , lymph node involvement , metastases , pattern of invasion and recurrence ( p<0.05).conclusions100a4 expression represents an important biomarker of prognostic significance that may be used to identify a subset of patients at high risk of invasion and metast

general remarks . all operations should be performed under an upright dissecting microscope ( leica ) and by using a surgical coagulator . the mice in the experimental and sham groups should be matched in age and weight to ensure comparability of the results . temperature , blood pressure , anesthesia and fluid administration should be stable and monitored during the entire experiment . c57bl/6 mice should be within an age range of 10 to 16 weeks and they should be gender - matched in the respective groups . anesthetize mice with sodium pentobarbital ( 70 mg / kg body weight i.p . ) and maintain anesthesia with approx . place mice on a temperature - controlled heated table ( rt , effenberg , munich , germany ) with a rectal thermometer probe attached to a thermal feedback controller to maintain body temperature at 37 c . place mice in a supine position on the surgery table , with the upper and lower extremities attached to the table with removable tape . in case of survival experiments proper aseptic techniques including clipping of hair , disinfection of skin with iodophors ( e.g. betadine ) , sterile handling of autoclaved instruments , and using masks cover the incision area with mineral oil to prevent inhalation of mouse hair by cutting the skin and thus preventing the risk of mouse hair allergies . perform midline laparotomy and incision of the linea alba up to the upper end so that you can easily expose the liver . if possible perform the incision with a coagulation electrode ( erbe , icc50 , tuebingen , germany ) to prevent bleeding . place the heating table with the mouse in a position that the tail is directed to the surgeon . that position provides you with the best setting to localize the triad and to place the suture . displace the intestine on the right side by using a wet cotton tip swab to expose the portal triad . slightly shift the median and left lobe towards the diaphragm and the right lobe towards the intestine . that will give you a clear view of the portal triad above the bifurcation of right , median / left lobes . once visually identified place the needle with the suture ( 7/0 nylon suture ; ethicon , norderstedt , germany ) via a needle holder under the left portal triad , including the hepatic artery , hepatic vein , and bile duct from the left to the right side of the mouse . a deeper stitch can injure the vena vaca inferior which is located under the triad . therefore hold the right liver lobes down with the forceps to identify the vena cava before placing the stitch . take the tip of the needle on the right side with a forceps and pull it gently out of the liver area without injuring the liver . attach to each end of the suture an eppendorf tube ( approximately 3 g , filled with water ) and place both suture ends over suture holders . place the suture holder distally of the stitch so that the suture does not interfere with the right triad . by applying the weights the triad will be immediately occluded , causing interruption of blood supply to the left and median lobes of the liver . successful occlusion can be easily confirmed by visual inspection of pale blanching of the ischemic lobes . in contrast , the change of color immediately disappears when the hanging weights are released from the poles and the liver is reperfused . this partial hepatic ischemia model avoids mesenteric congestion by preserving blood flow to the right liver lobes . keep the liver and intestine warm with a wet swab soaked with water at 37.0 c . in addition to keep body temperature stable cover the mice with commercially available food wrap after finishing surgery . perform ischemia as required per experimental protocol . after a defined ischemia time release the weights and reperfusion of the median and left liver lobes will start immediately . add approximately 400 l sodium chloride into the abdominal cavity 15 minutes before and directly after reperfusion . continue to substitute fluid every 30 minutes in an amount of 100 l to compensate fluid loss during the reperfusion time . for survival experiments the surgical wound is closed using continuous suture of the muscle wall and skin with a 4/0 suture . the following outcome parameters are recommended to determine liver injury following hepatic ischemia : determine liver enzymes ( e.g. alt , ast ) and liver histology ( h&e staining ) following liver ischemia and reperfusion . model of a hanging - weight system for liver ischemia : a hanging weight system has been established to induce liver ischemia of the left and median lobe . ( a ) therefore the caudate and right lobe were gently separated from the left lobe . the right lobe was then slightly shifted to clearly view the portal triad above the bifurcation of right , median and left lobes . ( b ) at the end of each suture a small weight ( 3 g eppendorf tubes filled with water ) was attached and applied . hepatic ischemia is visible by a color change of the left and median lobe from red to pale . this experimental design allows precise occlusion and reperfusion of the left hepatic triad by applying and releasing the weight load . effect of different liver ischemia times on alanine ( alt ) and aspartate ( ast ) aminotransferase in mice . partial portal triad ischemia was induced as indicated ( 0 - 60 min ischemia ) . after 2 hr of reperfusion , alt ( a ) and ast ( b ) were measured . c57bl/6 mice should be within an age range of 10 to 16 weeks and they should be gender - matched in the respective groups . anesthetize mice with sodium pentobarbital ( 70 mg / kg body weight i.p . ) and maintain anesthesia with approx . overdosing can significantly lower blood pressure and thus can alter the results . place mice on a temperature - controlled heated table ( rt , effenberg , munich , germany ) with a rectal thermometer probe attached to a thermal feedback controller to maintain body temperature at 37 c . place mice in a supine position on the surgery table , with the upper and lower extremities attached to the table with removable tape . in case of survival experiments proper aseptic techniques including clipping of hair , disinfection of skin with iodophors ( e.g. betadine ) , sterile handling of autoclaved instruments , and using masks a sterile gloves for the surgeon are required . cover the incision area with mineral oil to prevent inhalation of mouse hair by cutting the skin and thus preventing the risk of mouse hair allergies . perform midline laparotomy and incision of the linea alba up to the upper end so that you can easily expose the liver . if possible perform the incision with a coagulation electrode ( erbe , icc50 , tuebingen , germany ) to prevent bleeding . place the heating table with the mouse in a position that the tail is directed to the surgeon . that position provides you with the best setting to localize the triad and to place the suture . displace the intestine on the right side by using a wet cotton tip swab to expose the portal triad . slightly shift the median and left lobe towards the diaphragm and the right lobe towards the intestine . that will give you a clear view of the portal triad above the bifurcation of right , median / left lobes . once visually identified place the needle with the suture ( 7/0 nylon suture ; ethicon , norderstedt , germany ) via a needle holder under the left portal triad , including the hepatic artery , hepatic vein , and bile duct from the left to the right side of the mouse . a deeper stitch can injure the vena vaca inferior which is located under the triad . therefore hold the right liver lobes down with the forceps to identify the vena cava before placing the stitch . take the tip of the needle on the right side with a forceps and pull it gently out of the liver area without injuring the liver . attach to each end of the suture an eppendorf tube ( approximately 3 g , filled with water ) and place both suture ends over suture holders . place the suture holder distally of the stitch so that the suture does not interfere with the right triad . by applying the weights the triad will be immediately occluded , causing interruption of blood supply to the left and median lobes of the liver . successful occlusion can be easily confirmed by visual inspection of pale blanching of the ischemic lobes . in contrast , the change of color immediately disappears when the hanging weights are released from the poles and the liver is reperfused . this partial hepatic ischemia model avoids mesenteric congestion by preserving blood flow to the right liver lobes . keep the liver and intestine warm with a wet swab soaked with water at 37.0 c . in addition to keep body temperature stable cover the mice with commercially available food wrap after finishing surgery . perform ischemia as required per experimental protocol . after a defined ischemia time release the weights and reperfusion of the median and left liver lobes will start immediately . add approximately 400 l sodium chloride into the abdominal cavity 15 minutes before and directly after reperfusion . continue to substitute fluid every 30 minutes in an amount of 100 l to compensate fluid loss during the reperfusion time . for survival experiments the surgical wound is closed using continuous suture of the muscle wall and skin with a 4/0 suture . the following outcome parameters are recommended to determine liver injury following hepatic ischemia : determine liver enzymes ( e.g. alt , ast ) and liver histology ( h&e staining ) following liver ischemia and reperfusion . model of a hanging - weight system for liver ischemia : a hanging weight system has been established to induce liver ischemia of the left and median lobe . ( a ) therefore the caudate and right lobe were gently separated from the left lobe . the right lobe was then slightly shifted to clearly view the portal triad above the bifurcation of right , median and left lobes . ( b ) at the end of each suture a small weight ( 3 g eppendorf tubes filled with water ) was attached and applied . hepatic ischemia is visible by a color change of the left and median lobe from red to pale . this experimental design allows precise occlusion and reperfusion of the left hepatic triad by applying and releasing the weight load . effect of different liver ischemia times on alanine ( alt ) and aspartate ( ast ) aminotransferase in mice . partial portal triad ischemia was induced as indicated ( 0 - 60 min ischemia ) . after 2 hr of reperfusion , alt ( a ) and ast ( b ) were measured . the present study describes a technique of performing ischemia in a murine model by using a hanging - weight system for occlusion of the left hepatic portal triad . although it is an easy method to learn the following pitfalls should be taken into account . a very common mistake in the beginning is a deep stitch which includes the portal triad and the vena cava . the breathing and heart rate will slow down within 15 minutes due to the increased resistance and the decrease in returning blood to the heart resulting in an early death of the mice . the best way to prevent the including of the vena cava is to localize the vena cava before the stitch by holding the right lobes down with a forceps . another important issue is that the weights are not to heavy which can result in too much stress on the left and right portal trial which also stops blood flow to the right lobes . therefore the amount of water in the eppendorf vials should be adjusted to the mouse weight and the suture holders should be placed distally from the stitch location . on the opposite both mistakes are easily detectable by an additional color change to pale of the right lobe or insufficient color change of the median and left lobes , respectively . therefore the hepatic color change following applying the weights should be controlled every 10 minutes . therefore the right lobes should be held down with a bland forceps so that the tip of the needle can be easily seen and taken with a needle holder . if these pitfalls in the beginning are taken into account the hanging - weight system for hepatic ischemia provides a highly reproducible injury due to ischemia by minimizing the variability of liver damage associated with clamping of the portal triad . by using a hanging - weight system , the portal triad is only manipulated once throughout the entire surgical procedure , causing significantly less damage to the hepatic lobes . more tissue trauma occurs by removing and replacing vessel clamps , especially with reapplication of the clamp during multiple ischemia cycles like during ischemic preconditioning . the use of hanging weights that are in a remote location from the liver tissue , according to our observations , provides the advantage of reliable occlusion while preventing tissue trauma due to manipulation of the liver lobes by reapplication of a clamp . taken together , the present study provides feasibility of the hanging - weight system for portal triad occlusion during ischemia , minimizing the variability and limitations associated with clamping . thus this technique might be of interest for other investigators who consider studying hepatic ischemia .

acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation , hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate1 - 2 . therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies3 - 9 . specifically , conditional mice with tissue specific deletion of a gene ( cre , flox system ) provide insights into the role of proteins in particular tissues10 - 13 . because of the technical difficulty associated with manually clamping the portal triad in mice , we performed a systematic evaluation using a hanging - weight system for portal triad occlusion which has been previously described3 . by using a hanging - weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes , since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels . furthermore , the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved . furthermore , the portal triad is only manipulated once throughout the entire surgical procedure . as a result , procedures like pre - conditioning , with short times of ischemia and reperfusion , can be easily performed . systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine ( alt ) and aspartate ( ast ) aminotransferase serum levels3,9 . taken together , these studies confirm highly reproducible liver injury when using the hanging - weight system for hepatic ischemia and intermittent reperfusion . thus , this technique might be useful for other investigators interested in liver ischemia studies in mice . therefore the video clip provides a detailed step - by - step description of this technique .

hepatitis c virus ( hcv ) is a small , enveloped positive sense , single stranded rna virus that belongs to the genus hepacivirus and the family flaviviridae . it is estimated that 3% of the world s population ( 170 million people ) are infected with hcv ( 1 ) and its prevalence ranges from 0.2% to 40% in different countries ( 2 , 3 ) . hcv is a major cause of chronic viral hepatitis , liver cirrhosis and hepatocellular carcinoma . therefore it has important clinical , epidemiological and economic outcomes throughout the world especially in developing countries ( 4 , 5 ) . genotyping of hepatitis c virus is important because different genotypes have different infectivity and influencing the rate of progression of hcv infection to cirrhosis and hepatocellular carcinoma . besides various hcv genotypes for example , genotypes 1 and 4 are more resistant to interferon based therapies than genotypes 2 and 3 ( 68 ) . currently , hcv is classified into eleven genotypes ( 111 ) with sequence differences range from 30% to 50% that six of them are the major genotypes ( 1 to 6 ) ( 6 , 9 ) . within hcv genotype , several subtypes ( named as a , b , c , etc . ) can be defined by sequence differences range from 15% to 30% ( 10 , 11 ) . types 1a and 1b are the most common , accounting for about 60% of global infections . they predominate in northern europe and north america , and in southern and eastern europe and japan , respectively . genotype 4 is principally detected in the middle east , egypt , north and central africa . type 5 is mostly found in southern africa and genotypes 611 are distributed in asia ( 1216 ) . the determination of the hcv genotypes is important for the prediction of response to antiviral treatment . the distribution of hcv genotypes was reported from various regions of iran ( 1720 ) ; however , such studies were not conducted in arak city . therefore , this investigation was performed to determine the distribution of hcv genotypes in arak , central province of iran . in this retrospective study , 174 cases with chronic hcv infection [ positive result of hepatitis c antibody ( anti - hcv ) more than 6 months and positive hcv - rna ] referred to infectious center of valiasr hospital , arak ; central province of iran were enrolled . hcv infection was confirmed by positive hcv antibody ( anti - hcv ) and hcv - rna tests . all the cases were negative for hepatitis b surface antigen ( hbsag ) and human immunodeficiency virus ( hiv ) antibodies . a questionnaire was used to collect data such as age , sex and possible route of hcv transmission . a peripheral blood sample from each patient was collected in an edta containing sterile tube . anti - hcv and hbsag were tested by enzyme - linked immunosorbent assay ( elisa ) . the commercial enzyme immunoassay kits used were as follows : hbsag ( hepanosticka biomerieux , boxtel , the netherlands ) and anti - hcv ( biorad , segrate , italy ) . recombinant immunoblot assay ( riba innogenetics , ghent , belgium ) was employed to confirm anti - hcv reactivity . anti - hiv was determined by elisa ( mp biomedicals , illkirch , france ) ; with positive tests confirmed by western blot assay ( diaplus , san francisco , usa ) . viral rna was extracted from plasma sample using high pure viral nucleic acid kit ( roche diagnostics gmbh , mannheim , germany ) , according to the manufacturer s instructions . hcv genotyping was carried out using a commercial kit ( sacace , italy ) according to manufacturer s instruction . this kit is designed for the detection of genotypes 1a , 1b , 2 , 3a ( which are most prevalent hcv genotypes in iran ) by generating different size pcr products . the specificity and sensitivity of the kit are 100% and 1000 viral particles / ml respectively . for each patient two sets of pcr amplifications were performed in two separate tubes containing primers for either genotypes 1a+1b or genotypes 2 + 3a . 5 l of cdna was subjected to pcr amplifications using two sets of mixed primers included in the kit . the pcr profile was an initial denaturation at 95c for 5 min , followed by 42 cycles of 95c for 1 min , 68c for 1 min , 72c for 1 min and a final extension at 72c for 10 min . genotype 1a generates a 338 bp pcr product , genotype 1b , 2 and 3a generate 395 , 286 and 227 bp pcr products respectively . the pcr products were electrophoresed in a 2% agarose gel and stained with ethidium bromide . after determination of hcv genotypes , patients with genotypes 2 or 3 , received 800 mg ribavirin ( rbv ) daily in two divided doses for 24 weeks . patients with genotype 1 , received 1000 mg rbv daily if they weighed less than 75 kg and 1200 mg if over 75 kg for 48 weeks . sustained virologic response ( svr ) which is defined as undetectable hcv - rna using a highly sensitive assay ( real time polymerase chain reaction ) was determined in patients after 24 weeks of the end of treatment . the chi - square was used with the spss 16 package program for statistical analysis ( chicago , il , usa ) . data are presented as mean sd or , when indicated , as an absolute number and percentage . in this retrospective study , 174 cases with chronic hcv infection [ positive result of hepatitis c antibody ( anti - hcv ) more than 6 months and positive hcv - rna ] referred to infectious center of valiasr hospital , arak ; central province of iran were enrolled . hcv infection was confirmed by positive hcv antibody ( anti - hcv ) and hcv - rna tests . all the cases were negative for hepatitis b surface antigen ( hbsag ) and human immunodeficiency virus ( hiv ) antibodies . a questionnaire was used to collect data such as age , sex and possible route of hcv transmission . a peripheral blood sample from each patient was collected in an edta containing sterile tube . anti - hcv and hbsag were tested by enzyme - linked immunosorbent assay ( elisa ) . the commercial enzyme immunoassay kits used were as follows : hbsag ( hepanosticka biomerieux , boxtel , the netherlands ) and anti - hcv ( biorad , segrate , italy ) . recombinant immunoblot assay ( riba innogenetics , ghent , belgium ) was employed to confirm anti - hcv reactivity . anti - hiv was determined by elisa ( mp biomedicals , illkirch , france ) ; with positive tests confirmed by western blot assay ( diaplus , san francisco , usa ) . viral rna was extracted from plasma sample using high pure viral nucleic acid kit ( roche diagnostics gmbh , mannheim , germany ) , according to the manufacturer s instructions . hcv genotyping was carried out using a commercial kit ( sacace , italy ) according to manufacturer s instruction . this kit is designed for the detection of genotypes 1a , 1b , 2 , 3a ( which are most prevalent hcv genotypes in iran ) by generating different size pcr products . the specificity and sensitivity of the kit are 100% and 1000 viral particles / ml respectively . for each patient two sets of pcr amplifications were performed in two separate tubes containing primers for either genotypes 1a+1b or genotypes 2 + 3a . 5 l of cdna was subjected to pcr amplifications using two sets of mixed primers included in the kit . the pcr profile was an initial denaturation at 95c for 5 min , followed by 42 cycles of 95c for 1 min , 68c for 1 min , 72c for 1 min and a final extension at 72c for 10 min . genotype 1a generates a 338 bp pcr product , genotype 1b , 2 and 3a generate 395 , 286 and 227 bp pcr products respectively . the pcr products were electrophoresed in a 2% agarose gel and stained with ethidium bromide . after determination of hcv genotypes , patients with genotypes 2 or 3 , received 800 mg ribavirin ( rbv ) daily in two divided doses for 24 weeks . patients with genotype 1 , received 1000 mg rbv daily if they weighed less than 75 kg and 1200 mg if over 75 kg for 48 weeks . sustained virologic response ( svr ) which is defined as undetectable hcv - rna using a highly sensitive assay ( real time polymerase chain reaction ) was determined in patients after 24 weeks of the end of treatment . the chi - square was used with the spss 16 package program for statistical analysis ( chicago , il , usa ) . data are presented as mean sd or , when indicated , as an absolute number and percentage . a total of 174 patients with chronic hcv infection with mean age 37.510.24 ( range : 1876 years ) were enrolled in the study . 170 ( 97.7% ) of cases were male and 4 ( 2.3% ) were female . the main route of hcv transmission was injection drug use ( idu ) which was observed in 104 ( 59.8% ) of cases . other presumed routes of hcv transmission were needle stick in 12 ( 6.9% ) , blood and blood products transfusion in 5 ( 2.9% ) , incarceration in 4 ( 2.3% ) , heterosexual contact in 1 ( 0.6% ) , tattooing in 1 ( 0.6% ) , idu and tattooing in 7 ( 4% ) , idu and blood transfusion in 7 ( 4% ) , idu and incarceration in 6 ( 3.4% ) , idu and heterosexual contact in 3 ( 1.7% ) , tattooing and heterosexual contact in 2 ( 1.2% ) , idu , tattooing and incarceration in 2 ( 1.2% ) , idu , tattooing and heterosexual contact in 3 ( 1.6% ) and in 17 ( 9.8% ) the route of hcv acquisition was not identified . genotyping results demonstrated that subtype 3a 92 ( 52.9% ) was the most prevalent hcv type in arak , followed by subtype 1a 40 ( 22.9% ) and subtype 1ab 31 ( 17.8% ) . hcv genotypes distribution was not significantly associated with age and gender ( p=0.07 and 0.06 respectively ) . distribution of hcv genotypes and subtypes in arak city sustained virologic response was observed in 78 ( 83.8% ) of cases . svr was detected in 42/4 ( 91.3% ) of patients infected with genotype 3a versus 17/22 ( 77.3% ) of those infected with genotype 1a and 9/13 ( 69.2% ) of cases infected with genotype 1ab . epidemiological studies in different region of the world show the wide variation and regional differences in the distribution of hcv genotypes ( 21 ) . hcv genotyping is an important factor in clinical hcv treatment and investigation of hcv genotypes distribution is essential as prognostic factor in chronic hcv infection ( 11 , 22 ) . hcv genotypes are studied in different parts of iran ( 1720 ) . in this survey which is the first study of hcv genotypes distribution in arak city , central province of iran , we showed that subtype 3a ( 52.9% ) was the most prevalent hcv type , followed by subtype 1a ( 22.9% ) and subtype 1ab ( 17.8% ) . ( 23 ) on specimens from 16 provinces of iran revealed that type 3a was the most frequent hcv type ( 46.6% ) , followed by type 1 with rate of 43.2% ( including 1a and 1b with 25.73% and 17.47% for each respectively ) . reviewed the prevalence of hcv genotypes in iranian patients and reported that hcv subtype 1a was maximum in east azarbayjan and guilan respectively but minimum in west azarbayjan . prevalence of subtype 1b was 25% , 25% and 23% in kermanshah , zahedan and hormozgan respectively . prevalence of genotype 2 was 0.7% in tehran and 17.2% in kermanshah ( 24 ) . in a study by esmaeilzadeh et al . ( 17 ) in zanjan ( the northwest of iran ) , subtype 3a was the most current subtype in this city . in another study by omrani et al . ( 25 ) hcv genotype 3a with the prevalence rate of 48.1% was the most prevalent hcv type in west azerbaijan , northwest of iran . a study in shiraz , a city in southern part of iran revealed that the highest level of hcv infection belonging to type 3a followed by 1a ( 26 ) . additionally genotype 3 was the most common genotype in plasma and peripheral blood mononuclear cells ( pbmcs ) of hcv infected patients in jahrom city ( another city in southern part of iran ) ( 27 ) . besides , genotype 3a is the most frequent hcv genotype in isfahan province , iran . hcv subtypes 3a and 1a were determined in the hemodialysis patients living in tehran as the prevalent subtypes ( 28 ) . on the other hand genotype 1a was the more frequent hcv genotype in khorasan razavi province ( northeast of iran ) ( 29 ) . in another survey in southeast of iran , genotype 1 was the more prevalent hcv type followed by genotype 3 , 4 and 2 ( 30 ) . ( 41.7% ) followed by 3a , 2 and 4 were reported as most frequent hcv genotypes in khuzestan province , southwestern iran ( 18 ) . jahanbakhsh sefidi et al.(31 ) showed that in 2003 , genotype 1a was the most common hcv genotype in iran ( 47.8% ) but it decreased over time and hcv genotype 3a was increased in this period of time ( 30.1% in 2003 which was increased up to 39.6% in 2011 . ( 32 ) also reported that while hcv subtype 1a is predominant among hcv infected iranian subjects , subtype 3a is predominant among iranian injecting drug users . our results are in accordance with a recent study from yazd city , another town in central province of iran which showed that hcv genotype 3a was the predominant genotype followed by the subtypes 1a and 1b ( 33 ) and it was also compatible with the results reported from another parts of iran ( 17 , 2528 ) . on the other hand , genotype 3a is significantly associated with hcv transmission through injection drug use ( 34 ) , which is the main route of hcv transmission in our participants that was observed in 59.8% of cases . in our study , svr was more observed in patients infected with genotype 3a versus those infected with genotype 1a and 1ab , which was in accordance to this issue that genotype 3a revealed a great svr among interferon - treated patients , compared to genotype 1(68 ) . this study had limitations like small sample size and lack of the correlation between hcv genotypes and the severity of the disease among the studied population . this study showed that hcv subtype 3a was the most prevalent hcv type , followed by subtype 1a and subtype 1ab in arak , central province of iran . determination of hcv genotype distribution could affect the hcv therapeutic methods and duration and could increase the chance of successful treatment . investigation of hcv genotypes in different parts of the country is needed to facilitate treatment options and preventive strategies .

background and objectives : hepatitis c virus ( hcv ) infection is a worldwide concern and it is the major cause of liver disease . several genotypes of the hcv have been reported from different regions of the world . the determination of the hcv genotypes is important for the prediction of response to antiviral treatment and clinical outcomes . so , hcv genotyping in each region is of great importance . this investigation was performed to determine the distribution of hcv genotypes in arak city , central province of iran.patients & methods : in this cross sectional study , 174 cases with chronic hcv infection from arak city were enrolled . hcv infection was confirmed by positive results in hcv antibody ( anti - hcv ) and hcv - rna tests . hcv genotypes were determined using a pcr based genotyping kit.results:a total of 174 hcv infected patients with mean age of 37.510.24 years were enrolled . 97.7% of cases were male and 2.3% were female . the main route of hcv transmission was injection drug use ( idu ) which was observed in 59.8% of cases . genotyping results demonstrated that subtype 3a ( 52.9% ) was the most prevalent hcv type in arak , followed by subtype 1a ( 22.9% ) and subtype 1ab ( 17.8%).conclusion : this study showed that hcv subtype 3a was the most prevalent hcv type , followed by subtype 1a and subtype 1ab in arak , central province of iran . investigation of hcv genotypes in different parts of the country is needed to facilitate treatment options and preventive strategies .

small , autonomously folding peptides that display -sheet secondary structure in the absence of a specific tertiary context are useful tools for exploring intrinsic relationships between sequence and -sheet stability and for establishing characteristic spectroscopic signatures of this motif . in addition , autonomously folding systems have been valuable for probing noncovalent interactions involving post - translationally introduced units . engineered -sheets have been used to explore preferred modes of intermolecular -strand associations , phenomena that underlie amyloid formation ; such -sheet designs may ultimately lead to diagnostic or therapeutic tools for amyloid diseases . design of -sheet - forming peptides is challenging because the natural residues that promote this secondary structure are hydrophobic ( ile , val , phe , trp , tyr ) or only moderately polar ( thr ) . therefore , sequences intended to adopt -sheet conformations but not self - associate in aqueous solution must be rich both in -promoting residues and in solubility - promoting residues , the latter usually bearing charged side chains . the necessity of using distinct sets of residues for conformational propensity and solubility represents a significant design constraint . here we explore the hypothesis that -sheet propensity and peripheral charge can be combined in a single -amino acid residue . side chain branching adjacent to the backbone ( -branching ) , as in ile , val and thr , is correlated with high -sheet propensity ; therefore , we have synthesized and evaluated new amino acids that contain both a -branch point and an ionizable group in the side chain ( figure 1 ) . our initial design hypothesis focused on derivatives of threonine in which the side chain hydroxyl is used to form an ether linkage to a unit bearing an ionizable group . scheme 1 shows the synthesis of protected amino acid 3 from aziridine 1 , which was generated from l - threonine via well - precedented methods . have shown that nucleophiles can open closely related thr - derived aziridines in the presence of bf3 with high stereoselectivity , and this approach was useful in our case . the residue derived from 3 , which we designate to , was readily incorporated into synthetic peptides via fmoc - based solid - phase synthesis . to can be viewed as an analogue of lys that contains a -branch point in the side chain . we used 12-mer peptide i as a benchmark for evaluating the -sheet propensity of to and other new residues described below ( figure 1 ) . the conformational behavior of i was previously characterized via nmr . in aqueous solution this peptide adopts an antiparallel two - stranded -sheet conformation ( -hairpin ) with a reverse turn at the dpro - gly segment . d - proline was employed to promote a type ii reverse turn , which favors -sheet interactions between flanking strands composed of l - residues . nmr data indicate 68% population of the -hairpin conformation at 4 c in aqueous buffer ( ph 3.8 ) . peptide ii is the analogue of i in which lys-9 has been replaced with to . 2d nmr analysis suggested significant population of the -hairpin conformation at 4 c in aqueous buffer ( ph 3.8 ) . the noes observed for ii are consistent with adoption of the expected -hairpin conformation . for additional insight , we compared chemical shifts for protons ( ch ) of residues within i and ii . ch values are sensitive to secondary structure , with residues that participate in -helix displaying upfield shifts and residues that participate in -sheet showing downfield shifts relative to random coil values . figure 2a shows ch data , the deviation from the random coil chemical shift at each residue , for peptides i and ii . the lpro-6 diastereomers of i and ii were used to provide the random coil ch values because we have previously shown that replacing dpro with lpro abolishes -hairpin folding in i and comparable designs . for both i and ii , ch 0.2 ppm for the segments tyr-2 to val-5 and orn-8 to ile-10 , which is consistent with the expected locations of the two -strand segments . the consistent negative ch values for leu-11 presumably arise from the proximity of the tyr side chain to leu-11 ch in the -hairpin conformation . the near - zero values at dpro-6 and gly-7 are consistent with the expected turn , and the near - zero values at arg-1 and gln-12 are consistent with fraying at the termini . comparison of ch values for the ( a ) parent , ts , and to peptides and ( b ) parent and ts peptides . the ch values at or adjacent to the substitution can not be directly compared as the substitution changes the dynamic range . ch values at hydrogen bonding positions in the core of the peptide have been shown to most accurately reflect the population of the -hairpin . the ch comparison between i and ii suggests that the new to residue may have a slightly lower -sheet propensity than does lys . this surprising conclusion emerges because the absolute ch values for ii are smaller than those for i at most strand residues common to the two peptides . the unexpected behavior of to led us to re - evaluate our design hypothesis , which focused exclusively on -branching in the side chain . the side chain oxygen of thr can form an h - bond with a nearby backbone n h , which leads to thr backbone torsion angles ( and ) more compatible with the -helical than -sheet secondary structure . if the h - bond acceptor ability of the ether oxygen in to works against -sheet propensity , then we hypothesized that the desired conformational properties should be achieved by replacing this oxygen atom with sulfur , to generate ts . a protected -amino acid that could be used to incorporate ts residues was prepared as shown in scheme 1 ; the key step was use of trityl thiol to open the thr - derived aziridine . protected amino acid 5a allowed solid - phase synthesis of peptide iii , which contains ts in place of lys-9 of i. 2d nmr analysis of iii revealed interstrand noes consistent with significant population of the expected -hairpin conformation in aqueous buffer at 4 c . resonance overlap hindered identification of noes involving the tyr-2 side chain at this temperature , but at 10 c such noes could be detected ( figure 3 ) . cross - strand noes observed for peptide iii at 10 c in ph 3.8 aqueous buffer . ch data for iii ( figure 2a ) suggest that the extent of -hairpin formation is higher for this peptide than for ii , which contains the to residue , because the absolute value of ch is larger at each strand residue ( positions 25 and 811 ) for iii than for ii . moreover , the ch values are larger at most strand positions for iii relative to parent peptide i , which suggests that the ts residue stabilizes the -hairpin conformation relative to lys . we explored the scope of this design strategy by evaluating the residue ts , an analogue of ts that bears an acidic rather than a basic side chain along with a thioether - based -branch point . peptide iv , prepared using 5b , is an analogue of i in which glu-4 is replaced by ts . the noes observed at 4 c are consistent with the formation of the expected hairpin . ch data for iv ( figure 2b ) suggest that this peptide forms a more stable -hairpin than that formed by i. circular dichroism spectra of peptides i , iii , and iv ( phosphate buffer , ph = 7.0 ) are consistent with the formation of a hairpin and the stability trends observed by nmr . ch - based population analysis was undertaken in order to estimate the thermodynamic impact of replacing lys-9 with either to or ts or replacing glu-4 with ts . residues 3 , 5 , 8 , and 10 occupy hydrogen bonded positions within the -hairpin conformation adopted by i such peptides are presumed to equilibrate rapidly between -hairpin and unfolded states on the nmr time scale . the ch value measured at each indicator residue represents a populated - weighted average of the contributions from the fully unfolded state and the fully folded ( i.e. , -hairpin ) state . for each sequence , we use the lpro-6 diastereomer to estimate ch for each indicator residue in the fully unfolded state , and we use a macrocyclized analogue with dpro gly turns at both ends to estimate ch in the fully folded state . table 1 shows the -hairpin population deduced in this way at each of the four indicator positions in peptides i iv . the variation among the four values for each molecule is a measure of the intrinsic uncertainty associated with this quantification strategy , which involves independent analysis at four distinct sites within each peptide . despite this uncertainty , the data support the qualitative conclusion that replacing lys-9 of i with to ( ii ) has little effect on -hairpin population , while replacing lys-9 with ts ( iii ) or glu-4 with ts ( iv ) leads to enhanced -hairpin population . the g was calculated for each reporter position in the hairpin and then averaged . iv relative to parent i , and for iii relative to ii , based on the residue - specific population . this thermodynamic analysis shows that replacing lys with to , the basic thr derivative with an ether linkage at the branch point , fails to enhance -hairpin stability , which invalidates our original design hypothesis . in contrast , the analogous basic residue containing a thioether linkage at the branch point , ts , stabilizes the -hairpin conformation . although the gfold values for ii vs i and for iii vs i are not cleanly distinguished given the uncertainties in table 1 , direct comparison of iii vs ii shows unambiguously that ts enhances -hairpin stability relative to to . the magnitude of the -sheet stabilization provided by ts or ts relative to proteinogenic residues with unbranched side chains , lys and glu , respectively , is significant given that the natively folded state of a typical globular protein , containing a few hundred residues , is only 5 kcal / mol more stable than the unfolded state . it should be noted that differences between ch values ( bar heights ) from figure 2a and % folded values from table 1 are not directly comparable . each ch value in figure 2a is determined by the difference between the chemical shift ( ch ) for a given residue in one of three peptides ( i , ii or iii ) and the analogous chemical shift from a reference peptide that has l - proline in place of d - proline . the % folded values in table 1 are generated from an algebraic expression based on three chemical shifts , the two mentioned above and the ch value for the analogous cyclic peptide , which represents the fully folded state . since this last value can vary among macrocyclic peptides with different sequences , quantitative correlations between ch values ( figure 2a ) and % folded values ( table 1 ) may not be evident at specific residues , particularly at or adjacent to substitution positions . in addition , another factor may prevent such quantitative correlations : the relationship between changes in ch and changes in % folding is not linear . for these reasons , and to account for local factors that can influence ch values at particular residues , we draw conclusions based on measurements at multiple sequence positions . circular dichroism ( cd ) provides information on peptide folding that is intrinsically of lower structural resolution than the insights available from 2d nmr ; however , cd can be very useful for qualitative comparisons . cd data in the far - uv region ( 190250 nm ) arise largely from the backbone amide groups and therefore report on secondary structure . the far - uv signatures of peptides i , iii , and iv obtained in the buffer used for nmr studies , 100 mm acetate buffer , ph 3.8 , all manifest a minimum at 215 nm ( figure 5 ) , which is characteristic of -sheet secondary structure and therefore consistent with nmr data for these peptides . cd data for peptides ( a ) i , ( b ) iii , and ( c ) iv in phosphate buffer ( ph 7.0 ) or acetate buffer ( ph 3.8 ) . cd data for peptides ( d ) i , ( e ) iii , and ( f ) iv in phosphate buffer at 4 , 20 , and 37 c . for each peptide , the far - uv cd spectrum obtained in the nmr buffer is not significantly different from the spectrum obtained in 100 mm phosphate buffer , ph 7.0 ( figure 5a c ) , which indicates that -hairpin folding is not ph - dependent over this range . this observation is important because the glu or ts side chain carboxyl group is likely to be largely protonated at ph 3.8 , but fully deprotonated at ph 7.0 . varying temperature from 4 to 20 to 37 c does not significantly alter the far uv cd of i , iii or iv ( figure 5d f ) , which suggests that conclusions drawn from nmr analysis at low temperature are relevant to room or physiological temperatures . we turned to a different -hairpin system to explore the generality of the behavior observed in derivatives of peptide i for new residues to , ts and ts ( figure 6 ) . the design of peptide v was based on the sequence of a -hairpin that occurs at the n - terminus of ubiquitin ; a central dpro previously reported nmr data suggest that the expected -hairpin conformation is significantly populated in aqueous solution . we prepared analogues of v in which lys-15 is replaced with to ( vi ) or ts ( vii ) . the ch data indicate that replacing lys-15 with to does not lead to a significant change in -hairpin population , because the ch values for vi are within error of those for v at strand positions gln-2 to ser-7 ( figure 7 ) . in contrast , replacing lys-15 with ts results in consistently higher ch values among the n - terminal strand residues ( figure 7 ) . these conclusions are similar to those derived from comparisons among i - iii , which also differ at a single residue ( lys vs to vs ts ) . to evaluate ts in this -hairpin system , we evaluated viii , the derivative of v in which ile-3 has been replaced by glu . ch analysis indicates that this change causes a significant decline in -hairpin population , which may reflect two factors , the loss of -branching and the loss of a hydrophobic side chain . replacing glu-3 of viii with ts , to generate ix , leads to an increase in -hairpin population , as judged by ch data ; however , these data indicate that the ts residue in ix is not as effective as the ile residue of v in terms of stabilizing the -hairpin conformation . despite the apparently diminished -sheet propensity of the ts residue relative to the more hydrophobic ile residue , ts represents a useful design tool because one can match the charge provided by glu while enhancing the folding tendency . comparison of ch values for the parent , ts , and to peptides . the ch values at or adjacent to the substitution can not be directly compared as the substitution changes the dynamic range . the ch values at hydrogen bonding positions in the core of the peptide have been shown to most accurately reflect the population of the -hairpin . our design strategy is based on the assumption that new amino acid residues with a side chain that contains both a -branch point and an ionizable group will be more hydrophilic than the proteinogenic -branched residues ( thr , ile and val ) . to test this hypothesis , we evaluated the hydrophilicities of ts and ts along with selected proteinogenic residues , lys , glu , gly , thr and ile , using a previously described system . distribution coefficients are determined ( at equilibrium ) between equal volumes of octanol and aqueous buffer ( 100 mm phosphate , ph 7.0 ) . the parameter of comparison , , is normalized : the logarithm of the distribution coefficient of glycine , log(dglycine ) , is subtracted from the logarithm of distribution coefficient of the amino acid under consideration , log(damino acid ) to calculate for that amino acid . table 2 shows values measured for ts and ts and the selected proteinogenic residue . as we predicted , ts and ts cluster with lys and glu , all significantly preferring aqueous buffer relative to octanol . it is interesting to note that ts and glu are very similar on this scale , while ts is somewhat less hydrophilic than lys . in contrast , ile significantly prefers octanol to aqueous buffer , while thr is similar to gly . the data clearly show that ts and ts are significantly more hydrophilic that thr or ile . we have identified a new family of unnatural -amino acid residues featuring two properties , intrinsic conformational propensity and side chain charge , that have proven to be valuable for design of peptides that fold autonomously to -sheet secondary structure in aqueous solution . these two properties are not paired in any proteinogenic amino acid residue , which has made it challenging to design small peptides that adopt -sheet conformations but do not aggregate . high -sheet propensity is generally associated with -branching in a residue s side chain . our initial attempt to build charge and side chain branching into a new residue , involving an ether linkage at the branch point , was not successful . thioether - based branch points , on the other hand , lead to the desired properties . we have illustrated this approach with two new residues , ts and ts , which feature basic and acidic side chains , respectively . the versatility of the synthetic route will enable preparation of many related thioether - containing -amino acids .

proteinogenic amino acid residues that promote -sheet secondary structure are hydrophobic ( e.g. , ile or val ) or only moderately polar ( e.g. , thr ) . the design of peptides intended to display -sheet secondary structure in water typically requires one set of residues to ensure conformational stability and an orthogonal set , with charged side chains , to ensure aqueous solubility and discourage self - association . here we describe new amino acids that manifest substantial -sheet propensity , by virtue of -branching , and also bear an ionizable group in the side chain .

glucose meters have been used in the hospital setting for decades . traditionally glucose meters were used in the hospital to dose subcutaneous insulin for patients with diabetes when they were hospitalized . as even well - controlled diabetic patients will have their insulin needs , diet and caloric requirements change during periods of acute illness ; glucose must be measured frequently ( four or more times per day ) before meals and/or insulin dosing in the hospital . although most hospital laboratories offer a measurement of serum or plasma glucose , hospitals and healthcare systems find it both convenient and efficient to measure capillary whole blood glucose at the bedside in order to expedite insulin dosing . this can help insure that glucose values are taken before ( rather than after ) meals are consumed , as it is the pre - prandial blood sugar value that is most often used to dose insulin . in 2001 van den berghe and colleagues changed the landscape of glucose control in the hospital by studying the impact of tight glycemic control ( maintaining blood glucose between 80 - 110 mg / dl ) among critically ill patients ( both diabetic and non - diabetic ) after cardiovascular surgery . van den berghe s original study sought to determine whether closely controlling glucose levels in patients in a surgical intensive care unit ( icu ) would improve patient outcome . in the study 1500 patients were divided into two groups : one control group that received what was conventional treatment of hyperglycemia in the icu at that time ( subcutaneous or intravenous insulin to keep glucose levels less than 200 mg / dl ) , and an experimental group that received intravenous insulin to keep blood glucose at relatively normal levels of 80 - 110 mg / dl the experimental group that received intravenous insulin to keep blood glucose relatively normal had much better health outcomes than the control group ( mortality decreased 34% , renal failure 41% , bloodstream infections 46% ) . the outcomes were startling to critical care experts , and almost overnight changed the standard of care in critical care medicine from a relaxed attitude towards hyperglycemia in the icu to vigilant glucose monitoring and insulin treatment to maintain normal or near - normal blood glucose levels . subsequent studies found that depending upon the patient population ( medical vs. surgical icu ) , icu nutrition practices , and protocols to dose insulin and monitor glucose ; intensive glycemic control was of either benefit in only some icu patients or not beneficial at all . finally , in 2011 a multi - center trial called nice - sugar was performed to determine what level of glycemic control was optimal in the icu setting . unlike the preliminary studies done by dr . van den berghe , nice - sugar did not compare conventional treatment to more rigorous management of glycemic control ; as by that time some active management of glucose levels in the icu was standard of care . rather , nice - sugar compared two different glucose management strategies one aimed at controlling glucose levels among critically ill patients to near - normal levels ( similar to the van den berghe strategy ) and one that aimed for slightly higher ( 140 - 180 mg / dl ) glucose levels . nice - sugar , performed in over 40 medical centers , found that patients assigned to the higher ( < 180 mg / dl ) glucose target had significantly better health outcomes than those whose glucose target was near - normal ( 81 - 108 mg / dl ) . among the reasons why more moderate glucose targets may be beneficial to critically ill patients , all studies of intensive glucose control in the icu , including the original studies by dr . van den berghe , found that rates of hypoglycemia are higher among patients whose glucose levels are controlled actively with intravenous insulin . in fact , studies have shown that intravenous insulin therapy increases the rate of hypoglycemia among icu patients on average 5-fold . this is significant because even a single episode of hypoglycemia in the icu may increase the odds of death in the hospital up to two - fold . thus the need to control glucose levels in the icu must be balanced against the risk of hypoglycemia . while the original study ( showing the most positive outcomes ) by dr . van den berghe and colleagues used more accurate blood gas analyzers for all glucose measurements ; the subsequent studies often used less accurate glucose meters for measurement of blood glucose . this has fueled considerable controversy over whether glucose meters , originally intended for use in diabetic patients to monitor glucose and dose subcutaneous insulin , are accurate enough to manage intravenous insulin in critically ill hospitalized patients . traditionally , accuracy requirements for glucose meters were developed based upon the level of accuracy needed for safe and effective subcutaneous insulin dosing in the routine care of diabetes . these specifications are often visually displayed in an error grid , a tool developed by collecting the opinions of endocrinologists and other healthcare providers about the implications of various amounts of glucose measurement error on the safety and efficacy of subcutaneous insulin dosing . these error grid observations were codified in a set of guidelines issued by the international organization for standardization ( iso ) and clinical and laboratory standards institute ( clsi ) some years ago , and until recently used by some regulatory agencies as the measure of required glucose meter accuracy . one such commonly cited guideline , iso 15197 , required that 95% of glucose meter values fall within 15 mg / dl of the true or reference glucose value for serum glucose values < 75 mg / dl ; and 20% of the reference value for serum glucose values 75 mg / dl . because glucose meter use in the hospital has changed as glycemic control strategies have changed , most experts now feel that the original iso guideline is not appropriate as an accuracy guideline for hospital use glucose meters . to address these concerns , more stringent criteria for glucose meter accuracy have been proposed by both national academy of clinical biochemistry ( nacb ) and clsi . the guidelines are similar , and require 95% of glucose meter results to be within either 15 mg / dl ( nacb ) or 12 mg / dl ( clsi ) of reference glucose for glucose values < 100 mg / dl , and within 15% ( nacb ) or 12.5% ( clsi ) for glucose values 100 mg / dl ) several studies have documented that some glucose meters have limited accuracy when used on critically ill patients such as those on intravenous insulin in the icu . the degree to which glucose meters correlate with laboratory glucose measurement varies between glucose meter technologies ; and correlation in the hypoglycemic and hyperglycemic ranges is poor for some meters currently available . in addition , patients in the icu are on multiple medications , and often have abnormal hematocrit and/or oxygen tension , all of which may affect the performance of some glucose meters . finally , target glucose concentrations are narrower for this patient population than they are for patients using handheld meters to dose subcutaneous insulin , logically suggesting that improved accuracy of glucose measurement might be required . a number of studies have examined glucose meter accuracy and its impact on insulin dosing in the context of glycemic control , and concluded that glucose meters could not be safely and effectively used to manage critically ill patients on intravenous insulin in the icu . because studies examining glucose meter accuracy in the icu have been relatively small studies using different meters and reference methods , the larger question of the impact of glucose meter error on patient outcomes during glycemic control remains difficult to address . the primary manner this has been overcome is by utilizing simulation studies to model the effects of various levels of glucose meter error on insulin dosing decisions and glycemic control . boyd and bruns first established the use of simulation modeling as a tool to examine the relationship between glucose meter performance ( bias and precision ) and insulin dosing errors . the initial study was based upon glucose values and insulin doses used for conventional subcutaneous insulin dosing for diabetic patients . the authors used monte carlo simulation to relate glucose meter bias and imprecision to insulin dosing errors during conventional subcutaneous insulin dosing . they found that glucose meters available at that time had sufficient accuracy and precision to avoid large insulin dosing errors in the context of traditional subcutaneous insulin dosing regimens . another study , designed to specifically model glucose meter use during glycemic control in the icu , was based upon 29,920 observed glucose values among patients on intravenous insulin therapy in 2 icu units within one healthcare institution . as expected , most of the values were in a narrow range of glucose value ( 102 - 135 mg / dl ) , such that insulin dose would change with every 20 mg / dl glucose increment according to the insulin dosing protocol in use . the authors found that allowing 20% total error in glucose meter measurements ( previous iso 15197 criteria ) allowed for rare large ( 3 or more insulin dosing categories ) insulin dosing errors ; those that are most likely to produce hypoglycemia . decreasing allowable error to 15% eliminated large insulin dosing errors ; but still allowed for 2 - 5% of insulin dosing decisions to be in error by 2 insulin dosing categories . reducing error tolerance to 10% the authors concluded that 20% glucose measurement error was not safe and effective for intravenous insulin dosing protocols that sought to maintain glucose values at normal or near - normal concentrations ( tight glycemic control ) . after the publication of the nice - sugar study , many institutions changed the glucose target values for icu patients on intravenous insulin therapy to more moderate glucose values . to investigate whether glucose meter accuracy requirements for more moderate glycemic protocols differed from those suggested for tight glycemic control , the authors repeated the simulation studies using 25,948 observed glucose values in 1503 icu patients on a moderate glycemic control protocol ( 110 - 150 mg / dl target value ) . although the median glucose value was significantly higher among patients on moderate ( 134 mg / dl ) compared to tight ( 116 mg / dl ) glycemic control , most glucose values among patients on the moderate glycemic control protocol still fell into insulin dosing categories where insulin dose changed with every 20 mg / dl increment in glucose value . rates of insulin dosing errors as a function of meter bias and precision were nearly identical to those predicted for the population of patients on tight glycemic control . this suggests that the observed relationship between glucose meter and insulin dosing errors can be generalize to insulin infusion protocols where insulin dose changes with every 20 mg / dl change in glucose value . simulation models suggest that 20% error is too much for glucose meters used to manage patients on intravenous insulin therapy . because some studies of glucose meter accuracy in the icu observed that glucose meter error exceeded 20% when used on critically ill patients the simulation models have been used as evidence that glucose meters do not have the level of accuracy required for safe and effective management of critically ill patients placed on intravenous insulin ( glycemic control ) . only a small number of simulation studies have gone beyond relating glucose meter accuracy to insulin dosing errors ; and attempted to relate meter error to the short - term patient outcomes such as rates of hypoglycemia , rates of hyperglycemia , or glycemic variability ( rate and extent of change in glucose levels over time ) . one simulation model used a complex algorithm to predict the impact of glucose meter error over many days on rates of hypoglycemia , hyperglycemia and glycemic variability when glucose meter results were used to dose subcutaneous insulin in the context of diabetes self - management . the authors found that there was a threshold between 10 - 15% meter error that was predicted to result in increased incidences of hypoglycemia , hyperglycemia and increased glycemic variability . one additional study used simulation modeling to assess the impact of both glucose measurement frequency and precision on predicted rates of hypoglycemia in the context of glycemic control in the hospital . using hourly glucose monitoring to adjust insulin dose , the simulation model predicted that increasing imprecision above 10% cv would result in progressively increased rates of hypoglycemia ( glucose < 60 mg / dl ) . the same simulation models suggested that using hourly glucose monitoring rates of hyperglycemia ( > 160 mg / dl ) , time within intended target glucose range , and glycemic variability were all detrimentally affected when precision increased beyond 5 - 10% cv . these studies differed in the type of insulin dosing modeled ( subcutaneous vs. intravenous ) , glucose target ranges assumed , and frequency of glucose monitoring . however both raise concerns about the use of glucose meters to manage patients on intravenous insulin in the icu . both studies suggest a threshold effect of either glucose meter total erroror imprecision ; with a suggested minimum total error of 10 - 15% and imprecision of < 5% . because a number of previous studies demonstrated total error greater than 10 - 15% when glucose meters are used on icu patients , this has fueled concern about their use in this context . while studies of glucose meter use among critically ill patients have demonstrated both systematic differences ( generally positive bias ) and variability between glucose meter and laboratory glucose values , a few studies have concluded that the use of glucose meters during glycemic control may be appropriate . one study used parke s error grid analysis to assess the clinical impact of glucose meter errors when arterial , venous or capillary samples were used to dose glucose meters . these authors concluded that glucose meters may be appropriate for use in glycemic control protocols when arterial or venous ( but not capillary ) samples are used . however it is not clear whether use of the parke s error grid is appropriate for assessing the clinical impact of glucose meter errors in the context of intravenous insulin therapy during icu glycemic control protocols . another study also examined differences between glucose meter and laboratory glucose when either arterial , venous or capillary samples from critically ill patients were used . this study examined the number and magnitude of insulin dosing errors when glucose meter ( compared to laboratory glucose ) results were used to make insulin dosing decisions using the institutional glycemic control protocol ( target glucose 80 - 110 mg / dl ) . this study found that errors in the measurement of both venous catheter and capillary glucose resulted in more frequent large ( 2 or more insulin dosing categories ) dosing errors ; whereas use of arterial catheter whole blood on the glucose meter resulted in predominantly one category dosing errors . finally one study used consensus error grid and bland altman analysis to study whole blood glucose accuracy using several different devices ; and found that by limiting sample type to arterial blood that some glucose meters were accurate enough to be used during glycemic control in assessing the appropriateness of glucose meter use in the icu , choice of sample type is an essential consideration . a number of studies have demonstrated that capillary glucose can be highly inaccurate in patients in shock , or patients with edema or poor tissue perfusion several studies have also demonstrated systematic overestimation of glucose values when venous catheters are used to obtain venous whole blood for analysis on some glucose meter technologies . arterial whole blood is very likely the best sample choice for monitoring whole blood glucose in critically ill patients . in considering the evidence for and against use of glucose meters in the icu , one should pay special attention to sample source as a potential cause for poor glucose meter performance . other investigators have studied whether other factors may be more important than glucose monitor accuracy in determining the effectiveness of a glycemic control protocol . one study compared use of a standardized insulin infusion protocol to physician - directed intravenous insulin dosing in a mixed medical / surgical icu . use of the standardized infusion protocol reduced the rate of hypoglycemia from 16% to 4% , and also reduced the frequency of dextrose rescue . patients using the standardized protocol reached target glucose faster and maintained blood glucose in the target range ( 81 - 110 mg / dl ) longer . glucose in this study was monitored using capillary samples on a glucose meter , perhaps the least desirable sample for critically ill patients . even with this limitation , the study demonstrated that execution of a standardized infusion protocol can improve at least short - term outcomes ( hypoglycemia , time in therapeutic range ) . another study demonstrated that by using an insulin infusion protocol that focused on velocity of glucose change ( rather than absolute glucose levels ) , glucose meters could be used to maintain blood glucose in the range of 100 - 139 mg / dl with very little ( 0.3% of all glucose values < 60 mg / dl ) hypoglycemia . another investigator has described a collaborative approach to establishing both glucose target ranges and insulin infusion algorithms based upon practice and nursing leader opinions about what could be safely accomplished . using this approach they implemented an initial glycemic control protocol to keep glucose levels among critically ill patients below 140 mg / dl they used hourly capillary glucose meter and/or laboratory serum / plasma glucose for all patients on intravenous insulin and observed a rate of severe hypoglycemia ( glucose < 40 mg / dl ) of 0.38% . when staff in the icu was comfortable with the under 140 protocol , the target glucose range was decreased to 80 - 125 mg / dl with only a modest increase in severe hypoglycemia ( 0.92% ) . the authors concluded that by taking an incremental approach to glycemic control , starting with a higher target range and lowering the range only after staff demonstrated they could reliably execute the protocol , safe and effective glycemic control was possible using glucose meters for some monitoring . a more common approach to improving outcomes during glycemic control is to use information technology solutions to computerize insulin doses based upon trended ( rather than individual ) glucose values . this approach mitigates the risk of hypoglycemia from a single aberrant glucose meter value . using this approach one study demonstrated that rates of severe hypoglycemia were 4.25% when mostly capillary whole blood glucose meter values were used to dose insulin among 4588 critically ill patients managed on a glycemic control protocol with an 81 - 110 mg / dl target range . these authors went on to investigate causes of hypoglycemia among all incidents where glucose fell below 40 mg / dl the authors found that ~ 70% of hypoglycemic episodes could be attributed to delay in obtaining glucose measurement ; suggesting that human error ( rather than measurement error ) is responsible for the most insulin - induced hypoglycemia during traditional tight glycemic control protocols . the same authors compared the computerized infusion protocol to a paper - based protocol and found that using a computerized protocol improved the time in therapeutic range , mean blood glucose level , and percent of blood glucose measurements below 70 mg / dl .. finally a study over a one month period in three intensive care units at one institution found that using arterial whole blood to dose glucose meters , and relying upon consistent hourly glucose measurements performed by laboratory ( rather than nursing ) staff , rates of severe hypoglycemia were 1.4% despite a relatively low glucose target range of 80 - 130 mg / dl in addition , 86% of severe hypoglycemic episodes observed were due to protocol violations ( missed hourly glucose measurements or failure to change insulin infusion rate according to protocol instructions ) . when the glucose target range was changed to 110 - 150 mg / dl ( with no change in glucose meter used or measurement frequency ) , no episodes of hypoglycemia were observed in 211 patients over one month . a larger study ( three months , 1503 patients ) within the same icu units found a rate of severe hypoglycemia of 0.25 % . collectively these studies highlight several key points that must be considered before determining the appropriateness of glucose meters for managing glycemic control in the icu . the choice of sample type ( arterial whole blood preferred ) may be as or more important than the type of glucose monitor used for whole blood glucose measurement . glucose meters have been used in effective glycemic control protocols demonstrating both low rates of severe hypoglycemia and reliable glycemic control in the icu . elements of effective protocols are computerized ( rather than paper - based ) insulin dosing algorithms , collaboration and teamwork to determine the appropriate glucose target for a given hospital or icu population , and use of frequent ( often hourly ) arterial whole blood sampling for all patients on intravenous insulin . while many studies demonstrating poor performance of glucose meters in critically ill patients used older glucose meter technologies , newer technologies with improved accuracy have recently become available . some recent studies have demonstrated that newer glucose meter technologies can meet even the more stringent clsi poct12-a3 accuracy guidelines ( 12.5% for values above 100 mg / dl ) when used in the intensive care unit . meters that meet more stringent accuracy guidelines such as poct12-a3 would be performing within the 10 - 15% total error allowance predicted to minimize large insulin dosing errors in the context of icu glycemic control . with the improved performance of newer glucose meters , one might think that the issue of glucose meter accuracy in the icu was close to resolution . to add fuel to the ongoing controversy about glucose meter use in the icu , the food and drug administration ( fda ) released draft guidelines suggesting that improved accuracy was necessary for any future glucose monitors intended for hospital use . while the guidelines are still in draft form at the time of this review , fda draft guidance criteria suggested that 99% of glucose meter values should be within 10% of the reference or true glucose value . there is concern among some that tightening accuracy criteria to this level could impede the development of new meters and monitors , without improving the quality of care delivered in the icu during glycemic control . amidst this cloud of confusion and controversy surrounding glucose meter use in the icu , first and foremost , consider the entire glycemic control protocol in use within your institution , and the role that glucose meters play in the overall scheme of glycemic control . eliminating the use of glucose meters in support of intravenous insulin protocols , without first considering alternatives and implications , would almost certainly have an adverse effect on patient care . understand the effectiveness of the glycemic control protocol ( rates of hypo and hyperglycemia , time within intended glucose range ) as implemented , and the systematic issues that may be leading to adverse outcomes such as hypoglycemia . if the major issues are remembering to obtain glucose values in a timely manner to facilitate insulin dosing decisions , or communicating glucose results to providers in a timely manner , then changing glucose measurement devices ( especially away from the bedside ) would not be expected to improve outcome . if spurious glucose results have been observed in some icu patients , determine whether common interferences ( low hematocrit , some medications ) in the icu environment may be affecting the glucose meter technology in use . if user errors such as incorrect strip codes or under - dosing of strips are suspected ; consider switching to a glucose meter technology that reduces the likelihood of these errors and examining training and competency systems . hospitals and point of care programs should also consider the sample type ( capillary , arterial or venous whole blood ) routinely used for bedside glucose measurements , before making a decision to switch technologies or glucose measurement devices . if capillary sampling is being used as the predominant sample type , switching to arterial whole blood may improve measurement accuracy without requiring large changes in workflow or testing processes . finally , consider evaluating the accuracy of the device being used by comparing whole blood glucose meter values to laboratory serum or plasma glucose obtained from icu patients . if the vast majority of glucose meter values are not within 15% of lab glucose values , then it is likely that more accurate glucose measurements are both possible and desirable . several studies have documented that some glucose meters have limited accuracy when used on critically ill patients such as those on intravenous insulin in the icu . the degree to which glucose meters correlate with laboratory glucose measurement varies between glucose meter technologies ; and correlation in the hypoglycemic and hyperglycemic ranges is poor for some meters currently available . in addition , patients in the icu are on multiple medications , and often have abnormal hematocrit and/or oxygen tension , all of which may affect the performance of some glucose meters . finally , target glucose concentrations are narrower for this patient population than they are for patients using handheld meters to dose subcutaneous insulin , logically suggesting that improved accuracy of glucose measurement might be required . a number of studies have examined glucose meter accuracy and its impact on insulin dosing in the context of glycemic control , and concluded that glucose meters could not be safely and effectively used to manage critically ill patients on intravenous insulin in the icu . because studies examining glucose meter accuracy in the icu have been relatively small studies using different meters and reference methods , the larger question of the impact of glucose meter error on patient outcomes during glycemic control remains difficult to address . the primary manner this has been overcome is by utilizing simulation studies to model the effects of various levels of glucose meter error on insulin dosing decisions and glycemic control . boyd and bruns first established the use of simulation modeling as a tool to examine the relationship between glucose meter performance ( bias and precision ) and insulin dosing errors . the initial study was based upon glucose values and insulin doses used for conventional subcutaneous insulin dosing for diabetic patients . the authors used monte carlo simulation to relate glucose meter bias and imprecision to insulin dosing errors during conventional subcutaneous insulin dosing . they found that glucose meters available at that time had sufficient accuracy and precision to avoid large insulin dosing errors in the context of traditional subcutaneous insulin dosing regimens . another study , designed to specifically model glucose meter use during glycemic control in the icu , was based upon 29,920 observed glucose values among patients on intravenous insulin therapy in 2 icu units within one healthcare institution . as expected , most of the values were in a narrow range of glucose value ( 102 - 135 mg / dl ) , such that insulin dose would change with every 20 mg / dl glucose increment according to the insulin dosing protocol in use . the authors found that allowing 20% total error in glucose meter measurements ( previous iso 15197 criteria ) allowed for rare large ( 3 or more insulin dosing categories ) insulin dosing errors ; those that are most likely to produce hypoglycemia . decreasing allowable error to 15% eliminated large insulin dosing errors ; but still allowed for 2 - 5% of insulin dosing decisions to be in error by 2 insulin dosing categories . reducing error tolerance to 10% further reduced the rate of 2 category insulin dosing errors to less than 0.2% . the authors concluded that 20% glucose measurement error was not safe and effective for intravenous insulin dosing protocols that sought to maintain glucose values at normal or near - normal concentrations ( tight glycemic control ) . after the publication of the nice - sugar study , many institutions changed the glucose target values for icu patients on intravenous insulin therapy to more moderate glucose values . to investigate whether glucose meter accuracy requirements for more moderate glycemic protocols differed from those suggested for tight glycemic control , the authors repeated the simulation studies using 25,948 observed glucose values in 1503 icu patients on a moderate glycemic control protocol ( 110 - 150 mg / dl target value ) . although the median glucose value was significantly higher among patients on moderate ( 134 mg / dl ) compared to tight ( 116 mg / dl ) glycemic control , most glucose values among patients on the moderate glycemic control protocol still fell into insulin dosing categories where insulin dose changed with every 20 mg / dl increment in glucose value . rates of insulin dosing errors as a function of meter bias and precision were nearly identical to those predicted for the population of patients on tight glycemic control . this suggests that the observed relationship between glucose meter and insulin dosing errors can be generalize to insulin infusion protocols where insulin dose changes with every 20 mg / dl change in glucose value . simulation models suggest that 20% error is too much for glucose meters used to manage patients on intravenous insulin therapy . because some studies of glucose meter accuracy in the icu observed that glucose meter error exceeded 20% when used on critically ill patients the simulation models have been used as evidence that glucose meters do not have the level of accuracy required for safe and effective management of critically ill patients placed on intravenous insulin ( glycemic control ) . only a small number of simulation studies have gone beyond relating glucose meter accuracy to insulin dosing errors ; and attempted to relate meter error to the short - term patient outcomes such as rates of hypoglycemia , rates of hyperglycemia , or glycemic variability ( rate and extent of change in glucose levels over time ) . one simulation model used a complex algorithm to predict the impact of glucose meter error over many days on rates of hypoglycemia , hyperglycemia and glycemic variability when glucose meter results were used to dose subcutaneous insulin in the context of diabetes self - management . the authors found that there was a threshold between 10 - 15% meter error that was predicted to result in increased incidences of hypoglycemia , hyperglycemia and increased glycemic variability . one additional study used simulation modeling to assess the impact of both glucose measurement frequency and precision on predicted rates of hypoglycemia in the context of glycemic control in the hospital . using hourly glucose monitoring to adjust insulin dose , the simulation model predicted that increasing imprecision above 10% cv would result in progressively increased rates of hypoglycemia ( glucose < 60 mg / dl ) . the same simulation models suggested that using hourly glucose monitoring rates of hyperglycemia ( > 160 mg / dl ) , time within intended target glucose range , and glycemic variability were all detrimentally affected when precision increased beyond 5 - 10% cv . these studies differed in the type of insulin dosing modeled ( subcutaneous vs. intravenous ) , glucose target ranges assumed , and frequency of glucose monitoring . however both raise concerns about the use of glucose meters to manage patients on intravenous insulin in the icu . both studies suggest a threshold effect of either glucose meter total erroror imprecision ; with a suggested minimum total error of 10 - 15% and imprecision of < 5% . because a number of previous studies demonstrated total error greater than 10 - 15% when glucose meters are used on icu patients , this has fueled concern about their use in this context . while studies of glucose meter use among critically ill patients have demonstrated both systematic differences ( generally positive bias ) and variability between glucose meter and laboratory glucose values , a few studies have concluded that the use of glucose meters during glycemic control may be appropriate . one study used parke s error grid analysis to assess the clinical impact of glucose meter errors when arterial , venous or capillary samples were used to dose glucose meters . these authors concluded that glucose meters may be appropriate for use in glycemic control protocols when arterial or venous ( but not capillary ) samples are used . however it is not clear whether use of the parke s error grid is appropriate for assessing the clinical impact of glucose meter errors in the context of intravenous insulin therapy during icu glycemic control protocols . another study also examined differences between glucose meter and laboratory glucose when either arterial , venous or capillary samples from critically ill patients were used . this study examined the number and magnitude of insulin dosing errors when glucose meter ( compared to laboratory glucose ) results were used to make insulin dosing decisions using the institutional glycemic control protocol ( target glucose 80 - 110 mg / dl ) . this study found that errors in the measurement of both venous catheter and capillary glucose resulted in more frequent large ( 2 or more insulin dosing categories ) dosing errors ; whereas use of arterial catheter whole blood on the glucose meter resulted in predominantly one category dosing errors . finally one study used consensus error grid and bland altman analysis to study whole blood glucose accuracy using several different devices ; and found that by limiting sample type to arterial blood that some glucose meters were accurate enough to be used during glycemic control in assessing the appropriateness of glucose meter use in the icu , choice of sample type is an essential consideration . a number of studies have demonstrated that capillary glucose can be highly inaccurate in patients in shock , or patients with edema or poor tissue perfusion several studies have also demonstrated systematic overestimation of glucose values when venous catheters are used to obtain venous whole blood for analysis on some glucose meter technologies . arterial whole blood is very likely the best sample choice for monitoring whole blood glucose in critically ill patients . in considering the evidence for and against use of glucose meters in the icu , one should pay special attention to sample source as a potential cause for poor glucose meter performance . other investigators have studied whether other factors may be more important than glucose monitor accuracy in determining the effectiveness of a glycemic control protocol . one study compared use of a standardized insulin infusion protocol to physician - directed intravenous insulin dosing in a mixed medical / surgical icu . use of the standardized infusion protocol reduced the rate of hypoglycemia from 16% to 4% , and also reduced the frequency of dextrose rescue . patients using the standardized protocol reached target glucose faster and maintained blood glucose in the target range ( 81 - 110 mg / dl ) longer . glucose in this study was monitored using capillary samples on a glucose meter , perhaps the least desirable sample for critically ill patients . even with this limitation , the study demonstrated that execution of a standardized infusion protocol can improve at least short - term outcomes ( hypoglycemia , time in therapeutic range ) . another study demonstrated that by using an insulin infusion protocol that focused on velocity of glucose change ( rather than absolute glucose levels ) , glucose meters could be used to maintain blood glucose in the range of 100 - 139 mg / dl with very little ( 0.3% of all glucose values < 60 mg / dl ) hypoglycemia . another investigator has described a collaborative approach to establishing both glucose target ranges and insulin infusion algorithms based upon practice and nursing leader opinions about what could be safely accomplished . using this approach they implemented an initial glycemic control protocol to keep glucose levels among critically ill patients below 140 mg / dl they used hourly capillary glucose meter and/or laboratory serum / plasma glucose for all patients on intravenous insulin and observed a rate of severe hypoglycemia ( glucose < 40 mg / dl ) of 0.38% . when staff in the icu was comfortable with the under 140 protocol , the target glucose range was decreased to 80 - 125 mg / dl with only a modest increase in severe hypoglycemia ( 0.92% ) . the authors concluded that by taking an incremental approach to glycemic control , starting with a higher target range and lowering the range only after staff demonstrated they could reliably execute the protocol , safe and effective glycemic control was possible using glucose meters for some monitoring . a more common approach to improving outcomes during glycemic control is to use information technology solutions to computerize insulin doses based upon trended ( rather than individual ) glucose values . this approach mitigates the risk of hypoglycemia from a single aberrant glucose meter value . using this approach one study demonstrated that rates of severe hypoglycemia were 4.25% when mostly capillary whole blood glucose meter values were used to dose insulin among 4588 critically ill patients managed on a glycemic control protocol with an 81 - 110 mg / dl target range . these authors went on to investigate causes of hypoglycemia among all incidents where glucose fell below 40 mg / dl the authors found that ~ 70% of hypoglycemic episodes could be attributed to delay in obtaining glucose measurement ; suggesting that human error ( rather than measurement error ) is responsible for the most insulin - induced hypoglycemia during traditional tight glycemic control protocols . the same authors compared the computerized infusion protocol to a paper - based protocol and found that using a computerized protocol improved the time in therapeutic range , mean blood glucose level , and percent of blood glucose measurements below 70 mg / dl .. finally a study over a one month period in three intensive care units at one institution found that using arterial whole blood to dose glucose meters , and relying upon consistent hourly glucose measurements performed by laboratory ( rather than nursing ) staff , rates of severe hypoglycemia were 1.4% despite a relatively low glucose target range of 80 - 130 mg / dl in addition , 86% of severe hypoglycemic episodes observed were due to protocol violations ( missed hourly glucose measurements or failure to change insulin infusion rate according to protocol instructions ) . when the glucose target range was changed to 110 - 150 mg / dl ( with no change in glucose meter used or measurement frequency ) , no episodes of hypoglycemia were observed in 211 patients over one month . a larger study ( three months , 1503 patients ) within the same icu units found a rate of severe hypoglycemia of 0.25 % . collectively these studies highlight several key points that must be considered before determining the appropriateness of glucose meters for managing glycemic control in the icu . the choice of sample type ( arterial whole blood preferred ) may be as or more important than the type of glucose monitor used for whole blood glucose measurement . glucose meters have been used in effective glycemic control protocols demonstrating both low rates of severe hypoglycemia and reliable glycemic control in the icu . elements of effective protocols are computerized ( rather than paper - based ) insulin dosing algorithms , collaboration and teamwork to determine the appropriate glucose target for a given hospital or icu population , and use of frequent ( often hourly ) arterial whole blood sampling for all patients on intravenous insulin . while many studies demonstrating poor performance of glucose meters in critically ill patients used older glucose meter technologies , newer technologies with improved accuracy have recently become available . some recent studies have demonstrated that newer glucose meter technologies can meet even the more stringent clsi poct12-a3 accuracy guidelines ( 12.5% for values above 100 mg / dl ) when used in the intensive care unit . meters that meet more stringent accuracy guidelines such as poct12-a3 would be performing within the 10 - 15% total error allowance predicted to minimize large insulin dosing errors in the context of icu glycemic control . with the improved performance of newer glucose meters , one might think that the issue of glucose meter accuracy in the icu was close to resolution . to add fuel to the ongoing controversy about glucose meter use in the icu , the food and drug administration ( fda ) released draft guidelines suggesting that improved accuracy was necessary for any future glucose monitors intended for hospital use . while the guidelines are still in draft form at the time of this review , fda draft guidance criteria suggested that 99% of glucose meter values should be within 10% of the reference or true glucose value . there is concern among some that tightening accuracy criteria to this level could impede the development of new meters and monitors , without improving the quality of care delivered in the icu during glycemic control . amidst this cloud of confusion and controversy surrounding glucose meter use in the icu , what is the point of care program to do ? first and foremost , consider the entire glycemic control protocol in use within your institution , and the role that glucose meters play in the overall scheme of glycemic control . eliminating the use of glucose meters in support of intravenous insulin protocols , without first considering alternatives and implications , would almost certainly have an adverse effect on patient care . understand the effectiveness of the glycemic control protocol ( rates of hypo and hyperglycemia , time within intended glucose range ) as implemented , and the systematic issues that may be leading to adverse outcomes such as hypoglycemia . if the major issues are remembering to obtain glucose values in a timely manner to facilitate insulin dosing decisions , or communicating glucose results to providers in a timely manner , then changing glucose measurement devices ( especially away from the bedside ) would not be expected to improve outcome . if spurious glucose results have been observed in some icu patients , determine whether common interferences ( low hematocrit , some medications ) in the icu environment may be affecting the glucose meter technology in use . if user errors such as incorrect strip codes or under - dosing of strips are suspected ; consider switching to a glucose meter technology that reduces the likelihood of these errors and examining training and competency systems . hospitals and point of care programs should also consider the sample type ( capillary , arterial or venous whole blood ) routinely used for bedside glucose measurements , before making a decision to switch technologies or glucose measurement devices . if capillary sampling is being used as the predominant sample type , switching to arterial whole blood may improve measurement accuracy without requiring large changes in workflow or testing processes . finally , consider evaluating the accuracy of the device being used by comparing whole blood glucose meter values to laboratory serum or plasma glucose obtained from icu patients . if the vast majority of glucose meter values are not within 15% of lab glucose values , then it is likely that more accurate glucose measurements are both possible and desirable . simulation models have provided the best evidence available to relate glucose meter accuracy to insulin dosing errors during glycemic control in the icu . however they do not provide a way to measure the impact of glucose meter error on patient outcome . studies directly relating glucose monitor accuracy to glycemic control outcome ( mortality , infections , transfusions , etc ) or effectiveness ( hypoglycemia , hyperglycemia , time in therapeutic range ) are needed to understand the level of glucose meter accuracy required for management of critically ill patients on intravenous insulin therapy .

glucose meters are a fast and convenient way to measure circulating blood glucose . like many technologies in healthcare , the use of glucose meters within the hospital has evolved significantly over the last few decades . this change has been driven predominantly by changes in the approach to glycemic control for critically ill patients . both glycemic control in the intensive care unit ( icu ) , and use of glucose meters to manage insulin dosing during glycemic control , are likely to remain controversial topics in the years to come . this review will elaborate on the evidence for and against use of glucose meters in the icu to monitor glucose concentrations during glycemic control , and provide some tips for point of care programs on how to evaluate glucose monitors for this purpose .

some of the substances used for these dyes are either allergenic or have other side effects in some persons . 6 ) , the disodium salt of 3-hydroxy-4-[(4-methyl-2-sulfophenyl ) azo]-2-naphthalenecarboxylic acid , which is an anionic azo compound ( ci : 15850 ) , is one such substance . lithol rubine b is mutagenic because of intermediate reactions or by - products found in commercial samples . there are several methods for determining trace concentrations of lithol rubine b in cosmetic products . some use high - performance liquid chromatography ( hplc ) with ultraviolet visible ( uv / vis ) or mass spectrometry ( ms ) detection [ 26 ] , raman spectra , and voltammetry [ 8 , 9 ] . most methods for the quantitative detection and characterization of azo dyes use hplc analysis [ 1014 ] . uv visible spectrometry is a useful tool , but the azo dyes are largely affected by solvent influence , and require a photodiode array detector . detection using mass spectrometry is a more sensitive , but more expensive , approach and is not yet available for every laboratory . lc / ms has been recommended for analyzing disperse and low - molecular ( < 300 ) azo dyes , but it is not applicable to sulfonated azo dyes . when the mixture sample contains more than two azo dyes , the voltammetric waves of these dyes are seriously overlapped , which makes their quantification difficult without any preanalysis separation and purification . recent research on the electrodeposition of antimony ( sb ) focuses on ( a ) sb / sb2o3 particles for lithium - ion battery anodes ; ( b ) binary and ternary alloys for use in thermoelectric elements ; ( c ) the electrodeposition of sb on tin oxide ( sno2 ) electrodes , gold ( au ) electrodes , glassy carbon electrodes ( gces ) [ 19 , 20 ] , and mercury ( hg ) electrodes ; ( d ) one ternary film , ( antimony / tellurium ) ( sb2te3 ) , on a silver ( ag ) electrode . ag and metal - sb alloys [ 24 , 25 ] cause the reduction of electrocatalytic activity and oxidation in organic compounds . however , there are no published studies on using an electrocatalytic sensor with liquid chromatography - electrochemical detection ( lc - ec ) and flow - injection analysis ( fia ) determination of lithol rubine b in cosmetic products . directive 76/768/eec allows lithol rubine b to be used in cosmetic products as a coloring agent at a level of 0.22.0% . in the present study , we developed a more sensitive and low - cost method for detecting lithol rubine b in cosmetic products . we also report the development of a flow - through voltammetric sensor to catalyze the electroduction of lithol rubine b. the hplc system consisted of a hitachi model l-7110 pump with a rheodyne 7125 injection valve with a 20 l sample loop and was coupled with an eg&g parc 400 controlled potentiostat . the flow - through electrolysis cell was designed with the following electrodes : an ag / agcl/0.1 m kcl reference electrode ( bas ) , a platinum auxiliary electrode , and modified silver electrodes as working electrodes for detecting lithol rubine b azo dye . all solvents and the analyte were filtered through 0.45 m cellulose acetate and polyvinylidene fluoride ( pvdf ) syringe membrane filters , respectively . a chromatogram of lithol rubine b was acquired , and the peak height was calculated using an sisc chromatogram data integrator . lithol rubine b ( d&c red 6 ) pure dye content ( > 90% ) was purchased from unipure lc sensient cosmetic technologies / lcw ( milwaukee , wi , usa ) . a thin - film antimony electrode was produced using the following method . before the analysis , the silver wire electrode ( 4 cm long , 3 mm in diameter ) was mirror - polished sequentially with aqueous suspensions of 1.0 , 0.5 , and 0.05 m alumina . the electrode was then rinsed with deionized water and electrolytically plated with antimony ions ( 1.0 10 to 4.0 10 m ) from 10 ml of acetate buffer ( ph 4.5 ) . plating time was 8 min according to a potential scan of between 1.0 and 0 v ( versus ag / agcl ; at 10 mv / s ) . the antimony - modified silver electrode ( 0.3 mm diameter ) was constructed from a length ( ~8 cm ) of teflon tubing ( 1/32 in . , i.d . ; 1/16 in . , o.d . ) , inserted into one end of the teflon tube , and then sealed with acrylic resin ( struers ) . a small copper wire was placed at the other end of the teflon tube to allow an electrical connection to the antimony - modified silver wire electrode . the platinum wire , which served as a counter , and the ag / agcl wire , a reference electrode , the voltammetric detector , that is , the eluate , was fed to the antimony - modified silver wire electrode , which had been placed in an overflow vessel containing counter - electrodes and reference electrodes . the cosmetics ( rouge , lipstick , and nail polish ) contained oils ( mineral oil , petrolatum , and lanolin ) , ester ( isopropyl myristate ) , waxes ( paraffin and carnauba ) , and a powder base ( zinc oxide , titanium dioxide , inorganic pigments , and talc ) as the primary constituents of the base . the base was bound with the required color . raw samples received for analysis were converted into a form suitable for making useful measurements by pretreatment , separation , and preconcentration . taking into account the content of lithol rubine b azo dye in each sample , about 1.0 g of each sample was accurately weighed in a 50 ml beaker , diluted to about 10 ml with methanol , dissolved , and then stirred for 2 h. the mixture solution was concentrated , 9 ml of acetate buffer was added , and then the mixture was centrifuged at 6000 g for 30 min . the supernatant was transferred into a 10 ml calibrated flask ; acetate buffer was added to ensure that the flask contained 10 ml of liquid . the sample ( 1 ml ) was loaded onto a c18 cartridge and washed with 1 ml of ethyl acetate , and then the eluent was discarded . the resulting solution was extracted twice with methanol , and the organic layer was evaporated at 40c under nitrogen . the dried extract was reconstituted with 1 ml of methanol and filtered through 0.45 m membrane filters before hplc analysis . a stock standard sample solution was prepared by dissolving 10 mg of lithol rubine b in 10 ml of water , because lithol rubine b is a soluble sulfonated salt . working standard solutions in the range 0.051.2 mg / l were prepared from the stock standard solution . reversed - phase- ( rp- ) hplc was done on a phenomenex hyperclone c18 5 m ( 250 mm 4.6 mm ) column eluted with methanol - water ( 30 : 70 , v / v ) and acetonitrile - water ( 30 : 70 , v / v ) , respectively , containing 0.1 mm of k2hpo4 ( ph 4.08 ) as the mobile phase , at flow rates of 0.7 , 0.5 , and 0.3 ml / min . after the azo dye components in the cosmetics sample had been separated on the hyperclone c18 column , they were examined using an ultraviolet detector set at 235 nm . the electrochemical detector was operated at 0.6 v. a chromatograph was obtained with 20 l of the prepared sample solution and a standard solution under the operating conditions described above . azo linkages are reducible ; a typical two - electron , two - proton reduction was found for azobenzene . azobenzene undergoes a fast ecec reduction to hydrazobenzene which is then oxidized at approximately the same potential as azobenzene is reduced . azobenzene was reduced to the corresponding anilines using controlled potential coulometry since the initially formed intermediate hydrazobenzene decomposes to aniline . the proposed pathway for the electrochemical reduction of lithol rubine b is shown in scheme 2 . for both the gce and the sb / ag electrodes , good linearity was observed between the peak height ( current ) and the square root of the scan rate ( figure 1 ) . two different eluents , methanol - water ( 30 : 70 , v / v ) and acetonitrile - water ( 30 : 70 , v / v ) containing 0.1 mm of k2hpo4 ( ph 4.08 ) as the mobile phases of both solvent systems , were investigated at various flow rates . the retention times of acetonitrile - water were 9.8 , 5.3 , and 4.6 min for flow rates of 0.3 , 0.5 , and 0.7 ml / min , respectively . using methanol - water was inappropriate for determining lithol rubine b under our analytical conditions because the acetonitrile content in the mobile phase affects capacity factors and sensitivity . in most reversed - phase - hplc separations of highly unipolar colorants , broad , tailing peaks are obtained . a flow rate of 0.5 ml / min and acetonitrile - water were , therefore , used to determine lithol rubine b. the linear slope ( 5.3641 ) of the sb / ag electrode was higher than that ( 1.3568 ) of the gce ( figure 1 ) . lc - ecd chromatograms of lithol rubine b azo dye at various working electrodes ( a gce , a bare ag electrode , and an sb / ag electrode ) . figure 2 shows that the height of the reduction peak ( 0.3 g / ml ) of the sb / ag electrode was higher than those of the other electrodes ( gce : 1.0 g / ml ; bare ag : 0.3 g / ml ) . sb / ag film gave a better performance than the others did ; therefore , we used the sb / ag electrode to determine lithol rubine b levels in cosmetic products . to determine the optimum applied potential for electrochemical detection , after the lc analysis , hydrodynamic voltammograms were constructed for lithol rubine b ( figure 3 ) . the voltammetric detector was operated at 0.6 v. using the injection valve , 20 l of the prepared sample solution and of the standard solution were chromatographed under the operating conditions described above . the calibration curve showed good linearity over the range 0.01881.203 g / ml ; the regression equation was y = 430x + 150 , and the correlation coefficient was r = 0.9997 . the detection limit of our method was approximately 2.0 ng / ml . quantitation was based on the peak area of the sample . the operational stability of the bare ag and sb / ag sensors was tested and compared by continuously exposing them to the flow stream , and by monitoring the amperometric response ( at 0.6 v versus ag / agcl ) of acetonitrile - water ( 30 : 70 , v / v ) containing 0.1 mm k2hpo4 ( ph 3.97 ) over several hours of repetitive injections . scanning electron microscope ( sem ; at 15 kv ) images of 4 mm antimony ( sb ) particles distributed ( figure 4(a ) ) at the bare ag working electrode surface ( 0.3 mm i.d . ) as the working electrode in the flow cell after ( b ) 0 h and ( c ) 6 h. figure 4(c ) illustrates the stability of the sensor after 6 h of repetitive injections . the calibration plots obtained by plotting the peak area against the concentration of lithol rubine b show good linearity over the range 1080 mg / l . the regression equations were y = 1.17x + 0.387 ( correlation coefficient r = 0.9999 ) ; the range 0.301.2 mg / l and y = 202x + 10.0 ( correlation coefficient r = 0.9999 ) for lc - uv and lc - ed , respectively ( figures 5(a ) and 5(b ) ) . subsequently , we developed a simple and sensitive green electrochemical procedure for determining lithol rubine b in real samples . commercial lipstick was spiked with 0.1 , 0.2 , or 0.3 mg / l and then analyzed . the calibration plot ( figure 6 ) shows good linearity . because it is less expensive than lc - uv analysis , hplc should be done using a conventional variable wavelength detector to achieve some selectivity and close to the optimum sensitivity of all the colorants . thus , our proposed analytical method offers a valid and economical alternative to lc - uv detection of lithol rubine b. our proposed lc - ec method was used to determine lithol rubine b in cosmetic products . chromatograms of a comparison of lc - uv ( figure 7(a ) ) and lc - ec ( figure 7(b ) ) for lithol rubine b in cosmetics show that the sensitivity for the lithol rubine b investigated was about two orders of a magnitude higher with lc - ec than with lc - uv detection ; however , there were no significant differences in the values obtained using both types of analysis ( table 1 ) . we constructed an antimony - modified silver electrode to use as an electrocatalytic sensor liquid chromatography - electrochemical ( lc - ec ) detection and flow - injection analysis ( fia ) determination of lithol rubine b in cosmetics . the electrode not only exhibited catalytic activity toward this analyte , but also provided a stable , quantitatively reproducible performance in the chromatographic stream . thus , the proposed analytical method offers a valid , and economical , alternative to uv detection and mass spectrometry detection of lithol rubine b.

lithol rubine b ( lrb ; the disodium salt of 3-hydroxy-4-[(4-methyl-2-sulfophenyl ) azo]-2-naphthalenecarboxylic acid ) was detected using high - performance liquid chromatography with an electrochemical ( antimony film on silver ) detector ( hplc - ecd ) . for direct current ( dc ) mode , with the current at a constant potential , and measurements with suitable experimental parameters , a linear concentration from 0.125 to 1.80 g / ml was found . the detection limit of our method was approximately 2.0 ng / ml . an antimony - modified silver detector was used to demonstrate that lrb is electrochemically reduced in acidic media and to analyze commercial cosmetics to determine their lrb content . findings using hplc - ecd and hplc with an ultraviolet detector were comparable .

primitive neuroectodermal tumors ( pnets ) are highly malignant embryonal neoplasms of bone and soft tissues accounting for 4%17% of all pediatric soft tissue tumors . metastasis at presentation is quite common , with central pnets metastasizing to cranium and leptomeninges while peripheral pnets ( ppnets ) disseminating to lungs , bone , liver , and lymph nodes . primary pulmonary pnet is known to metastasize to pancreas , adrenal gland and ovaries while it metastasizing to brain is exceedingly rare . a 29-year - old female presented with a cough and right - sided chest pain of 1-month duration . computed tomography scan of chest [ figure 1 ] showed an 11.3 cm 11 cm 10 cm soft tissue mass lesion in right perihilar region with the loss of fat planes with esophagus , aorta , and diaphragm without chest wall or pleural involvement . biopsy showed atypical round to oval cells with scanty cytoplasm intermixed with respiratory epithelium [ figure 2 ] . immunohistochemistry ( ihc ) was positive for neuron - specific enolase ( nse ) [ figure 3 ] , synaptophysin , chromogranin , cd 99 , and vimentin . smooth muscle actin ( sma ) , epithelial membrane antigen ( ema ) , cytokeratin ( ck ) , leukocyte common antigen ( lca ) , cd 20 , cd 45 , thyroid transcription factor ( ttf-1 ) and desmin were negative , confirming the diagnosis of pnet lung . computed tomography scan ( axial section ) of thorax showing a large heterogeneously enhancing soft tissue mass lesion in the right perihilar region involving right lower lobe extending into the mediastinum with loss of fat planes with esophagus , aorta , and right crus of the diaphragm . there is no chest wall or pleural involvement biopsy from lung lesion showing small round cells with scanty cytoplasm with condensed chromatin and inconspicuous nucleoli ( h and e , 200 ) immunohistochemistry picture from lung lesion showing tumor cells positive for neuron specific enolase ( 200 ) magnetic resonance imaging ( mri ) brain and positron emission tomography scan showed localized disease . received chemotherapy vincristine , adriamycin , and cyclophosphamide alternating with ifosfamide plus etoposide ( vac / ie ) resulting in partial response ( pr ) to therapy . conformal radiotherapy ( rt ) was given to postchemotherapy residual volume to a dose of 60 ( gray ) gy in 30 fractions . post - rt patient experienced significant symptomatic relief with improved performance status though with a static disease . the patient was on follow up for 9 months when she presented with a headache , vomiting , seizures and left - sided hemiparesis . mri brain [ figure 4 ] showed an 8.6 cm 7.5 cm solitary lesion with areas of altered signal intensity at right periventricular and periatrial parietal lobe with significant perilesional edema suggestive of hemorrhagic metastatic deposit . she underwent right fronto - temporo - parietal craniotomy with excision of tumor and evacuation of hematoma . ihc was positive for synaptophysin [ figure 5a ] , chromogranin [ figure 5b ] , vimentin [ figure 5c ] , and cd 99 [ figure 5d ] with ki67 of 90% while negative for ck , ttf-1 , and melan - a which suggested metastasis from primary pnet lung . we treated her with whole brain rt ( wbrt ) to a dose of 30 gy in 10 fractions followed by oral pazopanib 800 mg once a day as a palliative intent . the patient is under follow up for over 1 year with a karnofsky performance status of 70% . magnetic resonance imaging brain ( a = coronal section and b = axial section ) showing a solitary lesion with areas of altered signal intensity at right periventricular and periatrial parietal lobe with significant perilesional edema suggestive of hemorrhagic metastatic deposit immunohistochemistry picture from brain metastasis showing tumor cells positive for ( a ) synaptophysin ( 100 ) , ( b ) chromogranin ( 200 ) , ( c ) vimentin ( 100 ) , ( d ) cd 99 ( 200 ) thoracic pnets are the most aggressive form of ppnets arising from chest wall or underlying lung pleura invading bone , lung or mediastinum in children and young adults . they are extremely aggressive with a dismal prognosis as patients may present with an upfront metastatic disease to contralateral lung , bone , bone marrow , lymph nodes , liver , pancreas , adrenals , and ovaries . primary lung pnet can often be misdiagnosed with a metastatic lung lesion , mucinous adenocarcinoma or squamous cell lung carcinoma . since pnets and other small round cell tumors share the same histological picture , ihc plays a vital role in their differentiation . cd 99 , vimentin and s100 are nonspecific while nse , synaptophysin and chromogranin confirm the diagnosis of pnet . ttf-1 is found in pulmonary adenocarcinomas and thyroid malignancies , ck for carcinoma , desmin for rhabdomyosarcoma , sma and ema for soft tissue sarcoma while lca , bcl-2 , cd 20 , cd 45 are for acute lymphoblastic lymphoma and leukemia . however , to confirm the diagnosis , amplification of ewing sarcoma breakpoint region-1 by situ hybridization techniques is required . according to javery et al . and previous other series on extraskeletal ewing sarcoma ( ees ) , lung is the most common site of metastasis followed by bone comprising 80% and 40% cases , respectively . huh et al . reported lymph nodes as the most frequent metastatic site ( 75.9% ) followed by bone , lung , peritoneum , pleura while least being brain ( 3.4% ) . the most prevalent primary sites were extremities , abdomen , pelvis , thorax , paravertebral space followed by head and neck current treatment recommendations for lung pnet and ppnets / ewing 's are same with vac / ie . rt concurrent with chemotherapy is preferred for the limited stage while sequential rt is beneficial for extensive lesions . we have treated two more cases of adult primary lung pnet although with a radiological pr but a significant symptomatic response . both patients are on follow - up for more than a year now without any evidence of disease progression or metastasis . for the management of brain metastasis from pnet lung , at present wbrt to a dose of 30 gy was exhibited as it is the standard of care in patients with brain metastasis which has shown superiority in preventing neurologic morbidity and mortality . pazopanib is an oral multikinase inhibitor of angiogenesis indicated mainly for progressive or chemotherapy refractory soft tissue sarcomas . although pazopanib is not the standard therapeutic option in ewing sarcoma , it has demonstrated its efficacy in refractory and ees metastatic cases . since ewing 's tumors and ppnets share similar genetic characteristics with reciprocal translocations of chromosomes 11 and 22 at ewsr1 , differing only in degree of neural differentiation with the former being more differentiated than latter , we , therefore , exhibited pazopanib to our patient with a palliative intent as she had progressed after standard chemotherapeutic regimen . the effectiveness of pazopanib by children 's oncology group ( cog ) nct01956669 phase-2 study and another multi - kinase inhibitor regorafenib by nct02048371 and nct02389244 phase-2 studies for refractory ewing sarcoma are underway , and the results are awaited . newer prognostic markers should be developed to identify primary pnet lung at risk of developing brain or distant metastases . a better understanding and interpretation of the molecular and biological mechanisms of this entity we recommend craniotomy and excision followed by wbrt for brain metastasis and oral pazopanib to check further dissemination as an incisive treatment guideline , though more prospective data favoring this recommendation is required .

primitive neuroectodermal tumors ( pnets ) are highly malignant neoplasms of embryonal origin manifesting in children and adolescents , rarely seen in adults . carcinoma lung with hemorrhagic metastasis to the brain is very common , but primary lung pnet with hemorrhagic brain metastasis is extremely uncommon . we hereby report a 29-year - old female diagnosed as pnet lung was treated with vincristine , adriamycin , and cyclophosphamide alternating with ifosfamide plus etoposide followed by radiotherapy ( rt ) . after 9 months , she developed hemorrhagic brain metastasis from pnet lung confirmed from tissue immunohistology postcraniotomy . received palliative whole brain rt followed by oral pazopanib resulting in significant improvement in performance status . a thorough review of literature reveals that our case may be the second case of primary lung pnet with hemorrhagic brain metastasis and also the first to be exhibited oral pazopanib resulting in a significant therapeutic effect to be reported in world literature till date .

pu - erh tea is a special tea species in china and has become one of the most popular beverages in southwestern china and southeast asian , owing to its special flavour properties and potential healthy benefits . it is originated from yunnan province ( china ) through a special post - fermentative process , using crude green tea prepared from the leaves of c. sinensis var . assamica as original materials . because pu - erh tea has a malty flavour and low - stimulation taste in tea infusions which may fit female appetite , recent years , interest in the flavor and the healthy properties and the related scientific investigations were increasing [ 3 , 4 ] . up to now , a little information on the relationship of the chemical composition to the flavor is available . volatile flavor component is one of the most important factors to influence the flavor , taste , and quality of pu - erh tea , in which the contents are different from the green and black tea because of different processing procedure and variety of species and cultivar . investigation on the components in green and black tea are reported elsewhere , yet few on those in pu - erh teas . in order to explore the influence of those components to flavor , taste , and quality , quantitative analysis of main volatile flavor components in pu - erh tea for analysis of volatile components or essential oils , several extraction techniques , including soxhlet extraction , liquid - liquid extraction ( lle ) , simutaneous distillation - solvent extraction ( sde ) , solid phase microextraction ( spme ) , and headspace microextraction ( hsme ) and so forth , had been used to diferent matices . soxhlet extraction is a classical method for decades in extraction of organic compounds from solid sample , and this apparatus has been developed to several types for special use . thorough extraction method because the organic phase cooled from condensation tube continuously passes through the target solid sample for hours . however , poor recovery commonly occurred for extraction of high - volatile or heat - labile compounds . lle is a conventional method for isolation of all boiling range volatile compounds , based on the compatibility of compounds with organic phase selected . the main disadvantage is solvent - consuming , tedious and , low - recovery for some target compounds . sde , proposed by godefroot and so forth , has been widely applied to analysis of volatile components in tea samples . although low recovery has been found for extracting the most volatile or heat - labile components , this technique has achieved higher recoveries and greater repeatability of volatile or semi - volatile and heat - stable components than other isolation technique such as spme or hsme when low water temperature in the circulating system was used . spme and hsme are relatively new techniques that are able to address the need for concentrating the components in the headspace [ 10 , 11 ] . both of them use a small piece of fused silica , on which a liquid or solid phase , similar to a gc stationary phase , has been coated to absorb the desired components and concentrate them on the fibre . thus the two techniques are more sensitive for the isolation of high volatile components than sde , while less sensitive and lower repeatability for the low volatiles than sde . conventionally , pu - erh tea is condensed to a pie - like tea - biscuit in the final processing procedure . extraction of the volatile flavor components will be different from the green and black tea . in order to select the best extraction technique for studying the volatile flavor components of pu - erh tea , a modified sde was evaluated for quantitative determination of the analytes using ethyl decylate as internal standard , and the two classical techniques , steam distillation - liquid / liquid extraction and soxhlet extraction , pu - erh tea samples were obtained from dayi limited incorporation ( menghai , yunnan , china ) . the sample was dried at 40c in electric oven for 6 h , ground to 3060 mesh , and sealed for use . the reference volatile chemicals were purchased from sigma ( st . louis , mo , usa ) . the stock solution was prepared by dissolving single solid / liquid standard in dichloromethane to an appropriate concentration depending on the content in pu - erh tea . ultra - pure water was obtained from a milli - q water purification system ( pall co , il , usa ) . the other solvents used in the test were all of analytical - grade and disdillated before use . dichloromethane and ethyl decylate were employed as solvent and internal standards , respectively . for each extraction , 15 g of tea sample , 10 g sodium sulphate , 100 l internal standard solution and 300 ml ultra - pure water were placed in a 1 l flask , 50 ml dichloromethane was in a 100 ml flask , and temperature of the circulating water system was operated at 8c . stream distillation was stopped after 2 h , while the solvent extraction was continued for a further 15 min . the extract was concentrated to 1 ml at 10c by a nitrogen - purge apparatus ( shanghai anpel scientific instrument co. ltd ) . the concentated solution was dehydrated with anhydrous sodium sulphate for at least 12 h , of which 2 l was injected to gc or gc - ms system for analysis . for sd - lle , 15 g of tea sample , 10 g sodium sulphate , 100 l internal standard solution and 500 ml ultra - pure water were placed in a 1 l distillation flask , respectively . the liquid was transferred to a 500 ml of separation funnel and then extracted three times ( 30 ml 3 ) using dichloromethane . the extracted organic phase was combined and concentrated to 1 ml at 30c by a nitrogen - purge apparatus after internal standard was added . 15 g of tea sample containing 100 l internal standard solution were placed in soxhlet 's apparatus and 50 ml of dichloromethane in an 150 ml distillation flask . at both ends of the sample in soxhlet 's apparatus , there are 2 cm - height of celite to help fix the sample . extraction was carried out at 50c for 2 hours , and extraction was concentrated to 1 ml at 30c by a nitrogen - purge apparatus after internal standard was added . the concentrated extracts were chromtographed by an hp 6890 series gc system ( agilent , usa ) . a 30 m 0.25 mm db-5 quartz capillary column ( supelco , bellefonte , pa , usa ) with 0.25 m film thickness was used to resolve the volatiles . temperature programming was as follows : initial oven temperature was set at 60c and kept for 3 min , then raised to 200c at a ramp of 4c / min and kept for 2 min ; to 210c at of 1c / min and kept for 2 min ; and finally , it was raised to 270c and kept for 7 min . nitrogen was used as carrier gas with column head pressure at 12.26 kpa in constant pressure mode . programming split / splitless injection temperature was set at 260c with split ratio of 10 : 1 and fid detector at 280c . auto system shimaszu qp 2010 gc - ms was employed for qualitative analysis to confirm the target components . the oven temperature was set at 50c and kept for 2 min , then raised to 60c at a ramp of 1c / min and kept for 2 min , to 200c at of 4c / min and kept for 2 min , and finally , to 270c at 10c / min and kept for 5 min . the mass spectrometry was operated at 200c in the electron impact mode ( 70 ev ) , scanning from m / z 40 to 600 in 0.3 s with an 0.2 s interval time of the scan ; the temperature of the gc - ms interface was 280c ; the voltage of the photoelectric multiplier tube ( pmt ) was 200 v. the mass spectral identifications of the target components were carried out by comparing to the nist 107 ( national institute of standards and technology , gaithersburg , usa ) mass spectral library as well as to wiley 6.0 ( wiley , new york , ny , usa ) mass spectral library . for gc separation of volatile components in tea samples , bp-20 sge column ( polar column , 30 m 0.25 mm i.d . film thickness 0.25 m ) was commonly used for quantitative analysis . in this work , film thickness 0.25 m ) was employed to separate the target components with temperature programming described above , and the chromatograms of standard compounds by gc - fid and gc - ms was shown in figures 1(a ) and 2(a ) . from the chromatograms , it can be seen each peak of the target compounds was baseline separated , and separating degree between two vicinity peaks was beyond 2 , confirming weak - polar capillary column can be used to separate the target components if gc separation condition was well optimized . according to gc - fid chromatogram , we calculated the relative factor of each of the standard compounds , it was shown in table 1 , and the relative factor was used to determination of the corresponding components in real pu - erh teas . figures 1(b ) and 2(b ) show the chromatograms of pu - erh raw tea that was obtained from gc - fid and gc - ms determination , in which the target peaks of real sample could be easily discerned and accurately quantified . the mass spectra of each target peaks in figure 2(b ) was checked by nist or wiley mass spectral library respectively , and campared with those in figure 2(a ) , confirming that each mass spectra of the components was the same as those of the corresponding standard compound , and no interference was appeared in the target peaks . comparing figures 1(b ) and 2(b ) , we found that both gc - fid and gc - ms separation methods can be applied to quantitative analysis of volatile flavor components in pu - erh teas . nevertheless , quantitative determination of the target components using gc - fid was more cost - saving than doing gc - ms , so the rest determinations for all of the samples were carried out in gc - fid . during sde operation , dichloromethane and diethylether are the most suitable extraction solvents for extraction of volatile components because of their weakly polar characteristics [ 15 , 17 ] . in this experimental , we select dichloromethane as extraction solvent for sde , due to its property of low combustion , and higher boiling point ( 40c ) than diethylether ( 35c ) thus liable to storage . zhu et al . had applied sde to extract volatile constitutes in green tea , confirming of 2 h was adequate to extract all the target compounds . initially , we operated sde apparatus as zhu et al . done , and found that there are about 6% of target components dwelling on the tea residue . this is because pu - erh tea was condensed to a pie - like shape , and the components were more difficult to release from tea matrix although the teas had been ground to 3060 mesh . then we modified this method by adding 10 g of sodium sulphate to the 1 l of steam flask containing 300 ml of water , and then operating sde device . after 2 h of sde and a further 15 min of the solvent extraction , the tea residue was re - extracted by sde , we found the area of the target peaks were less than 2% of the total . obviously , boiling point of water in steam flask was enhanced by adding sodium sulphate , accelerating the releasing velocity of target compounds . to confirm the repeatability , parallel the relative standard deviations ( rsd ) were in range of 1.44%12.6% , which were shown in table 3 . to check accuracy of sde for volatile flavor components , a known amount of standard solutions were added to aliquot of pu - erh tea . the adding level and corresponding recoveries were listed in table 3 . for quantitative extraction of volatile flavor components from complex matrices , common technique used is sde . because both liquid / liquid extraction and soxhlet extraction are classical techniques [ 8 , 10 ] , in this test , the modified classical techniques were compared with sde in extraction of the volatile flavor components from pu - erh tea in order to select the best one for sample preparation . the results were shown in table 2 . generally , sde was the best one among them , because the amounts of the components extracted were greater than those by employing sd - llc or soxhlet for the high - volatile components such as benzyl alcohol , linalool oxide , and linalool . this may attributes to the fact that sde was a closed and continuous extraction system , in which the target components can be furthermore , the temperature of the circulating water in sde was set at 8c , reducing the lose of the high - volatile components . as for extraction of the low - volatile components in pu - erh tea , such as 1,2,4-trimethoxylbenzene , sde is less poor compared with soxhlet extraction , probably due to the characteristics of high - boiling point and incompatibility with water steam . although sd - lle shares a same extraction principle with sde , the limitation of the technique is clear in table 2 . this is due to its open steam distillation system and following step - liquid - liquid extraction . during this procedure , not only high - volatile components such as benzyl alcohol , linalool oxide , and linalool and so forth had a chance to escape from the distillation system , but the analytes hardly transferred to organic phase . we could not confirm whether isomerization reaction was happened between the two compounds during extraction , and further investigation was beyond the work . table 2 shows that it can almost exhaustively extract all the 1,2,3- and 1,2,4-trimethoxylbenzene from pu - erh tea compared with sde , nevertheless it is very poor for benzyl alcohol , linalool oxide , linalool , phenethyl alcohol , and geraniol . these findings indicate soxhlet was suitable for high - boiling point ( low - volatile ) compounds such as essential oil . as for the trace high - volatile component , , we analyzed several tea samples , including four pu - erh ripe tea , four pu - erh raw tea , one green tea ( huangshan , anhui ) and one black tea ( qimen , anhui ) , and the contents of the target volatile components were shown in table 4 . it can be seen that the content of high volatile components , such as benzyl alcohol , linalool oxide , linalool in raw teas are higher than those in ripe teas , these findings are mainly due to the high temperature ( 4555c ) during the pile - fermentation process of ripe pu - erh teas , leading to the lose of these components . however , in raw teas , the content of 1,2,3- and 1,2,4-trimethoxylbenzene are much lower than those in ripe teas , this is because microbes play an important role in the synthesis of the two compounds during the pile - fermentation process of ripe pu - erh teas , which promote reaction procedure of methylation . generally , the contents of the volatile components in the green and the black teas are higher than those in pu - erh teas , except for 1,2,3- and 1,2,4-trimethoxylbenzene , which are not found in both of teas , probably due to lack of post - pile - fermentation process in manufacture , and linalool , lower than those in pu - erh raw teas due to different cultivar , variety of species and processing procedure . sde combined gc method was constructed for determination of volatile flavor components in pu - erh tea samples . 10 of volatile flavor components were quantitatively determined , and the recoveries and rsds were in the range of 66.4%109% and 1.44%12.6% , respectively . the method was compared with sd - lle and soxhlet extraction , comfirming sde was suitable for pu - erh teas among them . pu - erh raw tea and ripe tea samples were analyzed by the method , indicating the high - volatile components , such as benzyl alcohol , linalool oxide , and linalool , were rich in pu - erh raw teas , while the contents of 1,2,3- and 1,2,4-trimethoxylbenzene were much high in pu - erh ripe teas .

a simutaneous distillation extraction ( sde ) combined gc method was constructed for determination of volatile flavor components in pu - erh tea samples . dichloromethane and ethyl decylate was employed as organic phase in sde and internal standard in determination , respectively . weakly polar db-5 column was used to separate the volatile flavor components in gc , 10 of the components were quantitatively analyzed , and further confirmed by gc - ms . the recovery covered from 66.4%109% , and repeatability expressed as rsd was in range of 1.44%12.6% . sde was most suitable for the extraction of the anlytes by comparing with steam distillation - liquid / liquid extraction and soxhlet extraction . commercially available pu - erh tea samples , including pu - erh raw tea and ripe tea , were analyzed by the constructed method . the high - volatile components , such as benzyl alcohol , linalool oxide , and linalool , were greatly rich in pu - erh raw teas , while the contents of 1,2,3-trimethoxylbenzene and 1,2,4-trimethoxylbenzene were much high in pu - erh ripe teas .

we used data collected in the capital of chad , ndjamna , which in 2001 had a human population of 776,000 and a dog population of 23,600 ( 4 ) . dog rabies is endemic to chad ; before the vaccination campaigns , the prevalence of dog rabies was 1.41.7 cases/1,000 unvaccinated dogs ( 5,6 ) . we obtained direct observations of the association between compliance ( i.e. , percentage of dogs vaccinated ) and amount charged to owners from 2 pilot dog vaccination campaigns held in ndjamna in 2002 and 2006 ( 7 ; unpub . each campaign covered the same 3 city quarters , which had high - density dog populations ( 4 ) . only owned animals were vaccinated , but owned dogs comprise 90%99% of all dogs in ndjamna ( 7 ) . owners brought their animals ( dogs , cats , monkeys ) to 1 of 10 vaccination sites . in the 2002 campaign , vaccinations were free to owners ( 7 ) ; in the 2006 campaign , owners were charged 2,000 cfa francs ( us $ 3.88)/animal vaccinated ( unpub . ( exchange rate us $ 1 = 515.71 cfa francs as of february 2007 ; www.oanda.com/convert/classic ) for each campaign , the percentage of dogs vaccinated was estimated by using a capture recapture method ( 7 ; unpub . data ) . for the 2002 free - to - owners campaign , 71%87% ( 95% confidence interval [ ci ] 64%89% ; mean 79% ) of all dogs ( owned and unowned ) were vaccinated in 2 of the zones ( 1 zone per quarter ) included in the campaign ( 7 ) . for the 2006 campaign , in which owners were charged , the mean vaccination coverage among all dogs was estimated at 24% ( 95% ci 0.13%24.82% ) ( unpub . vaccination rates for owned dogs averaged only 78% and 25% in the 2002 and 2006 campaigns , respectively ( 7 ; unpub . , we used the latter estimates because we were interested in measuring owner compliance to charges for dog vaccination . additional observational data were obtained from a household survey conducted in 2001 ( 4 ) , which recorded that 19% of owned dogs were vaccinated against rabies . such vaccinations the charge for such vaccinations at the urban government - run veterinary clinic and the 3 private veterinary practices of ndjamna was 3,0005,000 cfa francs ( us $ 5.82$9.69 ) . we did not inflate the 2001 charges because we encountered problems identifying an appropriate conversion factor that considered veterinary medical services . during the 2006 campaign we surveyed dog owners by using a short questionnaire ( technical appendix ) . the survey was conducted in the vaccination zones ; households ( containing at least 1 animal ) were chosen randomly . the questions ( written in french ) were translated , when needed , into local languages by 4 interviewers . one question asked owners how much they were willing to pay for the vaccination of their animals . we graphed the 3 observational data points ( assuming a straight - line interpolation between points ) and the reverse cumulative probability of the owner - stated amounts that they would be willing to pay for their animal to be vaccinated against rabies ( figure ) . an initial statistical ( regression ) analysis of the relationship between the amounts that owners said they would pay and the variables collected during the survey ( table ) provided an adjusted r value of 0.07 ( data not shown ) . average probability of having a dog vaccinated against rabies by charge for vaccination : observed versus owner - stated values for vaccination . the observed values of charges to vaccinate an owned dog against rabies and probability of vaccination came from 3 sources . points a and b ( recording vaccination coverage for all owned dogs vs. costs charged ) come from 2 vaccination campaigns held in ndjamna in 2002 and 2006 , respectively . point c represents the midpoint of the range of recorded 2001 clinic charges in ndjamna for vaccinating a dog against rabies ( costs not adjusted for any potential inflation ) . the owner - stated amounts that they would be willing to pay for their dogs to be vaccinated against rabies came from a survey of 356 households , conducted in 2006 . interviewed households provided 356 questionnaires from which we estimated owner - stated willingness - to - pay for pet vaccination and calculated the resultant reverse cumulative probability of having their animal vaccinated . when asked how much they would be willing to pay , 5 ( 1% ) owners stated that they were against vaccination . we interpreted that response to indicate that such owners would , essentially , have to be paid to have their animals vaccinated . when the proposed cost of vaccination was < 1,500 cfa francs / animal vaccinated , owners were more likely to state that they would pay to have their pet vaccinated than they were to actually do it . the stated values and observed values were closest at 2,000 cfa francs ( 25% probability of animal being vaccinated ) ( figure ) . this finding was probably because the questionnaire was administered immediately after the campaign in which owners were charged 2,000 cfa francs / animal vaccinated . for > 2,000 cfa francs , the observed values indicated that compliance would be greater than that stated by owners responses to the willingness - to - pay question . the figure shows that to achieve a minimum of 70% of owned animals vaccinated , the maximum amount that could be charged would be 400 cfa francs ( us $ 0.78 ) ( observed values ) to 700 cfa francs ( us $ 1.36 ) ( owner - stated values ) . because the data shown in the figure reflect owned animals only , to get vaccination coverage up to 70% of all animals ( owned and stray ) , vaccination rates among owned animals would have to be > 70% . to attain these higher rates few studies have compared what members of the general public state they are willing to pay for a public health intervention with their actual observed behavior ( 8) . direct comparison between stated and observed behavior , as influenced by charges to owner , provides public health officials with an understanding of the reliability of owner surveys . furthermore , to maintain dog vaccination rates at the who - recommended rate of 70% , dog vaccination campaigns would have to be held every 16 years , which could reduce compliance . the survey was , by design , short , but a longer questionnaire may have allowed us to better identify why 75% of respondents did not wish to pay > 500 cfa francs ( us $ 0.97 ) . to achieve the who - recommended goal , public health officials can not charge owners more than the equivalent of 400 cfa francs ( us $ 0.78 ) . full - cost recovery concepts will not ensure that enough dogs are vaccinated in chad ( or , most likely , other african countries ) to interrupt rabies transmission in dogs in urban areas . clearly , to have > 70% of all dogs vaccinated , public health officials and policy makers must consider methods to substantially subsidize dog rabies vaccinations .

we estimated the association between amount charged and probability that dog owners in ndjamna , chad , would have their dogs vaccinated against rabies . owners would pay 400700 cfa francs ( us $ 0.78$1.36)/animal . to vaccinate > 70% of dogs , and thus interrupt rabies transmission , health officials should substantially subsidize these vaccinations .