Patent Application: US-36964009-A

Abstract:
a method is provided for diagnosing appendicitis in a patient that includes identifying at least one symptom of appendicitis in the patient and identifying the presence of at least one molecule differentially associated with appendicitis in a fluid or tissue sample of said patient . mrp - 8 / 14 and haptoglobin are examples of molecules differentially associated with appendicitis . devices and kits for performing the appendicitis assays of this invention are also provided . in one embodiment , the device is in a flow - through immunoassay format for testing blood samples . further , methods for screening for molecules differentially associated with appendicitis are provided that include the use of samples from patients being operated on for suspected appendicitis .

Description:
the vermiform appendix is recognized as a separate organ from the large and small intestines . it extends as a finger - like pouch from the base of the ascending colon , which is also called the cecum . the appendix , like the large intestine , is hollow and composed of the same three tissue layers . these three layers are a mucosa , muscularis and a serosa . the appendiceal lumen communicates with the lumen of the cecum via a round opening ( os ) through which the appendix adds its secretions to the fecal stream . these secretions are excess mucus produced from the appendiceal mucosa . in addition to containing mucus , the appendix also contains numerous bacteria common to the right colon . obstruction of the appendiceal lumen is the dominant factor causing acute appendicitis . while fecaliths are the usual cause of appendiceal obstruction , hypertrophied lymphoid tissue , inspissated barium from previous x - ray studies , vegetable and fruit seeds , and intestinal worms like ascarids can also block the appendiceal lumen . following luminal obstruction an escalating cycle of events ensues . the proximal obstruction of the appendix produces a closed - loop obstruction that blocks the normal flow of appendiceal mucus into the cecum . the continuing normal secretion of the appendiceal mucus very rapidly fills the luminal capacity of the appendix ( approximately 0 . 1 cc ). once the luminal capacity of the appendix is reached additional mucus production from the obstructed appendix rapidly elevates the intraluminal pressure within the organ . this elevated intraluminal pressure is exerted outward against the appendiceal wall and causes the appendix to distend . distention stimulates nerve endings of the visceral afferent pain fibers , producing vague , dull , diffuse pain in the midabdomen or lower epigastrium . peristalsis is also stimulated by the rather sudden distention , so that some cramping may be superimposed on the visceral pain early in the course of appendicitis . distention of the appendix continues , not only from continued mucosal secretion , but also from rapid multiplication of the resident bacteria of the appendix . as pressure in the organ increases , venous pressure within the appendiceal wall is exceeded . this rising intraluminal pressure then occludes capillaries and venules , but arteriolar inflow continues , resulting in engorgement and vascular congestion . distention of this magnitude usually causes reflex nausea and vomiting , and the diffuse visceral pain becomes more severe . the inflammatory process soon involves the serosa of the appendix and in turn parietal peritoneum in the region , producing the characteristic shift in pain to the right lower quadrant ( rlq ). the disease process is fairly advanced when pain is localized to the rlq . the mucosa of the gastrointestinal tract , including the appendix , is very susceptible to impaired blood supply . thus mucosal integrity is compromised early in the process , allowing bacterial invasion of the deeper tissue layers . this bacterial invasion leads to appendiceal destruction and systemic liberation of various bacterial toxins . fever , tachycardia , and leukocytosis develop as a consequence of this systemic release of dead tissue products and bacterial toxins . as progressive appendiceal distention rises , encroaching on the arteriolar pressure , ellipsoidal infarcts develop in the antimesenteric border of the appendiceal serosa . as distention , bacterial invasion , compromise of vascular supply and infarction progress , perforation occurs through one of the infarcted areas on the antimesenteric border . this perforation then releases the bacteria and its toxins into the abdominal cavity . appendicitis has been called the “ great imitator ,” as its symptoms are frequently confused with those of other conditions . this confusion stems from the nonspecific nature of the pain early in its course and the variability in how appendicitis progresses . pain in the right lower quadrant of the abdomen is the hallmark of appendicitis but this is not typically what the patient first perceives . when the appendiceal lumen first obstructs , the patient will have few if any symptoms because the appendiceal lumen has not yet had the chance to fill with mucus . the time required to fill the appendiceal lumen is proportional to the lumen volume available behind the obstruction . this is variable and unpredictable , as that volume is dependent upon the individual &# 39 ; s appendix size and precisely where the fecalith or other obstruction is located along that length . should the fecalith or other obstruction be close to the tip of the appendix the available volume is relatively small and the time to symptoms or perforation short . in contrast , the opposite will be true should the fecalith or other obstruction be near the base of the appendix and provide for the largest possible appendiceal volume . once the appendix begins to distend , the appendicitis patient will begin to experience a nonspecific discomfort usually in the mid portion of the abdomen . this discomfort can be easily confused with common ailments such as indigestion , constipation or a viral illness . continued appendiceal distention is also accompanied by some nausea and frequently vomiting . rarely is the vomiting severe or unrelenting , which reinforces the confusion with common ailments . later in the progression of appendicitis , inflammation will have progressed to the outermost layer of the appendix . this outmost layer is called the serosa and it touches the inner lining of the abdominal cavity called the peritoneum . this contact irritates the peritoneum , producing peritonitis that is perceived by the appendicitis patient as focal pain wherever the appendix is touching the peritoneum . this too can vary between different individuals . the appendix is most usually located in the right lower quadrant under an area known as mcburney &# 39 ; s point . mcburney &# 39 ; s point is a position on the abdomen that is approximately two - thirds of the distance from the anterior superior iliac spine in a straight line toward the umbilicus . the appendix can , however , reside in other locations in which case the peritonitis produced by the appendix will be in an atypical location . this again is a common factor producing an erroneous diagnosis and delays surgical treatment in cases of appendicitis . regardless of its location , if appendicitis is allowed to progress the organ will eventually perforate . this contaminates the abdominal cavity around the perforated appendix with bacteria producing a severe infection . this infection will usually lead to a localized intra - abdominal abscess or phlegmon and can produce generalized sepsis . to identify molecules differentially associated with appendicitis , a proteomic approach was used . a protein complex , mrp - 8 / 14 , that is present in appendix tissue in patients with acute appendicitis was identified . the highly correlative nature of this complex with appendicitis led us to examine mrp - 8 / 14 serum levels in patients with apparent appendicitis . mrp - 8 / 14 is significantly elevated ( p & lt ; 0 . 02 ) in patients with appendicitis as compared to levels in patients with apparent appendicitis yet having no appendiceal inflammation . the source of mrp - 8 / 14 in the serum is the inflamed appendix tissue . this is consistent with the known functions of mrp - 8 / 14 . the role of mrp - 8 / 14 in inflammation is not fully understood but it does seem to play a vital role in retaining leukocytes in microcapillaries . extracellular mrp - 8 / 14 interacts with endothelial cells by binding to heparin sulfate and specifically carboxylated glycans ( robinson et al ., 2002 ). the intracellular signal pathways and effector mechanisms induced by binding of mrp - 8 / 14 to endothelial cells are not well defined . however , interaction of mrp - 8 / 14 with phagocytes increases binding activity of the integrin receptor cd11b - cd18 . this is one of the major adhesion pathways of leukocytes to vascular endothelium ( ryckman et al ., 2003 ). it is believed that the mrp - 8 / 14 utilizes the receptor for advanced glycation end products ( rage ). a relative of mrp - 8 / 14 , s100a12 , is a specific ligand of rage expressed by endothelial cells and their interaction activates nf - kappa b binding in these cells ( hsieh et al ., 2004 ). the nf - kappa b binding subsequently induces expression of many proinflammatory molecules , such as various cytokines or adhesion molecules . thus , release and extracellular functions of s100 proteins represent a positive feedback mechanism by which phagocytes promote further recruitment of leukocytes to sites of inflammation . taken together , these proteins appear to play a role in a fundamental inflammatory response in certain inflammatory conditions , and are excellent markers of appendix tissue inflammation . neutrophils are white blood cells that are the first to migrate from the circulation into sites of inflammation . within neutrophils , constituting approximately 40 % of total cytosolic proteins is the mrp - 8 / 14 complex . this protein is specifically expressed only in cells of macrophage lineage , making blood monocytes and acutely activated macrophages other potential white blood cell sources of these proteins . mrp - 8 / 14 is not usually expressed in lymphocytes nor resident macrophages or those macrophages involved in chronic inflammation . these two proteins are also known to be independently expressed by mucosal epithelium in specific states of acute inflammation . in the case of appendicitis , the luminal obstruction and the resultant distention of the appendiceal wall triggers an inflammatory response . the circulating neutrophils are then recruited into the area , as are activated macrophages . while the expression of this protein complex is related to the activity of the macrophages in inflammation , the exact relationship between mrp - 8 / 14 and cellular activity is not fully known . what is known is that the intracellular distribution of mrp - 8 / 14 varies with the activation state of macrophages . normal macrophages contain the complexes in the cytosol , but once stimulated , mrp - 8 / 14 translocates from the cytosol to the cell membrane ( specifically with the proteins of the cytoskeleton ). this would imply that mrp - 8 / 14 may be related to cell movement , phagocytosis or inflammatory signal transduction . the roles of cellular movement and signal transduction may also explain why mrp - 8 / 14 is produced directly from vascular epithelium such as that lining the blood vessels within the appendix . regardless of its role in certain inflammatory conditions , mrp - 8 / 14 &# 39 ; s abundance within cells of acute inflammation makes it an excellent detector and monitor of acute appendicitis . the first step in the inflammatory process is the recruitment of neutrophils and macrophages to a specific site . in our study , the specific site is the appendix , where those mrp - 8 / 14 - containing cells will engage the offending stimulus . this engagement will usually result in mrp - 8 / 14 cell death and the liberation of mrp - 8 / 14 from either the cytosol or cell membrane into the patient &# 39 ; s circulation . at the same time , the mucosal linings of the appendix will start to produce and release mrp - 8 / 14 to facilitate macrophage migration or inflammatory amplification . this process will then escalate as increasing amounts of mrp - 8 / 14 cells are recruited by the appendicitis to ultimately release more mrp - 8 / 14 into the circulation . other examples of inflammatory states causing increases of extracellular mrp - 8 / 14 and the tendency of these increases of mrp - 8 / 14 to correlate with extent of inflammation are known . specifically , chronic bronchitis , cystic fibrosis and rheumatoid arthritis are all associated with elevated serum levels of mrp - 8 / 14 and the severity of these diseases is generally proportional to the serum levels of mrp - 8 / 14 detected . the physiological role of mrp - 8 / 14 makes it an ideal clinical marker for acute appendicitis . as patients with appendicitis are generally young and healthy , they generally produce a vigorous inflammatory response . this vigorous response is believed to liberate mrp - 8 / 14 in the earliest stages of the disease , which then escalates as appendicitis progresses . additionally , the diseases known to be associated with elevated levels of mrp - 8 / 14 are not common in this younger age group and usually do not produce symptomology similar to appendicitis . finally , as mrp - 8 / 14 is not located in nor associated with lymphocyte proliferation , this marker is not believed to be elevated in viral infections . this is an especially powerful advantage for diagnosing appendicitis , as viral infections are one of the most common imitators of appendicitis . haptoglobin was also identified in this invention as a useful marker for appendicitis . a differential proteomic screen of depleted serum identified haptoglobin as a marker for appendicitis . a second differential screen of appendix tissue confirmed that haptoglobin is upregulated in the appendix tissue of patients with appendicitis . this finding was confirmed by western blotting of tissue protein . in particular the alpha subunit isoforms were present only in diseased tissue . since haptoglobin is a plasma protein , it is highly valuable as a biomarker for appendicitis . the objective of this study was to identify a tissue - specific marker that could contribute to the decision matrix for diagnosing early acute appendicitis . a proteomic screen was used to identify a protein in the appendix specifically upregulated in acute appendicitis . mrp - 8 / 14 was identified as present both in the diseased appendix and in serum of acute appendicitis patients . specimen and serum collection . all patients enrolled in this study were treated according to accepted standards of care as defined by their treating physicians . prior to being approached for inclusion in our study , all patients were evaluated by a surgeon and diagnosed by that surgeon as having appendicitis . the treating surgeon &# 39 ; s plans for these appendicitis patients included an immediate appendectomy . the specifics of all treatments such as use of antibiotics , operative technique ( either open or laparoscopic ) were determined by the individual surgeon . exclusion criteria any patients with pre - existing chronic inflammatory diseases such as asthma , rheumatoid arthritis , inflammatory bowel disease , psoriasis , or neutropenia . pregnancy was also considered an exclusion criterion . an investigator counseled all patients about the study and informed consent was obtained . at the time of informed consent , the subject was assigned an identification number and non - personal demographic and clinical information was obtained ( age , sex , race , duration of symptoms , white blood count ( wbc ), results of imaging studies , etc ). at the time of surgery , following induction of general anesthesia , a whole blood sample ( 5 - 10 cc volume ) was obtained via peripheral venopuncture . this blood specimen was then placed on ice . as soon as possible , a small sample ( approximately 1 gram ) of inflamed appendix was taken from the pathologic specimen and also placed on ice . the iced blood specimens were then centrifuged for 20 minutes at 3000 rpm and the separated serum isolated . this isolated serum and the piece of appendix tissue were then stored separately , frozen at − 80 ° c . appendicitis tissue processing . appendix tissue from appendectomy patients was harvested and stored at − 80 ° c . until processed . individual tissue samples were ground into powder using a sterile mortar and pestle under liquid nitrogen . protein was extracted from tissue powder by incubating at 37 ° c . in extraction buffer ( 0 . 025m tris - base , 200 mm sodium chloride , 5 mm edta , 0 . 1 % sodium azide , ph 7 . 5 ). samples were centrifuged for 10 minutes at 14k rpm . supernatants were stored at − 80 ° c . until analysis . 2d gel analysis of extracted tissue samples . 2d gel analysis was performed on depleted serum samples and extracted tissue samples . isoelectric focusing ( ief ) and sds - page were performed according to the zoom ( invitrogen ) protocol for 2d gel analysis . equal quantities of protein were analyzed on each gel . comparisons between negative serum gel and positive serum gel were made to determine which proteins were present in positive samples and absent in negative samples . candidate gel spots were identified and submitted to maldi - tof protein identification analysis ( linden biosciences ). western blot analysis of extracted appendix tissue samples . samples ( 10 μg ) were subjected to standard laemmli sds - page and proteins were transferred to nitrocellulose membrane for western blot analysis using standard techniques with chemiluminescent detection . magic mark western standard ( invitrogen ) was used to determine molecular weight . mrp - 8 ( calgranulin a c - 19 , santa cruz , s . c .- 8112 ) was used in a 1 : 100 dilution in 0 . 5 × uniblock ( aspenbio , inc ) for primary antibody . the secondary antibody was peroxidase anti - goat igg ( h + l ), affinity purified ( vector , pi - 9500 ) in a 1 : 2000 dilution in uniblock . mr - 14 ( calgranulin b c - 19 , santa cruz , s . c .- 8114 ) was used in a 1 : 100 dilution in 0 . 5 × uniblock for primary antibody . the secondary antibody was peroxidase anti - goat igg ( h + l ), affinity purified ( vector , pi - 9500 ) in a 1 : 2000 dilution in uniblock . serum mrp - 8 / 14 determinations . serum levels of mrp - 8 / 14 were determined by elisa using a commercially available elisa ( buhlmann s100 - cellion s100 a8 / a9 ) according to the manufacturer &# 39 ; s protocol . identification of proteins present in appendix tissue from appendicitis patients . a differential proteomic analysis was performed on depleted serum samples with the goal of identifying proteins elevated in patients with acute appendicitis . the analysis involved comparing samples from normal patients versus patients with perforated appendices . blood samples were obtained immediately prior to surgery . a normal patient in this study is one that presented with abdominal pain , underwent surgery , and was found to have a normal appendix . normal and diseased appendix tissue was collected during surgery . the proteomic approach was to compare a pool of 4 normal samples with a pool of 4 appendicitis samples using two - dimensional electrophoresis . fig1 shows the 2d profile of proteins analyzed . comparison between the gels was performed and the most obvious difference is indicated in fig1 b as ap - 93 . based on the gel in fig1 , the molecular weight of ap - 93 is approximately 14 kilodaltons . the corresponding gel slice was analyzed by maldi - tof and a positive identification was made . the identification was based upon spectra of two tryptic peptides , nietiintfhqysvk [ seq id no : 1 ] and lghpdtlnqgefkelvr [ seq id no : 2 ]. the peptides correspond to the underlined residues in the following amino acid sequence of mrp - 14 ( genbank accession number p06702 ): the maldi - tof identification of ap - 93 as mrp - 14 was confirmed by the matching molecular weights . based on this data , mrp - 14 protein was more highly abundant in the diseased sample pool than in the normal sample pool . presence of mrp - 14 and mrp - 8 in diseased appendix tissue . in order to confirm the presence of mrp - 14 in diseased tissue , an anti - mrp - 14 antibody was used in western blotting of tissue extracts from individual normal and diseased appendices . fig2 shows the western blot data from 9 normal and 11 appendicitis samples . a 14 kilodalton band is present in every appendicitis sample . there is no detectable signal in the normal samples . this data confirms the proteomic screen data and shows that the protein is an indicator of diseased appendix tissue . since it is known that mrp - 8 exists as a dimer with mrp - 14 , tissue specimens were also examined for the presence of mrp - 8 . fig3 shows the western blot data using an anti - mrp - 8 antibody on the normal and diseased tissue samples . as expected , mrp - 8 is present in all of the diseased appendix samples and not detectible in the normal appendix tissue . these western blot data show that the mrp - 8 and mrp - 14 proteins are markedly more abundant in appendicitis than in normal appendix tissue . elevated serum levels of mrp - 8 / 14 patients with acute appendicitis . the high correlation between appendicitis and the presence of mrp - 8 / 14 in the appendix led us to examine the mrp - 8 / 14 levels in serum of those patients and other patients subsequently added to the study . the sera were collected before surgery , banked and analyzed after the disease status was known . mrp - 8 / 14 levels were measured using a sandwich elisa specific for the complex . table 1 lists serum mrp - 8 / 14 levels for 39 patients as determined by an elisa manufactured by hycult ( netherlands ) and available commercially through cell sciences , canton , mass . the amounts are given as fractions compared to an average level for patients in the study without appendicitis . note that all patients with appendicitis show a fold - increase of mrp - 8 / 14 over average normal levels . the procedure was conducted according to instructions accompanying the elisa product . the sample numbers do not correspond to the sample numbers shown in fig2 and 3 as the samples were renumbered . we have identified a protein complex that is present in the appendix and serum of appendicitis patients . based on the western blot data , the presence of mrp - 8 / 14 in appendix tissue is highly correlative with disease . furthermore , levels of mrp - 8 / 14 in serum are predictive of appendicitis . we presume that this increase is due to increased production of these proteins from systemic neutrophil infiltration of the appendix and possibly direct mucosal production of the proteins by the appendix itself . this study demonstrates that mrp - 8 / 14 is a useful clinical marker for acute appendicitis . after our discovery that mrp - 8 / 14 was a molecule differentially associated with appendicitis , our work was confirmed by the finding of power , c . et al ., 2004 and 2005 , who reported detection of this molecule in feces of patients having acute appendicitis . using a proteomic screen of serum and appendix tissue , we determined that haptoglobin is upregulated in patients with acute appendicitis . the alpha subunit of haptoglobin is an especially useful marker in screening for the disease . specimen and serum collection , appendicitis tissue processing , 2d gel analysis of extracted tissue samples , and western blot analysis of extracted appendix tissue samples were as described above in example 1 , except that for the western blot , affinity - purified anti - human haptoglobin ( rockland , 600 - 401 - 272 ) was used at a 1 : 5000 dilution in 0 . 5 × uniblock for the primary antibody ; and the secondary antibody was peroxidase anti - rabbit igg ( h + l ), affinity purified ( vector , pi - 1000 ) in a 1 : 5000 dilution in uniblock . identification of proteins present in appendix tissue from appendicitis patients . a differential proteomic analysis was performed on depleted serum samples with the goal of identifying proteins elevated in patients with acute appendicitis . the analysis involved comparing samples from normal patients versus patients with perforated appendices . blood samples were obtained immediately prior to surgery . a normal patient in this study is one that presented with abdominal pain , underwent surgery , and was found to have a normal appendix . normal and diseased appendix tissue was collected during surgery . the proteomic approach was to compare a pool of 4 normal samples with a pool of 4 appendicitis samples using two - dimensional electrophoresis . fig5 shows the 2d profile of proteins analyzed from serum depleted of igg and albumin . comparison between the gels was performed and the most obvious difference is indicated in fig5 b as ap - 77 . the protein in gel spot ap - 77 was digested with trypsin and analyzed by maldi - tof . the resulting two peptides have the following sequences : tegdgvytlnnekqwink [ seq id no : 4 ] and avgdklpeceaddgcpkppeiahgyvehsvr [ seq id no : 5 ]. the sequences were aligned with the alpha subunit of haptoglobin . the sequence of haptoglobin precursor ( genbank accession number np005134 ) is shown below with the tryptic fragments underlined . fig6 shows the two - dimensional electrophoresis profile comparison between diseased and normal appendix tissue proteins . two spots , ap - 91 and ap - 93 , were analyzed by maldi - tof and positive identifications were determined . ap - 91 protein was determined to be identical to ap - 77 , haptoglobin - alpha . elevated haptoglobin in diseased appendix tissue . in order to confirm the presence of haptoglobin in diseased tissue , an anti - haptoglobin antibody was used in western blotting of tissue extracts from individual normal and diseased appendices . fig7 shows the western blot data from 6 normal and 6 appendicitis samples . nearly every sample contained some level of the 38 kd beta subunit , however , there appeared to be an elevated level in cases of appendicitis . a & gt ; 20 kilodalton band is present in every appendicitis sample and absent from all of the normal tissue samples . this data confirms the proteomic screen data and shows that the protein is an indicator of diseased appendix tissue . the alpha subunit has higher specificity than the beta subunit . in variations of this example , fluid samples can include whole blood , serum , or plasma . the samples are whole blood collected from human patients immediately prior to an appendectomy . the specimens are placed on ice and transported to the lab . the blood is then processed by centrifugation at 3000 rpm for 15 minutes . plasma is then separated by pouring into another container upon performing an appendectomy , a patient is classified as having appendicitis ( ap ) or non - appendicitis ( nap ). the classification is based on clinical evaluation , pathology , or both as known in the art . for cases of appendicitis , the clinical condition is also characterized as either perforated or non - perforated . the samples from ap patients are optionally pooled and divided into aliquots . optionally , a pooled aliquot is treated so as to remove selected components such as antibodies and serum albumin . similarly , the samples from nap patients are optionally pooled and divided into aliquots with optional treatment to remove the same selected components . preferably the ap samples and nap samples are processed in a similar manner . next , the pooled aliquots of ap and nap samples are each subjected to two - dimensional gel electrophoresis as known in the art . the results of each sample type are compared with respect to the presence , absence , and relative expression levels of proteins . preferably , one detects a signal corresponding to a protein derived from an ap sample that is either absent or expressed at relatively lower levels in a nap sample . further characterization is performed for such an ap protein . the further characterization can include partial amino acid sequencing , mass spectrometry , and other analytical techniques as known in the art . a full length clone of the gene corresponding to the partial amino acid sequence can be isolated and expressed as a recombinant protein . the recombinant protein can be used as an antigen for detection . alternatively , a partial or complete recombinant protein can be used to induce or otherwise generate a specific antibody reagent , polyclonal or monoclonal . the antibody reagent is used in the detection of antigen in a patient so as to aid in appendicitis diagnosis . a combination of antigenic molecules can be employed in appendicitis diagnosis . tissue samples are collected from appendicitis ( ap ) and non - appendicitis ( nap ) patients . preferably the tissue is the appendix . the ap or nap tissues samples are optionally pooled so as to generate an ap tissue pool or an nap tissue pool . the ap and nap tissue samples are each used as a source for isolation of total rna and / or mrna . upon isolation , the ap - rna and nap - rna are maintained separately and used for preparation of cdna . a subtraction library is created using techniques available in the art . a cdna library is optionally amplified . the cdna library is treated so as to remove undesirable constituents such as highly redundant species and species expressed both in diseased and normal samples . examples of the techniques include those described by bonaldo et al . ( 1996 ) and deichmann m et al . ( 2001 ). upon generation of the subtraction library , one analyzes , isolates , and sequences selected clones corresponding to sequences differentially expressed in the disease condition . using molecular biology techniques , one selects candidates for recombinant expression of a partial or complete protein . such a protein is then used as an antigen for detection . alternatively , a partial or complete recombinant protein can be used to induce or otherwise generate a specific antibody reagent , polyclonal or monoclonal . the antibody reagent is used in the detection of antigen in a patient so as to aid in appendicitis diagnosis . it is envisioned that a combination of antigenic molecules can be employed in appendicitis diagnosis whole blood is drawn from a suspected appendicitis patient immediately prior to appendectomy . the specimens are placed on ice and transported to the clinical lab . the blood is processed by centrifugation at 3000 rpm for 15 minutes followed by separation of plasma from the sample by pouring into another container . during the step of pouring , the samples are evaluated with respect to viscosity . increased viscosity is indicative of appendicitis . approximately 80 % of samples corresponding to appendicitis cases demonstrate increased viscosity , whereas approximately none to less than 5 % of samples corresponding to non - appendicitis cases demonstrate increased viscosity . it is noted that the degree of increased viscosity can correlate with the severity of appendicitis . viscosity measurements can be conducted by visual observation or by using techniques known in the art . for example , a coulter harkness capillary viscometer can be used ( harkness j ., 1963 ) or other techniques ( haidekker m a , et al ., 2002 ). the presence of increased viscosity in plasma may be used in combination with other diagnostic techniques , for example with one or more of the following : physical examination , complete blood count ( cbc ) with or without differential , urinalysis ( ua ), computed tomography ( ct ), abdominal ultrasonography , and laparoscopy . all references throughout this application , for example publications , patents , and patent documents , are incorporated by reference herein in their entireties , as though individually incorporated by reference , to the extent each reference is at not inconsistent with the disclosure in this application . where the terms “ comprise ”, “ comprises ”, “ comprised ”, or “ comprising ” are used herein , they are to be interpreted as specifying the presence of the stated features , integers , steps , or components referred to , but not to preclude the presence or addition of one or more other feature , integer , step , component , or group thereof . the invention has been described with reference to various specific and preferred embodiments and techniques . however , it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention . it will be apparent to one of ordinary skill in the art that compositions , methods , devices , device elements , materials , procedures , techniques , and embodiments , and variations respectively thereof , other than those specifically described herein can be applied to the practice of the invention as broadly disclosed herein without resort to undue experimentation . all art - known functional equivalents are intended to be encompassed by this invention . whenever a range is disclosed , all subranges and individual values are intended to be encompassed . this invention is not to be limited by the embodiments disclosed , including any shown in the drawings or exemplified in the specification , which are given by way of example and not of limitation . aadland e , fagerhol m k . faecal calprotectin : a marker of inflammation throughout the intestinal tract . europ j gastroenterol hepatol 2002 ; 14 : 1 ahlquist d a , gilbert j a . stool markers for colorectal screening : future considerations . dig dis 1996 ; 14 ( 3 ): 132 - 44 . alic m . is fecal calprotectin the next standard in inflammatory bowel disease activity tests ?. 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