Patent Application: US-201314108437-A

Abstract:
determining a biological condition in a mammal by using a cartridge , having fluidic open channels , sealable after receiving a fluid specimen , passing any of the specimen through any of the channels , contacting any reagent stored in a chamber with the specimen in a reaction chamber inducing a reaction and forming a reaction product , a mechanical controller including first urging means applying a force externally onto the chamber to release the reagent , second urging means applying a removable force onto the channels thereby inducing fluidic movement in a first direction in the channels and upon removal of the force causing fluidic movement in an opposite direction , alignment means aligning a reading channel on the cartridge for a detection to take place , an optical reader detecting the reaction product in the reading channel , and a processor receiving data from the optical reader and processing the data to determine the biological condition .

Description:
in the detailed description , numerous specific details are set forth in order to provide a thorough understanding of the invention . however , it will be understood by those skilled in the art that these are specific embodiments and that the present invention may be practiced also in different ways that embody the characterizing features of the invention as described and claimed herein . international patent application publication no . wo2011 / 128893 to kasdan et al ., describes a device , system and method for rapid determination of a medical condition and is incorporated herein by reference . the microfluidic cartridges of the present invention may be any suitable cartridge as shown in the figures or any of the prior art cartridges described or cited herein , such as , but not limited to , those described in u . s . pat . no . d669 , 191 s1 , us20120266986 a1 , ep1846159 a2 , us2012275972 , wo11094577a , us2007292941a and ep1263533 b1 . reference is now made to fig1 , which is a simplified schematic illustration of an apparatus 100 , which comprises a cartridge 102 for detecting a biological condition , in accordance with an embodiment of the present invention . apparatus 100 comprises cartridge 102 and a number of chemical / biochemical reactants termed herein , treatment compositions 120 , 122 , 124 . the treatment compositions are adapted to react , at least in part , with biological specimen , such as a body specimen , to be introduced to the apparatus . the body specimen may be a bodily fluid such as , but not limited to , blood , serum , plasma , urine , saliva , cerebrospinal fluid ( csf ), serous fluid , peritoneal fluid and synovial fluid . additionally or alternatively , the body specimen may be a solid such as a hair , a tooth part , a bone part or a piece of cartilage . apparatus 100 comprises a specimen receiving element 118 , adapted to transfer the specimen to a sample composition chamber 104 . the sample composition chamber comprises one or more transfer elements 105 , adapted to transfer the specimen from the sample composition chamber to one or more other locations in the cartridge . in the non - limiting example shown in fig1 , transfer element 105 is a conduit in fluid connection with a treatment chamber 112 . additionally , the cartridge comprises a number of treatment composition chambers 106 , 108 , 110 , adapted to respectively house a corresponding number of treatment compositions 120 , 122 , 124 . these treatment compositions may be liquid , solid or combinations thereof . apparatus 100 is typically sold commercially as a system with the treatment compositions disposed therein . in some cases , the apparatus 100 may be adapted for a one - off test and may be disposable . in other cases , the apparatus may be re - used . a re - usable apparatus may be adapted to receive additional external compositions ( not shown ) or may have a plurality of treatment compositions , wherein only a portion is used for each test . the apparatus may be constructed and configured such that the treatment composition comprises proteins attached to a surface , such as to beads . a plurality of beads or other structural elements with proteins attached to their surfaces can be made by any one or more of the following methodologies :— simple attachment such as by adsorption via electrostatic or hydrophobic interactions with the surface , entrapment in immobilized polymers , etc . non - covalent or physical attachment ; covalent bonding of the protein to the bead surface biological recognition ( e . g ., biotin / streptavidin ). requires two steps : a first layer is formed by silane chemistry such that the surface presents a reactive group ( for example epoxy , amino , thiol , etc . ), and a second layer ( e . g ., the protein to be immobilized or a linker molecule ) is covalently attached via the immobilized reactive groups . covalent attachment to functionalized polymer coatings on the interior of the device or linkage to the free end of a self - assembled monolayer ( sam ) on a gold surface . the reaction type may include any one or more of antigen - antibody binding , sandwich ( such as antibody - antigen - antibody ), physical entrapment , receptor - ligand , enzyme - substrate , protein - protein , aptamers , covalent bonding or biorecognition . cartridge 102 further comprises at least one transfer element 107 , 109 , 111 in fluid communication with each respective of treatment composition chamber , each transfer element also being in fluid communication with treatment chamber 112 . these elements are typically microfluidic channels and may be designed for mixing , such as being tortuous in shape . various methodologies for transferring the contents of the treatment composition chambers and the sample composition chamber via the transfer elements to the treatment chamber may be employed , some of which are known in microfluidics technologies . these include air blowing , suction , vacuuming , mechanical transfer , pumping and the like . cartridge 102 further comprises at least one transfer element 113 in fluid communication with treatment chamber 112 and with an evaluation chamber 114 . optionally , evaluation chamber 114 is further in fluid communication with a transfer element 115 , adapted to remove the contents of the evaluation chamber for disposal outside the cartridge . alternatively , the evaluation chamber may have no external disposal means . table 1 shows some representative applications of apparatus 100 and methods of the present invention . reference is now made to fig2 , which is a simplified flow chart 200 of a method for detecting a biological condition , in accordance with an embodiment of the present invention . it should be understood that each of the steps of the method may take a predetermined period of time to perform , and in between these steps there may be incubation and / or waiting steps , which are not shown for the sake of simplicity . in a sample transferring step 202 , a sample , such as a bodily specimen is transferred from outside apparatus 100 via receiving element 118 into sample composition chamber 104 and then to the treatment chamber 112 . according to some embodiments , the volume of the specimen or sample is less than 200 μl , less than 100 μl , less than 50 μl , less than 25 μl or less than 11 μl . thereafter , treatment composition 120 is transferred via transfer element 107 to the treatment chamber in a composition transfer step 204 . in some cases , there may be a treatment composition disposed in the treatment chamber . depending on the nature of the treatment composition and sample / specimen type , there may be a requirement to mix or agitate the treatment chamber contents in an optional mixing step 206 . this may be performed by using a small stirbar ( not shown ) disposed in the chamber . additionally or alternatively , this may be effected by the fluid dynamics of system . additionally or alternatively , stirbars may be disposed in any of the other chambers in the apparatus . typically , the total sample volumes are in the range of 10 to 1000 μl , 100 to 900 μl , 200 to 800 μl , 300 to 700 μl , 400 to 600 μl , or 420 to 500 μl . according to some embodiments , the volume of the treatment composition chambers 106 , 108 , 110 ( also called blisters ) is from about 1 μl to 1000 μl . according to other embodiments , the volume of the specimen is from about 10 μl to 200 μl . according to other embodiments , the volume of the specimen is about 1 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 120 , 140 , 160 , 180 , 200 , 250 , 300 , 350 , 400 , 450 , or 500 μl . according to some embodiments , the volume of the treatment compositions 120 , 122 , 124 is at most about 500 μl . according to other embodiments , the volume of the specimen is at most about 200 μl . according to other embodiments , the volume of the specimen at most about 500 , 450 , 400 , 350 , 300 , 250 , 200 , 180 , 160 , 140 , 120 , 100 , 90 , 80 , 70 , 60 , 50 , 40 , 30 , 20 , 10 , or 1 μl . according to some embodiments , the volume of a reactant is at least about 1 μl . according to other embodiments , the volume of the specimen is from about 10 μl . according to other embodiments , the volume of the specimen is at least about 1 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 120 , 140 , 160 , 180 , 200 , 250 , 300 , 350 , 400 , 450 , or 500 μl . the sequence of transfer of the various treatment compositions may be important to the reaction sequence and is typically predefined . steps 204 - 206 may be performed , for example on treatment composition chamber 106 , thereafter on treatment composition chamber 108 and thereafter on treatment composition chamber 110 . in some cases , some of these steps may be performed concurrently . in a checking step 208 , it is ascertained whether all the compositions required for the sample treatment have been transferred to the treatment chamber . if any compositions remain , then steps 204 - 206 are performed on the subsequent treatment composition chamber ( s ). if no further treatment compositions require transfer , then the sample / specimen is transferred from chamber 104 into the treatment chamber . thereafter , in a second sample transfer step 210 , the sample is transferred from the sample composition chamber into the treatment chamber . according to some embodiments , step 210 may be performed before steps 204 - 208 . if required , an optional mixing step 212 to the contents of the treatment chamber may be performed . in a transferring step 214 , the contents of the treatment chamber are transferred to the evaluation chamber . the evaluation chamber 114 is configured and constructed for one or more evaluation steps 216 . these may include any of the following , or combinations thereof : a ) transfer of radiation there - through , b ) impinging radiation thereupon ; c ) detecting reflected , refracted , and / or transmitted radiation , d ) detecting emitted radiation ; e ) capturing one or more images thereof ; f ) performing image analysis on the captured images ; g ) measuring electrical characteristics of the treated specimen ; h ) impinging sonic energy thereon ; i ) detecting sonic energy therefrom ; and j ) analyzing the outputs of any one or more of the above steps . according to some embodiments , the cartridge is introduced into a system as described in international patent application publication no . wo2011 / 128893 to kasdan et al ., incorporated herein by reference . the results of the evaluation step are then outputted in a results outputting step 218 . according to some embodiments ; the apparatus may have on - board means for showing a result , such as a colorimetric strip ( not shown ). additionally or alternatively , the results are displayed in a display unit , separate and remote from apparatus 100 . reference is now made to fig3 , which is a simplified schematic illustration showing a methodology 300 for detecting a biological condition associated with a cd64 cell surface antigen , in accordance with an embodiment of the present invention . according to some embodiments , the method is carried out in the apparatus shown in fig1 and as described herein . a biological specimen , such as a blood sample , is aspirated via specimen receiving element 118 to sample composition chamber 104 , and then to treatment chamber 112 . the sample is typically of a volume in the range of 10 - 200 μl . the blood sample is typically whole blood recently removed from a patient . the whole blood comprises mainly red blood cells ( also called rbcs or erythrocytes ), platelets and white blood cells ( also called leukocytes ), including lymphocytes and neutrophils . increased number of neutrophils , especially activated neutrophils are normally found in the blood stream during the beginning ( acute ) phase of inflammation , particularly as a result of bacterial infection , environmental exposure and some cancers . a cocktail 304 comprising antibodies to cd64 and antibodies to cd163 is introduced to the treatment chamber ( see davis et al . ( 2006 )). each antibody type is typically tagged by a specific fluorescent tag . the contents of the chamber are incubated and / or mixed as is required to bind the activated blood neutrophils with the cd64 tagged antibody ( also called a marker ) to form activated neutrophils with cd64 marker 310 , and / or monocyte with a cd64 tagged antibody and a cd163 tagged antibody 312 . lymphocytes with no markers 314 are present in the contents , as well as unaffected rbcs 316 . thereafter , a lysis reagent or diluent 306 is introduced into treatment chamber 112 . in the case of a lysis reagent , it is adapted to lyse red blood cells to form lysed red blood cells 324 . additionally , reference / calibration beads 308 are added to the treatment chamber . these are used to calibrate the outputs , as is explained with reference to fig5 a - 5d hereinbelow . cd64 ( cluster of differentiation 64 ) is a type of integral membrane glycoprotein known as an fc receptor that binds monomeric igg - type antibodies with high affinity . neutrophil cd64 expression quantification provides improved diagnostic detection of infection / sepsis compared with the standard diagnostic tests used in current medical practice . cd163 ( cluster of differentiation 163 ) is a human protein encoded by the cd163 gene . it has also been shown to mark cells of monocyte / macrophage lineage . reference is now made to fig4 , which is a simplified flow chart 400 of a method for detecting a biological condition associated with a cd64 cell surface antigen , in accordance with an embodiment of the present invention . according to some embodiments , the method is carried out in the apparatus shown in fig1 and as described herein . in a first transferring step 402 , a biological specimen , such as a blood sample is aspirated via specimen receiving element 118 to sample composition chamber 104 . the sample is typically of a volume in the range of 10 - 200 μl . typically , the total sample volumes are in the range of 10 to 1000 μl , 100 to 900 μl , 200 to 800 μl , 300 to 700 μl , 400 to 600 μl , or 420 to 500 μl . according to some embodiments , the volume of the treatment composition chambers 106 , 108 , 110 ( also called blisters ) is from about 1 μl to 1000 μl . according to other embodiments , the volume of the specimen is from about 10 μl to 200 μl . according to other embodiments , the volume of the specimen is about 1 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 120 , 140 , 160 , 180 , 200 , 250 , 300 , 350 , 400 , 450 , or 500 μl . according to some embodiments , the volume of the treatment compositions 120 , 122 , 124 is at most about 500 μl . according to other embodiments , the volume of the specimen is at most about 200 μl . according to other embodiments , the volume of the specimen at most about 500 , 450 , 400 , 350 , 300 , 250 , 200 , 180 , 160 , 140 , 120 , 100 , 90 , 80 , 70 , 60 , 50 , 40 , 30 , 20 , 10 , or 1 μl . according to some embodiments , the volume of a reactant is at least about 1 μl . according to other embodiments , the volume of the specimen is from about 10 μl . according to other embodiments , the volume of the specimen is at least about 1 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 120 , 140 , 160 , 180 , 200 , 250 , 300 , 350 , 400 , 450 , or 500 μl . in an addition step 404 , a cocktail of tagged antibodies to cd64 and to cd163 is added to the treatment chamber 112 and is incubated with the blood sample . in the incubation phase of this step , the antibodies bind activated neutrophils with cd64 marker 310 , and / or monocytes activated with a cd64 tagged antibody and a cd163 tagged antibody 312 . in a lysis reagent addition step 406 , the lysis reagent is added to the treatment chamber and thereby lyses at least some of the rbcs in the chamber . at any suitable time , typically following lysis step 406 , reference beads are added to the contents of the treatment chamber in a reference bead adding step 408 . after a predefined period of time , an analysis step 410 is performed to analyze the fluorescent emission signatures from the contents . this is described in further detail with reference to fig5 a - 5d . according to some examples , the evaluation chamber 114 is constructed and configured to allow cells to pass through a reading zone 130 such that each cell passing therethrough is analyzed individually . the assay sensitivity is around 86 % and its specificity is around 87 % ( hoffmann , 2011 ). the time required to complete an assay using apparatus 100 of the present invention varies depending on a number of factors , with non - limiting examples that include described herein . in some embodiments , the time required to complete an assay is from about 0 . 5 to 100 minutes . in other embodiments , the time required to complete an assay is from about 1 to 20 minutes . in still other embodiments , the time required to complete an assay is from about 1 to 10 minutes . in some examples , the time required to complete an assay is from about 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 50 , 60 , 80 , or 100 minutes . reference is now made to fig5 a , which is a graphical output of a fluorescent detection assay of a non - activated neutrophil signature 500 associated with the method of fig3 - 4 , in accordance with an embodiment of the present invention . the non - activated tagged neutrophils each emit a signal 502 at wavelength w 1 of an intensity i 1 . the wavelengths shown in fig5 a - 5d represent a peak wavelength of waveband outputs detected , as are shown in fig7 - 11 . fig5 b shows a graphical output of a fluorescent detection assay of an activated neutrophil signature 510 , associated with the method of fig3 - 4 , in accordance with an embodiment of the present invention . each activated tagged neutrophil emits an activated neutrophil signature 512 at wavelength w 1 of an intensity i 2 . typically i 2 is greater than i 1 . in some cases the difference in signatures 512 and 510 may be detected by an image analysis , a fluorescent emission radiation count or by other qualitative or quantitative methods known in the art . the current example is not meant to be limiting . turning to fig5 c , there can be seen a graphical output of a fluorescent detection assay of a monocyte signature 520 , associated with the method of fig3 - 4 , in accordance with an embodiment of the present invention . the monocyte signature comprises a first signal 522 at a first wavelength w 1 of an intensity i 3 and a second signal 524 at a second wavelength w 2 of an intensity i 4 . fig5 d shows a graphical output of a fluorescent detection assay of a reference bead signature 530 , associated with the method of fig3 - 4 , in accordance with an embodiment of the present invention . the reference bead signature comprises a first signal 532 at a first wavelength w 1 of an intensity i 1 ( similar or equal to non - activated tagged neutrophils &# 39 ; signal 502 ) and a second signal 534 at a second wavelength w 3 of an intensity i 5 . this methodology enables the identification and quantification of activated neutrophils by intensity of signature 512 of the cd64 tag . monocytes are identified by the double signal signature 522 , 524 , acting as a positive control . reference beads are identified by the unique signal 534 at wavelength w 3 . the intensity of signal 532 at wavelength w 1 provides a reference level of cd64 for the comparison of intensity of 512 of the neutrophils . lymphocytes with no markers 330 ( fig3 ) act as a negative control and should provide no fluor signature , but may be detected by their scattering or other characteristics . further details of some embodiment of this assay procedure are described in u . s . pat . no . 8 , 116 , 984 and in davis , b h et al ., ( 2006 ). reference is now made to fig6 , which is a simplified flow chart of a method 600 for differentiating between different particles , in accordance with an embodiment of the present invention . the input to the processing is a time series from each of the channels in the eight channel photomultiplier array 601 . in addition , data from multiple scatter channels 609 is introduced . each fluorescent time series and scatter time series may be processed individually employing respective spectral cross - correlation algorithm 606 and scatter algorithm 607 to smooth it and minimize noise . two possible processing methods are boxcar averaging algorithm 602 and matched filtering algorithm 604 . in addition , groups of individual channels may be correlated to yield a multiple spectral cross - correlations 606 . one or more of these derived time series may be used to determine event locations . once an event is located in the eight channel time series the composition of that event in terms of known fluorophore signatures is determined using a minimum mean square error fit 610 . the event is now described in terms of its composition of known fluors . each event thus described is stored in an event store , i . e . memory , together with the data from the eight time series for that event and its description 612 . based on the fluor composition for each event in the data store , it is possible to determine the type of particle . for example , a neutrophil 616 is characterized by the single fluor attached to the cd64 antibody shown in fig5 as w 1 . thus events that are preponderantly characterized by the single fluor attached to the cd64 antibody are identified as neutrophils . similarly , monocytes 618 are characterized by fluors w 1 and w 2 so that an event with both of these fluor signatures is identified as a monocyte . similarly , a bead 620 is characterized by an event that has fluors w 1 and w 3 . lymphocytes 622 do not express significant fluorescence but are identified by their scatter as events . events that do not match any of the known combinations of the fluorophores are identified as rejects 626 . given the population of identified events , the median intensity of the neutrophil population and the median intensity of the bead population are determined . the ratio of the neutrophil median to the bead median is the desired leuko64 index . the positive control value is determined as the median intensity of the cd64 fluorophore bound to monocytes divided by the median intensity of the same fluorophore on the bead population . the negative control value is determined by the median intensity of the cd64 fluorophore bound to lymphocytes . these are the key steps in performing the leuko64 assay . fig7 is a graphical output 700 of fluorescence from reference beads in eight wavebands , in accordance with an embodiment of the present invention . this figure shows the smoothed signals from the eight channel pmt array for two reference beads . the amplitude for each waveband is shown on the same graph . the corresponding wavelength range is shown for each plot 702 , 706 , 708 , 710 , 712 , 714 , 716 , 718 in the legend box . the two fluorophores signatures present in this plot are 702 , 706 and 708 for fitc , which is the fluorophore attached to the cd64 antibody and 710 , 712 for starfire red , which is the fluorophore identifying the reference beads . reference is now made to fig8 , which is a graphical output 800 of data from fig7 after a first mathematical manipulation , in accordance with an embodiment of the present invention . fig8 shows the cross correlation of wave bands one two and three corresponding to wavelength 500 to 525 , 525 to 550 , and 552 to 575 nm . this cross correlation is computed by multiplying the boxcar smoothed time series corresponding to these wavelengths . this signal will have a high - value when an event containing the fitc fluorophore is present . fig9 is a graphical output 900 of data from fig7 after a second mathematical manipulation , in accordance with an embodiment of the present invention . fig9 shows the cross correlation of wave bands 3 , 4 and 5 corresponding to wavelengths 550 to 575 , 575 to 600 , and 600 to 625 nm . this signal will have a high - value when an event containing the pe fluorophore is present . fig1 is a graphical output 1000 of data from fig7 after a third mathematical manipulation , in accordance with an embodiment of the present invention . fig1 shows the cross correlation of wave bands 7 and 8 corresponding to wavelengths 650 to 675 , and 675 to 700 nm . this signal will have a high - value when an event containing the starfire red fluorophore is present . fig1 is a graphical output 1100 of an event locator , based on data from fig8 - 10 , in accordance with an embodiment of the present invention . fig1 shows the event locations determined from the cross correlations computed in fig8 , 9 and 10 . the solid fill area 1102 corresponds to the region where any of the cross correlations 802 , 902 and 1002 exceeded a predefined threshold . similarly , the solid fill area 1104 corresponds to the region where any of the cross correlations 804 , 904 and 1004 exceeded a predefined threshold . this then completes the event location process . reference is now made to fig1 , which is a scatterplot matrix 1200 of four fluor signatures 1210 , 1220 , 1230 and 1240 , showing four distinct event groups , in accordance with an embodiment of the present invention . fluor signature 1210 is the group of reference beads that contain the reference level of the f488 fluorophore ( f488 is the designation of a fluorophore with an emission spectrum identical to or equivalent to fluorescein isothiocyanate ( fitc )) and are identified by their starfire red ™ ( starfire red is the designation of a fluorophore or its emission spectrum , produced by bangs laboratory , 9025 technology dr . fishers , ind . 46038 - 2886 , usa ) embedded fluorophore . fluor signature 1220 is the group of monocytes tagged with the phycoerythrin ( pe ) fluorophore identified by the signature name pe488 ( exited by a laser at 488 nm ). ( pe488 is the designation of a fluorophore with an emission spectrum identical to or equivalent to phycoerythrin ( pe )). fluor signature 1230 is the group of lymphocytes tagged with the peaf488 ( alexa fluor ® 610 , life technologies corporation , 3175 staley rd . grand island , n . y . 14072 usa ) fluorophore . finally , fluor signature 1240 is the group of neutrophils tagged with the f488 ( alexa fluor ® 488 life technologies corporation , 3175 staley rd . grand island , n . y . 14072 usa ) fluorophore , which is the primary target group for analysis . reference is now made to fig1 a , which is a flowchart 1300 of a specific implementation of an algorithm for selecting groups of data from a scatterplot , in accordance with an embodiment of the present invention . the algorithm in fig1 a is a specific implementation of the general algorithm in fig1 b to select each of the groups 1210 , 1220 , 1230 and 1240 ( fig1 ) and determine specific parameter values in each of the groups . in a first ordering signature step 1304 the star fire red ( sfr ) signature is used to order ( from smallest sfr signature to largest ) the entire dataset of waveband and signature values 1302 . in a second step 1320 , an analysis of a histogram of an sfr signature values as shown in fig1 a to select the group 1210 . this is a small group 1404 at the upper end of group 1402 in the histogram 1400 in fig1 a . the next step is to remove this group from the overall dataset as shown in fig1 step 1322 . the removed group is the bead dataset 1324 . a dataset of waveband and signature values with bead dataset removed 1340 is then manipulated as follows . in an ordering step 1342 , the data is organized according to its pe ( phycoerythrin ) signature from smallest to largest pe ( phycoerythrin ) signature . in an analyzing pe histogram set step , 1344 , the data is manipulated to find a group corresponding to monocytes . in an extracting monocytes dataset step 1346 , a monocyte dataset of waveband and signature values 1348 is extracted . a dataset of waveband and signature values with beads and monocytes removed 1360 is then further processed as follows . set 1360 is organized according to its peaf ( peaf488 ) ( see above for beads and pe ) signature in an order according to peaf signature ordering step 1362 . in an analyzing peaf histogram to find a group corresponding to lymphocytes step 1364 , set 1360 is analyzed to determine if any of the data have behavior corresponding to lymphocytes . in an extraction step 1366 , a lymphocyte dataset of waveband and signature values 1368 is extracted from set 1360 and the remaining dataset is a dataset of waveband and signature values with bead , monocytes and lymphocytes removed 1380 . in an order by diodel signature step 1382 , dataset 1380 is analyzed according to a diodel signature ( see above ). dataset 1380 is then analyzed in an analyzing step 1384 to find a group of data having properties of neutrophils . in an extracting step 1386 , a group of data having properties of non - neutrophils 1388 is removed . a remaining group 1391 ( assumed to be neutrophils ) is used in a computing step 1392 to compute desired metric from the group parameters . reference is now made to fig1 b , which is a flowchart of a general implementation of an algorithm 1350 for selecting groups of data from a scatterplot , in accordance with an embodiment of the present invention . in a first ordering signature step 1305 a first signature is used to order the dataset of waveband and signature values 1303 . in a second step 1321 , an analysis of a histogram of a 1st signature values to find the group corresponding to 1 st signature 1325 , as exemplified in fig1 a to select the group 1210 . this is a small group 1404 at the upper end of group 1402 in the histogram 1400 in fig1 a . it should be noted that this is but one way to select the group and other methods employing additional data set values in combination may be used . the next step is to remove this group from the overall dataset as shown in fig1 b step 1323 . a removed group is a 1st signature dataset 1325 . a dataset of waveband and signature values with 1st dataset removed 1341 is then manipulated as follows . in an ordering step 1343 , the data is organized according to its 2nd signature . in an analyzing 2 nd signature histogram set step , 1345 , the data is manipulated to find a group corresponding to the 2 nd signature . in an extracting 2 nd signature dataset step 1347 , a 2 nd signature dataset of waveband and signature values 1349 is extracted . a dataset of waveband and signature values with 1 st and 2 nd signatures groups removed 1361 is then further processed as follows . set 1361 is organized according to its i th signature in an order according to i th signature ordering step 1363 . in an analyzing i th histogram to find a group corresponding to i th signature step 1365 , set 1361 is analyzed to determine if any of the data have behavior corresponding to the i th signature . in an i th signature extraction step 1367 , an i th signature dataset of waveband and signature values 1369 is extracted from set 1381 and the remaining dataset is a dataset of waveband and signature values with 1 st 2 nd and i th signature groups removed 1381 . in an order by n th signature step 1383 , dataset 1381 is analyzed according to an n th signature . dataset 1381 is then analyzed in an analyzing step 1385 to find a group of data having properties of not having nth signature properties . in an extracting step 1387 , a group of data having properties of non - nth signatures 1397 is removed . a remaining group 1395 ( assumed to be nth groups ) is used in a computing step 1393 to compute desired metric from the group parameters . fig1 a is a histogram 1400 of data of starfire red ( sfr ) signature values , in accordance with an embodiment of the present invention . fig1 b shows a plot 1450 of a polynomial 1452 and first derivative thereof 1456 and second derivative thereof 1458 of histogram 1400 shown in fig1 a , in accordance with an embodiment of the present invention . referring to fig1 b , the method of determining an upper group 1404 in fig1 a is as follows . a polynomial 1452 of sufficient degree is fitted to the histogram data 1454 ( as shown in fig1 a , set 1324 ) is shown in fig1 b . the first derivative 1456 and the second derivative 1458 of this polynomial are computed . a plurality of zeros 1460 of the first derivative are indicated by the square boxes along the zero line . a point where the polynomial is both maximum and has a zero derivative 1462 is indicated by the box with an x in it . this point in the histogram corresponds to the peak of the large group 1402 ( fig1 ). a next zero 1464 of the derivative of the polynomial corresponds to the end of the large group in the histogram . all points in the histogram above this value are in the small group . since the dataset has been ordered from smallest to largest based on the value of sfr488 , and the histogram horizontal axis is also ordered from smallest to largest value of sfr488 the point at which the large group ends is the value of sfr488 above which records in the sfr488 ordered dataset are to be removed and identified as the bead dataset 1324 of waveband and signature values as indicated in fig1 a . fig1 a is a histogram 1500 of data of pe488 signature values , in accordance with an embodiment of the present invention . fig1 b shows a polynomial fitted to the histogram in fig1 a as well as a corresponding first derivative 1556 and a second derivative 1558 , in accordance with an embodiment of the present invention . the records remaining in the dataset are now reordered using the pe488 signature from smallest to largest . histogram 1500 of the pe488 signature values 1502 is shown in fig1 a . again in this case , there is a small group 1504 to the right of the large group 1502 which corresponds to the desired monocyte population . fig1 b shows the polynomial 1552 fitted to data 1554 of histogram 1500 in fig1 a as well as the corresponding first and second derivatives . the upper group 1504 is determined in the same way as the upper group of the sfr488 histogram as was described previously . it should be noted that while in both of these cases only a one dimensional histogram was analyzed and used as the basis for selecting the desired population , multiple fields from each record in the dataset may be used to effect a group selection . as noted in fig1 , the monocyte group 1504 is removed from the dataset which now contains primarily lymphocytes , neutrophils and other particles such as un - lysed erythrocytes and other debris . fig1 a is a histogram 1600 of data 1602 of peaf488 signature values , in accordance with an embodiment of the present invention . fig1 b shows a polynomial 1652 fitted to histogram data 1654 from fig1 a as well as a corresponding first derivative 1656 and a second derivative 1658 , in accordance with an embodiment of the present invention . the records remaining in the dataset are now reordered using a peaf488 signature corresponding to lymphocytes . a histogram 1600 of the peaf488 signature is shown in fig1 a and the corresponding polynomial fit with its first and second derivatives are shown in fig1 b . the process outlined above is applied in this case as well to identify and remove a small group 1604 appearing at an upper end of the histogram , from a large group 1602 . the lymphocyte group is now removed as shown in fig1 a leaving a dataset 1380 which now contains primarily neutrophils and other particles such as unlysed erythrocytes and other debris . while neutrophils 1391 are tagged with a fluorophore with an f488 signature , other particles appear to express this signature because of the unbound fluorophore in solution . the other particles , however , are smaller than neutrophils , which now comprise the group with the largest forward scatter as measured by a diodel ( forward scatter detector ) channel . a histogram of the diodel channel is shown in fig1 a . fig1 a is a histogram 1700 of data of diode 1 channel signature values , in accordance with an embodiment of the present invention . fig1 b shows a polynomial 1752 fitted to data 1754 from the histogram in fig1 a , as well as a corresponding first derivative 1756 and a second derivative 1758 , in accordance with an embodiment of the present invention . as described above , an upper group 1704 ( fig1 a ) corresponding to larger particles , which are the neutrophils is selected . this completes the decomposition of the original dataset 1302 into the four distinct event groups ( 1324 , 1348 , 1368 , 1391 ) shown in fig1 a . within each group various parameters may be computed from the fields in the dataset . an example is shown in the following table . the observations column contains the name of the group . the nam column is the number of events in the group . the medug column is the median value of the signature for that group . for example in the sfr488 row the median sfr488 signature value is 978 . 72 . the medf488 column contains the median value of the f488 signature for the specified group . the medwaveband2 column contains the median value of the waveband2 values in the group . the medwaveband2n column contains the median value of the waveband2n values in the group . the index488 column contains the ratio of the medf488 value for the group to that of the sfr488 group . similarly , indexwaveband2 and indexwaveband2n are the ratios of the waveband2 and waveband2n medians for the group to that of the sfr488 group . although , specific groups corresponding to leukocyte subsets and a specific algorithm to compute a specific index based on these groups has been illustrated , one skilled in the art can use this basic approach whenever it is necessary to select groups from a dataset and compute numeric values based on parameters associated with these groups as shown in the general diagram of figure x . fig1 a - 18n is a sequential set of schematic drawings of the operation of an apparatus 100 ( fig1 ) for detecting a biological condition , in accordance with an embodiment of the present invention . in fig1 a , a blood sample 1801 enters a specimen receiving element 1818 and fills a chamber 1804 . in fig1 b , a blister 1820 comprising a treatment composition 120 ( fig1 ) is pressed and antibody cocktail is mixed with 10 microliters of the blood sample . in fig1 c , a mixing bellows 1815 is pressed and this effects mixing of the antibody cocktail and the 10 microliters of the blood sample in a first mixing chamber 1812 to form a first mixture 1803 . in fig1 d , the bellows is released and mixture 1803 is siphoned along a tortuous channel 1813 and into a second mixing chamber 1811 . upon release of the bellows , the first mixture returns from the second mixing chamber , back along the tortuous channel to the first mixing chamber . every time the bellows is pressed the mixture moves towards the second chamber and every time it is released , it returns , wholly or in part to the first chamber . this mixing may be performed multiple times . in fig1 e - 18g , a second composition blister 1822 is pressed , releasing a second composition 122 ( fig1 ), such as a lysis composition thereby forming a second mixture 1805 . the second mixture is mixed by pressing of bellows 1815 , the second mixture returns from the second mixing chamber , back along tortuous channel 1813 to the first mixing chamber . every time the bellows is pressed the mixture moves towards the second chamber 1811 and every time it is released , it returns , wholly or in part to the first chamber 1812 . this mixing may be performed multiple times . in fig1 h - 18j , a third blister 1824 is released comprising a third composition 124 ( fig1 ), such as a control reference , into the second mixing chamber , thereby forming a third composition 1807 . the third mixture is mixed by pressing of bellows 1815 , the third mixture returns from the second mixing chamber , back along tortuous channel 1813 to the first mixing chamber . every time the bellows is pressed the mixture moves towards the second chamber 1811 and every time it is released , it returns , wholly or in part to the first chamber 1812 . this mixing may be performed multiple times . in fig1 j - 18l , a reading bellows 1817 is pressed , which forces some of the third composition towards a reading cuvette 1830 . in fig1 m - 18n , particles 1860 from the third composition flow from the cuvette 1830 along a channel 1852 to a reading region 1850 . the cells pass through the reading region and are excited by one or more lasers 1862 , 1863 . at least one excitation laser beam 1864 impinges on cell 1860 and an emission beam 1866 is detected by a detector 1870 . in one example , this is cell emission fluorescence and detector 1870 is a spectrometer . reference is now made to fig1 , which is a simplified three dimensional front view of a system 1900 comprising a reader assembly 1901 and a cartridge 1911 for detecting a biological condition , in accordance with an embodiment of the present invention . shown in fig1 are outer view of the reader assembly 1901 and an inner view 1902 and cartridge 1911 . the cartridge is inserted in the reader assembly as shown . once the cartridge is inserted in the reader assembly all assay pre - analytical processing and analysis are performed automatically . results of the analysis are displayed on a user interface touch - screen 1915 , which is also used to control operation of the reader . the internal components of the reader assembly are seen in view 1902 , fig1 . reader assembly 1901 comprises an itx computer , 1922 , a galil motor controller , 1924 , an electronics power supply 126 , cartridge , 1911 , inserted into a cartridge handling unit ( chu ) 1928 and a forward scatter detector 1930 . not seen are the reader optics 1942 , a data acquisition board 1944 and a general electronics printed circuit board 1946 . a cartridge 102 ( fig1 ) is prepared for receiving a blood sample . the cartridge comprises a number of treatment composition chambers 106 , 108 , 110 , adapted to respectively house a corresponding number of treatment compositions 120 , 122 , 124 . these compositions are described in further detail in u . s . pat . no . 8 , 116 , 984 and in davis , b h et al ., ( 2006 )), incorporated herein by reference . in brief , reagent a comprises a mixture of murine monoclonal antibodies ( contains buffered saline ), reagent b — 10 × concentrated trillium lyse solution ( contains ammonium chloride ), reagent c — suspension of 5 . 2 μm polystyrene beads labeled with starfire red and fluorescein isothiocyanate ( fitc ), ( contains & lt ; 0 . 1 % sodium azide and 0 . 01 % tween 20 ). in a sample transferring step 202 ( fig2 ), a 10 ul blood sample , is transferred from outside apparatus 100 via receiving element 118 into sample composition chamber 104 and then on to treatment chamber 112 in a transferring step 214 . an antibody composition ( reagent a ) 120 comprising cd64 antibodies is transferred via transfer element 107 to the treatment chamber 112 in a composition transfer step 204 . these two steps combined with mixing step 206 take around four minutes using cartridge 102 of the present invention . a lysis buffer ( reagent b ) 122 is also added and mixed with the resultant mixed composition . this step and mixing all the compositions takes around three minutes using cartridge 102 of the present invention . reference beads ( reagent c ) 308 are added to the treatment chamber . the evaluation chamber 114 is configured and constructed for one or more evaluation steps 216 . according to some embodiments , the cartridge is introduced into a system as described in international patent application publication no . wo2011 / 128893 to kasdan et al ., incorporated herein by reference . this system has software associated therewith for computing the cd64 and cd163 indices on leukocytes . the results of the evaluation step are then outputted in a results outputting step 218 . according to this example , the time taken from the introduction of the small blood sample to obtaining an indication of sepsis is less than 15 minutes , typically around 10 minutes . from a user point of view , the following steps are performed : 1 ) the user adds drop of blood to the cartridge 102 and seals it . ( 10 μl are metered out by microfluidics ). 2 ) blister a ( 106 ) is pressed , releasing 100 μl of reagent a . mixing in the cartridge is controlled by the cartridge handling unit ( chu ), followed by a 4 - minutes incubation . 3 ) blister b ( 108 ) is pressed , releasing ˜ 250 μl of reagent b . mixing in the cartridge is controlled by the chu , followed by a 3 - 5 - minutes incubation . 4 ) magnetic stirbar is activated , stirring the bead suspension ( reagent c ). 5 ) blister c ( 110 ) is pressed , releasing 100 μl of reagent c . mixing in the cartridge is controlled by the chu . according to one example , reagent a is a mixture of murine monoclonal antibodies - diluted 1 : 5 in buffered saline ( pbs + 0 . 5 % bsa ); reagent b is a trillium lyse solution ( at working concentration ); reagent c is a suspension of 5 . 2 μm polystyrene beads labeled with starfire red and fitc , diluted 1 : 100 in pbs + 0 . 01 % tween 20 . 6 ) the sample is read by the optoelectronics core , and collected to the reading below . 7 ) data is analyzed automatically and result is presented . 8 ) the cartridge is disposed as biohazard . in the case of sepsis , by “ normalization ” is meant taking the ratio of the median of the target population fluorescence emission to the median of the reference bead population fluorescence emission . according to some embodiments , the readout may comprise an optoelectronics core , which enables identification and detection of fluorescent signals . the ccd in the core , used for focusing , can also be used to read chemiluminescent signals . the readout to user may also indicate where the result falls relative to reference ranges . the contents of these publications are incorporated by reference herein where appropriate for teachings of additional or alternative details , features and / or technical background . it is to be understood that the invention is not limited in its application to the details set forth in the description contained herein or illustrated in the drawings . the invention is capable of other embodiments and of being practiced and carried out in various ways . those skilled in the art will readily appreciate that various modifications and changes can be applied to the embodiments of the invention as hereinbefore described without departing from its scope , defined in and by the appended claims . assicot , marcel , et al . “ high serum procalcitonin concentrations in patients with sepsis and infection .” the lancet 341 . 8844 ( 1993 ): 515 - 518 . aulesa , c ., et al . “ validation of the coulter lh 750 in a hospital reference laboratory .” laboratory hematology 9 . 1 ( 2003 ): 15 - 28 . hawkins , robert c . “ laboratory turnaround time .” the clinical biochemist reviews 28 . 4 ( 2007 ): 179 . ault , kenneth a . “ flow cytometric measurement of platelet function and reticulated platelets .” annals of the new york academy of sciences 677 . 1 ( 1993 ): 293 - 308 . blajchman , morris a ., et al . “ bacterial detection of platelets : current problems and possible resolutions .” transfusion medicine reviews 19 . 4 ( 2005 ): 259 - 272 . bodensteiner , david c . “ a flow cytometric technique to accurately measure post - filtration white blood cell counts .” transfusion 29 . 7 ( 1989 ): 651 - 653 . cheson , bruce d ., et al . “ national cancer institute - sponsored working group guidelines for chronic lymphocytic leukemia : revised guidelines for diagnosis and treatment .” blood 87 . 12 ( 1996 ): 4990 - 4997 . christ - crain , mirjam , et al . “ effect of procalcitonin - guided treatment on antibiotic use and outcome in lower respiratory tract infections : cluster - randomised , single - blinded intervention trial .” lancet 363 . 9409 ( 2004 ): 600 - 607 . cristofanilli , massimo , et al . “ circulating tumor cells , disease progression , and survival in metastatic breast cancer .” new england journal of medicine 351 . 8 ( 2004 ): 781 - 791 . davis , bruce h ., et al . “ neutrophil cd64 is an improved indicator of infection or sepsis in emergency department patients .” archives of pathology & amp ; 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