Patent Application: US-79115204-A

Abstract:
the invention relates to the detection and determination of erythrocytopathies and hemoglobinopathies . the invention provides a method for distinguishing between subsets of red blood cells in a sample involving contacting the sample with at least a first marker reagent reactive with a first component of a red blood cell and with at least a second marker reagent reactive with a second component of a red blood cell and determining the reactivity of the marker reagents with the cells .

Description:
several alternative and more accurate screening methods to detect fmh using flow cytometry have been proposed and described . the first reports investigating the feasibility of using flow cytometry for fetal cell counting primarily relied upon the detection of the human d antigen on the cell surface of rbcs ( 10 , 11 , 12 , 13 ). these approaches all demonstrated greater sensitivity and precision than manual methods . however , the use of anti - rhd is applicable only to the clinical situations with rh - or d - antigen incompatibility and cannot be utilized in all cases of maternal trauma and suspected fmh . several other methods for flow cytometric detection of fetal cells in maternal peripheral blood have recently been described ( 14 , 15 , 16 , 17 ). the methods differ in their means of using various cellular fixation and permeabilization steps , in combination with the intracellular detection of hbf antigen using anti - hbf antibodies . the flow cytometric anti - hbf approach has several potential advantages and the hbf antigen allows broad application of flow cytometric fetal red blood cell detection to many clinical situations . furthermore , the anti - hbf method provides good correlation with the standard kleihauer - betke test of fetal cell detection , although with a much higher precision than the manual acid elution assay . however , our findings with the approach of one cell marker such as hbf indicated that this single - color assay was not precise enough for the enumeration of true fetal cells and could not rule out the inclusion of a low percentage of false - positive f cells . a small population of 2 to 8 % adult cells containing low amounts of hbf was observed . separation and a clear distinction of both populations of fetal hbf - containing cells and interfering adult f cells with a lower hbf content is very important in order to produce accurate data of true fetal cell frequency in maternal blood for fmh assessment or f cell measurements . our findings describe an alternative approach to flow cytometric quantification of fetal rbcs using antibodies to the intracellular located fetal hemoglobin ( hbf ) and carbonic anhydrase ( ca ), and an optimal intracellular staining technique . the method or procedure is based on the discrimination between fetal and adult red blood cells using specific anti - carbonic anhydrase ( ca ) polyclonal and monoclonal anti - hbf antibodies . the hbf antigen / protein is the best marker for the detection of fetal red blood cells , while the ca antigen / protein is a good marker for adult red blood cells and is almost absent in fetal red blood cells . the monoclonal and polyclonal antibodies used in the test are fluorochrome - conjugated for direct and indirect detection of hbf and ca markers in a dual - flow cytometry analysis . both antibody preparations were shown to be specific for hbf and ca , respectively , using the protocol as described in the material section . no positive cell staining was ever demonstrated without permeabilization of the cells . the preliminary results from the dual - color flow cytometric method were comparable to those in some previous reports describing anti - hbf data . the f cell percentages in whole blood of normal donors obtained during the study were also comparable to those in previous reports . our findings demonstrate that by adding a second unique cell marker such as carbonic anhydrase to the hbf flow cytometric method , it is possible to identify distinct populations of red blood cells . the new method is able to discriminate between true fetal cells and possible false - positive f cells , by shifting the small population of hbf - containing f cells from the fetal cell population to the larger population of ca - positive adult cells . the complete dual - color staining and analysis of up to five samples can be easily performed within 1 . 5 hours of blood collection . the accurate detection of hbf - containing fetal red blood cells in maternal blood circulation will aid to estimate the degree of feto - maternal hemorrhage ( fmh ) in women during pregnancy , and subsequently in the management of hemolytic disease of the newborn ( hdn ). finally , the new and sensitive flow cytometric method using a dual - fluorescent - staining procedure , will accurately identify and quantify both interfering maternal f cells and fetal red blood cell populations . in agreement with other studies ( 14 , 15 , 16 , 17 ), it represents a practical and technically superior alternative for the routine measurement of feto - maternal hemorrhage compared to the more subjective and manual kleihauer - betke test . whole blood of 0 . 5 to 1 ml from normal donors as well as cord blood was collected on edta . the samples were usually assayed on the same day or stored at 4 - 8 ° c . for one week before testing . monoclonal antibody nam16 - 2f4 ( mouse iggi ) specific for the gamma chain of human hbf was previously described ( 14 ) and purchased from bioatlantic ( nantes , france ). pure antibody preparations were directly conjugated to r - phycoerythrin ( r - pe ) following standard labeling procedures . its pe - conjugated iggi isotype control was purified and labeled using standard labeling procedures . polyclonal goat anti - human carbonic anhydrase was purchased from abcam while the fitc - conjugated anti - goat was obtained from sigma . lds - 751 dna - staining dye was purchased from molecular probes and stored at 4 ° c . until use . newborn calf serum was purchased from greiner . all other reagents were of analytical grade . ten microliters of whole blood or cord blood were resuspended into 100 μl of newborn calf serum ( ncs ) in pbs , after which the rbcs were fixed with 100 μl of 20 % paraformaldehyde in pbs , vortexed for five seconds , and incubated at room temperature ( rt ) for 30 minutes . after fixation , the cells were washed once with 2 ml of pbs with heparin and resuspended in 100 μl of pbs with heparin . for permeabilization of the rbcs , 100 μl of fixed cells were mixed thoroughly with 100 μl of 0 . 3 % sodium dodecyl sulfate ( sds ) in pbs with heparin and allowed to stand at rt for 3 minutes . to remove the sds , the cells were washed twice with 2 ml of pbs with heparin and suspended in 1 ml of the same solution . for immunophenotyping , 100 μl of the washed cell suspension was then mixed with either 50 μl anti - hbf - pe moab diluted at 40 μg / ml in pbs , 50 μl of anti - carbonic anhydrase poab diluted 1 - in - 500 , and 50 μl of lds - 751 , or 100 μl of the washed cell suspension was mixed with 50 μl of lds - 751 and 50 μl of pbs and 50 μl isotype control as a negative control . after an incubation of 15 minutes at rt in the dark , cells were washed once with 2 ml pbs - containing heparin . then 100 μl suspended cell solutions were both mixed with 50 μl of anti - goat igg - fitc and incubated at rt for 15 minutes . the cells were washed to remove the secondary labeled conjugate and finally resuspended in 0 . 5 ml of pbs with heparin , ready for flow cytometry . the isotype control was used instead of the anti - hbf moab for a negative control . sample acquisition was performed on a coulter epics xl mcl flow cytometer ( beckman - coulter , u . s . a .). the hbf and ca cells were counted by setting the autostop at 50 , 000 or 100 , 000 events , with the collection of measures of logfsc and logssc , and fluorescence signals of logfl1 , logfl2 and logfl4 as list mode files . data analysis was performed with software ( winlist , verity software , topsham , me ) on list mode files . the live gate on lds - 751 - negative cells was used to exclude possible interfering nuclei - containing white blood cells . the positive cutoff point was approximately at 0 . 5 % above negative population of isotype control staining cells . a dual - color flow cytometric method results in the simultaneous detection of two intracellular antigens and provides a convenient and rapid test that can be completed within 1 . 5 hours of blood collection . the use of paraformaldehyde as a fixative reagent and sodium dodecyl sulfate ( sds ) to permeabilize fixed rbcs resulted in low background staining , negligible hbf leakage , and minimal cell clumping . following fixation and permeabilization , both cell antigen markers could be detected with high fluorescence signals , which resulted in a clear distinction between different stained cell populations . an important aspect of the flow cytometric fetal cell count data is to exclude possible interfering adult hemoglobin containing rbcs and auto - fluorescent wbcs present from the region of fetal cell identification . the induced auto - fluorescent wbcs can be readily excluded from the fetal cell population by staining these nuclei - containing cells with a dna - staining dye such as lds - 751 as shown in fig1 . the imprecise separation of hbf - containing f cells from the true hbf - containing fetal cells is demonstrated in the cytograms of fig2 . the fact that it is difficult to set an accurate region on the fetal cells without the interfering f cells , leads to the overestimation of the true population of fetal hbf - containing cells . especially when the number of fetal cells is low ( 0 . 4 - 0 . 6 %), the true count of fetal cells in a background of contaminating f cells with variable amounts of hbf is not accurate . the flow cytometric results of the combination of the two different red blood cell markers hbf and ca , as presented in fig3 , demonstrated that most fetal rbcs with a high hbf content and no ca could be well separated from interfering adult f cells with a lower hbf content but containing high amounts of ca . adult rbcs with no hbf were , as expected , only positive for ca . another small but not unexpected population of fetal cells , with a high hbf content and lower ca , was detected next to the fetal cell population and also clearly separated from the f cells . both the populations of fetal cells combined together resulted in the true count of fetal rbcs in cord blood and mixtures of cord and adult blood . the linearity and precision of the dual - color flow cytometric method was studied in mixtures of cord blood and whole blood from non - pregnant adults . as shown in fig4 , excellent linearity for the method was observed by serial dilutions of mixtures of cord blood and adult blood ( n = 10 ), with a fetal cell frequency range of 0 to 5 %. precision of the method was determined by performing an analysis on the same preparation of mixtures of cord and adult blood within a five - day period . the precision of all the samples consistently resulted in a coefficient of variation ( cv ) of & lt ; 5 %. therefore , the new flow cytometric method demonstrated an excellent assay performance and was observed to be linear and precise both above and below the clinically important fetal cell frequency of approximately 0 . 6 %. linearity of measurement was determined for samples consisting of different ratios of cord blood mixed with normal adult blood in the range of 0 . 0 - 10 % positive cord blood cells ( hbf / ca ). nine months old cord blood consists of 85 % fetal rbcs . the linear regression method was used to plot the known expected values versus the observed values for the percent of double - labeled fetal cells determined by the double - fluorescent cytometric method . fig5 shows that the method provided herein for distinguishing between subsets of red blood cells in a sample is also advantageously used to monitor the efficacy of intrauterine blood transfusion . the cytograms demonstrate a clear distinction between red blood cell populations in an adult donor blood sample and in a fetal blood sample before and after a transfusion with the donor blood . our studies indicate that the use of a second antibody for the detection of ca , preferably in combination with the hbf marker or with another determinant of essentially fetal cells , provides a more detailed discrimination of the different fetal and adult rbc populations and results in an improvement in the ability to determine fetal rbc frequency . 1 . sebring , e . s ., polesky , h . f . 1990 . fetomaternal hemorrhage : incidence , risk factors , time of occurrence , and clinical effects . transfusion 30 : 344 - 357 . 2 . garratty , g ., and arndt , p . a . 1999 . applications of flow cytofluorometry to red blood cell immunology . cytometry ( communications in clinical cytometry ) 38 : 259 - 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