Chemistry: analytical and immunological testing – Composition for standardization – calibration – simulation,... – Preparation composition
Reexamination Certificate
2001-07-27
2003-06-03
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Composition for standardization, calibration, simulation,...
Preparation composition
C436S008000, C436S010000, C436S018000, C436S063000, C252S408100, C435S002000, C435S029000
Reexamination Certificate
active
06573102
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a lytic reagent composition for determination of nucleated blood cells in a blood sample. More specifically the lytic reagent composition enables differentiation of nucleated red blood cells from other cell types in a blood sample by a direct current impedance measurement. In addition, the lytic reagent composition can further be used for measuring total hemoglobin concentration of the blood sample.
BACKGROUND OF THE INVENTION
Normal peripheral blood contains mature red blood cells which are free of nucleus. Nucleated red blood cells (NRBCs), or erythroblasts, are immature red blood cells. They normally occur in the bone marrow but not in peripheral blood. However, in certain diseases such as anemia and leukemia, NRBCs also occur in peripheral blood. Therefore, it is of clinical importance to measure NRBCs. Traditionally, differentiation and enumeration of NRBC are performed manually. The process involves the smearing of a blood sample on a microscope slide and staining the slide, followed by manual visual analysis of the individual slide. The NRBC concentration is reported as numbers of NRBC per 100 white blood cells. Usually, 200 white blood cells and the numbers of NRBC present in the same region on a blood smear are counted and the numbers are divided by 2 to express the NRBC concentration as the numbers of NRBC/100 WBC. This approach is extremely time-consuming as well as being subjective to the interpretation of the individual analyzing the slide.
In recent years, several fluorescence flow cytometry methods have been developed for differentiating NRBCs. These methods utilizes specific nuclear staining technique to distinguish NRBCs from other cell types because it is difficult to differentiate NRBCs based on their electronic or optical properties.
U.S. Pat. No. 5,298,426 (to Inami et al.) discloses a fluorescence method for differentiating NRBCs. The method utilizes a two-step staining using a first fluid which is an acidic hypotonic fluorescent dye solution, and a second fluid which changes the osmolality and pH of the first fluid. Inami et al. teaches that the first fluid contains an erythroblast-staining dye that diffuses into nucleated red blood cells to specifically stain their nuclei, and then separating a group of NRBCs from other cell groups on a two-dimensional plot whereby the results of NRBC differentiation are computed.
U.S. Pat. Nos. 5,516,695 and 5,648,225 (to Kim et al) disclose a multipurpose lysing reagent system and a method of use for subclassification of nucleated blood cells. The lysing reagent comprises a non-quaternary ammonium salt, an aliphatic aldehyde, a non-phosphate buffer which is inert to the aliphatic aldehyde, and a nuclear stain. The method comprises the steps of lysing a blood sample with the lysing reagent, incubating the sample mixture at an elevated temperature, and determining the nucleated blood cells including NRBCs with an automated electro-optical hematology instrumentation.
U.S. Pat. No. 5,559,037 (to Kim et al) discloses a method for flow cytometric analysis of NRBCs and leukocytes. The method comprises lysis of red blood cells and NRBC cytoplasm from a whole blood sample to expose the NRBC nuclei to a vital nuclear stain and minimizing the permeation of the vital nuclear stain into the leukocytes and analyzing the sample by measuring fluorescence and two angles of light scatter. Since leukocytes are also nucleated cells, staining of these cells needs to be prevented to avoid interference to the fluorescence measurement. The preservation of leukocyte membrane and minimizing the permeation of the nuclear stain into the leukocytes are achieved by concurrently fixing the leukocytes with an aliphatic aldehyde during lysis of red blood cells. The aldehyde fixatives are known as hazardous chemicals. In addition, the method requires heating of the reagent to 42° C. in order to obtain the NRBC and leukocyte differentiations.
EP 1 004 880 A2 discloses reagents and a method for discrimination and counting of nucleated red blood cells. The reagents include a hemolytic agent for dissolving red blood cells, and conditioning white blood cells and NRBCs in a sample to be suitable for staining; and at least one fluorescent dye selected to stain white blood cells and NRBCs differentially. The method includes the steps of lysing red blood cells, staining white blood cells and NRBCs, assaying the sample by measuring at least one scattered light parameter, and at least one fluorescence parameter.
U.S. Pat. No. 5,874,310 (to Li et al) discloses reagents and a method for differentiation of nucleated red blood cells. The method includes exposing a blood sample to a lysing reagent system to lyse mature red blood cells and analyzing the sample in a flow cell by two low angle light scatter measurements to differentiate NRBCs from other cell types. The method further includes a concurrent differentiation of white blood cells using electronic and optical analysis, wherein the electronic analysis is a DC impedance measurement.
U.S. Pat. No. 5,917,584 (to Li et al) discloses a method for differentiation of nucleated red blood cells. The method includes lysing mature red blood cells in a blood sample; analyzing the sample in a flow cell by two angles of light scatter measurement to differentiate NRBCs from other cell types, wherein the second light scatter signal is a medium angle or a right-angle light scatter signal.
The above described methods enable differentiation and enumeration of NRBCs and leukocytes by fluorescence flow cytometry and light scatter measurements. However, fluorescence and light scatter measurements are complex and expensive detection methods.
Many current non-fluorescence automated hematology analyzers, such as Abbott Cell-Dyn® 3500, COULTER® Gen*S™, Bayer Advia*120®, and Sysmex™ NE-9000 are only able to provide a NRBC flagging for the possible presence of NRBCs in an analyzed blood sample when the instruments sense an increased amount of signals near red blood cell debris area of an obtained cell distribution histogram. However, such methods are prone to generate false positive flaggings because many other blood abnormalities can cause increased signals at the same area, such as platelet clumps and sickle cells, as well as red cell debris from insufficiently lysed blood samples. In these methods NRBCs are not distinctly identified. Instead, only a common NRBC sample distribution pattern in a histogram or a dotplot is recognized by the instrument which can be confused with a similar pattern generated by above-mentioned other causes. For the flagged samples, including false positive flags, re-examination of the sample with manual method is required in clinical laboratories.
Furthermore, a well known problem with NRBC containing samples is erroneous white blood cell count (WBC) reported by hematology analyzers on these samples. Since the nuclear volumes of NRBCs are close to those of white blood cells, the NRBCs are commonly counted as white blood cells on hematology analyzers which measure the sizes of blood cells, resulting an elevation of the WBC. Therefore, correction of NRBC contribution to the WBC reported from hematology analyzer is required for samples containing NRBC. Current practice in clinical laboratory is to subtract the numbers of NRBC obtained by manual count from the WBC reported by the hematology analyzers. This is time consuming and error prone.
In a different aspect, various lytic reagent compositions for analysis of white blood cells are known in the art. U.S. Pat. No. 5,618,733 (to Tsuji et al) discloses a reagent for analyzing leucocytes, which comprises an ionic surfactant; at least one organic compound having a hydrophobic group and an acidic group which has a negative charge in an aqueous solution for preserving leukocyte morphology by combining with a cationic component in leukocytes; a nonionic surfactant; and a buffer.
U.S. Pat. No. 4,528,274 (to Carter et al) discloses a lytic reagent for determination of at least two leukocyte populations in blood.
Li Jing
Li Yi
Alter Mitchell E.
Coulter International Corp.
Wallenhorst Maureen M.
LandOfFree
Lytic reagent composition for determination of nucleated... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Lytic reagent composition for determination of nucleated..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Lytic reagent composition for determination of nucleated... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3096383