Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-08-15
2003-12-30
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091100, C435S091200, C435S287200, C536S022100, C536S023100, C536S024300, C536S024310, C536S027310, C536S027310, C210S767000, C422S050000, C436S518000
Reexamination Certificate
active
06670128
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for isolating leukocytes and genetic materials therefrom. It also relates, in part, to a method for isolating genetic material, such as genomic DNA, from spent leukodepletion or leukoreduction filter devices in order to analyze the genetic material.
BACKGROUND OF THE INVENTION
Each year in the United States about 14 million transfusions of blood or blood components take place. There are three major blood products in transfusion medicine:
1. RED BLOOD CELLS (RBC, typically about 340 ml contained in 1 unit of donor blood)—the remaining red cell mass after most of the plasma is removed.
2. PLATELETS (typically 300 ml/1 unit of donor blood) or platelet concentrates (PCs, typically further concentrated to about 50 ml/1 unit of donor blood)—one platelet concentrate (one unit of random donor platelets) is derived from one unit of donor blood.
3. FRESH FROZEN PLASMA (FFP, 225 ml/1 unit of donor blood)—One unit of FFP can raise coagulation factor levels by 8% and fibrinogen by 13 mg/dl in the average patient.
Despite the increasing need for transfusions and the use of transfusion products, such use involves a number of risks. About 150,000 patients each year experience adverse reactions to such products. Such adverse reactions occur regardless of the type of blood transfusion a patient receives. Ninety percent of adverse transfusion reactions are caused by donor leukocytes contained in the transfusion products.
Further problems stem from Human Leukocyte Antigen (HLA) alloimmunization, in which the recipient is sensitized to antibodies contained in the transfusion product which can react, for example, to the recipient's leukocytes (HLA sensitization).
Where the recipient suffers from a non-hemolytic febrile transfusion reaction, the patient most frequently experiences fever, chills, and nausea due to white blood components contained in the transfusion product, to which the patients has antibodies (usually anti-HLA).
Other serious risks of the use of transfusion products include transmission and/or reactivation of cytomegalovirus (CMV), occurrence of graft-versus-host disease (GVHD), and the risk of viral transmissions. (HIV, HCV transmission are the most feared complications of transfusion.)
Certain precautions have been adopted in order to reduce the likelihood and/or severity of adverse reactions to transfusion products. Leukoreduction of blood products before transfusion into a patient is considered the most significant recent improvement in safety and purity of blood transfusion. Leukoreduction is the process of removing >99.9% of the white blood cells (WBC) from cellular blood components (red cells and platelets).
Leukodepletion (LD) (also known as leukoreduction) is a technique most commonly carried out by the filtration of whole blood or blood products to remove nucleated cells (leukocytes) from a donated sample required for transfusion. LD is a very potent measure employed by the transfusion medicine industry to avoid the risk of transferring disease from donor to patient and also as a prevention of adverse immuno-response to the donated blood.
The FDA has announced publicly that it will require that all cellular blood components transfused in the U.S be leukoreduced (leukodepleted) by the year 2002. Worldwide, ten countries, including Canada, Britain, France, Portugal, and Germany, have mandated universal leukocyte reduction, and 13 more, including Denmark, Italy, Japan, and New Zealand, are moving toward the practice.
As in any essential step of blood processing, the step of leukoreduction is subject to quality control. In order to label a component as leukocyte-reduced (leukoreduced), the American Association of Blood Bank Standards (19th ed) requires that the residual leukocyte content in the component must be <5×10
6
WBC/unit blood. European guidelines define leukocyte reduction as residual leukocyte content of <1×10
6
leukocytes/unit.
FDA guidelines state that quality control testing of leukocyte-reduced units should be performed on at least 1% of products (or 4/month for facilities preparing <400 units/month) and that 100% of tested units are required to contain <5×10
6
residual leukocytes/unit.
Most LD techniques employ a filter system that specifically captures leukocytes from blood, allowing the other desired blood components to pass. Specific leukocyte capture by filtration can either be carried out by affinity interaction of a leukocyte cell surface antigen such as P-selectin, CD44 or CD8, for example, or more commonly by a physical entrapment of the relatively larger leukocytes within the filter matrix of an LD device. In either case, an LD filter device, once used for the removal of leukocytes from a donated blood unit (600 ml), contains a very high concentration of leukocytes.
Potentially, assuming 100% leukodepletion, the spent LD filter device will contain between 36×10
8
and 6×10
9
cells. Each leukocyte is nucleated; i.e., it contains a nucleus that is the storage organelle for genomic DNA (gDNA), the molecular representation of an organism's genetic makeup. Therefore, a spent LD filter device containing many leukocytes will also carry the genetic makeup of the donor.
Genotyping is the discipline of identifying an individual's genome in relation to disease-specific alleles and/or mutations that occur as an effect of parental linkage. The rapid purification of human genomic DNA is an essential part of a genotyping process; the genomic DNA of an individual being the structural unit for the entire DNA sequence of every allele expressed.
Human genomic DNA cannot be directly sequenced. In order to carry out sequence analysis on regions of the chromosomes that may contain portions of mutation or disease specific sequences, selected portions are amplified, e.g., via polymerase chain reaction (“PCR”), and the amplified products are sequenced. The selected portions of the chromosomes that are amplified are dictated by the specific sequence of the primers used in the PCR amplification. The primer sets that are used in genotyping studies are commercially available and are representative for the chromosome under examination. If linkage studies identify that a disease-bearing sequence is on a particular chromosome, then many primer sets will be utilized across that chromosome in order to obtain genetic material for sequencing. The resultant PCR products may well represent the entire chromosome under examination. Due to the large length of chromosomes, many PCR reactions are carried out on the genomic DNA template from a single patient.
Human genomic DNA is currently purified by a variety of methods (Molecular Cloning, Sambrook et al. (1989)). Consequently, many commercial kit manufacturers provide products for such techniques, for example: AmpReady™ (Promega, Madison, Wis.), DNeasy™ (Qiagen, Valencia, Calif.), and Split Second™ (Roche Molecular Biochemicals, Indianapolis, Ind.). These products rely on the use of specialized matrices or buffer systems for the rapid isolation of the genomic DNA molecule.
Recently, microporous filter-based techniques have surfaced as tools for the purification of genomic DNA as well as a whole multitude of nucleic acids. The advantage of filter-based matrices are that they can be fashioned into many formats that include tubes, spin tubes, sheets, and microwell plates. Microporous filter membranes as purification support matrices have other advantages within the art. They provide a compact, easy to manipulate system allowing for the capture of the desired molecule and the removal of unwanted components in a fluid phase at higher throughput and faster processing times than possible with column chromatography. This is due to the fast diffusion rates possible on filter membranes. Nucleic acid molecules have been captured on filter membranes, generally either through simple adsorption or through a chemical reaction between complementary reactive groups present on the filter membrane or on a filter-bound ligand resulting
Baker Matthew
Butt Neil J.
Fomovskaia Galina N.
Fomovsky Mikhail A.
Smith Martin A.
Siew Jeffrey
Whatman Inc.
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