US20030215832A1 - Method for processing blood samples in order to produce DNA complex patterns for diagnostic applications - Google Patents

Method for processing blood samples in order to produce DNA complex patterns for diagnostic applications Download PDF

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US20030215832A1
US20030215832A1 US10/313,996 US31399602A US2003215832A1 US 20030215832 A1 US20030215832 A1 US 20030215832A1 US 31399602 A US31399602 A US 31399602A US 2003215832 A1 US2003215832 A1 US 2003215832A1
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blood cell
approximately
buffer
blood
tris
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Gloria Paz De Rodriguez
Gin Wu
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Paz De Rodriguez Gloria Elena Leon
Gin Wu
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/10Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/107497Preparation composition [e.g., lysing or precipitation, etc.]
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/10Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/108331Preservative, buffer, anticoagulant or diluent

Abstract

A method for processing blood samples in order to produce DNA complex patterns for diagnostic applications. The method includes the aggregation and deposition of the DNA complex from the blood of a human being in order to form a unique pattern which can be used as a medical diagnostic tool to identify a change in the body caused by a specific physiological or pathological condition. The method identifies a change in the body by comparing the pattern of a person before a specific physiological or pathological condition to the pattern of a person after the condition. The specific condition causing the change is then determined by identifying and associating the unique pattern after the condition to the specific condition or disease based on comparative appearance. The present invention can be used as a diagnostic tool to aid in the determination of, among other things, the sex of a human fetus within a few days after conception, and the presence of a cancerous condition in the earliest stage of the disease.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • The present application is a continuation of U.S. application Ser. No. 09/764,783, filed Jan. 17, 2001.[0001]
  • BACKGROUND OF THE INVENTION
  • 1. Field of Invention [0002]
  • This invention relates to the field of medical diagnostic techniques, and more specifically to the processing and preparation of blood samples to produce precipitated samples with unique and identifiable patterns. [0003]
  • 2. Background of Invention [0004]
  • Traditional disease diagnosis normally begins with a physical examination of the patient's symptoms and a review of the patient's medical and family histories. The diagnosis is usually confirmed as the patient's disease progresses and additional symptoms appear. A proper diagnosis may often times require that laboratory tests be performed in order to provide more detailed information about the patient's condition. In this regard, there are numerous specific tests for different diseases that have been developed and used in modern medicine. Some of the most common tests involve obtaining a blood sample from a patient and examining the sample in the laboratory for both the general and specific diagnostic purposes. Since the circulation of blood is the most significant biochemical transporting and exchanging system in the human body, most illnesses will produce or induce certain changes in the blood's content or its properties. It does not matter whether the human body is exposed to a toxin released from a pathogen or from a pathogen-induced immune response, or whether the body is under the influence of an endocrine system dysfunction or is experiencing nutritional or metabolic problems. All of the foregoing and any other abnormal conditions have the potential to induce significant changes in the blood. It is for this reason that blood samples have become the most commonly collected samples for the laboratory diagnosis and forensic evidence. [0005]
  • Macroscopically, the composition of blood is categorized into two major components: the cellular portion consisting of blood cells, and the fluid portion called plasma. Normally, a blood sample will start to coagulate after being extracted from a blood vessel. The coagulation process involves the aggregation of the blood cells to form a dark solid precipitation. The remaining liquid portion of the blood is called serum. If the blood sample is mixed with an anti-coagulant during or immediately after collection, the blood will not coagulate and will stay in fluid form. If the anti-coagulated blood is centrifuged or kept in a stationary state for a period of time, the blood cells will precipitate. The up-liquid portion of the anti-coagulated blood is the plasma. Depending on the test being conducted, the blood samples are normally collected and used in these three forms: the whole blood, plasma or serum. Most laboratory tests, however, focus on the serum component of coagulated blood. [0006]
  • There are many prior art technologies for detecting changes in blood components and relating the changes as a diagnostic tool to indicate the presence of different diseases. For example, an increase in a patient's white blood cell count generally indicates an inflammation occurring somewhere in the body. A low level hemoglobin number indicates anemia. Without proper medication, diabetes patients will show an abnormal blood glucose level. Therefore, the changes of the blood components are not only resulting from the changes of blood system itself but also can reflect many diseases on other parts of the body. Literally, hundreds of blood components have been identified and used to assist in diagnosing disease. There are still a significant portion of components and functions yet being identified. In this regard, the molecular changes of well-defined markers in the blood are readily detected for many well defined and understood diseases. Most of infectious diseases that induce the immune responses are easily identified by the detection of antibodies in the blood. Unfortunately, for many less-well defined physiological illnesses, there are no apparent changes in the body, nor in the blood, until the relatively late stages of the disease. Especially for many kinds of cancer, there is no good marker available. [0007]
  • Genes were originally understood to be the basic molecular unit which controlled inheritance. After the development of the advanced biochemical technology, the chemical structural unit of genes has been revealed to be nucleic acids. Except for a small number of RNA (ribonucleic acid) viruses, genes in all other living organisms are formed from DNA (deoxyribonucleic acid). DNA strands are extremely long and unbranched nucleic acid polymers that contain many different genes in each strand. In living cells, DNA strands fold tightly with certain proteins to form a compact nucleoprotein complex called chromatin. Normally, DNA strands in this form are not active. These sequestered genes can be activated, however, as a result of certain controlled mechanisms that are, in general, not well understood by today's science. Though some studies on the physical structure of the DNA double helix were done in the early stages of DNA analysis by biochemists, biochemistry and molecular biology research done today on the structure and functionality of DNA are mostly confined to an analysis of DNA after it has been almost completely isolated from its nucleoprotein complex. [0008]
  • During the later development stage of molecular biology, the identification of Enhancers, a kind of gene expression regulator, and the discovery of ribozymes have indicated that an analysis of the secondary or tertiary structure of entire nucleic acid complex, beyond just the linear sequence of the nucleic acid units, might have a potentially more important role in the analysis and understanding of gene regulation. The gene rearrangement process in the immune system also has indicated that gene regulation is involved in changes in substantial portions of DNA, instead of just an alteration of a few nucleic acids that only affected a very small portion of entire DNA sequence. Unfortunately, however, due to the extremely long and thin structure of DNA and its dense and compact nucleoprotein complex, it is extremely difficult to isolate the DNA without significantly altering its nucleoprotein complex. Indeed, standard DNA extraction procedures used in molecular biology research cause severe damage to the integrity of the DNA complex. These procedures strip off all of the DNA's associated proteins and related components in order to isolate the DNA. Unfortunately, all of the DNA complex's structure is demolished leaving DNA in an isolated and an approximately linear and uniform form. As a result, it is not possible on a macroscopic level to ascertain any structural difference between normal and altered DNA. [0009]
  • It would be greatly advantageous to overcome the foregoing problem in order to enable researchers to distinguish normal DNA from altered DNA by providing them with a method for processing and preparing blood samples to produce precipitated samples with unique and identifiable patterns so that they can compare the patterns associated with a normal DNA complex to the patterns associated with an abnormal DNA complex. [0010]
  • SUMMARY OF THE INVENTION
  • In order to overcome the technological barrier, the present invention provides a new and unique method for processing and preparing blood samples that isolates and preserves the structural integrity of DNA and its associated nucleoprotein complex (“DNA complex”). The method includes the aggregation and deposition of the DNA complex from the blood of a human being in order to form a unique pattern which can be used as a medical diagnostic tool to identify a change in the body caused by a specific physiological or pathological condition. The aggregation and deposition of the DNA complex from the blood results in a unique pattern. Changes in the body can be identified by comparing these patterns derived before a specific physiological or pathological condition to those derived after the condition. Further, the specific condition causing the change can be determined by identifying and associating the unique pattern after the condition to the specific condition or disease based on comparative appearance. The present invention can be used as a diagnostic tool to aid in the determination of, among other things, the sex of a human fetus within a few days after conception, and the presence of a cancerous condition in the earliest stage of the disease. In these and other contexts, an important advantage of the present invention is the ability to detect changes in the body during the early stages of a physiological or pathological condition without damaging the integrity of the DNA complex. [0011]
  • DESCRIPTION OF THE DRAWINGS
  • The present invention is further described by reference to the following drawings: [0012]
  • FIG. 1 is a diagrammatical illustration of the present method of processing human blood samples. [0013]
  • FIG. 2A is a diagram of the pattern formed using the present invention to process the blood from a pregnant woman carrying a female fetus for six (6) weeks. [0014]
  • FIG. 2B is a diagram of the pattern formed using the present invention to process the blood from a pregnant woman carrying a female fetus for seven (7) weeks. [0015]
  • FIG. 2C is a diagram of the pattern formed using the present invention to process the blood from a pregnant woman carrying a female fetus for eight (8) weeks. [0016]
  • FIG. 2D is a diagram of the pattern formed using the present invention to process the blood from a pregnant woman carrying a female fetus for nine (9) weeks. [0017]
  • FIG. 2E is a diagram of the pattern formed using the present invention to process the blood from a pregnant woman carrying a female fetus for ten (10) weeks. [0018]
  • FIG. 2F is a diagram of the pattern formed using the present invention to process the blood from a pregnant woman carrying female twins. [0019]
  • FIG. 3A is a diagram of the pattern formed using the present invention to process the blood from a pregnant woman carrying a male fetus for five (5) weeks. [0020]
  • FIG. 3B is a diagram of the pattern formed using the present invention to process the blood from a pregnant woman carrying a male fetus for seven (7) weeks. [0021]
  • FIG. 3C is a diagram of the pattern formed using the present invention to process the blood from a pregnant woman carrying a male fetus for nine (9) weeks. [0022]
  • FIG. 3D is a diagram of the pattern formed using the present invention to process the blood from a pregnant woman carrying a male fetus for ten (10) weeks. [0023]
  • FIG. 3E is a diagram of the pattern formed using the present invention to process the blood from a pregnant woman carrying a male fetus for eleven (11) weeks. [0024]
  • FIG. 4 is a diagram of the pattern formed using the present invention to process the blood of a healthy woman. [0025]
  • FIG. 5 is a diagram of the pattern formed using the present invention to process the blood of a woman with breast cancer. [0026]
  • FIG. 6 is a diagram of the pattern formed using the present invention to process the blood of a woman with cervical cancer.[0027]
  • DETAILED DESCRIPTION OF THE INVENTION
  • The new and unique method of processing human blood samples according to the present invention is illustrated diagrammatically in FIG. 1. [0028]
  • Initially, fresh human blood [0029] 10 is collected and placed in a first centrifuge tube 20, where it is mixed with an anticoagulant to form an anticoagulated blood mixture 30. The centrifuge tube 20 containing the anticoagulated blood mixture 30 is then placed in a centrifuge 40. Centrifugation causes the anticoagulated blood to separate into a top portion, containing blood plasma 50, which is removed and discarded, leaving behind blood cells 60 in centrifuge tube 20.
  • In a second centrifuge tube [0030] 21, a blood cell solution is prepared in accordance with the following steps: first, approximately two (2) volumes of Tris-buffer is placed into the centrifuge tube 21; second, approximately one (1) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, is added to centrifuge tube 21; and lastly, approximately four (4) volumes of the blood cells 60 are added to centrifuge tube 21. In each of the first two steps, the Tris-buffer consists of 0.5 M Tris, 0.2 M EDTA, 0.6% NaCl, having a pH of between 10.3 and 10.4.
  • The blood cell solution is then mixed well in the centrifuge tube [0031] 21 by strong vibration for approximately 30 seconds to form a first blood cell mixture 80, and then the mixture 80 is placed in the centrifuge 40 where the mixture is centrifuged at 11,000 rpm for approximately 10 minutes to produce a first liquid phase 90 and first blood cell debris 91.
  • Next, a phenol/chloroform solution [0032] 100, consisting of approximately one (1) volume of Tris-buffer saturated phenol, prepared as described above, and approximately one (1) volume of chloroform, is added to the centrifuge tube 21 containing the first liquid phase 90 and first blood cell debris 91. The contents in centrifuge tube 21 are then mixed well to produce a second blood cell mixture 110.
  • The centrifuge tube [0033] 21 containing the second blood cell mixture 110 is placed in centrifuge 40 and centrifuged at approximately 11,000 rpm for approximately 15 minutes to produce a second liquid phase 120 and second blood cell debris 130. The tube 21 containing the second liquid phase 120 and debris 130 is then placed in a beaker of ice 140 for approximately fifteen (15) minutes.
  • A slide [0034] 160 is then prepared by placing an acid alcohol sample 150 consisting of approximately 25 μl of freshly made 20% acid alcohol (i.e., ethanol containing 20% acetic acid) onto the top surface of the slide 160, and immediately adding a blood cell sample 170 consisting of approximately one (1) volume of the second liquid phase 120, which has been cooled, onto the center portion of the top surface of acid alcohol sample 150. The slide 160 is then maintained on a stable surface in order to allow acid alcohol sample 150 and blood cell sample 170 to dry on the slide at room temperature without any disturbance for 10 to 15 minutes. During the drying phase, the DNA complex contained within blood cell sample 170 aggregates in the acid alcohol sample 150 and deposits a unique pattern on slide 160.
  • The phenol does not strip off all of the nucleoprotein complex. Phenol by itself cannot strip off all of the complex (e.g., histone protein), albeit this can be done by treating the tissue with protease (e.g., protease K). The method of the present invention does not include this step. Rather, the present invention relies, in part, upon the fact that the DNA structure changes as part of a rearrangement process which takes place in bone marrow and which has been shown to be critical to the production of a vast variety of white blood cells with each variety containing a uniquely rearranged DNA structure. What is significant to the present invention is that after the mature white blood cells enter the blood stream, some of the cells produce specific clones as part of an immunological response to the exposure to an antigen, such as a physiological or pathological condition. If the physiological or pathological condition is significant (e.g., pregnancy or cancer), the immunological response produces large numbers of specific clones which are designed to protect the body from the antigen. The change in the DNA complex pattern of a healthy person after the person has been exposed to a physiological or pathological condition is most probably due to this substantial increase in the volume of specific white blood cell clones as compared to the volume of all other white blood cells in the blood. Since the method of the present invention preserves the structural integrity of the DNA complex, the DNA complex tends to aggregate during the deposition phase in an orderly manner, rather than in a random manner. This effect is enhanced by the change of surface tension during the acid alcohol drying process, a process similar to DNA combing effect. It is this orderly aggregation process that produces the unique patterns. [0035]
  • The foregoing has been established through extensive experiments provided that the relative concentrations of blood cells, Tris-buffer diluted phenol, Tris-buffer saturated phenol and chloroform are maintained. Moreover, it has been found that the resulting DNA complex patterns for any given blood sample from the same person with no physiological or pathological changes will exhibit practically identical shapes. However, physiological or pathological changes to that person impart repeatable changes in the patterns. Consequently, visual analysis of the patterns under an optical microscope has revealed that the patterns are useful as a diagnostic tool to assist in the determination of whether a particular human being has been exposed to a specific physiological or pathological condition. Changes in the patterns serve as an early indicator of the onset of a physiological or pathological condition. Moreover, in the experimental contexts to be described the type of change tends to indicate the type of physiological or pathological condition. [0036]
  • In one set of experiments, the inventors used the method of the present invention to produce DNA complex patterns of approximately 1,000 pregnant women, and the pattern results are shown in FIGS. 2A through 3E. FIGS. 2A through 3E are line drawings of selected representative DNA complex patterns derived from photographs taken through an optical microscope during the above-described experimentation. Generally, it was found that the DNA complex patterns for any given blood sample from the same person exhibited practically identical shapes. It was also found that the patterns were visually distinguishable in the earliest stages of pregnancy. [0037]
  • In each instance, the following procedure was utilized. After obtaining whole blood from a woman, the blood was placed in a centrifuge tube where it was mixed with an anti-coagulant to form an anti-coagulated blood mixture. The anti-coagulated blood is then centrifuged and the plasma was removed, leaving the blood cells in the tube. In another centrifuge tube, a blood cell solution was prepared in accordance with the following steps: first, two (2) volumes (5 μl) of Tris-buffer were placed into the centrifuge tube; second, one (1) volume (2.5 μl) of Tris-buffer saturated phenol, which was prepared by mixing re-distilled phenol with Tris-buffer, was added to the centrifuge tube; and lastly, four (4) volumes (10 μl) of the blood cells were added to the centrifuge tube. In each of the first two steps, the Tris-buffer consisted of 0.5 M Tris, 0.2 M EDTA, 0.6% NaCl, having a pH of between 10.3 and 10.4. The blood cell solution was then mixed well to produce a first blood cell mixture, and the mixture was centrifuged for ten (10) minutes at 11,000 rpm to produce a first liquid phase and a first blood cell debris, which formed as a precipitate in the tube. Next, a phenol/chloroform solution was prepared by mixing 2.5 μl of Tris-buffer saturated phenol, prepared as described above, with 2.5 μl chloroform, and the phenol/chloroform solution was added to the centrifuge tube containing the first liquid phase and first blood cell debris, which was mixed well to produce a second blood cell mixture. [0038]
  • The second blood cell mixture was then centrifuged for fifteen (15) minutes at 11,000 rpm to produce a second liquid phase and second blood cell debris, which formed as a precipitate in the tube. The centrifuge tube containing the second liquid phase and second blood cell debris was then removed from the centrifuge and placed in a beaker of ice for fifteen (15) minutes. [0039]
  • A slide was prepared by placing an acid alcohol sample of 25 μl of freshly prepared 20% acid alcohol (i.e., ethanol containing 20% acetic acid) onto the top surface of the slide, and a blood cell sample of 1.0 μl of the cooled second liquid phase was immediately added to the center of the top surface of the acid alcohol sample. The samples were then allowed to dry on the slide at room temperature without any disturbance for 10 to 15 minutes. After the samples had dried, the slide was viewed under an optical microscope. [0040]
  • Microscopic analysis of each sample from the blood of the pregnant women participating in the experiment revealed that the patterns that formed on the slide from women carrying a female fetus were readily distinguishable from the patterns formed from women carrying a male fetus. It was also discovered that the patterns were distinguishable in the earliest stages of pregnancy. The pattern diagrams shown in FIGS. 2A through 2F were obtained by using the present invention, as described in the experimental procedure above, to process a blood sample obtained from each of six (6) different pregnant women carrying a female fetus. The gestation period for the women carrying a female fetus was between six (6) and ten (10) weeks. Each of these patterns exhibits a single and approximately circular or polygonal ring shape, with the exception of FIG. 2F which exhibits a double ring shape formed from the blood of one of the women who was carrying twins. [0041]
  • By comparison, the pattern diagrams shown in FIGS. 3A through 3E were formed by using the present invention to process a blood sample obtained from each of five (5) different women carrying a male fetus. The gestation period for the women carrying a male fetus was between five (5) and eleven (11) weeks. Each of these patterns reveals either a generally linear pattern, or a linear pattern in combination with one or more elongated or collapsed ring patterns. [0042]
  • None of the patterns illustrated in FIGS. 3A through 3E, however, exhibits the circular or polygonal shapes exhibited by the patterns illustrated in FIGS. 2A through 2F. Thus, with regard to a pregnant woman, if the pattern is circular or polygonal in shape, the fetus is a female and if the pattern is linear or elongated in shape, the fetus is a male. Applicants' experiments with pregnant women and nonpregnant women also revealed that the DNA complex patterns for any given blood sample from the same person exhibited practically identical shapes. [0043]
  • For purposes of the present invention, the contemplated pattern recognition is actually a pattern characterization. It is not a quantitative measurement. There is no numeric number or data that results as the characterization is a subjective “yes” or “no”. Consequently, conventional statistical analysis will not support or reject the method, and has no place. As an example, consider the restriction mapping of DNA samples in forensic analysis. What matters is that the DNA fragment bands match, but the quantity has no meaning. Another example involves pathology inspection. In liver cirrhosis, for example, all damaged liver cells are different but they all share some similar characters. The similarity can be visually identified by pathologists. However, there is no place for a statistical analysis on these cells because the cell is irregular in shape, and quantitative analysis has no meaning for the disease. The question is whether there is a cirrhosis or not, and not the percentage of livers with cirrhosis. The present method involves the same qualitative diagnosis, not quantitative. [0044]
  • In another set of experiments, the inventors used the method of the present invention to produce DNA complex patterns from the blood of approximately 300 healthy women and 300 healthy men. The inventors also used the method of the present invention to produce DNA complex patterns of over 100 women with cervical cancer, and over 50 with breast cancer. The healthy female patterns were compared to the patterns from women with breast and cervical cancer. [0045]
  • FIG. 4 diagrammatically illustrates a typical pattern formed from a healthy woman, and FIG. 5 and FIG. 6 illustrate the patterns of a woman with breast and cervical cancer, respectively. [0046]
  • The pattern from the healthy women, as shown in FIG. 4 displays a generally linear, single continuous strand. Throughout most of the strand's length it is smooth and of uniform width. In some healthy persons the strands are straight and in some they form a nearly circular shape, yet in all cases they retain a smooth and uniform width. These smooth strand shapes are readily distinguishable from the irregular beaded and branched strand shapes in women with cancer. [0047]
  • The pattern formed from the blood cells of a woman with breast cancer, as illustrated in FIG. 5, reveals a generally polygonal shaped strand that is beaded and there is a substantial discontinuity on one side of the polygonal shaped strand. In all breast cancer samples there was a rough string with very obvious irregular width and always circular or near circular shape. Also, there were consistently two linked closed rings of unequal size. The shapes of the closed rings were irregular. It could be triangle, circular, polygonal, etc. The discontinuity appears as a branching pattern over from the polygonal shaped strand. [0048]
  • FIG. 6 is an exemplary aggregated DNA pattern of a female with cervical cancer. In all such samples there was always a closed ring with partially diffused string forming branches or mesh type distribution filling in the central space of the closed ring. Also, the shape of the rings was highly metamorphic (as was the breast cancer). This can be seen in FIG. 6 where the strand pattern that was formed appears to be generally smooth, but its shape is irregular, being neither linear, circular, nor polygonal, and there is a discontinuity or loop on one portion of the strand, and thinner strands form branches which extend from one end of the thicker strand to the other end and one thin strand extends across the interior of the irregularly shaped strand pattern. [0049]
  • It should be apparent that the foregoing analyses are qualitative pattern characterizations. They do not involve quantitative measurements. There is no numerical quantization of these patterns. The comparison is visual and the result is either “yes” or “no”, just as a pathologist would diagnose a biopsy. Therefore, conventional statistic analysis can not be applied. Still, the sample characteristics are consistently the same. [0050]
  • Based upon the consistent results of these experiments, it is apparent that the non-invasive method at the present invention may be used by biomedical researchers and technologists to identify physiological changes in the body of a pregnant woman in the earliest stages of her pregnancy, and to determine whether the changes are the result of carrying a male or female fetus. As described in detail above, applicants have identified two (2) predominate strand patterns from the blood of a pregnant woman which determine the sex of a fetus within a few weeks of conception. If the pattern is circular or polygonal in shape (composite FIG. 2), the fetus is a female and if the pattern is linear or elongated in shape (composite FIG. 3), the fetus is a male. Thus, the present method of processing human blood allows someone skilled in the art to readily recognize changes in the predominately smooth strands by using an optical microscope to detect the presence or absence of beads within a smooth strand, a loop within a strand and/or significant branching coming off of a smooth strand. [0051]
  • Similarly, the marked differences between the patterns of a healthy women and women with cancer indicates that the present invention may also be used to identify pathological changes in a human being. The inventor's experiments have correlated a beaded strand with some branching to the presence of breast cancer (see FIG. 5) and have correlated a looped strand with some branching with cervical cancer (see FIG. 6). These results are significant because they indicate that the method can be used as a diagnostic screen to indicate whether a person is being subjected to a significant pathological condition, warranting further medical evaluation and diagnostic techniques. In other words, the relatively simple blood test described in the specification can be used to tell a person whether or not he or she is healthy. If the test indicates the presence of disease, earlier detection would then be possible using more expensive and invasive techniques like an MRI, a CAT scan and a colonscopy. Naturally, a normal test result would provide a general sense of well being to the patient, possibly removing a generalized fear that he or she has cancer or some other serious disease. Again, the method of the present invention preserves the structural integrity of the DNA complex, the DNA complex tends to aggregate during the deposition phase in an orderly manner, rather than in a random manner. It is this orderly aggregation process that produces the unique patterns. [0052]
  • It should be further understood by those persons who are skilled in the art of biomedical research that the application of the present invention is not limited to its use in diagnosing the gender of a fetus, and in diagnosing breast and cervical cancer. Since the patterns which are used to form the diagnosis are formed from the DNA complex of the blood cells, the existence of all physiological and pathological conditions are potentially detectable using the method of the invention. Although current biomedical technology is not able to completely describe all of the physical changes that take place within the DNA complex of the blood cells when it is exposed to physiological and/or pathological conditions, it is known that changes do occur in the DNA complex of the blood cells in response to such conditions. As a result, the present invention's ability to compare a DNA complex pattern of a the blood cells of a healthy person to the pattern of a DNA complex which has reacted or changed in some manner compels the conclusion that the method may be used to diagnose the existence of all known diseases and to do so at an early stage of the disease. [0053]
  • Although the preferred method of the present invention involves the use of fresh blood as the source material to form the patterns, it will be understood by biomedical researchers that other cellular source materials may also be used to extract the DNA complex and used to form precipitated patterns. Finally, it will also be apparent to those skilled in the art that the method of the present invention, in addition to being applicable to human beings, is equally applicable to all other animals or living cells. Accordingly, the disclosure of the present invention is intended to be illustrative, but not limiting, of the scope of the invention. [0054]

Claims (18)

We claim:
1. A method of processing human blood samples to form a DNA complex strand pattern, comprising the steps of:
a. mixing a sample of blood containing plasma and blood cells with an anticoagulant to form an anti-coagulated blood mixture;
b. centrifuging the anti-coagulated blood mixture in order to separate the plasma from the blood cells;
c. preparing a first blood cell mixture in accordance with the following steps:
i. preparing approximately one (1) volume of Tris-buffer;
ii. adding approximately a half (½) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, to the approximately one (1) volume of Tris-buffer to produce a buffer diluted phenol; and
iii. adding approximately two (2) volumes of the blood cells to the buffer diluted phenol;
d. centrifuging the first blood cell mixture to form a first liquid phase and first blood cell debris;
e. preparing a second blood cell mixture by mixing the centrifuged first blood cell mixture and first blood cell debris with approximately a half (½) volume of chloroform and approximately a half (½) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer;
f. centrifuging the second blood cell mixture to form a second liquid phase and second blood cell debris;
g. cooling the second liquid phase and second blood cell debris thereby causing the structural components of the DNA complex within the second liquid phase to aggregate;
h. placing an acid alcohol sample consisting of approximately twelve and a half (12½) volumes of freshly made 20% acid alcohol on a slide; and
i. adding a blood cell sample consisting of approximately one fifth (⅕) volume of the cooled second liquid phase onto the center of the top surface of the acid alcohol sample and allowing both samples to dry at room temperature without any disturbance, whereby an aggregate of the DNA complex deposits a strand pattern on the slide.
2. A method of processing human blood samples to form a DNA complex strand pattern, comprising the steps of:
a. mixing a sample of blood containing plasma and blood cells with an anticoagulant to form an anti-coagulated blood mixture;
b. centrifuging the anti-coagulated blood mixture in order to separate the plasma from the blood cells;
c. preparing a first blood cell mixture in accordance with the following steps:
i. preparing approximately 5 ml of Tris-buffer;
ii. adding approximately 2.5 ml of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, to the approximately 5.0 ml of Tris-buffer to produce a buffer diluted phenol; and
iii. adding approximately 10 ml of the blood cells to the buffer diluted phenol;
d. centrifuging the first blood cell mixture to form a first liquid phase and first blood cell debris;
e. preparing a second blood cell mixture by mixing the centrifuged first blood cell mixture and first blood cell debris with approximately 2.5 ml of chloroform and approximately 2.5 ml of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer;
f. centrifuging the second blood cell mixture to form a second liquid phase and second blood cell debris;
g. cooling the second liquid phase and second blood cell debris thereby causing the structural components of the DNA complex within the second liquid phase to aggregate;
h. placing an acid alcohol sample consisting of approximately 25 ml of freshly made 20% acid alcohol on a slide;
i. adding a blood cell sample consisting of approximately 1.0 ml of the cooled second liquid phase onto the center of the top surface of the acid alcohol sample and allowing both samples to dry at room temperature without any disturbance, whereby an aggregate of the DNA complex deposits a strand pattern on the slide.
3. A method of determining the sex of a fetus comprising:
a. mixing a sample of blood containing plasma and blood cells with an anticoagulant to form an anti-coagulated blood mixture;
b. centrifuging the anti-coagulated blood mixture in order to separate the plasma from the blood cells;
c. preparing a first blood cell mixture in accordance with the following steps:
i. preparing approximately one (1) volume of Tris-buffer;
ii. adding approximately a half (½) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, to the approximately one (1) volume of Tris-buffer to produce a buffer diluted phenol; and
iii. adding approximately two (2) volumes of the blood cells to the buffer diluted phenol;
iv. mixing the first blood cell mixture;
d. centrifuging the first blood cell mixture to form a first liquid phase and first blood cell debris;
e. preparing a second blood cell mixture by mixing the centrifuged first blood cell mixture and first blood cell debris with approximately a half (½) volume of chloroform and approximately a half (½) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer;
f. centrifuging the second blood cell mixture to form a second liquid phase and second blood cell debris;
g. cooling the second liquid phase and second blood cell debris thereby causing the structural components of the DNA complex within the second liquid phase to aggregate;
h. placing an acid alcohol sample consisting of approximately twelve and a half (12½) volumes of freshly made 20% acid alcohol on a slide; and
i. adding a blood cell sample consisting of approximately one fifth (⅕) volume of the cooled second liquid phase onto the center of the top surface of the acid alcohol sample and allowing both samples to dry at room temperature without any disturbance, whereby an aggregate of the DNA complex deposits a strand pattern on the slide; and
J. determining that the sex of the fetus is female if the shape of the strand pattern is approximately circular or polygonal, or that the sex of the fetus is male if the shape of the strand pattern is generally linear or generally linear in combination with at least one elongated ring.
4. A method of determining the sex of a fetus comprising:
a. mixing a sample of blood containing plasma and blood cells with an anticoagulant to form an anti-coagulated blood mixture;
b. centrifuging the anti-coagulated blood mixture in order to separate the plasma from the blood cells;
c. preparing a first blood cell mixture in accordance with the following steps:
i. preparing approximately 5 μl of Tris-buffer;
ii. adding approximately 2.5 μl of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, to the approximately 5.0 μl of Tris-buffer to produce a buffer diluted phenol; and
iii. adding approximately 10 μl of the blood cells to the buffer diluted phenol;
iv. mixing the first blood cell mixture;
d. centrifuging the first blood cell mixture to form a first liquid phase and first blood cell debris;
e. preparing a second blood cell mixture by mixing the centrifuged first blood cell mixture and first blood cell debris with approximately 2.5 μl of chloroform and approximately 2.5 μl of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer;
f. centrifuging the second blood cell mixture to form a second liquid phase and second blood cell debris;
g. cooling the second liquid phase and second blood cell debris thereby causing the structural components of the DNA complex within the second liquid phase to aggregate;
h. placing an acid alcohol sample consisting of approximately 25 μl of freshly made 20% acid alcohol on a slide;
i. adding a blood cell sample consisting of approximately 1.0 μl of the cooled second liquid phase onto the center of the top surface of the acid alcohol sample and allowing both samples to dry at room temperature without any disturbance, whereby an aggregate of the DNA complex deposits a strand pattern on the slide; and
j. determining that the sex of the fetus is female if the shape of the strand pattern is approximately circular or polygonal, or that the sex of the fetus is male if the shape of the strand pattern is generally linear or generally linear in combination with at least one elongated ring.
5. The method of claim 3 or 4 in which the Tris-buffer consists of 0.5 M Tris, 0.2 M EDTA, 0.6% NaCl, having a pH of between 10.3 and 10.4.
6. The method of claim 3 or 4 in which the step of centrifuging the first blood cell mixture is performed for approximately ten (10) minutes at 11,000 rpm.
7. The method of claim 3 or 4 in which the step of centrifuging the second blood cell mixture is performed for approximately fifteen (15) minutes at 11,000 rpm.
8. The method of claim 3 or 4 in which the step of cooling the second liquid phase is performed by placing the second liquid phase on ice for approximately fifteen (15) minutes.
9. A method of detecting a change in the body of a human being caused by a pathological condition, comprising:
a. mixing a sample of blood from a human donor with an anticoagulant to form an anti-coagulated blood mixture;
b. centrifuging the anti-coagulated blood mixture in order to separate the plasma from the blood cells;
c. preparing a first blood cell mixture in accordance with the following steps:
i. preparing approximately one (1) volume of Tris-buffer;
ii. adding approximately a half (½) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, to the approximately one (1) volume of Tris-buffer to produce a buffer diluted phenol; and
iii. adding approximately two (2) volumes of the blood cells to the buffer diluted phenol;
iv. mixing the first blood cell mixture;
d. centrifuging the first blood cell mixture to form a first liquid phase and first blood cell debris;
e. preparing a second blood cell mixture by mixing the centrifuged first blood cell mixture and first blood cell debris with approximately a half (½) volume of chloroform and approximately a half (½) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer;
f. centrifuging the second blood cell mixture to form a second liquid phase and second blood cell debris;
g. cooling the second liquid phase and second blood cell debris thereby causing the structural components of the DNA complex within the second liquid phase to aggregate;
h. placing an acid alcohol sample consisting of approximately twelve and a half (12½) volumes of freshly made 20% acid alcohol on a slide;
i. adding a blood cell sample consisting of approximately one fifth (⅕) volume of the cooled second liquid phase onto the center of the top surface of the acid alcohol sample and allowing both samples to dry at room temperature without any disturbance, whereby an aggregate of the DNA complex deposits a strand pattern on the slide; and
j. using the strand pattern to detect a change in the body of the human donor.
10. A method of detecting a change in the body of a human being caused by a pathological condition, comprising:
a. mixing a sample of the human blood with an anticoagulant to form an anti-coagulated blood mixture;
b. centrifuging the anti-coagulated blood mixture in order to separate the plasma from the blood cells;
c. preparing a first blood cell mixture in accordance with the following steps:
i. preparing approximately 5 μl of Tris-buffer;
ii. adding approximately 2.5 μl of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, to the approximately 5.0 μl of Tris-buffer to produce a buffer diluted phenol; and
iii. adding approximately 10 μl of the blood cells to the buffer diluted phenol;
iv. mixing the first blood cell mixture;
d. centrifuging the first blood cell mixture to form a first liquid phase and first blood cell debris;
e. preparing a second blood cell mixture by mixing the centrifuged first blood cell mixture and first blood cell debris with approximately 2.5 ml of chloroform and approximately 2.5 ml of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer;
f. centrifuging the second blood cell mixture to form a second liquid phase and second blood cell debris;
g. cooling the second liquid phase and second blood cell debris thereby causing the structural components of the DNA complex within the second liquid phase to aggregate;
h. placing an acid alcohol sample consisting of approximately 25 μl of freshly made 20% acid alcohol on a slide; and
i. adding a blood cell sample consisting of approximately 1.0 μl of the cooled second liquid phase onto the center of the top surface of the acid alcohol sample and allowing both samples to dry at room temperature without any disturbance, whereby an aggregate of the DNA complex deposits a strand pattern on the slide; and
j. using the strand pattern to detect a change in the body of the human donor.
11. A method of detecting a change in the body of a human being caused by a pathological condition, comprising:
a. mixing a sample of blood from a human donor with an anticoagulant to form an anti-coagulated blood mixture;
b. centrifuging the anti-coagulated blood mixture in order to separate the plasma from the blood cells;
c. preparing a first blood cell mixture in accordance with the following steps:
i. preparing approximately one (1) volume of Tris-buffer;
ii. adding approximately a half (½) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, to the approximately one (1) volume of Tris-buffer to produce a buffer diluted phenol; and
iii. adding approximately two (2) volumes of the blood cells to the buffer diluted phenol;
iv. mixing the first blood cell mixture;
d. centrifuging the first blood cell mixture to form a first liquid phase and first blood cell debris;
e. preparing a second blood cell mixture by mixing the centrifuged first blood cell mixture and first blood cell debris with approximately a half (½) volume of chloroform and approximately a half (½) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer;
f. centrifuging the second blood cell mixture to form a second liquid phase and second blood cell debris;
g. cooling the second liquid phase and second blood cell debris thereby causing the structural components of the DNA complex within the second liquid phase to aggregate;
h. placing an acid alcohol sample consisting of approximately twelve and a half (12½) volumes of freshly made 20% acid alcohol on a slide;
i. adding a blood cell sample consisting of approximately one fifth (⅕) volume of the cooled second liquid phase onto the center of the top surface of the acid alcohol sample and allowing both samples to dry at room temperature without any disturbance, whereby an aggregate of the DNA complex deposits a strand pattern on the slide; and
j. detecting a change in the body of the human donor if the strand pattern comprises a strand which is not smooth throughout most of the strand's length.
12. A method of detecting a change in the body of a human being caused by a pathological condition, comprising:
a. mixing a sample of blood from a human donor with an anticoagulant to form an anti-coagulated blood mixture;
b. centrifuging the anti-coagulated blood mixture in order to separate the plasma from the blood cells;
c. preparing a first blood cell mixture in accordance with the following steps:
i. preparing approximately one (1) volume of Tris-buffer;
ii. adding approximately a half (½) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, to the approximately one (1) volume of Tris-buffer to produce a buffer diluted phenol; and
iii. adding approximately two (2) volumes of the blood cells to the buffer diluted phenol;
iv. mixing the first blood cell mixture;
d. centrifuging the first blood cell mixture to form a first liquid phase and first blood cell debris;
e. preparing a second blood cell mixture by mixing the centrifuged first blood cell mixture and first blood cell debris with approximately a half (½) volume of chloroform and approximately a half (½) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer;
f. centrifuging the second blood cell mixture to form a second liquid phase and second blood cell debris;
g. cooling the second liquid phase and second blood cell debris thereby causing the structural components of the DNA complex within the second liquid phase to aggregate;
h. placing an acid alcohol sample consisting of approximately twelve and a half (12½) volumes of freshly made 20% acid alcohol on a slide;
i. adding a blood cell sample consisting of approximately one fifth (⅕) volume of the cooled second liquid phase onto the center of the top surface of the acid alcohol sample and allowing both samples to dry at room temperature without any disturbance, whereby an aggregate of the DNA complex deposits a strand pattern on the slide; and
j. detecting a change in the body of the human donor if the strand pattern comprises a strand which has a plurality of beads and a substantial discontinuity with associated branching.
13. A method of detecting a change in the body of a human being caused by a pathological condition, comprising:
a. mixing a sample of blood from a human donor with an anticoagulant to form an anti-coagulated blood mixture;
b. centrifuging the anti-coagulated blood mixture in order to separate the plasma from the blood cells;
c. preparing a first blood cell mixture in accordance with the following steps:
i. preparing approximately one (1) volume of Tris-buffer;
ii. adding approximately a half (½) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, to the approximately one (1) volume of Tris-buffer to produce a buffer diluted phenol; and
iii. adding approximately two (2) volumes of the blood cells to the buffer diluted phenol;
iv. mixing the first blood cell mixture;
d. centrifuging the first blood cell mixture to form a first liquid phase and first blood cell debris;
e. preparing a second blood cell mixture by mixing the centrifuged first blood cell mixture and first blood cell debris with approximately a half (½) volume of chloroform and approximately a half (½) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer;
f. centrifuging the second blood cell mixture to form a second liquid phase and second blood cell debris;
g. cooling the second liquid phase and second blood cell debris thereby causing the structural components of the DNA complex within the second liquid phase to aggregate;
h. placing an acid alcohol sample consisting of approximately twelve and a half (12½) volumes of freshly made 20% acid alcohol on a slide;
i. adding a blood cell sample consisting of approximately one fifth (⅕) volume of the cooled second liquid phase onto the center of the top surface of the acid alcohol sample and allowing both samples to dry at room temperature without any disturbance, whereby an aggregate of the DNA complex deposits a strand pattern on the slide; and
j. detecting a change in the body of the human donor if the strand pattern comprises a strand which has a looped portion and a substantial discontinuity with associated branching.
14. A method of detecting a change in the body of a human being caused by a pathological condition, comprising:
a. mixing a sample of the human blood with an anticoagulant to form an anti-coagulated blood mixture;
b. centrifuging the anti-coagulated blood mixture in order to separate the plasma from the blood cells;
c. preparing a first blood cell mixture in accordance with the following steps:
i. preparing approximately 5 μl of Tris-buffer;
ii. adding approximately 2.5 μl of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, to the approximately 5.0 μl of Tris-buffer to produce a buffer diluted phenol; and
iii. adding approximately 10 μl of the blood cells to the buffer diluted phenol;
iv. mixing the first blood cell mixture;
d. centrifuging the first blood cell mixture to form a first liquid phase and first blood cell debris;
e. preparing a second blood cell mixture by mixing the centrifuged first blood cell mixture and first blood cell debris with approximately 2.5 ml of chloroform and approximately 2.5 ml of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer;
f. centrifuging the second blood cell mixture to form a second liquid phase and second blood cell debris;
g. cooling the second liquid phase and second blood cell debris thereby causing the structural components of the DNA complex within the second liquid phase to aggregate;
h. placing an acid alcohol sample consisting of approximately 25 μl of freshly made 20% acid alcohol on a slide; and
i. adding a blood cell sample consisting of approximately 1.0 μl of the cooled second liquid phase onto the center of the top surface of the acid alcohol sample and allowing both samples to dry at room temperature without any disturbance, whereby an aggregate of the DNA complex deposits a strand pattern on the slide; and
j. detecting a change in the body of the human donor if the strand pattern comprises a strand which is not smooth throughout most of the strand's length.
15. A method of detecting a change in the body of a human being caused by a pathological condition, comprising:
a. mixing a sample of the human blood with an anticoagulant to form an anti-coagulated blood mixture;
b. centrifuging the anti-coagulated blood mixture in order to separate the plasma from the blood cells;
c. preparing a first blood cell mixture in accordance with the following steps:
i. preparing approximately 5 μl of Tris-buffer;
ii. adding approximately 2.5 μl of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, to the approximately 5.0 μl of Tris-buffer to produce a buffer diluted phenol; and
iii. adding approximately 10 μl of the blood cells to the buffer diluted phenol;
iv. mixing the first blood cell mixture;
d. centrifuging the first blood cell mixture to form a first liquid phase and first blood cell debris;
e. preparing a second blood cell mixture by mixing the centrifuged first blood cell is mixture and first blood cell debris with approximately 2.5 ml of chloroform and approximately 2.5 ml of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer;
f. centrifuging the second blood cell mixture to form a second liquid phase and second blood cell debris;
g. cooling the second liquid phase and second blood cell debris thereby causing the structural components of the DNA complex within the second liquid phase to aggregate;
h. placing an acid alcohol sample consisting of approximately 25 μl of freshly made 20% acid alcohol on a slide; and
i. adding a blood cell sample consisting of approximately 1.0 μl of the cooled second liquid phase onto the center of the top surface of the acid alcohol sample and allowing both samples to dry at room temperature without any disturbance, whereby an aggregate of the DNA complex deposits a strand pattern on the slide; and
j. detecting a change in the body of the human donor if the strand pattern comprises a strand which has a plurality of beads and a substantial discontinuity with associated branching.
16. A method of detecting a change in the body of a human being caused by a pathological condition, comprising:
a. mixing a sample of the human blood with an anticoagulant to form an anti-coagulated blood mixture;
b. centrifuging the anti-coagulated blood mixture in order to separate the plasma from the blood cells;
c. preparing a first blood cell mixture in accordance with the following steps:
i. preparing approximately 5 μl of Tris-buffer;
ii. adding approximately 2.5 μl of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, to the approximately 5.0 μl of Tris-buffer to produce a buffer diluted phenol; and
iii. adding approximately 10 μl of the blood cells to the buffer diluted phenol;
iv. mixing the first blood cell mixture;
d. centrifuging the first blood cell mixture to form a first liquid phase and first blood cell debris;
e. preparing a second blood cell mixture by mixing the centrifuged first blood cell mixture and first blood cell debris with approximately 2.5 ml of chloroform and approximately 2.5 ml of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer;
f. centrifuging the second blood cell mixture to form a second liquid phase and second blood cell debris;
g. cooling the second liquid phase and second blood cell debris thereby causing the structural components of the DNA complex within the second liquid phase to aggregate;
h. placing an acid alcohol sample consisting of approximately 25 μl of freshly made 20% acid alcohol on a slide; and
i. adding a blood cell sample consisting of approximately 1.0 μl of the cooled second liquid phase onto the center of the top surface of the acid alcohol sample and allowing both samples to dry at room temperature without any disturbance, whereby an aggregate of the DNA complex deposits a strand pattern on the slide; and
j. detecting a change in the body of the human donor if the strand pattern comprises a strand which has a looped portion and a substantial discontinuity with associated branching.
17. A method of detecting a change in the body of a human being caused by a pathological condition, comprising:
a. mixing a sample of blood from a human donor with an anticoagulant to form an anti-coagulated blood mixture;
b. centrifuging the anti-coagulated blood mixture in order to separate the plasma from the blood cells;
c. preparing a first blood cell mixture in accordance with the following steps:
i. preparing approximately one (1) volume of Tris-buffer;
ii. adding approximately a half (½) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, to the approximately one (1) volume of Tris-buffer to produce a buffer diluted phenol; and
iii. adding approximately two (2) volumes of the blood cells to the buffer diluted phenol;
iv. mixing the first blood cell mixture;
d. centrifuging the first blood cell mixture to form a first liquid phase and first blood cell debris;
e. preparing a second blood cell mixture by mixing the centrifuged first blood cell mixture and first blood cell debris with approximately a half (½) volume of chloroform and approximately a half (½) volume of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer;
f. centrifuging the second blood cell mixture to form a second liquid phase and second blood cell debris;
g. cooling the second liquid phase and second blood cell debris thereby causing the structural components of the DNA complex within the second liquid phase to aggregate;
h. placing an acid alcohol sample consisting of approximately twelve and a half (12½) volumes of freshly made 20% acid alcohol on a slide;
i. adding a blood cell sample consisting of approximately one fifth (⅕) volume of the cooled second liquid phase onto the center of the top surface of the acid alcohol sample and allowing both samples to dry at room temperature without any disturbance, whereby an aggregate of the DNA complex deposits a strand pattern on the slide; and
j. using the strand pattern to detect a change in the body of the human donor.
18. A method of detecting a change in the body of a human being caused by a pathological condition, comprising:
a. mixing a sample of the human blood with an anticoagulant to form an anti-coagulated blood mixture;
b. centrifuging the anti-coagulated blood mixture in order to separate the plasma from the blood cells;
c. preparing a first blood cell mixture in accordance with the following steps:
i. preparing approximately 5 μl of Tris-buffer;
ii. adding approximately 2.5 μl of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer, to the approximately 5.0 μl of Tris-buffer to produce a buffer diluted phenol; and
iii. adding approximately 10 μl of the blood cells to the buffer diluted phenol;
iv. mixing the first blood cell mixture;
d. centrifuging the first blood cell mixture to form a first liquid phase and first blood cell debris;
e. preparing a second blood cell mixture by mixing the centrifuged first blood cell mixture and first blood cell debris with approximately 2.5 ml of chloroform and approximately 2.5 ml of Tris-buffer saturated phenol, prepared by mixing re-distilled phenol with Tris-buffer;
f. centrifuging the second blood cell mixture to form a second liquid phase and second blood cell debris;
g. cooling the second liquid phase and second blood cell debris thereby causing the structural components of the DNA complex within the second liquid phase to aggregate;
h. placing an acid alcohol sample consisting of approximately 25 μl of freshly made 20% acid alcohol on a slide; and
i. adding a blood cell sample consisting of approximately 1.0 μl of the cooled second liquid phase onto the center of the top surface of the acid alcohol sample and allowing both samples to dry at room temperature without any disturbance, whereby an aggregate of the DNA complex deposits a strand pattern on the slide; and
j. using the strand pattern to detect a change in the body of the human donor.
US10/313,996 2001-01-17 2002-12-06 Method for processing blood samples in order to produce DNA complex patterns for diagnostic applications Abandoned US20030215832A1 (en)

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