WO1985001960A1

WO1985001960A1 - Improved method of live blood cell analysis

Info

Publication number: WO1985001960A1
Application number: PCT/US1984/001673
Authority: WO
Inventors: James R. Privitera
Original assignee: Livcell Analysis, Inc.
Priority date: 1983-10-31
Filing date: 1984-10-17
Publication date: 1985-05-09
Also published as: EP0160038A1; AU3550684A

Abstract

A method of performing an analysis of a live blood cell preparation to detect and identify pathological conditions that are manifested by pathophysiological alterations of blood cells. The determination of cellular aberrations and inclusions is determined on a live blood cell preparation that are subjected to dark-field microscopic examination immediately after it has been withdrawn from a patient. The method utilizes a microscope with a video camera attachment thereby permitting simultaneously viewing of the live blood cell preparation by both the attending technician and the patient. Further, the invention involves a method of treatment that utilizes natural substances, vitamin and mineral supplements, and modified diets.

Description

IMPROVED METHOD OF LIVE BLOOD CELL ANALYSIS

BACKGROUND OF THE INVENTION The diagnosis of certain diseases or conditions of the body often depends to a large extent on microscopic examination of blood or bone marrow smears in which certain blood disorders appear that are indicative of a disease or condition. Although recent technical advances have essentially automated cell-counting procedures, the clinical hematology laboratory still depends on the visual inspection of smears under the microscope in order to discern blood disorders.

The gradual development of the techniques that led to the present day hematologic practice began over a century ago. Modern techniques basically involve the production of a blood smear on a slide, the differential staining of the slide, which is then followed by microscopic inspection and evaluation. The preparation of a smear on a slide generally involves the use of a "pre-cleaned" slide upon which a drop of blood or dilutent containing the cells is placed. The .cells are then uniformly spread over the slide generally by the use of a coverslip that is brought in contact with the slide and the cells with the slip being moved rapidly from one end of the slide to the other, thereby spreading the cells by capillary action along the line of contact of the slide and coverslip. The thickness of the smear is a function of the speed with which the coverslip is moved across the slide. The smearing technique must be such that in the final film, the cells generally lie separated side by side with some cells being aggregated in small rouleaux. Smears that are made too thick usually result in overstaining and some clumping, making microscopic analysis of the cells practically impossible. The acquisition of a proper smearing technique is essential to clinical hematology and can only be acquired by practical experience. The smears must then be dried rapidly in order to preserve the shape of the majority of the cells; drying may be' accomplished by the rapid movement of the slide, through the air, the use of previously heated slides, or the preparation of smears under an infrared lamp. Care must be taken in both the preparation of the smear and the drying in order to minimize the creation of artifacts making the appearance of cells in the smears different from those occuring naturally in the circulation. Once a properly made smear has been made and the smear appropriately dried, the preparation must then be fixed with a material such as methanol to prevent subsequent changes in the cells such as distortion or hemolysis when subjected to humidity or staining solutions. When the smear has been properly fixed, the

OMP WIP preparation is subjected to certain standard staining procedures. The most widely used staining procedure (Pappenheim) involves the use of May-Grunwald stain followed by a distilled water rinse then subsequently stained with dilute Giemsa solution. Generally, the staining technique must be adapted to the requirements of the individual laboratory based on the pH of the distilled water and the frequency of use and replacement of the staining stock solutions. Another common staining procedure is to use commercially available Wright's Stain. Other special staining and cytochemical (histochemical) methods are also available.

Once the slide has been properly prepared, a routine microscopic examination of the smear specimen is carried out under low objective power to obtain a rapid indication of the quantity and quality of the cells, which is then followed by a microscopic examination using both high dry lens and oil emersion lens to make the necessary cytological diagnosis. Considering the vast number of hematological studies that are performed daily, the development of automated techniques would seem eminent. However, at present, the developments with objectives of automatically reading the cells or entirely eliminating the need for smears, staining and microscopic examination, have met with only limited success. Except for standard cell counting instruments and instruments that have been proposed for the automatic spreading of blood on slides, basic clinical hematological study still involves tedious preparation of smears and the sensitive time-consuming steps of fixing and staining the slide preparation.

Typically, the preparation of a slide requires several hours, and must be accomplished in a laboratory established for that purpose. Therefore, the microscopic examination and evaluation must necessarily occur sometime after the blood has been withdrawn from the patient. Because of the time and special facilities necessary to prepare the slides and perform the microscopic examination, generally the attending physician does not perform the slide evaluation and, further, the results are generally not available until the following day and then presented in such a manner, typically by telephone, as to be useful only to the attending physician and, therefore, have less effect on the patient who does not have an opportunity to view the blood preparations. Thus, in view of the technical expertise required to prepare blood smears for hematological analysis, the necessity of specialized facilities, technicians, and materials, the fact that the attending physican does not perform the evluation of the blood because of the inconvenience, and, therefore, the impossibility of providing immediate results, diagnosis and prescribed treatment to the patient at the time the blood is drawn, it is highly desirable to provide a method by which a clinical hematological study can be done by an attending physican in the presence of a patient at the time the blood is drawn especially to enhance compliance.

SUMMARY OF THE INVENTION Accordingly, the present invention provides a method of detecting, diagnosing, and prescribing treatment for a variety of pathological conditions that are generally manifested in hematological abnormalities. In particular, the invention provides a method by which live blood cells can be subjected to microscopic examination immediately after blood has been withdrawn from the patient and dispensing with the expensive and time-consuming necessity of- preparing a standard blood smear slide. In addition, the method utilizes a microscope with a dark field condenser and with a video camera attachment thereby permitting simultaneous viewing of the live blood cell preparation by both the attending physician and the patient. The method thus permits the attending physician or techician to evaluate immediately the blood preparation, and provides an immediate feedback to the patient on the evaluation and diagnosis of the blood, while offering a visual presentation of the cells

OMPI that the attending physican may use to actually show the patient any abnormalities observed.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1-23 are photomicrographs of live blood cell preparations as viewed under conventional dark field illumination using a 100X oil emersion objective, except for Figure 3 which utilizes a 40X objective; and

Figures 24 and 25 are photomicrographs of sputum preparations viewed under conventional dark field illumination, utilizing a 100X oil emersion objective. DETAILED DESCRIPTION OF THE INVENTION The basis of the present invention described is the detection and identification of pathophysiologically altered cells in a live state. Although these morphological changes and inclusions in cell preparations can be visualized using standard blood smear preparations, the advent of phase contrast, dark field, fluorescent, electron, and scanning electron microscopy have made the identification of many morphological features of cells possible without the need for the more complicated and difficult slide preparation and staining procedures.

In particular, the invention uses dark field microscopy which profoundly enhances morphological characteristics of living cells such as size, shape, and type of cells, as well as major inclusions within the cell and abnormal constituents of the surrounding fluid. The invention has compiled a series of the more common pathophysiological states of living cells that can be identified by morphological changes or abnormal inclusions and has reinterpreted these findings to diagnose a pathological state or condition of the body manifested by the pathophysiological state of the living cells. More importantly, the evaluation of the live cell preparation is done within minutes after having withdrawn the blood from the patient and is accomplished in the presence of the patient such that the attending physician or technician may point out the differences between the normal state and any observed pathophysiological state of the cells. Therefore, the invention provides a blood examination procedure that may also be used by the attending physician to clearly illustrate to the patient abnormalities that may occur in the blood. The patient not only receives diagnostic information that can be readily understood, but the entire procedure of drawing the patient's blood, evaluation of the blood preparation, diagnosis, explanation of the diagnostic results to the patient, and the prescribing of a treatment for any abnormal conditions that may be found, may be accomplished within a short period of time such as less than one hour. The instrumentation used in the microscopic examination of the living cell preparation includes a standard bright field microscope (Bausch and Lomb, Model

DRT3Z1H) that has been equipped with 10X, 20X, 40X, and

100X (oil) planachromatic objectives, and a turret-type condenser which includes dark field capability. The microscope is additionally configured with a conventional video camera that is mounted on the microscope, the image of which-is displayed on a standard television set.

The procedure involved for obtaining a blood specimen and preparing the live blood cell preparation . involves obtaining a sample of blood from a patient using a standard finger prick. In particular, the patient's finger is cleaned well with an alcohol swab then wiped dry with lint-free tissue. Once the finger has been pricked with a lancet and a drop of blood has formed on the finger, the center of a precleaned glass slide is touched gently to the drop of blood taking care not to touch any portion of the finger. The slide is subsequently rotated such that the blood sample is now positioned on the top surface of the slide and a standard coverslip (22x40) is then placed on the slide over the sample of blood. Following standard microscopy techniques, a drop of emersion oil is placed between the slide and the condenser and a 10X objective is used to scan the blood preparation to ascertain that portion of the blood preparation where the blood appears the thinnest and the cells most evenly distributed. A drop of oil is then placed on the coverslip and the 100X oil emersion objective is used for the evaluation of the blood cell preparation, except when thrombocyte aggregations are to be viewed, wherein the 40X objection is occasionally used (without oil). Of course, those skilled in the art will recognize the procedure described above refers to the preparation of a blood sample and that the procedure can be used for the preparation of other samples such as sputum and urine. For the purpose of this application, the term blood sample shall be defined to include blood, sputum, urine, and other body fluid specimens.

The live blood cell preparation is then searched for abnormalities, each of which is separately discussed below:

1. Rouleaux Formation

In the circulation, mature red blood cells

(erythrocytes) have a general biconcave shape. Normally, these red blood cells both in circulation and in a live preparation, form rouleaux in which several red blood cells stack together similar to coins. While in normal plasma, the rouleaux formations are very short, they are markedly enhanced by the presence of high concentration of certain macromolecules, such as fibrinogen, IgM, igA, igG, dextran, and certain other long macromolecules. Rouleaux formations increase the red cell sedimentation rate, and this rate is often used as an indirect measure of fibrinogen, IgM, and other inducing agents. The finding of excessive rouleaux in the live preparation (Figure 1) is indicative that certain macroglobulin levels are high and that the most common causative agent should be sought depending on the excessivity of the rouleaux formation. Since red cells normally must squeeze through capillaries in single file in order to exchange oxygen for carbon dioxide, rouleaux formations would cause less oxygen to be transferred to the cell, therefore, resulting in fatigue. Conventional anti-coagulant therapy has been found helpful in reducing the degree of rouleaux formations. Hydrochloric acid (HC1) and niacin have been shown clinically to separate rouleaux formation, and these natural substances are used for the treatment of rouleaux formations in the present invention.

2. Erythrocyte Aggregation Erythrocyte aggregation, as can be seen in Figure

2, is similar to rouleaux formation except the condition is usually worse and rouleaux formation is often seen ,as clumps of red cells. This condition is obviously worse than rouleaux formation and has a more negative influence -li¬ on oxygen exchange.

Erythrocyte aggregation is caused by abnormal protein levels as described above in rouleaux formation. Similarly, therapy would be directed at reversing or controlling the particular disease entity. Niacin and HC1 are used in the treatment of red cell aggregation because of their ability to separate red blood cells.

3. Thrombocyte Aggregation Recently, thrombocyte (platelet) aggregation has been implicated in various heart conditions and strokes. For example, Barnett, H.J.M., et al. (1980), Randomized trial of therapy platelet anti-aggregants for threatened strokes. Observations on the pathogeneis, and natural history of threatened stroke, CMA Journal 112, 535; Kalendovsky, Zdenka, et al. (1975), Increased platelet aggreability in young patients with stroke. Archives of Neurology 32,12; Valzhakis, Nicolas, et al. (1979), Platelet aggregation in relationship to plasma catecholamines in patients With hypertension, Atherosclerosis 32,451; Mehta, Jawahar, et al. (1981), Platelet function in hypertension and effective therapy, American Journal of Cardiology, 47,331; Al-Mefty, Osama, et al. (1979), Transient ischemic attacks due to increased platelet aggregation and adhesiveness. Journal of Neurology 50,449; and Davis, James W., et al. (1978),

OMP Platelet aggregation, Adult-onset diabetes mellitus and coronary artery disease, JAMA 239, 732. Referring to Figure 3, thrombocyte aggregation can be easily discerned in the live cell preparation. A variety of natural substances have been found to counteract platelet aggregation, for example, vitamin B6, vitamin C, cod liver oil, vitamin E, garlic, onion, and linseed oil, as well as certain minerals such as magnesium and zinc. Treatment for platelet aggregation in the present invention would involve an individualized diet, vitamin, and mineral regimen,in accordance with the above discussion.

4. Spicules Spicule formations, as can be seen in Figure 4, appear as fibrous-type formations and are frequently associated with rouleaux formation or red cell aggregation, and generally consist of fibrin or alpha-2 macroglobulin, which are the heavier proteins in the blood. Spicule formation, and red cell aggregation, frequently occur with chronic degenerative diseases, and are clinically indicative of a weak liver, as with drug, or alcohol usage. The causative fact or factors for the formation of spicules should be sought. Appropriate treatment according to the present invention includes the use of hydrochloric acid and niacin. 5. Chylous Material The presence of chylous material in the live cell preparation, as indicated by the arrow in Figure 5, indicates an abnormally high level of blood fat. Since chylous material can be seen in the live blood cell preparation following meals that are high in fat content, this test is of importance only after a low-fat meal has been consumed. Assuming the chylous material is observed after a small low-fat meal, the test indicates that the patient has high blood fats and/or or a weak liver, that is, disability of the liver to clear fats. Treatment would be geared toward fat emulsification using substances such as lecithin, lipotrophic factors such as choline, inositol, and methionine, and instituting a modified diet which include foods low in saturated fats, such as fish and vegetables, and foods high in certain minerals such as iron, known to be helpful for liver function.

6. L-Forms L-Forms are embryonic bacteria that are indicative of infection when observed in the live blood cell preparation. As can be seen in Figure 6, L-forms are similar to the fat particles or chylous material as discussed above, but are generally larger. Patients having L-forms in their blood, as well as other infections discussed below, often have other characteristic conditions such as feeling low and frequently contracting

OMPI flus, colds, or sore throats.

If the infection is severe, as indicated by the quantity of bacteria observed, antibiotics may be used to treat the infection. If the use of antibiotics is not clinically indicated, treatment in the present invention involves the use of nutritional immune stimulators such as vitamins A, C, E, and B6, and other substances such as zinc, pantothenic acid, and thymus.

7. Rod Forms Rod forms are another type of infection that can be seen in the live blood cell preparation. As shown by the arrow in Figure 7, these bacteria appear almost exclusively outside the red blood cells. As indicated above with the L-forms, the type of treatment often depends on the quantity of rod forms that are observed.

8. Parasitized Red Blood Cells Another type of infection that can be seen in the live cell preparation are red blood cells that are infected usually by embryonic bacteria but also occassionally by rods or cocci (Figure 8). Similar accompanying symptoms are observed with parasitized red blood cells as seen with the other types of infection as discussed above. Also, the treatment of parasitized red blood cells is the same as that described in the L-forms and rod forms above and involve the use of antibiotics if the infection is severe or the use of nutritional immune stimulators with a less severe level of infection.

9. Protoplast Protoplasts, as shown in Figure 9, are believed by Dr. V. Livingston (Microbiology of Cancer, Compendium c, 1977) to be large forms of the pleomorphic "cryptocydes" microbe, and classified by Dr. Livingston under the family actinomycetales and have been found to stain intermittently acid fast. The appearance of protoplasts indicate multiple degenerative diseases such as scleroderma cancer or rheumatoid arthritis. Treatment in part includes the use of immunologic enhancing nutrients such as vitamins A, C, E, and B-6, and zinc, pantothenic acid, thymus, dimethylglycine, and betacarotene. Vaccines and more aggressive nutritional parenteral therapy may be indicated.

10. Plaque Plaque formations, as illustrated in Figure 10, are similar to protoplasts, the later being more transluscent. Plaque formation occurs coincidentally with heart disease and arteriosclerosis and correlates well with poor circulation. Treatment includes low saturated fat diet and supplements such as vitamin E, niacin, and chondroitin sulfate. 11. Red Crystals

OMPI The formation of red crystals, as illustrated in

Figure 11 and identified by characteristic red color (not shown), correlates with toxicity. Red crystals are frequently observed in the live cell preparation from patients involved in heavy drug use (as in the photomicrograph in Figure 11) and are also seen in patients with terminal cancer. These patients will frequently- also have a positive bowel toxemia test (urine indican) . The treatment of the present invention is to remove the toxic crystals and thereby eliminate the toxicity accompanying the crystal formation with digestive enzymes, acidophilous or yogurt, coupled with a conventional bowel cleanser. Vaccines and more aggressive nutritional parenteral therapy may be indicated. 12. Acanthocyte Percentage

Acanthocytes are red blood cells which have lost their discoid shape and which have formed on their surface spicules or burrs of uneven length irregularly distributed over the entire surface. As can be seen in Figure 12, in addition to the burr-like extensions on the acanthocyte, acanthocytes appear smaller than the normal red blood cells having assumed a more spheroidal shape.

The presence of acanthocytes in excess of 1 percent is a valuable diagnostic sign and can indicate certain pathological conditions such as abetalipoproteinemia, disorders of lipid metabolism, alcoholic cirrhosis, and in rare cases of neonatal or other acquired hepatitis. Further, the more deformation of the red blood cell surface, the worse the oxygen exchange at the cellular level. Therefore, if more than 1 percent of these types of red cells are observed in the live cell preparation, the reason for their occurrence should be sought. Treatment includes the use of antioxidants and a low saturated fat diet. 13. Neutrophilic Viability

A characteristic neutrophil, as shown in Figure 13, usually possesses three distinct nuclear segments or lobes. This cell is the primary defense to pyogenic organisms, phagocytizsing the alien invaders. Neutrophils represent more than one-half of the total leukocytes found in a normal live cell preparation, and the activity, and presumably the phagocytoability of the neutrophil, can be evaluated by observing their numbers in the live cell preparation. Further, as indicated by the research of Johnston, et al. (New England Journal of Medicine , August 12, 1982) oxygen is necessary for the phagocytic activity of the neutrophil. The greater the red cell clumping or aggregation, the more inactive the neutrophils appear; and it is assumed that less oxygen is present. Treatment would include hydrochloric acid, vitamins C and A, zinc and

OMPI other immune enchancers as well as dietary manipulation. 14. Hypersegmentation of Neutrophils The hypersegmentation of neutrophil nuclei as illustrated in Figure 14 wherein the neutrophils have five or more lobes and can be routinely diagnosed as megaloblastic anemia. With rare exceptions, megaloblastic anemia responds completely either to parenteral vitamin B-12 or to folate, these disease correlating well with folic acid deficiency (Bills T. and L. Spatz, Neutrophilic Hypersegmentation and Folate Deficiency, American Journal of Clinical Pathology, Vol. 68, 1977). In particular, the finding that any neutrophil has 5 or more nuclear segments, that more than 5 percent has more than 5 segments, or that the majority possess 4 or more segments, signifies megaloblastic hypersegmentation.

In addition, as illustrated by Figure 14 wherein the hypersegmented neutrophil appears as the large white cell in the center of the photomicrogragh, the appearance of ovalocytes (immediately right of the neutrophil) also commonly occurs with megaloblastic anemia.

15. Eosinophils

Eosinophils normally constitute about 5 to 10 percent of the granulocyte population and are normally identical in size to neutrophils. Although they are normally identified in standard size preparations by the

OMPI brilliant orange or red refractile granules that are peroxidase rich, they may also be identified using the microscopic technique of the present invention by their unique nuclear segmentation. As illustrated in Figure 15, the eosinophil being the large cell in the center of the photomicrograph, and comparing this eosinophil in Figure 15 with the neutrophil in Figure 14, it can be seen that the eosinophils generally have one or more typically two large round or potato-shaped lobes; they also appear much brighter than neutrophils.

The population of eosinophils is increased in allergy and parasitic involvement; also, certain tumors . such as eosinophilic granuloma also cause an increase in the production of eosinophils. The cause of the increase in eosinophils should be sought and treated accordingly. The present invention utilizes vitamins A, C, E, and B-6, and pantothenic acid as treatment.

16. Target Cells Target cells are red blood cells of broader diameter and diminished thickness, which have an artifactual bull's-eye appearance when viewed with dark field microscopy as in the present invention, and are illustrated by an arrow in Figure 16. Targeting is most uniform and striking in hemoglobin C disease and interhapatic or extrahapatic obstructive jaundice. In such diseases, target cells are obvious and very numerous, constituting over half the red blood cell population. Targeting can also be seen in normal individuals postsplenectomy and in rare individuals having a familial deficiency of lecithin-cholesteral acyltransferase. Further, in many other disorders, target cells are numerous but intermingled with other aberrant cells; such disorders, include sickle cell anemia, most thalassemic disorders, as well as a feature of iron deficiency (hypochromic) anemia. Once the other aberrant cells are identified, the disorder or anemia is treated using iron supplements.

17. Anisocytosis Anisocytosis is characterized by extreme variations in cell size as indicated in Figure 17; the majority of red cells are conspicuously macrocytic, although mingled with these in less numbers are cells about half-normal in size as indicated by the arrow in Figure 17. Typically, as the anemia worsens, the diverse cell population gravitates towards the extreme in sizes. Generally, this condition of different cell size is caused by a severe anemia such as hypochromic or megaloblastic anemia and is treated, as indicated above, with vitamin B-12, folate, iron supplements, and special diet. 18. Poikilocytosis

OMPI Morphologic heterogeneity that occurs in severly hypochromic red cells is often characterized by variations in shape (poikilocytosis) as well as variations in size

(anisocytosis), as indicated as above. These variations in the shape of red blood cells is illustrated in Figure 18 as indicated by the arrows. Recent work, however, by Bland, et al. (Effect of alpha-tocopherol on the rate of photohemolysis of human erythrocytes, Physiol. Chem. Physics 7:69, 1975) and Horwitt, et al. (Effects of limited tocopherol intake in man with relationship to erythrocyte hemolysis, Am. J. Clin. Nutr. 8:451, 1960) has shown that these cells had an increased tendency to hemolyze, since these cell membranes containing a quantity of unsaturated fat and other fats. The research of Dr. Bland was designed to determine if the cell wall could be protected from oxidative damage and subsequent deformation by treating the patient with antioxidants. Specificly, he administered 600 units of vitamin E which subsequently prevented the oxidated damage. Therefore, based upon this research, the present invention involves the treatment of the poikilocytic red blood cells using an antioxidant such as vitamin E thereby supplementing the apparent deficiency of normal blood antioxidants.

19. Ovalocytes As indicated above in test number 14,

OMPI hypersegmentation of neutrophils, the appearance of ovalocytes has also been associated with iron, folic acid, and B-12 deficiencies. A population of 15 to 25 percent ovalocytes in a live cell preparation, as indicated by the arrows in Figure 19, strongly indicates the presence of hypochromic microcytic anemia, and is generally treated as described above in hypersegmentation of neutrophils.

20. Macrocytes

The occurrence of macrocytes, which are cells that have diameter greater than 9 microns (the average diameter of red blood cell being 7.2 microns) and are indicated by the arrow in Figure 20, is- generally found in B-12 and folic acid deficiencies, myxedema, and in hemolytic anemias. Once the cause of this deformity is determined, based on the appearance of other cells in the live cell preparation, appropriate treatment is prescribed.

21. Microcytes

Microcytes are defined by a red blood cell having a diameter of less than 6 microns and frequently characterized with a mean corpuscular volume of less than 80 to 82 cubic microns. Microcytic cells generally have less hemoglobin than normal cells and they are therefore frequently seen in iron deficiency anemia and spherocytic and mediterranean hemolytic anemias. The microcyte typically appears as indicated by the arrow in Figure 21. Once the cause of the production of microcytes is determined based on the appearance of other aberrant cells in the live cell preparation as discussed above in the macrocyte test, appropriate treatment is prescribed.

22. Fungal Forms In Blood In addition to the detection and identification of pathophysiological conditions of blood, various tests of the present invention also involve the detection and identification of other forms of microorganisms both in. the blood and in sputum. Referring to Figure 22 as indicated by the arrow, there is shown a fungal organism in the live cell preparation. Natural immune stimulators such as biotin, zinc, and vitamins A and C are prescribed for treatment.

23-25. Sputum Preparation The method described in the invention for the preparation of blood for live cell analysis can be easily adapted to analyze samples of sputum for the presence of certain types of pathogenic microorganisms. For example, as shown in Figure 23, there is shown a fusiform of bacteria observed in a sample of sputum taken from the mouth of a patient with the Vincent's infection (trench mouth), gingiva-stomatitis. Treponema was also observed in the same preparation (Figure 24). Various types of other

OMPI fungal forms can also be observed in sputum samples, as illustrated by the arrows in Figure 25.

In summary, the present invention describes a method of performing an analysis of a live blood cell preparation to detect and identify pathological conditions that are manifested by pathophysiological alterations of blood cells. Further, the method dispenses with the expense of time-consuming preparation and staining of the conventional blood smear by utilizing dark-field microscopy thereby permitting the attending physician to examine and evaluate the preparation while the patient simultaneously views the same preparation. This serves both as a means of visually instructing the patient in the observed abnormalities and as a means of providing the patient with an immediate feedback and diagnosis of any observed pathological conditions requiring treatment. In addition, the present invention involves a method of treatment that utilizes natural substances, vitamin and mineral supplements, and modified diets.

Claims

What is claimed is: 1. The method of detecting and treating pathological conditions comprising the steps of: obtaining a blood sample from a patient; applying the sample to a microscope slide; covering the sample on the slide with a coverslip; viewing the sample on the microscope slide through a microscope to view the sample; displaying the microscope field so as to be directly viewed simultaneously by a technician and a patient; identifying blood cell aberations in the sample; informing the patient while viewing the displayed sample of any observed blood cell aberations; and prescribing diet supplements in response to the identification of predetermined cell aberations.

2. The method of claim 1 wherein the simultaneous viewing of the microscope field comprises displaying the microscope field on a video screen.

3. The method of claim 1 wherein the viewing of the microscope slide comprises the use of dark field

OMPI S IPO microscopy .

4. The method of claim 1 wherein said identification step comprises the observation of rouleaux formation of more than three red blood cells per formation.

5. The method of claim 1 wherein said identification step comprises the observation of erythrocyte aggregation.

6. The method of claim 1 wherein said identification step comprises the observation of thrombocyte aggregation.

7. The method of claim 1 wherein said identification step comprises the observation of spicule formations.

8. The method of claim 1 wherein said identification step comprises the observation of chylous material.

9. The method of claim 1 wherein said identification step comprises the observation of L-forms.

10. The method of claim 1 wherein said identification step comprises the observation of rod forms.

11. The method of claim 1 wherein said identification step comprises the observation of parasitized red blood cells.

12. The method of claim 1 wherein said identification step comprises the observation of protoplasts.

13. The method of claim 1 wherein said identification step comprises the observation of plaque.

14. The method of claim 1 wherein said identification step comprises the observation of red crystals.-

15. The method of claim 1 wherein said identification step comprises the observation of acanthocyte percentage in excess of 1 percent of the red blood cells.

16. The method of claim 1 wherein said identification step comprises the observation of the activity of neutrophils.

17. The method of claim 1 wherein said identification step comprises the observation of hypersegmentation of neutrophils, further comprising the observation of any neutrophil having more than five nuclear segments, more than 5 percent having five segments or more, or a majority of neutrophils possessing four or more segments.

18. The method of claim 1 wherein said identification step comprises the observation of eosinophils in excess of 5 - 10 percent of the granulocyte

OMPI population.

19. The method of claim 1 wherein said identification step comprises the observation of target cells.

20. The method of claim 1 wherein said identification step comprises the observation of anisocytes^*.

21. The method of claim 1 wherein said identification step comprises the observation of poikilocytes.

22. The method of claim 1 wherein said identification step comprises the observation of ovalocytes.

23. The method of claim 1 wherein said identification step comprises the observation of macrocytes.

24. The method of claim 1 wherein said identification step comprises the observation of microcytes.

25. The method of claim 1 wherein said identification step comprises the observation of microorganisms.

26. The method of claim 4 wherein said prescribing step comprises prescribing hydrochloric acid and niacin.

27. The method of claim 5 wherein said prescribing step comprises prescribing hydrochloric acid and niacin.

28. The method of claim 6 wherein said prescribing step comprises prescribing vitamins B-6, C, E and cod liver oil.

29. The method of claim 7 wherein said prescribing step comprises prescribing hydrochloric acid and niacin.

30. The method of claim 8 wherein said prescribing step comprises prescribing lecithin and low ' saturated fat diet.

31. The method of claim 9 wherein said prescribing step comprises prescribing vitamins A, C, E, and B-6.

32. The method of claim 10 wherein said prescribing step comprises prescribing vitamins A, C, E, and B-6.

33. The method of claim 11 wherein said prescribing step comprises prescribing vitamin A, C, E, and B-6.

34. The method of claim 12 wherein said prescribing step comprises prescribing vitamins A, C, E, and B-6, and zinc, pantothenic acid, thymus, dimethylglycine, and betacarotene. ^^TREX

OMPI

V- WIPO

35. The method of claim 13 wherein said prescribing step comprises prescribing low saturated fat diets and diet supplements vitamin E, niacin, and chondroitin sulfate.

36. The method of claim 14 wherein said prescribing step comprises prescribing acidophilous and conventional bowel cleansers.

37. The method of claim 15 wherein said prescribing step comprises prescribing antioxidants and a low saturated fat diet cleansers and lactobacillus.

38. The method of claim 16 wherein said prescribing step comprises prescribing vitamins C and A, zinc, and a special diet.

39. The method of claim 17 wherein said prescribing step comprises prescribing vitamin B-12 and folate.

40. The method of claim 18 wherein said prescribing step comprises prescribing vitamins A, C, E, and B-6, and pantothenic acid.

41. The method of claim 19 wherein said prescribing step comprises prescribing iron supplements.

42. The method of claim 20 wherein said prescribing step comprises prescribing vitamin B-12, folate, iron supplements, and a specialized diet.

43. The method of claim 21 wherein said prescribing step comprises prescribing vitamin E.

44. The method of claim 22 wherein said prescribing step comprises prescribing vitamin B-12 and folate.

45. The method of claim 23 wherein said prescribing step comprises prescribing folic acid and

B-12.

46. The method of claim 24 wherein said prescribing step comprises prescribing iron supplements.

47. The method of claim 25 wherein said prescribing step comprises prescribing vitamins A and C, zinc and biotin.