WO1992014149A1 - Apparatus and methods for using hemozoin - Google Patents

Abstract

Diagnostic and therapeutic methods employing birefringent markers of the type characterized by optical or electrical birefringence, such as hemozoin, a metabolite of protozoan parasites in the genus Plasmodium are disclosed. The invention also discloses diagnostic and therapeutic modalities employing birefringent markers complexed with a pharmacologically active and a cell-specific targeting carrier, such as a monoclonal antibody, affinity ligand, liposome, or the like, wherein hemozoin serves first, as a paramagnetic label to facilitate magnetic imaging or magnetic separation and second, as a birefringent marker to determine binding of the complex to a targeted site. Therapeutic modalities for the treatment of malarial infection are also disclosed.

Description

APPARATUS AND METHODS FOR USING HEMOZOIN

Background of the Invention

The present invention relates generally to diagnostic an therapeutic methods employing hemozoin, a metabolite of protozoa parasites in the genus Plasmodium . Four species of the Plasmodiu parasite are known to infect humans with malaria. Plasmodium viva causes tertian malaria; Plasmodium malariae , quartan malaria; Plasmodium ovale , ovale malaria; and Plasmodium falcipazum . cause the most severe, and sometimes fatal, form of the disease, know as pernicious, subtertian, malignant or estivoautumnal malaria.

The life cycle of the Plasmodium organism is well known. See, e.gr., Kissane, J.M. , ed. , Anderson ' s Pathology, 9th ed. , 446-447, 1990. Sporozoites of the malarial parasite invade and incubate i liver cells. The sporozoites evolve in and eventually lyse the liver cells, after which they are free to invade the red blood cells. Within the erythrocyte the parasite ingests hemoglobin and converts the heme portion to malarial pigment. Robbins, S.L., et al. , Pathologic Basis of Disease , 2 ed. , 459-462, 1979.

The most widely used diagnostic method is microscopic examination of thick and thin blood films to determine the existence of stained malarial pigment. This method typically requires about thirty to forty-five minutes in the preparation and examination of each sample and is associated with an error rate of about twenty-five percent.

It is important to note, at the outset, that the literature has, we believe, erroneously interchanged the terms "malarial pigment" and "hemozoin." For purposes of clarity and definition, we regard the term "malarial pigment" to be a brown-black amorphous matrix which is the consequence of hemoglobin digestion by the asexual Plasmodium parasite in the stages of maturing trophozoites, schizonts, merozoites, and gametocytes. Jamjoon, G. , JRev. Infect . Diε . , 10:1029-1034, 1988 has proposed malarial pigment serves as a nutrient and energy source through the gametocytes for the parasite in the sexual stages within the female Anopheles host. The term "hemozoin," as used herein, shall mean a linear polymer of oxidized heme molecules consisting of ferriprotoporphyrin IX with a +3 valence for the iron atom in the high spin mode, a having an adherent investing coat of protein. Hemozoin is bo birefringent and paramagnetic. The term "crystalline hemozoin, as used herein, shall mean hemozoin absent the investing coat protein. Crystalline hemozoin is also both birefringent a paramagnetic.

The literature has, however, focused on two distincti properties of hemozoin, i.e., birefringence under polarized lig and paramagnetic susceptibility, which have been demonstrated t facilitate diagnostic identification. Hemozoin is known to b birefringent under polarized light. Lawrence, C. , et al. , Am. J Clin. Path. , 86:360-363, 1986 has demonstrated that the hemozoi contains brilliantly birefringent granules within the pigment, whe blood smears were examined at 500 X magnification under polarize light. Birefringence was noted in stained and unstained smears Lawrence. et al. demonstrated that intracellular birefringen granules were detected at parasitemia levels as low as 0.0 percent. The anisotropic property of hemozoin was first shown b Schaudinn, F., ArJeiten Kaiεerlichen Gesundheitsant , 19:169-215 1903. Lawrence. et al. , however, appears to be the firs researcher to propose the use of the known anisotropic property o hemozoin for diagnosis of malaria.

Lawrence . et al.. however, clearly recognized that he proposed diagnostic method suffers from an inability to detec early ring forms of the parasite which may contain very little o no pigment. Additionally, spurious sources of birefringence, suc as dust particles on the slide, can result in false positiv indications. She does suggest that a negative^result should be followed by a careful search for early unpig ented ring forms in a stained smear. Prior to Lawrence, et al.. alternative diagnostic methods had been proposed, including dark field microscopy, Ja joon, J. Clin . Microbiol . , 17:717-721, 1983, reflex microscopy, Westphal, A., Zeitεchrift fiir Tropenmedizin und Paras itologie, 13:460-465, 1962, and spot hybridization, Franzen, L., et al., Lancet, 1:525-528, 1984. Jamioon suggests the utility of dark field microscopy for th detection of all forms of malarial parasites, including, the non pigment containing ring forms, in unstained blood films. Th principal advantage offered by the dark field microscopic metho proposed by Jamioon. is its ability to detect ring forms of th malarial parasites which contain much smaller amounts of aggregate hemozoin than the more evolved, highly pigments schizonts an gametocytes. The dark field process proposed by Jam oon requires however, that the thin film sample be prepared on a long cove slip, inverted, and mounted in glycerol while being raised from th slide by the support of two smaller cover slips. While thi arrangement avoids artifacts present on the surface of the slid and the mounting medium, by rendering them out of focus, it i cumbersome and inconvenient. Jamioon suggests that the propose dark field microscopic diagnostic method is more sensitive tha routine bright-field methods due to faster scanning of relativel larger volumes of blood under lower magnifications (X 250 to 400) .

The spot hybridization techniques of Franzen are tim consuming and require a DNA probe. While these techniques are o value in detecting malarial infection, they are not commonl available in third world countries where malaria is most prevalen and are not easily adapted to rapid screening off multiple samples.

The second property of hemozoin which has recognize diagnostic value is its paramagnetic susceptibility. Melville, D., et al., Nature , 255:706, 1975 employed a magnetic separato consisting of a polyethylene cylinder having an inflow and outflo arrangement mounted between the poles of a conventiona electromagnet. Stainless steel wire having a circular cros section and diameter of 251m was randomly packet into the cylinder.

A magnetic field of 1.75 T (17.5 kgauss) was used and an estimate magnetic field gradient of 8 X 10 T/m was established close t the wires. A sample of whole human blood was introduced into th cylinder. It was determined that approximately 70% of the re cells applied to the column were retained until the field was removed.

BSTITUTE SHEET Subsequently, Paul, F., Roath, S., et al. , Br. J. Haemat 38:273-280, 1978, noted that oxygenated hemoglobin, i.e oxyhe oglobin, contains iron i .n the low-spin ferrous state, Fe+2 a exhibits diamagnetic susceptibility. When, however, oxyhemoglob is oxidized to form methemoglobin, which contains iron in the hig spin ferric state, Fe⁺ , it exhibits paramagnetic susceptibilit Paul, F. , et al., Lancet, ii:70-71, 1981, subsequently exploit the difference in magnetic susceptibility between the reduc diamagnetic oxyhemoglobin and oxidized paramagnetic heme produc produced by the Plasmodium organism, as the basis for separati malaria infected red cells from whole blood.

Shortly after Paul, et al. published their work on th separation process, they published a bench top magnetic separato for concentrating Plasmodium infected red cells. Their magneti separator yielded a forty fold parasite concentration usin starting parasitemias of 0.01% and 0.1% infected red cells. Paul F., Melville, D. , et al., IEEE Trans. Mag. , MAG-17:2822-2824, 1981

Fairla b, et al., Mol . Biochem. Parasit. , 12:307-312, 198 proposed a magnetic method for purification of hemozoin. Fairlamb, et al. uses the paramagnetic susceptibility of hemozoi to separate hemozoin from lysed red cells. The lysed cells wer then loaded onto a magnetic separation apparatus. The describe magnetic separation apparatus consisted of a circular C-type Alnic permanent magnetic which generates a field strength of 0.7 Tesl between the poles. The separation chamber was packed wit stainless steel wool and inserted between the poles of th magnetic; an induced magnetic field is established in the stee wool. The lysed cells were run through the separation chamber an exposed to the magnetic field. The paramagnetic hemozoin magnetically binds to the steel wool in the presence of the magnetic field. Removal of the separation chamber from the magnetic field demagnetizes the steel wool and releases much of the magnetically bound hemozoin.

It is known, therefore, that hemozoin, oftentimes confusingly termed malarial pigment, is characterized by birefringence and paramagnetic susceptibility. The characteristics of birefringenc and paramagnetic susceptibility attributed to hemozoin are, i fact, attributable to the crystalline structural arrangement of th components comprising hemozoin. As early as 1956, Deegan, T. , e al., Ann. Top. Med . & Parasit . , 50:212-222, 1956, proposed that th "structure of malarial pigment" consisted of a complex of hemati and a nitrogenous compound. It appears well settled that hemozoi is composed of proteinaceous macromolecules bonded to iron III protoporphyrin IX by coordination bonding, van der Waals forces and hydrophobic interaction, but not by covalent bonding. Yamada, K.A., et al., Exper. Parasit . , 48:61-74, 1979. Transmission electron-microscopy of isolated digestive vacuoles of the parasite reveals a population of "roughly spherical, 1.5 to 2 lm (diameter) organelles, each with a classic lipid bilayer membrane surrounding a striking array of crystals." Goldberg, D.E., et al., Proc. Natl . Acad. Sci . USA, 87:2931-2935, 1990.

Co-pending U.S. Patent Application Serial No. 07/332,328 filed March 31, 1989, of which a co-inventor hereof is the inventor and which is hereby incorporated by reference, teaches it is known that a blood sample may be examined by magnetically concentrating parasitemia cells from whole blood, and examining the separated parasitemia cells to determine the presence of any birefringent hemozoin in the sample. This knowledge, however, provides only a diagnostic method.

Administration of chloroquine, which is believed to interfere with the parasitic degradation of hemoglobin into hemozoin (Yayon, A., et al., J. Biol . Chem . , 259:8163-8167, 1984), is currently the therapeutic method of choice. A number of alternative therapeutic methods have been suggested, including, sporozoite vaccines, immunogenic blockade of merozoite invasion of erythrocytes, immunogenic blockade of cytoadherence between a P. falciparum infected cell and the vascular endothelium, or transmission blocking vaccines which induce an immunogenic host response to parasitic gametocytes, or to development of zygotes and ookinetes in the mosquito. Miller, L.H. , et al., Science , 234:1349-1356, 1986 offers an excellent review of these various possible therapeutic methods. While this variety of therapeutic methods have been proposed, malaria therapy is still substantially limit to administration of chloroquine, which is believed to interfe with the parasites degradation of hemoglobin into hemozoin. Orji A.U. , et al., Science, 214:667-669, 1981.

The most sever infective parasite, Plasmodium falciparu causes death usually due to vascular occlusion in the cerebr capillaries. The parasitized erythrocytes are characterized knob protrusions of the erythrocyte membrane and an underlyi cytoskeleton. These cytoskeletal knobs cause cytoadherence betwee the erythrocyte membrane and the vascular endothelium. Miller, e al. These knobs seem to bind specifically to an endothelia receptor, proposed to involve thrombospondin, which, in turn, lead to accumulation of the red cells within the visceral and cerebra capillaries. Kissane, J.M. , ed. , Anderson ^rs Pathology, 9th ed. 446-447, 1990. Miller, et al. discloses the involvement of malarial protein of about 300 kDa, which has been identified on th surface of a P. falciparum infected red cell, as a possibl cytoadherent group based upon its reactivity to antisera sensitivity to tryptic cleavage at the surface of the red cel coupled with loss of cytoadherence, and that knob-less variant which are not cytoadherent do not express this surface protein There are, however, no known therapeutic methods to prevent bindin of the cytoskeletal knobs, present in an infected erythrocyte, t the vascular endothelium.

Finally, the use of magnetic albumin microspheres has bee explored as drug delivery vehicles. Widder, K. , et al., J. Pharm . Sci . , 68:79-82, 1979 was the first group to synthesis human seru albumin microspheres, average diameter of 1 lm, in which magnetit and doxorubicin were entrapped. Wider, et al. demonstrated tha the magnetic microspheres, when injected intravascularly, could b localized to a desired target site in vivo and in the presence of an externally applied magnetic field. It was found that the intra- arterial injection of the magnetic microsphere carrier-delivered doxorubicin resulted in the same target site concentrations as a 100 fold higher dose of free doxorubicin administered intravenously. Subsequent work by Widder, K.J. , Senyei, A.E., et al., J. Pharm . Sci . , 71:379-387, examined the use of liposomes having included natural or synthetic glycolipids, and the use o immunoglobulins to direct liposomes and an associated antitumo drug to a targeted site. Widder & Senvei again proposed the us of magnetic albumin microspheres as an alternative drug carrie targeted by application of an external magnetic field. Gupta P.K., et al., Intl . J. Pharm . , 43:167-177, 1988, have synthesize adriamycin-associated bovine serum albumin-magnetic BS microspheres and demonstrated the release of adriamycin may b controlled by altering their stabilization temperature. Arshady R. , J. Cont . Rel . , 14:111-131, 1990, offers an excellent review o the known methodologies for manufacturing albumin microspheres an microcapsules, and is hereby incorporated by reference. Thus, th chemistry for creating magnetic microspheres linked t pharmacologically active substances is well-known to those skille in the art. The biological fate of magnetite, ferrosoferric oxide, in vivo after carrier administration, is, however, unknown.

Summary of the Invention

Hemozoin's characteristics of birefringence and paramagneti susceptibility, render it an ideal candidate for incorporation int a drug delivery system. A use which has heretofore not been know or suggested. Because Qf the structure of hemozoin postulate herein, the crystalline hemozoin molecule offers a multitude o available binding sites, which may be linked to pharmacologicall active compounds, such as chemotherapeutic agents, to affinit ligands, to monoclonal antibodies or to DNA probes, in accordanc with well-known methodologies. The characteristics o birefringence and paramagnetic susceptibility, further permi diagnostic and therapeutic screening of body fluids or tissue to aid in determining the targeting and incorporation of the pharmacologically active compounds into the targeted site.

Accordingly, the present invention provides a diagnostic method which employs hemozoin or crystalline hemozoin bound to a targeting carrier, as a birefringent and paramagnetic label. The present invention also provides a related therapeutic method which employs hemozoin or crystalline hemozoin bound to a pharmacologically active substance, and bound to a targeting

^• SUBSTITUTE SHEET carrier, for delivering the pharmacologically active substances a targeted body area.

In order to optimize utility of hemozoin or crystalli hemozoin in the foregoing diagnostic and therapeutic methods, th present invention includes a purification methodology for purifyin hemozoin by degrading the proteinaceous material associated wit hemozoin, to yield crystalline hemozoin, which is substantiall free of an associated protein sheath.

The present invention further presents a novel means for usin hemozoin in the development and screening of monoclonal antibodie and DNA gene probes. Because of its birefringent and paramagneti properties, hemozoin is an ideal label for monoclonal antibodie and DNA probes. The cellular internalization of monoclona antibodies, as well as their cellular specificity and bindin affinity can be readily determined based upon magneti concentration of hemozoin bound cells and subsequent ligh microscopy under polarized light to determine birefringence.

Hemozoin, either in crystalline form or with an investin protein coat, therefore, acts as a label and can be attached t monoclonal antibodies, affinity ligands or bioactive substances, and will replace conventional fluorescent labels, radioactiv labels, and chromogen labels as presently used in immunoassays and immunohistochemistry. Moreover, because of its paramagnetic susceptibility, hemozoin may be used as a paramagnetic label to impart paramagnetic properties to nonmagnetic cells and bioactive substances. The imparted paramagnetic property permits the separation of cell sub-populations for diagnostic assays or in automated flow-through instrumentation, such as flow cytometry. The birefringent characteristics of a labeled cell or bioactive substance, either alone, or in combination with known fluorescent, radioactive or chro ogenic markers, will facilitate differential speciation of cell sub-populations.

Closely related to the foregoing diagnostic, therapeutic and developmental test methods, the present invention presents a novel kit for magnetic separation of hemozoin bound cells or cellular material in a sample or for diagnosing malarial infection b detecting the presence of hemozoin complex in a blood sample. Thi kit permits, for the first time, accurate diagnosis of malaria infection at the ring or early trophozoite (non-hemozoi containing) stage.

The present invention also presents a novel malaria therapeutic modality which is based upon blocking parasitemi degradation of hemoglobin into hemozoin, and, hence, destroying th parasite by obstructing the catabolism of hemoglobin or by blockin the aggregation of heme into hemozoin, thereby allowing the letha heme to injure the parasitic membrane and causing the parasite t self destruct.

These and other objectives, features and advantages of th present invention will become more apparent to those skilled in th art from the following more detailed description of the invention with reference to the preferred embodiments, thereof.

Brief Description of the Drawings

Figure 1 is a scanning electron micrograph, at 30,000 magnification, of aggregated hemozoin molecules with an investin protein coat.

Figure 2a is a scanning electron micrograph, at 250,000 magnification, of a single hemozoin molecule showing the investin protein coat and the underlying crystalline hemozoin.

Figure 2b is a negative stain scanning electron micrograph at 200,000 X magnification, of a hemozoin preparation treated wit 4M urea illustrating the investing protein coat removed from th underlying crystalline hemozoin molecule.

Figure 3a is a diagrammatic representation of the deduce molecular structure of a single hemozoin molecule.

Figure 3b is a diagrammatic representation of the deduce three dimensional molecular structure of a crystalline hemozoi polymer.

Figure 4 is a transmission electron micrograph, at 30,000 X magnification, of a malarial infected erythrocyt showing the development of intra-erythrocytic cytoskeletal knobs.

Figure 5 is a perspective view of a magnetic separation apparatus in accordance with the present invention.

Figure 6 is a diagrammatic illustration of an automated hematology multi-parameter analyzer which has been extended to diagnose malaria in accordance with the present invention.

Figure 7a is a micrograph illustrating binding specificity of hemozoin-linked CTMO-1 monoclonal antibody to mouse erythrocytes under light microscopy.

Figure 7b is the micrograph of Figure 7a taken under polarized light. Figure 8a is a micrograph illustrating binding specificity o hemozoin-linked rabbit anti-mouse monoclonal antibody to mous erythrocytes under light microscopy.

Figure 8b is the micrograph of Figure 8a taken under polarize light.

Figure 9 is a micrograph illustrating binding specificity o hemozoin-linked CTMO-1 monoclonal antibody to mouse B cells unde polarized light microscopy.

Detailed Description of the Preferred Embodiments The structure of hemozoin and crystalline hemozoin i important to the usefulness of hemozoin and crystalline hemozoi in the diagnostic and therapeutic methods of the present invention. Moore, G.A. , et al., Ann . Trop. Med. & Parasit . , 68:488-489, 1974, demonstrated by transmission electron microscopy that hemozoin ha a crystalline core comprised of layers of lattice work oriente parallel to the central axis of symmetry of the hemozoin. Figur 1 is the inventor's scanning electron micrograph of isolate hemozoin, clearly illustrating its linear structure havin dimensions of about 11 to 1.51 x 0.51. Moore. et al. describe hemozoin as having a periodicity for iron atoms at intervals of 10

(lnm). Ashong, I.P., et al., Tran . Royal . Soc . Trop. Med . Hyg. ,

83:167-172, 1989, suggests that glycine-rich proteins may for sheets between adjacent ferriprotoporphyrin molecules, which ar trapped by noncovalent hydrophobic attraction. These glycine-ric sheets may act as spacer molecules which give rise to the 10A (lnm) periodicity noted by Moore, et al. Alternative ligands oriented perpendicular to the plane of the pyrrole groups of each ferriprotoporphyrin IX molecule in the hemozoin molecule may include oxo, hydroxo, aquo, amide, phospho or disulfide.

Figures 2a and 2b are scanning electron micrographs taken by the inventors. As illustrated in Figures 2a and 2b, the ferriprotoporphyrin IX polymer has an intimately adherent investing coat of laminar proteins, from which there is a protruding portion of the underlying crystalline hemozoin. The molecular structure of crystalline hemozoin, underlying the investing coat of protein.

ITUTE SHEET offers numerous available binding sites at each of the thir hydrogen atoms and eight hydrocarbon chains, consisting of fo methyl, two vinyl and two propionate chains, on each heme molecu within the polymer. Hemozoin may be isolated from infected cel according to any one of a known variety of methodologies describ above, each of which are hereby incorporated by reference. F example, Fairlamb. et al. discloses the extraction of malari pigment from infected red cells by saponin lysis a centrifugation. The resulting pellet is ground in a morta Ground samples are withdrawn and diluted with buffer a centrifuged. The supernatant is loaded onto a magnetic separati device. Paramagnetic hemozoin in the sample binds to the ste wool in the magnetic separator. Removal of the magnetic fie releases the bound hemozoin for collection.

Removal of the investing protein coat increases t aggregation of the resulting crystalline hemozoin. Removal of t investing protein coat is achieved by treating it with pronase, mixture of proteolytic enzymes, at 37° C. in 0.1M TRIS-HC1 buffe at pH7.5. Due to its hydrophobic nature, crystalline hemozoin ha a marked tendency to aggregate in the presence of water. To preven aggregation, crystalline hemozoin may be maintained in sodiu dodecyl sulfate (SDS) , non-ionic detergents, or other cationic o anionic detergents except deoxycholate, may be associated wit uniformly charged species, which are readily disassociated, suc as by cations, or a uniform protein coating, such as albumin, ma be imparted on the crystalline hemozoin in accordance with know methodologies.

Figure 3a represents the deduced structure of a singl crystalline hemozoin molecule, i.e., the ferriprotoporphyrin I polymer. The structure illustrated in Figure 3a was deduced fro the known hemozoin's known characteristics of paramagneti susceptibility, birefringence and linear structure Ferriprotoporphyrin IX is known to have unique valence electro orbital configurations in the 3d subshell, which are characterize by five unpaired outer orbital electrons, each with identica orbital spins. These orbital spins are algebraically additive i each planar molecule of ferriprotoporphyrin IX and, therefore, throughout the linear polymer. This unique electron arrangeme is in compliance with both the Pauli Exclusion Principle and Hund' Rules.

These known characteristics indicate that the linearl arranged planar molecules of ferriprotoporphyrin IX ar periodically arrayed along a central axis of symmetry which passe through the central site of the Fe atoms in each molecule. Th spacer molecules such as that proposed by Ashonq. et al.. or othe spacer molecules such as those proposed herein, maintain th precise periodic distance between two facing pairs of th ferriprotoporphyrin IX molecules, which is consistent with th polymer's marked birefringence under polarized light. In addition the magnetic dipole of each Fe⁺ atom at the center of each plana ferriprotoporphyrin IX molecule may contribute to stabilization o the periodic distance between facing molecular pairs.

Each heme molecule contains thirty hydrogen atoms and eigh hydrocarbon chains, consisting of four methyl, two vinyl and tw propionate chains. It is believed that hydrophobic interaction and hydrogen bonds, mediated by the methyl, vinyl and propionat side chains of the tetrapyrrole ring of the ferriprotoporphyrin I molecule, may act with each other and the protein coat to furthe serve to stabilize the linear hemozoin polymer.

Molecular size calculations, however, fail to explain th disparity between the calculated dimensions of a single polymeri molecule of ferriprotoporphyrin IX, as illustrated in Figure 3a and the confirmed dimensions of hemozoin. Figure 3b illustrate the deduced structure of crystalline hemozoin. It is proposed tha each crystalline hemozoin molecule consists of a plurality o ferriprotoporphyrin IX polymers aligned in a three-dimensiona array. A plurality of ferriprotoporphyrin IX polymers form sheet in the X and Y axes, and overlying sheets of th ferriprotoporphyrin IX polymers stack to form the Z axis of th crystalline hemozoin. Within the X-axis, the arraye ferriprotoporphyrin IX polymers are thought to be axially aligne through the central Fe in each ferriprotoporphyrin IX molecule. In both of the Y and Z axes, the ferriprotoporphyrin IX polymer are thought to be laterally adjacent. The entire crystalli hemozoin molecule is stabilized by interactions between the si chains of each ferriprotoporphyrin IX molecule.

Chaotropic agents, such as urea, which have electric dipo moments greater than water (water = 1.8, urea = 4.6) perturba hydrophobic interactions. This has been confirmed with sickl erythrocytes by Nalbandian, R.M. , et al., The Amer. J. Med. Sci . 261:325-334, 1971; Nalbandian, R.M. , et al., Amer. J. Path. 64:405-412, 1971; and Nalbandian, R.M. , et al. , The Amer. J. Me Sci . , 261,309-324, 1971. Sherman, I.W., et al., J^". Protozool . 7:409-416, 1960 demonstrated that urea was important as a vehicl for solubilizing P. lophurae hemozoin. Nalbandian, R. , et al. Am. J. Hematol . , 15:147-151, 1983, demonstrated that Piraceta dislodged sickled cells from the endothelial wall by depolymerizin polymerized hemoglobin within the sickled erythrocytes. Each o the foregoing are hereby incorporated, in relevant part, b reference thereto. Other membrane active compounds which hav electric dipole moments greater than that of water, such a Piracetam (N00TR0PIL by UCB, Brussels, Belgium, 2-oxy-l pyrrolidin acetamide; electric dipole moment = 5.6), nicotinic acid, ascorbi acid or even chloroquine.

It is known that Plasmodium falciparum forms intra erythrocytic cytoskeletal knobs which specifically adhere to th post venule endothelium, ultimately resulting in vascula occlusion. Miller et al.. A preferred diagnostic and therapeuti strategy includes diagnosing Plasmodium falciparum infection b administering sub-therapeutic dosages of a membrane active compoun to a subject suspected of malarial infection. The subject's bloo levels are monitored to determine whether there is any shedding o the Plasmodium parasite into the blood stream. Once Plasmodiu infection is confirmed, therapeutic dosages of at least on membrane active compound may be given to have malariacidal effect. Alternatively, it is desirable to formulate a cocktail consistin of multiple membrane active compounds, each at lower relative molarities. A mixture of such agents reduces the possibility of adverse side effects from any single agent while gaining overall efficacy. A therapeutically effective dosage level will promote de polymerization or blockade of polymerization of ferriprotoporphyri to hemozoin, which will increase intra-erythrocytic levels of heme resulting in killing of the infected erythrocytes prior to maturit of the infection. There will also be reverse binding of th cytoskeletal knobs to the vascular endothelium as well a interference with merozoite invasion of healthy erythrocytes du to action of the chaotropic agents at the red cell membrane and/o interference with pinocytotic uptake by the red cell.

It has been found in Plasmodium falciparum tissue cultures, that treatment with 0.6M urea or 0.01M to 0.02M Piracetam exhibite malariacidal activity. Tables 1 and 2 detail the results of a fou day test of the malariacidal action of urea and Piracetam o Plasmodium falciparum . Plasmodium falciparum was plated into RPMI liquid growth medium with human serum and human red cells. The original samples were divided into two groups, Group 1 fo treatment with Urea and Group 2 for treatment with Piraceta (NOOTROPIL, UCB, Brussels, Belgium) . Group 1 was divided into fiv titers, a control, 0.2M, 0.4M, 0.6M,and 0.8M; Group 2 was divide into seven titers, a control, 0.02M, 0.04M, 0.06M, 0.08M, 0.1M an 0.2M. After twenty-four hours from inoculation, parasitemia dat was collected for four additional consecutive twenty-four hou periods.

• ET TABLE 1

Urea (M) Day 1 Day 2 Day 3 Day 4

TABLE 2

Piracetam (M) Day 1 Day 2 Day 3 Day 4

Urea therapy of malaria will not precipitate the clinica paroxysms of malarial crisis. The elevated and maintained BU levels will kill the parasites in situ within the erythrocyte which, in turn, will be removed from the systemic circulation b the reticulo-endothelial system. No pyrogenic or foreig substances are introduced into the blood, as occurs when th Plasmodium schizont stage perforates and releases merozoites int the blood precipitating a malarial crisis.

Since malarial patients suffer paroxysms of fever, perspiration, chills and rigors, the side effects of urea therapy, such as somnolence and sedation, are desirable. A marked diuresi will occur, but is easily managed by routing fluid and electrolyt management.

By way of example, and not intended to limit the scope of this preferred embodiment of the invention, UREAPHILE (Abbott Laboratories) , consisting of urea in 5% glucose/water, is administered intra-vascularly to a subject having confirmed malarial infection. BUN levels are regularly monitored until a BUN level of about 200 mg/% is achieved. The 200 mg/% BUN level maintained and the subject is observed until the clinical malari paroxysms have abated. Diuresis is managed by water a electrolyte balance regimen.

Once isolated, crystalline hemozoin may be chemically link to pharmacologically active substances, such as chemotherapeut agents, and formed into or in association with albumin microspher or microcapsules in accordance with the methods described Arshadv. Widder. et al. or Gupta, et al.. which are here incorporated by reference. Alternatively, the crystalli hemozoin-pharmacologically active substance complex may be boun to monoclonal antibodies or affinity ligands as an antigen-specifi targeting carrier. For example, Vernes, A., et al., Am. J. Trop Med. , 33:197-203, 1984, which is hereby incorporated by reference identified a mechanism of strain-specific blockade of Plasmodiu falciparum invasion of erythrocytes by human antibody reacting a the surface of merozoites. Perkins, M.E., J. Exp. Med. , 160:788 798, 1984 confirmed that merozoite binding is a receptor-mediate event and identified 130 kDa and 155 kDa glycophorin bindin proteins on the surface of the merozoite. Orlandi, P.A. , et al. Mol . Biochem . Parasit . , 40:285-294, 1990, also incorporated b reference, has characterized a 175 kDa Plasmodium falciparu erythrocyte binding antigen (EBA-175) by raising polyclona monospecific antibodies against EBA-175. Orlandi, et al demonstrated by comparative immunoprecipitations that EBA-175 i immunologically distinct from other well characterized merozoit surface antigens. These other merozoite surface antigens includ the 130 kDa glycophorin binding protein identified by Perkin an Pf200, the major merozoite surface glycoprotein which gives ris to several soluble processed products which interact with ligand at the surface of erythrocytes.

Thus, from the foregoing it is clear that monoclona antibodies have been devised which are specific to Plasmodiu falciparum . Examples of other known clinically relevant monoclona antibodies which have been developed include monoclonal antibodie with specificity for malignant mammary tumor, malignant melanoma breast cancer, cervical carcinoma, lung tumor, measles virus

^• Hemophiluε influenzae polysaccharide, pneumococcal polysaccharid influenza virus nucleoprotein and Mycobacterium leprae . T general hybridoma technology for yielding monoclonal antibodies well-described in the scientific literature. See, e .g. , Kohle G. , et al., Nature, 265:465-499, 1975 and Nowinski, R.C., et al. Science, 219:637-644, 1983 the original papers on hybrido technology; or Barrett, J.T., Textbook of Immunology. Introducti to Immunochemistry and Immunobiology , C.V. Mosby, St. Louis. IS 0-8016-0504-0, each of which are hereby incorporated by referenc

Thus, in accordance with a preferred embodiment of t invention, at least one of crystalline hemozoin having bound o chemically linked pharmacologically active substances, crystallin hemozoin formed with a uniform investing coat of protein, to whic there are linked pharmacologically active substances, crystallin hemozoin having chemically linked pharmacologically substances an formed into or in association with a microsphere or microcapsule is bound to a selected monoclonal antibody for targeted deliver of the pharmacologically active substance. The chemical linke between the crystalline hemozoin and the pharmacologically activ substance is stable until the complex binds to the surfac receptors of a target cell and is taken up by pinocytosis o membrane transport.

Similarly, in place of a monoclonal antibody, an affinit ligand specific for selected receptor types may be bound to a least one of crystalline hemozoin having bound or chemically linke pharmacologically active substances, or crystalline hemozoin forme with a uniform investing coat of protein, to which there are linke pharmacologically active substances, crystalline hemozoin havin chemically linked pharmacologically substances and formed into o in association with a microsphere or microcapsule.

Widder & Senvei, hereby incorporated by reference, note tha IgG and F(ab)₂ fragments have been covalently linked to liposome via oxidized glycolipids present in the bilayer of the liposome. Where F(ab)₂ specific for red cells were employed, a high degree of specificity for red cells was observed in vitro . Monoclonal antibodies were rendered hydrophobic and linked to the liposome with subsequent in vitro specificity, or were covalently linked v phosphatidylethanolamine in the liposome. Widder & Senve however, recognized that increased binding to the target cell do not ensure greater efficacy of the associated antitumor drug, ev in vitro.

Recently, U.S. Patent No. 4,885,172 disclosed that t glycoprotein streptavidin, which is a common biochemical linke can be coupled to any type of liposome, for the purpose of dr delivery. This patent further disclosed that streptavidin may additionally coupled to monoclonal antibodies of proteins, such Immunoglobulin G, to bioactive agents, including drugs, f therapeutic purposes, or dyes for diagnostic uses.

Similarly, the hemozoin polymer, either in crystalline for or with a substantially uniform investing protein coat, may b bound to other macromolecules, such as dyes, e .g. , acridine orange to assist in the diagnosis of disease states or for imagin applications.

The methyl, vinyl and propionate groups on eac ferriprotoporphyrin IX molecule within the hemozoin polymer serv as binding sites for attaching the polymer to carbohydrates of th heavy chain of monoclonal antibodies, amino groups on the heavy o light chains of monoclonal antibodies, to sulfhydryl groups on th monoclonal antibodies, amino groups, carboxylic acid groups o phosphate groups of the nucleic acids or their derivatives of gen probes, or reactant moieties, such as sulfhydryl groups, of variety of pharmacologically active substances. Binding hemozoi or crystalline hemozoin to a monoclonal antibody, liposome or t an affinity ligand, as well as to a drug, permits verification o the uptake of the drug by the targeted cell by i) magneti separation of the targeted cells and ii) light microscopy of th separated cells under polarized light to determine the presence o birefringent hemozoin. Hemozoin may also be bound t radionucleosides for medical imaging, chemotherapy and/o radiography. The association between hemozoin or crystalline hemozoin wit a monoclonal antibody or affinity ligand, and a drug, under th present invention, therefore, readily facilitates testing for th internalization of the monoclonals, specificity and affinity of th monoclonals, internalization and membrane trafficking in the cell and targeted delivery of the monoclonals, affinity ligands an associated drugs, both in vitro and in vivo. Additionally, thos skilled in the art will appreciate, from the foregoing, tha hemozoin, crystalline hemozoin or complexed hemozoin, will als serve as a paramagnetic and birefringent label for screening o monoclonal antibodies, purification of monoclonals and isotypin of monoclonal antibodies. Moreover, because of its paramagneti susceptibility, hemozoin, crystalline hemozoin, or complexe hemozoin may be used to give magnetic susceptibility to non magnetically susceptible cells.

The paramagnetic and birefringent properties of hemozoin an crystalline hemozoin facilitate use of the molecule as a extracorporeal diagnostic marker when linked to monoclona antibodies which are cell or species specific. In accordance wit an embodiment of the inventions, therefore, it is contemplated tha a class of birefringent markers which may be non-magnetic or naturally or derivitized to have magnetic properties such as diamagnetism or paramagnetism. Those skilled in the art will understand that hemozoin and crystalline hemozoin are but one example of potential members of this class. The class of birefringent materials for in vitro diagnostic use is characterized by either optical or electric birefringence and have available binding sites for binding to monoclonal antibodies, affinity ligands or liposomes. Where a particular birefringent material has magnetic properties, it will have additional utility as a means for separating bound cells from a cell sample in the presence of an applied magnetic field, which permits sequestration and concentration of the cell sample.

The use of birefringent materials bound to cell or species specific monoclonal antibodies, affinity ligands, liposomes, gene probes etc. , can replace or supplement the use of fluorphores in fluorescence microscopy. Fluorescence microscopy is a widely used, expensive diagnostic technique which requires the diagnostici have relatively expensive system requiring a the generation of filtered specific high frequency stimulating wave length of lig in the u.v. range, and a series of filters which sequentially pa longer wave lengths of light to avoid injury to the observer. contrast, use of birefringent markers requires only the use of polarizing filter with a light microscope.

The use of optically or electrically birefringent marke coupled with site directed monoclonal antibodies, gene probe affinity ligands, etc. facilitates diagnosis of the presence neoplastic cells, circulating cellular elements, i . e erythrocytes, leukocytes, platelets, cell surface antigen bacteria, fungi, parasites, viruses, histoarchitectural tiss components, e . g. , elastin, collagen, amyloid varieties, endotheli cells, reticuloendothelial cells, or the like.

When tagged with a birefringent marker, the presence of particular molecular structure in a sample may be verified und polarized light microscopy, irrespective of whether the sample a wet cell preparation, frozen tissue section, formalin or alcoh fixed tissue section, thin or thick blood smears, such as f malaria or sickle cell diagnosis, and pap smears. Non-exclusi examples of current diagnostic tests which will benefit from u of birefringent markers include i) malaria diagnosis, whi currently employs thick and thin blood smears; ii) blood groupi and subgrouping, which is currently accomplished by agglutinati techniques; iii) HLA typing, which is currently performed fluorescence microscopy; iv) paternity testing, which is mo commonly accomplished by agglutination techniques; v) diagnost coagulopathy which is evidenced by the presence or absence platelet/endothelial surface active factors; and pap smears.

Examples of known birefringent materials suitable for use diagnostic markers, in accordance with the present inventio include polyurethane fibers, polypropylene, nylon beads or fibers frozen agarose gel, amylose complexes with polar and non-pol compounds which form crystalline complexes, polyethylene or ult high molecular weight-low molecular weight polyethylen polyethylene blend gels, polyamide spherulites an poly(aminocarboxylie acid) type polyamide spherulites, fibrin polydiethylsiloxane, proteoglycan, hyaluronic acid, collagen, variety of polypeptides, including, without limitation, poly-γ benzyl- -glutamate, sodium dodecyl sulfate complexes o transferrin, bovine serum albumin, fumarase, ovalbumin chymotrypsinogen, pepsin, β-lactoglobulin, hemoglobin or myoglobin Stein, et al . , Ann . Rev. Phyε . Chem. 24:207-34 (1973) provides good review of the optical properties, and in particular th birefringent properties, of various polymers and is hereb incorporated by reference.

In addition to the birefringent property, it may be desirabl to employ a marker with paramagnetic properties to facilitat magnetic separation. Such markers may be naturally paramagnetic, such as hemozoin, crystalline hemozoin, or fibrin, or may b derivitized by attachment of the birefringent marker to a magneti bead or marker, such as by attachment to a magnetized particle, as taught by U.S. Patent No. 4,910,148 to Kvalheim, disclosing cellular attachment to magnetic particles; PCT application No. WO 9101368 to Mechaelsen, disclosing hapten and anti-hapten bound to agarose beads and magnetic particles; PCT application No. WO 9006045 to Homes, disclosing attachment of nucleic acid probes to paramagnetic particles for hybridization assays and oligonucleotide sequencing; PCT application no. WO 9006044 to Homes, disclosing production of cDNA by contacting mRNA with an magnetic support; and Homes PCT application WO 9006042, disclosing the use of magnetic particles for carrying a 5• attached DNA probe during the polymerase chain reaction, each of which is hereby incorporated by reference.

Additionally, as exemplified by U.S. Patent No. 4,331,784 to Ishibashi, incorporated hereby by reference, it is known to bind polybutadiene-coated polystyrene particles to antibody proteins to serve as a support for immunological diagnostics. Thus, binding of polymers to antibodies is known in the art and binding of magnetic particles to various biological material is known in the art. By selecting an optically or electrically birefringe particle from the class of birefringent particles meeting t above-mentioned criteria, and coupling the birefringent partic with a monoclonal antibody with or without an accompanyi paramagnetic particle, the present invention affords those in th art a significant advance over the current methods of choice fo in vitro diagnostics, i . e . , fluorescence microscopy.

The following examples of uses for hemozoin are merel intended to illustrated uses and are not intended to be, nor shoul they be construed to be, limiting of the scope or spirit of th invention.

EXAMPLE 1

Monoclonal antibodies which are either species-specific and/o stage-specific for each species, for a known disorder, are prepare according to known techniques. The monoclonal antibodies are mixe with either crystalline hemozoin, or hemozoin prepared with substantially uniform investing protein coat to form a hemozoin monoclonal antibody complex. A blood sample is drawn from subject. The blood sample is mixed with the hemozoin-monoclona antibody complex and the mixture is passed through a magneti separation column in the presence of a magnetic field. The colum is washed with buffer to remove loosely entrapped material, and th column is removed from the magnetic field. Bound hemozoin-labele monoclonal antibodies are collected and examined under a ligh microscope using polarized light to determine the presence of an birefringent hemozoin. The diagnosis is scored.

EXAMPLE 2

A hemozoin-gene probe complex is prepared by mixing a selecte gene probe with crystalline hemozoin or crystalline hemozoin havin a substantially uniform investing protein coat. A blood sampl is drawn from a subject. The erythrocytes are separated from th blood sample by centrifugation. The erythrocytes are lysed b saponin lysis to release Plaεmodium, if any, in the sample. DN in the sample is isolated and bound to non-magnetic affinity beads.

E SHEET Double stranded DNA bound to the affinity beads is denatured b heat, and the denatured DNA is mixed with the hemozoin-gene prob complex. The hemozoin-gene probe complex is hybridized to DNA, an the hybridized DNA is passed through a magnetic separation colum exposed to a magnetic field. Washing with buffer removes debri from the column. The column is removed from the magnetic field an the hemozoin bound DNA, if any, is collected. The collected DN may then be processed according to known amplification techniques

EXAMPLE 3 A hemozoin-gene probe complex is formed as described i Example 2. Cells or cell cultures are collected and lysed b saponin lysis. The cellular DNA is bound to cellulose affinit paper, and heat denatured. The bound cellular DNA is exposed t the hemozoin-gene probe complex and the DNA-hemozoin-gene probe i hybridized. The cellulose affinity paper is washed to remove non specifically bound DNA, and the paper is examined for the presenc of birefringent hemozoin.

EXAMPLE 4

A cell membrane-specific recognition protein is selected, e.g. , Plaεmodium falciparum 175 kDa erythrocyte binding antigen (EBA-175) , is bound to either crystalline hemozoin or crystalline hemozoin having a substantially uniform investing protein coat. A pharmacologically active agent, e . g. , a pro-drug, is bound to the hemozoin. The hemozoin, recognition protein, drug complex is injected intravenously into a subject, and after twenty-four hours, a blood sample is taken. The blood sample is passed through a magnetic separation column in the presence of a magnetic field. The column is washed to elute the column of debris, and the column is removed from the magnetic field and magnetically bound red blood cells are collected. The collected sample is viewed by light microscopy under polarized light to determine the efficacy of the drug delivery system by determining the localization of birefringent hemozoin within the sample.

EXAMPLE 5 Hemozoin was purified from a lysate of Plasmodium falcipar refrigerated at 4°C. The lysate was centrifuged for 25 min.

20°C and 40,000 g. to form a pellet. The pellet was washed wi

0.1% sodium dodecyl sulfate (SDS) until the optical density of t supernatant was 0.0186.

1 ml of hemozoin lysate (O.D. 2.0313 at 630 nm) was spun 40,000 g for 25 min at 20°C, the resulting pellet was re-suspend in 0.6 ml phosphate buffered saline (PBS) pH 7.2. The suspensi was washed once with about 0.5 ml of PBS pH 7.2 and centrifug under the same temperature and conditions, the pellet was r suspended in 0.1 ml of 0.85% NaCl. A first control group Laboratory mice were immunized i.p. with 0.2 ml each of the Na suspension.

A second group was immunized i.p. with 0.2 ml each of solution consisting of the hemozoin pellet from the lysate and 0 ml CTMO-1 monoclonal antibody (5μg/ml, Lederle) , an antibody to surface component of ovarian or breast cancer. The mixture w stored overnight at 4°C, then centrifuged at 40,000g for 15 mi at 20°C. The resulting pellet was re-suspended with the 0.1 CTMO-1 monoclonal antibody for 1 hour, centrifuged under identic conditions and re-suspended in with 0.1 ml of CTMO-1 monoclon antibody. The re-suspension was stored overnight at 4°C.

A third group was immunized i.p. with 0.2 ml each of solution consisting of the hemozoin pellet from the lysate and 0 ml of rabbit anti-mouse erythrocyte (RAME) monoclonal antibo

(5μg/ml) . The mixture was stored overnight at 4°C, th centrifuged at 40,000g for 15 min at 20°C. The resulting pell was re-suspended with the 0.1 ml RAME monoclonal antibody for hour, centrifuged under identical conditions and re-suspended with 0.1 ml of RAME monoclonal antibody. The re-suspension w stored overnight at 4°C.

Blood samples were withdrawn from mice in each of the thr groups, and examined under a light microscope with and witho polarized light. Jn vivo specificity was determined by visualizi the birefringent hemozoin bound to the red cells in the bloo samples, as exemplified by Figures 7a, 7b, 8a and 8b.

EXAMPLE 6

In vitro specificity of hemozoin-linked monoclonal antibod was evaluated by mixing the hemozoin-SDS suspension with i) lOμ of RAME and 20μl of naive mouse blood (1.6 x IO⁶ RBCs) ; ii) lOμl o CTMO-1 and 20μl of naive mouse blood; and iii) lOμl hemozoin-SD with 20μl of naive mouse blood. Each mixture was allowed reaction time of 30 min, and evaluated under non-polarized an polarized light microscope and qualitatively scored for binding. The mixture samples were refrigerated at 4°C for four hours an qualitatively evaluated for binding. Two subsequent refrigeratio and re-evaluation periods were conducted.

In vitro binding specificity was noted for the RAME-hemozoi complex and the CTMO-1-hemozoin complex, but the control SDS hemozoin exhibited no binding, with subsequent cell lysis after the first refrigeration and storage period. Results similar to those depicted in Figures 7a,b and 8a,b, were noted.

In accordance with the preferred embodiments of the invention, and with particular reference to Figure 5, there is provided a novel magnetic separation apparatus 10. Magnetic separation apparatus 10 consists generally of a steel-jacketed permanent magnet 12 which defines a pole gap 14. A separation chamber 20 is disposed within the pole gap to expose the separation chamber 20 to the magnetic flux generated by the magnet. The separation chamber 20 is packed with magnetic beads or iron filings 16 to a packing density which permits fluid flow through the separation chamber 20. It is preferable to employ a separation chamber 20 having a generally rectilinear or elliptical cross-section. The use of magnetic beads and/or iron filings enormously increases the magnetic surface area and, therefore, the resultant magnetic dipoles between the pole gap, when compared to the prior art technique of random packing of stainless steel wire. Moreover, by configuring the separation chamber 20 to have a generally rectilinear or elliptical cross-sectional shape, the peripheral areas of the chamber, i.e., those closer the chamber wall, a packed with a volume of magnetic beads and/or iron filings, whi closely corresponds to that in the more central areas of t chamber. In this manner, a minimal pole gap distance may maintained and, therefore, the maximal magnetic flux density, whi varies inversely with the size of the pole gap, is achieved.

Alternatively, by providing an extremely narrow pole gap a separation chamber, e.g., about 1-101, and by utilizing a magn having a high magnetic flux, it is possible to sequest paramagnetic hemozoin or paramagnetic beads within the magnet field without packing the separation chamber with magnetic bea or filings. In this manner, it is solely the magnetic field whi sequesters the paramagnetic particles within the separati chamber.

The magnet 12 may be configured as a C-type magnet, b according to the preferred embodiment contemplated for t invention, magnet 12 consists of a pair of rectilinear magnets with the pole gap 14 being maintained by bilateral steel shims 18 Each magnet 12 is contemplated to have dimensions of 6.5 cm x 1. cm x 1.5 cm, with a pole gap of 1.5 mm being maintained by th bilateral steel shims 18. This provides a very compact, high flu magnet which is well suited to field use in remote areas wher automated diagnostic equipment is unavailable. Those skilled i the art will understand that these stated dimensions ar representational, and are not to be construed as limiting the scop of the invention.

It has been found that rare earth alloys, e . g. , neodymium vanadium alloy, provides optimum magnetic flux and should b employed to form the magnet 12. It is well known that the magneti flux density may be increased depending upon the alloy used t produce the magnet. Moreover, it is known that steel jacketin enhances the magnetic flux density even more.

Thus, in accordance with the preferred embodiments of th present invention, and with reference to the best mode fo practicing the methods of the invention, there is provided a steel jacketed rare earth alloy permanent magnet and a flattene separation chamber packed with magnetic beads and/or iron filings This combination dramatically increases the magnetic flux densit within the small area of the separation chamber lumen, and afford a strong magnetic environment for sequestration of paramagneti material flowing through the separation chamber. In practicing th diagnostic and therapeutic methods, set forth above, it has bee found advantageous to employ the above-described magneti separation apparatus to concentrate hemozoin bound material fro the sample.

There is provided, therefore, a diagnostic kit, consisting o the magnetic separation apparatus 10, as described above, plurality of pre-packed separation chambers 20, packed wit magnetic beads and/or iron filings, and an external peristalti pump (not shown) to provide a fluid flow force to move a sampl through the separation chamber. This it will further be provide with appropriate washing buffer solutions, which may be added t the sample and which will be used to wash the separation chambe of extraneous non-magnetically attached material. The buffers wil be provided in the range of pH 6-9, and may, for example consis of borate, phosphate, carbonate or the like. Buffer concentrations will generally range from about 0.01 to about 0.5M. The technicia will provide a light microscope properly fitted with a polarized light source, for examination and diagnostic scoring of the sample.

Those skilled in the art will further understand that an automated flow-through diagnostic test system may be constructed, utilizing techniques already existent in the art, for the diagnosis of the presence of hemozoin labeled material in a sample. For example. Coulter counters, as disclosed in U.S. Patent No. 2,656,508, count blood cells and differentiate types of blood cells, and may be easily adapted to provide counts of hemozoin- labeled cells within a sample.

Finally, in accordance with the various above-described diagnostic methodologies, there is provided an automated system consisting of an improved automated continuous flow hematology multi-parameter analyzer as illustrated with reference to Figu 6. The hematology multi-parameter analyzer 50 of the inventi provides a three-mode blood analysis system. As with convention multi-parameter hematology analyzers, such as the Coulter counte the analyzer 50 of the invention is capable of performing standa blood analysis, including several parameters of white blood coun red blood count, and platelet data and providing a print out that data. However, in accordance with the invention, there also provided a manifolded system consisting of a primary three-w manifold 52 and a secondary three-way manifold 54. Prima manifold 52 bifurcates or directs a specimen blood flow 56 receiv from an input source to either of the standard blood analysis mo 58 or to a malarial diagnostic mode 60. As provided by the prese invention, malarial diagnostic mode 60 provides input to t secondary three-way manifold 54. Secondary three-way manifold 5 in turn, bifurcates or directs a specimen blood flow 56 to eith of an outlet mode for external intact cellular analysis 62 or a lysed blood cell mode 64 for detecting the presence of hemozo polymers in the sample. Samples withdrawn from the outlet mode are ultimately utilized for external microscopic slide examinati of the malarial cells. Samples directed to the lysed blood ce mode 64 are exposed to a plurality of analytical sub-systems 7 which consist of appropriate sensors and reagents for the detecti of structural and/or molecular features or components of hemozoi For example, analytical sub-systems 70 may consist of birefringence sensor having a polarized light source for examinin the sample for the presence of hemozoin. The birefringence senso will be coupled to a computer programmed with a birefringenc pattern recognition library, which will record valid patter signals on an attached printer. Additionally, analytical sub systems 70 may consist of a ferrimagnetic electron spin resonanc and a magnetometer which will sense the presence of the Fe characteristic of ferriprotoporphyrin IX, thereby also determinin the presence of hemozoin in the sample. Furthermore, analytica sub-systems 70 may consist of the magnetic separator devic illustrated in Figure 6, described above, adapted for use in a automated system by providing an electromagnet controlled by a D coil, which can be pulsed on and off in response to sensing th presence of a sample in the separation chamber, to provide sequestering and separation of paramagnetic particles or hemozoi in the sample, and having associated washing and bufferin solutions to clear the separation chamber for a subsequent sample Nomarski optics, gene probes and/or monoclonal antibodie techniques, as described above, may also be employed as discret analytical sub-systems 70. The above-noted birefringent and/o paramagnetic markers other than hemozoin or crystalline hemozoin, may be used with the manual separation and/or automatic diagnosti system as markers in conjunction with gene probes, monoclonal antibodies, affinity ligands or other cell-specific materials.

While the invention has been fully described with reference to certain preferred embodiments thereof, it will be recognized and understood, by those skilled in the art, that many variations are possible without departing from the scope and spirit of the invention. It will be further understood that the scope of the invention is intended to cover all changes and modifications of the invention, as disclosed herein for the purposes of illustration, which do not constitute departures from the spirit and scope of the invention.

Claims

We Claim:

1. An in vitro method for diagnosing a biological conditio comprising the steps of: obtaining a sample of at least one of a body fluid tissue, from a subject; providing at least one of a monoclonal antibody, ge probe or affinity ligand, specific for the biological condition; providing a birefringent marker characterized by at leas one of optical birefringence under polarized light or electric birefringence in an applied electric field, and a binding affinit for the at least one of a monoclonal antibody, gene probe o affinity ligand; mixing said at least one monoclonal antibody, gene prob or affinity ligand with said birefringent marker, thereby bindin said at least one monoclonal antibody, gene probe or affinit ligand to said birefringent marker to form a birefringent labele vector; exposing said sample to said birefringent labeled vector and binding said birefringent labeled vector to cells in the sampl exhibiting the biological condition sought to be diagnosed; washing said exposed sample to remove unboun birefringent labeled vector; and viewing said exposed sample under polarized ligh microscopy to determine the presence of birefringent in the expose sample, thereby providing a positive or negative diagnosis for th biological condition.

2. The diagnostic method according to Claim 1, wherein sai step of providing a birefringent marker, further comprises the ste of selecting at least one a natural or synthetic polymer.

3. The diagnostic method according to Claim 2, wherein sai step of selecting at least one of a natural or synthetic polyme further comprises selecting said polymer from the group consistin of polyurethane, polypropylene, nylon, agarose amylose polyethylene, polyamide, poly(aminocarboxylie acids) , fibrin polydiethylsiloxane, proteoglycan and hemozoin.

4. A biological marking compound, comprising: A. a birefringent marker characterized by at least on of optical birefringence under polarized light or electrica birefringence in an applied electric field; and B. at least one cellular-specific bioactive agent bound to said birefringent marker, said bioactive agent bein selected from the class consisting of gene probes, monoclona antibodies, immunoglobulins, affinity ligands, pro-drugs liposomes, fluorophores, and chromogens.

5. The compound according to Claim 4, wherein sai birefringent marker further comprises hemozoin.

6. The compound according to Claim 4, further comprising a least one linking molecule binding said at least one cellular specific bioactive material to said birefringent marker.

7. A method for evaluating delivery of a bioactive compoun to a targeted site, comprising the steps of: A. complexing a birefringent marker with at least one cell-specific targeting agent, and the bioactive compound to be delivered, said birefringent marker being characterized by at least one of optical birefringence under polarized light or electrical birefringence in an applied electric field; B. administering said complex to a subject; C. recovering cells from the targeted site of the subject; D. determining the presence of birefringent marker associated with said targeted cells and the location of said birefringent marker with respect to said targeted cells to evaluate delivery of said bioactive compound to said targeted cells.

8. The method according to Claim 7, wherein said complexing step further comprises the step of binding hemozoin to at least one cell-specific bioactive agent from the group consisting of gen probes, monoclonal antibodies, immunoglobulins, affinity ligands, pro-drugs, liposomes, fluorphores and chromogens.

9. A diagnostic kit for evaluating the presence o paramagnetic materials in a sample, comprising: A. at least one magnet defining a pole gap; and B. a separation chamber disposed within said pole gap; and C. pump means for providing a sample fluid flow into an through said separation chamber.

10. The diagnostic kit according to Claim 9, wherein sai separation chamber further comprises a tubular member having a substantially rectilinear cross-sectional shape.

11. The diagnostic kit according to Claim 10, wherein said separation chamber is packed with at least one of magnetic beads or iron filings.

12. The labeled species-specific gene probe according to Claim 35, wherein said hemozoin further comprises crystalline hemozoin substantially free of bound proteins.

13. The labeled species-specific gene probe according t Claim 35, wherein said hemozoin further comprises a substantiall uniform investing coat of protein and said DNA probe is bound t said substantially uniform investing coat of protein.

14. The labeled species-specific gene probe according t Claim 37, wherein said substantially uniform investing coat o protein further comprises albumin.

15. An assay employing the labeled species-specific gene probe of Claim 35, comprising the steps of: A. isolating and denaturing DNA from host cells; B. immobilizing said isolated and denatured DNA on an immobilization medium; C. exposing said immobilized DNA to a gene probe complexed with a birefringent marker, said birefringent marker characterized by at least one of optical birefringence under polarized light or electrical birefringence in an applied electric field; D. hybridizing said immobilized DNAwith said complexed gene probe; E. removing un-hybridized complexed gene probe; and F. examining said immobilizationmediumunder polarized light to determine the presence of the birefringent marker.

16. The method of Claim 15, wherein said immobilizing step further comprises the step of selecting said immobilizing medium from the group consisting of non-magnetic beads, non-magnetic microspheres, cellulose paper, cellulose beads, or a plastic matrix.

17. A method for screening monoclonal antibodies, comprising the steps of: A. selecting at least one monoclonal antibody sought to be tested; B. complexing a birefringent marker with said at leas one monoclonal antibody to be tested to form a birefringent marker monoclonal antibody complex, said birefringent marker characterize by at least one of optical birefringence under polarized light o electrical birefringence in an applied electric field; C. exposing said birefringent marker-monoclona antibody complex to a host comprising at least one of a host cell, host cell culture, a plurality of different host cells or plurality of different host cell cultures; D. recovering cells or cellular material from sai exposed host; and E. determining the presence of birefringent hemozoi associated with said separated cells and the location of sai birefringent marker with respect to said separated cells.

18. The method according to Claim 17, wherein said step of complexing a birefringent marker with a monoclonal antibody further comprises the step of providing hemozoin as the birefringent marker and complexing said hemozoin with said at least one selected monoclonal antibody.

19. The method according to Claim 18, further comprising the step of magnetically separating hemozoin bound host cells prior to the identification step.

20. A malarial diagnostic method for Plasmodium parasitemia, comprising the steps of: A. selecting at least one biocompatible membrane active compound having an electric dipole moment greater than that of water; B. administering said selected at least one biocompatible membrane active compound to a subject; and C. monitoring the subject for shedding of the Plasmodium parasite indicative of Plasmodium infection.

21. The method of Claim 20, wherein said step of selecting at least one biocompatible membrane active compound further comprises the step of selecting said at least one biocompatib membrane active compound from the group consisting of ure ascorbic acid, nicotinic acid, chloroquine and piracetam.

22. The method of Claim 20 or Claim 21, wherein said step administering said at least one biocompatible membrane acti compound further comprises the step of administering said compou in solution having a concentration in the range of about 0.01M about 10M.

23. The method of any of Claims 20 to 22, wherein said st of administering said at least one biocompatible membrane acti compound further comprises the steps of: A. administering an aqueous solution of about fi percent urea; B. monitoring the subject's blood urea nitrogen leve until a clinically therapeutic BUN level of at least about 200 mg is achieved; C. managing diuresis by administering electrolytes an water; and D. monitoring the subject for the abatement of malaria paroxysms.

24. The method of any of Claims 20-23, wherein said step o determining the presence of Plasmodium parasitemia furthe comprises the steps of: A. obtaining a blood sample from the subject; B. magnetically separating infected erythrocytes fro non-infected erythrocytes in the blood sample; and C. examining said separated infected erythrocytes unde polarized light to determine the presence of birefringen particles.

25. The method of any of Claims 20 to 24, wherein said ste of administering said selected membrane active compound furthe comprises the steps of: A. speciating the suspected Plasmodium infection determine the presence of Plasmodium falciparum by administeri sub-therapeutic dosages of said at least one selected membra active compound, thereby interfering with formation of intr erythrocytic cytoskeletal knobs and reversing binding of said kno to the vascular endothelium characteristic of Plasmodium falciparu infection; and B. determining the presence of elevated levels o hemozoin in at least one blood sample withdrawn from the subject.

26. The method of Claim 26, wherein said step of determinin the presence of elevated levels of hemozoin further comprises th steps of: A. magnetically separating infected erythrocytes fro non-infected erythrocytes in said at least one blood sample; and B. examining said separated infected erythrocytes unde polarized light to determine the presence of birefringen particles.

27. In an apparatus for analyzing blood samples of the typ for analyzing white blood count, red blood count and platele count, wherein the improvement comprises a molecular and structura diagnostic system, said molecular and structural diagnostic syste further comprising: A. first switching means for directing a flow of a least one blood sample to said molecular and structural diagnosti system; B. an intact cellular microscopy system comprising o a sample outlet for examining whole cell samples microscopy; ( i cell lysis and lysate analysis system; and C. second switching means for receiving said flow o at least one blood sample from said first switching means an directing said flow of at least one blood sample to at least on of said intact cellular microscopy system or to said cell lysis an lysate analysis system.

28. The apparatus according to Claim 28, wherein said cel lysis and lysate analysis system further comprises at least one o a birefringence sensor, electron spin resonance, magnetometer, Nomarski optical sensor, a magnetic separator a DNA gene probe, a least one monoclonal antibody or at least one DNA intercolatin dye.