Wear, 142 (1991) 135-149 135

Modification of ferrography method for analysis of lymphocytes and bacteria

M. Zborowski, P. S. Malchesky, S. R. Savon, R. Green, G. S. Hall and Y. Nose Department of Art(ficial Organs, Laboratory of Hematology and Microbiology, The

Cleveland Clinic Foundation, One Clinic Center, 14, 9500 Euclid Avenue, Cleveland, OH 441955132 (U.S.A.)

(Received March 5, 1990; accepted July 30, 1990)

Abstract

Problems associated with the analysis of low magnetically susceptible or diamagnetic biological particles in ferrography are related to the unoptimal fluid dynamics, the low magnetic field strength and gradients and the lack of effective magnetizing agents. Flow geometry optimization in slide ferrography has been undertaken with the specific aims of obtaining a thinner flow channel, better-defined fluid flow geometry, uniform distribution of particles within the flowing volume, containment of the flowing liquid sample, exposure of all flowing particles to the magnetic field, and differentiation of deposition layers formed by different forces, i.e. magnetic, gravitation and flow related. These specific aims were attained by adopting a novel slide scheme which at present reduces the fluid flow thickness by tenfold and increases the peak relative magnetostatic force by up to three orders of magnitude, compared with the conventional slide ferrography. The novel slide ferrography scheme, combined with the use of the strongest permanent magnet available, is better suited to the analysis of biological particles than conventional ferrography is. Where native mononuclear blood cells showed no response to the magnetic field when treated with erbium, mononuclear cells which had been cultured for as short as 24 h and as long as 72 h show magnetically induced deposition after erbium treatment. Non- specific labeling with cationized ferritin causes the magnetic deposition of all cells studied, i.e. native and cultured human lymphocytes, and mouse lymphoma cells (YAC-1). Modified slide ferrography shows that the bacterium Eschmichiu coli has a high afTini@ for erbium ions and becomes readily magnetized and separated. Observations of bacterial magnetic deposition are possible in unstained slide mounts using scattered light. Scattered-light intensity correlated with the bacterial cell concentration in the samples analyzed by ferrography.

1. Introduction

Ferrography is a method of particle separation onto a glass slide based upon the interaction between an external magnetic field and the magnetic moments of the particles suspended in a flow stream. If the particles are not ferromagnetic, it may be possible to render them magnetic through the use of paramagnetic agents. The Grst published use of ferrography was in 1972 by Siefert and Westcott [ 1 ] for industrial applications. Industrial applications have included the monitoring of wear debris in oil or grease

0043-1648/91/$3.50 0 Elsevier SequoiaPrinted in The Netherlands

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lubricants and hydraulic systems and gas stream monitoring of non-lubricant- washed components [ 2, 31. On-line systems have been developed in some selected applications [ 41. Ferrography has proven itself in the early wear detection and in prescription preventative maintenance for the non-cata- strophic downtime of man-made machines.

The experience of ferrography in biomedical applications is very limited and consists primarily of the experience of a limited number of investigations on the wear in natural and prosthetic joints [5-lo] and the limited experience on other biological fluids [ 11, 121, such as erythrocyte and white blood cell separations, and that of a few bacterial strains [ 131. However, the use of magnetic fields for the separation of cells has been described and applied with various approaches [ 141. It has been demonstrated that leukocytes can be separated by first allowing them to phagocytize magnetic particles [ 151, to bind to magnetic microspheres [ 17,181, or to rosette erythrocytes containing paramagnetic methemoglobin [ 19, 201. A further method of conferring mag- netic susceptibility to cells or biological molecules is through antibodies and biomolecules coupled to ferritin, a paramagnetic protein containing iron [ 16, 18, 211.

This paper focuses on ferrography, modified for a better separation and analysis of biological particles on a glass slide. A primary advantage of the modified ferrography separator, compared with other types of magnetic separator such as high gradient magnetic filters, is that it preserves the separated material on a glass slide in a form suitable for further routine cytological analysis. Specific objectives of this paper are to describe and evaluate the magnetic separation system applicable to the analysis of biological particles, to evaluate magnetizing agents in the separation of blood-borne or derived white blood cells and to describe the preliminary evaluation of bacterial quantification.

2. Equipment design evaluation

2.1. original industrial slide fmography analyzer Separation of particles on a ferrography slide depends on their volume

V, the volume magnetic susceptibility xm, the magnetic field intensity B, the magnetic field strength H, and the gradient grad B of the magnetic field intensity. The magnetostatic force acting, for example, in the x coordinate direction, i.e. F,, J, is given as

Corresponding formulae apply to y and z coordinates [ 221. In Fig. 1 is illustrated the industrial slide ferrograph analyzer. Most biological particles are by nature diamagnetic and therefore require

attachment of a magnetizing agent to make them susceptible to an external magnetic field. Therefore, before the suspension of biological particles is

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‘116” I D Tygon tubing

Aluminum casing

Fig. 1. Industrial slide ferrograph analyzer.

processed in ferrography, a solution of a cation of a high magnetic dipole moment is added to the sample. The cations bind to the particle to be separated and increase their magnetic susceptibility.

The overall force exerted on a cell by a magnetic field is the resultant of the difference of the magnetic susceptibility xceu of the cell and the magnetic susceptibility Xm&ium of the medium [ 7 ] as expressed by the equation

Xm = Xceu - Xmedium (2)

As expressed by the two above equations, the overall force acting on the biological magnetized particle can be increased by (1) increasing the cell’s magnetic susceptibility xceu, (2) increasing the difference ,y,, between the magnetic susceptibilities of the cell and medium, (3) increasing the magnetic field B and (4) increasing the magnetic field gradients.

Separation of biological particles in the original industrial ferrograph analyzer calls in general for a three-step procedure.

(a) The particulate elements must be endowed with a magnetic sus- ceptibility by mixing them with a solution of a cation with a high magnetic dipole moment, usually an element of the rare earth series (e.g. Era’).

(b) The sample must be delivered onto the glass slide surface with the use of a mechanical device (a roller pump) (see Fig. 1). The slide is placed in a slightly inclined position on top of a permanent magnet which is shaped so as to create a strong and nonuniform magnetic Aeld along the flow path. The magnetic particles in the fluid, as they flow, are pulled by the magnetostatic force of the magnet and they are distributed along the flow path on the slide according to their size, their relative position in the flowing stream and the geometry of force field F,.

(c) The outflowing sample must be removed from the slide by a manually adjusted drainage tube. The end step of the process requires removal of the

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residual volume of the medium left on the slide. In the original industrial procedure this was accomplished by using a rinsing solution. In biological applications, rinsing removes all the weakly attached deposition and was therefore substituted by a controlled tilting of the magnet and removal of the excess fluid by wicking at the sample exit point.

2.2. Typical results of biological particles separation using industrial

f~w-why Figure 2 illustrates the processed ferromagnetic particle deposition

(uniform latex particle, magnetic) (Seragen Diagnostics, IN) and cultured human lymphocytes in 5 mM ErCla solution (paramagnetic), in the industrial ferrograph analyzer. It was determined that most organic materials separate very weakly under the chosen experimental conditions; the very light deposition of cells along the slide center-line in Fig. 2(b) compared with that for ferromagnetic particles in Fig. 2(a) should be noted. Apart from magnetically induced deposition, it has been noted that gravity and flow related deposition may be also prominent, e.g. in the case of processed human lymphocytes

(a) (b)

Fig. 2. Examples of typical differences in industrial slide ferrograph analyzer separation between ferromagnetic and biological particles. (a) Uniform latex particles, magnetic. The concentration of deposition along the line of the strongest magnetic interaction, as indicated by an arrow should be noted. The circles surrounding the deposition are artifacts of covering the slide with a protective cover slip. (b) Human lymphocytes, cultured, in 5 mM ErCl, in saline. The diffuse character of the deposition showing only slight accumulation of particles along the center-line, as indicated by the arrows, should be noted.

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(Fig. 3(a)). In most cases in the analysis of biological particles the use of a control sample run outside the magnetic field was required to distinguish between the rna~etic~y induced deposition from that induced by gravi@ and flow-related forces. An example of human lymphocytes processed outside the magnetic field (other parameters of separation being held constant) is presented in Fig. 3(b). The freshly drawn human lymphocytes in 5 mM ErCl, in saline are not magnetically separated as a characteristic magnetic deposition line along the center axis of the slide is not present in Fig. 3(a). A heavy deposition was noted along the barrier region in Fig. 3(a), formed during residual fluid volume withdrawal from the slide at the end step of the ferrogram preparation.

2.3. Mod@ed fmograph anul~zm- applicable to triological particle s~arat~

The original ferrography apparatus has been significantly modified to adapt it to much more exacting requirements of biological separation and

(4 Fig. 3. Two examples of deposition caused by gravity, flow and surface forces acting on cells, using industrial slide ferrography of erbium-treated (5 mM final) human lymphocytes. In (a) deposition appears to be primarily flow related, with cells concentrated along the pathway barriers (as indicated by the arrows), the areas of decreased velocity. The absence of any pattern indicative of magnetic deposition, i.e. a central line, should be noted. Deposition in (b) appears to be primarily the result of gravity, creating an almost homogenous coverage of the pathway surface. (b) was processed without a magnet.

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analysis compared with the industrial standarde. dome of the characteristics of biological particles compared with that of the industrial particles, relevant in ferrographical separation, are presented in Table 1. The following mod- ifications of the ferrograph analyzer were made.

(a) Magnetic field intensity and magnetic field gradients were significantly increased by introducing the strongest permanent magnets available on the market (Nd-Fe-B, Permag Co., Toledo, OH), by using thicker magnet pole pieces for better conduction of the magnetic flux (3 in), and by positioning the flow chamber flat against the magnet surface, thereby increasing the magnetostatic force acting on the particles.

@) The sensitivity of the evaluation of the magnetic deposition was significantly improved by orienting the sample flow to intersect the junction at an angle or perpendicular, rather than parallel to the junction of the pole pieces, thus freeing the deposition pattern from the interference of gravity and flow-related deposition.

(c) The geometry of the sample flow, as related to the magnetic field’s highest intensity line, allows for a comparison of the amount of the deposition directly above the junction of the pole pieces and outside it using scattered- light measurements. Thus the amount of the deposition may be correlated with the intensity of scattered light used for quantification of the magnetic deposition.

The summary of the design modifications is presented in Fig. 4, and major characteristics of both the industrial and the biological ferrograph analyzer are summarized in Table 2. By using stronger magnets the mag- netostatic force (relative) was increased sevenfold, and by using a modified flow channel the average distance of flowing particle to the bottom slide was decreased about tenfold. Considering that the magnetostatic force acting on a magnetic dipole decreases as the inverse cube of the distance of the

TABLE 1

Comparison of physical characteristics of representative industrial ferrous iine particles and biological particles

Physical characteristic Industrial particle Biological particle

Size (pm) Material Specific gravity (g cm-‘) Magnetic susceptibility Surface charge Fragility Rigidity Magnetizing agents required

0.1-10” Ferrous solids 5.18 Ferromagnetic Yes No Yes No’

2-lob Organic 1.07d Diamagnetic Yes Yes No Yes

“Machine wear. bBacteria, blood cells. ‘Ferrous oxide. dLymphocytes. ‘Except for non-ferrous particles.

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Fluid Volume ----_

(4

Fluid Volume -

Pieces (3/4”)

@)

Fig. 4. Comparison of arrangements of flow and magnetic field between (a) original industrial slide ferrograph analyzer and (b) modified ferrograph analyzer applicable for biological particles separation and analysis. Drawings not to scale.

magnetic dipole from the field source [22], it may be estimated that the flow channel design modihcations of the industrial ferrograph separator increased the force of magnetic interaction with the flowing particles about (1 /(l /lo))’ or lo3 times. Combined modifications of the flow channel design and the magnets increased the force up to 7 X lo3 times.

3. Evaluation of particle separation in modified ferrography

3.1. Evaluation of wmgnetizing agents in separation of blood-borne or derived white blood cells

The magnetizing agents used in this study are presented in Table 3. Because of their net positive charge, these agents bind non-specifically to negatively charged sites on the cell. Magnetic labeling was carried out by a 1:J dilution of sample with a magnetizing agent just prior to processing.

The evaluation of the mononuclear cells consisted of the following steps: cell separation and resuspension, production of ferrograms, and ferrogram staining and analysis. Each mononuclear cell fraction for evaluation was

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TABLE 2

Comparison of major parameters of original and modiied ferrography analyzers

Parameter

Peak magnetic field intensitya (T)

Peak magnetic field gradient (average)b (T m-l)

Peak relative magnetic force (average) (T” m - ‘)

Flow channel cross-section

Average distance to the bottom slide’ (mm)

Average flow velocity (cm s-r)

Original Modified

0.19 0.42

17.5 48.0

2.60 14.00

0 3 mm = 0.3 mm 6mm 6mm

1.27 0.15

0.044 0.555

“Measured along the pole pieces junction using a gaussmeter model 810, and a probe model STBS0402 (F.W. Bell Inc., Columbus, OH). The tip of the probe was held pe~endic~~ to the surface of the pole pieces. bCalculated for a distance increment of 0.5 cm from the junction of the pole pieces. ‘Equal to j.l-t_ dx dgiA, where A is the flow cross-section area and x and 21 are particle coordinates within this cross-section area with x being the distance from the bottom slide.

TABLE 3

Magnetizing agents

Paramagnetic ingredient

Description Solution

Atomic weight, 167.26. Rare earth element of lanthanide series. Calcium competitor in biological systems. Complexing and cell- aggregating agent. Strongly paramagnetic, atomic susceptib~ity 3 X lo4 cgs units. Binds to anionic groups

10 mM Err&’ in 0.9% NaCl in water

Fe&tin Molecular weight, 750 000. Iron-containing protein. Protein shell 100~1200 nm in diameter. Ferric hydroxide core containing 2000-3000 Fe atoms. Pammagnetic. Binds to anionic groups

0.02% cationized horse spleen ferritinb in HEPES bufferC

BErCl,-6Hz0, Alfa Products, Morton Thiokol Inc., Danvers, MA. bHorse spleen ferritm coupled with N~dimet;hyl-l,3-propanediamin‘e, Siia Chemical Co., St. Louis, MO. c~-2-Hydro~ethylpiper~~e-~-2-eth~es~oni~ acid (HEPES), Sigma Chemical Co., St. Louis, MO.

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prepared by centrifugation using LeucoPREP cell separation tubes (Bec- ton-Dickinson) according to the manufacturer’s specifications. In brief, the cell suspension was drawn into heparinized vacuum tubes, and mononuclear cells were concentrated within 2 h of the drawing time. Each type of sample was transferred into pre-centrifuged LeucoPREP tubes and centrifuged. A cell count was then made of the mononuclear layer, diluted if necessary to achieve the desired cell concentration of about 10’ ml-’ and then processed.

Slides were stained using a prepared Wright-Giemsa stain (Volu-Sol, Las Vegas, NV), examined directly for deposition patterns and evaluated by light microscopy for cell identification and mo~holo~ preservation.

Three types of mononu~le~ cell fraction were ~ves~gate~ l~pho~~es from freshly drawn human whole blood; l~phoc~es that had been similarly isolated and then cultured for 24 or 48 h; cells from an established line of cultured mouse lymphocytes that had been virally transformed (YAC-1 cells).

Lymphocytes were cultured according to an established protocol. Cultured lymphocytes, both human and mouse, were grown suspended in commercially prepared RPM1 1640 medium, a sterile solution of buffers, salts and nutrients, to which had been added 1% glutamine, 1% sodium pyruvate and other additions as specified. These cells were incubated at 37 “C in a humidified COz-controlled atmosphere. Human lymphocytes, after separation from whole blood, were placed in sterile six-well dishes with RPM1 1640 which contained 10% heat-~acti~ted fetal calf serum, and 1% ~tibiotics in addition to the generalized formulation specified above. These cells were incubated for 24, 48 or 72 h respectively. Cultured mouse lymphoma cells were generously supplied by Dr. J. H. F’inke, Department of General Medical Sciences, the original murine (mouse) cells having been transformed by Moloney murine leukemia virus many generations previously. These murine cells, called YAC- 1, were grown in RPM1 1640 containing 10% fetal calf serum, 1% non- essential ammo acids, 0.5% HEPES and 4 X 10w5% P-mercaptoethanol, in addition to the general formulation. They were incubated in 10% COz and subcultured three times weekly.

EscWchia coli was obtained in suspensions in saline. Suspensions were kept at room temperature ihroughout ferrographic processing and were diluted to obtain cell concentrations lo’-lo5 ml-‘. The protocol for bacterial separation in slide ferrography was similar to that used for the mononuclear white blood cell studies. Only erbium was used as the magnetizing agent.

The amount of magnetic deposition of bacteria on the slide was measured by the difference between the intensity of the scattered light of the magnetic deposition area and that of the area outside it. The scattered-light intensity was measured using an adapted densitometer from the ferroscope analyzer, Sohio Predictive Maintenance Services, Solon, OH. Scattered-light intensity histograms were made using an x-21 recorder with the 2( input coupled to the light intensity display of the ferroscope, and the z input obtained from

144

a linear 10 rev full-scale potentiometer fed by a 12 V d.c. power supply. The potentiometer was directly coupled to the longitudinal position of the ferroscope stage and a linear relationship was achieved between the lon- gitudinal position along the slide and x position of recorder’s pen. Magnetic deposition, as observed in scattered light, appeared as peaks in the x-y graphs, and the heights of those peaks were used as a measure of the quantity of the deposition.

4. Results

The effects of gravity and flow-related forces on the deposition of circulating (freshly drawn) lymphocytes in industrial slide ferrography are presented in photographs of the ferrograms in Fig. 3. The ferrogram in Fig. 3(a) was made in the presence of the magnetic field and compared with the ferrogram in Fig. 3(b) prepared outside the magnetic field. Particulate elements deposited in this latter ferrogram are entirely related to the gravity and flow forces.

The results of the human lymphocyte (circulating and cultured) separation in the modified ferrograph analyzer using 5 mM ErCl, solution are illustrated by photographs of magnetic deposition in Figs. 5(a) and 5(b). They show the difference between the cell separation in circulating and in cultured human lflphocytes. The results of circulating human Iymphocyte separation in 0.01% solution of cationized ferritin are illustrated in Fig. 5(c). It may be noted that cationized ferritin is a better magnetizer of circulating lym- phocytes than erbium solution is. Figure 6 illustrates deposition of YAC-1 cells in erbium and in ferritin solutions. Table 4 summarizes the findings.

The results of bacterial quantification on the ferrograph slides, as measured by the intensity of ‘scattered light (relative), are presented in Table 5. They show a correlation of bacterial cell concentration in the processed suspension with the intensity of the scattered light.

5. Discussion

To improve the analytical capabilities of the slide ferrography in the studies of biological particles, a modified ferrography scheme was developed. Modified fe~o~aphy, combined with the use of the strongest permanent magnet available and a slanted orientation of the flow chamber, is better suited to analysis of biological samples than standard single slide ferrography is. It increases the interaction of cells with the magnetic field by causing all particles to flow near the points of highest magnetic field strength and produces higher and more specific deposition. Also, it permits direct cytological evaluation of the separated material which is not available in other magnetic separation methods such as high gradient magnetic filtration.

The results of mouse and human cultured mononuclear cell separation in the industrial slide ferrography using 10 mM ErCl, in 0.9% saline as the

(a> @I (cl

Fig. 5. Comparison of modified ferrography separation of human l~pho~es under wing conditions. (a) Lymphocytes freshly isolated from whole blood, labelled with 5 n-&I ErC&. (b) Lymphocytes labelled with 5 mM ErCl, after 48 h in culture; magnetic deposition is indicated by the arrows. The loss of a clear deposition pattern near the pathway exit is a result of complications in the removal of sample fluid. (c) Lymphocytes freshly isolated from whole blood, labelled with 0.01% cationized horse spleen ferritin.

magnetizing agent showed the tendency of the cells to concentrate along the area of the strongest magnetostatic force, i.e. the center-line of the sample pathway, downstream. The findings are similar to those reported previously by Russell et at. [ 131 ~thou~ these workers note cell accumulation upstream rather than downstream along the center-line of the sample flow pathway. This difference may result from a change in the last step of the industrial ferrography analysis procedure in the present study, namely the substitution of rinsing the deposition on the slide by tilting the magnet and wicking the excess fluid.

Differences between magnetic cell separation in cultured and in circulating lymphocytes were noted. No such differences were reported in other studies on the magnetic separation of living cells [ 12-16, 19-201. These differences may reflect living-cell reaction to variations in its environment. The lymphocyte is the blood cell specialized to discriminate between “self” and foreign protein macromolecules. Lymphocytes identify ~~010~~~~ foreign material and initiate a sequence of events that leads to the kilhng or neu~tion of

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Fig. 6. ~ornp~~~ of fe~o~a~~~ separation of cukwe mouse ~~h~rna c&s, YAC-I: (a) in 5 mM &CL3 in saliie; (b) in 0.02% cationized horse spfeen ferritin.

Cell type Magnetic eel separation for the foIh+ng rn~e~~~g agents”

F Er

NumM ~pb~c~~ Giititig Yf% No Cultured Yes Yes

Mouse lymphoma YAC-1 cultllred Yes Yes

aF, 0.02% caticmized horse spleen ferritin in liEPE2 buffer; Er, 10 mM Ed& in 0.9% N&l in xvater.

this material. Cells in culture are no fonger under the same re~~ti~~ as in the intact immme system and they may grow or undergo changes at a Werent rate from their respective counterparts in the body [23]. A cell that is actively growing will be effecting active trrutsport o;f nutrient material for synthesis whkb could result in greater uptake of Er@+.

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TABLE 5

Scattered-light mean intensity readings (relative) at varying E. coli concentrations as measured in the modified ferrography slides

Concentration Scattered-light Residence time Sample (cells ml ‘) intensity (relative) of samples in number

over magnetic deposition magnetic field (mean&-standard deviation) (mean f standard deviation)

10s 73+41 2 mm 27 s&2 min 24 s 7 10’ 6.8 + 5.5 2 min 07 sfl min 37 s 6 106 3.6* 1.4 2 mm 30 sfl min 31 s 5 1Oj 2.0+ 1.2 1 min 49 s*51 s 8

Separation of human lymphocytes in 5 mM ErCl, solution using modified ferrography confirmed the difference between native and cultured cells. Mononuclear cells which have been cultured for as short a time as 24 h showed magnetic deposition whereas native mononuclear cells showed no magnetic response when treated with erbium (see Fig. 5).

Non-specific labeling with cationized ferritin produces the magnetic deposition of both native lymphocytes and YAC cells (see Figs. 5(c) and 6(b)) and in this respect is different from erbium labeling. Magnetic separation of YAC-1 cells was also observed in 5 mM ErC& solution (Fig. 6(a)) which is similar to the finding that cultured human lymphocytes separate in erbium solution, as YAC-1 are cultured cells. The results of magnetic separation of mononuclear blood cells tagged with cationized ferritin obtained in this study are similar to those reported [ 161 on the kinetics of red blood cells tagged with ferritin in a magnetic field. Studies by other workers of the magnetic removal of cells using ferritin attached through an antibody may serve as an example of a cell-type specific magnetizing agent which may be used in further studies on ferrography separation of biological particles [ 211.

Separation of a specific type of bacterium, E. coli, in modified ferrography using 10 mM ErC& as the magnetizing agent con6rm.s the previous report on bacteria ‘separation in industrial ferrography [ 131. Modified ferrography analysis allowed for quantification of bacterial deposition using the scattered- light technique which was found adequate for the cell concentration as low as 10’ ml-’ and possibly, after necessary improvements of the flow chamber to make it dimensionally stable, for as low as 10’ cells ml- ‘.

6. Conclusions

The modified ferrography analysis technique developed in this study was found to be better suited to analysis of biological particles, represented by mononuclear blood cells and bacterial cells, than industrial ferrography analysis is. The chemistry of Er3+ and the ferritin reaction with the cell membrane surface groups allow for a paramagnetic tagging of a wide variety of cells

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(human, animal and bacterial) in different media. The results showed that there is a differential binding of Er3+ to native and cultured cells. Correlation between the concentration of bacterial cells in suspension and that in the magnetic concentrate on the analytical substrate glass suggests the possibility of early detection of bacterial cells in body fluids.

Acknowledgment

This work was supported by BP America Inc. and the State of Ohio Thomas A. Edison Program.

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