- Email: [email protected]
Influence of an inhomogeneous magnetic field on erythrocyte aggregation mechanism—an analysis by He–Ne laser aggregometer
Journal of Magnetism and Magnetic Materials 252 (2002) 412–414
Influence of an inhomogeneous magnetic field on erythrocyte aggregation mechanism—an analysis by He–Ne laser aggregometer Sanjay Jayavanth*, Megha Singh Biomedical Engineering Division, Indian Institute of Technology, Madras 600 036, India
Abstract The influence of the inhomogeneous magnetic field (IMF) on the erythrocytes and their aggregates, while sedimenting in a glass chamber under gravity, is analyzed. The aggregation data are acquired by using online He–Ne laser aggregometer and are represented in terms of various dynamic parameters. The analysis shows that the sedimentation of the erythrocytes and their aggregates is accelerated in the presence of the IMF. Thus, this technique could be applied in the study of blood samples in diseases with altered hemoglobin properties. r 2002 Elsevier Science B.V. All rights reserved. Keywords: Erythrocytes; Aggregation; Inhomogeneous magnetic field
1. Introduction The influence of inhomogeneous magnetic fields (IMF) on blood is due to the paramagnetic property associated with the iron atom of hemoglobin in deoxygenated erythrocytes [1]. This property is employed to capture the red cells [2], separate the malarial parasitized erythrocyte [3], and to change the erythrocytes sedimentation rate [4]. Erythrocyte aggregation is a reversible dynamic process where in cells form chain-like structures in the presence of plasma proteins. As the application of the IMF may affect the aggregation process, the objective of the present study is to analyze the influence of the IMF on this mechanism under gravitational field.
mentation of aggregates. For computation of the aggregation parameters this signal was digitized and acquired by the PC. The experiments with IMF were conducted with the sample chamber sandwiched between two coaxially held ring magnets by mutual attraction (Fig. 2). 2.1. Magnetic field calculation The distribution of magnetic field and its gradient with respect to the chamber was measured by thin Hall probe [6] (Fig. 3). The effective force acting on the erythrocytes and their aggregates, due to field gradient was calculated using the equation [1,2] Fmag ¼ wV ðH dH=dhÞave ;
ð1Þ
6
2. Material and methods Fig. 1 shows the block diagram of He–Ne laser aggregometer [5]. Laser light was passed through the erythrocyte suspension in plasma, placed in a glass chamber. The transmitted intensity (TI) was detected by the photodiode-amplifier configuration during the sedi*Corresponding author.
SI units, the magnetic susceptwhere w=3.88 10 ibility of deoxygenated erythrocytes, V is the volume of erythrocytes (m3) and ðH dH=dhÞave is the average gradient of the magnetic field along the height of the chamber (2.8 109 A2/m2/m). 2.2. Sample preparation Fresh blood samples were obtained by venepuncture from healthy subjects (n ¼ 10) in citrate phosphate
0304-8853/02/$ - see front matter r 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 8 8 5 3 ( 0 2 ) 0 0 6 7 6 - 5
S. Jayavanth, M. Singh / Journal of Magnetism and Magnetic Materials 252 (2002) 412–414
413
trough of the TI signal correspond to minimum (Nmin ) and the maximum number of cells (Nmax ) in the OV, respectively. The variation in the number of cells (Nmax 2Nmin ) indicates the change in aggregate size, which is thresholded at 3000. For further analysis the variations in the TI signal are divided into four categories. These data are presented in terms of following parameters: Aggregate size index (ASI): indicates the instantaneous change in the aggregate size, given by the ðDNÞ ¼ Nmax 2Nmin : DNmax represents maximum aggregate size and DTmax represents the sedimentation time corresponding to DNmax measured as the time width at the base of the given fluctuation. Total number of fluctuations (TNF): include all the fluctuations in the TI observed during the movement of the formed aggregates. The process initiation time (PIT): indicates the time required for the first fluctuation of the order over 3000 to occur in TI from t ¼ 0: The process completion time (PCT): indicates the total time required for the completion of the sedimentation process from the beginning till the maximum TI (I0 ). Effective number of cells (ENC): indicates the number of cells present in the OV at any instant of time and was calculated from the ASI variation. Further details of this analysis are given elsewhere [4,5].
Fig. 1. Block diagram of aggregometer.
Fig. 2. Sample chamber with ring magnets.
3. Results and discussions Fig. 3. Distribution of magnetic field (H) and its gradient (H dH=dh) along the height (h) of glass chamber.
dextrose, as an anticoagulant (10:1.4). Each sample was centrifuged at 3000 RPM for 20 min. Thereafter, the plasma was separated and the buffy layer on top of the cells was discarded. From each sample a suspension of erythrocytes of 5% hematocrit (Hct.) was prepared in the plasma and was used for aggregation measurement with and without the presence of IMF, at room temperature (25711C).
Fig. 4 shows the variation in the erythrocyte aggregation and sedimentation through the OV. The TI varies with time from the minimum to maximum associated with fluctuations. This is because initially the cells are monodisperse and with the formation and sedimentation of aggregates the mean intensity rises gradually. In the presence of the IMF, the maximum TI is attained faster compared to that of the control sample (without the IMF). This means that the mobility of the cells and their
2.3. Data analysis The aggregation data were analyzed for a set of parameters that describe the sequence of changes in the aggregation. The number of cells in the observed volume (OV) at any instant, based on Beer’s law, is given by Nt ¼ ð1=aX ÞlnðI0 =It Þ;
ð2Þ 1
where a is attenuation coefficient (0.772 cm for the sample at 5% Hct.), X is the mean thickness of erythrocyte (4 mm), I0 is the TI with plasma alone and It is the TI of the sample at any instant ‘t’. The peak and
Fig. 4. Aggregation curves of erythrocyte suspension of a healthy subject with and without the IMF.
414
S. Jayavanth, M. Singh / Journal of Magnetism and Magnetic Materials 252 (2002) 412–414
Table 1 shows the aggregation parameters for samples with and without the influence of IMF. The change in PCT is highly significant. The DNmax shows that the maximum aggregate size is higher in case of the sample with IMF. The DTmax under the influence of IMF is lesser, indicating that the formed aggregates are of larger size which sediment faster. This is further supported by the TNF which is lesser compared to that of the control sample. Due to the high initial concentration of cells, no significant change in the PIT of both the samples is observed. Fig. 5. Variation in magnetic force acting on the aggregates as represented by the ENC.
4. Conclusion Table 1 Aggregation parameters of samples with and without IMF (n ¼ 10) Parameter
Without IMF
With IMF
P value
DNmax DTmax (s) TNF PIT (s) PCT (min)
2416077916a 10.971.5 13977356 1.4470.7 30.87877.4
2434979137 10.20071.8 13087315 1.240070.5 28.97477.3
0.46 0.09 0.15 0.25 0.006
The influence of the IMF on erythrocytes and their formed aggregates is primarily due to the paramagnetic property of the deoxygenated erythrocytes. The force due to the field gradient accelerates the sedimentation process and is proportional to the effective number of cells in the aggregate. This technique could further be combined with imaging of these aggregates, to analyze the cellular mechanisms in hemoglobin-affected erythrocytes.
Details of abbreviations are given in the text. a Mean7SD.
References
formed aggregates is enhanced under the influence of the IMF. This is further supported by the variation in the PCT. Using Eq. (1) the force acting on the cells and their aggregates of a given volume (obtained from the ENC data) due to the IMF was calculated. Fig. 5 shows that the magnetic force increases with the increase of aggregate volume as represented by the ENC. This force is primarily responsible for the enhanced mobility of the aggregates.
[1] M. Okazaki, N. Maeda, T. Shiga, J. Eur. Biophys. 14 (1987) 139. [2] D. Maleville, F. Paul, S. Roath, IEEE Trans. Magn. 11 (1975) 1701. [3] F. Paul, et al., IEEE Trans. Magn. 17 (1981) 1701. [4] M. Singh, M. Kumaravel, J. Cell. Eng. 1 (1995) 21. [5] J. Sanjay, M. Singh, Proceedings of the International Conference on Biorheology, Sofia, October 2000, p. 13. [6] S. Swarnamani, M. Singh, IEEE Trans. Biomed. Eng. 30 (1983) 70.
Influence of an inhomogeneous magnetic field on erythrocyte aggregation mechanism—an analysis by He–Ne laser aggregometer Sanjay Jayavanth*, Megha Singh Biomedical Engineering Division, Indian Institute of Technology, Madras 600 036, India
Abstract The influence of the inhomogeneous magnetic field (IMF) on the erythrocytes and their aggregates, while sedimenting in a glass chamber under gravity, is analyzed. The aggregation data are acquired by using online He–Ne laser aggregometer and are represented in terms of various dynamic parameters. The analysis shows that the sedimentation of the erythrocytes and their aggregates is accelerated in the presence of the IMF. Thus, this technique could be applied in the study of blood samples in diseases with altered hemoglobin properties. r 2002 Elsevier Science B.V. All rights reserved. Keywords: Erythrocytes; Aggregation; Inhomogeneous magnetic field
1. Introduction The influence of inhomogeneous magnetic fields (IMF) on blood is due to the paramagnetic property associated with the iron atom of hemoglobin in deoxygenated erythrocytes [1]. This property is employed to capture the red cells [2], separate the malarial parasitized erythrocyte [3], and to change the erythrocytes sedimentation rate [4]. Erythrocyte aggregation is a reversible dynamic process where in cells form chain-like structures in the presence of plasma proteins. As the application of the IMF may affect the aggregation process, the objective of the present study is to analyze the influence of the IMF on this mechanism under gravitational field.
mentation of aggregates. For computation of the aggregation parameters this signal was digitized and acquired by the PC. The experiments with IMF were conducted with the sample chamber sandwiched between two coaxially held ring magnets by mutual attraction (Fig. 2). 2.1. Magnetic field calculation The distribution of magnetic field and its gradient with respect to the chamber was measured by thin Hall probe [6] (Fig. 3). The effective force acting on the erythrocytes and their aggregates, due to field gradient was calculated using the equation [1,2] Fmag ¼ wV ðH dH=dhÞave ;
ð1Þ
6
2. Material and methods Fig. 1 shows the block diagram of He–Ne laser aggregometer [5]. Laser light was passed through the erythrocyte suspension in plasma, placed in a glass chamber. The transmitted intensity (TI) was detected by the photodiode-amplifier configuration during the sedi*Corresponding author.
SI units, the magnetic susceptwhere w=3.88 10 ibility of deoxygenated erythrocytes, V is the volume of erythrocytes (m3) and ðH dH=dhÞave is the average gradient of the magnetic field along the height of the chamber (2.8 109 A2/m2/m). 2.2. Sample preparation Fresh blood samples were obtained by venepuncture from healthy subjects (n ¼ 10) in citrate phosphate
0304-8853/02/$ - see front matter r 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 8 8 5 3 ( 0 2 ) 0 0 6 7 6 - 5
S. Jayavanth, M. Singh / Journal of Magnetism and Magnetic Materials 252 (2002) 412–414
413
trough of the TI signal correspond to minimum (Nmin ) and the maximum number of cells (Nmax ) in the OV, respectively. The variation in the number of cells (Nmax 2Nmin ) indicates the change in aggregate size, which is thresholded at 3000. For further analysis the variations in the TI signal are divided into four categories. These data are presented in terms of following parameters: Aggregate size index (ASI): indicates the instantaneous change in the aggregate size, given by the ðDNÞ ¼ Nmax 2Nmin : DNmax represents maximum aggregate size and DTmax represents the sedimentation time corresponding to DNmax measured as the time width at the base of the given fluctuation. Total number of fluctuations (TNF): include all the fluctuations in the TI observed during the movement of the formed aggregates. The process initiation time (PIT): indicates the time required for the first fluctuation of the order over 3000 to occur in TI from t ¼ 0: The process completion time (PCT): indicates the total time required for the completion of the sedimentation process from the beginning till the maximum TI (I0 ). Effective number of cells (ENC): indicates the number of cells present in the OV at any instant of time and was calculated from the ASI variation. Further details of this analysis are given elsewhere [4,5].
Fig. 1. Block diagram of aggregometer.
Fig. 2. Sample chamber with ring magnets.
3. Results and discussions Fig. 3. Distribution of magnetic field (H) and its gradient (H dH=dh) along the height (h) of glass chamber.
dextrose, as an anticoagulant (10:1.4). Each sample was centrifuged at 3000 RPM for 20 min. Thereafter, the plasma was separated and the buffy layer on top of the cells was discarded. From each sample a suspension of erythrocytes of 5% hematocrit (Hct.) was prepared in the plasma and was used for aggregation measurement with and without the presence of IMF, at room temperature (25711C).
Fig. 4 shows the variation in the erythrocyte aggregation and sedimentation through the OV. The TI varies with time from the minimum to maximum associated with fluctuations. This is because initially the cells are monodisperse and with the formation and sedimentation of aggregates the mean intensity rises gradually. In the presence of the IMF, the maximum TI is attained faster compared to that of the control sample (without the IMF). This means that the mobility of the cells and their
2.3. Data analysis The aggregation data were analyzed for a set of parameters that describe the sequence of changes in the aggregation. The number of cells in the observed volume (OV) at any instant, based on Beer’s law, is given by Nt ¼ ð1=aX ÞlnðI0 =It Þ;
ð2Þ 1
where a is attenuation coefficient (0.772 cm for the sample at 5% Hct.), X is the mean thickness of erythrocyte (4 mm), I0 is the TI with plasma alone and It is the TI of the sample at any instant ‘t’. The peak and
Fig. 4. Aggregation curves of erythrocyte suspension of a healthy subject with and without the IMF.
414
S. Jayavanth, M. Singh / Journal of Magnetism and Magnetic Materials 252 (2002) 412–414
Table 1 shows the aggregation parameters for samples with and without the influence of IMF. The change in PCT is highly significant. The DNmax shows that the maximum aggregate size is higher in case of the sample with IMF. The DTmax under the influence of IMF is lesser, indicating that the formed aggregates are of larger size which sediment faster. This is further supported by the TNF which is lesser compared to that of the control sample. Due to the high initial concentration of cells, no significant change in the PIT of both the samples is observed. Fig. 5. Variation in magnetic force acting on the aggregates as represented by the ENC.
4. Conclusion Table 1 Aggregation parameters of samples with and without IMF (n ¼ 10) Parameter
Without IMF
With IMF
P value
DNmax DTmax (s) TNF PIT (s) PCT (min)
2416077916a 10.971.5 13977356 1.4470.7 30.87877.4
2434979137 10.20071.8 13087315 1.240070.5 28.97477.3
0.46 0.09 0.15 0.25 0.006
The influence of the IMF on erythrocytes and their formed aggregates is primarily due to the paramagnetic property of the deoxygenated erythrocytes. The force due to the field gradient accelerates the sedimentation process and is proportional to the effective number of cells in the aggregate. This technique could further be combined with imaging of these aggregates, to analyze the cellular mechanisms in hemoglobin-affected erythrocytes.
Details of abbreviations are given in the text. a Mean7SD.
References
formed aggregates is enhanced under the influence of the IMF. This is further supported by the variation in the PCT. Using Eq. (1) the force acting on the cells and their aggregates of a given volume (obtained from the ENC data) due to the IMF was calculated. Fig. 5 shows that the magnetic force increases with the increase of aggregate volume as represented by the ENC. This force is primarily responsible for the enhanced mobility of the aggregates.
[1] M. Okazaki, N. Maeda, T. Shiga, J. Eur. Biophys. 14 (1987) 139. [2] D. Maleville, F. Paul, S. Roath, IEEE Trans. Magn. 11 (1975) 1701. [3] F. Paul, et al., IEEE Trans. Magn. 17 (1981) 1701. [4] M. Singh, M. Kumaravel, J. Cell. Eng. 1 (1995) 21. [5] J. Sanjay, M. Singh, Proceedings of the International Conference on Biorheology, Sofia, October 2000, p. 13. [6] S. Swarnamani, M. Singh, IEEE Trans. Biomed. Eng. 30 (1983) 70.