Monitoring red blood cell aggregation with nuclear magnetic resonance

Biochimica et Biophysica Acta, 805 (1984) 123-126

123

Elsevier

BBA Report BBA 10022

M O N I T O R I N G R E D B L O O D CELL A G G R E G A T I O N W I T H N U C L E A R M A G N E T I C RESONANCE MARK D. HERBST a,b and J.H. G O L D S T E I N a

Department of Chemistry and b School of Medicine, Emory University, Atlanta, GA 30322 (U.S.A.) (Received January 20th, 1984)

Key words: Erythrocyte," Cell aggregation," Water transport; NMR," Dextran; (Human blood)

We present a new application of nuclear magnetic resonance to monitor rouleaux formation in static blood suspensions of physiological hematocrit. The method measures the apparent mean residence time of water inside the rouleaux and yields the kinetics and the extent of red cell aggregation in units of cells per aggregate.

H u m a n red blood cells suspended in plasma or in artificial media containing certain macromolecules (e.g., dextran) tend to aggregate into long cylindrical formations called rouleaux. This report describes and utilizes a simple pulsed N M R technique for quantifying red blood cell aggregation. This new method, an extension of a previously used N M R procedure for measuring erythrocyte water transport, is based on the fact that the mean residence time of water inside red cells depends on the mean water volume to surface area ratio of the aggregates. Since aggregation increases this ratio, the basic water transport measurement can be modified to provide a measure of the extent of aggregation. The instrumentation employed can also be utilized for various other clinical tests, such as estimation of the viscosity of plasma or other fluids [1] and characterization of possibly malignant tissue [2]. While other techniques are available for measuring red cell aggregation into rouleaux, the N M R method is uniquely capable of providing both the kinetics and the extent of aggregation as well as water transport rates for single erythrocytes and r o u l e a u x in s u s p e n s i o n s of p h y s i o l o g i c a l hematocrit. Direct microscopy [3-5] can yield 0167-4889/84/$3.00 © 1984 Elsevier Biomedical Press

quantitative results, but only in dilute suspensions (1% hematocrit). Light transmission [6] or reflection methods [7,9] can measure relative aggregation of cells at various flow rates, but the results are qualitative and may be affected by hematocrit variation. Erythrocyte sedimentation rate (ESR) studies are inexpensive, but give only qualitative information about rouleau formation which is very sensitive to hematocrit and plasma viscosity [3,4]. Viscosity measurements at various shear rates [3] can be performed quickly, but the concentration of cells and the plasma viscosity exert a large effect on the results. In the literature, there are several detailed descriptions of the basic N M R technique for measuring any erythrocyte water transport [9-11], and our own version has been described previously [12]. In brief, the method employs a radiofrequency pulse to transiently label the protons in an erythrocyte suspension placed in a magnetic field. The label decays (relaxes) slowly inside the cells, but quickly outside the cells, because of the addition of the paramagnetic relaxation agent, Mn / +. By using two-compartment exchange equations, the composite relaxation curve can be analyzed to provide the mean residence time (~'a)

124 of water inside the cells. The extension of the technique requires a method for dispersing the labile aggregates. This was accomplished with a stirring paddle which was fashioned from a six-inch piece of polyethylene tubing by heating one end, pinching it closed, and then trimming the pinched end to the shape of a paddle. The open end of the tubing fit snugly onto an eight-inch long stainless steel syringe needle which was inserted into a spare air-driven N M R tube spinner mounted above the sample in the N M R magnet. Tile paddle was lowered into the blood sample, and stirring was controlled with a pinch clamp on the air inflow tubing. Complete dispersion without hemolysis was obtained after about 10 s of stirring, and zero-time measurements were made as soon as stirring ceased. Subsequent measurements were timed to within 1 s accuracy. The transverse magnetization decay curves were obtained with a Spin-Lock CPS-2 pulsed spectrometer (Spin-Lock, Port Credit, Ontario, Canada) equipped with a custom-built accessory which sampled the midpoints of the echoes from the Carr-Purcell-Meiboom-Gill pulse sequence [12]. Blood samples from healthy adults were obtained by ordinary venipuncture into heparinized Vacutainers. The final heparin concentration was about 10 U.S.P. units/ml blood. The whole blood was centrifuged at 1000 x g for 5 min at room temperature, and the supernatant was separated and centrifuged again at 1000 x g for 5 min at room temperature to remove any white cells that may have been taken with the plasma. The buffy coat was discarded. A portion of the packed cells was washed three times in isotonic 27 : 1 NaC1/KC1. Unwashed cells were resuspended in plasma, in plasma diluted 50% with isotonic 27 : 1 N a C 1 / K C I containing 4% ( w / v ) human serum albumin (Sigma, St. Louis, MO, U.S.A.), or in undiluted plasma which contained 5% ( w / v ) Dextran T 40 (Pharmacia, Piscataway, N J, USA). Washed cells were suspended in the isotonic N a C l / K C l / h u m a n serum albumin solution with or without 2% (w/v) Dextran T40 or Dextran T 70 (Pharmacia). Each of these six cell suspensions was prepared at a hematocrit between 40 and 45%. The suspending media contained approx. 4.4 mM MnC12 after addition of a small volume of isotonic 100 mM

MnC12 to each suspension. The transverse relaxation times of the suspending media were between 1.9 and 2.4 ms, and all measurements were performed at room temperature. The observed ~- values obtained for erythrocyte~ suspended in N a C 1 / K C 1 / h u m a n serum albumin did not change significantly over time, while those obtained for erythrocytes in plasma increased rapidly at short times, and then much more slowly at longer times. Dilution of the plasma by 50% or the presence in plasma of 5% (w/v) Dextran T 40 caused za to increase rapidly at first, but to level off at a value lower than that obtained in undiluted plasma. These results are illustrated in Fig. 1. At 2% (w/v) Dextran in N a C 1 / K C l / h u m a n serum albumin Dextran T 40 did not produce an increase in ~a values over time, but Dextran T 70 produced a curve similar to that obtained in undiluted plasma. These results are also illustrated in Fig. 1. The following discussion, based on the use of a new model, explains how aggregation into rouleaux affects the observed r, values, and how the average number of cells per aggregate can be estimated from these results. For simplicity, the model neglects the presence of trapped volume between cells in a rouleau. This trapped volume leads to a dependence of the observed aggregate ~-~ on the concentration of manganese in the suspending medium. Higher concentrations of manganese cause cells in a rouleau to appear as discrete exchanging units, because labeled water relaxes quickly in the intercellular trapped volume [13]. At low concentrations of manganese, the ~ of the aggregate behaves as if there were no trapped volume. The mean residence time of water inside monodispersed erythrocytes (r,) is related to the diffusional permeability (Pd) of the cell membrane by the following equation [3]:

llVw~

v~= - - - -

PdAc)

(1)

where Vw is the average volume of water inside a cell, and A c is the average surface area of a cell membrane. The same relationship applies to the mean resi-

125 dence time of water in a cell aggregate of n cells, %(n), if the v o l u m e / a r e a ratio for an average n-aggregate is used. 1

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(3)

By combining eq. 1, 2 and 3, a relationship between the aggregate % and the single-cell % is obtained. =

[ n - (n---n 1)2C ] ra

(4)

Rearranging Eq. 4 and introducing explicit time dependence of n and % leads to the following expression

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where %(1, 0) is identical with the previous single cell %, obtained at zero time. The best value of C is 0.3, which was estimated from geometrical considerations of p h o t o m i c r o g r a p h tracings of rouleaux [5] and recalculation of published dextran adsorption data [8]. Eq. 5 was used to convert experimental ~', values into the values of n presented in Fig. 2. It is from graphs of this type that aggregation rates may be obtained. The experimental part of this study employed various condition which were selected for the purpose of assessing the reliability of this N M R method for measuring red cell aggregation. For example, we were able to demonstrate that dilution of the plasma by 50% decreased the rate and the extent of red cell aggregation. This result is in accord with the fact that aggregation of cells in plasma is dependent on the concentration of macromolecules capable of forming bridges between cells which overcome intercellular repulsive forces [5]. In addition, 5% ( w / v ) Dextran T-40 in undi-

:~

13

120

Time (s)

Fig. 1. The time dependence of the mean residence time of water inside human erythrocytes ('ra) at room temperature immediately following disaggregation in various suspending media; ~, plasma; +, 50% diluted plasma; ×, plasma with 5% (w/v) de×tran T 40; rn, isotonic 27:1 NaCI/KC1 with 4% (w/v) human serum albumin; v, 2% (w/v) Dextran T 40; A, 2% (w/v) Dextran T 70. luted plasma was seen to inhibit aggregation. This is also consistent with previous findings that Dextran T-40 decreases aggregation of red cells in plasma by increasing cell surface potential and by decreasing the solubility of fibrinogen, a bridging macromolecule [14]. Finally, the aggregating ability of 2% ( w / v ) Dextran T 40 was much less than that of 2% ( w / v ) Dextran T 70, a difference also cited in the literature [15]. The conclusions of this study are: (1) red cell aggregation can be monitored easily and quantitatively by simple N M R methods and instrumentation, and (2) the results are in general agreement with previously reported observations regarding red cell aggregation, which provides additional confidence in the reliability of this N M R method. The importance of red cell aggregation has been extensively discussed, especially by Dintenfass [16]. The availability of a new, reliable, N M R - b a s e d technique will provide an additional route to further application of red cell aggregation data. This study was supported in part by a grant from the National Institutes of Health. The authors wish to express their gratitude to Mr. Keith Runyan and Dr. Mary Kimberly for their contributions

126 I

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Fig. 2. The time dependence of the average rouleau size immediately following disaggregation. The symbols are the same as in Fig. 1.

during the early stages of this work, and to Dr. Robert Long, Jr. for his expert technical assistance. One of the authors (M.D.H.) was a trainee in the Medical Scientist Training Program, U.S. Public Health Service Grant No. GM07415, during this study.

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