TUBES Red blood cells (erythrocytes) constituting 45% of blood volume significantly affect the flow properties of blood. Flow properties of blood in turn are among the most important determinants of flow in circulatory system, hence the perfusion of all tissues, including the vital organs. Erythrocytes form aggregates under low flow conditions or at stasis. Erythrocyte aggregation interferes with the re- establishment of blood flow following stasis or continuation of flow at lower rates, increasing flow resistance. Erythrocyte aggregation tendency is increased in case of inflammatory conditions including infections and rheumatoid diseases, metabolic disorders and cardiovascular diseases. The degree of enhancement in erythrocyte aggregation correlates with the severity of the diseases process. Quantification of erythrocyte aggregation is therefore clinically important. There are commercially available instruments specialized for the quantification of erythrocyte aggregation, manufactured by various companies

Red blood cells aggregate with a time course determined by both their intrinsic properties and the suspending phase (plasma) properties, after the establishment of appropriate conditions of aggregation (i.e., following transition to stasis after flow). Alterations in these properties during diseases processes (e.g., increased plasma fibrinogen concentration) affect the time course and the size of formed aggregates. Aggregation is a reversible process. Erythrocyte aggregates are dispersed to individual erythrocytes under sufficiently high shear forces and participate in flow independently from each other.

Various methods have been used to quantitate erythrocyte aggregation. The oldest and most widely used clinical method for quantification of erythrocyte aggregation is the determination of "erythrocyte sedimentation rate" (ESR). A strong correlation between the parameters reflecting erythrocyte aggregation and ESR have been reported by a number of investigators (Ozdem, S., H. S. Akbas, L. Donmez, and M. Gultekin. 2006. Comparison of TEST 1 with SRS 100 and ICSH reference method for the measurement of the length of sedimentation reaction in blood. Clinical Chemistry and Laboratory Medicine 44:407-412; Hardeman, M. R., M. Levitus, A. Pelliccia, and A. A. Bouman. 2010a. Test 1 analyzer for determination of ESR. 1. Practical evaluation and comparison with the Westergren technique. Scand. J. Clin. Lab. Invest. 70: 21-25). However, ESR is not a preferred test for quantification of erythrocyte aggregation for the following reasons: 1) ESR is an indirect indicator of aggregation. 2) It is not sensitive and accurate enough. 3) It requires at least an hour to be completed. 4) It does not provide any information about the time course of aggregation process. Alternative methods have been developed for more detailed analysis of erythrocyte aggregation. The principle of monitoring light transmitting and reflecting properties of anticoagulated blood samples have been used in the devices manufactured for the measurement of erythrocyte aggregation, during the last 30 years. Recording the time course of optical properties (light reflectance or transmittance) of blood during erythrocyte aggregation is known as "syllectrometry", and this record (time course) is known as "syllectogram" (Zijlstra, W. G. 1958. Syllectometry, a new method for studying rouleaux formation of red blood cells. Acta.Phys.Pharm.Neerl. 7: 153-154). A syllectogram should be recorded under special conditions, for a proper reflection of aggregation process:

1) Erythrocyte aggregates are totally dispersed in to individual erythrocytes, by applying sufficiently high shear forces (disaggregation).

2) Erythrocyte aggregation is started by sudden stop of flow (or shift to a lower rate of flow)

3) Recording (analysis) of light transmittance or reflectance is started with this sudden change in flow conditions. Various approaches have been applied in medical instruments measuring erythrocyte aggregation to obtain these special conditions (especially for adequate disaggregation). Shearing the blood sample between two surfaces by rotating one of them (Schmid-Schonbein, H. 1979. Apparatus for measuring the aggregation rate of particles. US patent: 4135819), pumping blood through special micro- channels at sufficiently high flow rates (Shin, S., J.H. Nam, J.X. Hou, J.S. Suh. 2009. A transient, micro fluidic approach to the investigation of erythrocyte aggregation: The threshold shear-stress for erythrocyte disaggregation. Clin. Hemorheol.Microcirc. 42: 117-125) or stirring by a tiny magnetic bar in a special chamber (Shin, S., Y. Yang, and J. S. Suh. 2007. Microchip-based cell aggregometer using stirring-disaggregation mechanism. Korea-Australia Rheology Journal 19: 109-115) are among the methods used for disaggregation. Specially designed and manufactured measurement chambers should be used to apply these methods during the measurement of erythrocyte aggregation. Shin et al. proposed that externally vibrating the whole measurement chamber can be used for disaggregation (Shin, S., M. S. Park, Y. H. Ku, and J. S. Suh. 2006. Shear-dependent aggregation characteristics of red blood cells in a pressure-driven microfluidic channel. Clin.Hemorheol.Microcirc. 34:353-361), however this approach could not be proven to be effective for aggregation measurements. Some of these measurement chambers and associated disaggregation mechanisms are integral parts of the instruments measuring erythrocyte aggregation. Such disaggregation systems should be cleaned from blood sample and dried following each measurement. Some instruments utilize specially designed, disposable measurement chambers.

Using disposable disaggregation mechanisms in instruments developed for measuring erythrocyte aggregation has several advantages: 1) Cleaning of disaggregation mechanisms (cone-plate, plate-plate, concentric cylinder) from blood generates biohazard risk. It is essential to minimize this risk in modern clinical analyses systems. 2) Cleaning and drying of the measurement- disaggregation systems those are integral parts of the instruments are time consuming. Using disposable measurement chambers may prevent such problems. Tomita proposed a method of measuring erythrocyte aggregation using a transparent vinyl tube of 2.6 mm diameter and 300 mm length (Tomita M, 1989. Apparatus for measuring aggregation rate of whole blood reod blood cells. US patent No: 4822568). Disaggregation is achieved by continuous pumping blood of blood through this tube, using a syringe. The transparent tube can be used as a disposable unit, however the system requires large amount of blood to be pumped through, to obtain the adequate flow for complete disaggregation. Being able to perform the measurements using a small sample size is an important advantage for routine clinical analysis. Shin et al. developed a measurement chamber having a miniature stirrer for disaggregation; this metallic stirrer is rotated by a magnetic rotor for disaggregation (Shin, S., Y. Yang, and J. S. Suh. 2007. Microchip-based cell aggregometer using stirring-disaggregation mechanism. Korea-Australia Rheology Journal 19: 109-115). However, disposable measurement chamber of currently available measurement systems require special design and manufacturing, therefore relatively expensive.

The invention includes the method of measuring erythrocyte aggregation using low cost, glass capillary tubes utilized for other purposes in clinical laboratories (microhematocrit tube), as a disposable measurement chamber. A novel disaggregation mechanism has been developed for effective disaggregation in disposable glass capillaries. The measurement of erythrocyte aggregation can be performed using ~40 microliter of blood obtained by finger prick. The invention has been explained by the aid of figures included. The content of these figures are as follows:

Figure 1. Disaggregation mechanism including miniature selenoid and piston moving the blood sample back and forth in the glass capillary and the photometric measurement system. Figure l .a. Alternative mechanism for disaggregation; moving the piston using a miniature electric motor.

Figure 2. Flow chart of the erythrocyte aggregation measurement according to the invention.

Figure 3. Light transmittance time course (syllectogram) following disaggregation according to the invention.

Figure 4. Calculation of parameters reflecting static and dynamic properties of erythrocyte aggregation using light transmittance time course.

Figure 5. Evidence for the effectiveness of disaggregation according to the invention; comparison of light transmittance during disaggregation achieved according to the invention and by flow at various rates, induced using a syringe pump.

Figure 6. Influence of blood sample hematocrit on the aggregation index measured according to the invention.

Figure 7. Influence of blood sample hematocrit on light transmittance during disaggregation according to the invention.

Items used in the figures are identified with numbers corresponding to:

1. Miniature selenoid

2. Piston

3. Cylinder

4. Connecting element

5. Adaptor for the glass capillary

6. Glass capillary (microhematocrit tube; 1 mm id, 75 mm length)

7. Blood sample (filling the mid portion of the capillary)

8. Light source

9. Phototransistor

10. Supporting element for the glass capillary

11. Temperature controlled box (maintained at 37 °C) 12. Driver circuit

13. AD/DA converter

14. Electronic amplifier (for conditioning the signal from phototransistor)

15. Computer

16. Miniature electric motor

The invention includes the photometrical measurement of erythrocyte aggregation using disposable glass capillaries (6) (microhematocrit tube) widely utilized in clinical laboratories and disaggregation mechanism enabling such measurements.

The apparatus is presented schematically in Figure 1. The flow chart related to the method of erythrocyte aggregation measurement according to the invention is presented in Figure 2.

A method for the measurement of erythrocyte aggregation comprises the steps of,

- half filling the heparinized glass capillary (6) (microhematocrit tube) with blood (7)

- placing the capillary (6) into the adapter (5) in a temperature controlled box (11),

- moving the blood (7) column back and forth by vibrating the air column in the cylinder (3) by the solenoid (1),

- digitizing and recording the light transmittance through the blood-filled capillary (6), after the stopping of vibration and

- integrating the light transmittance-time curve to calculate the red blood cell aggregation index.

The apparatus to disperse existing aggregates in the blood samples (disaggregation) as the initial step of the measurement of erythrocyte aggregation by monitoring light transmittance of the blood sample comprising,

-a glass capillary (6) which is filled with a blood sample (7), -a temperature controlled box (11) into which glass capillary (6) is located, -an adaptor (5) which is located in the temperature control box (11) and connects to the glass capillary (6),

-a supporting element (10) for the glass capillary (6),

-a connecting element (4) which is connected to the adaptor (5) from one side,

-a cylinder (3) which is connected to the other side of the connecting element (4),

-a piston (2) which moves in the cylinder (3) and generates pressure pulses in the cylinder (3) and glass capillary (6),

-a miniature solenoid (1) which actuates the piston (2),

-a driver (12) which controls the forward movement of the miniature solenoid (1),

-a light source (8) which illuminates the glass capillary (6),

-a phototransistor (9) which records the light intensity and located on the opposite side of the capillary (6) and

-a computer (15) which records the light intensity values received from the phototransistor (9), via an AD/DA converter (13) and an electronic amplifier (14). Glass capillary (microhematocrit tube) (6) which has 1 mm internal diameter and 75 mm length is half-filled with blood (7). This sample (7) can be obtained directly from a finger prick as the standard microhematocrit tubes (glass capillary) (6) are coated with an anticoagulant (heparin). Alternatively, blood samples (7) obtained using a syringe for other purposes can also be used.

Blood sample (7) is moved to the middle part of the microhematocrit tube (6), by slightly raising one side and attached to the special adaptor piece (5) which tightly fits to the capillary (6), in the box (11) of with about 40 x 80 x 15 mm dimensions with the temperature maintained at 37 °C. This adaptor (5) is connected to a miniature cylinder (3) via a connecting element (4). The internal diameter and length of the cylinder (3) are approximately 2 and 8 mm, respectively. The pistons (2) tightly fitting to the cylinder (3) is moved by a selenoid (1). Each activation of solenoid (1) moves the piston (2) about 2 mm, while it returns back to original position when the current is turned off, by the aid of a spring. This back and forth movement of the piston (2) generates sudden positive and negative pressure pulses in the cylinder (3) and the capillary (6) which is pneumatically connected to it. It has been observed that each activation-deactivation cycle of solenoid (1) results in 8 mm back and forth displacement of blood (7) in the glass capillary (6).

An alternative method has been presented in Figure 1. a for the movement of piston (2) using a miniature electric motor (16), instead of the solenoid (1).

The activation cycle of the solenoid (1) is controlled by a driver circuit (12) which is in turn driven by a digital computer (15) via a suitable interface (AD/DA converter, 13). If an electric motor (16) is used to move the piston (2), than the driver circuit (12) controls the rotational speed of the motor (16). The disaggregation apparatus can run at frequencies between 2 - 50 per second (i.e., number of back and forth movements per second). However, the studies on the prototype of the system revealed that the optimum disaggregation conditions can be achieved with the activation of selenoid 5 times per second (at 5 Hz frequency),

Following the placement of the glass capillary (6) loaded with blood (7), it is illuminated by a light source (8) and the light intensity recorded by the phototransistor (9) located on the opposite side of the capillary (6) is started to be recorded on the digital computer (15) via the interface (AD/DA, 13), following the signal conditioning (14). A general-purpose, desktop or a laptop computer can be used for this purpose. Alternatively, all the control, data collection and calculations can be done using a specially programmed microprocessor. During the initial phase of the measurement procedure controlled by the computer (15), the piston (2) is moved at the desired frequency to disperse all erythrocyte aggregates existing at stasis. This movement of the piston (2) vibrates the air column in the cylinder (3) and connecting elements (4), forcing the blood sample (7) located in the middle portion of the capillary (6) to move at the same frequency. This back and forth movement of blood (7) in the capillary (6) generates the sufficiently high shear stresses to disperse erythrocyte aggregates into individual cells.

Following the disaggregation of the blood sample (7) by back and forth movements, the computer (15) stops the movement of piston (2) , which also results in the sudden stop of the movement of blood sample (7) in the capillary (6).

The computer (15) continues to record the light transmittance (i.e., light intensity recorded by the phototransistor (9) for appropriate length of time. The light intensity recording process is ended after the predetermined length of time and the aggregation index is calculated by the computer (15) using the light transmittance time course (syllectogram) (Figure 3).

As shown in Figure 3, light transmittance through the blood sample exhibits a sudden decrement following the stop of the movement of blood sample (disaggregation process). The reason for the decrement in light transmittance is the return of erythrocyte shape to normal from the elongated form achieved to accommodate the shear forces and transition to a random distribution from a status of orientation to flow stream lines, both occurring during the disagggregation phase of the measurement procedure. This re-distribution is completed within a second following the disaggregation period and followed by aggregation of erythrocytes with a typical time course. Erythrocyte aggregation is characterized by increased light transmittance (Figure 3). Light transmittance reaches to a minimum in the fraction of a second following the stop of the movement of blood, which is then followed by an increment in transmitted light intensity with a characteristic time course. The time course of light transmittance following the above-mentioned minimum reflects the clinically important aggregation properties of erythrocytes (Zijlstra, W. G. 1958. Syllectometry, a new method for studying rouleaux formation of red blood cells. Acta.Phys.Pharm.Neerl. 7: 153-154.; Dobbe, J. G. G., G. J. Streekstra, J. Strackee, M. C. M. Rutten, J. M. A. Stijnen, and C. A. Grimbergen. 2003. Syllectometry: the effect of aggregometer geometry in the assessment of red blood cell shape recovery and aggregation. IEEE Trans. Biomed. Eng. 50:97-106). Aggregation indexes reflecting these properties can be calculated by integrating the initial (5 or 10 second) parts of the light transmittance time course (Baskurt, O. K., H. J. Meiselman, and E. Kayar. 1998. Measurement of red blood cell aggregation in a "plate -plate" shearing system by analysis of light transmission. Clin.Hemorheol.Microcirc. 19, no. 4:307-314; Bauersachs, R. M., R. B. Wenby, and H. J. Meiselman. 1989. Determination of specific red blood cell aggregation indices via an automated system. Clin.Hemorheol. 9: 1-25; Kiesewetter, H., H. Radtke, R. Schneider, K. Mussler, A. Scheffler, and H. Schmid-Schonbein. 1982. The mini erythrocyte aggregometer: a new apparatus for the rapid quantification of the extent of erythrocyte aggregation. Biomed.Tech. (Berlin) 28:209-213; Hardeman, M. R., J. G. G. Dobbe, and C. Ince. 2001. The laser-assisted optical rotational cell analyzer (LORCA) as red blood cell aggregometer. Clin.Hemorheol.Microcirc. 25: 1-11; Shin, S., Y. Yang, and J. S. Suh. 2007. Microchip-based cell aggregometer using stirring-disaggregation mechanism. Korea-Australia Rheology Journal 19: 109-115). This method of calculation providing the area below the light transmittance time course (syllectogram) curve (Area B, Figure 4) has been successfully used to assess erythrocyte aggregation in many clinical and experimental studies. This approach is also being used to express the results obtained by the majority of instruments developed for measuring erythrocyte aggregation. The measurement of erythrocyte aggregation according to the invention can be completed within 30 seconds, including disaggregation period, recording of light transmission and calculation of aggregation index. Erythrocyte aggregation can be evaluated in more detail by recording light transmittance time course for longer periods (e.g., 120 seconds). Additional parameters reflecting the extent and time course of erythrocyte aggregation can be calculated using such curves (Figure 4). Amplitude (AMP, the total magnitude of the change in light intensity during the measurement period), aggregation half time (Ti _/2 , time required to reach the level corresponding to 50% of AMP, and aggregation index (AI, the ratio of the area below the light transmittance time course curve to the sum of the areas above and below the curve) are among the parameters used by instruments developed previously. Additionally, the light transmittance time course curve can be fitted to the double-exponential equation given below (Hardeman, M. R., J. G. G. Dobbe, and C. Ince. 2001. The laser- assisted optical rotational cell analyzer (LORCA) as red blood cell aggregometer. Clin.Hemorheol.Microcirc. 25: 1-11):

I _t = a + b - e ^~t/Tfas ' + c · e ^~t/Tslow

The time constants (T _fast and T _s i _ow ) can be used to mathematically express the time course of erythrocyte aggregation.

The same mathematical approaches can be used to analyze the light transmittance time courses obtained according to the invention.

In order to obtain reliable and accurate results by the instruments developed for measuring erythrocyte aggregation, a mechanism which is effective for complete disaggregation should be used at the initial phase of the measurement (Baskurt, O. K., M Uyuklu, P. Ulker, M. Cengiz, N. Nemeth, T. Alexy, S. Shin, M. R. Hardeman, and H. J. Meiselman. 2009c. Comparison of three instruments for measuring red blood cell aggregation. Clin.Hemorheol.Microcirc. 43:283-298). For this reason, the effectiveness of the disaggregation mechanism developed according to the invention has been evaluated by comparing the light transmittance measured during disaggregation according to the described invention with the light transmittance measured during disaggregation achieved by pumping the same blood samples through the same glass capillaries in a wide range of flow rates, using a positive-pressure pump. Figure 5 demonstrates the light transmittance levels as a function of shear rate corresponding to various flow rates generated by the pump (black squares). Light transmittance increases as shear rate decreases below 200 s ^"1 shear rate. This increment in light transmittance with decreased shear rate is due to increased aggregation under lower shear forces. That means, erythrocyte aggregates exist in this shear rate zone. Erythrocyte aggregates become smaller as shear rate increases reaching to a minimum at about 170 s ^"1 , indicating that this shear rate level corresponds to complete disaggregation. Therefore, light transmittance level at this shear rate characterizes the complete disaggregation. Light transmittance slightly increased at shear rates above this critical level, which can be explained by the shape change and orientation of erythrocytes with higher shear forces. Increasing shear rate up to 7000 s ^"1 did not cause any further increment in light transmittance above that shown in Figure 5. The white square in Figure 5 demonstrates the light transmittance level measured during disaggregation with the periodic back and forth movement of blood sample, according to the invention. This light transmittance level is very close to that measured during complete disaggregation induced by pumping the blood through the same capillary (as explained above). Therefore, it can be deduced from this experiment that the disaggregation mechanism according to the invention can generate shear forces sufficient for complete disaggregation in the blood sample under investigation. However, it is not possible to estimate the magnitude of shear forces generated during this period, since a continuous flow regime does not develop with this mechanism. Erythrocyte aggregation is influenced by the hematocrit (the volume fraction of erythrocytes in blood). Figure 6 demonstrates the aggregation indexes (the area under the light transmittance time course curve) measured using the system according to the invention for blood samples with hematocrit adjusted to 20-60%. This dependence of aggregation indexes on hematocrit of the sample has been reported by a number of investigators. This dependence underlines the necessity for a standardization of measured aggregation indexes according to the hematocrit of the sample. The light transmittance of the blood sample during disaggregation process (i.e., the period during which the blood sample is moved back and forth in the glass capillary, according to the invention) reflects the hematocrit value (Figure 7) and hemoglobin oxygen saturation. If the influence of hemoglobin oxygen saturation is omitted or reduced to negligible levels, light transmittance level during disaggregation can be used to estimate the sample hematocrit using a regression equation. It has been observed that the sensitivity of light transmittance to oxyhemoglobin saturation significantly decreased as the wavelength of the light used for measurement approaches to 800 nm (the isosbestic point for hemoglobin and oxyhemoglobin). Therefore, it is strongly recommended that a light source at 800 nm wavelength (or above) should be used for aggregation measurement. In that case, hematocrit value estimated using the light transmittance data obtained during disaggregation can be used to correct aggregation indexes according to the hematocrit of the sample under investigation.

Developed method may provide the basis for the development of an instrument for fast and easy assessment of aggregation which does not need cleaning. The advantages of this instrument include:

1. Utilization of disposable glass capillaries (microhematocrit tube) as the disposable unit of the instrument significantly decreases the measurements costs. This has been achieved by the development of a novel disaggregation method, including movement of blood sample back and forth in the glass capillary by a miniature piston.

2. The measurement can be performed using about 40 microliters of blood. This sample can be obtained by a finger prick; therefore there is no need for a venipuncture for the sampling. The sample size in some of the currently used devices for the same purpose might be as high as 1 milliliter.

3. Currently available instruments developed for the measurement of erythrocyte aggregation include disaggregation mechanisms that need to be cleaned after the measurement. This necessity introduces a biohazard risk to the measurement procedure. Additionally, cleaning and drying procedures prolong the total measurement time. There is no need for cleaning with the system developed according to the invention, as disposable glass capillaries are used.

An instrument developed according to the invention, having the above listed superiorities against the currently available instruments might be widely used in clinical medicine. Erythrocyte aggregation strongly correlates with erythrocyte sedimentation rate (ESR) which is commonly used for evaluating the severity of inflammatory-infectious events and rheumatoid diseases. Therefore, a fast, easy to use and relatively less expensive method can also be used in physician's offices. The erythrocyte aggregation measurement can be completed within a minute using the methods according to the invention, while the equivalent ESR test takes at least an hour to be completed.