Effectiveness of Erythrocyte Morphology Observation as an Indicator for the Selection and Qualification of Blood in a Mechanically Induced Hemolysis Test

Abstract

Background: This study was conducted to confirm the reliability of an in vitro mechanically induced hemolysis test (ISO 10993-4:2017), which is essential for ensuring the safety of blood pumps. Methods: For appropriate anticoagulant selection, porcine blood was prepared in anticoagulant citrate dextrose solution A (ACD-A), heparin, and citrate phosphate dextrose adenine (CPDA-1), respectively, according to the ASTM F1830 standard. Anticoagulant-treated porcine and bovine blood were circulated in a mock circulatory loop (MCL) for 6 h to observe the rate of plasma-free hemoglobin (pfHb) and RBCs with morphological integrity. Results: A morphological loss of red blood cells (RBCs) was observed over time. While there were differences in morphological loss depending on the anticoagulant, no consistent trend could be identified. The pfHb concentration was significantly higher in bovine than in porcine blood. Conversely, the number of RBCs with morphological integrity decreased over time in both, but the ratio of RBCs with morphological integrity was similar across all timepoints. Conclusions: The percentage of RBCs with morphological integrity can be used as a reliable indicator for the interpretation of mechanically induced hemolysis results in different blood types. Furthermore, the reliability of the in vitro mechanically induced hemolysis test (ISO 10993-4:2017) was assessed.

1. Introduction

The international standard ISO 10993-4:2017 [1], as a method of evaluating the safety of medical devices, explores the hemolytic test. Hemolysis depends on material properties, including blood–material exposure time and surface energy, morphology, and chemistry. For evaluating the interaction of blood and medical devices, hemolysis may depend on local mechanical forces (mechanically induced hemolysis) and biochemical factors (material-induced hemolysis) [1]. In particular, mechanically induced hemolysis—caused by fluid dynamic factors such as flow rate, turbulence, and non-physiological shear forces generated by mechanical interactions between pumps and blood—is a risk factor that should be evaluated when developing medical devices as it can cause anemia or interfere with the blood-clotting system [2,3].

ISO 10993-4:2017 also provides considerations for in vitro tests to evaluate hemolysis. In vitro test methods can measure the amount of plasma hemoglobin at very low levels that cannot be measured in vivo. Dynamic testing under clinical use conditions to evaluate the influence of the medical device on the test environment, mechanical–physical interaction of materials and blood, clinically relevant use conditions (blood flow rate, rpm, pressure, and exposure time), intended use, and hemodynamic factors of hemolysis should be considered [1]. Hemocompatibility tests, such as mechanically induced hemolysis, have repeatability and reproducibility limitations; management difficulties, including physiological characteristics of test blood and blood collection and storage procedures, are a potential cause [4,5]. However, a detailed testing protocol was not provided.

The American Society for Testing and Materials (ASTM) F1830-19 standard [6] aims to standardize whole blood collection and preparation for the American Society for Testing and Materials (ASTM) F1841-19 evaluation standard [7] for the analysis of hemolysis in vitro. Hematological (red blood cell (RBC), WBC, platelet counts, hematocrit, and total hemoglobin concentration) and physiological blood parameters should be within the acceptable normal range, but information regarding these parameters is lacking. In addition, it is recommended that sufficient volumes of anticoagulants, such as citrate dextrose solution A (ACD-A), citrate phosphate dextrose adenine (CPDA-1), and heparin, and healthy large animal blood be used for testing [6]. However, specific information on how to determine blood conditions for test application and the differences in blood preservation techniques between anticoagulants is not provided. The evaluation of mechanically induced hemolysis may be affected by the donor species, sex, age, fasting, method of harvesting, anticoagulant properties, period of storage, biochemical state of the blood, and hemoglobin and hematocrit blood levels [8,9].

Application of human blood is a potential strategy for overcoming interspecies differences during testing; however, securing the volume required is difficult, and regulatory challenges, such as Institutional Review Board (IRB) approval, may hamper product safety evaluation processes. Therefore, some studies have compared blood characteristics in vitro using various animal blood samples as alternatives to human blood [10,11,12,13,14,15,16].

The ASTM F1841-19 standard requires a hemolysis evaluation comparison by simultaneously performing tests between test and control groups using blood from the same animal within 48 h of collection [7]. In general hemolysis studies, the plasma-free hemoglobin (pfHb) concentration is used as an indirect evaluation indicator for hemolysis. However, for mechanically induced hemolysis, confirming test repeatability is limited in hemolysis evaluation using a single evaluation indicator because the initial state of the individual RBC can affect the hemolysis result significantly. Consequently, it is necessary to apply additional indicators to explain the hemolysis caused by various blood conditions by directly observing morphological changes such as structural damage to RBC membranes [17,18,19,20,21,22,23].

The structural organization of the cytoplasmic membrane contributes to mechanical integrity by responding quickly to external fluid shear stress throughout the circulatory life of RBCs. The degree of RBC membrane loss and the resulting increase in cell sphericity are used as indicators of severity for some human genetic anemia disorders [24,25].

The aim of the present study was to evaluate the performance of a self-developed in vitro mock circulatory loop system model and to confirm the validity of the test method by introducing a morphological indicator that changes RBC shape from normal biconcave disc-shaped cells with a concave center to a spherical shape. Furthermore, the reliability of the in vitro mechanically induced hemolysis test (ISO 10993-4:2017) was assessed.

2. Materials and Methods

2.1. Materials and Reagents

ACD-A was purchased from CEPHAM Life Sciences (Fulton, MD, USA). CPDA-1, heparin, and Drabkin’s reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) and bovine blood were purchased from Gibco (Grand Island, NY, USA) and Innovative Research (Novi, MI, USA), respectively.

2.2. Preparation of Experimental Blood

The Korea Research Institute of Bioscience and Biotechnology (Republic of Korea) and Cronex (Cheongju, Republic of Korea) anesthetized porcine and collected cardiac blood in containers containing anticoagulants, and we purchased the blood. Laboratory animal blood anticoagulated with ACD-A (1:5 v/v), CPDA-1 (1:7 v/v), or heparin (final concentration approximately 4000–6000 USP units/L), in accordance with the ASTM F1830 standard, were compared. Blood with an anticoagulant was refrigerated until testing.

Table 1. Summary of experimental animals.

Case 1 Porcine	Cronex, sus scrofa, 3 months, Female
Case 2 Porcine	KRIBB, sus scrofa, 8 months, Female

contains a summary of the experimental animals used.

2.3. Observation of RBCs

Microscopic analyses of RBCs were performed using an Inverted Phase Contrast Microscope (ECLIPSE Ts2; Nikon, Tokyo, Japan). RBCs (10 μL) were resuspended in 990 μL of PBS and transferred to a hemocytometer. Only normal RBCs (biconcave disc-shaped) were counted, and cell concentration was determined by multiplying the counted cells by the dilution factor. Two slides were made per sample. To ensure inter-rater reliability, the slides were reviewed by two or more individuals [26,27].

2.4. Mock Circulatory Loop System Model Design

The mock circulatory loop (MCL) was constructed based on the model presented in ASTM F1841-19 for an in vitro hemolysis test (Figure 1) [7]. The MCL included a polyvinyl chloride (PVC) reservoir and tube lines, blood pumps, heat exchangers, sensors, and adjustable clamp resistors. To remove all hemolytic variables excluding those produced by the pump, the internal diameter change of the blood tube was minimized, and the reservoir was designed and manufactured to minimize congestion based on the reservoir module proposed by Olia et al. [28] The upper part of the medical blood bag (Green TF Bag; Doowon Meditec, Yongin-si, Republic of Korea) was diagonally heat-sealed, the inlet port was tilted parallel to the angle, and the outlet port was applied to a funnel-shaped form at the bottom to enable easy mixing of the blood in the reservoir. Medical PVC Tygon^® tubes (ND-100-65, 3/8″ O.D.; Saint-Gobain Performance Plastics, Saint-Quentin-Fallavier, France) commonly used in CPB were used.

Measurement and control equipment were set up to confirm the fluid state in the tube and achieve the desired clinical conditions. A pressure gauge (P-MAT; PendoTECH, Princeton, NJ, USA) was placed 10 cm from the pump inlet and outlet ports to record the total pressure head (PΔ = Pin-Pout). To measure the flow rate, ultrasonic flow probes (ME9PXL; Transonic Systems Inc., Ithaca, NY, USA) and flow meters (TS410; Transonic Systems Inc.) were placed at the pump inlet, and the flow rate and pressure head were adjusted using a throttle clamp resistor. To prevent heat-related blood cell damage, the temperature was maintained at 36 ± 2 °C using a water bath and Liebig’s condenser combined with a PVC tube. A hose barb temperature sensor (Single-Use Temperature SensorsTM; PendoTECH) was used to measure the temperature inside the tube without interfering with the fluid path. To minimize blood responses from loop components in contact with blood, such as connectors and sampling ports, medical consumables with guaranteed biocompatibility were used.

2.5. Mechanically Induced Hemolysis

Using anticoagulant-treated whole blood, mechanically induced hemolysis was evaluated with a roller (L/S^® Analog Drive with Cytoflow TM Pump Head; Masterflex, Germany) and centrifugal pumps (Rotaflow RF32; Maquet Cardiopulmonary AG, Gelsenkirchen, Germany). The tests were performed according to ASTM F1841. Before the test, the blood was warmed to 37 °C, and its condition was confirmed. The inside of the ASTM F1841 test loop was primed with PBS before filling with blood; all air was removed. To simulate clinical conditions, the roller operating speeds were 400 rev/min. By adjusting the throttle clamp, the pump pressure head and flow rate were set to 80 ± 10 mm Hg and 5.0 ± 0.2 L/min, respectively. The priming volume was 400 mL. Blood was continuously circulated in the loop for a total of 6 h, and 5 mL of blood samples was collected at 1 h intervals through the sampling port for analysis. Considering the blood stagnated in the sampling port, the first 1 mL of collected blood was discarded. Some blood samples were collected and observed for morphological changes using a microscope (ECLIPSE Ts2; Nikon). The collected blood samples were centrifuged at 2000× g for 15 min (D2012 plus; DLAB Scientific, Beijing, China). The separated supernatant was re-centrifuged at 13,000× g for 15 min to collect plasma, which was used for hemolysis analysis.

2.6. Hemolysis Analysis

Plasma hemoglobin was measured using the cyanmethemoglobin method. The collected plasma was diluted with Drabkin’s reagent (1:5), and the absorbance was read at 540 nm after 15 min using a spectrophotometer (HITACHI U-2900; Hitachi High-Technologies Co., Tokyo, Japan). The pfHb levels were quantified using the following equation:

$$pfHb\left(\raisebox{1ex}{$\mathrm{m}\mathrm{g}$}\!\left/ \!\raisebox{-1ex}{$\mathrm{d}\mathrm{L}$}\right.\right)={Abs}_{540\mathrm{n}\mathrm{m}}\times F\times \frac{1}{D}$$

(1)

where Abs_540nm is the average absorbance of the test plasma, F is the slope of the determined hemoglobin reference solution curve, and D is the dilution ratio of Drabkin’s reagent. Quantified data were plotted with the pfHb curve, according to sampling time, and a linear regression analysis was performed. To explain the change in pfHb, the normalized hemolytic index (NIH) was calculated using the following equation:

$$NIH\left(\raisebox{1ex}{$\mathrm{g}$}\!\left/ \!\raisebox{-1ex}{$100\mathrm{L}$}\right.\right)=\frac{∆pfHb\times V\times \frac{\left(100-Hct\right)}{100}}{Q\times ∆T\times 1000}$$

(2)

where ΔpfHb is the change in the pfHb concentration (mg/dL) for the sampling interval, V is the volume of blood in the loop (mL), Hct is the hematocrit, Q is the flow rate (L/min), and ΔT is the sampling time interval (min). The blood sampled at each time point was observed under a microscope, and the number of normal-shaped RBCs was counted to confirm trends over time.

2.7. Statistics

Statistical analyses of the data generated were performed using paired statistical testing between the matched individual subject and device tests, and values are indicated as mean ± standard deviation (SD). The statistical significance was set at p < 0.05.

3. Results

3.1. Changes in RBC Shape According to Storage Time

In order to identify morphological changes in porcine blood RBCs, the appropriate selection of anticoagulants becomes necessary. The anticoagulants ACD-A, CPDA-1, and heparin recommended by ASTM F1830 were used. RBC morphology was observed immediately after collection, and morphological changes were observed over time. Blood containing anticoagulants was stored in the refrigerator, and a sample was removed for observation at each time period.

In Case 1 porcine, all anticoagulants showed marked morphology decreases after 24 (31–72%) and 72 h (10–22%) (Figure 2A). Similarly, all anticoagulants showed marked morphology decreases after 24 (38–62%) and 72 h (7–11%) in Case 2 porcine (Figure 2B). There were notable differences between anticoagulants at some observation points for both cases; however, no trend was confirmed.

3.2. Test Condition Conformation

Simulated clinical environment settings remained constant, and no leaks or blood clots occurred during the 6 h blood circulation. Flow rate and pressure changes remained constant for each pump (Figure 3). The average blood temperature of the centrifugal pump (37.29 ± 0.16 °C) was significantly higher than that of the roller pump (35.96 ± 0.17 °C), under the same constant temperature conditions (p < 0.05).

3.3. Mechanically Induced Hemolysis Analysis in Bovine and Porcine Blood

Using bovine and porcine anticoagulant-treated blood, mechanically induced hemolysis was evaluated with a roller pump. In both bovine and porcine blood, pfHb concentrations increased over time, while the number of RBCs with morphological integrity decreased (

Table 2. Quantitative results of the hemolysis tests.

		Time (Hours)						Slope	R2	Hct
		1	2	3	4	5	6
Porcine (n = 3)	ΔpfHb (mg/dL)	11.4 ±5.1	22.8 ±8.8	34.7 ±14.7	47.7 ±19.5	57.9 ±22.3	74.0 ±29.7	12.1	0.997	22 ±3.2%
	Percent of normocytes (%)	72.4 ±7.3	51.7 ±12.5	40.7 ±8.9	26.9 ±8.3	19.5 ±4.6	11.9 ±4.1	14.1	0.943
Bovine (n = 3)	ΔpfHb (mg/dL)	181.6 ±27.5	312.4 ±20.6	493.9 ±51.4	575.7 ±30.2	768.4 ±58.1	909.7 ±56.4	148.8	0.995	43 ±1.7%
	Percent of normocytes (%)	69.8 ±10.9	61.4 ±12.4	50.6 ±15.0	43.6 ±7.4	36.4 ±11.3	14.7 ±4.6	12.2	0.943

and Figure 4). The pfHb increase rate was considerably higher in bovine blood than in porcine blood. However, there was no significant difference in the ratio of RBCs with morphological integrity or the rate of decrease between bovine and porcine blood.

3.4. Mechanically Induced Hemolysis Analysis in Roller and Centrifugal Pumps

Using anticoagulant-treated whole bovine blood, mechanically induced hemolysis was evaluated with roller and centrifugal pumps (Figure 5A). Linear regression analysis showed that the coefficient of determination (R2) was 0.995 and 0.982 for roller and centrifugal pumps, respectively. In both pumps, the ΔpfHb in the blood sample was linear and increased constantly, with no change in test conditions such as temperature. The ΔpfHb slope of the roller pump (148.79 ± 7.98 mg/dL) was significantly higher than that of the centrifugal pump (63.98 ± 16.60 mg/dL; p < 0.05). The NIH was 0.111 ± 0.006 g/100 L and 0.049 ± 0.012 g/100 L for the roller and centrifugal pumps, respectively (Figure 5B).

The morphological changes and the percentages of morphologically intact RBCs were analyzed (Figure 5C,D). At 6 h, the normal-shaped RBCs decreased by 85.3 ± 4.6% and 84.7 ± 8.67% for the roller and centrifugal pumps, respectively. In both pumps, the normal-shaped RBC percentages decreased similarly over time.

4. Discussion

Hemolysis can be defined as damage to the cytoplasmic membrane of RBCs due to mechanical or chemical factors in the environment (in vivo or extracorporeal), resulting in lysis and content release into plasma. In the context of blood and medical device interactions, hemolysis may occur due to local mechanical forces (mechanically induced hemolysis) and biochemical factors (material-induced hemolysis). Particularly, mechanically induced hemolysis, caused by forced blood circulation in medical devices, is a risk factor that should be considered during the development of medical devices [1].

The present study confirmed the comparative indicators of RBC morphology and quantitative hemolysis level and the suitability of the evaluation system using in vitro MCL based on ASTM F1841-19 (a standardized method approved by the FDA). Furthermore, we designed a hemocompatibility evaluation model that could be used as an interpretation index for the repeatability and reproducibility limitations for the application of mechanically induced hemolysis results.

The evaluation of mechanical-induced hemolysis in blood pumps requires loop systems to simulate the clinical environment. Effort has been made previously to minimize hemolytic variables. Horobin et al. [29] applied resistance to 150 mm tubing to minimize hemolysis caused by the local pressure of the tube when controlling the flow rate and pressure head of the pump. Gräf et al. [30] minimized the effect of fluid inertia by narrowing the distance between the atrium and ventricle to reduce hemolysis caused by blood flow in a simulated circulatory loop for pulsatile total artificial heart testing.

We constructed a loop system according to ASTM F1841-19 to evaluate mechanically induced hemolysis. To minimize hemolytic variables, changes in the internal diameter were minimized, and an optimized reservoir was designed to prevent distortion caused by blood congestion. Although the blood temperature was controlled to exclude thermal factors generated in the system, the blood equilibrium temperature of the centrifugal pump was higher than that of the roller pump under the same temperature-control conditions. This implies that the heat generated in the centrifugal pump system, including the motor, is higher than that generated in the roller pump system. According to Lepock et al. [31], RBCs exposed to high temperatures experience increased hemolysis because of thermal denaturation. Nevertheless, the hemolysis level in the centrifugal pump in the present study was lower than that of the roller pump, which implies that the heat-related hemolysis effect was limited in our test system and that the evaluation system was properly deployed.

We also observed the morphological changes of RBCs as auxiliary indicators for the evaluation of hemolysis in medical devices. The ASTM F1841-19 and F1830-19 standards specify that “blood is generally used within 48 h of blood draw, including the time for transport. Best consistency may be expected when blood is used within 24 h; older blood may only provide reliable comparison in paired studies.” [6,7]. However, we attempted to confirm the test method by reflecting the contents of the standard through a small number of repetitions as a limitation section. Specific information on how to determine blood conditions for test application and the differences in blood preservation techniques between anticoagulants is not provided. We confirmed morphological changes in RBCs using porcine blood based on ASTM F1830-recommended anticoagulants ACD-A, CPDA-1, and heparin.

After sampling, anticoagulated porcine blood continued to exhibit morphological losses, including loss of the biconcave disc-shaped RBC. Even within 24 h of collection, a wide range of morphological loss was observed. The differences are most likely due to the anticoagulant applied, but consistent differences could not be confirmed. Due to molecular defects in some skeletal proteins constituting the RBC membrane, the RBC surface area per volume is reduced, and the shape changes to a sphere. Spherical RBCs are easily destroyed, resulting in hemolysis; defects in the RBC cytoplasmic membrane cause morphological losses at an early stage [24]. Furthermore, RBC morphological changes were directly observed, and the possibility of applying additional indicators that could explain hemolysis according to various blood conditions was confirmed.

pfHb concentration was confirmed as an index for evaluation in mechanically induced hemolysis. In both bovine and porcine anticoagulant-treated blood circulated in a roller pump for 6 h, the pfHb concentration increased over time; the increased rate of pfHb was notably higher in bovine blood than in porcine blood, reflecting the condition of the blood, including hematocrit. In contrast to the increase in pfHb, the number of RBCs with morphological integrity continued to decrease over time in both porcine and bovine blood.

The percentage of normal RBCs over time showed a negative correlation with pfHb; however, the absolute increase in pfHb showed a significant difference between the roller and centrifugal pumps under identical initial blood conditions. This implies that the amount of hemoglobin released into plasma through damaged RBC membranes varies depending on the degree of structural damage, based on the pump operation principle.

The results confirm that it is difficult to estimate the degree and pattern of RBC structural damage accurately with a single indicator, such as pfHb concentration, and to reflect the variability of blood, considering the morphological characteristics (as RBC integrity indicators) are more sensitive than pfHb concentration. This indicates that the percentage of RBCs with morphological integrity is consistent with mechanically induced hemolysis results compared to pfHb and can be used as a reliable index when interpreting the results. Combining pfHb concentration and RBC morphological integrity observed under an optical microscope showed that RBC morphological characteristics are an effective auxiliary indicator for the selection of test blood and interpretation of hemolysis test results, and can be used as an index for interpreting the repeatability and reproducibility limitations for the application of mechanically induced hemolysis results.

It is essential to ensure blood conditions are suitable for testing to avoid inaccurate test results and large fluctuations that cause side effects that can threaten the safety of medical device users. Consideration of RBC morphological indicators is a convenient method for estimating blood integrity, thereby acting as a primary preventive measure to minimize variability in results.

5. Conclusions

When evaluating the in vitro blood circulation loop system described in ASTM F1841, blood condition screening is necessary to avoid large variations and inaccurate test results, which have adverse effects that may threaten the safety of the medical device user. Particularly, when selecting animal blood (replacing human blood) and the optimal anticoagulants for evaluating the blood compatibility of medical devices, it is essential to consider the initial condition of the animal blood and the anticoagulant characteristics. Observing RBC morphology is a convenient method for estimating blood integrity, and can be a primary preventive measure to minimize data variation in the evaluation of hemolysis in blood pumps. The correlation between pfHb concentration and RBC number observed by an optical microscope can be applied as an evaluation index in blood compatibility tests.