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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2016 Jul 5;97(3):285–292. doi: 10.1111/iep.12182

New approaches in tail‐bleeding assay in mice: improving an important method for designing new anti‐thrombotic agents

Max Seidy Saito 1,2, André Luiz Lourenço 1,2, Hye Chung Kang 2, Carlos Rangel Rodrigues 3, Lucio Mendes Cabral 4, Helena Carla Castro 1, Plínio Cunha Satlher 1,4,✉
PMCID: PMC4960579  PMID: 27377432

Summary

This report describes a modified, simple, low‐cost and more sensitive method to determine bleeding patterns and haemoglobin concentration in a tail‐bleeding assay using BALB/c mice and tail tip amputation. The cut tail was immersed in Drabkin's reagent to promote erythrocyte lysis and haemoglobin release, which was monitored over 30 min. The operator was blinded to individual conditions of the mice, which were treated with either saline (NaCl 0.15m), DMSO (0.5%) or clinical anti‐thrombotic drugs. Our experimental protocols showed good reproducibility and repeatability of results when using Drabkin's reagent than water. Thus, the use of Drabkin's reagent offered a simple and low‐cost method to observe and quantify the bleeding and rebleeding episodes. We also observed the bleeding pattern and total haemoglobin loss using untreated animals or those under anti‐coagulant therapy in order to validate the new Drabkin method and thus confirm that it is a useful protocol to quantify haemoglobin concentrations in tail‐bleeding assay. This modified method provided a more accurate results for bleeding patterns in mice and for identifying new anti‐thrombotic drugs.

Keywords: anti‐thrombotic agents, bleeding time, tail‐bleeding assay

Thrombotic and cardiovascular diseases are the first cause of death worldwide (Yusuf & McKee 2014; WHO, 2013). Currently, there are several research groups working with the improvement of the treatment of these diseases by developing new anti‐thrombotic drugs with higher efficacy and lower side effects compared to current therapeutics. For these types of study, the assays that allow the evaluation of bleeding time are essential and important (Baron & Kamath 2014; Chapman & Yuen 2014; El‐Hayek et al. 2014).

Murine models are one of the current preclinical models for testing new anti‐haemostatic or anti‐thrombotic drugs. They usually involve tail amputation and a careful control of different interference factors such as mice strains, body temperature and blood pressure (Stella et al. 1975; Dejana et al. 1979; Greene et al. 2010). According to the literature it is difficult to establish a common protocol for testing and compare new anti‐thrombotic drugs (Herbert & Bernat 1993; Tanaka et al. 1998; Elg et al. 1999; Gadi et al. 2009; Greene et al. 2010; James & Ogirima 2011; Liu et al. 2012; Song & Wang 2012; Frattani et al. 2013; Chelucci et al. 2014). As a result, bleeding time assays vary considerably between laboratories, which hinders fair comparison of new drug prototypes and may eventually cause the loss of potential drugs (Greene et al. 2010; Liu et al. 2012).

A bleeding time assay in humans was first described in 1901 by Millian. Due to the importance of evaluating bleeding patterns in clinical patients, this bleeding test was widely disseminated amongst investigators both for clinical and for research purposes and is still traditionally used (Ivy et al. 1935; Borchgrevink & Waaler 1958; Greene et al. 2010). O′Brien defined the bleeding time as the time between the infliction of a small standard cut and the very first moment when the bleeding stops (O'Brien 1951). It is directly related to the interaction between platelets and damaged vessel wall, leading to the formation of a haemostatic plug (Liu et al. 2012).

Currently, bleeding induction assays in mice involve (i) a small incision (template model) (Stella et al. 1975; Dejana et al. 1979; Chelucci et al. 2014) or (ii) total amputation of the tip of the tail (transection model) (Dejana et al. 1979; Herbert & Bernat 1993; Tanaka et al. 1998; Elg et al. 1999; Gadi et al. 2009; Greene et al. 2010; Liu et al. 2012; Song & Wang 2012; Frattani et al. 2013). Dejana and co‐workers compared these two techniques and showed that the bleeding time is significantly longer in the transection model (Dejana et al. 1979). This model was sensitive to anti‐aggregant, anti‐coagulation and fibrinolytic drugs, mainly due to the different types of damaged vessels and resulting vascular injury (Caprino et al. 1973; Davis et al. 1974; De Clerck & Goossens, 1976; Minsker & Kling 1977; Dejana et al. 1979), allowing a broad spectrum of analytical observations.

Bleeding monitoring in the transection model is performed by (i) blotting the tip of the tail in filter paper at predetermined time period (s) until bleeding ceases (filter paper method) (Herbert & Bernat 1993; Tanaka et al. 1998; Grüner et al. 2004; Mangin et al. 2006; Gadi et al. 2009; Nieswandt et al. 2011) or (ii) immersing the tail into tubes containing isotonic solutions or water, usually at 37°C (immersion method) (Elg et al. 1999; Broze et al. 2001; Gushiken et al. 2009; Liu et al. 2012; Song & Wang 2012; Frattani et al. 2013). In the filter paper method, the operator may compromise the assays during blotting by pushing the injury against the paper and traumatizing the tail, wrongly inducing longer bleeding times (Greene et al. 2010). Due to this risk, the immersion technique is the most accepted technique nowadays (Greene et al. 2010).

The immersion technique is usually performed either at room temperature (23°C) or at 37°C (Elg et al. 1999; Gadi et al. 2009; Liu et al. 2012; Song & Wang 2012; Frattani et al. 2013). However, the temperature of the solution may interfere in the assay as bleeding times detected at 37°C can be significantly shortened due to the higher blood flow in comparison with a room temperature counterpart (Dejana et al. 1979). Other factors that may affect the assay include the following: (i) variation in distance of tail transection (2, 3, 5 or 10 mm) (Dejana et al. 1979; Herbert & Bernat 1993; Tanaka et al. 1998; Elg et al. 1999; Gadi et al. 2009; Greene et al. 2010; Liu et al. 2012; Song & Wang 2012; Frattani et al. 2013), (ii) sharpness of the scalpel and/or surgical knife inducing crush injury and (iii) instrument used (razor blade, scalpel, surgical knife, retractable knife) (Greene et al. 2010). Currently, there is no standardization in the distance of tail transection reported by the literature; however, it is well known that a bigger transection causes more blood loss (Greene et al. 2010). Thus, to avoid crush injury, the tail should be cut with a fixed downwards force (Leidenmuehler et al. 2009; Greene et al. 2010).

According to Liu and co‐workers, bleeding time does not vary in mice treated with clopidogrel at different doses, whereas bleeding volume differs significantly (Liu et al. 2012). This infers that bleeding time alone is insufficient to determine the potential haemorrhagic profile of a drug and points to the need of complementary assays to obtain more accurate and valid results. The current literature also shows several studies regarding transection and template models, describing limitations of using bleeding time evaluation alone (Greene et al. 2010; Liu et al. 2012).

In this work, we described an improved method to determine haemoglobin (Hb) concentration and total Hb loss by modifying the current immersion method in mice with the additional inclusion of Drabkin's reagent as a lysing agent. This modification may improve the identification of new anti‐thrombotic drugs, allowing a better comparison of their therapeutic potential compared to the current molecules that are in clinical use.

Material and methods

Animals and compounds evaluated

Male and female BALB/c mice weighing between 20–25 g (4–5 weeks of age) were provided by NAL‐UFF (Laboratory Animals Nucleus – Federal Fluminense University). We used NaCl 0.5m (calcium chloride) and DMSO 0.5% (vehicle dimethyl sulfoxide 0.5% – SIGMA®, St. Louis, MO, USA) as controls and acetylsalicylic acid (10 mm/kg – SIGMA®), a platelet anti‐aggregant drug and rivaroxaban (1 mm/kg – Bayer®), an anti‐coagulant drug, to verify interference in bleeding. Arabic Gum (SIGMA®) at 5% concentration was used for the suspension of these compounds prior to the oral gavage. Drabkin's reagent (catalog number: K023) and standard Hb (catalog number: K029) were obtained from Bioclin/Quibasa®, (Belo Horizonte, Minas Gerais, Brazil).

Ethical approval

All experiments were approved by the CEUA‐UFF (Ethical Committee of Animal Use) under a protocol 146/13 and were in accordance with the COBEA (Brazilian College of Animal Experimentation; 2012) (COBEA, 1991) and CARE (Committee on Animal Research and Ethics; 2012) (CARE, 2012) rules.

Tail‐bleeding assay with Drabkin's reagent

Liu and co‐workers (Liu et al. 2012) proposed an assay with tail amputation and immersion in isotonic saline to observe the bleeding volume. In this work, we propose a new approach to this method to measure the Hb concentration after tail amputation (Figure 1).

Figure 1.

Figure 1

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Modified Bleeding time protocol using Drabkin's reagent.

The body weight of these animals was determined, and, after 1‐h fasting, compounds were administered orally. One hour postadministration, we anaesthetized the animals with a solution of ketamine and xylazine (100 and 16 mg/kg, respectively), as proposed by Sathler and co‐workers (Sathler et al. 2014). Animals were placed in a supine position to continuously verify the possible lethality or cessation of anaesthesia during the experiment (30 min). A distal 5‐mm segment of the tail was amputated at 90° degree vertically with an in‐house device (patent pending) with fixed force (F = 1.4N) to reduce bias (i.e. operator amputation force). After amputation, the position was changed to vertical orientation and blood was collected in microtubes containing 1.5 ml of Drabkin's reagent. This reagent lyses the erythrocytes and the released Hb reacts irreversibly with potassium cyanide and potassium ferricyanide. Thus, Hb was oxidized to the stable pigment cyanmethaemoglobin (Bors et al. 2011; Acker et al. 2012). Over a total of 30 min, the microtube containing both bleeding tail and Drabkin's solution was gently homogenized and 500 μl was transferred every 5 min to five wells of a 96‐well plate (100 μl for each well) (Figure 1). The quantity of Drabkin's reagent was reset to 1.5 ml after each transfer. Absorbance was measured spectrophotometrically using a microplate reader at 420 nm (SpectraMax® 190 – Molecular Devices®), and Hb quantification was obtained using the equation:

Hb quantity(g/dl)=sample absorbance×calibration factor

The calibration factor was obtained as the ratio between a known standard concentration and the relative standard absorbance, as described by the manufacturer. We used three groups of three animals for each compound, and at the end of experiment, animals were euthanized by anaesthesia overdose as described by (Liu et al. 2012). Additionally, we performed the tail‐bleeding assay by collecting blood in microtubes containing 1.5 ml of water, following the same protocol for comparison.

Bleeding assay comparison

A standard Hb curve with known Hb concentration was used as a control for comparison between water and Drabkin's reagent as an immersion solution. We also explored two different optical densities (OD – 420 and 540 nm). Therefore, we prepared four different dilutions with equal concentration (10 μl of standard in 2.5 ml water or Drabkin's reagent; 20 μl in 5.0 ml; 30 μl in 7.5 ml and 40 μl in 10 ml) to determine the reproducibility and to verify the repeatability. The assay was performed in three consecutive days and the Hb standard ratio was 1:250 to ensure optimal reproducibility as proposed by manufacturer's methodology.

Statistical analysis

The statistical analysis was performed with anova followed by Tukey's test, with a significant value of P ≤ 0.05 in the spss 14.0® (Armonk, New York, USA) for Windows. All results were expressed as the mean ± standard deviation or otherwise specified.

Results

The comparison between water and Drabkin's reagent in the tail‐bleeding assays showed higher Hb concentrations in water (18.30 g/dl) than in Drabkin's reagent (11.25 g/dl) at 420 nm (Table 1). The reproducibility evaluation revealed the same pattern and results, with higher concentrations when using water (20.21 g/dl) as reagent over Drabkin's reagent (12.00 g/dl). The control evaluations showed that all animals bleed soon after the total incision of the distal 5‐mm segment of the tail in all experiments, and none of the animals died during the procedure. Altogether, the results support the repeatability factor of our protocol.

Table 1.

Comparison of the standard haemoglobin concentrations for the analysis of the repeatability and reproducibility. The measurement refers to successive measurements in three different days of standard haemoglobin in Drabkin's reagent provided by the manufacturer, water or Drabkin's reagent

Parameter Repeatability/Reproducibility
Drabkin's reagent (Manufacturer) Water Drabkin's reagent
Average concentration (g/dl) 11.33/11.18 18.30/20.21 11.25/12.00
Standard deviation (g/dl) 0.37/0.16 1.12/4.42 0.56/0.61
Coefficient of variation (%) 3.27/1.43 5.20/3.52 3.56/1.53
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Interestingly, the assays showed a higher deviation and higher coefficient of variation when performed in water than in the Drabkin's reagent. These data support inclusion of Drabkin's reagent to achieve an optimized measurement of total Hb loss. Importantly, measurements of the detectable Hb concentrations in water were affected by OD and the dilution used with higher values obtained at 420 nm with the first dilution (10 μl in 2.5 ml). Differently, all results for Drabkin's assays showed no significant variation regardless of OD or dilution used, which reinforces the reproducibility of the method (Figure 2 and Table 1).

Figure 2.

Figure 2

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Comparison of standard haemoglobin concentration using different optical densities and dilution conditions for water and Drabkin's reagent. Standard dilution in water showed the highest values for 420 and 540 nm with the same pattern in different dilutions compared with Drabkin's reagent.

The new proposed experimental conditions for Drabkin‐tail‐bleeding assay revealed the controls (DMSO 0.5% and NaCl 0.15m) with maximum Hb concentration in the very beginning of the test (15.3 g/dl and 11.9 g/dl, respectively) with a gradual decreasing bleeding pattern during the 30‐min period. Animals treated with acetylsalicylic acid (10 mm/kg) showed a rebleeding episode after an interval lasting between 10 to 15 min, causing a remarkable increase in the Hb concentration. Similarly, animals treated with the anti‐coagulant drug (rivaroxaban – 1 mm/kg) showed a higher increase in the total Hb concentration after 15 min, also showing an accentuated rebleeding episode (Figure 3). Importantly, tail‐bleeding assay exhibited higher levels of released Hb when using water pointing to the increase in the bleeding pattern (Figure 4).

Figure 3.

Figure 3

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Comparison of the haemoglobin concentration using different time assays for evaluating platelet anti‐aggregant (ASA – acetylsalicylic acid) and anti‐coagulant (rivaroxaban) effects. Bleeding assay was performed by tail tip amputation and then immersing the tail in 2.5 ml of Drabkin's reagent. Platelet anti‐aggregant (ASA – acetylsalicylic acid) and anti‐coagulant (rivaroxaban) showed a significant increase in haemoglobin concentration compared with the negative controls.

Figure 4.

Figure 4

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Comparison of the haemoglobin concentration in water or Drabkin's reagent for different time periods.

The data from total Hb loss assays showed similar results for DMSO 0.5% and NaCl 0.15m, the negative controls (33.2 g/dl ± 6.4 and 30.5 g/dl ± 5.4, respectively) without significant differences. Animals treated with rivaroxaban showed an elevated Hb concentration (167.8 g/dl ± 1.4) followed by acetylsalicylic acid (59.5 g/dl ± 11.6), displaying a rebleeding pattern directly related to the total Hb loss (Figure 5). We also evaluated the influence of Drabkin's reagent in plasma coagulation tests (prothrombin time – PT and activated partial thromboplastin time – aPTT) and platelet aggregation assays using multiple activators (ARA – 500 μm; ADP – 3 μm; epinephrine – 2 μm; collagen – 5 μg/ml), and no statistical changes were detected (Figure not show).

Figure 5.

Figure 5

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Comparison of the total haemoglobin loss when using platelet anti‐aggregant (acetylsalicylic acid) and anti‐coagulant (rivaroxaban) after 30 min. Bleeding assay was performed by tail tip amputation and then immersing the tail in 2.5 ml of Drabkin's reagent. Platelet anti‐aggregant (acetylsalicylic acid) and anti‐coagulant (rivaroxaban) showed a significant increase in haemoglobin loss and *P < 0.05.

Discussion

Designing new anti‐thrombotic drugs is a challenge that still requires easy, fast and low‐cost methods and protocols for evaluating them (Yusuf & McKee 2014; El‐Hayek et al. 2014). At present, bleeding assay for murine models have significant limitations (Greene et al. 2010; Liu et al. 2012). The nature of these assays poses several bias possibilities, and this is in turn reflected in the different techniques associated with numerous protocols that compromise the current comparison and analysis of results and outcomes. Currently, the attempt to standardize these methods has failed thus requiring that further attention needs to be paid to this subject (Liu et al. 2012). Due to the improved accuracy and efficacy in the measurement of Hb concentrations, Drabkin's reagent use has been recommended for human blood by The International Committee for Standardization in Hematology (International Committee for Standardization in Haematology, 1967). This reagent is able to lyse erythrocytes and further oxidize Hb to cyanmethaemoglobin, a stable pigment, through the reaction with potassium cyanide and potassium ferricyanide (Bors et al. 2011; Acker et al. 2012; Thomson, 2013).

Thus, in this study, we proposed a new modification of the current method of assay in the mouse model by performing the assay of transection of the mouse tip of the tail followed by immersion in Drabkin's reagent. We have monitored the bleeding pattern continuously in order to determine the rebleeding and Hb concentration different periods. This approach differs from that of Liu et al. (2012), who reported the use of isotonic saline and the verification of bleeding volume only.

Our protocol design was determined in order to perform the assay at room temperature (23°C). This was to reduce bias, such as temperature‐driven vasodilation normally observed at 37°C. Additionally, this modified assay is able to evaluate different types of vessels (artery, vein and capillary) and offers more sensitivity to different patterns of bleeding (Liu et al. 2012).

The comparison between water and Drabkin's reagent in tail‐bleeding assays revealed that higher Hb concentrations are found when using water as an immersion solution. Unlike offering greater sensitivity, the overall Hb concentrations obtained using water surpassed the average concentration of the Hb standard by almost twofold. In the drug discovery field, this is an important issue as the biological pattern of new drug candidates is still unknown. Thus, a misinterpretation may lead to the exclusion of real and potential molecules with promising in vitro and in vivo activities. Water is capable of lysing erythrocytes (Johnston et al. 1993); however, the high Hb concentrations detected in water assays differ from the real Hb concentration. Such feature may be due to the different absorption rates of the system, caused by different forms of free Hb. Thus, bleeding assay with water also showed high values of standard deviation and coefficient of variation when compared to Drabkin's reagent in consecutive measurements or measurements made in three different days, which are indicative of poor precision (repeatability and reproducibility).

The analysis of our new modified protocol also revealed a variation in Hb concentration when standard Hb was diluted in different water volume ratios in contrast to Drabkin's reagent. This result reinforced the smaller variation and better reproducibility observed for Drabkin's reagent‐treated samples when compared to water despite different dilutions or OD selected. Overall, the evaluation of Drabkin's reagent as the immersion solution in consecutive measurements or in different days showed improved sensitivity, repeatability and reproducibility in comparison with previous approaches, with small changes in average concentrations, standard deviation and coefficient of variation.

According to Bowie and Owen (Bowie & Owen 1980), bleeding time is closely related to the primary haemostasis, which associates platelet interaction with vessel wall and formation of an haemostatic plug. Thus, it is an important factor to be considered when evaluating new anti‐thrombotic drugs. Johansen and co‐workers (Johansen et al. 2008) described an automated system with cameras and software for monitoring bleeding time. The measurement of Hb concentration showed promising results but failed due to the Hb instability, often found in downstream oxidized forms. In this work, we showed that the normalization of Hb oxidative content to cyanmethaemogoblin can improve the detection of total Hb released.

In this work, we also challenged the Drabkin modified method by evaluating a platelet anti‐aggregant (acetylsalicylic acid) and an anti‐coagulant drug (rivaroxaban). Our results showed a higher bleeding profile for both platelet anti‐aggregant and anti‐coagulant drugs in comparison with the controls, in accordance with the reports using the traditional immersion method with water (Altman et al. 2012; De Berardis et al. 2012; Angiolillo et al. 2013; Connolly et al. 2013; Field et al. 2013; El‐Hayek et al. 2014; Thomas et al. 2014). However, it is difficult to establish a fair comparison between our findings and the previous reports as none of them quantified total Hb loss through a concentration‐dependent curve. Liu and co‐workers (Liu et al. 2012) also described the increased bleeding time with the onset of rebleeding patterns in animals treated with these anti‐thrombotic drugs; however, this study also lacks a quantitative analysis of Hb loss.

The inclusion of Drabkin's reagent in the traditional tail‐bleeding assay allows the quantification of total Hb concentration in different periods of time and total Hb concentration after 30 min. These data pointed to a valid modified method to quantify blood loss after drug administration and aid to the current efforts for standardizing tail‐bleeding methodologies to reduce bias and get more accurate results (Greene et al. 2010; Liu et al. 2012).

The prolonged bleeding profile observed herein for acetylsalicylic acid is in agreement with the literature (Beaumont et al. 1955; Evans et al. 1968; Mielke et al. 1969; De Gaetano et al. 1971; Praga et al. 1972; Altman et al. 2012; De Berardis et al. 2012; Angiolillo et al. 2013; Connolly et al. 2013; Field et al. 2013; El‐Hayek et al. 2014; Thomas et al. 2014). Similar to our observations, anti‐coagulants such as rivaroxaban show higher bleeding profile when compared to a platelet anti‐aggregant treatment (Fareed et al. 2012; Kennedy et al. 2012; Bacchus & Crowther 2013; Scaglione, 2013). According to the literature and reinforced by our results, the high Hb concentrations observed in acetylsalicylic acid assays are mainly due to the rebleeding patterns, whereas rivaroxaban showed the highest value due to its ability to directly inhibit the prothrombinase‐complex‐bound forms of activated factor X (Fareed et al. 2012; Fawole et al. 2013). This is particularly important as high amounts of thrombin are required for an efficient clot formation and bleeding cessation (Fareed et al. 2012; Broomhead & Mallett 2013).

Furthermore, Sarratt et al. (2005) showed that mice lacking platelet‐collagen receptors exhibit prolonged tail‐bleeding times corroborating with several studies using newer compounds such as aegyptin (Calvo et al. 2010), tablysin‐15 (Ma et al. 2011), nitrophorin 2 (Mizurini et al. 2010). These molecules interfere in thrombus formation blocking collagen or contact pathway with no increase in blood loss when evaluated by the classical method of tail transection. In contrast, alboserpin (Calvo et al. 2011), DU‐176b (Fuhugori et al. 2008) and BMS‐654,457 (Wong et al. 2015), which are factor Xa inhibitors, showed prolonged bleeding times using the same technique, corroborating with our results, different from platelet anti‐aggregant drugs that showed different bleeding patterns.

According to our results, the Drabkin method can provide an accurate and precise description of bleeding patterns in vivo with a new perspective on quantitative analysis of Hb loss. Currently, the bleeding profiles caused by available drugs are only studied using bleeding time assays with filter paper, which calls for a better standardization of the method (Herbert & Bernat 1993; Tanaka et al. 1998; Grüner et al. 2004; Mangin et al. 2006; Gadi et al. 2009; Nieswandt et al. 2011).

In conclusion, we described a modified method using a simple and low‐cost approach, which is capable of measuring Hb concentration in stable form for different periods. We suggest that it could be a very useful tool for the identification of new anti‐thrombotic molecules in drug development researches.

Author contributions

Saito, MS participated in literature research, writing of the manuscript and structuring the figures. Lourenço, AL participated in literature research, manuscript revision and structuring the figures. Rodrigues, CR participated in concept of the manuscript. Cabral, LM participated in concept of the manuscript. Hye Kang participated in concept and revision of the manuscript. Castro, HC participated in concept and revision of the manuscript. Sathler, PC participated in concept and revision of the manuscript.

Sources of funding

We thank Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the financial support and fellowships.

Conflict of interest

The authors declare no conflict of interest.

All listed authors met ICMJE authorship criteria.

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