Methods of diagnosing hemolytic anemia and hypoxia using blood diagnostic indicators in cattle

Abstract

The present invention relates to a method for diagnosing hemolytic anemia and hypoxia using blood diagnostic indicators of cattle. According to the present invention, a method for diagnosing hemolytic anemia and hypoxia using blood diagnostic indicators of cattle can be provided, comprising a blood analysis step of obtaining blood information by analyzing the blood of cattle through a blood test, and a judgment step of determining the health status of the cattle regarding hemolytic anemia and hypoxia by comparing the blood information with blood diagnostic indicators.

Description

Technology Field

[0001] The present invention relates to a method for diagnosing hemolytic anemia and hypoxia using blood diagnostic indicators of cattle. More specifically, it relates to a method for diagnosing hemolytic anemia and hypoxia using blood diagnostic indicators of cattle, which can identify changes in anaerobic metabolites such as L-Lactate produced due to hypoxia in cattle, particularly Hanwoo cattle, and thereby provide blood diagnostic indicators that can immediately and rapidly diagnose hemolytic anemia and hypoxia in cattle at livestock farms. Background Technology

[0003] L-Lactate is a substance produced by anaerobic metabolism in the body, mainly in skeletal muscle, red blood cells, the brain, skin, and renal medulla. Since it is usually removed by the liver and kidneys through the buffering action of cellular buffers, an increase in the body due to accumulation is not observed in healthy conditions. However, it is known that if oxygen delivery to organs and cells in the body is reduced due to severe hemolytic anemia, anaerobic metabolism is induced by the resulting hypoxia, and as a result, the concentration of L-Lactate in the body increases significantly.

[0004] Meanwhile, while there are various major causes of economic damage to cattle farms, a particularly significant issue is disease, which leads to the death of cattle or growth disorders resulting from chronic conditions. Furthermore, when acute hemolytic anemia is induced in cattle due to various causes, it can lead to severe clinical symptoms related to the anemia or result in very high mortality rates.

[0005] Conventional methods for diagnosing hematological diseases in cattle have mainly used genetic testing methods (RT-PCR or real-time RT-PCR), and when whole blood is used, blood cell components can interfere with the PCR reaction, so blood-specific gene extraction kits or red blood cells have been separated (removed) from blood samples and used for testing.

[0006] However, since the aforementioned genetic testing method requires transporting blood samples to a research facility for testing, difficulties arose in detecting hematological diseases in cattle as L-Lactate levels changed over time in blood collected from animals with hemolytic anemia and hypoxia.

[0007] Therefore, in order to reduce the mortality rate caused by acute hemolytic anemia and related hypoxia in cattle, it is necessary to develop indicators that can immediately diagnose hemolytic anemia and hypoxia in livestock farms based on changes in related blood markers such as L-Lactate. Prior art literature

[0009] Korean Published Patent Application No. 10-2023-0015938 Prediction of Sepsis Symptoms (Jan. 31, 2023) Korean Registered Patent Application No. 10-2098083 Serum Preparations (April 1, 2020) The problem to be solved

[0010] In order to solve the above-mentioned problems, the purpose of the present invention is to provide a method for diagnosing hemolytic anemia and hypoxia using a blood diagnostic indicator of a bovine according to an embodiment of the present invention, which involves collecting whole blood and plasma from the blood of a bovine and analyzing blood information including L-Lactate using a kit, thereby diagnosing hemolytic anemia and hypoxia in a bovine more efficiently and quickly, and enabling early prevention or treatment measures for the said disease. means of solving the problem

[0012] To solve the above problem, the present invention includes a blood analysis step for obtaining blood information by analyzing the blood of a cow through a blood test according to an embodiment of the present invention, and a judgment step for determining the health status of the cow regarding hemolytic anemia and hypoxia by comparing the blood information with a blood diagnostic indicator. The blood analysis step may obtain one or more blood information among the number of RBCs, HCT ratio, hemoglobin (Hb) concentration, reticulocytes, monocytes, total bilirubin concentration, and whole blood L-lactate concentration by analyzing the blood of the cow through a blood test.

[0013] In addition, the blood analysis step can be performed within at least 30 minutes after the blood of the cow is collected.

[0014] In addition, the above judgment step may include a health status judgment step for determining the health status of the cow by comparing the blood information with the blood indicators of a normal cow when the cow is normal.

[0015] Additionally, the above judgment step may include a disease state judgment step for determining the state of hemolytic anemia and hypoxia of the cattle by comparing the blood information with blood diagnostic indicators of a normal cattle when the cattle have symptoms of either hemolytic anemia or hypoxia.

[0016] In addition, the disease state determination step may determine hemolytic anemia and hypoxia based on one or more of the following cases: when one or more of the values ​​of RBC (106 / μL), HCT (%), and Hb (g / dL) in the blood information are decreased compared to the blood diagnostic indicator of a normal cow; when one or more of the values ​​of Reticulocyte (103 / μL) and Indirect bilirubin (mg / dL) in the blood information are increased compared to the blood diagnostic indicator of a normal cow; and when the value of whole blood L-lactate (mmol / L) in the blood information is increased compared to the blood diagnostic indicator of a normal cow.

[0017] In addition, the disease state determination step is such that if the whole blood L-lactate in the blood information of a cow having symptoms of either hemolytic anemia or hypoxia has a strong negative correlation with HCT(%); the whole blood L-lactate in the blood information is RBC(10 6 Hemolytic anemia and hypoxia can be determined based on one or more of the correlation cases between blood parameters, such as when there is a negative correlation with Hb (g / m / m / dL) and when whole blood L-lactate in the blood information has a positive correlation with indirect bilirubin (mg / dL). Effects of the invention

[0019] The method for diagnosing hemolytic anemia and hypoxia using blood diagnostic indicators of cattle according to an embodiment of the present invention analyzes blood information of normal cattle and cattle with hemolytic anemia and hypoxia to provide data-based blood diagnostic indicators of normal cattle and blood diagnostic indicators of cattle with hemolytic anemia and hypoxia, thereby enabling the assessment of the health status of cattle to prevent hemolytic anemia and hypoxia at an early stage or to establish treatment measures.

[0020] Accordingly, this has the effect of reducing the incidence of hemolytic anemia and hypoxia, as well as mortality rates.

[0021] In addition, it is believed that this can be applied to the development of kits for the treatment and prevention of hemolytic anemia and hypoxia. Brief explanation of the drawing

[0023] FIG. 1 is a flowchart schematically illustrating a method for diagnosing hemolytic anemia and hypoxia using a blood diagnostic indicator of a cow according to an embodiment of the present invention. Figure 2 is a graph showing the results of an analysis comparing the hematological parameters of healthy cattle and cattle with hemolytic anemia. Figure 3 is a graph showing the results of an analysis comparing the correlation between blood parameters of healthy cattle and cattle with hemolytic anemia. Specific details for implementing the invention

[0024] The following description of the present invention with reference to the drawings is not limited to specific embodiments and allows for various modifications and various embodiments. Furthermore, it should be understood that the content described below includes all modifications and substitutions that fall within the spirit and scope of the present invention.

[0025] In the following description, terms such as "first," "second," etc., are used to describe various components and are not limited in their meaning; they are used solely for the purpose of distinguishing one component from another.

[0026] Identical reference numbers used throughout this specification indicate identical components.

[0027] The singular expressions used in the present invention include the plural expressions unless the context clearly indicates otherwise. Furthermore, terms such as "comprising," "having," or "having" described below should be interpreted as specifying the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0028] Hereinafter, with reference to FIGS. 1 to 3, a method for diagnosing hemolytic anemia and hypoxia using a blood diagnostic indicator of a bovine according to an embodiment of the present invention will be described in detail.

[0030] FIG. 1 is a flowchart schematically illustrating a method for diagnosing hemolytic anemia and hypoxia using a blood diagnostic indicator of a cow according to an embodiment of the present invention.

[0032] Referring to FIG. 1, a method for diagnosing hemolytic anemia and hypoxia using a blood diagnostic indicator of a cow may include a blood collection step (S1), a blood analysis step (S2), and a judgment step (S3).

[0033] Specifically, the blood collection step (S1) may collect blood by collecting blood from a cow to determine its health condition, hemolytic anemia, and hypoxia.

[0034] Here, cattle may refer to Hanwoo, beef cattle, Wagyu (Australia), Angus (USA), and dairy cows, etc., and in this specification, Hanwoo will be used as an example for explanation.

[0035] In addition, whole blood of a cow can be collected using a syringe containing a heparin solution, such as an ABGA syringe, in the blood collection step (S1), and this whole blood can be used to obtain blood information by separating plasma or serum in the blood analysis step (S2).

[0036] The blood analysis step (S2) may be a step of obtaining blood information by analyzing the blood collected in the blood collection step (S1) through a blood test.

[0037] Here, blood testing may confirm an increase in L-Lactate levels by measuring whole blood or serum blood samples after attaching a strip using a portable L-Lactate meter (StatStrip Lactate Xpress Meter), but is not limited thereto.

[0038] In addition, in the blood analysis step (S2), kits and devices may be used to measure the number of RBCs, HCT ratio, hemoglobin (Hb) concentration, reticulocytes, monocytes, total bilirubin concentration, etc., in addition to the L-Lactate values ​​described above.

[0039] At this time, the blood test can measure blood information immediately after collection, within at least 30 minutes, thereby preventing an increase in the concentration of lactate when stored at room temperature for a long time. Additionally, if it takes a long time to measure, inactivated sodium can be added to the collected blood to block lactate formation, or a blood collection tube containing fluoride oxalate can be used to increase accuracy.

[0040] Through this, one or more blood information such as the number of RBCs, HCT ratio, hemoglobin (Hb) concentration, reticulocytes, monocytes, total bilirubin concentration, and whole blood L-lactate concentration can be obtained, and mean erythrocyte volume (MCV), mean hemoglobin concentration in the mean erythrocytes (MCTC), composition ratios of platelets, white blood cells (WBC), neutrophils, lymphocytes, eosinophils, and basophils, and concentrations of direct and indirect bilirubin and total bilirubin can also be obtained.

[0041] The quantitative value of the composition of each blood information is 10 6 / μL, 10 3 Concentrations in units of / μL and g / dl can be determined for each composition, but are not limited thereto.

[0042] The judgment step (S3) can determine the health status of cattle regarding hemolytic anemia and hypoxia by comparing the blood information obtained in the blood analysis step (S2) with blood diagnostic indicators.

[0043] Here, blood diagnostic indicators may include normal cattle blood diagnostic indicators and disease blood diagnostic indicators. Here, the disease may be a hematological disease and, most preferably, may refer to hemolytic anemia and hypoxia in cattle.

[0044] More specifically, the judgment step (S3) may include one or more of a health status judgment step and a disease status judgment step. Such judgment steps may be optionally configured according to the user's needs.

[0045] First, the health status determination step can determine the health status of the cattle by comparing the blood information obtained in the blood analysis step (S2) with the blood diagnostic indicators of normal cattle. At this time, the cattle may be in a normal state, i.e., normal cattle, in which hemolytic anemia and hypoxia have not occurred.

[0046] Meanwhile, the health status assessment stage is not limited to this; to assess the health of cattle from a more diverse perspective, disease blood diagnostic indicators may also be utilized to determine the cattle's health status.

[0047] The blood diagnostic indicators for normal cattle and diseased cattle may include the blood reference values ​​for normal cattle and the blood reference values ​​for cattle showing hemolytic anemia, as shown in Table 1 and Figure 2 below.

[0048] The health status analysis step can determine hemolytic anemia and hypoxia based on one or more of the following cases: when one or more of the values ​​of RBC (106 / μL), HCT (%), and Hb (g / dL) in the blood information are decreased compared to the normal cattle blood diagnostic indicators; when one or more of the values ​​of Reticulocyte (103 / μL) and Indirect bilirubin (mg / dL) in the blood information are increased compared to the normal cattle blood diagnostic indicators; and when the value of whole blood L-lactate (mmol / L) in the blood information is increased compared to the normal cattle blood diagnostic indicators.

[0049] This allows for the early detection and prevention of internal hypoxia caused by hemolytic anemia.

[0050] In addition, the health status analysis step may provide blood information deviating from the normal blood standard values ​​of cattle from the acquired blood information as blood abnormality information if the cattle's health status is determined to be abnormal.

[0051] Such blood abnormality information may include blood compositions that deviate from the standard values ​​of normal cattle blood in the blood information, or numerical differences between the standard values ​​and the blood compositions that deviate from each standard value.

[0052] The disease state determination step can determine the state of hemolytic anemia and hypoxia in cattle (cattle) exhibiting symptoms of either hemolytic anemia or hypoxia by comparing the blood information obtained in the blood analysis step (S2) with the blood diagnostic indicators of normal cattle. In other words, depending on the degree of difference between the blood information and the blood diagnostic indicators of normal cattle, the state of hemolytic anemia and hypoxia can be classified more specifically into mild, severe, or early, middle, or late stages.

[0053] Specifically, the disease state determination step may determine hemolytic anemia and hypoxia based on one or more of the following cases: when one or more of the values ​​of RBC (106 / μL), HCT (%), and Hb (g / dL) in the blood information are decreased compared to the blood diagnostic indicator of a normal cow; when one or more of the values ​​of Reticulocyte (103 / μL) and Indirect bilirubin (mg / dL) in the blood information are increased compared to the blood diagnostic indicator of a normal cow; and when the value of whole blood L-lactate (mmol / L) in the blood information is increased compared to the blood diagnostic indicator of a normal cow.

[0054] In addition, hemolytic anemia and hypoxic conditions can be determined based on the magnitude of the increase or decrease in blood information corresponding to one or more of the above cases.

[0055] In addition, hemolytic anemia and hypoxic conditions can be determined by further utilizing the correlation between whole blood L-Lactate levels and blood parameters in the blood information.

[0056] More preferably, in the disease state determination step, if the whole blood L-lactate in the blood information of a cow with symptoms of either hemolytic anemia or hypoxia has a strong negative correlation with HCT(%), the whole blood L-lactate in the blood information is RBC(10 6Hemolytic anemia and hypoxia can be determined based on one or more of the correlation cases between blood parameters, such as when there is a negative correlation with Hb (g / m / μL) and Hb (g / dL), and when whole blood L-lactate in the blood information has a positive correlation with indirect bilirubin (Indirect bilirubin (mg / dL)).

[0057] In this way, the judgment information regarding the health status or the judgment information regarding hemolytic anemia and hypoxia derived in the judgment step (S3) can be provided to the user.

[0058] The above-described judgment method is preferably performed more quickly and efficiently by the user using a kit as described in the blood analysis step (S2) above, but it is not necessarily limited to this, and may be implemented in various ways, such as automatically through the system when blood information is input into a terminal including an application.

[0060] As described above, the method for diagnosing hemolytic anemia and hypoxia using blood diagnostic indicators of cattle according to an embodiment of the present invention analyzes the blood information of normal cattle and cattle with hemolytic anemia, respectively, and provides data-based blood diagnostic indicators for normal cattle and diseased cattle, thereby enabling the assessment of the health status of cattle within a barn to prevent hemolytic anemia and hypoxia at an early stage or to establish treatment measures.

[0061] Accordingly, it is believed that this has the effect of reducing the incidence and mortality rates of hemolytic anemia and hypoxia, and can be applied to the development of kits for the treatment and prevention of hemolytic anemia and hypoxia.

[0063] The present invention will be explained in more detail below using experimental examples based on the embodiments described above, but the present invention is not necessarily limited to these embodiments and experimental examples.

[0065] [Example 1] Normal cow

[0066] Fifteen normal Hanwoo cattle were selected as 'normal cattle', and blood was collected from each normal cattle. The collected blood was centrifuged to separate the serum, and the ratios and quantitative values ​​of the components in the blood were determined through blood tests.

[0067] In particular, L-Lactate levels were confirmed by measuring whole blood or serum blood samples after attaching the strip using a portable L-Lactate meter (StatStrip Lactate Xpress Meter).

[0068] The above blood components were analyzed by calculating the mean and standard deviation of the quantitative values ​​for each cow.

[0069] Hereinafter, Example 1 will be referred to as 'normal cow'.

[0071] [Example 2] Hemolytic anemia

[0072] Twenty-seven Hanwoo cattle with an HCT (%) of 26% or less were selected as hemolytic anemic cattle, and blood was collected from each normal cattle. The collected blood was centrifuged to separate the serum, and the ratios and quantitative values ​​of the components in the blood were determined through blood tests.

[0073] In particular, L-Lactate levels were verified by measuring them in the same way as those of normal cattle.

[0074] The above blood components were analyzed by calculating the mean and standard deviation of the quantitative values ​​for each cow.

[0075] Hereinafter, Example 2 will be referred to as 'hemolytic anemia'.

[0077] [Experimental Example 1] Quantitative values ​​and reference values ​​of blood composition in normal cattle and cattle with hemolytic anemia

[0078] The mean and standard deviation of the quantitative values ​​of the blood components of normal cattle and hemolytic anemic cattle, respectively, were calculated and are shown in Table 1 below. In particular, among the quantitative values ​​of the blood components of normal cattle and hemolytic anemic cattle, respectively, RBC (10 6 / μL), HCT (%), Hb (g / dL), Reticulocyte (10 3 The levels of indirect bilirubin (mg / dL) and L-lactate (mmol / L) were compared using the graph in Figure 2.

[0079] In Figure 2, normal cattle were referred to as 'Normal', and cattle with hemolytic anemia were referred to as 'Hemolytic anemia'.

[0081] Normal cow Hemolytic anemia p-value RBC(10 6 / μL) 7.0±1.0 (6.5-7.6) 4.0±1.2 (3.5-4.5) 0.000 HCT(%) 36.4±5.3 (33.4-39.4) 22.0±3.2 (20.6-23.3) 0.000 Hb(g / dL) 11.7±1.6 (10.8-12.7) 7.0±1.2 (6.5-7.5) 0.000 MCV(fL) 52.2±6.1 (48.8-55.7) 59.5±15.7 (53.0-66.0) 0.507 MCHC(g / dL) 32.3±1.2 (31.6-33.0) 31.8±1.7 (31.1-32.5) 0.006 Reticulocyte(10 3 / μL) 0.9±0.6 (0.5-1.2) 6.50±10.2 (2.3-10.7) 0.011 Platelet(10 3 / μL) 220±54 (189-251) 137±64 (110-163) 0.001 WBC(10 3 / μL) 15.8±2.7 (14.2-17.7) 13.5±3.0 (11.4-14.6) 0.056 Neutrophil(10 3 / μL) 5.6±1.2 (4.9-6.4) 2.2±1.0 (1.5-2.4) 0.000 Lymphocyte(10 3 / μL) 7.8±2.2 (6.5-9.1) 7.8±1.5 (6.8-8.5) 0.048 Monocyte(10 3 / μL) 0.9±0.2 (0.8-1.1) 3.0±1.0 (2.4-3.4) 0.000 Eosinophil(10 3 / μL) 1.5±1.2 (0.9-2.2) 0.4±0.4 (0.2-0.6) 0.000 Basophil(10 3 / μL) 0.1±0.2 (-0.7-0.2) 0.1±0.1 (0.0-0.1) 0.305 Total bilirubin (mg / dL) 0.11±0.0 (0.09-0.12) 0.63±0.6 (0.22-0.81) 0.000 Direct bilirubin (mg / dL) 0.10±0.0 0.13±0.1 (0.09-0.14) 0.001 Indirect bilirubin (mg / dL) 0.01±0.0 (-0.01-0.02) 0.50±0.6 (0.13-0.67) 0.000 Whole blood, L-lactate (mmol / L) 0.6±0.3 (0.5-0.9) 2.3±0.7 (2.0-2.7) 0.000

[0082] As shown in Table 1 and Figure 2, RBC (10) of hemolytic anemic cattle compared to healthy cattle 6 It can be confirmed that the levels of HCT (%) and Hb (g / m 3 It can be confirmed that the levels of indirect bilirubin (mg / dL) and (mg / dL) significantly increased at p-values ​​of 0.011 and 0.000, and it can be seen that the parameter values ​​are more widely distributed in hemolytic anemia compared to normal cattle. In addition, it can be confirmed that L-lactate (mmol / L) significantly increased at p-value 0.000.

[0083] Among these, the health status can be determined using a normal cow blood diagnostic indicator that includes the blood standard values ​​of a normal cow. If the blood information obtained in the blood analysis step (S2) deviates from the blood standard values ​​of a normal cow, it can be determined that the cow has an abnormal health status, i.e., hemolytic anemia and hypoxia.

[0085] [Experimental Example 2] Analysis of the correlation between blood parameters of normal cattle and hemolytic anemic cattle

[0086] Among the quantitative values ​​of blood components of normal cattle and hemolytic anemic cattle, respectively, RBC (10 6 / μL), HCT (%), Hb (g / dL), MCV (fL), Reticulocyte (10 3 The correlation between the quantitative values ​​of indirect bilirubin (mg / dL) and L-lactate (mmol / L) was analyzed.

[0087] The results regarding the correlation between each blood information parameter of hemolytic cattle are shown in Table 3 below, and among the graphs shown in Figure 3 below, black represents the correlation between blood parameters of normal cattle, and red represents the correlation between blood parameters of hemolytic cattle.

[0089] RBC (10 6 / μL) HCT (%) Hb (g / dL) MCV (fL) Reticulocyte (10 3 / μL) Indirect bilirubin (mg / dL) L-lactate (mmol / L) RBC 1 HCT 0.751 ** 1 Hb 0.871 0.954 1 MCV -0.908 -0.456 -0.654 1 Reticulocyte -0.625 ** -0.747 ** -0.737 ** 0.488 ** 1 Indirect bilirubin -0.764 ** -0.920 ** -0.913 ** 0.534 ** 0.819 ** 1 L-lactate -0.393 -0.738 ** -0.649 ** 0.094 0.402 0.569 ** 1 *;p<0.05, **;p<0.01

[0090] Referring to Figure 3, the L-Lactate (mmol / L) level of a normal cow is RBC (10 6 It was confirmed that a positive correlation was observed with HCT (%), Hb (g / dL), and MCV (fL), and reticulocytes (10 3 It can be confirmed that indirect bilirubin (mg / dL)) and indirect bilirubin (mg / dL) did not show a significant correlation. However, as shown in Table 2 and Figure 3, the L-Lactate (mmol / L) levels of cattle with hemolytic anemia are RBC (10 6 It was confirmed that there was a significant negative correlation with L-Lactate (mmol / L), HCT (%), and Hb (g / dL), and in particular, a strong negative correlation was observed between L-Lactate (mmol / L) levels and HCT (%).

[0091] In addition, since it can be confirmed that there is a positive correlation between L-Lactate (mmol / L) and indirect bilirubin (mg / dL), hemolytic anemia and hypoxia can be determined by analyzing the correlation between blood parameters in the blood information of randomly selected cattle.

[0093] The embodiments of the present invention described above are not implemented only through devices and / or methods, but may also be implemented through a program for realizing a function corresponding to the configuration of the embodiments of the present invention, a recording medium on which the program is recorded, etc., and such implementation can be easily implemented by a person skilled in the art to which the present invention belongs from the description of the embodiments described above.

[0094] Furthermore, although embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims also fall within the scope of the present invention.

Claims

Claim 1 The method comprises a blood analysis step for obtaining blood information by analyzing the blood of a cow through a blood test, and a judgment step for determining the health status of the cow regarding hemolytic anemia and hypoxia through the blood information, wherein the blood analysis step obtains one or more blood information among RBC count, HCT ratio, hemoglobin (Hb) concentration, reticulocyte, monocyte, total bilirubin concentration, direct bilirubin concentration, indirect bilirubin concentration, and whole blood L-lactate concentration by analyzing the blood of the cow through a blood test, and the judgment step includes a disease state judgment step for determining the state of hemolytic anemia and hypoxia of the cow, wherein the disease state judgment step determines, if the cow has symptoms of either hemolytic anemia or hypoxia, whether the indirect bilirubin level of the cow is 0.13 mg / dL or higher If whole blood L-lactate concentration is 2.0 mmol / L or higher; if whole blood L-lactate has a strong negative correlation with HCT (%); if whole blood L-lactate is RBC (10 6 A method for diagnosing hemolytic anemia and hypoxia characterized by diagnosing hemolytic anemia or hypoxia in cases corresponding to one or more of the following: when there is a negative correlation with Hb (g / m / μL) and Hb (g / dL); and when whole blood L-lactate has a positive correlation with indirect bilirubin (mg / dL). Claim 2 A method for diagnosing hemolytic anemia and hypoxia using a bovine blood diagnostic indicator, wherein, in claim 1, the blood analysis step is performed within at least 30 minutes after collecting the bovine blood. Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete

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