A fully automatic screening method for coagulation factor VIII inhibitors

Abstract

The present invention discloses a fully automatic screening method for coagulation factor VIII inhibitors. The FVIII inhibitor screening method of the present method is fully automated, simple, fast and accurate. Unlike the traditional manual method which is cumbersome and time-consuming, the present method can directly load the centrifuged specimen onto the machine, and the detection time is short, the results are more objective, the repeatability is stronger, the quality control is easier, and the influence of non-objective factors can be more effectively eliminated.

Description

Technical Field

[0001] The present invention relates to a method for judging a blood sample, and in particular to a method for fully automatic screening of coagulation factor VIII inhibitors. Background Art

[0002] According to the diagnosis and treatment standards for hemophilia, the use of complementary therapy to infuse coagulation factor preparations can effectively prevent bleeding symptoms in hemophilia patients, but about 10% to 15% of hemophilia A patients and 1% to 3% of hemophilia B patients have inhibitors to coagulation factor VIII and coagulation factor IX. For these patients, it becomes very difficult to simply infuse coagulation factors for hemostasis management, so the treatment plan needs to be changed in time. In addition, hemophilia patients who are positive for coagulation factor inhibitors are more likely to develop severe conditions. Therefore, from the perspective of choosing an appropriate treatment plan, it is extremely important to accurately detect coagulation factor inhibitors as early as possible.

[0003] At present, the qualitative method for screening the coagulation factor VIII inhibitors in the blood of hemophilia A patients is the activated partial thromboplastin time (APTT) correction test, and the quantitative detection methods include Bethesda, Nijmegen, Chromogenic and fluorescence immunoassay (FLI), etc. However, the detection of FVIII inhibitors using the above traditional methods takes more than 4 hours, and the operation is very cumbersome, with too many steps and too complicated. If an error occurs in any link in the middle, it will affect the final result, which is not conducive to large-scale promotion and popularization. In addition, if antiphospholipid antibodies such as lupus anticoagulant (LA) are present in the sample to be tested, they will react with the phospholipid components in the detection reagent to inhibit the coagulation process, thereby interfering with the detection of FVIII inhibitors and affecting the results. Summary of the invention

[0004] The object of the present invention is to overcome at least one disadvantage of the prior art and to provide a fully automatic screening method for factor VIII inhibitors.

[0005] The technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a method for fully automatic screening of coagulation factor VIII inhibitors, comprising the following steps:

[0007] 1) APTT test: directly detect the APTT value of the blood sample to be tested;

[0008] 2) APTT-I test: add standard plasma to the blood sample to be tested to obtain mixed solution 1, incubate at 36-38°C for 58-62 seconds, add the same volume of APTT reagent as that of mixed solution 1, and then add the same volume of calcium reagent as that of mixed solution 1 after 178-182 seconds to detect the clotting time, i.e., the APTT-I value;

[0009] 3) APTT-P test: Add standard plasma to the blood sample to be tested to obtain mixed solution 2, incubate at 36-38°C for 418-422 seconds, add the same volume of APTT reagent as mixed solution 1, and then add the same volume of calcium reagent as mixed solution 1 after 178-182 seconds to detect the clotting time, i.e., the APTT-P value;

[0010] 4) Calculate the APTT-D value and APTT-CI value according to the following formula:

[0011] APTT-D= (APTT-P) - (APTT-I);

[0012] APTT-CI= (APTT-D) 3 ×APTT × (APTT-I) × (APTT-P)÷10 6 ;

[0013] 5) Judgment criteria: When APTT-D ≥ 1.6 or APTT-CI ≥ 0.55, the sample is judged to be positive for coagulation factor VIII inhibitor.

[0014] In some examples, the volume ratio of the blood sample to be tested and the standard plasma is (3.9-4.1):1.

[0015] In some examples, the incubation temperature of the APTT-I test is 37°C.

[0016] In some examples, the incubation condition of the APTT-P test is 37°C.

[0017] In some examples, the blood sample to be tested is plasma.

[0018] In some examples, the calcium reagent is a calcium chloride solution.

[0019] In some cases, when both the APTT-I value and the APTT-P value are greater than 100 s, the sample needs to be diluted and retested.

[0020] In a second aspect, the present invention provides an automatic screening device for coagulation factor VIII inhibitors, comprising a memory and a processor, wherein the memory is used to store at least one program, and the processor is used to load the at least one program to execute the method described in the first aspect.

[0021] In some instances, the automatic screening device may also include a sample adding system for adding samples to be tested, an incubation system for APTT-I test and APTT-P test, a detection system for detecting coagulation time, and a system for storing, processing and calculating APTT-D and APTT-CI values.

[0022] In a third aspect, the present invention provides a storage medium storing processor-executable instructions, wherein the processor-executable instructions are used to execute the method described in the first aspect when executed by the processor.

[0023] The beneficial effects of the present invention are:

[0024] The FⅧ inhibitor screening method of this method is fully automated, simple, fast and accurate. Unlike the traditional manual method which is cumbersome and time-consuming (generally 2-4 hours), this method can directly load the centrifuged specimen onto the machine, and the detection time is short (less than 20 minutes), the results are more objective, the repeatability is stronger, the quality control is easier, and the influence of non-objective factors can be more effectively eliminated. DETAILED DESCRIPTION

[0025] The disclosure below provides many different embodiments or examples for implementing different schemes of the present invention. As reagents for APTT determination, for example, there are Coagpia (registered trademark) APTT-S (manufactured by Sekisui Medical Co., Ltd.), ThromboCheck APTT-SLA (manufactured by Sysmex Corporation) or APTTliquid RD (manufactured by Roche). Unless otherwise specified, the reagents and equipment used in the present invention are all commercially available conventional products.

[0026] The blood sample to be tested in the embodiment of the present invention has a platelet count of less than 10×10 9 / L of platelet-poor plasma, anticoagulated with sodium citrate.

[0027] Calcium ions are required in the measurement sample for blood coagulation, and in the present embodiment, a 0.025 mol / L calcium chloride aqueous solution is used for the preparation of the measurement sample.

[0028] The sample can be measured manually or by a fully automatic measuring device. Examples of such a device include the CS series of fully automatic blood coagulation measuring devices (Sysmex Corporation).

[0029] The representative method of the traditional method for qualitative testing of factor VIII inhibitors is the APTT mixed correction test. The specific experimental steps are as follows: Take more than 20 healthy people's plasma with normal results of four coagulation tests and mix them in equal amounts to make normal mixed plasma (NP). The patient specimen (PP) is tested on the machine to obtain APTT1, the normal mixed plasma is tested on the machine to obtain APTT2, and the patient plasma and normal mixed plasma are mixed in equal amounts of 1:1 to make mixed plasma (MIX) and tested on the machine to obtain APTT3. After all PP, NP, and MIX are placed in a 37°C environment and incubated for 2 hours, they are tested again on the machine to obtain APTT4, APTT5, and APTT6, and after incubation for 2 hours, PP and NP are mixed in equal amounts again and tested on the machine to obtain APTT7.

[0030] The results are interpreted as follows:

[0031] 1. Both APTT3 and APTT6 are corrected: indicating simple coagulation factor deficiency and no inhibitors;

[0032] 2. APTT3 correction and APTT6 extension exceeding the critical value: indicating the presence of time-temperature-dependent inhibitors;

[0033] 3. Both APTT3 and APTT6 are prolonged:

[0034] (1) APTT6 is longer than APTT7 and does not exceed the corresponding critical value, indicating the presence of inhibitors that are not time- and temperature-dependent;

[0035] (2) APTT6 was prolonged beyond the corresponding critical value compared with APTT7, suggesting the existence of time-temperature-dependent inhibitors.

[0036] 4. APTT4 / 5 / 7 is significantly longer than APTT1 / 2 / 3: This indicates that there are unstable factors in the experimental process, and the experimental failure leads to unreliable results. It is necessary to eliminate the interference and conduct the incubation experiment again.

[0037] The representative method of the traditional method of quantitative test of factor VIII inhibitor is the Bethesda test, which detects the ability of patient plasma to inactivate FVIII in normal plasma. One Bethesda (BU / mL) unit is defined as the FVIII inhibitor titer that can inactivate 50% of FVIII activity in NP. The specific experimental steps are as follows:

[0038] Experimental steps: Patient plasma (PP) of different dilutions (1:1, 1:2, 1:4, etc.) diluted with imidazole buffer at pH 7.4 was mixed with equal amounts of normal mixed plasma (normal mixed plasma NP treated with imidazole buffer), and incubated at 37°C for 2 hours. The FVIII activity in each mixed plasma was determined, and the value was divided by the FVIII activity of the control plasma after equal amounts of imidazole buffer and normal mixed plasma (NP) were mixed and incubated at 37°C for 2 hours to obtain the percentage of residual FVIII activity. Finally, the corresponding antibody titer can be found through the inhibitor titer standard table. Example

[0039] The specific steps of this method are: using a fully automatic coagulation analyzer to test the plasma of the subject on the machine for APTT, and setting up two new test procedures on the machine: APTT immediate correction test (APTT-I) and APTT post-incubation correction test (APTT-P). The specific steps are as follows:

[0040] (1) APTT-I test: add 10 μL of standard human plasma (SHP) to 40 μL of the subject's plasma, incubate at 37°C for 60 seconds, then add 50 μL of APTT reagent, and then add 50 μL of CaCl2 reagent after 180 seconds to detect the APTT-I coagulation time;

[0041] (2) APTT-P test: add 10 μL of standard plasma (SHP) to 40 μL of the subject's plasma, incubate at 37°C for 420 seconds, then add 50 μL of APTT reagent, and then add 50 μL of CaCl2 reagent after 180 seconds to detect the APTT-P coagulation time;

[0042] (3) The instrument test results are automatically transmitted to the laboratory information system (LIS). Two calculation formulas are set in the LIS to calculate the APTT difference (APTT-D) and the APTT correction index (APTT-CI). The specific formulas are as follows:

[0043] APTT-D = (APTT-P) - (APTT-I)

[0044] APTT-CI= (APTT-D) 3 ×APTT × (APTT-I) × (APTT-P)÷10 6

[0045] (4) Judgment criteria: The cut-off value of APTT-D is 1.6, and the cut-off value of APTT-CI is 0.55. When APTT-D ≥ 1.6 or APTT-CI ≥ 0.55, it is positive for FVIII inhibitor. The higher the APTT-D and APTT-CI values, the higher the titer of FVIII inhibitor. When both APTT-I and APTT-P are greater than 100 seconds, it is recommended to appropriately dilute the sample to be tested and then test it.

[0046] The presence of factor inhibitors in the sample was determined based on the Cut-off value, and then the Bethesda (BU / mL) units of FⅧ factor inhibitors in the sample were detected using the traditional method. The results of the two methods were compared, as shown in Table 1.

[0047] Table 1

[0048] APTT APTT-I APTT-P FⅧ inhibitor (BU / mL) APTT-CI APTT-D The method of the present invention determines Sample 1 25.2 29.1 29.6 0.00 0.00 0.50 Negative Sample 2 35.7 36.7 38.1 0.00 0.14 1.40 Negative Sample 3 180.0 45.1 46.0 0.00 0.27 0.90 Negative Sample 4 65.6 44.3 44.6 0.00 0.00 0.30 Negative Sample 5 118.4 40.8 46.4 1.40 39.36 5.60 Positive Sample 6 108.3 43.3 47.7 1.05 19.05 4.40 Positive Sample 7 113.3 42.6 45.0 0.95 3.00 2.40 Positive Sample 8 32.0 28.0 28.9 0.00 0.02 0.90 Negative Sample 9 25.0 28.2 28.7 0.00 0.00 0.50 Negative Sample 10 53.8 42.7 42.9 0.00 0.00 0.20 Negative

[0049] In addition, the presence of lupus anticoagulant (LA) may interfere with the detection of FⅧ inhibitors, resulting in false positives. Samples 11 to 20 are all from subjects with positive lupus anticoagulants, but experiments have shown that the present invention has an anti-interference effect on patients with positive LA, and the results are shown in Table 2 below.

[0050] Table 2

[0051] APTT APTT-I APTT-P FⅧ inhibitor (BU / mL) APTT-CI APTT-D The method of the present invention determines Sample 11 131.6 47.8 45.6 0.15 -3.05 -2.20 Negative Sample 12 49.5 43.8 44.2 0.00 0.01 0.40 Negative Sample 13 96.8 41.0 41.6 0.00 0.04 0.60 Negative Sample 14 100.1 44.1 43.4 0.05 -0.07 -0.70 Negative Sample 15 76.7 39.0 38.5 0.30 -0.01 -0.50 Negative Sample 16 85.8 38.5 38.5 0.00 0.00 0.00 Negative Sample 17 106.2 42.7 43.0 0.10 0.01 0.30 Negative Sample 18 74.2 41.8 44.8 1.15 3.75 3.00 Positive Sample 19 99.3 118.0 120.9 161.28 34.55 2.90 Positive Sample 20 107.9 57.3 89.8 8.40 19059.10 32.50 Positive

[0052] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A fully automatic screening method for factor VIII inhibitors for non-medical purposes, characterized in that: The following steps are involved: 1) APTT test: directly detect the APTT value of the blood sample to be tested; 2) APTT-I test: Add standard plasma to the blood sample to be tested to obtain mixed solution 1, incubate at 37°C for 58-62 seconds, add the same volume of APTT reagent as that of mixed solution 1, and then add the same volume of calcium reagent as that of mixed solution 1 after 178-182 seconds to detect its clotting time, i.e., APTT-I value; 3) APTT-P test: add standard plasma to the blood sample to be tested to obtain mixed solution 2, incubate at 37°C for 418-422 seconds, add the same volume of APTT reagent as that of mixed solution 2, and then add the same volume of calcium reagent as that of mixed solution 2 after 178-182 seconds to detect its clotting time, i.e., APTT-P value; 4) Calculate the APTT-D value and APTT-CI value according to the following formula: APTT-D= (APTT-P) - (APTT-I); APTT-CI= (APTT-D) 3 ×APTT × (APTT-I) × (APTT-P)÷10 6 ; 5) Judgment criteria: When APTT-D ≥ 1.6 or APTT-CI ≥ 0.55, the sample is judged to be positive for coagulation factor VIII inhibitor. When both the APTT-I value and the APTT-P value are greater than 100 s, the sample needs to be diluted and retested; Wherein, the volume ratio of the blood sample to be tested and the standard plasma is (3.9-4.1):

1.

2. The fully automatic screening method according to claim 1, characterized in that: The blood sample to be tested is plasma.

3. The fully automatic screening method according to claim 1, characterized in that: The calcium reagent is calcium chloride solution.

4. An automatic screening device for coagulation factor VIII inhibitors, characterized in that: The invention comprises a memory and a processor, wherein the memory is used to store at least one program, and the processor is used to load the at least one program to execute the method according to any one of claims 1 to 3.

5. A storage medium storing instructions executable by a processor, characterized in that: The processor-executable instructions are used to perform the method according to any one of claims 1 to 3 when executed by the processor.