Author: Cui Ming
Unit: Department of Medical Laboratory, Affiliated Hospital of Nantong University
APTT's Chinese called Activated Partial Thromboplastin Time, is a member of the traditional four-item coagulation and is clinically primarily used to understand the potential bleeding risk.
APTT
First, the detection principle of APTT
APTT test requires the participation of two reaction reagents, reagent 1 is mainly activators (laminated acid, silicone, etc.), phospholipids (rabbit brain phospholipids, soybean phospholipids), etc., reagent 2 is calcium chloride, responsible for providing Ca2+ to the reaction system.
The first stage of the whole reaction is 100 μl of reagent 1 and 100 μl of plasma mixed, the activator activates the coagulation factor XII., and its downstream factors (XI., IX., VIII.) are activated successively, at this time, due to the lack of calcium ions in the reaction system, the IX.-VIII.-phospholipid-calcium complex cannot be generated, and it is impossible to further activate factor X.
Immediately followed by the addition of reagent 2, calcium ions were obtained in the reaction system at this time, whereby the IX.-VIII.-phospholipid-calcium complex was generated, the coagulation waterfall was opened, with the X.-V.-phospholipid-calcium complex formed, and then activated factor II ( prothrombin) to form thrombin, thrombin hydrolyzed fibrinogen (factor I) to form fibrin, the sample transmittance changed, the instrument read the transmittance value every 0.1 seconds, and then according to the instrument's conversion scheme (omitted), the detection results (seconds) were reported.
Figure 1 APTT detection schematic
Various factors that affect APTT results
In short, any obstacle or component deletion involving any link in the APTT detection response process will lead to the prolongation of aptt results. What are some of them? Let's take a look!
Differences in activators and phospholipids in reagent 1
At present, the activators and phospholipid components used by different brands of reagents around the world are not the same, and even the activators and phospholipid components of the same brand but different generations of reagents are not the same, which leads to the difference between the initiation link (XII. activation) of the APTT reaction of different reagents and the two important complexes (IX.-VIII.-phospholipid-calcium complex, X.-V.-phospholipid-calcium complex).
Therefore, different brands of instruments (using their own reagents) are tested for the same specimen, and different test results will be obtained. Therefore, there is no comparison between the test results of different brands of instruments, and it will be completely possible for a specimen to detect the result for 30 seconds on the A instrument and 50 seconds on the B instrument.
Even the test results of the same brand of 1st and 2nd generation reagents are not exactly the same, so when the reagent changes between generations, the reference interval should be adjusted accordingly.
Deficiency of contact factors PK, HMWK, XII
The lack of these contact factors will cause the entire APTT reaction process to not occur from the beginning, just as the car will not start up, it will not drive forward at all, in this case the APTT result will certainly be prolonged or even undetectable (and the degree of lack).
Factor IX and factor VIII are deficient
This is familiar to everyone and is also the most clinically concerned aspect. Factor VIII deficiency often means congenital or acquired hemophilia A and partial vascular hemophilia, while factor IX deficiency corresponds to hemophilia B. When factor VIII is deficient, the defect of VWF needs to be excluded.
Factor XI deficiency
Lack of factor XI, formerly known as hemophilia C. Due to the difference between the genetic pattern and clinical manifestations of hemophilia A and B, it is now called factor XI deficiency.
X., V., II., I. lack
Different degrees of quantity deficiency or qualitative defect of coagulation factors X., V., II., and I. will lead to different degrees of prolongation of APTT test results.
Differences in calcium ions (factor IV).
The concentration of calcium ions in the APTT reaction system is too low, which will also lead to the prolongation of the detection results; the concentration is too high will lead to the shortening of the detection results.
illustrate
Our department has encountered several rare cases this year, 3 cases are briefly introduced.
Female patients, 32 years old, pregnant early, requested termination of pregnancy and hospital admission. In 2008, she gave birth to a son, in 2013 she had an abortion, and in 2014 she underwent a "lower uterine caesarean section" due to "fetal distress". Plain and healthy.
At the patient's present visit, the first coagulation image detection APTT was 91.1 seconds, other indicators were normal, considering that the patient had no other special course and medication history, the next step was to carry out APTT correction test, and the correction test results showed that the Rosner index was 2.35% (judging criteria).
A further confirmation test, the test of coagulation factor, yielded XI. 0.4%.
Clinically, according to the recommendations of the Hematology Consultation, 400-600ml of plasma are transfused daily, and termination of pregnancy can be considered within 10 seconds after APTT is significantly shortened. After 3 days of plasma transfusion, APTT was reduced to 41.4 seconds and the pregnancy was terminated by abortion. Intraoperative bleeding is about 10 ml. No abnormalities were discharged from hospital after 1 day.
Factor XII deficiency
Female patient, 39 years old, menopause for 26 + 4 weeks, pregnancy supervision. Previous history of hepatitis B, tenofovir is taking. Yu is nothing special. The coagulation imaging test at this visit showed apttogram of 82 seconds, and the APTT correction test was correctable, suggesting the possibility of endogenous factor deficiency.
Further confirmation test, our hospital coagulation factor test, the results of VIII., IX., XI. are normal, it is recommended that they go to the outer hospital for other endogenous coagulation factor test, and then in the Jiangsu Provincial Blood Research Institute test found that it is xiI. factor deficiency (0.5%).
Acquired hemophilia A?
The male patient, 67 years old, presented to the emergency department for acute pain, was presented with CT showing that the right iliopsoas muscle mass was occupied with massive exudation, and the right lumbar major muscle morphology was full with exudation. MRI suggests: lumbar degeneration, L3/4 disc bulging, L4-S1 disc herniation. Subcutaneous oozing from the lower back. Right iliopsoasparaburosces muscle strengthening lesions, further investigation is recommended.
Coagulation imaging test showed that the main abnormality was a significant prolongation of APTT, and the blood routine: WBC 15×109/L, HGB 94g/L, platelet 241×109/L, CRP 100.6 mg/L, TP 52 g/L, LDH (dry chemical method) 323U/L, the rest was basically normal.
APTT correction test: APTT was 50.4 sec after immediate 1:1 mixing, not corrected, and 90.5 sec after 2 h of incubation, showing time temperature dependence characteristics. Endogenous coagulation factor test results are as follows
Combining the results of coagulation, APTT correction test and endogenous coagulation factor, the presence of factor VIII inhibitors was suspected, so a confirmation test was followed by a confirmatory test, factor VIII inhibitor test, which was 42.6 BU/mL with a high titer factor VIII inhibitor. Unfortunately, the patient will visit our hospital in the future.
A few further points
1. When using different brands of testing instruments and reagents, there is no comparability between APTT test results, so the same hospital tries to use the same testing platform. Based on the main clinical purpose of excluding clotting defects, APTT tests that are sensitive to coagulation factor deficiency are required.
2. In addition to the difference in phospholipid components in the reagent affecting the APTT result, there are two situations that affect the phospholipid in the APTT reaction system (and thus affect the APTT result).
First, the centrifugation of the specimen, the laboratory needs to ensure that the result is platelet-less plasma (< 10×109/L) after centrifugation, because the membrane of platelets is rich in phospholipids.
Second, when there are substances such as antiphospholipid antibodies in the patient's plasma, the most common is lupus-like anticoagulants, which will bind to the phospholipids in the reaction system, resulting in prolonged APTT results and thus misleading clinical diagnosis and treatment (because these substances play a similar anticoagulant effect in in vitro detection experiments, and more pro-coagulation in the body, so that the patient is in a state of easy thrombosis).
3. Different from the in vitro participation in APTT detection experiments, the contact factors PK, HMWK, XII. are basically not involved in the in vivo coagulation process, but are more involved in the fibrinolytic activation process. Therefore, the prolongation of APTT caused by the lack of these factors does not mean bleeding, and there may be fibrinolysis problems.
4. When confirming the lack of congenital factor VIII. factor, it is necessary to detect VWF (vascular hemophilia factor), because VWF in vivo and inside and outside are the protectors of factor VIII.
5. Compared with hemophilia B, patients with hemophilia A are more likely to produce factor VIII inhibitors due to the use of exogenous factor VIII. preparations, which in turn leads to a decrease in factor VIII. and is manifested as APTT prolongation. In patients with acquired hemophilia A, due to the production of factor VIII antibodies in vivo, the results of the APTT test in vitro are certainly prolonged, and the clinical bleeding may be more serious.
6. Similar to the deficiency of factor XII. factor XI., the lack of factor XI. will also be manifested as APTT prolongation, but the degree of its lack and the correlation between the bleeding performance in the clinic are not consistent. In severe deficiency, there is not even bleeding, but "post-traumatic delayed bleeding" may occur.
According to the theory of cell coagulation, xi factor plays a generalist role in the body, physiological hemostasis does not completely rely on factor XI. it is a weak predictor of bleeding, which is completely different from factor VIII. and IX., and factor XI. plays an important role in pathological thrombosis. In vivo, factor XI is more involved in antifibrinolytic, and factor XI continues to produce thrombin after the formation of a clot, thereby indirectly activating TAFI (thrombin-activated fibrinolytic inhibitor) and inhibiting plasmolysis.
Deficiency of 7., X., V., II., I. can be congenital, but acquired factors lead to more cases. Acquired antibodies to factor V or factor II cause APTT to be prolonged and patients have bleeding manifestations, which is not clinically common.
More common factors in the clinic are the use of drugs, such as heparin and low molecular weight heparin (the target is activated factor II and X., the effect varies according to the size of the drug molecule), saban drugs (the target is activated X.), dabigatran ester (the target is activated factor II), Agatroban (the target is activated factor II) and so on. The use of these drugs affects the corresponding activation factors, which can cause APTT to be prolonged or even undetectable. In addition, sepsis, severe trauma, etc., when heparinoid substances are produced in the body, can also lead to aptts.
8. For factor II and I. factors, when these two coagulation factors are lacking or disturbed, the thrombin time (TT) is more sensitive than APTT, that is to say, the TT prolongation is greater, and even when hereditary abnormal fibrinogenemia is presented, it is only manifested as the prolongation of TT. This is related to the detection system of TT and APTT, and the thrombin time (TT) test is designed as a interference test.
9. The problem involving calcium ions (IV. factor) is mainly that the amount of blood collected by the specimen should be accurate. When the amount of blood collected is small, it will lead to an inappropriate ratio of plasma and anticoagulants in the specimen, and the anticoagulants are relatively too much, which will bind more calcium ions, so that the concentration of calcium ions in the APTT reaction system is not enough, and then the APTT is prolonged. Therefore, the amount of blood collected from the specimen should meet the requirements, which is a pre-analysis factor.
In addition, it is rare that when the patient has a high hematocrit (such as polycythemia, newborn, etc.), there are more cells and less plasma in the same volume of whole blood specimens, and the amount of anticoagulant in the collection blood vessel needs to be reduced to obtain accurate APTT results. Also, when the patient has hypercalcemia, if the calcium ion is high, and the amount of anticoagulant in the collection blood vessel is fixed, the calcium ion in the APTT reaction system will be high, and then the APTT will be shortened.
10. There is also a pre-analysis factor, when the blood draw is not smooth, resulting in a clot in the specimen, generally if there is a small clot, because the activation of the coagulation factor has been initiated at this time, it is manifested as a low APTT test result, and when there is a large clot, because the coagulation factor has been excessively consumed when forming a clot, the APTT test result is prolonged at this time. This condition is similar to the early hypercoagulable phase and the medium-term consumable hypocoagulable phase of DIC. So the blood draw must be smooth.
11. The formation of blood clots in the body requires fibrinogen transformation into conjugate fibrin to form a stable thrombus and then reliably stop bleeding, which requires the participation of XIII coagulation factor (factor 13). However, in vitro APTT tests do not need to finally form cross-linked fibrin as the response endpoint, that is, factor XIII is not required to participate in the reaction, which means that APTT does not reflect whether factor XIII is deficient or not. As a result, APTT results are normal when factor XIII alone is deficient, and factor XIII deficiency can lead to life-threatening bleeding. Although the deficiency of clotting factor XIII is rare, it is necessary to have this concept in our hearts.
To sum up: Under the premise of excluding patients from using anticoagulants such as heparin, when the unexplained cause of APTT is significantly beyond the normal control, the APTT correction test can be considered, and it is recommended to determine it in combination with the clinical manifestations and clinical needs of patients.
The APTT correction test is a screening test that, if corrected, indicates a lack of clotting factors and does not correct the presence of factor inhibitors or antiphospholipid interference. Then, according to the results of the screening test, selective confirmation tests are carried out, such as coagulation factor detection, coagulation factor inhibitor detection, lupus-like anticoagulant detection, anti-β2 glycoprotein I antibody, anticardiolipin antibody and so on.
Source: Department of Medical Laboratory, Affiliated Hospital of Nantong University
Edited by: Yeah Reviewer: Xiao Ran