CN113813454B - Method for collecting blood of organ donation donor - Google Patents

Abstract

The invention discloses a method for collecting blood of an organ donation donor, which comprises the following steps: 1) placing the perfusion tubes into the abdominal aorta and the portal vein of the organ donation donor, placing the perfusion tubes into the inferior vena cava from the tube placing plane of the abdominal aorta, and finally connecting each perfusion tube with an autologous blood recovery machine; 2) washing a blood storage tank and a pipeline of the autologous blood recovery machine in the step 1) by an anticoagulant, and then collecting donor blood until hemodynamics instability or blood oxygen saturation reduction occurs in the donor; 3) taking the blood obtained in the step 2), centrifuging, washing and storing in a blood preservation solution III. Under the condition of ensuring that the function of the transplanted organ is not influenced, the invention safely finishes the collection of the donor red blood cell suspension by the autologous blood recovery machine, and the obtained donor red blood cell suspension is infused to a receptor, thereby playing the role of partially replacing allogeneic red blood cell suspension and effectively avoiding the occurrence of transfusion complications.

Description

Method for collecting blood of organ donation donor

Technical Field

The invention relates to a method for collecting blood from organ donation donor.

Background

Because of the reduction of citizens who donate blood without compensation, medical institutions in various regions face a serious challenge of blood supply crisis, and because patients who receive liver transplantation operations are end-stage liver patients, the blood coagulation function of the patients is poor, the operations are complex, sufficient blood supply is often needed as guarantee, otherwise, the patients face a great risk. Therefore, in order to avoid the waste of organ resources, it is imperative to find a method that can alleviate the crisis of blood supply.

At present, when the liver and kidney organs of the organ donation donor are obtained, a set of standardized collection organ donation donor blood recycling technology is lacked, so that precious blood resources are wasted, the organ donation recipient is exposed to the risk of insufficient and safe blood supply, and the survival rate of recipient patients is finally influenced.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for collecting blood from an organ donor, which collects blood when organs of the organ donor which are dead in brain and/or dead in heart are obtained, comprising the following steps:

1) placing the perfusion tubes into the abdominal aorta and the portal vein of the organ donation donor, then additionally placing the perfusion tubes into the inferior vena cava along the tube placing plane on the abdominal aorta, and finally connecting each perfusion tube with an autologous blood recovery machine;

2) washing a blood storage tank and a pipeline of the autologous blood recovery machine in the step 1) by taking an anticoagulant, and collecting donor blood until hemodynamics instability or blood oxygen saturation reduction occurs in the donor;

3) taking the blood obtained in the step 2), centrifuging, washing and then preserving in blood preserving fluid III.

Further, the dosage of the anticoagulant during the washing in the step 2) is 500-1000 ml, and 500ml is preferable.

Further, the anticoagulant is normal saline containing heparin; the concentration of the normal saline containing heparin is 25000U heparin/500 ml normal saline.

Further, the centrifugation in step 3) is performed by using an autoblood recovery machine at a speed of 5600rpm until free hemoglobin, white blood cells, plasma, platelets and anticoagulant are removed.

Further, the washing in the step 3) is washing by using a ringer's solution; the volume ratio of the ringer's solution to the centrifuged erythrocytes is 3-5: 1, preferably 4: 1.

further, the preservation temperature in the step 3) is 2-8 ℃, preferably 4 ℃, and the temperature is shaken every 30 minutes during preservation.

Further, the systolic blood pressure is maintained above 70mmhg when the collection organ donates donor blood.

Still further, the organ donor donates liver and/or kidney.

The invention also provides a method for simultaneously obtaining blood and organs of a brain-dead and/or heart-dead organ donor, which comprises the following steps:

a. collecting donor blood by the method;

b. when the donor blood is collected until hemodynamics instability or oxyhemoglobin saturation of the donor appears, cold perfusion is carried out immediately, and crushed ice is put into the liver periphery, the abdominal cavity and the right thoracic cavity;

c. cold perfusing until the lavage liquid is clear, and obtaining the organ.

Further, cold perfusion is to cut the superior and inferior vena cava of the liver close to the right atrium, and then to perfuse HTK liquid with the temperature of 2-8 ℃, preferably HTK liquid with the temperature of 4 ℃ through the abdominal aorta and the portal vein perfusion tubes respectively; the filling pressure of the HTK solution is 80-120 mmHg, and preferably 100 mmHg.

The method for collecting Donor Blood from organ of the invention, under the condition of ensuring that the function of transplanted organ is not affected, collects Donor Red Blood Cell suspension safely by autoblood collector, and transfuses the obtained Donor Red Blood Cell suspension to recipient, namely, adopts Donor specific Red Blood Cell Transfusion (DRBCST). In emergency, the DRBCT can partially replace the allogeneic erythrocyte suspension, and most patients receiving liver transplantation can meet the transfusion principle, so the occurrence of transfusion complications can be effectively avoided. Therefore, the application of DRBCT to DDLT can ensure that liver transplantation and/or kidney transplantation operation can be normally carried out under the condition of insufficient blood supply, and avoid discarding the liver or kidney supply due to insufficient blood supply.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 blood collection pattern during liver acquisition

Detailed Description

EXAMPLE 1 Collection of blood from an organ donated donor according to the invention

1) Respectively placing the perfusion tubes into the abdominal aorta and the portal vein of the brain death patient with donated organs, additionally placing the perfusion tubes into the inferior vena cava along the tube placing plane of the abdominal aorta, and finally connecting each perfusion tube with an autologous blood recovery machine;

2) taking 25000U of heparin as anticoagulant/500 ml of normal saline, flushing a blood storage tank and a pipeline of the autologous blood recovery machine in the step 1), and collecting donor blood until hemodynamics instability or blood oxygen saturation reduction occurs to the donor (the systolic pressure is maintained to be more than 70mmhg during blood collection);

3) after blood collection was completed, the cells were centrifuged by an autoblood collection machine at a speed of 5600rpm to remove free hemoglobin, leukocytes, plasma, platelets, and anticoagulant, and then the erythrocytes were washed with 1000ml of ringer's solution per 250ml, and finally the obtained erythrocytes were transferred to blood preservation solution III and stored in an environment of 4 ℃ and shaken once every 30 minutes.

The advantageous effects of the present invention are described below by way of test examples.

Test example 1 study of the Process of collecting, treating and storing donor blood according to the present invention

First, research method

The safety and efficacy of DRBCT application to DDLT (organ donated liver transplantation after the deceased of citizens) was evaluated by comparing short-term clinical results of liver transplant recipients receiving DRBCT (donor-specific red blood cell transfusion) or allogeneic blood transfusion only (blood bank stock blood) during the same period of surgery. The research is approved by the ethical committee of the Huaxi hospital of Sichuan university and registered in the Chinese clinical laboratory center (ChiCTR 2000041375) to meet the ethical criteria in the declaration of Helsinki. Written informed consent was obtained from each organ donor's family and recipient.

1. Donor inclusion criteria:

all organ donors were defined as chinese class I donors. Screening social history and medical history of all donors is completed, and the screening specifically comprises residence history, travel history, metallurgical history, environmental exposure history, past infection history, drug abuse history and the like. We also performed bacterial, fungal culture on donor blood samples, urine samples, sputum samples, and collection of donor blood samples for Metagenomic next-generation sequencing (mNGS) to detect any potential infections prior to liver harvest. In addition, we have performed Cytomegalovirus (CMV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), syphilis, Human Immunodeficiency Virus (HIV) serological antibody detection, and nucleic acid molecule detection of CMV, HBV, HCV, HIV. Blood collection was performed on organ donors who met the following criteria:

(ii) the donor is older than 18 years of age.

② the donor family agrees to blood donation.

And ③ donors only donate liver and kidney.

And fourthly, the result of blood culture of the donor is negative.

Donator sputum culture negative or donator sputum culture positive but no evidence of infection in clinical manifestation, laboratory examination and imaging examination (except multiple drug-resistant bacteria).

Sixthly, the result of the CMV-DNA of the donor is negative.

Seventhly, donation to transfused infections including HIV, HBV, HCV and syphilis infections.

Testing mNGS of donor blood sample to find out no infection of bacteria, fungi, virus and parasite.

The length of stay in the Intensive Care Unit (ICU) of the donor is not more than 7 days.

2. Receptor inclusion exclusion criteria

All adult patients who received DDLT during the epidemic at western hospital, university of sichuan, were enrolled in the study, but excluded recipients who received non-routine liver transplants, including ABO blood group incompetent liver transplants, secondary liver transplants, split liver transplants, multi-organ combination transplants, and the like. In addition, recipients with signs of infection during 1 week prior to surgery were excluded from the study.

3. DRBCT on-going criteria

DRBCT is performed by the recipient when the inclusion exclusion criteria above are met, with the following requirements:

blood resources in blood banks are in tension.

② donor and acceptor blood cross matching test is negative.

③ according to guidelines, the receptor is at low risk (D-/R-) or moderate risk (D +/R +) of CMV infection, based on the serological state (D/R) of CMV in the donor receptor.

And fourthly, the hemoglobin in the receptor operation is lower than 70g/L or the massive hemorrhage in the operation exceeds 1000ml or the hemodynamics instability caused by the hemorrhage occurs in the operation.

4. Process for collecting, treating and storing donor blood

All blood collection of the present invention is performed at the time of organ harvest. As shown in fig. 1, organ donors were first placed in a supine position, draped, sterilized, and monitored for blood pressure, heart rate, and respiration; performing a large 'cross' incision on the abdomen, ligating the distal end of the abdominal aorta close to the bifurcation of the common iliac artery, and arranging a perfusion tube at the proximal end of the abdominal aorta about 2cm above the bifurcation of the common iliac artery; and (3) finding the superior mesenteric vein trunk, ligating the superior mesenteric vein trunk at the far end, cutting the near end, inserting a portal perfusion tube, and completing the placement of the abdominal aorta and the portal perfusion tube. Then, the inferior vena cava catheterization is carried out on the abdominal aorta catheterization plane, and then the inferior vena cava catheterization is connected with an autoblood recovery machine (P3000, Beijing Jingjing medical equipment Co., Ltd.) to rapidly collect donor blood. The blood reservoir and tubing were pre-flushed with 500ml of anticoagulant (25000U heparin/500 ml saline) prior to blood collection. The whole collection process is continued until the donor is hemodynamically unstable or the blood oxygen saturation is reduced, and then the donor immediately enters a cold perfusion process (namely, the upper and lower vena cava of the liver is cut off at the level close to the right atrium, and HTK liquid is perfused at 4 ℃ through an abdominal aorta and a portal vein perfusion tube, crushed ice is rapidly put into the periphery of the liver, the abdominal cavity and the right thoracic cavity to rapidly cool the liver, and the lavage pressure of the HTK perfusion liquid is 100mmHg according to experience, the perfusion liquid flowing out is clear, or 2000 ml of the HTK liquid is perfused through the abdominal aorta, and the perfusion is stopped after 1000ml of the HTK liquid is perfused through the portal vein). Cold perfusion is the key to ensure the function recovery of transplanted organs after organ transplantation, and the blood of a donor is safely collected under the condition of ensuring that the function of the transplanted organs is not influenced. We completed blood collection within 3 minutes as much as possible and maintained donor systolic pressure above 70 mmhg.

After completion of blood collection, centrifugation was performed at 5600rpm using an autologous blood collection system to remove free hemoglobin, leukocytes, plasma, platelets, anticoagulant, and the like as much as possible. The centrifuged erythrocytes were washed with 1000ml of ringer's solution per 250 ml. Finally, the obtained red blood cells were transferred to blood preservation solution III and stored in an environment of 4 ℃ with shaking every 30 minutes.

In the recipient surgery, donor-specific red blood cell transfusion DRBCT is performed if there is a transfusion indication, and white blood cells and impurities and the like in blood are further filtered using a white blood cell filter (FTS-RC 102, duby bio-medical science co.

5. Cross matching test procedure

Firstly, two clean test tubes are taken and divided into a main side tube and a secondary side tube. Then, a coagulated polyamine medium reagent (Kyoto Biotechnology Co., Ltd.) was prepared.

Adding 2 drops of blood plasma of a patient and 1 drop of 3% -5% erythrocyte suspension of a donor into a main side tube; 2 drops of donor plasma and 1 drop of 3% -5% erythrocyte suspension from the patient were added to the secondary lateral tube.

③ respectively adding 0.65ml of Low ionic solution (Low ionic medium) into the main side tube and the secondary side tube, and shaking the tube to mix them evenly.

Fourthly, 2 drops of the condensed amine solution (Polybrene reagent) are respectively added into the main side tube and the secondary side tube, and the tube is shaken to be evenly mixed.

Fifthly, using a centrifuge to centrifuge at 3400rpm for 10 seconds and then removing the supernatant.

And sixthly, slightly shaking the main side tube and the secondary side tube to observe whether the erythrocytes are agglutinated or not. If no agglutination reaction occurs, the above steps are repeated.

Seventhly, 2 drops of suspension (Resuspending) are respectively added into the main side tube and the secondary side tube, and the tubes are shaken to be uniformly mixed. If the coagulation is dispersed within 60 seconds, the coagulation is caused by the coagulation amine solution, and the matching result is the coincidence. If the agglutination does not disperse within 60 seconds, it indicates that the agglutination is caused by the specific binding of the erythrocyte antigen and antibody, and the matching result is not matched. If the result is suspicious, further observation under a microscope is required. The results of the primary side tube and the secondary side tube are both consistent, and the DRBCT can be carried out.

6. Blood quality analysis after treatment by autoblood recovery machine

After each collection, centrifugation, washing, etc. of donor blood, 2ml of donor blood was drawn and subjected to blood quality analysis by a blood gas analyzer (cobas b 123 full-automatic blood gas analyzer, roche). Then, blood gas analysis was randomly performed on 100 bags of the allogeneic erythrocyte suspension. And comparing and analyzing the pH value, the potassium ion concentration, the sodium ion concentration, the chloride ion concentration and the lactic acid content of the two groups of blood so as to evaluate the quality of the blood treated by the autoblood recovery machine.

7. Observation outcome

All patients received at least 30 days of post-operative follow-up.

The primary endpoint was the occurrence of an infectious complication within 30 days after transplantation. The occurrence, severity and time of any infections (bacteria, fungi and viruses) in both groups were recorded in detail. Infection is defined according to the standards set forth by the american society of transplantation.

Secondary endpoints include other transfusion-related complications, 30-day patient and graft survival, post-operative Total Bilirubin (TB), post-operative glutamate-pyruvate transaminase (ALT), post-operative Alkaline phosphatase (ALP), post-operative Aspartate Aminotransferase (AST), post-operative glutamyl transferase (GGT), post-operative Lactate Dehydrogenase (LDH), post-operative International Normalized Ratio (INR), post-operative Creatinine (CREA), and changes in post-operative t lymphocyte subpopulations of recipients. The definition of transfusion-related complications has been described in the related review.

Second, clinical application

1. The method comprises the following steps:

during the period of 5-11-2020, the receptors receiving DRBCT were included in the DRBCT group according to the study procedure described above and their clinical characteristics were compared to receptors receiving only a suspension of allogeneic red blood cells from a blood bank to assess the safety and efficacy of DRBCT.

2. As a result:

a total of 56 patients were enrolled in the study, 23 in the DRBCT group and 33 in the control group of allogeneic red blood cell suspensions. The patients in the DRBCT group were not observed to develop transfusion-related complications, and the incidence of infection within 1 month after surgery was similar to that in the control group (21.7% vs 18.1%,P= 1.000). The median number of allogeneic erythrocyte suspension transfusions from the blood bank during the operation was 4U (0-8U, IQR) in patients in the DRBCT group due to the factors of transfusing donor-derived erythrocytes, while the difference was 7U (4-10U, IQR) in the control group (statistical significanceP = 0.034). In addition, the peak level of the early aspartate Aminotransferase (AST) after surgery in the DRBCT group was significantly lower than that in the control group: (P = 0.031), and the AST level was also significantly lower than the control group for the first 2 days after surgery: (P =0.019，P =0.045）。

3. And (4) conclusion:

the application of DRBCT to DDLT is a safe and effective measure which can relieve the condition of insufficient blood supply, and can reduce the AST level of patients after liver transplantation to a certain extent, thereby providing safety guarantee for life-saving transplantation operation of patients with end-stage liver diseases.

In conclusion, the method for collecting blood of an organ donation donor finishes the preparation of the donor red blood cell suspension by the autologous blood collection machine, and the application of DRBCT to DDLT can ensure that liver transplantation and/or kidney transplantation operations can be normally carried out under the condition of insufficient blood supply, thereby avoiding the phenomenon that the liver or kidney is discarded due to insufficient blood supply.

Claims (11)

1. A method of collecting blood from an organ donated donor, comprising: the method is used for collecting blood when organs of brain death and/or heart death organ donations donors are obtained, and specifically comprises the following steps:

1) respectively placing the perfusion tubes into the abdominal aorta and the portal vein of the organ donation donor, then additionally placing the perfusion tubes into the inferior vena cava along the tube placing plane of the abdominal aorta, and finally connecting each perfusion tube with an autologous blood recovery machine;

2) washing a blood storage tank and a pipeline of the autologous blood recovery machine in the step 1) by using 500-1000 ml of anticoagulant, and collecting donor blood until hemodynamics instability or blood oxygen saturation reduction occurs in the donor;

3) centrifuging the blood obtained in the step 2), washing with a ringer's solution, and preserving in a blood preservation solution III at 2-8 ℃, wherein the volume ratio of the ringer's solution to the centrifuged erythrocytes is 3-5: 1.

2. the method of claim 1, further comprising: the dosage of the anticoagulant during the washing in the step 2) is 500 ml.

3. The method of claim 1, further comprising: the anticoagulant is normal saline containing heparin; the concentration of the normal saline containing heparin is 25000U heparin/500 ml normal saline.

4. The method of claim 1, wherein: and 3) centrifuging by using an autoblood recovery machine at the speed of 5600rpm until free hemoglobin, white blood cells, plasma, platelets and anticoagulant liquid are removed.

5. The method of claim 1, wherein: and 3) the volume ratio of the ringer's solution to the centrifuged red blood cells is 4: 1.

6. the method of claim 1, further comprising: the temperature for the preservation in step 3) was 4 ℃ and the preservation period was shaken every 30 minutes.

7. The method of claim 1, further comprising: the systolic blood pressure is maintained above 70mmhg when the collection organ donates donor blood.

8. The method according to any one of claims 1 to 7, wherein: the organ donor donates liver and/or kidney.

9. A method of simultaneously obtaining brain-dead and/or heart-dead organ donated donor blood and organs, comprising: it comprises the following steps:

a. collecting donor blood according to the method of any one of claims 1 to 7;

b. when donor blood is collected until hemodynamics is unstable or the blood oxygen saturation degree is reduced, cold perfusion is carried out immediately, and crushed ice is put into the periphery of the liver, the abdominal cavity and the right thoracic cavity;

c. and (5) performing cold irrigation until the lavage fluid is clear, and obtaining the organ.

10. The method of claim 9, further comprising: the cold perfusion is to cut off the superior and inferior vena cava of the liver near the right atrium, and then perfuse HTK liquid with the temperature of 2-8 ℃ through the abdominal aorta and the portal vein perfusion tube respectively; the filling pressure of the HTK liquid is 80-120 mmHg.

11. The method of claim 10, further comprising: the temperature of the HTK liquid is 4 ℃; the perfusion pressure of the HTK solution is 100 mmHg.

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