CA2059791C - In vitro method to determine the safety of modified hemoglobin blood substitutes for human prior to clinical use - Google Patents
In vitro method to determine the safety of modified hemoglobin blood substitutes for human prior to clinical use Download PDFInfo
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Abstract
The present invention relates to an in vitro method to determine the safety of modified hemoglobin blood substitutes for human subjects prior to their clinical usages, wherein the method is based on complement activation reaction from adding modified hemoglobin blood substitutes to a human plasma sample and comprises the steps of: a) obtaining at least one plasma sample from at least one human subject by i) taking a blood sample and immediately centrifugating; and ii) separating the centrifuged blood sample of step i) and retaining the supernatant plasma; b) mixing the plasma of step ii) with the modified hemoglobin blood substitutes or control-ringer in a weight/volume ratio of about 4:1; c) incubating for a time sufficient to allow for a complement activation reaction to occur; d) adding the product of step c) to an appropriate volume of saline in an EDTA tube; and e) analyzing the degree of complement activation by analysis of the product of step d); thereby determining the safety of the modified hemoglobin blood substitutes relative to the human subject plasma sample based on the detection of complement activation.
Description
_~_ ~~~~~ ~F ~~~ ~~~~~~~~
An in vitro method to determine the safety of modified hemoglobin blood substitutsa for human prior to clinical use.
~AG:FCGi2~ZTND ~F °PHE Tldi7~I~'.L'IOid Blood is a living tissue; transfusion of it or of its cellular components from a donor to a recipient is a form of transplantation. About 11 ~to ~.2 million transfusions are given yearly in the United States, and 7.0 the number is steadily increasing. The decision to transfuse is a clinical judgment that requires weighing the possible benefits and known ha2ards with alternative treatments. A transfusion not specifically indicated is contraindicated.
In the United States, regulations for collecting storing, and transporting blood and its components are established by the Federal Drug Agency (FDA), and sometimes also by state or local health authorities.
The American National Red Cross and American Association of Blood Banks also have stanr~ards affecting their respective systems. Screening a donor includes a health interview, testing for vemoglobin (Hb), and taking the temperature, pulse rate, and blood pressure (BP).
Before use, blood must be classified for suitability. This includes ABO and Rh typing, antibody screening, STS, a test for hepatitis B surface antigen (HBsAg}, and tests to detect antilbodies to the virus that causes acquired immunodeficiency syndrome (AIDS}.
The container label and the federally required Circular of Information give the results of these testy and important information and cautions and should be consulted by physicians using blood transfusions.
The various components of blood can be separated, concentrated, and stored individually for precise replacement of patient needs.
Red blood cells (RBCs} are transfused to replace Hb or OZ carrying capacity, including blood lost at surgery, and to prime extracorporeal circuits. When volume expansion is required, other fluids can be used concurrently or ~eparateiy.
Frozen-thawed RBCs are costly and mainly used for patients who have multiple blood group antibodies or antibodies to high frequency antigens.
wasted RBCs (by continuous-flow washing) are free of almost all traces of plasma and of most white blood cells (wBCs) and platelets. They are suitable for patients who have severe reactions to plasma (eg, severe allergies or IgA immunization), or for those who have leucocyte antibodies and repeated :Febrile transfusion reactions.
The four ABO blood types are determined by testing far the presence or absence of A and B antigens an the RBCs using Anti-A and Anti--B reagents (forward or cell typing) , and by testing for Anti-A and Anti-B in the serum using A and B reagents RBCs (serum or reverse typing) .
Where are several reactions than accompany or follow intravenous administration of blood or blood components.
Hemolytic r~aaici~aas Hemolysis can result from blood group inco~npatibiaity, incompatible plasma or serum, hemolyzed or fragile RBCs. Incompatibility is the most frequent cause of hemolysas despite advances in blood typing and tesaing.
~'ebxi7.e reactions Reactions consisting of chills, fever with a rise of at least 1°C, and sometimes headache and back pain, rarely progressing to cyanosis and shock. Tn some patients, after many transfusions or pregnancies, leucocyte antibodies appear in response to antigens .of transfused or fetal WBCs.
Allergic reactions Reactions due to hypersensitivity of the patient to 7.0 an unknown component in donor blood are common, usually ' due to allergens in donor plasma, or, less often, to antibodies from an allergic donor.
Disease itransmission Virus hepatitis may follow the infusian of whole blood, plasma, or other products prepared from human blood; notably AHF and factor T~ concentrate.
Acquired immunodeficiency syndrome (AIDS): The causative agent of this condition has been identified as human immunadeficiency virus (HIV); epidemiologic evidence indicates that it is infectious and blood-borne. A dew patients with ATDS, not belonging to any of the known high-risk groups, have a history of w~a~'~~~.
-. 5 ..
receiving blaod products and their disease is considered "transfusion-associated".
Cytomegalovirus (CMV) can be transmitted by leucocytes in transfused blood. Usually its effects are either absent or mild, and need cause no concern.
Bacterial infection: Despite careful preparation, from 2 ~0 5 0 of all blood drawn contains a few bacteria, presumably from the donor's skin.
Malaria is transmitted easily by infected donor blood. HIany donors are unaware that they have malaria, certain varieties of which may be latent and transmissible for 20 to 15 yearn.
Syplaillis may be transmitted by fresh blood from a donor with the disease, but the incidence is very rare.
Modified hemoglobin Red blood cell contains hemoglobin which carries oxygen to tissues as required. Red blood cell is the best transfusion material at present: Fiowevar, as described above, it does.prssent a number of problems.
In particular; donor blood can be in short supply, _ ~ _ especially in major disaster situation or during war.
Red blood cell transfusions also rec~aire crossmatching.
They have a very short storage tune, unless they are stored by some expensive and complex means. During the last few years, the potential problem of the AIDS
transmission through blood transfusion has evolved.
As a result, a number of centres have investigated the potential uses of hemoglobin extracted from red blood cells. Rxtracted hemoglobins which are transfused to a patient are rapidly removed from the patient circulating blood; they do not carry and release oxygen efficiently and they can be toxic. Unfortunately, hemoglobin cannot be used as such for transfusion.
Because of these reasons, hemoglobin have to be modified before it can be used as a blood product.
Presently, there are two major groups of modified hemoglobin; 1) the encapsulated hemoglobin and 2) the crosslink~d hemoglobin (PROC. 1983 2nd TNTL. SYMP. BLOOD
SUBS., San Francisco, R.P.,Geyer and G.J. Nemo, Alan R.
Liss Inc. Publisher, New York, pp. 7,-X68; PROC. 1987 3rd TNTL. SYMP. BLOOD SUBS., T.M.S. Chang and R.P. Geyer, 1988, Maxcel Dekker Publisher, USA, pp. 1--708; PROC.
1989, SYMP. BLOOD SUB. BIOMAT., AR'f. CELLS AND ARI'.
ORGANS, R. Winslow and T.M.S. Chang, 1990, vol. 18, pp.
133-°366; ABSTRACTS 1991 4th INTL. S3tMP. BLOOD SZJBS.
BIOMAT., ART. CELLS AI~TD IMMOBI. BIOTECH., T.M.S. Chang and R.P. Geyer, 1991, vol. 19, pp. 299--520).
Crosslinked hemoglobin is subdivided into polyhemoglobin, conjugated hemoglobin and intramolecular crosslinked hemoglobin.
Animal studies carried out in many centres showed that these modified hemoglobin are effective in animal for resuscitating animals which would have otherwise died from severe bleeding. Other areas of applications for red blood cell blood substitutes were also demonstrated. The first preliminary clinical trial in a very small number of patients was approved by the FDA
and completed in the United States in 1989. Preliminary report appears to be safe. However, in March of 1990, the FDA called a meeting to announce that there are observed unexplained intense and severe reactions in clinical trials carried out.
A~iimal s8fety studies slot valid f~r ~itx~ata~1 This is surprising since animal studies carried out by many groups showed that modified hemoglobins are safe and without adverse effects. The animal result does not _ g _ compare to human study. Reactions in human are different to animal reactions, especially in immunological type of reactions including anaphylactic reactions, antigen-antibody reactions, allergic reactions.
In other words if a product is immunologically safe in animals, it is not necessarily safe in humans. There is an urgent need to fill the gap between animals study arid phase I clinical trial in human patients. A
preclinical screening test for modified hemoglobin, after its safety has been demonstrated in animals, is needed before it can be tested in patients.
Furthermore, screening of industx-ial production batches is needed. There are variations from person to person 25 in human ianmunological reactions.
The infusion of large amount of modified hemoglobin as blood substitute can potentially result in hypersensitivity and anaphylactic reactions, antibady-antigen reactions and other. Therefore, very careful efficacy and safety animal studies of modified hemoglolain blood substitute are required before phase I
of the clinical trial is started. However, one major problem is to select the right type of animals for testing before clinical trial, since one need to be sure that response in human will be the Came as in the test animals.
Another important area which is being developed is S non-human sources of hemoglobin. human hemoglobin is theoretically the most optimal source because there is not as much immunological problems as with Cad blood cells transfusion, but human hemoglobin could be in short supply because it has to come from donor x>lood.
There is considerable recent researches conducted to determine whethex bovine hemoglobin can be used to prepare modified hemoglobin. extensive development is also conducted on recombinant human hemoglobin from microorganisms and even animals. The impor~ancs of an in vitro human preclinical screening test will increase considerably if these types of hemoglobin are used to prepare modified hemoglobin for human use, sinne the result of animal safety studies is not necessary the same as human.
Furthermore, even if a given hemoglobin blood substitute product is without adverse side effects in a small number of phase T Clinical trial patients, there may still be some patients who may be more sensitive to this hemoglobin product. Presently, there exists no screening test to detect patients with potential severe hypersensitivity to hemoglobin products in large scale clinical trials and later on in routine clinical applications.
In vitro screening test specific fore human response It would be highly desirable to be provided with an in vitro screening test which would be based on using human blood or plasma to determine the safety of modified hemoglobin blood substitutes for human prior to clinical use. This type of test would provide a bridge between the animal testing and 'the human clinical trial of modified hemoglobin blood substitutes., Further, it would be highly desirable to be provided with an in vitro screening test so specific that one cauld determine the safety of madified hemoglobin blood substitutes for a particular patient, who is to receive the blood substitutes, prior to his actual clinical use of the blood substitutes.
Finally, it would be highly desirable to be provided with an in vitro screening test for screena.ng industrial productions of modified hemoglobin blood ~~~~ ~'~
_ 11 _ substitutes to rule out potential. problems before starting the clinical trial of the blood substitute by a population of patients or human subjects.
~tlP~,it7t OF' '.~'~iE II~T~IQ'fION
Surprisingly and in accordance with the present invention, there is provided an in vitro method to determine the safety of modified hemoglobin blood substitutes for human before their clinical trial in human. The in vitro method of the present invention i,s based on the effect of modified hemoglobin blood substitute on complement activation when added to human plasma.
The present invention relates ~o an in vitro method to determine the safety of modified hemoglobin blood substitutes for human subjects prior to their clinical usages, wherein the method is based on complement activation reaction from adding modified hemoglobin blood substitutes to a human plasma sample and includes the steps of: a) obtaining at leash one p~.asma sample from at least one human subject by: i) tak.ing a blond sample aa~d immediately centrifuging: and ii) separating the centrifuged blood sample of step i) and retaining the supernatant plasmas b) mixing plasma of step ii) with the modified hemoglobin blood substitutes or the control-ringer in a weight/volume ratio of about 4z1~ c) incubating for a time sufficient to allow for a complement activation reaction to occurs d) adding the product of step c) to an appropriate volume of saline in an EDTA tube and e) analyzing the degree of complement activation by analysis of the product of step d)~
thereby determining the safety of the mollified hemoglobin blood substitutes relative to the human subject plasma sample based on the detection of complement activation.
In accordance with the present invention, there is provided an in vitro screening method which is based ora using human blood or plasma to determine the safety of modified hemoglobin blood substitutes for human prior to clinical use: Thus method provides a bridge between the animal testing and the human clinical trial of modified hemoglobin blood substitutes.
In accordance with the present invention, there is provided an in vitro screening method so specific that one can determine the safety of modified hemoglobin blood substitutes for a particular patient, who is to receive the blood substitutes] prior to his actual clinical use of the blood substitutes.
1n accordance with the present invention, there is provided an in vitro method for screening industrial productions of modified hemoglobin blood substitutes to follow: (1) the different steps of industrial production, (2) different batches produced and (3) screening before the packaging the final product for human use.
The in vitro method of the present invention can also be used for human preclinical trial studies and for screening before human clinical use of modified hemoglobin blood substitutes.
Most of t~xe problems related to potential hypersensitivity reactions, anaphylactic reactions, effects due to antibody--antigen Complexes, and ethers could be detected in vitro by the method of the present invention.
Other advantages of the present invention will be readily illustrated by referring to the following description.
BRIEF' DESCR3P~.°~oN ~F' TfIE l3R~t~~N~~
Figure 1 is diagxam scheme of the complement activation pathway.
Figure 2 is a graph of the results of Table 1 below.
DE'fAI~EI9 ~ES~RI~TT~N of THE 7CNVErITI~N
The best in vitro test before clinical trial in human is the method of the present invention which uses the human plasma. The in vitro method of the present invention is based on the degree of complement activation of human plasma upon the addition of the modified hemoglobin blood substitutes.
In the preclinical test provided by the method of the present invention, a plasma sample is obtained from a human subject and modified hemoglobin is added to the plasma sample. Then, the sample may be analyzed fox C3a level to analyze possible C3 complement activation.
Using the method of the present invention, one can test the hemoglobin product to detexmins if it Causes any complement aC~tivation in huanan plasma; it is the next closest test to actual injection into human.
_ I5 _ Trace amount of blood cell membrane material may be present during the extraction of hemoglobin from red blood cells, and it can be incorporated into the modified hemoglobin preparation. This trace amount of blood cell membrane material may not be detectable using the animal testing procedure, whereas upon infusion into a patient it can cause adverse reactions, including complement activation.
The method of the present invention can also test for different 'types of antibody-antigen complexes. It can also test for the contamination of the modified hemoglobin preparation including endotoxin, trace fragments of microorganisms. It can also detect contaminants resulted from large scale production of modified hemoglobin, including chemicals, trace amount of some polymers which can cause complement activation, emulsifying agents and some types of organic solvents.
The most important feature of the method of the present invention is that before clinical testing in patients, one can take plasma from each individual potential participant in clinical trial and test their plasma with modified hemoglobin used for clinical trial and therefore, foresee the reactions of each individual - ~6 -person. Thereafter, before clinical trial is initiated, one already knows which patient would have reaction and hence would not subject the patient to the test.
The in vitro method of the present invention involves collecting heparinised blood sample, centrifuging the collected blood sample, separating the centrifuged blood sample and retaining the supernatant plasma. The blood sample may be taken from a given subject or a given population of subjects. The blood sample collected may contains 202U of heparinfml of blood and may be centrifuged at a range of about 3000 to 55008 and at about 0 to 4°C for about 15 to ~0 minutes;
and is preferably centrifuged at 55008 and 2°C for ZO
minutes.
It is possible to either proceed with the subsequent steps of the method of the present invention or to freeze the heparinised plasma at about -30 to -70°C until use, preferably at -70°C.
Then, it is followed by mixing the plasma with the modified hemoglobin blood substitutes or the control-ringer in a weight/volume ratio of about 4:1, which could be 400 lambda of the plasma and 100 lambda of modified hemoglobin blood substitutes samples.
Then, the mixture is incubated for a time sufficient to allow for a complement .activation reaction to occur, which preferably consists in incubating in a shaker at about 5 to 100 rpm and about 20 to 37°C for about 15 minutes to 3 hours and more preferably incubating in a shaker at 60 rpm and 37°C for 1 hour.
The incubated product is added to an appropriate volume of saline in an EDTA tube to stop the reaction, preferably about 0.5 to 3m1 of saline, more preferably 1.6m1 of saline.
It is possible to either proceed with the subsequent steps of the method of the present invention on to freeze the heparinised plasma at about. -30 to -70°C until use, preferably at ~70°C.
Then, it is followed by determining the degree of complement activation by analysis of the incubated product. The complement activation analysis is conducted by measuring the level of at least one member of the group consisting of C3, C4, C3a, C3b, C3c and C5 using a standard technigue, preferably measuring C3a by radioimmunoassay~ whereby the safeisy of the modified hemoglobin blood substitutes relai~ive to the human subject plasma sample is determined.
Figure 1 shows the complement activation pathway.
In order to determine the degree of conversion of C3 into C3a, one can measure the change in the level of C3, C4, C3a, C3b, C3c and C5. The more conversion of C3 to C3a, ttie more unsafe is the blood substitute product from the human plasma sample taken.
The method of the present invention, based on the complement activation analysis allows the screening of levels, at least sufficient to cause complement activation in human subjects, of endot0xin, cell membrane material, organic solvents, polymers and of chemical emulsifiers present in the modified hemoglobin blood substitutes.
The method of the present invention can be used for testing industrial batches of modified hemoglobin blood substitutes which comprises using a plasma sample or pool oaf plasma samples from human subjects.
An in vitro method to determine the safety of modified hemoglobin blood substitutsa for human prior to clinical use.
~AG:FCGi2~ZTND ~F °PHE Tldi7~I~'.L'IOid Blood is a living tissue; transfusion of it or of its cellular components from a donor to a recipient is a form of transplantation. About 11 ~to ~.2 million transfusions are given yearly in the United States, and 7.0 the number is steadily increasing. The decision to transfuse is a clinical judgment that requires weighing the possible benefits and known ha2ards with alternative treatments. A transfusion not specifically indicated is contraindicated.
In the United States, regulations for collecting storing, and transporting blood and its components are established by the Federal Drug Agency (FDA), and sometimes also by state or local health authorities.
The American National Red Cross and American Association of Blood Banks also have stanr~ards affecting their respective systems. Screening a donor includes a health interview, testing for vemoglobin (Hb), and taking the temperature, pulse rate, and blood pressure (BP).
Before use, blood must be classified for suitability. This includes ABO and Rh typing, antibody screening, STS, a test for hepatitis B surface antigen (HBsAg}, and tests to detect antilbodies to the virus that causes acquired immunodeficiency syndrome (AIDS}.
The container label and the federally required Circular of Information give the results of these testy and important information and cautions and should be consulted by physicians using blood transfusions.
The various components of blood can be separated, concentrated, and stored individually for precise replacement of patient needs.
Red blood cells (RBCs} are transfused to replace Hb or OZ carrying capacity, including blood lost at surgery, and to prime extracorporeal circuits. When volume expansion is required, other fluids can be used concurrently or ~eparateiy.
Frozen-thawed RBCs are costly and mainly used for patients who have multiple blood group antibodies or antibodies to high frequency antigens.
wasted RBCs (by continuous-flow washing) are free of almost all traces of plasma and of most white blood cells (wBCs) and platelets. They are suitable for patients who have severe reactions to plasma (eg, severe allergies or IgA immunization), or for those who have leucocyte antibodies and repeated :Febrile transfusion reactions.
The four ABO blood types are determined by testing far the presence or absence of A and B antigens an the RBCs using Anti-A and Anti--B reagents (forward or cell typing) , and by testing for Anti-A and Anti-B in the serum using A and B reagents RBCs (serum or reverse typing) .
Where are several reactions than accompany or follow intravenous administration of blood or blood components.
Hemolytic r~aaici~aas Hemolysis can result from blood group inco~npatibiaity, incompatible plasma or serum, hemolyzed or fragile RBCs. Incompatibility is the most frequent cause of hemolysas despite advances in blood typing and tesaing.
~'ebxi7.e reactions Reactions consisting of chills, fever with a rise of at least 1°C, and sometimes headache and back pain, rarely progressing to cyanosis and shock. Tn some patients, after many transfusions or pregnancies, leucocyte antibodies appear in response to antigens .of transfused or fetal WBCs.
Allergic reactions Reactions due to hypersensitivity of the patient to 7.0 an unknown component in donor blood are common, usually ' due to allergens in donor plasma, or, less often, to antibodies from an allergic donor.
Disease itransmission Virus hepatitis may follow the infusian of whole blood, plasma, or other products prepared from human blood; notably AHF and factor T~ concentrate.
Acquired immunodeficiency syndrome (AIDS): The causative agent of this condition has been identified as human immunadeficiency virus (HIV); epidemiologic evidence indicates that it is infectious and blood-borne. A dew patients with ATDS, not belonging to any of the known high-risk groups, have a history of w~a~'~~~.
-. 5 ..
receiving blaod products and their disease is considered "transfusion-associated".
Cytomegalovirus (CMV) can be transmitted by leucocytes in transfused blood. Usually its effects are either absent or mild, and need cause no concern.
Bacterial infection: Despite careful preparation, from 2 ~0 5 0 of all blood drawn contains a few bacteria, presumably from the donor's skin.
Malaria is transmitted easily by infected donor blood. HIany donors are unaware that they have malaria, certain varieties of which may be latent and transmissible for 20 to 15 yearn.
Syplaillis may be transmitted by fresh blood from a donor with the disease, but the incidence is very rare.
Modified hemoglobin Red blood cell contains hemoglobin which carries oxygen to tissues as required. Red blood cell is the best transfusion material at present: Fiowevar, as described above, it does.prssent a number of problems.
In particular; donor blood can be in short supply, _ ~ _ especially in major disaster situation or during war.
Red blood cell transfusions also rec~aire crossmatching.
They have a very short storage tune, unless they are stored by some expensive and complex means. During the last few years, the potential problem of the AIDS
transmission through blood transfusion has evolved.
As a result, a number of centres have investigated the potential uses of hemoglobin extracted from red blood cells. Rxtracted hemoglobins which are transfused to a patient are rapidly removed from the patient circulating blood; they do not carry and release oxygen efficiently and they can be toxic. Unfortunately, hemoglobin cannot be used as such for transfusion.
Because of these reasons, hemoglobin have to be modified before it can be used as a blood product.
Presently, there are two major groups of modified hemoglobin; 1) the encapsulated hemoglobin and 2) the crosslink~d hemoglobin (PROC. 1983 2nd TNTL. SYMP. BLOOD
SUBS., San Francisco, R.P.,Geyer and G.J. Nemo, Alan R.
Liss Inc. Publisher, New York, pp. 7,-X68; PROC. 1987 3rd TNTL. SYMP. BLOOD SUBS., T.M.S. Chang and R.P. Geyer, 1988, Maxcel Dekker Publisher, USA, pp. 1--708; PROC.
1989, SYMP. BLOOD SUB. BIOMAT., AR'f. CELLS AND ARI'.
ORGANS, R. Winslow and T.M.S. Chang, 1990, vol. 18, pp.
133-°366; ABSTRACTS 1991 4th INTL. S3tMP. BLOOD SZJBS.
BIOMAT., ART. CELLS AI~TD IMMOBI. BIOTECH., T.M.S. Chang and R.P. Geyer, 1991, vol. 19, pp. 299--520).
Crosslinked hemoglobin is subdivided into polyhemoglobin, conjugated hemoglobin and intramolecular crosslinked hemoglobin.
Animal studies carried out in many centres showed that these modified hemoglobin are effective in animal for resuscitating animals which would have otherwise died from severe bleeding. Other areas of applications for red blood cell blood substitutes were also demonstrated. The first preliminary clinical trial in a very small number of patients was approved by the FDA
and completed in the United States in 1989. Preliminary report appears to be safe. However, in March of 1990, the FDA called a meeting to announce that there are observed unexplained intense and severe reactions in clinical trials carried out.
A~iimal s8fety studies slot valid f~r ~itx~ata~1 This is surprising since animal studies carried out by many groups showed that modified hemoglobins are safe and without adverse effects. The animal result does not _ g _ compare to human study. Reactions in human are different to animal reactions, especially in immunological type of reactions including anaphylactic reactions, antigen-antibody reactions, allergic reactions.
In other words if a product is immunologically safe in animals, it is not necessarily safe in humans. There is an urgent need to fill the gap between animals study arid phase I clinical trial in human patients. A
preclinical screening test for modified hemoglobin, after its safety has been demonstrated in animals, is needed before it can be tested in patients.
Furthermore, screening of industx-ial production batches is needed. There are variations from person to person 25 in human ianmunological reactions.
The infusion of large amount of modified hemoglobin as blood substitute can potentially result in hypersensitivity and anaphylactic reactions, antibady-antigen reactions and other. Therefore, very careful efficacy and safety animal studies of modified hemoglolain blood substitute are required before phase I
of the clinical trial is started. However, one major problem is to select the right type of animals for testing before clinical trial, since one need to be sure that response in human will be the Came as in the test animals.
Another important area which is being developed is S non-human sources of hemoglobin. human hemoglobin is theoretically the most optimal source because there is not as much immunological problems as with Cad blood cells transfusion, but human hemoglobin could be in short supply because it has to come from donor x>lood.
There is considerable recent researches conducted to determine whethex bovine hemoglobin can be used to prepare modified hemoglobin. extensive development is also conducted on recombinant human hemoglobin from microorganisms and even animals. The impor~ancs of an in vitro human preclinical screening test will increase considerably if these types of hemoglobin are used to prepare modified hemoglobin for human use, sinne the result of animal safety studies is not necessary the same as human.
Furthermore, even if a given hemoglobin blood substitute product is without adverse side effects in a small number of phase T Clinical trial patients, there may still be some patients who may be more sensitive to this hemoglobin product. Presently, there exists no screening test to detect patients with potential severe hypersensitivity to hemoglobin products in large scale clinical trials and later on in routine clinical applications.
In vitro screening test specific fore human response It would be highly desirable to be provided with an in vitro screening test which would be based on using human blood or plasma to determine the safety of modified hemoglobin blood substitutes for human prior to clinical use. This type of test would provide a bridge between the animal testing and 'the human clinical trial of modified hemoglobin blood substitutes., Further, it would be highly desirable to be provided with an in vitro screening test so specific that one cauld determine the safety of madified hemoglobin blood substitutes for a particular patient, who is to receive the blood substitutes, prior to his actual clinical use of the blood substitutes.
Finally, it would be highly desirable to be provided with an in vitro screening test for screena.ng industrial productions of modified hemoglobin blood ~~~~ ~'~
_ 11 _ substitutes to rule out potential. problems before starting the clinical trial of the blood substitute by a population of patients or human subjects.
~tlP~,it7t OF' '.~'~iE II~T~IQ'fION
Surprisingly and in accordance with the present invention, there is provided an in vitro method to determine the safety of modified hemoglobin blood substitutes for human before their clinical trial in human. The in vitro method of the present invention i,s based on the effect of modified hemoglobin blood substitute on complement activation when added to human plasma.
The present invention relates ~o an in vitro method to determine the safety of modified hemoglobin blood substitutes for human subjects prior to their clinical usages, wherein the method is based on complement activation reaction from adding modified hemoglobin blood substitutes to a human plasma sample and includes the steps of: a) obtaining at leash one p~.asma sample from at least one human subject by: i) tak.ing a blond sample aa~d immediately centrifuging: and ii) separating the centrifuged blood sample of step i) and retaining the supernatant plasmas b) mixing plasma of step ii) with the modified hemoglobin blood substitutes or the control-ringer in a weight/volume ratio of about 4z1~ c) incubating for a time sufficient to allow for a complement activation reaction to occurs d) adding the product of step c) to an appropriate volume of saline in an EDTA tube and e) analyzing the degree of complement activation by analysis of the product of step d)~
thereby determining the safety of the mollified hemoglobin blood substitutes relative to the human subject plasma sample based on the detection of complement activation.
In accordance with the present invention, there is provided an in vitro screening method which is based ora using human blood or plasma to determine the safety of modified hemoglobin blood substitutes for human prior to clinical use: Thus method provides a bridge between the animal testing and the human clinical trial of modified hemoglobin blood substitutes.
In accordance with the present invention, there is provided an in vitro screening method so specific that one can determine the safety of modified hemoglobin blood substitutes for a particular patient, who is to receive the blood substitutes] prior to his actual clinical use of the blood substitutes.
1n accordance with the present invention, there is provided an in vitro method for screening industrial productions of modified hemoglobin blood substitutes to follow: (1) the different steps of industrial production, (2) different batches produced and (3) screening before the packaging the final product for human use.
The in vitro method of the present invention can also be used for human preclinical trial studies and for screening before human clinical use of modified hemoglobin blood substitutes.
Most of t~xe problems related to potential hypersensitivity reactions, anaphylactic reactions, effects due to antibody--antigen Complexes, and ethers could be detected in vitro by the method of the present invention.
Other advantages of the present invention will be readily illustrated by referring to the following description.
BRIEF' DESCR3P~.°~oN ~F' TfIE l3R~t~~N~~
Figure 1 is diagxam scheme of the complement activation pathway.
Figure 2 is a graph of the results of Table 1 below.
DE'fAI~EI9 ~ES~RI~TT~N of THE 7CNVErITI~N
The best in vitro test before clinical trial in human is the method of the present invention which uses the human plasma. The in vitro method of the present invention is based on the degree of complement activation of human plasma upon the addition of the modified hemoglobin blood substitutes.
In the preclinical test provided by the method of the present invention, a plasma sample is obtained from a human subject and modified hemoglobin is added to the plasma sample. Then, the sample may be analyzed fox C3a level to analyze possible C3 complement activation.
Using the method of the present invention, one can test the hemoglobin product to detexmins if it Causes any complement aC~tivation in huanan plasma; it is the next closest test to actual injection into human.
_ I5 _ Trace amount of blood cell membrane material may be present during the extraction of hemoglobin from red blood cells, and it can be incorporated into the modified hemoglobin preparation. This trace amount of blood cell membrane material may not be detectable using the animal testing procedure, whereas upon infusion into a patient it can cause adverse reactions, including complement activation.
The method of the present invention can also test for different 'types of antibody-antigen complexes. It can also test for the contamination of the modified hemoglobin preparation including endotoxin, trace fragments of microorganisms. It can also detect contaminants resulted from large scale production of modified hemoglobin, including chemicals, trace amount of some polymers which can cause complement activation, emulsifying agents and some types of organic solvents.
The most important feature of the method of the present invention is that before clinical testing in patients, one can take plasma from each individual potential participant in clinical trial and test their plasma with modified hemoglobin used for clinical trial and therefore, foresee the reactions of each individual - ~6 -person. Thereafter, before clinical trial is initiated, one already knows which patient would have reaction and hence would not subject the patient to the test.
The in vitro method of the present invention involves collecting heparinised blood sample, centrifuging the collected blood sample, separating the centrifuged blood sample and retaining the supernatant plasma. The blood sample may be taken from a given subject or a given population of subjects. The blood sample collected may contains 202U of heparinfml of blood and may be centrifuged at a range of about 3000 to 55008 and at about 0 to 4°C for about 15 to ~0 minutes;
and is preferably centrifuged at 55008 and 2°C for ZO
minutes.
It is possible to either proceed with the subsequent steps of the method of the present invention or to freeze the heparinised plasma at about -30 to -70°C until use, preferably at -70°C.
Then, it is followed by mixing the plasma with the modified hemoglobin blood substitutes or the control-ringer in a weight/volume ratio of about 4:1, which could be 400 lambda of the plasma and 100 lambda of modified hemoglobin blood substitutes samples.
Then, the mixture is incubated for a time sufficient to allow for a complement .activation reaction to occur, which preferably consists in incubating in a shaker at about 5 to 100 rpm and about 20 to 37°C for about 15 minutes to 3 hours and more preferably incubating in a shaker at 60 rpm and 37°C for 1 hour.
The incubated product is added to an appropriate volume of saline in an EDTA tube to stop the reaction, preferably about 0.5 to 3m1 of saline, more preferably 1.6m1 of saline.
It is possible to either proceed with the subsequent steps of the method of the present invention on to freeze the heparinised plasma at about. -30 to -70°C until use, preferably at ~70°C.
Then, it is followed by determining the degree of complement activation by analysis of the incubated product. The complement activation analysis is conducted by measuring the level of at least one member of the group consisting of C3, C4, C3a, C3b, C3c and C5 using a standard technigue, preferably measuring C3a by radioimmunoassay~ whereby the safeisy of the modified hemoglobin blood substitutes relai~ive to the human subject plasma sample is determined.
Figure 1 shows the complement activation pathway.
In order to determine the degree of conversion of C3 into C3a, one can measure the change in the level of C3, C4, C3a, C3b, C3c and C5. The more conversion of C3 to C3a, ttie more unsafe is the blood substitute product from the human plasma sample taken.
The method of the present invention, based on the complement activation analysis allows the screening of levels, at least sufficient to cause complement activation in human subjects, of endot0xin, cell membrane material, organic solvents, polymers and of chemical emulsifiers present in the modified hemoglobin blood substitutes.
The method of the present invention can be used for testing industrial batches of modified hemoglobin blood substitutes which comprises using a plasma sample or pool oaf plasma samples from human subjects.
2~~"~~~.
~ 19 d The method of the present invention may be used for the screening of large populations of patients before clinical trial or usage.
There are different potential factors in hemoglobin preparations which can cause complement activation.
These most likely include endotoxins and cell membrane materials such as some types of phospholipids or membrane antigens. Chemicals such as surfactants and organic solvents can also increase complement activation. This further shows the need for an overall screening test as a step before the actual use of hemoglobin preparations in human. The method of the present invention can avoid potential clinical problems not detected by animal safety studies or by more specific tests for specific substances. The in vitro method of the present invention is useful in this regard.
A similar test has been used sometimes for screening gamma-globulin production for human use.
2p However, this test is based on complete domplement hemolytic activity (CH50) in guinea pig serum. Guinea pig serumP tk~ough very sensitive to complement activation, may not reflect the same type of response as in human.
The in vitro method of the present invention of using human plasma gives a closer response to that of human. It is especially useful when the plasma of the same patient ~aho is to receive the hemoglobin preparation can be used for screening. This allows for a very specific screening test of the specific patient to the exact specific modified hemoglobin preparation.
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
~X~M~hE I:
riTeasx~rem~nt of C~a to datex~ntim~e the safety of a modified h~mbglt~bi~a blo~d substz.~tute sample Blood is obtained by clean venous puncture from human volunteers into 50 ml polypropylene (Sastedt) heparinised tubas (10 IU heparinfml of blood). The plasma is immediately separated by centrifugation at 5500g at 2°C for 20 minutes, the plasma is decanted and f~.o~en in separate portions at -70°C. Serum is not used because coagulation initiates complement activation.
EDTA is not used as an anticoagulant, because it interferes with complement activation reactions.
Remoglobin Solutions For this example, two (2) hemoglobin solutior~s are used; solutions A and B. Solution A does not cause significant complement activation. Solution B causes complement activation. Solutions A and B are used in the following mi~ttures for testing (1) A 100; (2) A 75~
+ B 25%; (3) A 50% + B 50d; (4) 25~ + B 750; (5) B 1005.
Bffeets of ~remoglobin solutions on hu4man plasma Immediately before use, the plasma sample is thawed. 400 lambda of the plasma is pipetted into 4 m1 sterile polypropylene tubes (Fischer). 100 lambda of pyrogen free saline (or Ringer Lactate) for injection is added to the 400 lambda of human plasma as control. 100 lambda of one of the test solutions including hemoglobin or modified hemoglobin is added to one of the other tubes containing 400 lambda of human plasma.
The reaction m~.xtures are incubated at 37°C at s0 rpm for 1 hour in a dab-Line Orlbit Environ Shakerm (Fisher Scientific, Montreal, Canada). After 1 hour the r~~~'~~.
-° 22 _ reaction is stopped by adding 0.4 ml of the reaction mixture to 1.~ ml of sterile saline in a 2 ml EDTA
sterile tube (Becton Dickenson). The samples are immediately stored at -70°C until analyzed.
The base line control level of complement activation will vary with source of human plasma axtd how it is obtained. Therefore a control base line level must be used for each analysis. Furthermore, all control and test studies should be carried out in triplicate.
c~mpaemea~t a.otivatx9n analysis : c3a measurement The analytical kit for human complement activation designed for G3a qualitative measurements is purchased from Amersham Canada. The method of analysis is the same as that of 'the instructions in the kit with 2 minor modifications. Centrifugation is carried out at 10,0008 for 20 minutes. After the final step of inversion, the inside wall of the tubes is carefully blotted with Q-tips.
2 ~ ~nclotaxa.n Measurement Endotoxin measurements are based on the Limulus Amebocyte Lysate "PYROTELL°' Test (Catalogue x.00-5 ~°) ...,,\
(Associates of Cape Cod, Woods Hole, Mass, USA) with a sensitivity of 0.03 EU/ml. For hemoglobin, this or other tests which are not based on colour changes can be used.
Measured aQmplement activation after aclditioxr of hemoglobin Using the method of the present invention as described herein, the measured C3a levels (ng/ml) in plasma are: control, 1,980~ 280; Zymosan, 20,000;
Hemoglobin preparation A, 2,227~ 6173 Hemoglobin preparation B, 4,967~ 153; A 75% + B 25%, 3,967~ 270; A
50 % + B 50%, 4, 553~ 517: A 25% + B 75%, 4, 9201- 430. The results obtained are summarized in Table 1 and illustrated in Figure 2.
a ~~B~~
x~xoo~o~zx pro coa~~x~~r~rr~ ac~a~~~aox (a~~) Tes'~ solutioa~ C8a n~g/na~. (~seara ~ B.D.
) Control 1,980 280 2ymosan >20,000 A 100% 2,227 617 A 75% + B 25% 3,967 t 270 A 50% + B 50% 4,553 517 A 25% + B 75% 4,920 430 B 100% 4, 967 -~ 153 Hemoglobin preparation A does nat cause significant increases in C3a complement activation. Hemoglobin preparation B causes significant increase in C3a compliment activation. Serial dilution o$ Hemoglobin preparation B in Hemoglobin preparation A continue to cause the same degree of C3a c~mpl~ment activation.
This is not due to C3 exhaustion because Zymosan resulted in C3a of greater 'than 20,OOOng/ml. This demonstrates that the in vitro method of the present invention can detect compliment activation even at low concentrations of the hemaglobin preparation. Factors which cause complement activation may include endotoxin, toluene extractable factors like l:~pids, and organic solvents.
Efxeots of hemog~.obar~ pr~pa.rx~tao:ns on complems~at activation ~.n human plasma.
Manipulation of human plasma during collection, freezing, thawing, and procedures of the screening test were enough to result in some C3a complement activation.
Using the 'procedure described above, a control level of 1,980 ~ 280 ng/ml is obtained. This emphasizes the importance of a simultaneous control sample. After addition of hemoglobin preparation A to human plasma the C3a concentration is 2,227~ 617 ng/ml. This is not significantly different from control C3a level of 1,980~
280 ng/ml, when control Ringer Lactate solution (pyrogen free solution for injection) is added to human plasma.
Hemoglobin solutian B on the other hand, when added to humani plasma, results in significant increase in C3a level to 4,967~ 7.53 ng/ml. Diluting solution B with solution A (A 250 ~ B 75%. A 50% * 8 50~J and A 75a ~ B
25%~ still results in the same degree o~ C3a complement activation. At a dilution of A 75 0 -~ B 25~ the C3a complement activation though significantly lowered, is still nearly as much as those for the higher concentrations. Thus, the degree of complement activation is nat quantitatively proportional to the relative concentration of B and A. This is not because of the exhaustion of C3, since Zymosan when added to human plasma resulted in C3a level of 20,000 ng/ml.
Thus, in vitro the method of the pa-esent invention is sensitive enough to detect the factor or factors responsible for complement activation even at much lower concentration.
EXAMPT~E Tx Effects of endotoxin on complement activation in human plasma It has been demonstrated that endotoxin in saline when added to human plasma a~t concentrations of as low as 0.50 EU/ml causes significant increases in C3a complement activation as measured by the above procedure. ~Iodifiad hemoglobin with significant endotoxin levels also increases C3a level in the above screening test. Thus it is likely that endotoxin is one of the possible factors responsible for complement activation in human plasma.
However, endotoa~in is only one of the many factors in hemoglobin preparation which can cause complement activation. For example, hemoglobin solutions which ,..-.
27 _ contain insufficient endotoxin by itself to cause complement activation are tested. Nevertheless, although some of these do not cause significant C3a complement activation, some other ;still increase C~a complement activation when added to human plasma.
Therefore, the question arises as to whether (1) hemoglobin at high concentration interferes with the measurements of endotoxin in hemoglobin preparations:
(2) in addition to endo~toxin, there are other factors in the hemoglobin preparations which cause complement activation. The following studies are carried out.
Effects of hemoglobin on ex~dot~xin measurex~e~t~
Different concentrations of endotoxin are measured in pyrogen free saline. Then the same concentrations of endotoxin are added to 10 g/dl hemoglobin solution which already contains 0.48 EU/ml of endotoxin. The results are summarized in enable 2.
_ 28 _ Solutions Amount present Measured amount (EU/ml) (EU/ml) Control saline 0 0 Saline (0.25 EU/ml) 0.25 0.25 Saline (0.50 EU/ml) 0.50 0.50 Saline (1.00 EU/ml) 1.00 1.00 Hemoglobin (0.48 EU/ml) 0.48 0.48 Hemoglobin (0.725 EU/ml) 0.725 0.725 Hemoglobin (0.980 EU/ml) 0.980 0.970 Hemoglobin (1.48 EU/ml) 1.40 1.460 The above assay shows that hemoglobin does not interfere with endatoxin measurements based on the LAL
°'PYROTELL°' Test.
2 9 °-Effects of endotoxxn "~free~° toluene e;~traated he~oglobi.n on C3a oomple~tent aotivation in hum2dn plasma The above results show that high concentrations of hemoglobin does not interfere with the measurement of endotoxin in hemoglobin preparations (Table 2). This means that in the endotoxin °'free'° hemoglobin preparations discussed earlier, complement activation must be due to another factor or factors. Thus, in addition to endotoxin, there could be at least another to factor. For instance, hemoglobin solution prepared by hemolysis followed only by centrifugation caused C3a complement activation when added to human plasma.
After toluene extraction and crystallisation, the resulting hemoglobin solution no longer causes C3a complement activation. Toluene lipid extracted stroma-free hemoglobin with endotoxin level of less than 0.24 EU/ml when added to human plasma does not cause a significant increase in C3a. Endotoxin level of lass than 0.24 EU/ml is the approved U.S.P. level for intravenous infusion fluids. Modified hemoglobin prepared from this lipid extracted hemoglobin solution does not result in sa.gnificant increase in C3a level when added to human plasma.
'While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modification and this application is intends:d to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows from the scope of the appended claims.
~ 19 d The method of the present invention may be used for the screening of large populations of patients before clinical trial or usage.
There are different potential factors in hemoglobin preparations which can cause complement activation.
These most likely include endotoxins and cell membrane materials such as some types of phospholipids or membrane antigens. Chemicals such as surfactants and organic solvents can also increase complement activation. This further shows the need for an overall screening test as a step before the actual use of hemoglobin preparations in human. The method of the present invention can avoid potential clinical problems not detected by animal safety studies or by more specific tests for specific substances. The in vitro method of the present invention is useful in this regard.
A similar test has been used sometimes for screening gamma-globulin production for human use.
2p However, this test is based on complete domplement hemolytic activity (CH50) in guinea pig serum. Guinea pig serumP tk~ough very sensitive to complement activation, may not reflect the same type of response as in human.
The in vitro method of the present invention of using human plasma gives a closer response to that of human. It is especially useful when the plasma of the same patient ~aho is to receive the hemoglobin preparation can be used for screening. This allows for a very specific screening test of the specific patient to the exact specific modified hemoglobin preparation.
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
~X~M~hE I:
riTeasx~rem~nt of C~a to datex~ntim~e the safety of a modified h~mbglt~bi~a blo~d substz.~tute sample Blood is obtained by clean venous puncture from human volunteers into 50 ml polypropylene (Sastedt) heparinised tubas (10 IU heparinfml of blood). The plasma is immediately separated by centrifugation at 5500g at 2°C for 20 minutes, the plasma is decanted and f~.o~en in separate portions at -70°C. Serum is not used because coagulation initiates complement activation.
EDTA is not used as an anticoagulant, because it interferes with complement activation reactions.
Remoglobin Solutions For this example, two (2) hemoglobin solutior~s are used; solutions A and B. Solution A does not cause significant complement activation. Solution B causes complement activation. Solutions A and B are used in the following mi~ttures for testing (1) A 100; (2) A 75~
+ B 25%; (3) A 50% + B 50d; (4) 25~ + B 750; (5) B 1005.
Bffeets of ~remoglobin solutions on hu4man plasma Immediately before use, the plasma sample is thawed. 400 lambda of the plasma is pipetted into 4 m1 sterile polypropylene tubes (Fischer). 100 lambda of pyrogen free saline (or Ringer Lactate) for injection is added to the 400 lambda of human plasma as control. 100 lambda of one of the test solutions including hemoglobin or modified hemoglobin is added to one of the other tubes containing 400 lambda of human plasma.
The reaction m~.xtures are incubated at 37°C at s0 rpm for 1 hour in a dab-Line Orlbit Environ Shakerm (Fisher Scientific, Montreal, Canada). After 1 hour the r~~~'~~.
-° 22 _ reaction is stopped by adding 0.4 ml of the reaction mixture to 1.~ ml of sterile saline in a 2 ml EDTA
sterile tube (Becton Dickenson). The samples are immediately stored at -70°C until analyzed.
The base line control level of complement activation will vary with source of human plasma axtd how it is obtained. Therefore a control base line level must be used for each analysis. Furthermore, all control and test studies should be carried out in triplicate.
c~mpaemea~t a.otivatx9n analysis : c3a measurement The analytical kit for human complement activation designed for G3a qualitative measurements is purchased from Amersham Canada. The method of analysis is the same as that of 'the instructions in the kit with 2 minor modifications. Centrifugation is carried out at 10,0008 for 20 minutes. After the final step of inversion, the inside wall of the tubes is carefully blotted with Q-tips.
2 ~ ~nclotaxa.n Measurement Endotoxin measurements are based on the Limulus Amebocyte Lysate "PYROTELL°' Test (Catalogue x.00-5 ~°) ...,,\
(Associates of Cape Cod, Woods Hole, Mass, USA) with a sensitivity of 0.03 EU/ml. For hemoglobin, this or other tests which are not based on colour changes can be used.
Measured aQmplement activation after aclditioxr of hemoglobin Using the method of the present invention as described herein, the measured C3a levels (ng/ml) in plasma are: control, 1,980~ 280; Zymosan, 20,000;
Hemoglobin preparation A, 2,227~ 6173 Hemoglobin preparation B, 4,967~ 153; A 75% + B 25%, 3,967~ 270; A
50 % + B 50%, 4, 553~ 517: A 25% + B 75%, 4, 9201- 430. The results obtained are summarized in Table 1 and illustrated in Figure 2.
a ~~B~~
x~xoo~o~zx pro coa~~x~~r~rr~ ac~a~~~aox (a~~) Tes'~ solutioa~ C8a n~g/na~. (~seara ~ B.D.
) Control 1,980 280 2ymosan >20,000 A 100% 2,227 617 A 75% + B 25% 3,967 t 270 A 50% + B 50% 4,553 517 A 25% + B 75% 4,920 430 B 100% 4, 967 -~ 153 Hemoglobin preparation A does nat cause significant increases in C3a complement activation. Hemoglobin preparation B causes significant increase in C3a compliment activation. Serial dilution o$ Hemoglobin preparation B in Hemoglobin preparation A continue to cause the same degree of C3a c~mpl~ment activation.
This is not due to C3 exhaustion because Zymosan resulted in C3a of greater 'than 20,OOOng/ml. This demonstrates that the in vitro method of the present invention can detect compliment activation even at low concentrations of the hemaglobin preparation. Factors which cause complement activation may include endotoxin, toluene extractable factors like l:~pids, and organic solvents.
Efxeots of hemog~.obar~ pr~pa.rx~tao:ns on complems~at activation ~.n human plasma.
Manipulation of human plasma during collection, freezing, thawing, and procedures of the screening test were enough to result in some C3a complement activation.
Using the 'procedure described above, a control level of 1,980 ~ 280 ng/ml is obtained. This emphasizes the importance of a simultaneous control sample. After addition of hemoglobin preparation A to human plasma the C3a concentration is 2,227~ 617 ng/ml. This is not significantly different from control C3a level of 1,980~
280 ng/ml, when control Ringer Lactate solution (pyrogen free solution for injection) is added to human plasma.
Hemoglobin solutian B on the other hand, when added to humani plasma, results in significant increase in C3a level to 4,967~ 7.53 ng/ml. Diluting solution B with solution A (A 250 ~ B 75%. A 50% * 8 50~J and A 75a ~ B
25%~ still results in the same degree o~ C3a complement activation. At a dilution of A 75 0 -~ B 25~ the C3a complement activation though significantly lowered, is still nearly as much as those for the higher concentrations. Thus, the degree of complement activation is nat quantitatively proportional to the relative concentration of B and A. This is not because of the exhaustion of C3, since Zymosan when added to human plasma resulted in C3a level of 20,000 ng/ml.
Thus, in vitro the method of the pa-esent invention is sensitive enough to detect the factor or factors responsible for complement activation even at much lower concentration.
EXAMPT~E Tx Effects of endotoxin on complement activation in human plasma It has been demonstrated that endotoxin in saline when added to human plasma a~t concentrations of as low as 0.50 EU/ml causes significant increases in C3a complement activation as measured by the above procedure. ~Iodifiad hemoglobin with significant endotoxin levels also increases C3a level in the above screening test. Thus it is likely that endotoxin is one of the possible factors responsible for complement activation in human plasma.
However, endotoa~in is only one of the many factors in hemoglobin preparation which can cause complement activation. For example, hemoglobin solutions which ,..-.
27 _ contain insufficient endotoxin by itself to cause complement activation are tested. Nevertheless, although some of these do not cause significant C3a complement activation, some other ;still increase C~a complement activation when added to human plasma.
Therefore, the question arises as to whether (1) hemoglobin at high concentration interferes with the measurements of endotoxin in hemoglobin preparations:
(2) in addition to endo~toxin, there are other factors in the hemoglobin preparations which cause complement activation. The following studies are carried out.
Effects of hemoglobin on ex~dot~xin measurex~e~t~
Different concentrations of endotoxin are measured in pyrogen free saline. Then the same concentrations of endotoxin are added to 10 g/dl hemoglobin solution which already contains 0.48 EU/ml of endotoxin. The results are summarized in enable 2.
_ 28 _ Solutions Amount present Measured amount (EU/ml) (EU/ml) Control saline 0 0 Saline (0.25 EU/ml) 0.25 0.25 Saline (0.50 EU/ml) 0.50 0.50 Saline (1.00 EU/ml) 1.00 1.00 Hemoglobin (0.48 EU/ml) 0.48 0.48 Hemoglobin (0.725 EU/ml) 0.725 0.725 Hemoglobin (0.980 EU/ml) 0.980 0.970 Hemoglobin (1.48 EU/ml) 1.40 1.460 The above assay shows that hemoglobin does not interfere with endatoxin measurements based on the LAL
°'PYROTELL°' Test.
2 9 °-Effects of endotoxxn "~free~° toluene e;~traated he~oglobi.n on C3a oomple~tent aotivation in hum2dn plasma The above results show that high concentrations of hemoglobin does not interfere with the measurement of endotoxin in hemoglobin preparations (Table 2). This means that in the endotoxin °'free'° hemoglobin preparations discussed earlier, complement activation must be due to another factor or factors. Thus, in addition to endotoxin, there could be at least another to factor. For instance, hemoglobin solution prepared by hemolysis followed only by centrifugation caused C3a complement activation when added to human plasma.
After toluene extraction and crystallisation, the resulting hemoglobin solution no longer causes C3a complement activation. Toluene lipid extracted stroma-free hemoglobin with endotoxin level of less than 0.24 EU/ml when added to human plasma does not cause a significant increase in C3a. Endotoxin level of lass than 0.24 EU/ml is the approved U.S.P. level for intravenous infusion fluids. Modified hemoglobin prepared from this lipid extracted hemoglobin solution does not result in sa.gnificant increase in C3a level when added to human plasma.
'While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modification and this application is intends:d to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows from the scope of the appended claims.
Claims (19)
1. An in vitro method to determine the safety of modified hemoglobin blood substitute for human subjects prior to their clinical usages, wherein said method is based on complement activation reaction from adding said modified hemoglobin blood substitutes to a human plasma sample and comprises the steps of:
a) obtaining at least one plasma sample from at least one human subject by:
i) taking a blood sample and immediately centrifugating; and ii) separating said centrifuged blood sample of step i) and retaining the supernatant plasma;
b) mixing said plasma of step ii) with said modified hemoglobin blood substitutes or control-ringer in a weight/volume ratio of about 4:1;
c) incubating for a time sufficient to allow for a complement activation reaction to occur;
d) adding the product of step c) to saline in an EDTA tube: and e) analyzing the degree of complement activation by analysis of the product of step d);
wherein an increase in complement activation is indicative of an increase in toxicity of said modified hemoglobin blood substitute relative to said human subject plasma.
a) obtaining at least one plasma sample from at least one human subject by:
i) taking a blood sample and immediately centrifugating; and ii) separating said centrifuged blood sample of step i) and retaining the supernatant plasma;
b) mixing said plasma of step ii) with said modified hemoglobin blood substitutes or control-ringer in a weight/volume ratio of about 4:1;
c) incubating for a time sufficient to allow for a complement activation reaction to occur;
d) adding the product of step c) to saline in an EDTA tube: and e) analyzing the degree of complement activation by analysis of the product of step d);
wherein an increase in complement activation is indicative of an increase in toxicity of said modified hemoglobin blood substitute relative to said human subject plasma.
2. The method of Claim 1, wherein step a) further comprises:
- freezing said supernatant plasma of step a) for storage; and - thawing said frozen supernatant plasma immediately prior to conducting step b).
- freezing said supernatant plasma of step a) for storage; and - thawing said frozen supernatant plasma immediately prior to conducting step b).
3. The method of Claim 1, which further comprises:
- freezing said product of step d) for storage; and - thawing said frozen product of step d) immediately prior to conducting step e).
- freezing said product of step d) for storage; and - thawing said frozen product of step d) immediately prior to conducting step e).
4. The method of Claim 2, which further comprises:
- freezing said product of step d) for storage; and - thawing said frozen product of step d) immediately prior to conducting step e).
- freezing said product of step d) for storage; and - thawing said frozen product of step d) immediately prior to conducting step e).
5. The method of Claim 1, wherein said blood sample of step i) contains 10IU of heparin/ml of blood and is centrifuged at a range of about 3000 to 5500g and at about 0 to 4°C for about 15 to 30 minutes.
6. The method of Claim 1, wherein said blood sample of step i) contains 10IU of heparin/ml of blood and is centrifuged at 5500g and at 2°C for 20 minutes.
7. The method of Claim 1, wherein step b) consists in mixing about 400 lambda of said plasma of step ii) and 100 lambda of the modified hemoglobin blood substitutes or control-ringer.
8. The method of Claim 1, wherein step c) consists in incubating in a shaker at about 5 to 100 RPM and about 20 to 37°C for about 15 minutes to 3 hours.
9. The method of Claim 1, wherein step c) consists in incubating in a shaker at 6C RPM and 37°C
for 1 hour.
for 1 hour.
10. The method of Claim 1, wherein step d) consists in adding the product of step c) to about 0.5 to 3ml of saline.
11. The method of Claim 1, wherein step d) consists in adding the product of step c) to about 1.6ml of saline.
12. The method of Claim 2, wherein said supernatant plasma is freezed at about -30 to -70°C.
13. The method of Claim 2, wherein said supernatant plasma is freezed at -70°C.
14. The method of Claim 1, wherein the complement activation analysis of step e) is conducted by measuring the level of at least one member of the group consisting of C3, C4, C3a, C3b, C3c and C5.
15. The method of Claim 14, wherein the level of a member is measured using radioimmunoassay technique.
16. The method of Claim 1, wherein the complement activation analysis allows the screening of levels, at least sufficient to cause complement activation in human subjects, of endotoxin, cell membrane material, organic solvents, polymers and of chemical emulsifiers present in the modified hemoglobin blood substitutes.
17. The method of Claim 1, wherein the plasma sample is taken from a given subject or a given population of subjects.
18. The use of the method of Claim 1 for testing industrial batches of modified hemoglobin blood substitutes which comprises using a plasma sample or pool of plasma samples from human subjects.
19. The use of the method of Claim 1 for the screening of large population of patients before clinical trial or usage.
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