Immunologic Changes After Blood Transfusion Patients Undergoing Vascular Surgery Louis A. Fernandez,
FRCPC, J. Michael MacSween, FRCPC, Choong K. You, Max Gorelick, MD, Halifax. Nova Scotia, Canada
Immunologic changes after blood transfusions cannot be studied ethically in normal individuals. We therefore studied two comparable groups of patients with atherosclerotic cardiovascular disease who received similar drug treatment and experienced a similar degree of surgical trauma, except that one group received an average of 2.5 units of packed red cells at one time period during surgery. We conducted immunologic tests preoperatively and 5, lo,45 to 60,90, 180, and 360 days postoperatively. There was no significant difference in all indices tested preoperatively between the two groups. Five and 10 days postoperatively, the absolute numbers of CD3, CD4, CD8, and B cells decreased in both groups; however, the decrease was significantly greater in the transfused group than in the nontransfused group 5 days postoperatively. There was no significant difference in these parameters between the two groups at other time periods tested. At 5 and 10 days postoperatively, the lymphocyte responses to phytohemagglutinin, concanavalin A, and allogeneic lymphocytes in autologous serum were decreased in both groups. However, at 60 days postoperatively, the responses of the nontransfused group became significantly increased, whereas those of the transfused group remained relatively unchanged. By days 90, 180, and 360, the lymphocyte responses of the nontransfused group had dropped to levels seen at earlier time intervals and were comparable to responses in the transfused group. There were no significant differences between the groups in the number of T-cell colonies formed, the number of immunoglobulin-producing cells obtained, and the lymphokine responses (migration inhibitory factor/migration stimulation factor) at all times tested. The major immunologic perturbations attributed to blood transfusions were an exaggerated decrease in the numbers of circulating lymphocytes, particularly those with markers associated with T-helper cells, and failure to demonstrate a rebound increase in the proliferative response 45 to 90 days later.
From the Departments of Medicine (LAF, JMM, MG) and Surgery (CKY), Dalhousie University, Camp Hill Medical Center, Halifax, Nova Scotia, Canada. This work was supported by a grant from the Canadian Red Cross Society Blood Transfusion Service. Requests for reprints should be addressed to Louis A. Femandez, FRCPC, Camp Hill Hospital, 1763 Robie Street, Halifax, Nova Scotia, Canada B3H 3G2. Manuscript submitted February 6, 1990, and accepted in revised form June 25,199O.
in
FRCPC,
o any patient, the benefit of judicial use of blood T transfusion by far exceeds any possible harm. However, there are widely acceptable risks associated with blood transfusion therapy that may include allergic reactions to blood products, transfusion-induced bacterial and viral infections, and reactions to mismatched blood [I]. Viable lymphocytes are found in whole blood for as long as 22 days [2,3]; therefore, transfusion of blood into immunocompromised individuals may lead to graft-versushost reaction. Blood transfusion prior to transplantation has a favorable effect on kidney graft survival [4], and it has also been reported that, compared with nontransfused patients, transfused patients with operable colorectal cancer with similar Dukes’ stage had shorter recurrence-free survival [5,q. These clinical observations probably depend upon immunologic changes that occur in the recipient after blood transfusion. The immunologic changes that result from blood transfusions have been studied by several investigators in patients with disease, some of whom were clinically transfused [7-91. It is difficult to extrapolate the results of these studies to other conditions, since the diseases may have contributed to the immunologic changes that were observed. Studies have been done in patients with gastrointestinal bleeding secondary to acid pepsin disease assuming that these patients were not immunologically compromised; however, these studies have been of short duration (7 to 10 days) [IO]. Since blood transfusions cannot be given ethically to normal individuals, the changes that occur secondary to blood transfusion need to be studied in a patient population in need of blood, and, thus, even in patients who need blood, it would be unethical to do a randomized study. Additionally, the disease state should not significantly alter immune responses. The patient’s blood should be available for testing before and after blood transfusions are given, and therefore it would be preferable to study an elective group of patients. Preferably, the blood should be given on only one occasion rather than repeatedly. We therefore chose to study the short- and long-term immunologic changes after blood transfusion in patients with atherosclerotic cardiovascular disease who were undergoing surgery for vascular bypass grafts. The same underlying disease was present in patients who did or did not receive blood, and atherosclerosis has not been associated with clearly defined abnormalities of the immune system [II]. PATIENTS
AND METHODS
Our study group consisted of 38 patients: 17 received blood at one point in time during surgery and were followed for as long as 12 months afterwards whereas 21 did
THE AMERICAN JOURNAL OF SURGERY
VOLUME 163 FEBRUARY 1992
263
FERNANDEZETAL
not undergo transfusion. Of the 21 patients who did not receive transfusions, 15 were women and 6 were men. The mean age was 62 years, with a range from 41 to 80 years. The majority of these patients were taking an average of two drugs each, usually sulfinpyrazone and/or hypoglycemics. All the patients had either a femoralpopliteal or cross-over femoral graft except one who had an aortic-femoral graft. The mean anesthetic time was 162 f 9 minutes. The 17 transfused patients, 11 men and 6 women, received an average of 2.5 units of packed red cells. The mean age was 66 years, with a range of 49 to 82 years. The average number of drugs taken by these patients was 2.8, which were usually sulfinpyrazone, oral hypoglycemics, and a diuretic. Eight patients underwent femoral-popliteal grafts, and nine aortic-femoral bypass grafts. The mean anesthetic time was 194 f 15 minutes. Patients who developed sepsis or hypotension postoperatively or who were taking antihypertensives or other medications, particularly @blockers and calcium channel blockers, were not included in our series. The following immunologic indices were documented: lymphocyte surface markers, lymphocyte responses to mitogens and allogeneic lymphocytes in autologous and human AB serum, lymphokine responses, T-cell colony formation, and numbers of immunoglobulin-producing cells. These indices were determined preoperatively and 5, 10, 45 to 60, 90, 180, and 360 days postoperatively. Lymphocyte surface markers: The total and differential peripheral white blood cell counts were measured in the routine hematology laboratory. In addition, suspensions of peripheral blood mononuclear cells were stained with fluorescein-conjugated monoclonal antibodies to OKT3, OKT4, and OKT8 antigens as previously described [ 121. Thus, percentages and absolute numbers of T cells and T-cell subsets were calculated. B cells were identified by the presence of membrane immunoglobulins stained with fluorescein-conjugated polyvalent antibody to p heavy chain determinants, and the percentage and absolute numbers were determined. Responses to mitogens and in mixed lymphocyte reactions (MLR): Lymphocyte responses to mitogens
and allogeneic cells were evaluated as previously described [13,14]. Briefly, 25,000 mononuclear cells were suspended in 200 PL medium RPM1 1640 containing 10% AB sera or autologous sera, 2 mm/L L glutamine, antibiotics, and various concentrations of phytohemagglutinin (PHA) or concanavalin A (Con A) and placed in microtiter wells. Similar suspensions were used in oneway mixed lymphocyte reactions with stimulation provided by 25,000 mononuclear cells from a panel of unrelated individuals, which had been pulsed for 30 minutes with 25 pg/mL mitomycin. These cell suspensions were placed in a 5% carbon dioxide (CO*) atmosphere for 4 days for mitogenic stimulation and for 7 days for mixed lymphocyte reactions. The evening before harvesting, 0.5 &i of tritiated thymidine, specific activity 18.2, was added to each well. The next day the cells were harvested onto glass filters using a Titertec Harvester (Flow Laboratories, Mississauga, Ontario, Canada). The filters were then placed in vials containing scintillation fluid, and radioactivity was determined in a beta scintillation spectrometer. 264
THE AMERICAN
JOURNAL
OF SURGERY
VOLUME
Lymphokine responses: As described previously [15], aliquots of 4 X 106 peripheral blood mononuclear cells in 1-mL endotoxin-free medium RPM1 1640 were pulsed for 2 hours at 37OC with 10 pg/mL Con A or with medium alone as a control. The cells were washed three times with fresh medium, resuspended in medium RPM1 1640 without serum, and incubated for 24 hours at 37OC in 5% CO2. The supernatants were then recovered, mixed with an equal volume of fresh medium, and assayed for macrophage migration assays using the human monocytoid cell line U937 as indicator cells. The ratio of migration areas produced by cells exposed to m&control supernatants was multiplied by 100 to give a migration index. All assays were performed in triplicate. On the basis of previous observations, migration indices of 90 or less were considered to be migration inhibitory factor (MIF) responses, whereas indices of 110 or greater were considered to be migration stimulation factor (MStF) responses. T-cell colonies: The methodology for T-cell colony formation has been described previously [16]. Briefly, lo6 mononuclear cells were suspended in 1 mL of medium RPM1 1640 containing 20% human AB serum, 0.2% PHA-P (DIFCO; Baxter Diagnostic Corp., Toronto, Ontario, Canada), 2 mm/L L glutamine, 0.3% wt/vol agarose, and antibiotics. One-milliliter aliquots of this suspension were plated in 35 X lo-mm Petri dishes and placed in a 5% CO2 atmosphere at high humidity for 6 days. Colonies were enumerated and were identified as containing T cells by their ability to spontaneously rosette with sheep erythrocytes and the absence of surface immunoglobulin. Immunsglohulin-producing cells: The number of immunoglobulin-producing cells was enumerated in the reverse hemolytic plaque assay as previously described [ Z7]. Briefly, mononuclear cells in RPM1 1640, fetal calf serum, and antibiotics were stimulated with 10 pg/mL pokeweed mitogen. After 6 days of culture, the cells were washed twice, and 100 PL suspensions were mixed with 25 PL of packed sheep erythrocytes combined with protein A, 0.8 mL of 0.5% agarose (Sigma, Sigma Chemical Co., St. Louis, MO), 25 rL of antiserum to human immunoglobulin with specificity for EL,(Y,or y heavy chains in a dilution of 1:4 (Dako, California) and 25 PL guinea pig complement (Cedarlane Laboratories Ltd., Ontario, Canada) previously adsorbed with sheep erythrocytes. Aliquots of 0.2 mL were then placed in Petri dishes and covered by a cover slip. The Petri dishes were placed in an incubator for 4 hours, and then the cells secreting specific immunoglobulins were identified by viewing the plates under a microscope and identifying the zone of lysis around individual lymphocytes. Statistical measurements were done using the analysis of variance. The procedure tests equality between population means for a range of population group. The procedure involves the determination of the F statistic from which the hypothesis can be tested at various confidence levels. RESULTS Lymphocyte surface markers:
Preoperatively, the absolute numbers of CD3, CD4, CD8, and B cells were
163
FEBRUARY
1992
:.6
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e1.2P =: s g 1.0-
\
c0~0.011
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\
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FRCPC, J. Michael MacSween, FRCPC, Choong K. You, Max Gorelick, MD, Halifax. Nova Scotia, Canada
Immunologic changes after blood transfusions cannot be studied ethically in normal individuals. We therefore studied two comparable groups of patients with atherosclerotic cardiovascular disease who received similar drug treatment and experienced a similar degree of surgical trauma, except that one group received an average of 2.5 units of packed red cells at one time period during surgery. We conducted immunologic tests preoperatively and 5, lo,45 to 60,90, 180, and 360 days postoperatively. There was no significant difference in all indices tested preoperatively between the two groups. Five and 10 days postoperatively, the absolute numbers of CD3, CD4, CD8, and B cells decreased in both groups; however, the decrease was significantly greater in the transfused group than in the nontransfused group 5 days postoperatively. There was no significant difference in these parameters between the two groups at other time periods tested. At 5 and 10 days postoperatively, the lymphocyte responses to phytohemagglutinin, concanavalin A, and allogeneic lymphocytes in autologous serum were decreased in both groups. However, at 60 days postoperatively, the responses of the nontransfused group became significantly increased, whereas those of the transfused group remained relatively unchanged. By days 90, 180, and 360, the lymphocyte responses of the nontransfused group had dropped to levels seen at earlier time intervals and were comparable to responses in the transfused group. There were no significant differences between the groups in the number of T-cell colonies formed, the number of immunoglobulin-producing cells obtained, and the lymphokine responses (migration inhibitory factor/migration stimulation factor) at all times tested. The major immunologic perturbations attributed to blood transfusions were an exaggerated decrease in the numbers of circulating lymphocytes, particularly those with markers associated with T-helper cells, and failure to demonstrate a rebound increase in the proliferative response 45 to 90 days later.
From the Departments of Medicine (LAF, JMM, MG) and Surgery (CKY), Dalhousie University, Camp Hill Medical Center, Halifax, Nova Scotia, Canada. This work was supported by a grant from the Canadian Red Cross Society Blood Transfusion Service. Requests for reprints should be addressed to Louis A. Femandez, FRCPC, Camp Hill Hospital, 1763 Robie Street, Halifax, Nova Scotia, Canada B3H 3G2. Manuscript submitted February 6, 1990, and accepted in revised form June 25,199O.
in
FRCPC,
o any patient, the benefit of judicial use of blood T transfusion by far exceeds any possible harm. However, there are widely acceptable risks associated with blood transfusion therapy that may include allergic reactions to blood products, transfusion-induced bacterial and viral infections, and reactions to mismatched blood [I]. Viable lymphocytes are found in whole blood for as long as 22 days [2,3]; therefore, transfusion of blood into immunocompromised individuals may lead to graft-versushost reaction. Blood transfusion prior to transplantation has a favorable effect on kidney graft survival [4], and it has also been reported that, compared with nontransfused patients, transfused patients with operable colorectal cancer with similar Dukes’ stage had shorter recurrence-free survival [5,q. These clinical observations probably depend upon immunologic changes that occur in the recipient after blood transfusion. The immunologic changes that result from blood transfusions have been studied by several investigators in patients with disease, some of whom were clinically transfused [7-91. It is difficult to extrapolate the results of these studies to other conditions, since the diseases may have contributed to the immunologic changes that were observed. Studies have been done in patients with gastrointestinal bleeding secondary to acid pepsin disease assuming that these patients were not immunologically compromised; however, these studies have been of short duration (7 to 10 days) [IO]. Since blood transfusions cannot be given ethically to normal individuals, the changes that occur secondary to blood transfusion need to be studied in a patient population in need of blood, and, thus, even in patients who need blood, it would be unethical to do a randomized study. Additionally, the disease state should not significantly alter immune responses. The patient’s blood should be available for testing before and after blood transfusions are given, and therefore it would be preferable to study an elective group of patients. Preferably, the blood should be given on only one occasion rather than repeatedly. We therefore chose to study the short- and long-term immunologic changes after blood transfusion in patients with atherosclerotic cardiovascular disease who were undergoing surgery for vascular bypass grafts. The same underlying disease was present in patients who did or did not receive blood, and atherosclerosis has not been associated with clearly defined abnormalities of the immune system [II]. PATIENTS
AND METHODS
Our study group consisted of 38 patients: 17 received blood at one point in time during surgery and were followed for as long as 12 months afterwards whereas 21 did
THE AMERICAN JOURNAL OF SURGERY
VOLUME 163 FEBRUARY 1992
263
FERNANDEZETAL
not undergo transfusion. Of the 21 patients who did not receive transfusions, 15 were women and 6 were men. The mean age was 62 years, with a range from 41 to 80 years. The majority of these patients were taking an average of two drugs each, usually sulfinpyrazone and/or hypoglycemics. All the patients had either a femoralpopliteal or cross-over femoral graft except one who had an aortic-femoral graft. The mean anesthetic time was 162 f 9 minutes. The 17 transfused patients, 11 men and 6 women, received an average of 2.5 units of packed red cells. The mean age was 66 years, with a range of 49 to 82 years. The average number of drugs taken by these patients was 2.8, which were usually sulfinpyrazone, oral hypoglycemics, and a diuretic. Eight patients underwent femoral-popliteal grafts, and nine aortic-femoral bypass grafts. The mean anesthetic time was 194 f 15 minutes. Patients who developed sepsis or hypotension postoperatively or who were taking antihypertensives or other medications, particularly @blockers and calcium channel blockers, were not included in our series. The following immunologic indices were documented: lymphocyte surface markers, lymphocyte responses to mitogens and allogeneic lymphocytes in autologous and human AB serum, lymphokine responses, T-cell colony formation, and numbers of immunoglobulin-producing cells. These indices were determined preoperatively and 5, 10, 45 to 60, 90, 180, and 360 days postoperatively. Lymphocyte surface markers: The total and differential peripheral white blood cell counts were measured in the routine hematology laboratory. In addition, suspensions of peripheral blood mononuclear cells were stained with fluorescein-conjugated monoclonal antibodies to OKT3, OKT4, and OKT8 antigens as previously described [ 121. Thus, percentages and absolute numbers of T cells and T-cell subsets were calculated. B cells were identified by the presence of membrane immunoglobulins stained with fluorescein-conjugated polyvalent antibody to p heavy chain determinants, and the percentage and absolute numbers were determined. Responses to mitogens and in mixed lymphocyte reactions (MLR): Lymphocyte responses to mitogens
and allogeneic cells were evaluated as previously described [13,14]. Briefly, 25,000 mononuclear cells were suspended in 200 PL medium RPM1 1640 containing 10% AB sera or autologous sera, 2 mm/L L glutamine, antibiotics, and various concentrations of phytohemagglutinin (PHA) or concanavalin A (Con A) and placed in microtiter wells. Similar suspensions were used in oneway mixed lymphocyte reactions with stimulation provided by 25,000 mononuclear cells from a panel of unrelated individuals, which had been pulsed for 30 minutes with 25 pg/mL mitomycin. These cell suspensions were placed in a 5% carbon dioxide (CO*) atmosphere for 4 days for mitogenic stimulation and for 7 days for mixed lymphocyte reactions. The evening before harvesting, 0.5 &i of tritiated thymidine, specific activity 18.2, was added to each well. The next day the cells were harvested onto glass filters using a Titertec Harvester (Flow Laboratories, Mississauga, Ontario, Canada). The filters were then placed in vials containing scintillation fluid, and radioactivity was determined in a beta scintillation spectrometer. 264
THE AMERICAN
JOURNAL
OF SURGERY
VOLUME
Lymphokine responses: As described previously [15], aliquots of 4 X 106 peripheral blood mononuclear cells in 1-mL endotoxin-free medium RPM1 1640 were pulsed for 2 hours at 37OC with 10 pg/mL Con A or with medium alone as a control. The cells were washed three times with fresh medium, resuspended in medium RPM1 1640 without serum, and incubated for 24 hours at 37OC in 5% CO2. The supernatants were then recovered, mixed with an equal volume of fresh medium, and assayed for macrophage migration assays using the human monocytoid cell line U937 as indicator cells. The ratio of migration areas produced by cells exposed to m&control supernatants was multiplied by 100 to give a migration index. All assays were performed in triplicate. On the basis of previous observations, migration indices of 90 or less were considered to be migration inhibitory factor (MIF) responses, whereas indices of 110 or greater were considered to be migration stimulation factor (MStF) responses. T-cell colonies: The methodology for T-cell colony formation has been described previously [16]. Briefly, lo6 mononuclear cells were suspended in 1 mL of medium RPM1 1640 containing 20% human AB serum, 0.2% PHA-P (DIFCO; Baxter Diagnostic Corp., Toronto, Ontario, Canada), 2 mm/L L glutamine, 0.3% wt/vol agarose, and antibiotics. One-milliliter aliquots of this suspension were plated in 35 X lo-mm Petri dishes and placed in a 5% CO2 atmosphere at high humidity for 6 days. Colonies were enumerated and were identified as containing T cells by their ability to spontaneously rosette with sheep erythrocytes and the absence of surface immunoglobulin. Immunsglohulin-producing cells: The number of immunoglobulin-producing cells was enumerated in the reverse hemolytic plaque assay as previously described [ Z7]. Briefly, mononuclear cells in RPM1 1640, fetal calf serum, and antibiotics were stimulated with 10 pg/mL pokeweed mitogen. After 6 days of culture, the cells were washed twice, and 100 PL suspensions were mixed with 25 PL of packed sheep erythrocytes combined with protein A, 0.8 mL of 0.5% agarose (Sigma, Sigma Chemical Co., St. Louis, MO), 25 rL of antiserum to human immunoglobulin with specificity for EL,(Y,or y heavy chains in a dilution of 1:4 (Dako, California) and 25 PL guinea pig complement (Cedarlane Laboratories Ltd., Ontario, Canada) previously adsorbed with sheep erythrocytes. Aliquots of 0.2 mL were then placed in Petri dishes and covered by a cover slip. The Petri dishes were placed in an incubator for 4 hours, and then the cells secreting specific immunoglobulins were identified by viewing the plates under a microscope and identifying the zone of lysis around individual lymphocytes. Statistical measurements were done using the analysis of variance. The procedure tests equality between population means for a range of population group. The procedure involves the determination of the F statistic from which the hypothesis can be tested at various confidence levels. RESULTS Lymphocyte surface markers:
Preoperatively, the absolute numbers of CD3, CD4, CD8, and B cells were
163
FEBRUARY
1992
:.6
1 1.4-
e1.2P =: s g 1.0-
\
c0~0.011
\
z
\
i z? .8-
\
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_";i/
I \
I I
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