A randomized trial comparing the use of fresh and stored platelets in the treatment of bone marrow transplant recipients Shanwell A, Larsson S, Aschan J, Ringden 0. A randomized trial comparing the use of fresh and stored platelets in the treatment of bone marrow transplant recipients. Eur J Haematol 1992: 49: 77-81.
1
Abstract: Patients undergoing allogeneic bone marrow transplantation (BMT) were randomized into two groups. One group (n = 21) received single donor platelet concentrates (PC) that were as fresh as possible, the other group (n = 18) received single donor PC stored for 2 to 5 days. Actual mean storage times for PC were 1.12 f 1.25 (mean 5 SD) and 2.67 5 1.30 d, respectively (p < 0.001). The total need for platelets during 60 d after BMT in patients receiving fresh PC was 22.1 & 17.8 x 10’ platelets/liter blood volume/d, and for stored PC 29.2 f 19.2 x lo9 platelets/liter blood volume/d (n.s.). A multiple regression analysis of the data showed no correlation between PC storage time and the PC requirement (p = 0.85). Posttransfusion corrected count increment (CCI) at 1 hour was 10.4 k 5.1 for PC stored 0-1 d, 10.3 f 7.0 for PC stored 2-3 d, and 11.4 k 9.2 for PC stored 4-5 d. The corresponding CCI, at 18 h were 6.5 5 4.4, 5.4 3.3 and 6.8 k 4.6. We conclude that there is no major difference between fresh and stored single donor PC.
Introduction
Bone marrow transplantation (BMT) is a wellestablished form of therapy for hematological malignancies, severe aplastic anemia and some metabolic disorders (1-3). Conditioning with high-dose chemotherapy (with or without total body irradiation), destroys hematopoiesis. During the pancytopenic period that follows, the patient needs frequent blood product support before stable engraftment occurs several weeks after BMT (4). To prevent hemorrhages, the patient requires frequent platelet transfusions. The number of platelet transfusions is dependent on whether the patient is sensitized by previous transfusions and therefore refractory to random donor platelets, severe infections, graftversus-host disease (GvHD) and hemorrhages ( 5 , 6 ) . The present investigation was undertaken to evaluate whether patients treated with the freshest platelets possible would need less platelet transfusions than those receiving platelets stored for several days. Material and methods Patients
All patients underwent allogeneic BMT at Huddinge Hospital. Excluded from the trial were patients with
A. Shanwell’, S. Larsson’, J. Aschanb and 0. Ringden Stockholm Blood Transfusion Service, Department of Clinical Immunology and Department of Transplantation Surgery, Huddinge Hospital, Stockholm, Sweden
a
Key words: platelet transfusions - apheresis platelets - storage time - corrected count increment - bone marrow transplantation Correspondence: Agneta Shanwell, Blood Transfusion Center, K42, Huddinge Hospital, 141 86 Huddinge, Sweden Accepted for publication 1 1 May 1992
foreseeable platelet refractoriness, e.g. those with lymphocytotoxic antibodies, and those with endstage leukemia (> 25 % blasts in marrow). Forty-four consecutive patients were randomized into two groups, one intended to receive platelet concentrates (PC) stored for less than 24 h (n = 21) and the other group PC stored for 2 to 5 d (n = 18). At randomization the groups were stratified into children (age < 18 years) and adults ( 2 18 yr). Five patients were excluded because of the presence of lymphocytotoxic antibodies at the time of BMT. In the group receiving fresh platelets, 6 patients were children; 2-16 yr of age with acute leukemia in 3 cases and metabolic disorders in 3. Fifteen patients were adults with acute leukemia in 8 cases, chronic myeloid leukemia in 6 and multiple myeloma in 1. In the group receiving stored platelets, 9 were children; 2-16 yr of age with acute leukemia in 5 cases, nonHodgkin’s lymphoma and aplastic anemia in 1 case each, and metabolic disorders in 2 cases. Nine patients were adults with acute leukemia in 2 cases, chronic myeloid leukemia in 6 and Hodgkin’s disease in 1 case. HLA-identical siblings were used as marrow donors for all but 5 recipients (2 HLAnonidentical related donors, 1 in each group, and 3 unrelated HLA-matched donors in the group receiving stored platelets). The two groups were similar
Shanwell et al. regarding recipient mean age, donor mean age, sexmatch, ABO compatibility, marrow cell dose and the use of leukocyte-depleted blood products. Details regarding transplantation procedure have been previously described (3, 7, 8). Blood-transfusionpolicy
All blood products were prepared from donors who lacked cytomegalovirus (CMV) antibodies. Before transfusion, blood products were irradiated with 15 Gy. Red blood cells (RBC) were given to keep hemoglobin above 70 g/l. Single donor platelets were transfused when the patient’s platelet counts fell below 30 x 109/l. Granulocyte transfusions were given to patients affected by high fever (> 39°C) non-responsive to antibiotics with, and sometimes without, a positive blood culture. High fever in combination with a local bacterial infection or severe mucositis which was increasing in size were also indications for granulocyte transfusion. Granulocytes were only instituted if white blood cells (WBC) were 0.2 x 109/l. If the donor had a minor or major ABO incompatibility with the recipient, RBC of blood-group 0 were used; otherwise red cells of the recipient’s ABO blood group were used. Plasma was always fresh frozen and of blood-group AB. Blood-group 0 platelets were routinely used. Only Rh-compatible blood products were transfused. Blood products
Red blood cells. RBC were prepared from 4501111 whole blood. After centrifugation (3000 g; 1.75 min) of the fresh whole blood unit, the platelet-rich plasma and 40 ml of buffy coat layer were carefully removed. The red cells were then suspended in 100 ml SAGMAN solution (877 mg sodium chloride, 16.9 mg adenine, 8 19 mg glucose anhydride, and 525 mg mannitol in 100 ml of water; Baxter SA, Le Chatre, France). Platelet concentrates. Single donor PC were collected using a blood cell separator, CS-3000, (Baxter R&D, Europe) with a closed system apheresis kit for extended platelet storage (4R-22-30). Details about the apheresis procedure were published previously (9). Immediately after the apheresis procedure, the platelets were resuspended and 100 in1 extra autologous donor plasma was added (final volume, about 350 ml). Each PC unit was divided equally in two 1000-ml containers (PL 732) and stored for 8 to 120 h at 22°C regulated temperature on a flatbed shaker, oscillating at approximately 70 cycles per min (Linear Platelet Reciprocator Model LPR-3, 78
Melco Engineering Corp., Glendale, CA). One PC unit contained (mean ~frSD) 456 ~fr94 x 10’ platelets in approximately 350 ml plasma (n = 273). The number of contaminating WBC has previously been found to be 195 & 160 x 109/unit (9). Granulocyte concentrates. Single donor granulocyte concentrates (GC) were collected on the blood cell separator CS-3000, using the manufacturer’s method for granulocyte preparation. The donors were given hydrocortisone 100mg i.v. prior to donation. Hydroxyethylstarch (HES) was used as a sedimenting agent. The volume of a G C was approximately 210 ml/unit. The numbers of WBC were 15.9 5 5.1 x 109/unit (n = 42; granulocytes 9.7 2 3.8 x lO’/unit and mononuclear cells 6.2 ~fr2.3 x 109/unit). The number of platelets was 495 89 x lO’/unit (n = 42) and the erythrocyte volume fraction was 17 _+ 4 % (n = 33). G C were generally transfused within 6-8 h. Leukocyte-depleted blood products. Nine patients received leukocyte-depleted blood products in order to prevent allo-immunization. RBC and PC were then filtered through a PALL-filter (RC 100 or PL 100, respectively; Mediwest, Askim, Sweden). No saline rinsing was carried out. When PC are filtered through a PALL-filter without saline rinsing, a loss of 13.1% (range 6-20.5%) of platelets occurs (10). Platelet and white blood cell count
Platelet and WBC count in PC and GC was performed on a cell counter (Model Z B I ,Coulter Electronics, Ltd, Luton, UK). Lymphocytotoxic antibody detection
Lymphocytotoxic antibody screening was carried out at the time of BMT and repeated later if allo-immunization was suspected. The two-step complement-dependent lymphocytotoxicity test was used against a panel of 12 selected donors carrying the most frequently occurring HLA class I antigen s. Statistical analysis
For comparison of population means, Students t-test for unpaired observations was used. As the number of PC required is dependent on patient’s blood volume (b.v.) standardization for this factor was undertaken using the formula:
Platelets/b.v./day
Number of platelets transfused =
b.v. (L) x observed days
Fresh versus stored platelets Number of platelets transfused is the sum of platelets in all P C transfused per patient during the study. In 34% the actual number of platelets in the PC was not known, then the mean value ( = 465 x lo9) was used. We estimated the blood volume by the formulas 5.51 x h + 0.0437 x w - 7.12 (for men), 2.37 x h + 0.0455 x w - 2.25 (for women) and 0.08 x w (for children; 2-15 yr of age) where h = height in m and w = weight in kg. The patients were observed for 60 d after BMT. Nine patients died within this period and they were observed until death. We used linear regression analysis to see what factors influenced on the PC requirement. A multiple regression analysis was carried out, using PC storage time on the number of PC transfusions (after standardization for patient's size and observed days), controlling for different background factors. The background factors included in the multiple analysis were chosen because oftheir own correlation (p < 0.2) to the need for platelet transfusions. Some of the patients needed G C during the aplastic phase after BMT. Since a G C contains an amount of fresh platelets comparable with a fresh PC, we chose to account for both PC transfusions and the sum of PC and G C transfusions. Post-transfusion corrected count increment (CCI) at 1 h (CCI,,,) and the following day (CCI,8,,; 1224 h after PC transfusion) were determined using the formula:
CCI
Patient platelet count increment ( i09/i) x BSA(m2) =
Number of platelets in the PC (10")
BSA = Body Surface Area
Table 1 , Comparison of factors that may influence on the need of platelet transfusion after BMT in patients receiving fresh and stored platelets
Time to engraftment (days)' GVHD grade 0-11 Ill-IV CMV-infection Sepsis No. patients on Amphotericin B medication No. patients who died 1 6 0 days after B M l RBC total number units/patient No. patients receiving GC No. patients with lymhocytotoxic antibodies during the study
Fresh platelets
Stored platelets
15k3 20 1 1 11 12 5 5.4k3.7 4
1724ns 16 2 3 7 9 4 7.9k7.3"' 5
2
1
* WBC> 0.2X 109/1 " No significant difference (p>O.O5)
Patients transfused with fresh PC received a mean of 22.1 17.8 x lo9 platelets/b.v./d and those receiving stored PC 29.2 19.6 x lo9 platelets/b.v./d. The difference was insignificant (p = 0.24) (Table 2). When the number of platelets in G C was included, the corresponding figures were 29.3 29.1 x lo9 and 35.8 & 27.6 x lo9, respectively (n.s.). Patients who received fresh platelets required PC or P C + G C transfusions for 26 & 15 d after BMT, the corresponding time for the group who received stored platelets was 32 17 d (n.s.). See Table 2. The aim of providing patients in the respective groups with fresh/stored PC, was not fully achieved. We do not prepare apheresis platelets on Saturdays and Sundays at Huddinge Hospital, so if a patient randomized to receive fresh PC needed PC transfusions on a Sunday or Monday morning the PC transfused had been stored for 2-3 d. Of the 174 PC given to the patients receiving fresh PC, 67% were
Results
Conditions that may influence the amount of platelet transfusions required, such as time to engraftrnent, septicemia, use of amphotericin B, CMV infection and graft-versus-host disease (GvHD) were similar in patients receiving fresh and stored platelets (Table 1). The amount of RBC and granulocyte transfusions needed was also the same in both groups. In the group receiving fresh platelets, 5 patients died in the 60-d post-BMT period, i.e. at 11, 21, 28, 40 and 57 d after BMT. One patient died of cerebral hemorrhage; the peripheral platelet count was 52 x 10y/lthe day she died, and she had received her last PC transfusion 10 d earlier. Four patients died within 60 d post-BMT in the group receiving stored PC, i.e. at 30,3 1, 52 and 53 d after BMT.
Table 2. Comparison of platelet transfusion requirements in 8MT patients receivingfresh and stored PC (MeanfSD)
Fresh
Stored
platelets
platelets
21
18
PC storage time (days)
1.12k 1.25
2.67&1.30**
PC/patient total Platelets/b.v,/day ( X lo9]
7.9k3.7 22.1k17.8
13.9&10.5" 29.2f 19.6"s
(PCtGCl/patient, total Platelets in (PCtGC)/ b.v./day ( X 10')
10.3k9.2
l6.9f 14.2"'
29.3t29.1
35.8 f 27.6" '
Days after 8MT to last PC transfusion
26f15
32k17ns
No. of patients
~
** Significant (p
1
Abstract: Patients undergoing allogeneic bone marrow transplantation (BMT) were randomized into two groups. One group (n = 21) received single donor platelet concentrates (PC) that were as fresh as possible, the other group (n = 18) received single donor PC stored for 2 to 5 days. Actual mean storage times for PC were 1.12 f 1.25 (mean 5 SD) and 2.67 5 1.30 d, respectively (p < 0.001). The total need for platelets during 60 d after BMT in patients receiving fresh PC was 22.1 & 17.8 x 10’ platelets/liter blood volume/d, and for stored PC 29.2 f 19.2 x lo9 platelets/liter blood volume/d (n.s.). A multiple regression analysis of the data showed no correlation between PC storage time and the PC requirement (p = 0.85). Posttransfusion corrected count increment (CCI) at 1 hour was 10.4 k 5.1 for PC stored 0-1 d, 10.3 f 7.0 for PC stored 2-3 d, and 11.4 k 9.2 for PC stored 4-5 d. The corresponding CCI, at 18 h were 6.5 5 4.4, 5.4 3.3 and 6.8 k 4.6. We conclude that there is no major difference between fresh and stored single donor PC.
Introduction
Bone marrow transplantation (BMT) is a wellestablished form of therapy for hematological malignancies, severe aplastic anemia and some metabolic disorders (1-3). Conditioning with high-dose chemotherapy (with or without total body irradiation), destroys hematopoiesis. During the pancytopenic period that follows, the patient needs frequent blood product support before stable engraftment occurs several weeks after BMT (4). To prevent hemorrhages, the patient requires frequent platelet transfusions. The number of platelet transfusions is dependent on whether the patient is sensitized by previous transfusions and therefore refractory to random donor platelets, severe infections, graftversus-host disease (GvHD) and hemorrhages ( 5 , 6 ) . The present investigation was undertaken to evaluate whether patients treated with the freshest platelets possible would need less platelet transfusions than those receiving platelets stored for several days. Material and methods Patients
All patients underwent allogeneic BMT at Huddinge Hospital. Excluded from the trial were patients with
A. Shanwell’, S. Larsson’, J. Aschanb and 0. Ringden Stockholm Blood Transfusion Service, Department of Clinical Immunology and Department of Transplantation Surgery, Huddinge Hospital, Stockholm, Sweden
a
Key words: platelet transfusions - apheresis platelets - storage time - corrected count increment - bone marrow transplantation Correspondence: Agneta Shanwell, Blood Transfusion Center, K42, Huddinge Hospital, 141 86 Huddinge, Sweden Accepted for publication 1 1 May 1992
foreseeable platelet refractoriness, e.g. those with lymphocytotoxic antibodies, and those with endstage leukemia (> 25 % blasts in marrow). Forty-four consecutive patients were randomized into two groups, one intended to receive platelet concentrates (PC) stored for less than 24 h (n = 21) and the other group PC stored for 2 to 5 d (n = 18). At randomization the groups were stratified into children (age < 18 years) and adults ( 2 18 yr). Five patients were excluded because of the presence of lymphocytotoxic antibodies at the time of BMT. In the group receiving fresh platelets, 6 patients were children; 2-16 yr of age with acute leukemia in 3 cases and metabolic disorders in 3. Fifteen patients were adults with acute leukemia in 8 cases, chronic myeloid leukemia in 6 and multiple myeloma in 1. In the group receiving stored platelets, 9 were children; 2-16 yr of age with acute leukemia in 5 cases, nonHodgkin’s lymphoma and aplastic anemia in 1 case each, and metabolic disorders in 2 cases. Nine patients were adults with acute leukemia in 2 cases, chronic myeloid leukemia in 6 and Hodgkin’s disease in 1 case. HLA-identical siblings were used as marrow donors for all but 5 recipients (2 HLAnonidentical related donors, 1 in each group, and 3 unrelated HLA-matched donors in the group receiving stored platelets). The two groups were similar
Shanwell et al. regarding recipient mean age, donor mean age, sexmatch, ABO compatibility, marrow cell dose and the use of leukocyte-depleted blood products. Details regarding transplantation procedure have been previously described (3, 7, 8). Blood-transfusionpolicy
All blood products were prepared from donors who lacked cytomegalovirus (CMV) antibodies. Before transfusion, blood products were irradiated with 15 Gy. Red blood cells (RBC) were given to keep hemoglobin above 70 g/l. Single donor platelets were transfused when the patient’s platelet counts fell below 30 x 109/l. Granulocyte transfusions were given to patients affected by high fever (> 39°C) non-responsive to antibiotics with, and sometimes without, a positive blood culture. High fever in combination with a local bacterial infection or severe mucositis which was increasing in size were also indications for granulocyte transfusion. Granulocytes were only instituted if white blood cells (WBC) were 0.2 x 109/l. If the donor had a minor or major ABO incompatibility with the recipient, RBC of blood-group 0 were used; otherwise red cells of the recipient’s ABO blood group were used. Plasma was always fresh frozen and of blood-group AB. Blood-group 0 platelets were routinely used. Only Rh-compatible blood products were transfused. Blood products
Red blood cells. RBC were prepared from 4501111 whole blood. After centrifugation (3000 g; 1.75 min) of the fresh whole blood unit, the platelet-rich plasma and 40 ml of buffy coat layer were carefully removed. The red cells were then suspended in 100 ml SAGMAN solution (877 mg sodium chloride, 16.9 mg adenine, 8 19 mg glucose anhydride, and 525 mg mannitol in 100 ml of water; Baxter SA, Le Chatre, France). Platelet concentrates. Single donor PC were collected using a blood cell separator, CS-3000, (Baxter R&D, Europe) with a closed system apheresis kit for extended platelet storage (4R-22-30). Details about the apheresis procedure were published previously (9). Immediately after the apheresis procedure, the platelets were resuspended and 100 in1 extra autologous donor plasma was added (final volume, about 350 ml). Each PC unit was divided equally in two 1000-ml containers (PL 732) and stored for 8 to 120 h at 22°C regulated temperature on a flatbed shaker, oscillating at approximately 70 cycles per min (Linear Platelet Reciprocator Model LPR-3, 78
Melco Engineering Corp., Glendale, CA). One PC unit contained (mean ~frSD) 456 ~fr94 x 10’ platelets in approximately 350 ml plasma (n = 273). The number of contaminating WBC has previously been found to be 195 & 160 x 109/unit (9). Granulocyte concentrates. Single donor granulocyte concentrates (GC) were collected on the blood cell separator CS-3000, using the manufacturer’s method for granulocyte preparation. The donors were given hydrocortisone 100mg i.v. prior to donation. Hydroxyethylstarch (HES) was used as a sedimenting agent. The volume of a G C was approximately 210 ml/unit. The numbers of WBC were 15.9 5 5.1 x 109/unit (n = 42; granulocytes 9.7 2 3.8 x lO’/unit and mononuclear cells 6.2 ~fr2.3 x 109/unit). The number of platelets was 495 89 x lO’/unit (n = 42) and the erythrocyte volume fraction was 17 _+ 4 % (n = 33). G C were generally transfused within 6-8 h. Leukocyte-depleted blood products. Nine patients received leukocyte-depleted blood products in order to prevent allo-immunization. RBC and PC were then filtered through a PALL-filter (RC 100 or PL 100, respectively; Mediwest, Askim, Sweden). No saline rinsing was carried out. When PC are filtered through a PALL-filter without saline rinsing, a loss of 13.1% (range 6-20.5%) of platelets occurs (10). Platelet and white blood cell count
Platelet and WBC count in PC and GC was performed on a cell counter (Model Z B I ,Coulter Electronics, Ltd, Luton, UK). Lymphocytotoxic antibody detection
Lymphocytotoxic antibody screening was carried out at the time of BMT and repeated later if allo-immunization was suspected. The two-step complement-dependent lymphocytotoxicity test was used against a panel of 12 selected donors carrying the most frequently occurring HLA class I antigen s. Statistical analysis
For comparison of population means, Students t-test for unpaired observations was used. As the number of PC required is dependent on patient’s blood volume (b.v.) standardization for this factor was undertaken using the formula:
Platelets/b.v./day
Number of platelets transfused =
b.v. (L) x observed days
Fresh versus stored platelets Number of platelets transfused is the sum of platelets in all P C transfused per patient during the study. In 34% the actual number of platelets in the PC was not known, then the mean value ( = 465 x lo9) was used. We estimated the blood volume by the formulas 5.51 x h + 0.0437 x w - 7.12 (for men), 2.37 x h + 0.0455 x w - 2.25 (for women) and 0.08 x w (for children; 2-15 yr of age) where h = height in m and w = weight in kg. The patients were observed for 60 d after BMT. Nine patients died within this period and they were observed until death. We used linear regression analysis to see what factors influenced on the PC requirement. A multiple regression analysis was carried out, using PC storage time on the number of PC transfusions (after standardization for patient's size and observed days), controlling for different background factors. The background factors included in the multiple analysis were chosen because oftheir own correlation (p < 0.2) to the need for platelet transfusions. Some of the patients needed G C during the aplastic phase after BMT. Since a G C contains an amount of fresh platelets comparable with a fresh PC, we chose to account for both PC transfusions and the sum of PC and G C transfusions. Post-transfusion corrected count increment (CCI) at 1 h (CCI,,,) and the following day (CCI,8,,; 1224 h after PC transfusion) were determined using the formula:
CCI
Patient platelet count increment ( i09/i) x BSA(m2) =
Number of platelets in the PC (10")
BSA = Body Surface Area
Table 1 , Comparison of factors that may influence on the need of platelet transfusion after BMT in patients receiving fresh and stored platelets
Time to engraftment (days)' GVHD grade 0-11 Ill-IV CMV-infection Sepsis No. patients on Amphotericin B medication No. patients who died 1 6 0 days after B M l RBC total number units/patient No. patients receiving GC No. patients with lymhocytotoxic antibodies during the study
Fresh platelets
Stored platelets
15k3 20 1 1 11 12 5 5.4k3.7 4
1724ns 16 2 3 7 9 4 7.9k7.3"' 5
2
1
* WBC> 0.2X 109/1 " No significant difference (p>O.O5)
Patients transfused with fresh PC received a mean of 22.1 17.8 x lo9 platelets/b.v./d and those receiving stored PC 29.2 19.6 x lo9 platelets/b.v./d. The difference was insignificant (p = 0.24) (Table 2). When the number of platelets in G C was included, the corresponding figures were 29.3 29.1 x lo9 and 35.8 & 27.6 x lo9, respectively (n.s.). Patients who received fresh platelets required PC or P C + G C transfusions for 26 & 15 d after BMT, the corresponding time for the group who received stored platelets was 32 17 d (n.s.). See Table 2. The aim of providing patients in the respective groups with fresh/stored PC, was not fully achieved. We do not prepare apheresis platelets on Saturdays and Sundays at Huddinge Hospital, so if a patient randomized to receive fresh PC needed PC transfusions on a Sunday or Monday morning the PC transfused had been stored for 2-3 d. Of the 174 PC given to the patients receiving fresh PC, 67% were
Results
Conditions that may influence the amount of platelet transfusions required, such as time to engraftrnent, septicemia, use of amphotericin B, CMV infection and graft-versus-host disease (GvHD) were similar in patients receiving fresh and stored platelets (Table 1). The amount of RBC and granulocyte transfusions needed was also the same in both groups. In the group receiving fresh platelets, 5 patients died in the 60-d post-BMT period, i.e. at 11, 21, 28, 40 and 57 d after BMT. One patient died of cerebral hemorrhage; the peripheral platelet count was 52 x 10y/lthe day she died, and she had received her last PC transfusion 10 d earlier. Four patients died within 60 d post-BMT in the group receiving stored PC, i.e. at 30,3 1, 52 and 53 d after BMT.
Table 2. Comparison of platelet transfusion requirements in 8MT patients receivingfresh and stored PC (MeanfSD)
Fresh
Stored
platelets
platelets
21
18
PC storage time (days)
1.12k 1.25
2.67&1.30**
PC/patient total Platelets/b.v,/day ( X lo9]
7.9k3.7 22.1k17.8
13.9&10.5" 29.2f 19.6"s
(PCtGCl/patient, total Platelets in (PCtGC)/ b.v./day ( X 10')
10.3k9.2
l6.9f 14.2"'
29.3t29.1
35.8 f 27.6" '
Days after 8MT to last PC transfusion
26f15
32k17ns
No. of patients
~
** Significant (p
