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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3605-3612
TRANSFUSION MEDICINE
Effect of leukocyte compatibility on neutrophil increment after
transfusion of granulocyte colony-stimulating factor-mobilized
prophylactic granulocyte transfusions and on clinical outcomes after
stem cell transplantation
Douglas R. Adkins,
Lawrence T. Goodnough,
Shalini Shenoy,
Randy Brown,
Jennifer Moellering,
Hanna Khoury,
Ravi Vij, and
John DiPersio
From the Department of Internal Medicine, Division of Bone Marrow
Transplantation and Stem Cell Biology; the Department of Pathology,
Division of Laboratory Medicine; and the Department of Pediatrics,
Division of Hematology/Oncology, Washington University School of
Medicine, St Louis, MO.
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Abstract |
The primary limitations of granulocyte transfusions
include low component cell dose and leukocyte incompatibility.
Component cell dose improved with granulocyte colony-stimulating factor (G-CSF) mobilization, and the transfusion of
G-CSF-mobilized, human leukocyte antigen (HLA)-matched
granulocyte components resulted in significant, sustained absolute
neutrophil count (ANC) increments. However, the effect of leukocyte
compatibility on outcomes with G-CSF-mobilized granulocyte
transfusions is unclear. The objectives were to determine the effect of
leukocyte compatibility on ANC increments and selected clinical
outcomes after transfusion of prophylactic, G-CSF-mobilized
granulocyte components into neutropenic recipients of autologous
peripheral blood stem cell (PBSC) transplants. Beginning on transplant
day 2, 23 evaluable recipients were scheduled to receive 4 alternate-day transfusions of granulocyte components apheresed from a
single donor given G-CSF. G-CSF was also given to recipients after
transplantation. Recipient ANC was determined before and sequentially
after each granulocyte transfusion to determine the peak ANC increment.
Leukocyte compatibility was determined at study entry only by a
lymphocytotoxicity screening assay (s-LCA) against a panel of
HLA-defined cells. Eight recipients had positive s-LCA. On
days 2 and 4, the mean peak ANC increments after granulocyte
transfusion were comparable between the cohorts with positive and
negative s-LCA. However, the mean peak ANC increments on day 6 (246/µL vs 724/µL; P = .05) and day 8 (283/µL vs
1079/µL; P = .06) were lower in the cohort with positive
s-LCA, in spite of the transfusion of comparable component cell doses.
Adverse reactions occurred with only 5 of 87 (5.7%) granulocyte
transfusions and were not associated with leukocyte compatibility test
results. Platelet increments, determined 1 hour after granulocyte
transfusion, were comparable between the cohorts. Although the 2 cohorts received PBSC components with similar CD34+ cell
doses, the cohort with a positive s-LCA had delayed neutrophil engraftment and a greater number of febrile days and required more days
of intravenous antibiotics and platelet transfusions. Leukocyte
incompatibility adversely affected ANC increments after the transfusion
of G-CSF-mobilized granulocyte components and clinical outcomes after
PBSC transplantation.
(Blood. 2000;95:3605-3612)
© 2000 by The American Society of Hematology.
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Introduction |
Granulocyte transfusions improved the survival rates of
neutropenic dogs with Pseudomonas sepsis.1 In
preclinical models, granulocyte component cell dose was a critical
determinant of neutrophil increments and of survival.1
Leukocyte incompatibility also affected outcomes, resulting in poorer
absolute neutrophil count (ANC) increments and reduced granulocyte
migration and function.2-4 In a meta-analysis of randomized
human trials, both component cell dose and leukocyte compatibility were
important determinants of clinical efficacy of granulocyte
transfusions.5 In human trials of granulocyte components
collected without granulocyte colony-stimulating factor (G-CSF)
mobilization, leukocyte incompatibility resulted in poorer ANC
increments and reduced granulocyte migration and
function.6-8 Unfortunately, the development of
alloimmunization after granulocyte transfusions was
common.9,10
The benefit of G-CSF to mobilize granulocytes includes increased
component cell dose by a factor of 2- to 5-fold over
corticosteroids.11 In addition, G-CSF inhibits apoptosis
and prolongs survival of neutrophils in vitro12 and, thus,
may further sustain neutrophil increments after transfusion. We and
others11,13,14 demonstrated that the transfusion of
G-CSF-mobilized, human leukocyte antigen (HLA)-matched granulocytes
resulted in significant and sustained ANC increments. Mean peak ANC
increments between 631/µL and 1195/µL were observed after
granulocyte transfusions administered to recipients of
non-alloimmunized neutropenic bone marrow transplants.13 The duration of post-transfusion recipient mean ANC at or above the
baseline (before transfusion) value was 25 to 37 hours.13 Such granulocytes were also functional, as demonstrated by the localization of indium-labeled granulocytes to sites of inflammation for at least 48 hours after transfusion.15
Granulocyte component cell dose improved with the use of G-CSF as the
mobilization agent; however, the effect of leukocyte compatibility on
outcomes with G-CSF-mobilized granulocyte transfusions is unclear. The
objectives of this trial were to determine the effect of leukocyte
compatibility on ANC increments and clinical outcomes after the
transfusion of prophylactic G-CSF-mobilized granulocyte components
into neutropenic recipients of autologous PBSC transplants. For each
recipient, 4 granulocyte components were to be collected from a single
donor. Granulocyte components were transfused into recipients on
transplant days 2, 4, 6, and 8. Leukocyte compatibility between donor
and recipient was determined by lymphocytotoxicity assays, HLA-A and
HLA-B typing, and leukoagglutination cross-match. The results of this
trial are reported herein.
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Patients and methods |
Donor and recipient eligibility
The protocol was approved by the Institutional Review Board of
Washington University School of Medicine and accrued 25 donor-recipient pairs between March 1997 and July 1998. Informed
consent was required for entry of recipients and donors into the study.
Eligibility criteria for donors included age greater than 15 years, ANC
1500/µL or greater, platelet count 100 000/µL or greater,
hematocrit 30% or greater, and, if female, negative pregnancy test
result. Every donor was ABO-compatible with, and a first-degree
relative (biologic sibling, parent, or adult offspring) of, a
recipient. Otherwise, donors satisfied standard blood donation criteria
as determined by the U.S. Food and Drug Administration and as
interpreted by the Blood Bank director. Recipients between the ages of
15 and 70 years who were undergoing autologous PBSC transplantation for malignant disease were eligible. Stored autologous PBSC products for
each recipient had to contain a minimum of 2 × 106
CD34+ cells/kg actual body weight for subsequent
transfusion. The PBSC products of 1 recipient contained
1.4 × 106 CD34+ cells/kg, but the recipient
was permitted to participate in this protocol. Recipients had to be
free of active infection at study entry.
Prestudy evaluation and leukocyte compatibility tests
Within 7 days of initiation of the transplant-conditioning regimen,
donors and recipients underwent baseline history taking and physical
examination. Blood was obtained for complete blood count with
differential, ABO and Rh typing, and infectious serology testing, as
previously reported.13 Type and cross of donor and recipient blood were performed to confirm ABO compatibility.
At study entry only and before the conditioning regimen was initiated,
leukocyte compatibility between donor and recipient was determined by 4 methods. HLA-A and HLA-B typing of donor and recipient were performed
using a standard serologic typing technique,16 and a match
grade was assigned from 0 (0 of 4 HLAs matched) to 4 (4 of 4 HLAs
matched). A screening lymphocytotoxicity assay (s-LCA) was performed on
each recipient. Serum samples from each of the recipients were tested
for class I and II HLA antibodies against T- and B-cell targets by
standard microlymphocytotoxicity testing with a panel of 24 to 30 cells.17 Recipient serum was added to a standard panel of
screening cells and incubated to allow antigen-antibody interaction.
Rabbit serum was added to the cell-serum suspension as a source of
complement. Cell membrane injury that resulted from an interaction
between antibody-bound cells and complement was visualized by the
cells' inability to exclude a vital dye (ethidium bromide;
Mallinckrodt, Paris, KY), and the percentage of cell death was scored
from 1 (cell viability same as control) to 8 (essentially all cells
killed). Scores of 6 or greater indicated the presence of an HLA
antibody. Panel reactivity was calculated as percent reactive antibody:
number of positive panel cells divided by total number of panel cells times 100. Positive s-LCA was defined as percent reactive antibody greater than or equal to 10%. The sensitivity of the technique was
enhanced by using the Amos 3-wash method and by the addition of
anti-human globulin before the addition of complement.18 Appropriate positive and negative controls were concurrently performed. Using the same methods, a lymphocytotoxicity cross-match assay (c-LCA)
between recipient serum and donor cells was performed and scored as
positive or negative. A leukoagglutination cross-match was performed by
standard methods using recipient serum and granulocyte-enriched cell
suspension obtained from defibrinated blood of the donor.19 Appropriate positive and negative controls were concurrently performed. Reactions were scored based on clumping according to the following scale: 0 or negative (no visible microscopic agglutination), 1+ (10%
to 20% agglutination), 2+ (30% to 40% agglutination), 3+ (50% to
80% agglutination), and 4+ (90% agglutination).
Study design
To mobilize granulocytes, donors were given G-CSF at 10 µg/kg
subcutaneously 8 to 12 hours before each of 4 scheduled granulocyte apheresis collections. Leukapheresis of the donor began the evening (5 PM) of post-transplant day 1 and was repeated the mornings (9 AM) of days 4, 6, and 8. Venous access for each
apheresis procedure was by peripheral vein-to-peripheral vein
technique. Granulocyte collections were performed with an automated
continuous-flow blood cell separator by means of Standard Procedure 2 (CS-3000 Plus; Baxter, Deerfield, IL) modified by the use of an
interface offset setting of 35.20 For each collection, 7 L
was processed by continuous-flow centrifugation at a rate of
approximately 50 mL per minute. Clotting was prevented and red cell
sedimentation was facilitated by the use of sodium citrate (30 mL of
46.7% trisodium citrate) in 500 mL of 6% hydroxyethylstarch (McGraw
Laboratories, Glendale, CA) to the donor line at a ratio of 1 part
anticoagulant to 10 to12 parts blood.
To evaluate the cellular composition of each leukapheresis component,
the white blood cell (WBC) count and differential were determined on a
3-mL sample, by using a counter (Coulter, Hialeah, FL). The total
number of granulocytes in each leukapheresis component was calculated
on the basis of these data and the total component volume. Each
component was irradiated (2500 cGy, 137 Cs source) before transfusion
to the recipient, to prevent transfusion-associated graft-versus-host disease.
Donors were evaluated daily for any side effects caused by either the
G-CSF or the leukapheresis procedures. Donor monitoring, toxicity
grading, and criteria for removal of donors from the study were
previously reported.13 On the day of each scheduled leukapheresis, the donor's hematocrit and platelet count had to be
30% or greater and 75 000/µL or greater, respectively, for the
donor to be eligible for the procedure.
Recipients received transplant conditioning regimens consisting of
high-dose multi-agent chemotherapy only (n = 15 patients) or with
total body irradiation (n = 10 patients) given over 4 to 6 days,
followed by infusion of thawed autologous PBSC products on transplant
day 0. Beginning 4 hours after PBSC transplantation, each recipient
received G-CSF at 5 µg/kg subcutaneously and given daily until the
ANC recovered to greater than or equal to 1500/µL for 3 consecutive days.
Within 10 hours of completion of the first leukapheresis procedure and
within 2 hours of completion of subsequent leukapheresis procedures,
the apheresis components were transfused into the recipient over the
course of 1 hour. An extended interval between completion of
leukapheresis and component transfusion was required for the first
procedure to permit the completion of infectious serology tests on the
component to determine whether it was acceptable for transfusion
according to American Association of Blood Banks' (AABB)
guidelines.21 Apheresis components collected on subsequent days were transfused promptly if results of infectious serology tests
on the immediately prior component were acceptable, as recommended by
AABB guidelines.21 Based on this schedule, granulocyte
components were transfused into recipients on day 2 in the early
morning and on days 4, 6, and 8 in the early afternoon. The
recipient's ANC and platelet count were determined immediately before
each component transfusion (the baseline) and then at 1, 4, 8, 12, 24, 36, and 48 hours after each component transfusion.
Routine medications were given to recipients 15 to 30 minutes before
each granulocyte component transfusion to prevent transfusion reactions
(fever and hives); they included acetaminophen (650 mg by mouth) and
diphenhydramine (25 mg by mouth or intravenously [IV]). During and
after each granulocyte transfusion, recipients were observed and vital
signs were monitored for any evidence of acute toxicity resulting from
the transfusion. If fever (temperature 38.3°C or higher) or
respiratory symptoms (cough, shortness of breath, or chest tightness)
developed during or within 1 hour of a transfusion, hydrocortisone (100 mg IV) was also given to that recipient before subsequent transfusions.
If fever or respiratory symptoms developed with 2 transfusions and
after the addition of hydrocortisone, no further transfusions were
given. Respiratory distress requiring supplemental oxygen (caused by a
decline in O2 saturation to less than 90%) or hypotension
(systolic blood pressure less than 90 mm Hg) during or within 1 hour of
a transfusion was also an indication not to administer subsequent
scheduled components.
Supportive care
Prophylactic antibacterial and antifungal agents were not given to
recipients. Infection was presumed in recipients with fever (temperature 38.3°C or higher) when the ANC was less than
1000/µL, and it was managed initially with empiric vancomycin (1 g IV
bid) and cefepime (1 g IV tid) after evaluation (by examination, blood cultures, and other indicated tests), followed by amphotericin B (0.5 mg/kg per day) if febrile neutropenia persisted or recurred after 3 or
more days of antibacterial therapy. Amphotericin B was administered 8 to 12 hours before or after the transfusion of granulocyte
components to reduce the likelihood of adverse pulmonary reactions
reported when the 2 were given concurrently.22 Documented
bacteremia, tissue (eg, pneumonia), or catheter-related infections were
treated with a 14-day course of antimicrobials (minimum, 7 days, IV).
Recipients received prophylactic irradiated, WBC-filtered, single-donor
platelet concentrate transfusions if the platelet count was less than
10 000/µL and 2 U WBC-filtered packed red cells if the hemoglobin
level was less than 8.0 g/dL. Blood counts with differentials and
platelet counts were performed daily during the hospital stay and BIW
afterward until the platelet count was greater than 50 000/µL after transplantation.
Study endpoints and statistical analysis
The primary objective of this phase 2 study of 25 donor-recipient
pairs was to determine the effect, if any, of leukocyte compatibility
on peak ANC increments (over baseline) after the transfusion of
prophylactic G-CSF-mobilized granulocyte components into neutropenic
recipients of autologous PBSC transplants. Because apheresed
granulocyte components contain large numbers of
platelets,13 the absolute and corrected platelet count
increments (CCI) were also determined 1 hour after the granulocyte
transfusions, and the effect of leukocyte compatibility on these
outcomes were assessed. CCI was calculated according to the following
formula23: CCI = [absolute increment × body surface
area (m2)] / [number of transfused platelets × 1011].
Secondary endpoints of the study were to determine the effect of
leukocyte compatibility on selected clinical outcomes in PBSC
recipients including the number of febrile days, platelet transfusion
requirements, hematologic recovery, and adverse granulocyte transfusion
reactions (including fever, respiratory distress, and hypotension).
The kinetics of the recipient ANC and platelet count with transfusions
of G-CSF-mobilized granulocyte components on transplant days 2, 4, 6, and 8 were assessed by descriptive statistics (mean, range), as were
clinical outcomes. Comparisons between groups of data were performed
using either an unpaired t test or a Mann-Whitney U
test, as appropriate, with P = .05 defined as significant.
Statistical analysis was performed using a software program
(Statview SE + Graphics; Abacus Concepts, Berkeley, CA).
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Results |
Donor and recipient characteristics
Twenty-five donor-recipient pairs were accrued to this study, of
which 2 were nonevaluable because of poor venous access and the
inability to perform apheresis in 1 donor and a side effect of G-CSF in
1 donor. An anaphylactoid reaction developed in the latter donor within
1 hour of the first dose of G-CSF and has been reported in detail
elsewhere.24 The donor recovered without complications and
did not undergo apheresis or receive additional G-CSF injections. The
relationship of donor to recipient was sibling in 12 pairs, adult
offspring in 11, and parent in 2. Donors' mean age and weight were 34 years (range, 16 to 57 years) and 84.3 kg (range, 56.8 to 133.3 kg),
respectively, and the male-to-female ratio was 14:11. Recipients'
diagnoses included lymphoma (11 patients), breast cancer (9 patients),
myeloma (4 patients), and acute leukemia (1 patient). Recipients' mean
age and weight were 47 years (range, 19 to 64 years) and 79.8 kg
(range, 54.9 to 116.9 kg), respectively, and the male-to-female ratio
was 11:14.
Donor response to G-CSF and leukapheresis
Of the 23 evaluable donors, the G-CSF doses to be given on
transplant days 1, 3, 5, and 7 were administered to 23, 23, 22, and 20 donors, respectively. Four scheduled doses of G-CSF were not
administered to 3 donors as a result of a decision to discontinue subsequent scheduled granulocyte collections because of the development of adverse transfusion reactions in 3 recipients with prior infusions of granulocyte components. Of the 23 evaluable donors, the
leukapheresis procedures scheduled to occur on days 1, 4, 6, and 8 were
performed in 22, 23, 22, and 20 donors, respectively. Five scheduled
leukapheresis procedures were not performed either because of adverse
transfusion reactions in recipients with prior infusions of granulocyte
components (4 patients) or with a red blood cell component given just
before a scheduled granulocyte transfusion (1 patient).
After a single dose of G-CSF, the mean donor ANC increased from
3770/µL (2072 to 8458/µL) at baseline to 14 764/µL (3568 to 22 081/µL) just before the first leukapheresis on day 1. The mean donor ANC before the subsequent leukapheresis procedures increased from
27 785/µL (16 687 to 47 830/µL) on day 4; to 30 748/µL
(15 333 to 52 628/µL) on day 6; to 45 384/µL (26 396 to
64 490/µL) on day 8. The mean donor platelet count decreased from
215 000/µL (158 000 to 312 000/µL) at baseline to 135 000/µL
(102 000 to 231 000/µL) before the fourth scheduled leukapheresis
on day 8. The mean donor hemoglobin remained stable throughout (data
not shown).
Cellular composition of granulocyte components
The granulocyte cell dose of components increased with each
successive leukapheresis procedure (Table
1). The mean granulocyte cell dose
(×1010) of components collected on day +1 and
transfused on day +2 was 5.6 (1.9-9.3), whereas the component cell dose
collected and transfused on day +8 was 9.9 (1.8-16.5). The platelet
dose of granulocyte components decreased with each successive
leukapheresis procedure (Table 2). The mean
component platelet dose (× 1011) was 4.1, 3.3, 2.7, and
2.4 in components collected on days +1, +4, +6, and +8, respectively.
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Table 2.
Effect of lymphocytotoxicity screening assay on platelet
increments 1 hour after granulocyte transfusions
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ANC increments after granulocyte transfusions
ANC increments (over baseline) observed in the 23 evaluable
recipients after transfusions of G-CSF-mobilized granulocyte
components are shown in Table 1. Overall, the mean ANC increments were
of borderline significance and were sustained for a short term after each of the 4 scheduled days of granulocyte transfusions. For the
entire group, the peak mean ANC increment was 1191/µL at hour 4 after
transfusion on day 2; 477/µL at hour 8 after transfusion on day 4;
462/µL at hour 8 after transfusion on day 6; and 616/µL at hour 1 after transfusion on day 8. The ANC increments observed at 36 and 48 hours after transfusion of the granulocyte components on day 8 most
likely represented endogenous recovery of neutrophils rather than
increments resulting from the granulocyte transfusions.
For each recipient, the peak ANC increment occurred at different
intervals after the granulocyte transfusions. Thus, when considering
this variable, the mean peak ANC increment for the entire group was
1410/µL (109 to 3551/µL) on day 2; 541/µL (0 to 1561/µL) on day
4; 537/µL (0 to 2895/µL) on day 6; and 734/µL (0 to 4100/µL) on
day 8 (Table 1). In comparison with peak ANC increments observed after
the transfusion of granulocyte components on day 2, granulocyte
transfusions administered on subsequent days resulted in lower peak ANC
increments in spite of the transfusion of larger granulocyte component
cell doses.
Leukocyte compatibility
Table 3 summarizes the results of the
leukocyte compatibility tests. Eight of the 23 recipients had a
positive s-LCA, whereas only 1 of 23 recipients had a positive c-LCA
against donor cells. In the leukoagglutination cross-match, no clumping
was observed in the 20 evaluable tests. HLA-A and HLA-B typing
demonstrated that 19 of 23 donor-recipient pairs were 2 of 4 antigen
matches. Only 1 donor-recipient pair was an HLA 4/4 antigen match.
Given the results of the leukocyte compatibility tests, the effect of leukocyte compatibility on ANC increments after the transfusion of
G-CSF-mobilized granulocyte components was analyzed based on a
positive or negative s-LCA only. With each of the other 3 tests of
leukocyte compatibility, similar analysis could not be performed because of the skewing of most or all data points into 1 potential outcome for each test.
Effect of s-LCA on ANC increments after granulocyte transfusions
The relationship between the results of the s-LCA and the mean peak
ANC increment after transfusions of G-CSF-mobilized granulocyte components is shown in Table 4. On days 2 and 4, the mean peak ANC increments were comparable between the patient
cohorts with a positive and a negative s-LCA. However, on days 6 and 8, the mean peak ANC increments after granulocyte transfusion were lower in the cohort with a positive s-LCA in comparison with the cohort with
a negative s-LCA. These differences were of borderline significance (P = .05 and P = .06 for days 6 and 8, respectively). The poorer peak ANC increments observed on the latter 2 days of granulocyte transfusions in the cohort with a positive s-LCA
(day 6, 246 vs 724/µL, P = 0.05; day 8, 283 vs 1079/µL,
P = .06) occurred even though comparable granulocyte
component cell doses were administered to the 2 cohorts.
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Table 4.
Effect of lymphocytotoxicity screening assay on mean
peak ANC increment after transfusions of G-CSF-mobilized granulocyte
components
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Effect of s-LCA on platelet increments after granulocyte
transfusions
The granulocyte components contained approximately the number of
platelets present in a unit of single-donor apheresed platelet components and resulted in significant platelet increments after transfusion (Table 2). Within the limits of the study design, a
positive s-LCA did not predict for poorer absolute platelet count
increments or CCI at 1 hour after transfusion with granulocyte components administered on days 2, 4, 6, or 8. On each of the 4 days of
granulocyte transfusions, comparable absolute platelet increments and
CCI occurred 1 hour after transfusion between the recipients with a
positive s-LCA and those with a negative s-LCA (Table 2). The platelet
doses in granulocyte components were not significantly different
between these 2 patient cohorts.
Adverse reactions to granulocyte transfusions
Adverse reactions to granulocyte transfusions occurred in 3 of the
23 evaluable recipients. Each adverse reaction resolved without serious
or lasting complications. In one recipient, febrile reactions developed
with granulocyte transfusions given on days 2 and 4, and subsequent
scheduled granulocyte transfusions were withheld. In another recipient,
febrile reactions and hives developed with granulocyte transfusions
given on days 4 and 6, and the subsequent scheduled granulocyte
transfusion was withheld. Respiratory distress (shortness of breath,
wheezing) developed in 1 patient, who required supplemental oxygen for
low O2 saturation (89%) with the granulocyte transfusion
given on day 6, and the transfusion scheduled on day 8 was withheld.
Thus, of the 87 granulocyte transfusions administered, 5 (5.7%)
were associated with adverse reactions.
There was no apparent association between the results of leukocyte
compatibility tests and the development of adverse reactions to
granulocyte transfusions. Of the 3 recipients in whom adverse transfusion reactions developed, none had a positive s-LCA. Of the 20 recipients in whom adverse transfusion reactions did not develop, 8 had
a positive s-LCA. All 3 recipients with adverse transfusion reactions
were matched with the donor at 2 of the 4 HLA loci and had negative
results in the c-LCA and leukoagglutination assays.
Effect of s-LCA on hematologic recovery and clinical outcomes
Hematologic recovery was delayed in the recipients of granulocyte
transfusions with a positive s-LCA, in spite of transplantation of PBSC
components with comparable CD34+ cell doses between the
cohorts with a positive and a negative s-LCA (Table
5). The terminal portion of neutrophil
recovery was significantly longer in recipients with a positive s-LCA
than in those with a negative s-LCA, a finding that could not be
explained by differences in duration of G-CSF administration. The mean
duration to achieve an ANC greater than 1000/µL and an ANC greater
than 1500/µL was 5.7 and 7.2 days longer in recipients with a
positive s-LCA. G-CSF was administered for 17 (range, 12 to 30) days
and 14.2 (range, 11 to 18) days in the recipients with a positive s-LCA
and a negative s-LCA, respectively (P = .10). The absolute mean number of days to platelet recovery was longer in recipients with
a positive s-LCA than in those with a negative s-LCA, though the
difference was not statistically significant.
Between days 2 and 10, the interval during which granulocyte
transfusions were administered, the number of febrile days was significantly greater in the recipients with a positive s-LCA (Table
5). During this interval, recipients with a positive s-LCA experienced
a mean of 6.3 (range, 5 to 8) febrile days compared with 4.1 (range, 0 to 6) febrile days in recipients with a negative s-LCA
(P = .01). Recipients with a positive s-LCA also required more days of IV antibiotics and amphotericin B and more platelet transfusions than those with a negative s-LCA (Table 5). Between day 2 and neutrophil engraftment, recipients with a positive s-LCA received
10.5 (range, 8 to 13) days of IV antibiotics compared with 7.3 (range,
0 to 11) days of IV antibiotics for recipients with a negative s-LCA
(P = .01). This difference could not be explained by a
greater proportion of the positive s-LCA cohort on antibiotics at the
initiation of granulocyte transfusions on day 2 because the number of
days on IV antibiotic administration from the start of the preparative
regimen until day 1 was similar between the 2 cohorts. Three patients
received amphotericin B after transplantation, and all doses were given
between days 6 and 11. The number of days of amphotericin B
administered to the positive s-LCA cohort was 1.3 (range, 0 to 5 days),
whereas the negative s-LCA cohort did not require amphotericin B
(P = .02). The numbers of single-donor apheresed platelet
transfusions administered between day 2 and the day of platelet
engraftment were 7.6 (range, 1 to 18 days) and 3.4 (range, 0 to 10 days) in recipients with a positive and a negative s-LCA, respectively
(P = .05).
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Discussion |
In preclinical models and human trials, the primary limitations of
granulocyte transfusions included low component cell dose and leukocyte
incompatibility.25 Compared with other agents, mobilization
of granulocyte components with G-CSF improved component cell
dose.11 In addition, transfusion of HLA-matched and
G-CSF-mobilized granulocyte components into non-alloimmunized,
neutropenic recipients resulted in significant, sustained ANC
increments.11,13,14 It can be speculated that the
transfusion of G-CSF-mobilized granulocyte components with large cell
doses may result in significant ANC increments in recipients with
preformed leukocyte antibodies by a saturation effect or through
some as yet unknown mechanism. However, the effect of leukocyte
compatibility on outcomes with G-CSF-mobilized granulocyte
transfusions is unclear.
In this study, the presence of HLA antibodies in recipient serum at
baseline (before transfusion), as determined by a positive s-LCA,
predicted for poorer peak ANC increments with the latter 2 of 4 G-CSF-mobilized granulocyte transfusions administered on alternate
days beginning day 2 to patients who underwent autologous PBSC
transplantation. On days 2 and 4, the mean peak ANC increments after
granulocyte transfusion were comparable between the cohorts with a
positive and a negative s-LCA; however, on days 6 and 8, the mean peak
ANC increments after granulocyte transfusion were lower in the cohort
with a positive s-LCA. This observation should be confirmed in a larger
study, given that the latter differences in peak ANC increments were of
borderline statistical significance and that the sample size of the
study was small. Previous studies in humans demonstrated an adverse
effect of alloimmunization on outcomes with granulocyte transfusions
collected at steady state or after mobilization with agents other than
G-CSF. In the presence of preformed leukocyte antibodies directed
against donor cells, Graw et al26 and Goldstein et
al8 observed significantly lower WBC increments and
recoveries after transfusion into neutropenic recipients of granulocyte
components obtained from chronic myelogenous leukemia donors.
McCullough et al15 observed a significant reduction in the
intravascular recovery of normal donor granulocytes transfused into
recipients with preformed anti-leukocyte antibody and noted that
incompatible granulocytes failed to localize to sites of infection. The
latter finding was confirmed by Dutcher et al7 in a study
of larger numbers of patients. These outcomes in human trials of
granulocyte transfusions mirrored those observed in animal studies, in
which the presence of alloimmunization also resulted in poorer ANC
increments, reduced granulocyte migration, shortened recipient survival
time, and increased frequency of infection.2-4,27 Our
observations support the hypothesis that the adverse effect of
leukocyte incompatibility on outcomes with granulocyte transfusions
applies also to components mobilized with G-CSF. Within the range of
cell doses (1.3 to 16.5 × 1010) transfused in this
study, it appears unlikely that the larger granulocyte component cell
doses that occur with G-CSF mobilization can overcome the limitation of
leukocyte incompatibility. Although even larger component cell doses
may effectively address the problem of leukocyte incompatibility, a
collection of such components would require higher doses of G-CSF, the
addition of other mobilization agents, or more effective techniques to
collect granulocytes. These requirements may expose donors to greater
risks or more side effects. A more practical strategy may be to select
leukocyte-compatible donors or to focus further research of
G-CSF-mobilized granulocyte transfusions on patients who are not alloimmunized.
Preclinical studies clearly established that to ensure the survival of
neutropenic dogs with Pseudomonas sepsis, transfused granulocyte components must contain a minimum cell dose, 1 of which
resulted in significant ANC increments after transfusion.1 Lower component cell doses resulted in no ANC increments and no clinical benefit. These data support the notion that significant post-transfusion ANC increments may be a key condition of demonstrating reproducible improvements in clinical outcomes with granulocyte transfusions in humans and that variables that reduce the ANC increment
would likely adversely influence the efficacy of granulocyte transfusions. Our observations of an adverse effect of leukocyte incompatibility on post-transfusion peak ANC increments may have implications regarding the design and analysis of clinical studies attempting to determine the efficacy of G-CSF-mobilized granulocyte transfusions.
Neutropenic recipients of autologous PBSC transplants transfused with
G-CSF-mobilized granulocyte components on alternate days over 1 week
had significant peak ANC increments after transfusion if the baseline
recipient serum s-LCA and c-LCA were negative, even when the donor and
recipient were HLA mismatched. Recipients with a baseline serum
positive s-LCA and negative c-LCA had significant peak ANC increments
after G-CSF-mobilized granulocyte transfusions given on days 2 and 4;
however, poor peak ANC increments occurred in the same recipients after
granulocyte transfusions given on days 6 and 8. The mechanism of this
observation is unclear, but it may be explained by the rapid
development of donor leukocyte-specific alloimmunization in recipients
with a baseline serum positive s-LCA that did not occur in recipients
with a negative baseline s-LCA. Comparable peak ANC increments in the
positive and the negative s-LCA cohorts after granulocyte transfusions
given on days 2 and 4 likely occurred because only 1 of the 23 evaluable recipients had a positive c-LCA at baseline against donor
cells. If this hypothesis is supported by the results of future planned studies, in which recipient serum samples will be serially monitored after transfusion, the presence of a baseline positive s-LCA and a
negative c-LCA appeared to predict for rapid development of donor
leukocyte-specific alloimmunization after transfusion of G-CSF-mobilized granulocyte transfusions. Conversely, alloimmunization after granulocyte transfusion was unlikely to develop in recipients with negative s-LCA and c-LCA, at least in the 1-week interval during
which the transfusions were administered. Our results do not address
whether such recipients would later demonstrate evidence of
donor-specific alloimmunization. However, results of small studies in which recipients were given either prophylactic or therapeutic granulocyte transfusions mobilized with dexamethasone found
that alloimmunization developed frequently and was associated with the
number of prior granulocyte transfusions.9,10
A number of laboratory methods have been used to assess
leukocyte compatibility between granulocyte donors and
recipients.28These methods include lymphocytotoxicity
assays against donor cells or against a panel of HLA-defined cells,
leukoagglutination cross-match, granulocyte-specific antibody assays,
and HLA typing. Several studies examined the effect of each method on
outcomes, with granulocyte transfusions mobilized with agents other
than G-CSF, and reported mixed and occasionally contradictory results. If the baseline recipient lymphocytotoxicity cross-match and
leukoagglutination assays were negative, Graw et al26
observed poorer WBC recovery after granulocyte transfusion in
recipients who were HLA disparate with donors. Goldstein et
al8 observed an adverse effect of preformed leukocyte
antibodies as determined by leukoagglutination and screening
lymphocytotoxicity assays on outcomes after granulocyte transfusions.
Dutcher et al7 demonstrated that alloimmunization, as
defined by a positive lymphocytotoxicity cross-match with the donor, a
positive leukoagglutination assay, or both, prevented the migration of
transfused granulocytes to sites of infection. In contrast to these
reports, McCullough et al6 found that granulocyte
agglutinating antibodies, but not granulocytotoxic or lymphocytotoxic
antibodies, adversely affected transfused granulocytes. In our study,
though we observed a significant relationship between the results of
the baseline recipient s-LCA and peak ANC increments after
G-CSF-mobilized granulocyte transfusions, we were unable to further
elucidate the effect, if any, of the results of the baseline recipient
c-LCA, leukoagglutination assay, or donor-recipient HLA disparity.
Determination of which method or combination of methods more accurately
predicts leukocyte compatibility between donor and recipient is an
important area for future investigation, even with G-CSF-mobilized
granulocyte transfusions.
Compared with granulocyte transfusions, alloimmunization is perhaps
better understood with platelet transfusions. The mechanism of
alloimmunization to platelets usually results from the development of
antibodies to class I HLA antigens in
response to leukocytes contaminating platelet
transfusions.29 Granulocytes express class I, but not class
II, HLA antigens, and several studies provide evidence to support that
antibodies to class I HLA antigens also cause alloimmunization to
granulocyte transfusions.7-10 With this information in
mind, we anticipated in this study that the presence of HLA antibodies
in recipient serum, as determined by a positive s-LCA, would also
result in evidence of alloimmunization against contaminating platelets
transfused in the granulocyte component. However, a positive s-LCA did
not predict for poorer platelet increments at 1 hour after transfusion
of granulocyte components on either of the 4 days they were
administered. After the final scheduled granulocyte transfusion, it is
possible that alloimmunization to platelets occurred more frequently in
the cohort with a positive s-LCA, but this was not prospectively
addressed in this study. In addition, studies with larger numbers of
patients may be required to show a significant effect of the s-LCA
result on alloimmunization against platelets contained in granulocyte components.
Granulocyte transfusions are associated with a number of potential
adverse reactions, including fever, chills, hives, respiratory distress, or hypotension. In several reports, adverse reactions to
granulocyte transfusions were associated with the presence of leukocyte
antibodies.9,10 In this study, adverse reactions to
G-CSF-mobilized granulocyte transfusions occurred in only 3 of the 23 evaluable recipients and in only 5.7% of all granulocyte transfusions
administered. This compares to a prior report in which the frequency of
adverse reactions to G-CSF-mobilized granulocyte transfusions that
were HLA matched was 3.4%.13 In contrast to previous
reports,9,10 we observed no obvious association between the
results of baseline leukocyte compatibility tests and development of
adverse reactions to granulocyte transfusions. However, all recipients
in our study were premedicated with acetaminophen and diphenhydramine.
The frequency of adverse reactions may have been greater if no
premedications had been given.
In this study, the presence of HLA antibodies in recipient serum at
baseline, as determined by a positive s-LCA, predicted for delayed
neutrophil engraftment after autologous PBSC transplantation, in spite
of the transplantation of PBSC components with comparable CD34+ cell doses between the cohorts with a positive and a
negative s-LCA. Because neutrophil engraftment occurred 9 or more days after transplantation and after the final granulocyte transfusion, the
more significant ANC increments observed in the negative s-LCA cohort
with the latter 2 granulocyte transfusions did not confound the
interpretation of neutrophil engraftment in this cohort and cannot
explain our unique observation. The mechanism of the adverse effect of
a positive s-LCA on neutrophil engraftment after autologous transplantation is unclear but may be explained by the positive s-LCA
cohort reflecting a group of patients who were more heavily pretreated.
Such patients would likely have received more prior blood product
transfusions, which may explain why patients in this cohort had
preexisting HLA antibodies. The presence of preexisting HLA antibodies
in the positive s-LCA cohort was also associated with a greater number
of febrile days, days of IV antibiotics, and platelet transfusions
compared with the cohort with a negative s-LCA. Additional studies of
larger numbers of patients should be performed to confirm the adverse
effect of a positive s-LCA on neutrophil engraftment and on clinical
outcomes. If these observations are confirmed, the s-LCA may be used to
risk-stratify patients who undergo autologous PBSC transplantation
based on the likelihood for adverse clinical outcomes with the
transplantation procedure.
The data from this study support the conclusion that leukocyte
incompatibility adversely affects ANC increments after G-CSF-mobilized granulocyte transfusions and clinical outcomes after PBSC
transplantation. Future trials designed to determine the clinical
efficacy of G-CSF-mobilized granulocyte transfusions should consider
the potential adverse effect of leukocyte incompatibility on outcomes.
Strategies effective in reducing the incidence of alloimmunization
after platelet transfusion, such as ultraviolet irradiation, should be
evaluated with G-CSF-mobilized granulocyte transfusions.23
| |
Footnotes |
Submitted October 8, 1999; accepted February 1, 2000.
Supported in part by a research grant from Barnes-Jewish Hospital.
Reprints: Douglas R. Adkins, Department of Internal Medicine,
Division of Bone Marrow Transplantation and Stem Cell Biology,
Washington University School of Medicine, 660 S. Euclid Avenue, Campus
Box 8007, St Louis, MO 63110-1093.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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