Transfusion related acute lung injury in the critically ill: prospective nested case-control

study

Ognjen Gajic1

Rimki Rana1

Jeffrey L. Winters2

Murat Yilmaz1

Jose L. Mendez1

Otis B. Rickman1

Megan M O’Byrne3

Laura K. Evenson1

Michael Malinchoc 3

Steven R DeGoey2

Bekele Afessa1

Rolf D. Hubmayr1

S. Breanndan Moore2

1Division of Pulmonary and Critical Care Medicine 2Division of Transfusion Medicine

3Division of Biostatistics

Mayo Clinic College of Medicine

Rochester, Minnesota

Address correspondence to: Ognjen Gajic, M.D. Mayo Clinic 200 First Street SW Rochester, MN 55905 Telephone: 507-255-6051 Fax: 507-255-4267 E-mail: gajic.ognjen@mayo.edu

This study was supported by NHLBI 1 K23 HL087843-01A1 and the grant from National Blood Foundation, NBF 2004-05 Running Title: TRALI in the Critically Ill Descriptor Number: 2 Word Count: 2,328

This article has an online data supplement, which is accessible from this issue's table of content

online at www.atsjournals.org

AJRCCM Articles in Press. Published on July 12, 2007 as doi:10.1164/rccm.200702-271OC

Copyright (C) 2007 by the American Thoracic Society.

1

ABSTRACT

Rationale: Acute lung injury (ALI) that develops six hours after transfusion (TRALI) is the

leading cause of transfusion-related mortality. Several transfusion characteristics have been

postulated as risk factors for TRALI, but the evidence is limited to retrospective studies.

Objective: To compare patient and transfusion risk factors between the patients who do and do

not develop ALI

Methods: In this prospective cohort study, consecutive transfused critically ill patients were

closely observed for development of ALI. Donor samples were collected from the transfusion

bags. Risk factors were compared between patients who did develop ALI following transfusion

and transfused controls, matched by age, gender and admission diagnosis.

Measurements and main results: Seventy-four out of 901 transfused patients developed ALI

within 6 hours after transfusion (8%). Compared to transfused controls, ALI patients were more

likely to have sepsis (37% vs 22%, p=0.016), and a history of chronic alcohol abuse (37% vs

18%, p=0.006). When adjusted for patient characteristics, transfusion of plasma from female

(odds ratio-OR 5.09, 95%CI 1.37-18.85), rather than male donors (OR 1.60, 95%CI 0.76 to

3.37), number of pregnancies among the donors (OR 1.19, 95%CI 1.05 to 1.34), number of

donor units positive for antigranulocyte (OR 4.85, 95%CI, 1.32-17.86) and HLA class II

antibodies (OR 3.08, 95%CI 1.15-8.25), and the concentration of lysophosphatidylcholine in the

donor product (OR 1.69, 95%CI 1.10 to 2.59) were associated with development of ALI.

Conclusions: Both patient and transfusion risk factors determine the probability of ALI after

transfusion. Transfusion factors represent attractive targets for prevention of ALI

Abstract Word Count 250

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Keywords: Fresh-frozen plasma; Platelet transfusion, Transfusion; Outcome study; Pulmonary edema; Respiratory Distress Syndrome, Acute; Female; Blood Donors

3

INTRODUCTION

Since the original description twenty years ago (1), Transfusion-Related Acute Lung Injury

(TRALI) has emerged as the most important cause of morbidity and mortality resulting from

blood transfusion (2-4), in part due to the decrease in morbidity and mortality from other

adverse effects of transfusion. While the exact etiology is not known, uncontrolled clinical

studies and animal models have suggested an important role of donor derived anti-leukocyte

antibodies as well as biologic response modifiers accumulated during blood storage (5-9). Anti-

leukocyte antibodies (anti HLA class I, class II and antigranulocyte) are known to be commonly

present in the plasma of donors exposed to the foreign tissue during previous pregnancy or

transfusion and have been implicated in multiple case series and case reports (1, 3-5). Indeed,

anti-HLA antibodies have been detected in up to 25% of multiparous women donors (10).

TRALI is thought to be under-diagnosed and under-reported (4, 5, 11), particularly in critically

ill patients who often have multiple risk factors for acute lung injury (ALI). It is possible that

current TRALI cases reported to the blood bank represent the "tip of the iceberg" and that

transfusion factors play a mechanistic role in many more patients with ALI, as one of "multiple

hits" required for the full expression of this syndrome (12-14). Indeed, transfusion (massive) has

long been recognized as one of the most important ALI risk factors (15). Submassive transfusion

of all blood products, and particularly high plasma volume products have been recently identified

as independent risk factors for development of ALI in the critically ill(16, 17). In animal studies

the presence of additional ALI risk factors has been found to modulate both the development and

severity of TRALI (multiple-hit hypothesis) (8, 9). The lack of standardized definition, however,

posed a significant limitation on previous clinical studies(3, 4).

Two recent expert consensus panels have proposed a standardized TRALI definition (3, 4).

Using the proposed definition, we reported retrospective data showing a much higher incidence

4

of TRALI in critically ill patients than previously suggested (18, 19). In this prospective study

we aimed to determine the incidence, risk factors and outcome of ALI which develops 0-6 hours

after the transfusion of blood products in a cohort of patients admitted to the medical intensive

care unit (ICU). Using a matched case-control design, we compared types of blood

products transfused by donor and storage characteristics, the prevalence of anti-leukocyte

antibodies (anti-granulocyte, anti-HLA class I and class II)(1, 5) and the amount of biologic

response modifiers (interleukin 8, IL8 and lysophosphatidil choline, Lyso-PC)(20) in the samples

of donor blood units given to patients who did or did not develop ALI after transfusion.

Some of the results of this study have been previously reported in the form of an abstract.(21)

MATERIALS AND METHODS

In this two year prospective cohort study, all patients transfused in a medical ICU of a tertiary

care medical center were closely observed for 24 h after transfusion. Detailed methods are

provided in online data supplement. The Institutional Review Board approved the study protocol.

Patients who refused research authorization were excluded. Monitoring logs reflecting

cardiopulmonary function, chest radiographs and arterial blood gases were reviewed and

followed until 24 hours after each transfusion allowing for identification of patients who had any

worsening of respiratory status 0-24 hours after the transfusion (see online data supplement).

Expert intensivists blinded to specific transfusion factors, subsequently reviewed all clinical data

and assigned the diagnosis of ALI within 6 hours of transfusion (suspected or possible TRALI)

based on the standard clinical definition (4, 22, 23). Inter-observer agreement was measured

using Kappa statistics. Two of the reviewers (JLM and OBR) re-reviewed the discordant cases

together and resolved any disagreements.

In the nested case control part of the study the patients’ and transfusion factors were compared

between patients who developed ALI after transfusion and transfused controls matched by age

5

±10 years, gender and admission diagnostic group (19). Predictor variables were grouped as

follows:

1. ALI risk factors prior to transfusion: Severity of illness was determined by calculating

Acute Physiology and Chronic Health Evaluation (APACHE III) scores(24) prior to

transfusion. Sepsis and pneumonia were defined according to standard clinical criteria

(25, 26). Aspiration was defined as witnessed or suspected aspiration of gastric contents

into the airways. Chronic alcohol abuse was defined as a known diagnosis of chronic

alcoholism or a previous admission for alcohol detoxification or alcohol withdrawal(27)

2. Transfusion factors: Units given 0-6 hours prior to the development of ALI (in cases) or

0-6 hours after the beginning of the first transfusion (controls) were considered associated

units. The exposure time for each control was matched to the exposure time of the

corresponding case (see online data supplement). The data on type of blood product,

storage age and donor gender were collected from the institutional transfusion database.

Female donors were contacted to obtain pregnancy history unless available from the

medical record of the donor. Transfused plasma volume was calculated based on the

average plasma content in specific blood products (19). Fresh frozen plasma (FFP) and

platelet transfusions were considered high plasma volume components(28).

3. Donor plasma samples: Throughout the study period transfusion bags and tubing were

collected at the end of each transfusion and stored for subsequent testing. Anti HLA class

I and class II antibodies were measured by multiplexed, microsphere-based flow

cytometric assay, ABScreenTM PRA (One Lambda, Inc, Canoga Park, CA) (29).

Antigranulocyte antibodies were detected by standard indirect immunofluorescence

method (30). LysoPC was measured by high performance lipid chromatography (31).

Interleukin (IL)-8 was measured using standard ELISA Quantikine HS Immunoassay

6

(R&D Systems Inc. Minneapolis, MN). If insufficient amount of plasma was obtained

from the transfusion bag, antibody (but not LysoPC and IL8) testing was performed on

available donor product or donor sample from subsequent donation.

Statistical Analysis: Paired parametric and non parametric testing was used in univariate

analyses as appropriate. We anticipated that approximately 80% of cases, and 40% of controls,

will have received at least one unit of donor blood that tests positive for anti-leukocyte

antibodies (6, 20). It was determined that a 1:1 matched case/control study with 50 cases and 50

controls would provide >80% power to detect a significant difference in the number of positive

antibody tests between groups using a two-sided, alpha=0.05 level test. Conditional logistic

regression was used to compare specific transfusion factors after the adjustment for pre-

transfusion ALI risk factors. SAS statistical software was used for all the analysis (SAS Version

9, SAS Institute Inc., Cary, North Carolina).

RESULTS

We prospectively observed 901 critically ill patients who were transfused in the medical ICU

over the two year study period (see outline of the study in Figure 1). Among 6588 blood product

units transfused during the ICU stay 3383 (51%) were packed RBCs, 2728 (41%) FFP, 306 (5%)

platelets and 171 (3%) cryoprecipitate. Waste plasma samples were stored from 3813 (57%)

transfusions for subsequent laboratory studies. Seventy-four (8%) patients developed ALI within

6 h of transfusion (Figure 1). Inter-observer agreement was moderately good (kappa 0.6).

Tables 1 describes clinical and transfusion characteristics of patients who developed ALI after

transfusion and matched controls. Gastrointestinal bleeding was the most common reason for

ICU admission (Table 1). Compared to patients who had no respiratory worsening after

transfusion, patients who developed ALI were more likely to have sepsis, liver disease and a

history of chronic alcohol abuse (Table 1).

7

ALI cases were more likely to have received plasma-rich blood products (FFP or platelets). They

were also more likely to have received blood products from female donors and larger volumes of

plasma from female donors (Table 1). Five cases (7%) and 4 controls (5%) received massive

(≥10 units) transfusion during the exposure period. Donors of patients who developed ALI had a

higher number of pregnancies and tested positive for anti-leukocyte antibodies (Table 1 and 2)

more often. There was no difference in IL8 or storage age of red cell products between patients

who developed ALI and matched controls. The concentration of LysoPC was significantly higher

in blood products given to ALI cases than controls (Table 2). At least one of the associated units

was tested for anti-leukocyte antibodies on 82% of cases and 80% of controls. LysoPC and IL8

were tested on at least one associated unit in 73% of cases and 78% of controls (Table 2).

Table 3 provides the unadjusted and adjusted ALI odds ratios for specific transfusion risk

factors. When adjusted for baseline APACHE III scores, sepsis and alcohol abuse in the

conditional logistic regression analysis, several transfusion factors remained associated with the

development of ALI (Table 3). The results were similar after additional adjustment for baseline

liver disease (Table E2, online data supplement). The distribution of transfusion factors were

similar in subgroups of patients who had significant additional ALI risk factors (“possible

TRALI”) and those who did not (“suspected” TRALI), Table E1, online data supplement..

Of 74 patients who developed ALI, 58 were treated with mechanical ventilation (48 invasive, 10

noninvasive) and 16 were treated with oxygen supplementation via face mask. Median duration

of mechanical ventilation was 3.6 (1.6 to 7.1) days. Twenty seven cases and 27 controls were

mechanically ventilated at the time of transfusion. Additional 31 cases were started on

mechanical ventilation (26 invasive, 5 noninvasive) because of development of ALI. Hospital

mortality was higher in patients who developed ALI (41%) than in matched controls (23%),

p<0.01), see also online data supplement.

8

DISCUSSION

The principal findings of our study are the following: 1) In a cohort of critically ill medical

patients, the development of ALI shortly after transfusion was more common than usually

appreciated and an order of magnitude more common than what is reported to blood banks 2)

Both underlying patient characteristics (“first hit”) and specific transfusion factors (“second hit”)

were associated with the development of ALI after blood transfusion. 3) Our results support both

of the proposed TRALI mechanisms as well as general multiple hit model of TRALI and ALI.

To our knowledge, this is the first time the recommended standardized clinical definition (3, 4,

22) was implemented in a prospective study of TRALI. In critically ill medical patients admitted

to a tertiary care medical center, clinically defined TRALI appears to be more common than

previously thought, supporting the general notion that TRALI is grossly under-recognized and

under-reported (3-5). Rather than concentrating on clinically suspected TRALI reactions

reported to the blood bank, we prospectively examined consecutive transfused critically ill

medical patients. Patients who developed ALI after transfusion often had other important ALI

risk factors not related to transfusion itself, sepsis and a history of chronic alcohol abuse. The

fact that potentially modifiable transfusion risk factors (such as donor gender, parity and

alloimmunization) are commonly associated with development of ALI may have important

implications on prevention and treatment of ALI in critically ill medical patients. Observational

data already suggest a decrease in incidence of ALI in patients treated with less liberal

transfusion policy (32, 33). Moreover, preliminary data from UK suggest a significant decrease

in postoperative ALI associated with a decreased use of FFP from female donor plasma(34).

Our results to some extent support both of the main proposed mechanisms for TRALI (6, 7, 35,

36) as well as the overall multiple-hit paradigm in ALI development (13, 14). Both the presence

of anti-leukocyte antibodies and the concentration of bioactive lipid factors in the donor product

9

were higher in patients who developed ALI than in matched controls both with and without

adjustment for baseline characteristics (“first hit”). Although the small sample size precludes

wider inferences, the closer look at the distribution of antibodies and LysoPC across different

blood products (Tables E3 and E4, online data supplement) reveals a trend towards more

antibody positive units given to ALI patients vs controls across each of the blood products (RBC,

FFP, platelets). On the other hand, a higher concentration of LysoPC seems to merely reflect the

larger number of high plasma volume products (platelets and FFP) given to ALI patients vs

controls. Our study design did not allow us to determine the sensitivity and specificity of

laboratory diagnosis of TRALI, nor to assess the specific etiologic (as opposed to a biomarker)

role of anti-leukocyte antibodies and LysoPC.

The association between infusion of plasma from female donors and the subsequent development

of lung injury is an intriguing finding of our study and has important implications with regards to

the etiology and prevention of TRALI. Our findings support the results of the small randomized

trial by Palfi et al, in which the plasma transfusion from multiparous female donors lead to

worsening in oxygenation and increased inflammatory response(35). The United Kingdom blood

system recently nearly eliminated female donors from the production of high plasma volume

products (FFP) and the first reports suggest a significant decrease in TRALI reactions reported to

the national transfusion surveillance system (SHOT-serious hazards of transfusion) (37). AABB

TRALI working group recently recommended similar guidelines for North American Blood

Centers, with a plan to eliminate transfusion of high plasma volume products from donors at high

risk of leukocyte alloimunization (including previously pregnant female donors) by November of

2008 (28). Since this intervention may be associated with costs and/or shortage of blood products

in some blood centers, further studies will have to assess if benefit of this intervention outweighs

the costs.

10

Relatively high mortality of our suspected or possible TRALI cases is comparable to a report

from Wallis et al (38) and our recent retrospective study in the critically ill (19), but higher than

in some other epidemiological studies (6). It is important to emphasize the characteristics of the

population studied: medical ICU patients in a tertiary care medical center, a group with

inherently high mortality rate. The observed mortality may or may not be attributable to TRALI

as these critically ill patients had multiple additional risk factors for both ALI and poor outcome.

We need to point out limitations of our study design. The study was conducted in a single

medical ICU in tertiary care center and, while internal validity may be high, external validity is

limited. In addition, the observational nature of the study does not allow independent estimation

of the cause and effect relationship between the predictors and outcome. Residual confounding

is always an issue in observational studies but is unlikely to fully explain our observed

differences in donor product characteristics (anti-leukocyte antibodies from previously pregnant

female donors and the concentration of LysoPC). Since there is no way that clinicians could have

specifically ordered “male” or “low LysoPC product in our institution, indication bias should be

less of an issue. While the trend towards higher amount of total plasma could explain some of the

observed differences it is again unlikely to explain the association between the donor gender and

pregnancy and the development of ALI.

Important limitation of our study is that we did not test for corresponding leukocyte antigens and

were not able to determine the specificities of donor antibodies. We did not perform additional

testing or treatment of the sera (e.g. adsorption of HLA Class I antibodies with platelets) to

confirm if our GIF+ samples were indeed directed against granulocyte specific antigens.

Because the fresh granulocyte panels used for the assay could not be selected to include all

possible granulocyte antigens, the potential exists that antibodies directed toward uncommon

granulocyte antigens would not be detected. Therefore, we could not distinguish between the

11

etiologic, rather than a biomarker role of specific antibodies. In a number of units given to both

cases and controls we were unable to extract an adequate sample for laboratory analysis. While

the proportion of units tested for anti-leukocyte antibodies was similar in cases and controls,

somewhat higher proportion of control units were tested for IL 8 and LysoPC. This difference, if

anything, would make it less likely to detect a significant difference by chance alone.

While the differences in pre-transfusion risk factors to some may point to the limitation of our

matching procedure, we were able to distinguish important, but not as well appreciated patient

risk factors, i.e. chronic alcohol abuse. Moss et al previously reported a strong independent

association between alcohol use and development of ALI (27). Laboratory experiments suggest

the depletion of antioxidant glutathione stores associated with chronic alcohol abuse as the most

plausible mechanistic explanation (39). Since a neutrophil mediated oxidative injury is the

hallmark of TRALI (7, 8, 36, 40), it seems plausible that antioxidant depletion may be associated

with greatly increased risk of this type of ALI.

In conclusion, by prospectively applying the standardized clinical definition, we have found a

high incidence of suspected and possible TRALI among transfused critically ill medical patients.

The association between specific donor and transfusion characteristics and subsequent

development of ALI has important implications relative to both the etiology and prevention of

this syndrome. Ongoing multicenter studies in transfused patients with and without underlying

critical illness will allow us to further elucidate the incidence and mechanisms of TRALI and the

effect of potential preventive strategies.

12

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Neutrophils and their Fc gamma receptors are essential in a mouse model of transfusion-related

acute lung injury. J Clin Invest 116(6):1615-23.

FIGURE LEGENDS

Figure 1. Outline of the study. ALI = Acute Lung Injury, TACO = Transfusion Associated

Circulatory Overload

Table 1 Demographics, admission diagnostic groups pre-transfusion ALI risk factors and transfusion characteristics of patients who developed ALI after transfusion and matched controls

ALI n=74

Matched Controls n=74

P value

Age (years), median (IQR) 64 (52 to 78) 61 (53 to 73) - Female gender, n (%) 37(50%) 37(50%) - ICU admission diagnoses -

Gastrointestinal, n (%) 28 (38%) 28 (38%) Respiratory, n (%) 18 (24%) 18 (24%)

Cardiovascular, n (%) 17 (23%) 17 (23%) Genitourinary, n (%) 6(8%) 5 (7%)

Hematology, n (%) 4 (6%) 5 (7%) Metabolic, n (%) 1 (1%) 1 (1%)

Baseline APACHE III score 61 (44 to 75) 57 (45 to 81) 0.586 Sepsis, n (%) 27 (37%) 16 (22%) 0.016 Pneumonia, n (%) 5 (7%) 7 (9%) 0.683 Aspiration, n (%) 8 (11.4%) 13 (18.6%) 0.225 History of heavy alcohol use, n (%) 27 (36.5%) 13 (17.6%) 0.006 Liver disease, n (%) 20 (27.4%) 11(15.1%) 0.039 Pancreatitis, n (%) 5 (6.8%) 1 (1.4%) 0.102 DIC, n (%) 10 (13.9%) 5 (6.9%) 0.132 Patients receiving RBC transfusion, n (%)

46 (62.2%) 54 (73%) 0.144

Patients receiving high plasma volume components (FFP or platelet), n (%)

44 (59.5%) 27 (36.5%) 0.006

Average RBC storage age (days) (N=35 matched pairs), median (IQR)

22.9 (17 to 31) 22.9 (15 to 30) 0.801

Number of associated units, median (IQR)*

3 (1 to 5) 2 (1 to 3) 0.063

RBC 1 (0 to 2) 2 (0 to 2) 0.77 FFP 1 (0 to 4) 0 (0 to 1) 0.08 Platelet** 0 (0 to 0) 0 (0 to 0) 0.06

Number of units from female donors, median (IQR)

1 (1 to 2) 1 (0 to 2) 0.016

Number of pregnancies among donors (n=68 matched pairs), median (IQR)

2 (0 to 5) 0 (0 to 3) 0.030

Amount of plasma (L), median (IQR) 0.27 (0.07 to 1.03) 0.07 (0.07 to 0.32) 0.047 Amount of plasma from female donors (L), median (IQR)

0.16 (0.04 to 0.5) 0.04 (0 to 0.1) 0.013

Amount of plasma (L) from female donors with at least one pregnancy (n=68 matched pairs), median (IQR)

0.04 (0 to 0.25) 0 (0 to 0.07) 0.038

RBC; red blood cells, FFP fresh frozen plasma, ALI acute lung injury, IQR interquartile range, DIC disseminated intravascular coagulation, APACHE Acute physiology and chronic health evaluation * Units transfused within 0-6 hours of the development of ALI in cases and 0-6h after the first transfusion in controls **Of 16 platelet units given to cases, 10 were aphaeresis platelets and 6 were pooled platelets; of 6 platelet units given to controls, 3 were aphaeresis platelets and 3 were pooled platelets

Table 2.Specific laboratory testing of donor samples that were given to patients who developed ALI after transfusion and matched controls*

ALI

Matched Controls

P value

IL8 (pg/dL), median (IQR), (n=47 matched pairs)

2.5 (2.5 to 6.4) 2.5 (2.5 to 7.7) 0.448

LysoPC 16 umol/L, median (IQR), (n=47 matched pairs)

46 (21 to 109) 25 (18 to 72) 0.004

LysoPC 18 umol/L, median (IQR), (n=47 matched pairs)

15 (4.9 to 33) 7.25 (2.5 to 19) 0.005

Number of antibody + tests, median (IQR)**, (n=52 matched pairs)

1 (0 to 2) 0 (0 to 1) 0.014

Received any HLA class I + units, n (%), (n=52 matched pairs)

21 (40.4%) 12 (23.1%) 0.072

Received any HLA class II + units, n (%), (n=52 matched pairs)

17 (32.7%) 10 (19.2%) 0.108

Received any GIF + units, n (%), (n=52 matched pairs)

13 (25%) 4 (7.7%) 0.020

Percentage of associated units tested for anti-leukocyte antibodies, median (IQR)

67% (24 to 100%) 90% (33 to 100%) 0.341

Percentage of associated units tested for IL8 and LysoPC, median (IQR),

50% (0 to 100%) 67% (33 to 100%) 0.036

*HLA class I, II and GIF antibodies were analyzed in 52 matched pairs, IL8 and LysoPC were analyzed in 47 matched pairs **If the associated unit tested positive for more than one antibody (for example both HLA1 and HLA2) each of these was counted as a positive test. GIF granulocyte immunofluorescence, HLA human leukocyte antigen, LysoPC lysophosphatidyl choline, ALI acute lung injury, IQR interquartile range

Table 3 Transfusion-related risk factors for acute lung injury: *unadjusted and **adjusted for baseline APACHE III score, sepsis and chronic alcohol abuse

Variable OR (95% CI)* P-Value OR (95% CI)** P-ValueAny high plasma volume components (FFP or platelets)

2.55 (1.27,5.11) 0.009 2.78 (1.21,6.38) 0.016

Number of units 1.09 (0.99,1.20) 0.081 1.11 (0.99,1.25) 0.086 Number of units from female donors 1.30 (1.03,1.66) 0.029 1.51 (1.08, 2.12) 0.016 Amount of male plasma (L) 1.55 (0.79,3.06) 0.202 1.60 (0.76,3.37) 0.215 Amount of female plasma (L) 3.23 (1.17,8.91) 0.024 5.09 (1.37,18.85) 0.015 Amount of plasma from female donors with at least one pregnancy (L)

4.41 (1.00,19.55) 0.050 9.48 (1.38,65.35) 0.022

Number of pregnancies among donors 1.11 (1.00,1.22) 0.047 1.19 (1.05,1.34) 0.007 Number of HLA class I + units 1.81 (0.97,3.38) 0.061 1.70 (0.94,3.09) 0.098 Number of HLA class II + units 1.93 (0.88,4.28) 0.103 3.08 (1.15,8.25) 0.025 Number of GIF + units 4.19 (1.22,14.32) 0.023 4.85 (1.32,17.86) 0.018 Mean LysoPC 16 (per 10 umol/L) 1.16 (1.04,1.30) 0.011 1.16 (1.02,1.32) 0.022 Mean LysoPC 18 (per 10 umol/L) 1.58 (1.10,2.26) 0.013 1.61 (1.08,2.38) 0.018

OR odds ratio, CI confidence interval, RBC red blood cell, FFP fresh frozen plasma, GIF granulocyte immunofluorescence, HLA human leukocyte antigen, LysoPC lysophosphatidyl choline, APACHE Acute Physiology and Chronic Health Evaluation, ALI acute lung injury For continuous variables odds ratios were calculated per each unit of measurement: for each additional unit transfused, for each additional liter of plasma (1L of plasma corresponds to a usual dose of ~ four units of FFP), for each 10 umol/L increase in LysoPC)

Figure 1.

Transfusion related acute lung injury in critically ill medical patients: prospective

nested case-control study

Online Data Supplement

Ognjen Gajic Rimki Rana

Jeffrey L. Winters

Murat Yilmaz

Jose L. Mendez Otis B. Rickman

Megan M O’Byrne

Laura K. Evenson

Michael Malinchoc

Stever R DeGoey

Bekele Afessa

Rolf D. Hubmayr

S. Breanndan Moore

Materials and Methods:

Prospective screening and case identification: Figure E1 outlines the prospective review

of monitoring logs used to identify respiratory worsening after transfusion. Respiratory

worsening was defined as the sustained (more than two hours) change in two out of three

respiratory monitoring parameters: 1) continuous arterial oxygen saturation measured by

pulse oximetry (decrease of 5% or below 90%), 2) oxygen supplementation (increase in

FiO2 by 10% or more) and 3) respiratory rate (increase by 10 or above 30 per minute).

ALI was defined as acute onset (within 6 h of transfusion) of hypoxemia: arterial oxygen

tension to fraction of inspired oxygen ratio (PaO2/FIO2) <300 mm Hg and bilateral

infiltrates on chest radiography, in the absence of left atrial hypertension as the principal

cause of pulmonary edema (1).

Experts used standardized algorithm to assign the probability of acute lung injury (ALI)

and TRALI as opposed to hydrostatic pulmonary edema including transfusion associated

circulatory overload (TACO) (2). The patient’s history, physical exam, echocardiogram,

electrocardiogram, biomarkers of myocardial injury, digital portable chest radiographs,

fluid balance, and data from invasive monitoring were integrated with the clinical course

and response to therapy in order to determine whether the onset of acute pulmonary

edema in these patients was mainly due to hydrostatic (TACO) or permeability (ALI)

mechanism. The experts further classified ALI patients into “possible” and “suspected”

TRALI based on the presence or absence of clear temporal relationship to another non-

transfusion related ALI risk factor (sepsis, overdose, aspiration) (3, 4).

Sample collection and testing:

Throughout the study period, transfusion bags and tubing were collected at the end of the

transfusion by the bedside nurse and sent to the transfusion laboratory by the hospital

pneumatic tube system (Figure E2). The remaining blood product was extracted,

centrifuged at 3000 rpm for 10 minutes and the supernatant stored at -70C for subsequent

analysis. Units given 0-6 hours prior to ALI (in cases) and 0-6h after the onset of first

transfusion were considered associated units and were subsequently tested for anti-

leukocyte antibodies, IL-8 and LysoPC. The exposure time for each control was matched

to the exposure time of the corresponding case.

Anti HLA Class I and Class II antibodies were identified using flow cytometry analysis

of antibody binding to antigen coated microbead plates (5). ABScreenTM PRA (One

Lambda, Inc, Canoga Park, CA) allows combination of up to 100 different beads in one

suspension for a single test. Corresponding antibodies present in the test sample bind to

the antigens and then are labeled with R-Phycoerythrin (PE)-conjugated goat anti-human

IgG. The LABScan 100TM flow analyzer detects the fluorescent emission of PE from

each bead and the accompanying software assists in detection of the proteins of interest.

Testing for antigranulocyte antibodies was performed at the Mayo Clinic Tissue Typing

Laboratory using standard indirect immunofluorescence method (6). After incubation of

the sample with paraformaldehyde the presence of specific antibody is detected by the

addition of a fluorescein labeled antihuman globulin and examination with a fluorescent

microscopy. Serum was not absorbed against platelets and therefore a positive result

could represent antibodies directed toward granulocyte specific antigens or antibodies to

HLA Class I. The panel of fresh granulocytes used for the GIF studies were not selected

to include a full complement of granulocyte antigens.

IL-8 was measured using standard ELISA Quantikine HS Immunoassay (R&D Systems

Inc. Minneapolis, MN). In a quantitative sandwich enzyme immunoassay, the specimen

is added onto a microplate pre-coated with monoclonal antibody. After washing away

unbound substances and addition of enzyme-linked specific polyclonal antibody and the

substrate, the enzyme activity is measured which is proportional to the amount of the

cytokine bound in the first step

Lysophosphatidylcholines (LysoPC) have been described as important biologic response

modifiers which stimulate neutrophils by increasing concentration of intracellular

calcium(7). LysoPC 18 (18 acyl carbon) and lyso PC 16 (16 acyl carbon) were

determined using standard high performance lipid chromatography (HPLC)(8)

Remaining plasma was extracted with 3 mL butanol after the addition of 1 µmol/L 17:0

LysoPC as internal standard. After vortexing and centrifugation, the upper phase was

collected, evaporated under nitrogen at 50°C, and reconstituted in 200-µL of methanol.

HPLC was performed by autosampler injection of 5 µL of the reconstituted specimen

onto a Phenomenex C8 50x2 mm column. The mobile phase (methanol: 100 mM

ammonium acetate: formic acid, 90: 10: 0.05 was pumped over the HPLC analytical

column at the rate of 300 µL/min. LysoPC elute apart from the bulk of the specimen

matrix at a retention time of approximately 1.5 minutes. 16:0 and 18:0 LysoPC were

quantitated using 17:0 LysoPC as internal standard from calibration over a concentration

range 0.1-5.0 µmol/L.

Transfused plasma volume was calculated based on the average plasma content in

specific blood products: 250 mL for fresh frozen plasma (FFP) and platelets, 150 mL for

cryoprecipitate and 37 mL for packed red blood cells (RBCs) (4).

To assure similar exposure time to possible risk factors between cases and control the

exposure time for each control was matched to the exposure time of the corresponding

case. That means if the earliest unit given to the TRALI case was x hours prior to the

onset of TRALI then the control recipient had his/her transfusion data tabulated for the

same number of hours subsequent to the first transfusion

Results

Table E 1 demonstrates a comparison of ALI patients who did (“possible TRALI”) or did

not (“suspected TRALI”) have a temporal relationship to another risk factor for acute

lung injury (2).

Table E2 presents the association between the transfusion risk factors and ALI after

adjustment for baseline liver disease in addition to chronic alcohol abuse, sepsis,

APACHE III scores in a conditional regression analysis

Table E3 and E4 present the distribution of antibody tests and biologic modifier levels

between cases and controls across different blood products (FFP, RBC, Platelets)

Figures E3 and E4 present the amount of different blood products and amount of donor

plasma from female vs male origin in transfused critically ill patients who developed no

or mild worsening, ALI (TRALI, possible TRALI and ALI 6-24h after transfusion) or

hydrostatic pulmonary edema (TACO-transfusion related circulatory overload). While

the amount of RBCs was similar across all patient subgroups, non-RBC blood products

were given proportionally more often to patients with mild worsening and those who

developed pulmonary edema (TRALI, possible TRALI and TACO) within 6 hours after

transfusion (Figure E3).. The patients who developed TRALI and other forms of

pulmonary edema associated with transfusion received a larger amount of plasma.

(Figure E4)

Table E5 presents a post-hoc multivariate logistic regression analysis for mortality. When

adjusted for age, APACHE III, sepsis and liver disease, ALI was not independently

associated with hospital mortality (table E5).

References

E1. Bernard, G. R., A. Artigas, K. L. Brigham, J. Carlet, K. Falke, L. Hudson, M.

Lamy, J. R. Legall, A. Morris, and R. Spragg. 1994. The American-European Consensus

Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial

coordination. Am J Respir Crit Care Med 149(3 Pt 1):818-24.

E2. Gajic, O., M. A. Gropper, and R. D. Hubmayr. 2006. Pulmonary edema after

transfusion: how to differentiate transfusion-associated circulatory overload from

transfusion-related acute lung injury. Crit Care Med 34(5 Suppl):S109-13.

E3. Kleinman, S., T. Caulfield, P. Chan, R. Davenport, J. McFarland, S. McPhedran,

M. Meade, D. Morrison, T. Pinsent, P. Robillard, and P. Slinger. 2004. Toward an

understanding of transfusion-related acute lung injury: statement of a consensus panel.

Transfusion 44(12):1774-89.

E4. Rana, R., E. R. Fernandez-Perez, S. A. Khan, S. Rana, J. L. Winters, T. G.

Lesnick, S. B. Moore, and O. Gajic. 2006. Transfusion-related acute lung injury and

pulmonary edema in critically ill patients: a retrospective study. Transfusion 46(9):1478-

83.

E5. Pei, R., J. Lee, T. Chen, S. Rojo, and P. I. Terasaki. 1999. Flow cytometric

detection of HLA antibodies using a spectrum of microbeads. Hum Immunol

60(12):1293-302.

E6. Jiang, A. F., and P. Lalezari. 1975. A micro-technique for detection of leukocyte

agglutinins. J Immunol Methods 7(1):103-8.

E7. Silliman, C. C., D. J. Elzi, D. R. Ambruso, R. J. Musters, C. Hamiel, R. J.

Harbeck, A. J. Paterson, A. J. Bjornsen, T. H. Wyman, M. Kelher, K. M. England, N.

McLaughlin-Malaxecheberria, C. C. Barnett, J. Aiboshi, and A. Bannerjee. 2003.

Lysophosphatidylcholines prime the NADPH oxidase and stimulate multiple neutrophil

functions through changes in cytosolic calcium

10.1189/jlb.0402179. J Leukoc Biol 73(4):511-524.

E8. Silliman, C. C., K. L. Clay, G. W. Thurman, C. A. Johnson, and D. R. Ambruso.

1994. Partial characterization of lipids that develop during the routine storage of blood

and prime the neutrophil NADPH oxidase. J Lab Clin Med 124(5):684-94.

Table E1. Characteristics of patients who developed ALI after transfusion: “suspected”

versus “possible” TRALI as determined by post-hoc expert assessment

Suspected TRALI

n=28

Possible TRALI

n=46

P value

Age (years) 62 (52 to 79) 60 (54 to 71) 0.42

Female gender 18 (64) 19 (41) 0.06

APACHE III score before the

transfusion

50 (33 to 63) 67 (54 to 80) <0.01

Sepsis 5 (18) 22 (48) <0.01

Aspiration 5 (18) 3 (7) 0.15

Pancreatitis 2 (7) 3 (7) 0.94

Alcohol abuse 14 (50) 13 (28) 0.06

Liver disease 9 (32) 11 (24) 0.48

DIC 3 (11) 7 (16) 0.55

WBC count before 9.6 (8 to 15) 11.4 (6 to 18) 0.91

WBC count after 9.9 (6 to 15) 11.5 (11 to 18) 0.92

Any RBC 15 (54) 31 (67) 0.24

Any FFP 16 (57) 23 (50) 0.55

Any Platelet 8 (29) 5 (11) 0.07

Average RBC storage (days) 24 (15 to 29) 21 (17 to 30) 0.78

# of Units 4(2 to 6) 3 (1 to 5) 0.28

Any Units from female

donors

18 (64) 23 (55) 0.52

# Units from female donors 1 (1 to 2) 1 (1 to 2) 0.85

# Units from female donors

with at least one pregnancy

1 (0 to 2) 1 (0 to 2) 0.56

Number of donor pregnancies 2 (0 to 8) 1 (0 to 5) 0.28

Median amount of plasma (L) 0.66 (0.1 to 1.1) 0.25 (0.1 to 1.0) 0.12

Median amount

of female plasma (L)

0.25 (0.1 to 0.5) 0.04 (0.04 to 0.5) 0.10

Median amount of plasma

from female donors with at

least one pregnancy

0.06 (0 to 0.5) 0.04 (0 to 0.3) 0.23

Received any HLA I + units 12 (44) 13 (38) 0.64

Received any HLA II + units 9 (33) 11 (32) 0.94

Received any GIF + units 7 (26) 6 (18) 0.43

Received any Ab+ units 15 (56) 16 (47) 0.51

# of Antibody + tests (N=52)* 1 (0 to 2) 0 (0 to 2) 0.56

Mean IL8 (pg/dL) (N=47) 2.5 (3 to 12) 2.5 (3 to 6) 0.54

Mean LPC16 umol/L(N=47) 79 (29 to 111) 43 (21 to 95) 0.16

Mean LPC18 umol/L (N=47) 21 (8 to 38) 13 (4 to 30) 0.17

Mechanically ventilated

Invasive

Noninvasive

15 (54)

5 (18)

33 (73)

5 (11)

0.224

Hospital mortality 10 (36) 20 (44) 0.46

*If the associated unit tested positive for more than one antibody (for example both

HLA1 and HLA2) each of these was counted as a positive test.

Table E2 Transfusion-related risk factors for acute lung injury adjusted for baseline liver disease, APACHE III score, sepsis and chronic alcohol abuse

Variable OR (95% CI)** P-ValueAny high plasma volume components (FFP or platelets)

2.55 (1.1,5.9) 0.029

Number of units 1.13 (0.99,1.29 0.064 Number of units from female donors 1.53 (1.06, 2.20) 0.021 Amount of male plasma (L) 1.87 (0.83, 4.21) 0.131 Amount of female plasma (L) 5.52 (1.34,22.79) 0.018 Amount of plasma from female donors with at least one pregnancy (L)

17.9 (1.72 ,186) 0.016

Number of pregnancies among donors 1.20 (1.06,1.37) 0.005 Number of HLA class I + units 1.69 (0.93,3.08) 0.088 Number of HLA class II + units 3.19 (1.18,8.62) 0.022 Number of GIF + units 5.04 (1.35,18.85) 0.018 Mean LysoPC 16 (per 10 umol/L) 1.19 (1.02,1.38) 0.023 Mean LysoPC 18 (per 10 umol/L) 1.76 (1.11,2.80) 0.017

OR odds ratio, CI confidence interval, RBC red blood cell, FFP fresh frozen plasma, GIF granulocyte immunofluorescence, HLA human leukocyte antigen, LysoPC

lysophosphatidyl choline, APACHE Acute Physiology and Chronic Health Evaluation, ALI acute lung

injury

Table E3 Distribution of antibody tests between cases and controls across different associated blood products (RBC. Platelet, FFP)

Control ALI product

Not tested 19 20 Tested 60 65 # positive 8 17

HLA I

% positive 13.3 26.2 Not tested 19 20 Tested 60 65 # positive 4 9

HLA II

% positive 6.7 13.8 Not tested 19 20 Tested 60 65 # positive 4 8

RBC

GIF

% positive 6.7 12.3 Not tested 2 5Tested 1 9# positive 0 1

HLA I

% positive 0.0 11.1 Not tested 2 5Tested 1 9# positive 1 1

HLA II

% positive 100.0 11.1 Not tested 2 5Tested 1 9# positive 0 3

Platelets

GIF

% positive 0.0 33.3

Control ALI Not tested 11 71 Tested 30 56 # positive 5 13

HLA I

% positive 16.7 23.2 Not tested 11 71 Tested 30 56 # positive 6 11

HLA II

% positive 20.0 19.6 Not tested 11 71 Tested 30 56 # positive 0 4

FFP

GIF

% positive 0.0 7.1 Not tested 32 96 Tested 91 130 # positive 13 31

HLA I

% positive 14 24 Not tested 32 96 Tested 91 130 # positive 11 21

HLA II

% positive 12 16 Not tested 32 96 Tested 91 130 # positive 4 15

All products

GIF

% positive 4 12

Table E4 Distribution of IL8 and LysoPC between cases and controls across different associated blood products (RBC. Platelet, FFP)

Control ALI product

Not tested 22 30 Tested 52 52

IL 8, pg/dL

Median 2.50 2.50 Not tested 22 29 Tested 52 53

LPC16, umol/L

Median 19.00 21.00 Not tested 22 29 Tested 52 53

RBC

LPC18, umol/L

Median 5.00 6.00 Not tested 2 5Tested 0 8

IL 8, pg/dL

Median 8.00 Not tested 2 5Tested 0 8

LPC16, umol/L

Median 213.50 Not tested 2 5Tested 0 8

Platelets

LPC18, umol/L

Median 78.00 Not tested 7 77 Tested 25 41

IL 8, pg/dL

Median 2.50 2.50 Not tested 7 77 Tested 25 41

LPC16, umol/L

Median 103.00 104.00 Not tested 7 77 Tested 25 41

FFP

LPC18, umol/L

Median 33.00 31.00

Control ALI Not tested 32 124 Tested 79 103

IL 8, pg/dL

Median 2.50 2.50 Not tested 32 123 Tested 79 104

LPC16, umol/L

Median 29.00 36.50 Not tested 32 123 Tested 79 104

All products

LPC18, umol/L

Median 8.00 10.00

Table E5 Risk factors for hospital mortality in a multivariate logistic regression analysis

OR 95% CI p-value

ALI 2.16 0.98 4.88 0.059

Age 1.03 1.00 1.06 0.049

APACHE III 1.02 1.00 1.04 0.019

Sepsis 2.07 0.89 4.85 0.091

Liver disease 2.79 1.07 7.47 0.037

OR odds ratio, ALI acute lung injury, CI – confidence interval, APACHE – Acute Physiology and Chronic Health Evaluation

Figure E1 Snapshot of a monitoring log identifying worsening in respiratory status after

transfusion

FIO2

RR

MABP

O2 sat

FFP

Figure E2 Outline of the transfusion sample collection in the medical ICU

Figure E3 Type of blood products given to the patients who developed ALI (TRALI and

possible TRALI), TACO, mild worsening, ALI 24h after the transfusion, matched

controls and other controls

Figure E4 The amount of male vs female donor plasma given to the patients who

developed ALI (TRALI and possible TRALI), TACO, mild worsening, ALI 24h

after the transfusion, matched controls and other ontrols