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ifferential White Cell Count Relationships With Human Cardiacllograft Rejection

.G. Healy, R.W.G. Watson, J.M. Fitzpatrick, and A.E. Wood

ABSTRACT

The focus of research in allograft rejection has targeted the lymphocyte, with littleattention given to the neutrophil. Recent data indicate that a perioperative neutrophilinflux into the cardiac allograft influences early rejection. Factors that influence neutrophiltransendothelial migration might offer predictive markers of rejection. We explored therelationship between the number of circulating neutrophils in heart transplant recipientsand the development of rejection. Differential white cell counts were obtained prior totransplantation and concurrently with subsequent endomyocardial rejection surveillancebiopsies for 53 heart transplant recipients undergoing 410 biopsies. Preoperative differ-ential white cell counts had no relationship with rejection. In the first 3 months aftertransplantation, no relationship was found between contemporary differential white cellcounts and rejection. However, more than 3 months following surgery, rejection gradepositively correlated on univariate analysis with neutrophil counts and the usage ofcyclosporine, prednisolone, and mycophenolate. There was no relationship with eosino-phils or lymphocytes. Multivariate analysis demonstrated a persistent relationship amongrejection severity, neutrophil count, and prednisolone usage. A significant positiveassociation of higher steroid usage with higher rejection grades must reflect efforts to treatpatients with rejection. The significant association of higher neutrophil counts with higherrejection severity might suggest a pathological contribution to rejection. However, giventhe neutrophilia response to acute steroid administration, we must conclude that theneutrophil association was related to steroid administration. The absence of a relationshipbetween white cell counts and rejection suggests that functional rather than antiprolifera-

tive strategies may offer the greatest therapeutic potential.

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LLOGRAFT REJECTION contributes to cardiactransplantation mortality and morbidity.1 Rejection

esearch has focused on the contribution of the lymphocyte,hich is fundamental to the development of allograft

ejection. However, the immune system is multifaceted andhe composite immune effect is the result of interactionsmong many cell classes. The neutrophil is the largestomponent of the white cell population and the firsteukocyte to infiltrate the allograft. Neutrophils are ob-erved on histological analysis of hyperacute allograft rejec-ion, humoral rejection, and in high grades of chronicejection.2,3 The neutrophil can cause direct tissue damagend may influence subsequent lymphocyte activation.4 Re-ent data indicate that an early perioperative neutrophilnflux into a human cardiac allograft is associated withigher grades of rejection upon the first postoperative

ndomyocardial biopsy.5 Therefore, factors that influence I

041-1345/07/$–see front matteroi:10.1016/j.transproceed.2006.10.226

18

eutrophil transendothelial migration might offer predic-ive markers of rejection and therapeutic targets. To date,reoperative measurement of the neutrophil adhesion pro-ein CD11b has been associated with postoperative rejec-ion severity.5 The degree of neutrophil transendothelialigration might not only be influenced by adhesion mole-

ules, but also by the number of circulating neutrophils. In

From the Prof Eoin O’Malley National Centre for Cardiotho-acic Surgery (D.G.H., A.E.W.), Department of SurgeryR.W.G.W., J.M.F.) Mater Misericordiae University Hospital,ccles St, Dublin, Ireland; and Conway Institute of Biomolecularnd Biomedical Research (D.G.H., R.W.G.W., J.M.F.), Universityollege Dublin, Belfield, Dublin, Ireland.Address reprint requests to David Healy, Cardiac Surgery,ater Misericordiae University Hospital, Eccles St, Dublin 7,

reland. E-mail: cardiothoracic@eircom.net

© 2007 by Elsevier Inc. All rights reserved.360 Park Avenue South, New York, NY 10010-1710

Transplantation Proceedings, 39, 218–222 (2007)

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DIFFERENTIAL WHITE CELL COUNT 219

his study we sought to explore the relationship between theumber of circulating neutrophils in heart transplant recip-

ents and the development of cardiac allograft rejection.

ETHODS

ata on endomyocardial biopsy rejection grade and contemporaryifferential white cell counts was obtained retrospectively from 53atients. These patients were consecutive attendees at the heartransplantation outpatient clinics who had consented to participaten an institutional study approved by our ethics committee. Rejec-ion surveillance at this center was performed with a percutaneousndomyocardial biopsy as a day case procedure. Differential whiteell counts generated from venous samples were processed using anutomated blood analyser (Bayer Advia 120). It is routine practiceo take a venous sample for a full blood count and drug concen-ration on the morning of the biopsy. Where an endomyocardialiopsy was performed during an inpatient hospital stay, bloodamples taken within 24 hours of the procedure were accepted. Theiopsy specimen was read by a pathologist and graded using theriginal ISHLT standard.6 The maintenance immunosuppressionegimen for heart transplantation in this center consists of cyclo-porine, azathoprine/mycophenolate, and prednisolone. Drug us-ge was recorded by monitoring the trough cyclosporine levels withtarget range of 100 to 200 ng/mL and by recording the dose of

rednisolone, azathioprine, and mycophenolate as well as theatient’s weight.Comparisons were performed between individual cell classes and

llograft endomyocardial biopsy rejection grades at three timeoints: (1) comparison of the preoperative differential white cellount and rejection grade at the first and second postoperativeiopsies, (2) comparison between contemporary differential whiteell counts and rejection grade of biopsies performed within therst 3 months after surgery, and (3) comparison between contem-orary differential white cell counts and rejection grade of biopsieserformed more then 3 months after transplantation. The contri-ution of the immunosuppressive agents was also assessed asotential confounding variables. Statistical correlations were ini-ially screened with a Spearman rank order correlation coefficient.his was subsequently developed with a more complex binary

ogistic regression model. Binary outcome comparison for low-rade (ISHLT grade � 2) and high-grade (ISHLT � 3A) endo-yocardial biopsies was performed using a univariate model with

n alpha level set at 0.15. Further multivariate logistic regressionnalysis of factors positive on univariate analysis was then per-ormed with an alpha level set at 0.05. Standard receiver operatingharacteristic curve analysis was used to define the optimumhreshold for the differential diagnosis of ISHLT grade � 3A

Table 1. Preoperative Differenti

n Biopsy WCC Neutrophil Ly

3 0 9.9 (�4.7) 6.7 (�4.7) 10 1A 9.2 (�2.2) 6.0 (�1.5) 17 1B 8.3 (�0.7) 5.4 (�0.4) 28 2 10.5 (�3.7) 8.5 (�4.2) 12 3A 9.7 (�4.8) 6.7 (�4.6) 23 3B 7.4 (�3.2) 4.9 (�2.8) 1

Differential white cell counts were generated from full blood counts performiopsies performed following transplantation. The comparison with the first biophe cell count of any white cell type. Data displayed with means and standard

ejection. a

ESULTS

total of 410 endomyocardial biopsies with contemporaryifferential blood counts were analysed from 53 patients.here were 43 men and the mean age overall was 44.6�15.4) years. The mean number of biopsies per patientas 7.4 and the mean interval since transplantation 5.12ears (�3.7). We first compared preoperative differentialhite cell counts with rejection grade at the first and secondiopsies, since we wanted to determine whether the numberf neutrophils present at the initial reperfusion phase

nfluenced early rejection. Previous data had shown thatreoperative neutrophil adhesion molecule expression cor-elated with rejection upon the first postoperative biopsy.owever, there was no significant association between

eutrophil, lymphocyte, or eosinophil counts preoperativelynd rejection upon the first or second biopsies (Table 1).e then analyzed differential blood counts taken concur-

ently with endomyocardial biopsies in the 3 months follow-ng surgery, observing no significant relationship betweeneutrophil counts and the severity of rejection (Table 2). Inddition, no relationships were seen between cellularlasses such as total lymphocyte or eosinophil counts andejection severity. The mean prednisolone dose at the timef biopsies in the first month was 0.355 mg/kg (�0.179).However, when measured more than 3 months following

urgery, the neutrophil count (Spearman rank correlation,� .046, R � 0.178) positively correlated with increasing

ejection severity (Table 3). However, no such relationshipas seen with the total white cell count (Spearman rankorrelation, P � .132, R � 0.10), lymphocyte count (Spear-an rank correlation, P � .481, R � 0.070), or eosinophil

ount (Spearman rank correlation, P � .134, R � 0.094).urther univariate binary logistic regression analysis of theifferential white cell count and immunosuppression treat-ents in relation to high grades of rejection (�3A) identi-

ed four variables that were associated with higher rejec-ion grades: azathioprine or mycophenolate, prednisolone,nd neutrophil count. No significance was observed withymphocyte or eosinophil counts.

These variables were then included in a multivariateogistic regression analysis model, which identified neutro-hil count (logistic regression, P � .006) and prednisoloneose (logistic regression, P � .047) as significant factors

ite Cell Counts and Rejection

cyte Eosinophil Basophil Monocyte

.6) 0.13 (�0.) 0.07 (�0.04) 0.63 (�0.26)

.7) 0.58 (�1.0) 0.07 (�0.05) 0.58 (�0.15)

.4) 0.16 (�0.1) 0.06 (�0.02) 0.58 (�0.22)

.7) 0.21 (�0.1) 0.08 (�0.02) 0.63 (�0.25)

.6) 0.20 (�0.1) 0.10 (�0.04) 0.57 (�0.22)

.1) 0.15 (�0.1) 0.03 (�0.03) 0.41 (�0.30)

ior to transplantation and compared to the first and second endomyocardialhown here. No significant associations were seen between rejection grade andtions, n � 53.

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ostics of neutrophil count and prednisolone dose foundhat these two variables were independent (tolerance �.965). The mean prednisolone dose at the time of endo-yocardial biopsy after 3 months was 0.11 mg/kg (�0.09).sing neutrophil count greater then 6 � 109/L as a tool to

etect rejection severity of grade 3A or higher, neutrophilounts gave a sensitivity of 40% and a specificity of 77%,hich is inferior to current rejection detection techniques.

ISCUSSION

ince allograft rejection is an immunological process, it isntuitive to observe immune function to detect the occur-ence of rejection and to aid in the development of treat-ent strategies. Contemporary immunosuppressive strate-

ies target the lymphocyte, and yet rejection remains alinical problem. This suggests that exclusively lymphocyte-riented treatments are not the panacea. Previous work byur group has shown an early neutrophil migration into theardiac allograft, which is proportional to the rejectionrade at the first postoperative biopsy.5 Based on thisbservation, we further explored factors that might influ-nce the ability of neutrophils to migrate into the allograft,bserving that the preoperative expression of the neutro-hil adhesion protein CD11b was associated with subse-uent rejection severity.5 In this study we examined anotheractor that may influence neutrophil transendothelial mi-ration, namely the circulating neutrophil count.

Total leukocyte counts were raised following heart trans-lantation and persisted for 7 days.7 The largest componentf the total white cell count is the neutrophil. In animalodels of cardiac transplantation, they are the first leuko-

ytes to accumulate in the cardiac allograft.8,9 They reach a

Table 2. Early Postoperative Differe

n Biopsy WCC Neutrophil Lym

9 0 10.1 (�4.2) 9.0 (�3.9) 0.71 1A 8.9 (�3.9) 7.2 (�3.5) 1.15 1B 9.7 (�3.0) 8.8 (�3.1) 0.87 2 9.6 (�4.0) 8.1 (�4.1) 1.01 3A 11.0 (�4.6) 9.5 (�4.8) 1.04 3B 11.4 (�2.8) 9.4 (�2.2) 1.11 4 3.14 2.84

Differential white cell counts were generated from full blood counts performedfter cardiac transplantation. These counts were compared with the result of trade and the cell count of any white cell type. Data displayed with means an

Table 3. Late Postoperative Differe

n Biopsy WCC Neutrophil Ly

9 0 6.63 (�2.1) 4.78 (�2.0) 1.11 1A 6.78 (�2.4) 4.50 (�1.8) 1.25 1B 7.18 (�2.5) 5.46 (�2.6) 1.15 2 7.47 (�2.3) 5.45 (�1.8) 1.39 3A 6.95 (�2.9) 5.70 (�3.2) 1.23 3B 8.22 (�2.8) 6.37 (�3.5) 1.1

Differential white cell counts were generated from full blood counts performed

iopsy performed more than a 3 months following transplantation. These countsssociations were seen between rejection grade and the neutrophil cell count. Data

eak concentration at 6 hours following transplantation andecrease by 50% at 24 hours.8 Animal models of transplan-ation have demonstrated lower rejection severity and pro-onged survival with strategies that inhibit neutrophil trans-ndothelial migration.10

In our analysis we assessed neutrophil counts and otheromponents of the differential white cell count. Neutrophilounts taken at the preoperative stage did not correlateith rejection upon the first biopsy. This observation was of

nterest in the context of previous findings that preoperativeeutrophil CD11b correlated with rejection severity uponhe first postoperative biopsy,5 suggesting that neutrophilumbers at the time of implantation are not relevant toejection development, but rather that aspects of neutrophilunction may be more important. In the first 3 months aftermplantation, neutrophil counts did not correlate withimultaneous rejection biopsy results. The absence of aorrelation between neutrophil count and rejection in thearly postoperative period may reflect surgical trauma andigh-dose steroid regimens. All patients were treated with aigh-dose steroid regimen until their treatment was individ-alized following the first postoperative endomyocardialiopsy. However, neutrophil counts on the day of biopsiesaken more than 3 months after surgery showed a signifi-ant association with rejection severity. The significantelationship seen between rejection and neutrophil countseyond 3 months contradicts conventional explanations ofhe role of the neutrophil in chronic rejection.3 Neutrophilsre rarely seen on endomyocardial biopsies at this late timerom surgery. At this late stage, the patient has recoveredrom the surgical trauma, and glucocorticoid doses haveeen tapered. Assessment with logistic regression analysis

White Cell Counts and Rejection

yte Eosinophil Basophil Monocyte

.6) 0.05 (�0.09) 0.03 (�0.02) 0.49 (�0.25)

.9) 0.08 (�0.09) 0.03 (�0.03) 0.46 (�0.26)

.67) 0.04 (�0.03) 0.04 (�0.03) 0.49 (�0.24)

.05) 0.07 (�0.12) 0.03 (�0.03) 0.47 (�0.28)

.62) 0.07 (�0.05) 0.05 (�0.07) 0.49 (�0.23)

.03) 0.09 (�0.10) 0.02 (�0.02) 0.57 (�0.44)0 0 0.08

e morning of a surveillance endomyocardial biopsy performed in first 3 monthsomyocardial biopsy. No significant associations were seen between rejectiondard deviations, n � 138.

White Cell Counts and Rejection

cyte Eosinophil Basophil Monocyte

.58) 0.09 (�0.06) 0.04 (�0.02) 0.48 (�0.20)

.62) 0.12 (�0.08) 0.04 (�0.02) 0.48 (�0.19)

.68) 0.10 (�0.09) 0.04 (�0.03) 0.53 (�0.28)

.82) 0.13 (�0.08) 0.05 (�0.03) 0.58 (�0.24)

.69) 0.11 (�0.07) 0.05 (�0.03) 0.52 (�0.21)

.50) 0.11 (�0.09) 0.06 (�0.06) 0.41 (�0.11)

e morning of, or within the previous 24 hours of, a surveillance endomyocardial

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displayed with means and standard deviations, n � 272.

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DIFFERENTIAL WHITE CELL COUNT 221

dentified persistent significant relationships between rejec-ion and both neutrophil counts and steroid dose. We donow that acute administration of prednisolone, even atoses of 5 to 10 mg, is associated with a delay in neutrophilpoptosis and an acute increase in neutrophil counts.11,12

he positive relationship between neutrophil count andejection may simply be a consequence of the use of higheroses of oral steroids in patients with higher rejectionrades. Attempts to separate these two significant associa-ions with colinear diagnostics suggest that these variablesave independent relationships with rejection severity.owever, knowing the effects of glucocorticoids on neutro-

hil numbers, in this context it is difficult to mathematicallyeparate their contributions to rejection. Instead, an inter-ention trial would be required. Interestingly the effect ofepeated chronic administration of prednisolone on neutro-hil count is uncertain. An alternative explanation for this

ate correlation is that we have observed a marker ofejection rather then a participant. The rise in neutrophilount may instead reflect an ongoing inflammatory process.bdallah et al have reported positive correlations between

irculating tumor necrosis factor-�, interleukin-6, and sub-equent rejection.13

There are few other reports on differential white cellounts and cardiac rejection. Trull et al found no relation-hip between neutrophil count and rejection when evaluat-ng only the first 3 months following surgery.14 Instead, theyeported a significant relationship between eosinophilounts and the development of rejection.14 A similar rela-ionship between eosinophils and early rejection has beeneported in liver and renal transplantation.15,16 In our studyo relationship was observed between eosinophil countsnd rejection severity. The reasons for this difference areot certain. Trull et al’s study looked at the first 3 monthsollowing transplantation, analyzing 383 biopsies. They ob-erved that 19% of their biopsies were grade 3A or higher;ur patient population had a 26% rate of high-gradeiopsies. Trull et al accepted blood measurements up to 3ays prior to the performance of the biopsy, and 42% of theosinophil counts in our study were above the cutoff pointf 0.06 � 109/L used by Trull et al to detect rejection.herefore, a combination of different populations, his-

opathological analyses, and blood processing may haveontributed to this difference. In further studies that groupsed eosinophil levels in heart transplantation patients touide steroid treatment and successfully reduced rejectionvents.17 It would seem that they found eosinophils to be aseful bioassay for steroid dosing and perhaps compliance.teroids reduce eosinophil counts in vivo, most likely byccelerating apoptotic rates.18

Cytoimmunological monitoring of the immune system’sbility to initiate and maintain a rejection process has so farocused on lymphocyte parameters.19 In this study webserved no relationship between the total circulating lym-hocyte population and rejection. However, analysis ofotal lymphocyte counts may mask significant shifts in

ymphocyte subpopulations. Others have focused on the

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unctional measurements of lymphocyte activity. The per-entage of activated lymphocytes, lymphocyte proliferation,nd the presence of breakdown products of lymphocyte-ediated tissue damage have been associated with the

evelopment of cardiac allograft rejection.20–22 Quantifica-ion of the activity of the recipient’s lymphocytes againstonor antigen-presenting cells has shown good specificity forejection.23 These results, together with our observation of noorrelation between rejection severity and the total number ofymphocytes, suggested that lymphocyte-mediated myocardialamage was mediated by an alteration in lymphocyteunction or subpopulations rather than in the total popula-ion of circulating lymphocytes.

The net inflammatory effects of neutrophils is not onlyependent on the number of neutrophils present but alsoheir functional activation. The putative mechanism ofction of the clinically successful immunosuppressive agentyclosporine involves T-cell suppression; however, there isncreasing evidence of suppressive effects on neutrophilunction.24,25 Glucocorticoids also have effects on neutro-hil activity, causing a decrease in the production of oxygenree radicals and a delay in spontaneous apoptosis.11 In thiseport the increase in neutrophil count with higher rejec-ion grades was paralleled by increased steroid usage.teroids are effective in the management of rejection andet cause an acute neutrophilia, suggesting that neutrophilumbers may not be as important as their functionalctivity. An important distinction between cellular numbersn the circulation and functional activity was seen here dueo no association between rejection and neutrophils, lym-hocytes, or eosinophils. This observation has implicationsor therapies based on antiproliferative strategies. Selectiveunctional strategies may prove more rewarding in the longerm.

CKNOWLEDGMENTS

e wish to acknowledge the aid of SPSS Ireland Ltd. whorovided statistical support on a commission basis.

EFERENCES

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222 HEALY, WATSON, FITZPATRICK ET AL

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17. Trull A, Steel L, Sharples L, et al: Randomized trial of bloodosinophil count monitoring as a guide to corticosteroid dosagedjustment after heart transplantation. Transplantation 70:802,00018. Matsukara M, Yamada H, Yudate T, et al: Steroid-induced

hanges of eosinophils in atopic dermatitis. Int Arch Allergymmunol 114(suppl 1):51, 1997

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ytoimmunologic monitoring as an adjunct in monitoring rejectionfter heart transplantation: results of a 6 year follow-up in heartransplant recipients. J Heart Lung Transplant 13:869, 1994

21. Ansari A, Mayne A, Sundstrom J, et al: Frequency ofypoxanthine guanine phosphoribosyltransferase (HPRT-) T cells

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