Leucocyte transfusions from rhG-CSF or prednisolone stimulated donors for treatment of severe infections in immunocompromised neutropenic patients

Leucocyte transfusions from rhG-CSF or prednisolone

stimulated donors for treatment of severe infections

in immunocompromised neutropenic patients

C. PETE RS,1 M. MINKOV,1 S. MATTHES-MARTIN,1 U. POÈ T SCHGE R,1 V. WITT,1 G. MA NN,1 P. HOÈ CKER,2

N. WOREL,2 J. STARY,3 T. KLI NGEBIE L4

AN D H. GA DNE R1 1St Anna Children's Hospital, Vienna, Austria,

2University Clinic for Blood Group Serology and Transfusion, Vienna, Austria, 3University Hospital Motol,

Prague, Czech Republic, and 4University Clinic for Paediatrics, TuÈbingen, Germany

Received 20 January 1999; accepted for publication 14 June 1999

Summary. Sepsis in profound neutropenia after chemo-therapy is associated with high mortality despite appropriateantibacterial or antifungal treatment. In a prospective phaseI/II study we evaluated the feasability and ef®cacy ofleucocyte transfusions (LT) in patients with malignanciesor haematological disorders who were suffering from severebacterial or fungal infection during therapy-related bonemarrow aplasia. 30 patients with severe neutropenia andclinical signs of life-threatening sepsis not responding toadequate treatment, received LT from rhG-CSF-stimulatedfamily donors or from prednisolone-primed volunteers. Atotal of 301 LT were administered. The median number of LTper patient was seven (range three to 65), the medianduration of LT treatment was 8 d (range 2±35). The whitecell count (WBC), absolute neutrophil count (ANC) and

lymphocyte count of the concentrates from rhG-CSF-stimulated donors were signi®cantly higher than thosefrom prednisolone-primed volunteers (P�0´0001). Despitethe critical condition of the patients, LT were generally welltolerated. Only 39 (12´9%) LT were associated with adversereactions. The transfusion of leucocytes collected bycontinuous ¯ow leukapheresis from both rhG-CSF andprednisolone stimulated donors resulted in a measurableincrement of the peripheral leucocyte and ANC counts in ourpatients. On day 100 after the ®rst LT, 20/30 patients werealive with complete clearance of the infection.

Keywords: infection, leucocyte, transfusion, granulocytes,rhG-CSF.

Bacterial and fungal infections occurring in immunocom-promised patients, particularly those with prolonged severeneutropenia, represent a frequent cause of morbidity andmortality (Kusne et al, 1988; Denning, 1994). Althoughcurrent therapeutic approaches use combinations of broad-spectrum antibiotics, antifungal agents and haemopoieticcytokines, septic complications are still a serious problem inthese patients (Link et al, 1994) and clearance of theinfectious pathogen is often possible only in the presence ofan adequate number of neutrophils. Previously, transfusionof granulocytes has been used as a supportive measurementto bridge the interval until haemopoietic regeneration.Glucocorticoids have generally been used for stimulationand mobilization of neutrophils in healthy donors in the past

(Dale et al, 1975). The ef®cacy of these granulocytetransfusions was limited by the inability to collect anadequate number of granulocytes (Alavi et al, 1977), theshort half-life of the collected neutrophils (Colotta et al,1993), infusion-associated toxicity (Wright et al, 1981) andthe potential of transfusion-associated cytomegalovirus(CMV) infections in immunocompromised seronegativepatients (Bowden, 1995). There is evidence that theadministration of rhG-CSF to donors produces a dose-dependent and sustained rise of circulating granulocytesand enables the collection of a large number of maturemyeloid cells by leukapheresis (Bensinger et al, 1993; Casparet al, 1993). Furthermore, it has been shown that in vitrocytokine incubation of granulocytes signi®cantly reducesapoptosis and preserves chemotaxis, C. albicans killing andsurface-receptor expression (Rex et al, 1995). In a smallgroup of patients successful migration of neutrophils toin¯ammation sites could be observed (Adkins et al, 1997).

British Journal of Haematology, 1999, 106, 689±696

689q 1999 Blackwell Science Ltd

Correspondence: Dr Christina Peters, Bone Marrow TransplantationUnit, St Anna Children's Hospital, Kinderspitalgasse 6, A-1090

Vienna, Austria. e-mail: [email protected].

Based on this current knowledge we conducted a multi-centre trial to test the tolerability and ef®cacy of rhG-CSF- orprednisolone-elicited leucocyte transfusions in immunocom-promised neutropenic patients in whom the control of life-threatening infections by means of conventional methodshad failed.

MATERIALS AND METHODS

Study design. An open prospective phase I/II trial wasconducted between April 1996 and April 1998 after approvalfrom the ethics committees of the participating centres. The aimswere: (i) to evaluate the ef®cacy and tolerability of differentstimulation modalities for leucocyte yield from healthy donors,(ii) to evaluate the feasibility and tolerability of leucocytetransfusions in critically ill patients, and (iii) to achieve sustainedperipheral WBC (>1 ´ 109/l) and ANC (>0´5 ´ 109/l) countsin neutropenic patients with severe infections.

Patients. Subjects of the study were immunocompromisedneutropenic patients who met the following inclusioncriteria: diagnosis of a haematological disorder or malig-nancy; insuf®cient response to an appropriate antibacterialand respectively, antifungal therapy; ANC <0´2 ´ 109/ldespite stimulation with G-CSF; the haemopoietic regenera-tion or engraftment took >3 d (evidence of bone marrowaplasia was proved by bone marrow biopsy); deteriorationof the infection combined with progressing organ dys-function. Progressing organ dysfunction was de®ned aspresence of two or more of the following criteria: O2-dependency (FiO2 >0´3), hypotension and tachycardia,catecholamine demand (dopamine > 5 mg/kg/min), creati-nine >132´6 mmol/l, bilirubin >68 mmol/l, Glasgow-Coma-Scale 11-6. Patients with chronic uncontrollable state ofshock and neutropenia due to malignant bone marrowin®ltration were not eligible for the study. Two patientswere permitted to enter the study with ANC >0´2 ´ 109/land short duration of anti-infectious treatment becausethey experienced fulminant septicaemia shortly afterapplication of high-dose chemotherapy and a long periodof bone marrow aplasia was expected.

Characteristics of 30 evaluated patients are presented inTable I.

The infecting agents (Table II) were bacteria in 17patients, proved by positive blood cultures and/or culturesof the infected sites. 13 patients presented with fungalinfections, nine of them with aspergillosis and four withCandida infection. Aspergillosis presented with typical radi-ological ®ndings in chest X-rays in 8/9 patients; 2/9 showedmultiple lesions of the brain on MRI and 1/9 patientsdeveloped necrosis of the palate. Aspergillus species werespeci®ed by needle biopsy or after surgical excision in sevenpatients and by post-mortem examination in the twopatients with cerebral aspergillosis.

The different infecting pathogens, the extent of infectionand the patient's additional risk factors are summarized inTable II.

Donors. A donor was eligible for rhG-CSF-primed leucocytecollection if the following requirements had been met:relative of the patient, ABO compatibility, signed informedconsent, adequate peripheral venous access, normal physicalexamination, normal blood counts, negative HIV andhepatitis serology and a negative venereal disease test.Donor white cells were not tested against the recipient'sserum for the presence of HLA and granulocyte antibodies,nor was HLA typing performed. Among all the donors noadult HLA-identical sibling was available who could havedonated leucocyte concentrates. A negative donor CMV-serology was required if the recipient was CMV negative. Adose of 5 mg/kg rhG-CSF was applied subcutaneously (s.c.) tothe donor the evening before the ®rst collection andcontinued on a daily basis until the leukaphereses werecompleted. The rhG-CSF used in this study was ®lgrastim(NeupogenÒ, Amgen, Thousand Oaks, Calif.). In two casesmore than one cycle (®ve donations) was accepted due to thepersistent leucocyte transfusion dependency of the patient. Ifno appropriate donor for rhG-CSF stimulation could befound, LT were collected from prednisolone-stimulated

q 1999 Blackwell Science Ltd, British Journal of Haematology 106: 689±696

690 C. Peters et al

Table I. Patient characteristics (n�30).

Age at ®rst LT (years) 10 (2±23)

Sex (no. of patients)

Males 18 (60%)

Females 12 (40 %)

Body weight (kg) 28´5 (10±88)

Body surface (m2) 0´95 (0´33±1´93)

Underlying disease (no. of patients)

Leukaemia 20

Solid tumours 6

Other 4

Cause of neutropenia (no. of patients)

Chemotherapy 11

Stem cell transplantation 18

Allo-MSD 4Allo-MUD/HLA-mismatched family donor 10

Autologous 4

Severe aplastic anaemia 1

Days of neutropenia (< ANC 0´2 ´ 109/l)

before ®rst LT 12 (0±52)

Days of G-CSF application before ®rst LT 8´5 (0±45)

Days of infection before ®rst LT 8 (1±28)

Additional complications (no. of patients)Hypotension requiring catecholamines 21

Pathological kidney/liver function 14

Mechanically assisted ventilationand haemo®ltration/dialysis (ICU) 6

Transfusion duration/patient (d) 8 (2±35)

No. of LT/patient 7 (3±65)

No. of patients No. of LT/patient

LT sourcerhG-CSF-primed 17 11 (4±16)

Prednisolone-primed 4 6 (3±23)

Both 9 7 (3±65)

Abbreviations: LT: leucocyte transfusions; MSD: HLA-matched

sibling donor; MUD: HLA-matched unrelated donor; ICU: intensivecare unit.

691Leucocyte Transfusions for Septic Neutropenic Patients


volunteers. All these donors were recruited from the regularvolunteer donor pool of the local blood banks. They wereregistered on an institutionally approved protocol, andappropriate consent forms were signed. All donors werescreened for HIV, hepatitis A, B and C, and CMV. Furthermore aphysical examination was performed and a full bloodinvestigation including a neopterin test had to show normalvalues. An intravenous dose of 50±75 mg (<1´75 m2 bodysurface) or 100 mg (>1´75 m2 body surface) prednisolonewas given 2 h before leucocyte harvest. Leucocyte donationsfrom unrelated prednisolone-primed individuals wererestricted to one donation. A questionnaire on adverseside-effects of stimulation and leukapheresis was completedby the donors after completing the harvest procedure.

All aphereses were performed using peripheral venousaccess. Leucocyte concentrates were collected on a contin-uous-¯ow blood cell separator (Spectra, Cobe laboratories,Lakewood, Col., U.S.A.) or CS 3000 Plus (Baxter, Deer®eld,Ill.). 30 ml trisodiumcitrate was mixed with 500 ml of 6%hydroxyethylstarch and infused in a 1:12 (citrate�HES:whole blood) ratio.

A median of 6´4 litres (range 4´5±8´3) of blood wasprocessed per procedure. Duration of collection was median120 min (range 80±180).

Leucocyte concentrates. All leucocyte concentrates wereirradiated with 30 Gy and infused over 35±60 min througha standard transfusion set without a ®lter within 6 h afterleukapheresis in order to avoid leucocyte clotting andcytokine release. Amphotericin B was applied at intervalsof at least 2 h. LT were given once or twice daily. The patientsreceived premedication with antihistamines and antipyre-tics, in addition to 5 mg/kg rhG-CSF (i.v.) on the days LT wereperformed. Pre-de®ned criteria for LT discontinuation were:infection control, de®ned by stabilization of laboratory andclinical signs of septicaemia or systemic in¯ammatoryresponse syndrome, negative blood cultures, bone marrow

regeneration or engraftment with peripheral WBC >2´0 ´109/l or ANC >1 ´ 109/l at least 16 h after the last LT. Otherindications to stop LT were progressive multiorgan failure orunacceptable side-effects in the donor or the patient.

Statistical analysis. The Kaplan-Meier method was used toestimate the probability of a patient to reach an ANC>500 ´ 109/l and the probability of surviving 100 d follow-ing the ®rst leucocyte transfusion. Log-rank test was used toexamine differences between subgroups in survival curves.

The increase in the patient's WBC from the start of theleucocyte transfusions was analysed with Wilcoxon's signedrank test for prednisolone- or rhG-CSF-elicited leucocytetransfusions.

RESULTS

PatientsThe patients diagnoses included leukaemia (n�20), solidtumours (n�6), X-linked lymphoproliferative disease (n�1),severe aplastic anaemia (n�1), Fanconi anaemia (n�1) andfamilial haemophagocytic lymphohistiocytosis (n�1). Causesof neutropenia were chemotherapy for malignancy (n�11),allogeneic stem cell transplantation (n�14), autologous stemcell reinfusion (n�4) and severe aplastic anaemia (n�1). Thedays with a peripheral ANC <0´2 ´ 109/l were a median of 12(range 0±52). Evidence of infection was present for a medianof 8 d (range 1±28) before the ®rst LT. Two patients who didnot meet the previously mentioned criteria had been includedas an exception: one patient with newly diagnosed ALL withPseudomonas cellucitis and fulminant sepsis. The secondpatient had just ®nished high-dose chemotherapy for rhabdo-myosarcoma when he experienced tumour rupture and had toundergo large abdominal rescue surgery.

Twenty-eight of 30 patients had interventional treatmentwith intravenous rhG-CSF before the initiation of LT.21 patients presented with hypotension requiring requiring

Table II. Underlying infection and concomitant diseases.

Extent of infection*

Total no. Focal (ÿ) Focal (�)² Additional risk factorsInfecting pathogen of patients (no. of patients) (no. of patients) (no. of patients)³

Bacterial 17

Pseudomonas spp. 7 2 5 (2V, 3C)

E. coli 1 ± 1 (1C)Acinetobacter 1 ± 1 (Lips)

Coagulase-negative staphylococci 6 4 2 (2C) (2) TEN; (1) AV, HD; (1) myocarditis

Streptococci 1 ± 1 (1L)Enterocci 1 1 ± (1) ARDS, AV

Fungal 13

Aspergillus spp. 9 ± 9 (1P, 8L, 2B) (1) AV, HD; (1) GvHDCandida 4 4 ± (2) AV, HD; (1) VOD

* All patients had systemic infection with (focal�) or without (focalÿ) focal lesions.

² Localization: B, brain; C, cellulitis; CVL, central venous line tunnel; L, lungs, P, palate; V, vulvar.³ Complications: TEN, toxic epidermal necrosis; AV, mechanical assisted ventilation; HD, haemodialysis; ARDS, adult respiratory distress

syndrome; VOD, veno-occlusive disease.

catecholamines due to septicaemia. 14 patients hadpathological kidney and/or liver function tests >W.H.O.toxicity grade II. Six patients required mechanical ventila-tion and haemodialysis or ®ltration before LT were initiated.Patient characteristics are summarized in Table I.

Seventeen patients (57%) received median 11 (range 4±16)LT only from rhG-CSF-stimulated donors only. Four patients(13%) were transfused with median 6 (range 3±23) concen-trates from prednisolone-stimulated volunteers. Nine patients(30%) received median 7 (range 3±65) concentrates from bothsources. The median duration of treatment with LT per patientwas 8 d (range 2±35). The median number of LT per patientwas 7 (range 3±65) (Table I). If the increment of the patient'sANC was not >500 (generally due to high body weight of thepatient) 1 h after LT we asked for a second leucocyteconcentrate from related or volunteer unrelated donors.

An average of 0´5 ´ 109 PMN/kg (range 0´02±10´3)patient's body weight was given in one leucocyte transfu-sion. A signi®cantly higher amount of leucocytes and PMNwas given with rhG-CSF-elicited concentrates in comparison

to prednisolone-elicited concentrates (median 2´06 ´ 109

PMN/kg versus median 0´25 ´ 109 PMN/kg, P�0´0001).There was a median daily increment of 1´60 ´ 109/l WBC

(1´46 ´ 109/l ANC) after rhG-CSF-elicited LT and a mediandaily increment of 0´43 ´ 109/l WBC (0´45 ´ 109/l ANC) inthe peripheral blood after transfusion of prednisolone-elicitedLT (Fig 1). The probability to reach an ANC >0´5 ´ 109/lwithin 5 d was 83% (Fig 2).

LT were generally well tolerated by the recipientsindependent of the source of LT. Adverse side-effects wereobserved during 39 LT (12´9%).

The most common adverse reactions were fever, chills andshortness of breath during the LT. There was one severeacute respiratory reaction requiring transfusion disconti-nuation after a rhG-CSF-elicited LT. One patient experienceda rupture of the spleen during physiotherapy 1 h after LT.In 14 patients who received LT after allogeneic stem celltransplantation no higher incidences or more severe coursesof acute or chronic GVHD could be observed. The side-effectsdocumented during the administration of 301 leucocyteconcentrates in 30 patients are summarized in Table III.

On day 100 after the ®rst LT 14/17 patients with bacterialinfections were alive and showed no evidence of the infectionwhich had been the indication for the LT. Two patients died


692 C. Peters et al

Fig 1. Patients' absolute neutrophil count 22 h

after ®rst leucocyte transfusion from

prednisolone-primed unrelated or rhG-CSF-stimulated related donors. ANC: absolute

neutrophil count. The increment of patients'

ANC was signi®cantly higher after rhG-CSF-elicited LT.

Fig 2. Patients' probability to reach >500 ANC and >1000 WBC

(´109/l). ANC: absolute neutrophil cell count; WBC: white blood cellcount.

Table III. Tolerability of leucocyte tranfusions (LT).

No. of episodes

(n�76/301)Adverse side-effect (% of LT)

All side-effects 39 (12´9%)

Fever (T>38´58C) 23 (7´6%)

Chills 9 (3´0%)Shortness of breath 3 (1´0%)

Hypoxaemia, bronchospasm 1 (0´3%)

Erythema 2 (0´7%)

Spleen rupture? 1 (0´3%)



of multiorgan failure and one patient of overwhelmingPseudomonas sepsis. In the group of nine patients withinvasive aspergillosis ®ve patients had a complete clearanceof infection. Treatment consisted not only of amphotericin B(partly liposomal preparations) and itraconazole, but in ®veof the patients surgical resection of the lesions was performedafter stabilization of the septic condition. Six patients withfungal infections died: two patients with generalized invasiveaspergillosis including multiple lesions of the brain, onepatient with bacterial septicaemia and pulmonary aspergil-losis, one patient with Candida sepsis and multiorgan failuredue to X-linked lymphoproliferative syndrome. Anotherpatient with generalized Candida sepsis died with coexistinghepatic veno-occlusive disease (VOD) and multiorgan failure.All patients who showed an improvement of the underlyinginfection after discontinuation of LT had a stable orincreasing WBC and ANC. The reasons for discontinuationof LT are summarized in Table IV. The actual survival on day100 after initiation of LT was 70%.

Univariate analysis showed no statistically signi®cantin¯uence on outcome between patient's age, weight, under-lying disease, causes and duration of neutropenia and,infection duration. A signi®cantly better survival wasobserved in patients with bacterial infections compared tothose with fungal infections (P�0´033) and in patientswithout severe additional risk factors such as multiorgandysfunction syndrome, graft-versus-host disease or veno-occlusive disease (P�0´046). The source of LT did notin¯uence the outcome signi®cantly.

DonorsThe total number of leucocyte donors was 165. 39 donorswere stimulated with rhG-CSF and 126 received predni-solone. The median number of rhG-CSF-elicited leucocytedonations was four (range one to 15).

2±4 h after prednisolone application the median WBC was7´95 ´ 109/l (range 3´9±16´5) with ANC of 4´54 (range1´7±14´4). In comparison, after an average of 12 hfollowing the ®rst rhG-CSF application the median WBCcount of the donors was 22´86 (range 4´36±63´4) and theANC was 15´82 (range 3´15±44´27). The platelet countbefore the ®rst leukapheresis was median 255 ´ 109/l (range169±398) in the prednisolone-stimulated donors and

median 195 (range 117±410) in the rhG-CSF-stimulatedgroup.

The side-effects of cytokine administration and of apher-esis in donors were mild and did not exceed W.H.O. grade IItoxicity. Bone pain, myalgia and arthralgia were the mostcommonly reported side-effects. One donor developed mildfever and two donors acquired erythema. Two femaledonors developed weight gain and mild peripheral oedema4 d after daily rhG-CSF application and donations whichdisappeared soon after discontinuation of cytokine applica-tion. No adverse reactions were reported after prednisoloneapplication.

Transfusion products. A total of 301 leucocyte transfusionswere performed. 175 (59%) were from rhG-CSF-stimulatedfamily donors and 126 (41%) products were fromprednisolone-primed volunteers.

In the FACS-analysis concentrates of both sourcescontained an average of 80% neutrophils, 7% monocytes,5% CD3�, 3% CD4�, 2% CD8�, 1% CD19, 1% NK and 1%other cells. The ANC count was signi®cantly higher in thecytokine-elicited concentrates compared to the prednisolone-elicited: 45´3 (range 8´6±143´8) and 13´4 (range 1´5±49´4), respectively (P�0´0001) (Fig 3 and Table V).

Fig 3. Absolute neutrophil cell count of leucocyte concentrates

following prednisolone or rhG-CSF stimulation.

Table IV. Reason for LT discontinuation, outcome on day 100.

Bacterial Fungal Total

(n�17) (n�13) (n�30)

Infection control 14 (82%) 5 (38%) 19

Regeneration/engraftment 2 (12%) 3 (24%) 5

No bene®t 1 (6%) 5 (38%) 6

Survival with clearance of infection 14 survivors 7 survivors

(day 100 pSU) 0´82 6 0´09 0´54 6 0´14|��{z��} 21

P�0´082 0´70 6 0´08

pSU: probability of survival.

The content of WBC and ANCs showed a constant and closecorrelation. The absolute content of monocytes and lympho-cytes in the concentrates from rhG-CSF-stimulateddonors was signi®cantly higher than those fromprednisolone-stimulated volunteers (P�0´001). Thecomposition of the leucocyte concentrates is summarizedin Table V.

DISCUSSION

In recent years the administration of broad-spectrumantibiotics has greatly reduced mortality attributable tobacterial infections. However, in immunocompromisedpatients with additional risk factors such as long-lastingneutropenia, sepsis and pneumonia the outcome remainspoor (Maschmeyer et al, 1994; Elting et al, 1997). Allpatients in our study had clinical and laboratory evidence ofsepsis in addition to a profound neutropenia proved by bonemarrow biopsy. The infection was sometimes aggravated byrenal and pulmonary failure, severe skin problems or organtoxicity after chemotherapy for malignant disease, e.g.hepatic VOD. Six patients were treated in intensive careunits and were on assisted ventilation and haemodialysis.

It is notable that particular patients who suffered from anoverwhelming bacterial sepsis experienced complete clear-ance of the infection. None of the patients with Pseudomonascellulitis had to undergo surgical debridement. The mostremarkable experience was that of a patient who received 65LT during adult respiratory distress syndrome (ARDS),assisted ventilation, haemodialysis and life-threateningrhabdomyolysis due to enterococcal septicaemia. Presently,2 years after this event, the young women is in completeremission of relapsed AML and has only mild neurologicaland organ impairment.

Patients with invasive aspergillosis lacking haematologicalrecovery have a high mortality rate despite antifungaltreatment and the maximum available supportive care(Morrison et al, 1994). To our knowledge, there has notbeen a prospective randomized study dealing with theadministration of LT in patients with an invasive fungalinfection. A retrospective study (Bhatia et al, 1994) did not

®nd any bene®t for neutropenic patients with fungalinfections. However, only 15% of all transfusion productswere analysed for the administered dose of granulocytes. Theoverall survival of patients with invasive noncandidalinfections was 29% in the transfused group and 23%in the nontransfused group. A pilot trial (Dignani et al,1997) on the ef®cacy of rhG-CSF-elicited white blood celltransfusions in patients with neutropenia-related fungalinfections reported favourable responses in 11/15 adultpatients receiving a median of eight transfusions containinga median of 41 ´ 109 neutrophils. Nevertheless, 3 monthsafter starting leucocyte transfusions only three patients werealive; in contrast, 5/9 patients survived invasive aspergillosisin our trial.

Some study patients showed an impressive rapid haemo-poietic recovery or engraftment after only few LT. Afterinitiation of LT an impressive stabilization of overwhelmingsepsis was observed together with bone marrow regenera-tion or engraftment of the transplanted stem cells. Althoughthey all received interventional haemopoietic growth factorsthere were no signs of myeloid proliferation before LT eitherin peripheral blood or in bone marrow. One possibleexplanation might be a cascade-like phenomenon forcytokine secretion resulting in a proliferation of the patient'sstem cells following LT (Saarinen et al, 1995).

A retrospective study of alloimmunization to HLA andneutrophil antigens in 18 patients with chronic granulo-matous disease who had also received repeated granulocytetransfusions revealed WBC antibodies in sera from 14/18patients (Stroncek et al, 1996). In our trial WBC antibodieswere tested infrequently; however, most patients requiringfurther blood products had adequate cell increments. Thismight be due to the severe immunosuppression followingchemotherapy and irradiation. Nonetheless, careful screen-ing for WBC antibodies is indicated, particularly after adversereactions. None of our patients received LT from an HLA-identical donor. Therefore we can not comment on whetheran inadequate increment of patient's peripheral WBC wasin¯uenced by HLA disparities. A special task of our study wasto evaluate the acute side-effects related to LT in patientswith multiple organ dysfunction. In particular, those who


694 C. Peters et al

Table V. Composition of leucocyte concentrates.

Parameter Total rhG-CSF-primed Prednisolone-primed

No. of leucocyte concentrates 301 175 126Volume (ml) 207 (55±548) 200 (60±548) 209 (55±285)

Absolute WBC (´109/l) 30´9 (3±149´8) 53´7 (3±149´8) 19´3 (4´2±53´1)

Neutrophils 25´0 (1´5±143´8) 45´3 (8´6±143´8) 13´4 (1´5±49´4)Monocytes 0´7 (0±10´7) 1´8 (0±10´7) 0´4 (0±3´6)

Lymphocytes 4´9 (0±20´6) 5´5 (0±20´6) 4´0 (0´2±19´9)

Platelets (´1011) 1´04 (0´12±6´23) 0´90 (0´12±2´58) 2´55 (0´177±6´23)

Erythrocytes (ml) 30 (6±103) 29 (7±103) 30 (6±62)Dose of ANC/concentrate/kg (´109) 0´5 (0´02±10´3) 2´06 (0´12±10´3) 0´25 (0´02±1´88)

Dose of PMN/concentrate/body surface (´109) 15 (0´6±309) 61´8 (3´6±309) 7´5 (0´6±56´4)

WBC: white blood cell count; ANC: absolute neutrophil count; PMN: polymorph neutrophils.



received amphotericin B were closely monitored for pulmon-ary side-effects during and after LT. In some patients weobserved some adverse reactions during LT, in agreementwith others (Dutcher et al, 1989). Two serious adverse eventswere observed in connection with LT. One patient developedan acute pulmonary transfusion reaction. Unfortunately HL-A antibodies and granulocyte antibodies from the donoragainst the recipient could not be tested and therefore thereason for this reaction remains unclear. Another patientwho had received interventional LT experienced a rupture ofthe spleen; nevertheless, he survived this event with rapidtransfusions of red cells and prompt surgery. Two patientshad mild shortness of breath and/or tachypnoea during LTwhich did not require special therapeutic intervention.

Routine testing of coagulation parameters (partial throm-boplastin time, ®brinogen, D-dimers) did not reveal signs ofhyper®brinolysis in any patients receiving LT.

Two patients experienced cerebral haemorrhage >24 hafter the last LT, both with generalized invasive aspergillosisfor which this complication is well described (Takahashi et al,1998).

Our observations suggest that LT obtained by centrifuga-tion techniques irrespective of the stimulation modalitycan be administered safely even in patients with pulmon-ary complications and even after a short interval ofamphotericin B application. However, great care andattention must be given when administering this modalityto severely ill patients.

Leucocyte concentrates were transfused as soon aspossible after harvesting and irradiation to avoid cytokinerelease. In contrast to other recommendations (Tobias &Schleien, 1991), the leucocyte concentrates were infusedquickly (35±60 min) since we observed leucocyte clottingduring slower infusion. In most circumstances this wastolerated well. Nevertheless, careful monitoring of vitalparameters during and after LT are warranted.

Adverse side-effects in donors from both stimulation andleukapheresis procedures were mild and con®rmed theresults described elsewhere (Price, 1995).

Although the literature concerning infection therapy withleucocytes refers to these concentrates mainly as `granulo-cyte concentrates, we feel they should be termed `leucocyteconcentrates' because they consist of only 80% granulocytes.Whether other cell fractions in¯uence infection therapy(e.g. monocytes) should be a subject for future studies.

The median WBC, ANC and lymphocyte count of theconcentrates obtained with similar apheresis procedureswere signi®cantly higher after stimulation of donors withrhG-CSF when compared to those who received predni-solone. This con®rmed observations made by others that asigni®cantly higher yield was found in normal subjects afterstimulation with rhG-CSF than with dexamethasone (Lileset al, 1997). Although the median WBC and ANC wasremarkably lower in products harvested from prednisolone-primed volunteers, the latter could be enough to maintainthe neutrophil count >0´5 ´ 109/l in children. Nonetheless,rhG-CSF seems to be a much more effective agent for raisingthe ANC than glucocorticoids and might be the moreappropriate stimulant for neutrophils in healthy donors.

Very recently it has been demonstrated (Dale et al, 1998)that the combination of dexamethasone and rhG-CSFenabled collection of a mean of 77´4 ´ 109 PMN with near-normal functional properties.

As a minimum, a daily infusion of 2±3 ´ 1010 PMN (not<1 ´ 1010) should be given to patients with severe persistentneutropenia (Strauss, 1993). Our median cell dose perconcentrate was 1´9 ´ 1010/m2 body surface. When patientswith a high body weight did not receive enough PMNs withone concentrate, a second harvest of leucocytes wasorganized for the same day. We transfused LT once or twicedaily (also on weekends) to reach or maintain peripheralneutrophil counts >1 ´ 109/l WBC or 500 ´ 109/l ANC inthe peripheral blood. 83% of our patients achieved andmaintained an ANC >0´5 ´ 109/l within 5 d, whichappeared to be the critical level for controlling infections.

Jendiroba et al (1998) have described recently that G-CSFgiven every other day to healthy donors is as effective as adaily G-CSF administration for the recruitment of granulo-cytes, which makes the mobilization procedure morecost-effective.

In conclusion, leucocyte transfusions are feasible incritically ill patients with severe neutropenia and bacterialor fungal infections. The side-effects of stimulation andapheresis are tolerable for the donors. The signi®cantbene®cial effect of LT for the control of severe infections inpatients unresponsive to adequate conventional treatmentshould be studied further within comparable patient groupsin a randomized manner.

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Leucocyte transfusions from rhG-CSF or prednisolone stimulated donors for treatment of severe infections in immunocompromised neutropenic patients