quality assessment for auto transplant

Bone Marrow Transplantation (2001) 27, 463–470 2001 Nature Publishing Group All rights reserved 0268–3369/01 $15.00

www.nature.com/bmt

Special report

A European reference protocol for quality assessment and clinicalvalidation of autologous haematopoietic blood progenitor and stem cellgrafts

S Serke and HE Johnsen1Department of Haematology-Oncology, Humbolt-Universitat, Berlin, Germany; and 2Department of Haematology, Herlev Hospital,University of Copenhagen, Denmark

Summary:

Recently, the regulatory authorities have begun to showinterest in haematopoietic stem cell products. On a pro-fessional rather than a regulatory basis, the Inter-national Society for Hematotherapy and Graft Engin-eering (ISHAGE) has established the Foundation for theAccreditation of Haematopoietic Cell Therapy(FACHT), which has drawn up guidelines for standardsand accreditation of such activity. In Europe, the regu-latory environment with regard to haematopoietic stemcell grafts, processing and storage are currently lessstringent. However, in 1998 the European JointAccreditation Committee Euro-ISHAGE/EBMT(JACIE) prepared a regulatory document ‘Standardsfor Blood and Marrow Progenitor Cell Collection, Pro-cessing and Transplantation’ which was approved bythe EBMT General Assembly. The major objectiveswere to promote quality of medical and laboratorypractice in haematopoietic progenitor cell transplan-tation. The standards extend and detail the pre-existingactivity of EBMT centres including all phases of collec-tion, processing and administration of these cells. Thisis the platform for the proposed reference protocol forCD34! cell enumeration and clinical validation of qual-ity assessment to ensure that appropriate standards ofwork and product quality are established and will bemaintained. Bone Marrow Transplantation (2001) 27,463–470.Keywords: CD34; flow cytometry; standardization; refer-ence protocol; haematopoietic stem cell transplantation;prediction of engraftment

Current indications for high-dose therapy as adjuvant treat-ment are now being applied to newly diagnosed patients.This development has required evidence-based quality andsafety assessment of haematopoietic stem cell grafts and

Correspondence: Dr HE Johnsen, Stem Cell Laboratory, Department ofHaematology, Herlev Hospital, University of Copenhagen, 2730 Herlev,DenmarkReceived 14 July 2000; accepted 2 January 2001

introduced a new institution in medicine, the stem cell lab-oratory. In most cases this speciality has evolved from orwithin hematological research laboratories. However, theincreased routine technologies applied in quality evalu-ation, ex vivo manipulation and safety assessment in stemcell handling, naturally places this activity in transfusionmedicine in close collaboration with the clinic.In 1998, the European Joint Accreditation Committee

Euro-ISHAGE/EBMT (JACIE) prepared a regulatory docu-ment ‘Standards for Blood and Marrow Progenitor CellCollection, Processing and Transplantation’,1 which wasapproved by the EBMT General Assembly. In the sameyear the EBMT Board established a sub-committee on‘Quality Assessment of Haematopoietic Stem Cell Grafts’with the aim within the EBMT centres to: (1) Analyse theexisting situation regarding quality assessment of stem cellgrafts; (2) identify major problems in quality assessmentsand establish scientific protocols; (3) describe guidelinesfor the handling of auto- and allografts; (4) publish resultsof sub-committee studies; and (5) contribute a document tothe EBMT operational manual.The current situation regarding CD34! cell enumeration

has been analysed and published as an European Surveyon flow cytometric determination of CD34-expressingcells.2 The problems in quality assessment were disclosedduring the first sub-committee meeting held at the annual1999 EBMT meeting. One major concern was paragraphD4.000 on quality management in the standard documentstating: ‘D4.130 A nucleated cell count shall be performedfor any component after collection and after any subsequentprocessing (if applicable). D4.131 CD34! cell count shallbe performed. D4.132 The target should be to transfuse aminimum of 2 " 106 CD34! cells per kg body weight, butlower numbers may be acceptable in specific cases. (Thisdoes not apply to bone marrow or cord blood)’.1The 1999 subcommittee meeting concluded that the

numbers given in paragraph D4.132 were inappropriate asno convincing data exist from single- or multicentre studiesto document a common protocol for CD34! cell enumer-ation and strategy for clinical validation of numbers. Thisreflects a problem in preparing guidelines for qualityassessments and is the background for this proposal of aEuropean Reference Protocol on CD34! cell enumeration

European reference protocolS Serke and HE Johnsen

464

Bone Marrow Transplantation

by flow cytometry and a strategy for its validation by clini-cal end-points.

Laboratory analysis of progenitor and stem cells

First step in quality assessment was CD34standardisation

Culture assays of colony-forming cells were originally per-formed, but due to the inconvenience of this method severallaboratories focused on flow cytometric analysis of CD34!

cells3,4 in quality assessment and the first steps were takenin 1991 by Serke of Berlin, Siena of Milan and Fritsch ofVienna.5–8 This development was promoted by the farsee-ing Mulhouse Group when in early 1992, they hosted theFirst European Workshop on Stem Cell Determination andStandardization. The resulting Milan/Mulhouse manualis still a valuable procedure reference in laboratorypractice.9,10The subsequent years have resulted in several publi-

cations about the standardisation of flow cytometry analy-sis11–25 as well as reports on the clinical implication of theenumeration of CD34! cells.24,26–37 The CD34 antigen isstage-specific and identifies cells in the early stages ofhaemopoietic differentiation. This population, therefore,contains progenitors committed to the myeloid, erythroid,megakaryocytic and lymphoid lineages, as well as primitiveprogenitors and stem cells capable of long-term reconsti-tution.38–41 Enumeration of CD34! cells has been shown tobe useful in the procedure of stem cell mobilisation andharvest from blood for transplantation and it seemsinformative for the prediction of fast or delayed three-lin-eage engraftment and blood cell recovery following high-dose therapy.5,6,26–29We do not yet know the minimal safe number of CD34!

cells needed for clinical engraftment of all lineages, as thismay vary depending on the stem and progenitor cell subsetcomposition in a given patient or autograft.42–46 However,we do know that a graft content of more than 5–10 million

Table 1a Overall median number of CD34+ cells from nine studies of haematological recovery following autologous stem cell reinfusion

References Overall median CD34! Number of patients Overall median days to Overall median dayscells "106/kg studied (range) ANC #0.5 " 109/l to platelet #20 " 109/l

(range) (range) (range)

26, 30–37 6.0 (4.3–10.6) 1688 (50–642) 11 (2–93) 12 (0–1000!)

Table 1b Haematological recovery in subgroups of patients reinfused with varying low number of CD34! cells

References No. of CD34! cells Number of patients Overall median days to Overall median daysANC #0.5 " 109/l to platelet #20 " 109/l

(95% CI) (95% CI)

55 $1 " 106/kg 37 15 (11–21) 30 (22–43)26, 53, 54, 56 $2 " 106/kg 159 15 (13–18) 17 (14–20)28, 49–52, 58–60 $2.5 " 106/kg 116 13 (11–15) 17 (14–21)61 $3 " 106/kg 32 12 (9–18) 22 (16–32)48 $5 " 106/kg 16 11 (10–13) 21 (15–40)

CD34! cells per kg body weight is safe, resulting in fastrecovery of ANC and platelets before days 14 and 21,respectively, in a major fraction of patients and, mostimportant, only have a minor risk for engraftment fail-ures.26,28,47–54 In a survey of 1600 patients from nine pub-lished papers, including a minimum of 50 patients each, itis concluded that the overall median time to ANC and plate-let recovery is 11 days (2–93) and 11 days (0–1000!),respectively. From 15 studies with information of reinfusedlow numbers (Table 1a and b), it is concluded that no defi-nite lower level exists to document groups of patients athigh risk for prolonged recovery, based on CD34 numbersbelow 1 million/kg,55 2 million/kg,26,53–57 2.5 million/kg,28,49–52,58–60 3 million/kg61 or 5 million/kg.48 From suchdata, it is obvious that we will never obtain an exact numberof CD34! cells delineating an insufficient or safe graft. Wehave to reconsider these terms and change exact numbersinto probabilities of obtaining clinical efficacy and avoidingtoxicity evaluated by proper end-points (vide infra).

The final step in quality assessment is clinical validation

In general, new technologies, moving from the laboratorybench to the clinic, have to pass different stages before theyare validated. In parallel with therapeutic studies passingphase I–IV, the four different phases believed to be neces-sary and informative in clinical validation of, for example,CD34! cell enumeration as described in Table 2. In thefirst phase, the CD34 technique was established in the lab-oratory and analysed for specificity, sensitivity, reproduci-bility and accuracy.5–10,16–26 The subsequent second phase,documented a likely clinical influence by single centresanalysing retrospective material/data.27–34,36,37 The thirdphase prepared convincing single centre prospective evalu-ation51–58 evolving into the most important phase four, amulticentre prospective evaluation based upon importantclinical end-points (Table 2). Ideally, phases II–III docu-ment the usefulness, convincing one or more centres to par-ticipate in a phase IV validation trial, which however in this


465Table 2 The phases for clinical validation of a laboratory techniquewith special emphasis on CD34 enumeration

Phases Studies References

First phase The technique established 5–10and determination of 16–26precision and accuracy

⇓Second phase Retrospective, single center 27–37

data analysed for directclinical influence

⇓Third phase Prospective, single/few 51–58

center studies evaluated forclinical outcome

⇓Fourth phase Prospective, multicenter

study including relevant, andmore extended clinicalendpoint as proposed

case has not yet been performed. In an attempt to establish aphase IV study within the EBMT we propose the EuropeanReference Protocol for quality assessment by CD34! cellenumeration with clinical validation based on end-pointsrelevant for autograft-mediated supportive therapy (videinfra).

A European Reference Protocol on CD34! cellenumeration (Table 3)

Recently, several reports have presented protocols on flowcytometry enumeration and several companies have

Table 3 The European Reference Protocol for CD34 enumeration inblood and leukapheresis products

Time of analysis 0–24 h after end of harvest, kept at roomtemperature, no agitation

Cell counting Leukocyte count on a 1:10 PBS dilutedproduct twice (two different samples) byan automated analyser

Test volume 100 %l blood or leukapheresis product(about 0.5–1.0 " 106 cells per test)

Test antibody CD34 class III antibody, appropriatelytitrated and a pan-CD45 antibody,appropriately titrated.

Control antibody Defined by the userAntibody dilution Defined by an appropriate titrationIncubation Defined by the user, normally 10–15 minErythrocyte lysis Ammonium chloride-based RBS lysing

solution before antibody incubationWashes No wash necessary, free to useFixation Not necessary, free to use if analysis is

done within 4 hAnalysis A minimum of 50 000 cellular events –

debris eliminated by live gate on CD45!

cellsCD34 estimation Total number in leukapheresis product per

kg patient weight based on(1) CD34% and total number of CD45!

cells in product or(2) volumetric measurement


announced standard kits for absolute enumeration ofCD34! cells.11–25 These protocols seem different at severallevels depending on the designer and none has been provedsuperior to the others, if it fulfills ‘the state-of-the-art’ rec-ommendation. The major conclusion from the work doneover the last decade is that training diminishes the interlab-oratory variations with only minor differences betweenfirst- and second-line strategies. The most important changein relation to the simple Milan/Mulhouse protocol is therecommendation of a ‘lyse no wash’ technique. This tech-nique eliminates any potential cell loss caused by cell wash-ing which has been shown to reduce the number ofCD34! events.62One necessary step in such a strategy exemplified by this

European protocol (Table 3) is to add the pan-CD45 anti-body in order to be able to quantify leukocytes and dis-criminate these from erythroblasts and non-lysed erythro-cytes in the live gate, ie in obtaining an accuratedenominator for the CD34% calculation.13,15,17,63 The num-ber of CD34! events present in the low SSC region is stillcalculated on the basis of a minimum population in therange of 50 to 100 events positively stained by a CD34class III antibody.5,6,10,15,64The percentage of CD34! cells can then be calculated by

the number of CD34! events divided by the denominator(&CD45! leukocytes). The total number of CD34! cellsper volume can subsequently be calculated by multipli-cation of the CD34% and the leukocyte count obtainedfrom a haematology analyzer (the dual platform technique).However, a more accurate determination may require avolumetric measurement added into the analysis which canbe obtained, for example, by introducing a known numberof reference particles per volume of sample65 or by scan-ning an exact volume66–68 – the so-called single platformtechnique.63 These methods have the advantage of beingindependent of variation in leukocyte enumeration by thehaematology analyser, but precision in pipetting the samplevolume to get a correct absolute count is important.CD34 class III antibodies with sufficient activity, inde-

pendent of fluorochrome conjugation can be used if theyhave been appropriately titrated before use. The isotypecontrol should also be selected after sufficient testing. ClassI and II antibodies are not recommended.9,17,39,40The most important step, however, may be choosing the

red blood cell lysing reagent as this is not an ‘innocentby-stander’ in sample preparation. The different chemicals,particularly fixatives, may affect the cell and the stainingintensity.Introduction of such a reference protocol (Table 3) will

allow each stem cell laboratory to choose their own meth-odology, including commercial kits available on the mar-ket. The consequence may be that it will be impossible todiminish interlaboratory variation in future analysis. On theother hand, it may be that various methods used in differentlaboratories in accordance with a reference protocol willproduce interlaboratory variations, which are comparableby a strictly standardised method. Future bench workshopsmay give the answer to these questions.However, it is an open question whether such work will

be worth the effort as the benefit obtained by standardisedanalysis may be decreased or even eliminated by graft pro-


466


cessing differences including thawing/freezing, ex vivo cul-ture, purging, selection, etc. At the same time moreimportant clinical questions are present regarding the pre-diction of ‘poor mobilisers’ and the prediction of ‘insuf-ficient autografts’ with reduced efficacy and increased riskof side-effects following graft reinfusion (Table 4).Laboratories wanting to shift from one method to another

should compare the old method with a new one by theBland and Altman method based on graphical techniques,simple calculations and assessment of repeatability, asrecommended in a recent report from this committee.68

Clinical end-points and objectives in supportivetherapy

Engraftment and haematopoietic recovery are not theonly clinical end-points in supportive therapy

Supportive reinfusion of haematopoietic stem and progeni-tor cells following marrow ablative therapy ultimately aimsto re-establish haematopoiesis following an initial recoveryof end-stage blood circulating cells to a level necessary forreducing the risk of side-effects, such as infections, bleed-ing or anaemia. This level has traditionally been #0.5 "109 neutrophils/l, #20 " 109 platelets/l and #2% ! 0.2" 1012/l reticulocytes. So far no levels have been estab-lished for recovery of the immune system, ie B cells, Tcells, NK cells and monocytes.Consequently, engraftment has most often been evalu-

ated by time to three-lineage recovery by calculating thetime-dependent probability of reaching the above given lev-els following blood cell nadir after transplantation. A sim-ple description of the observed data can be performed bymeans of Kaplan–Meier plots. The prognostic value of dif-ferent factors as, for example, CD34! cell numbers, subsetnumbers as well as other factors of proposed importancecan be evaluated by log-rank test, for example. However,this strategy for analysis was established in the early daysof allogeneic and autologous bone marrow transplantationand needs to be re-evaluated in actual practice. The use of

Table 4 Proposed objectives, end-points and grading for clinical validation of quality assessment of haematopoietic grafts

Objectives End points Grading

Primary To study the efficacy of stem cell graft Days of hospitalisation, interventional Good:reinfusion antibiotic and antifungal treatment and $21 days in hospital and no

transfusion of blood components transfusionsPoor:#21 days in hospital or on continuousantibiotic or antifungal treatment

Secondary To evaluate toxicity following stem Common toxicity (CTC) as defined by Good: toxicity grade 0–1cell graft reinfusion WHO or derived references (ECOG, Poor: toxicity grade 3–4

SWOG, etc) for mucositis, dermatitis For haematological toxicity: see Tableand enteritis, as well as grading of 4haematological toxicity

Tertiary To evaluate safety following stem cell Regimen-related death or disease Good:graft reinfusion recurrence Alive and in complete remission day

90Poor: Death or disease progression

blood progenitor grafts and growth factor administrationmay have changed the initial correlation between blood cellrecovery and clinical events. Health, economic and life-quality considerations need to be included in assessment ofsupportive haematopoietic cell therapy. Of less importancemay be the side-effects, eg haematological toxicity, asdefined by time to three lineage recovery or engraftment.We propose a change to assessing the probabilities ofobtaining primary, secondary or tertiary end-points. Suchan assessment will allow us to handle each patient individu-ally in daily practice by predicting, not only the efficacy,but also the risk of side-effects as described in Table 4.

Proposed clinical end-points in supportive therapy

In the last decade, hundreds of reports have based theirconclusions about quality assessment on surrogate markersand as suggested it seems to be time for a move towardsevaluation on the basis of clinically relevant factors. Suchdata, although published from single centres, are not avail-able from multicentre trials and have to be generated in aprospective manner.It is worth mentioning that the introduction of peripheral

blood autografting has not changed the risk of documentedinfections compared to the use of conventional bone mar-row grafts, although a faster neutrophil recovery is substan-tially documented.69,70 Furthermore, the risk of thrombocy-topenic bleeding has neither been evaluated nor formallydocumented as being reduced by an increase of blood plate-lets from below to above 10, 20 or 50 " 109 platelets/lduring recovery.30It needs to be considered in future clinical trials whether:

(1) Primary end-points should be events documenting effi-cacy of clinical importance, ie the risk of severe bleedingand infections including antibiotic administration and trans-fusion of blood components as well as time in hospital.(2) Secondary end-points should be an evaluation of tox-icity in accordance with, for example, Common ToxicityCriteria (CTC),71 including a time-dependent grading ofhaematological toxicity. (3) Tertiary end-points should be


467the risk of regimen-related death or disease progressionwithin the first 90 days following graft reinfusion.The proposed objectives, end-points and grading for

clinical validation are given in Table 4. It takes into con-sideration actual clinical practice, as well as knowledge inbiology. A time-dependent grading of efficacy is proposedwith day 21 as the acceptable maximum time in hospital,which together with antibiotics, antifungal or transfusiontherapy delineates three groups: a good outcome forpatients discharged before day 22 with no transfusion ther-apy; a poor outcome for patients who stay in hospital morethan 21 days on continuous therapy; and finally an inter-mediate group of patients who are discharged before day22, but receive transfusions or interventional (notprophylactically) antibiotics/antifungal therapy.Concerning toxicity, the proposed grading system is

time-independent with outcome assessed according to theWHO recommendation for grading of organ toxicity asgood if toxicity is grade 0–1, poor if grade 3–4 and asintermediate if grade 2–3, and can vary depending on theorgan involved. By tradition haematological toxicity has tobe time-dependent and an evaluation is proposed in Table5.Finally, evaluation of mortality and disease recurrence indi-cate good outcome if patients are alive with no diseaserecurrence up to day 90 and poor if the high-dose therapyis followed by death or relapse before day 90. An inter-mediate group of patients are delineated as ‘not good’ or‘not poor’.A statistical evaluation would require firstly a judgment

of the actual end-point as good, acceptable or poor. Appro-priate statistical methods would then be applied to give thebest possible description of a correlation between the factorunder consideration and the probabilities of specific end-points as for example described in Figure 1.

Table 5 Proposed time-dependent grading of haematological toxicity (see Table 3)

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Figure 1 For patients undergoing haematopoietic stem cell transplan-tation, the Figure shows the estimated relative proportion of patients withthe end-point in question (see Table 4) graded as ‘Good’, ‘Acceptable’ or‘Poor’ and analysed for influence of the given risk factor, eg CD34 num-ber. At any number of CD34! cells transplantated one can read the riskfor poor or good outcome of the supportive therapy.

Future directions

Quality assessment of haematopoietic stem cell grafts

The success of stem cell laboratories during the last decadeis documented by the elimination of the risk of engraftmentfailure – not by the introduction of PBSPC grafting, butvery likely due to improved quality assessment procedures.This has been achieved by hard work on a very simpletechnology allowing us to identify patients at risk ofengraftment failure, due to either lack of mobilisation orobtaining an insufficient harvest.47Future flow cytometry techniques should comply with

the basic recommendations given in this report (Table 3)and be validated clinically by carefully planned prospectivemulticentre trials on efficacy and toxicity (Table 4). Wewould like to stress that this proposal does not intend to


468


change or compete with guidelines for enumeration recentlypublished.17,63,72,73 On the contrary, the European ReferenceProtocol attempts to cover all techniques providing thebasis for starting prospective multicentre studies. Hope-fully, when such data are available, the international com-munity will reach a consensus on guidelines and referenceprotocols for supportive cell therapy in an attempt to makehigh-dose therapy a safe and cost-beneficial procedure. Atthat time we finally may be able to fulfil the paragraphD4.000 on Quality Management in the EBMT/ISHAGEstandard.1 Much work has to be done in EBMT multicentreregistration studies to obtain that goal.

References

1 The Joint Accreditation Committee of ISHAGE-Europe andEBMT. Standards for blood and marrow progenitor cell pro-cessing, collection and transplantation. 1998.

2 Serke S, Johnsen H, Huhn D et al. European survey of theflow cytometric determination of CD34-expression cells.Cytotherapy 1999; 1: 343–347.

3 Krause DS, Fackler MJ, Civin CI et al. CD34: structure,biology, and clinical utility. Blood 1996; 87: 1–13.

4 Civin CI, Strauss LC, Brovall C et al. Antigenic analysis ofhematopoiesis III. A hematopoietic progenitor cell surfaceantigen defined by a monoclonal antibody raised against KG-1a cells. J Immunol 1984; 133: 157–165.

5 Serke S, Sauberlich S, Huhn D. Multiparameter flow-cyto-metrical quantitation of circulating CD34(!)-cells: correlationto the quantitation of circulating haemopoietic progenitor cellsby in vitro colony-assay. Br J Haematol 1991; 77: 453–459.

6 Siena S, Bregni M, Brando B et al. Flow cytometry for clinicalestimation of circulating hematopoietic progenitors for auto-logous transplantation in cancer patients. Blood 1991; 77:400–409.

7 Fritsch G, Emminger W, Buchinger P et al. CD34 analysis inperipheral blood correlates with colony forming capacity – anupdate. Prog Clin Biol Res 1992; 377: 531–536.

8 Ravagnani F, Siena S, Bregni M et al. Methodologies to esti-mate circulating hematopoietic progenitors for autologoustransplantation in cancer patients. Haematologica 1991; 76(Suppl. 1): 46–49.

9 Hematopoietic Stem Cells. The Mulhouse Manual. In: WunderE, Sovalat H, Henon P, Serke S (eds). Alpha Med Press: 1994,pp 1–317.

10 Wunder E, Sovalat H, Fritsch G et al. Report on the EuropeanWorkshop on Peripheral Blood Stem Cell Determination andStandardization – Mulhouse, France, 6–8 and 14–15 February1992. J Hematother 1992; 1: 131–142.

11 Inaba T, Shimazaki C, Ashihara E et al. Two-color flow cyto-metric analysis of CD34-positive peripheral blood stem cellsmobilized by recombinant human granulocyte-colony stimul-ating factor for autotransplantation. Prog Clin Biol Res 1992;377: 561–567.

12 Read EJ, O’Shaughnessy JA, Yu MY et al. Flow cytometricquantitation of circulating hematopoietic progenitor cells inbreast cancer patients on chemotherapy. Prog Clin Biol Res1992; 377: 523–530.

13 Bender JG, Williams SF, Myers S et al. Characterization ofchemotherapy mobilized peripheral blood progenitor cells foruse in autologous stem cell transplantation. Bone MarrowTransplant 1992; 10: 281–285.

14 Di Nicola M, Siena S, Bregni M et al. Quantization of CD34!

peripheral blood hematopoietic progenitors for autografting incancer patients. Int J Artif Organs 1993; 16 (Suppl.5): 80–82.

15 Bender JG, Unverzagt KL, Walker DE et al. Characterizationof CD34! cells mobilized to the peripheral blood during therecovery from cyclophosphamide chemotherapy. Prog ClinBiol Res 1992; 377: 575–582.

16 Brecher ME, Sims L, Schmitz J et al. North American multi-center study on flow cytometric enumeration of CD34! hema-topoietic stem cells (see comments). J Hematother 1996; 5:227–236.

17 Sutherland DR, Anderson L, Keeney M et al. The ISHAGEguidelines for CD34! cell determination by flow cytometry.International Society of Hematotherapy and Graft Engineer-ing. J Hematother 1996; 5: 213–226.

18 Serke S, Arseniev L, Watts M et al. Imprecision of countingCFU-GM colonies and CD34-expressing cells. Bone MarrowTransplant 1997; 20: 57–61.

19 Macey MG, McCarthy DA, van Agthoven A et al. Howshould CD34! cells be analysed? A study of three classes ofantibody and five leucocyte preparation procedures. J ImmunolMeth 1997; 204: 175–188.

20 Lowdell MW, Bainbridge DR. External quality assurance forCD34 cell enumeration – results of a preliminary national trial.Royal Microscopical Society Clinical Flow Cytometry GroupQA Schemes. Bone Marrow Transplant 1996; 17: 849–853.

21 Urashima M, Ohkawara J, Hoshi Y et al. Peripheral bloodprogenitor cell transplantation estimated by three-colour(CD34, HLA-DR, CD33) flow cytometry. Acta Haematol1994; 92: 23–28.

22 Knape CC. Standardization of absolute CD34 cell enumer-ation (letter, comment). J Hematother 1996; 5: 211–212.

23 Lumley MA, McDonald DF, Czarnecka HM et al. Qualityassurance of CD34! cell estimation in leucapheresis products.Bone Marrow Transplant 1996; 18: 791–796.

24 Remacha AF, Martino R, Sureda A et al. Changes in reticulo-cyte fractions during peripheral stem cell harvesting: role inmonitoring stem cell collection. Bone Marrow Transplant1996; 17: 163–168.

25 Johnsen HE, Knudsen LM. Nordic flow cytometry standardsfor CD34! cell enumeration in blood and leukapheresis pro-ducts: report from the second Nordic Workshop. Nordic StemCell Laboratory Group (NSCL-G). J Hematother 1996; 5:237–245.

26 Johnsen HE, Baech J, Nicolaisen K et al. Validation of theNordic flow cytometry standard for CD34! Cell enumerationin blood and autografts: report from the Third Workshop. JHematother 1999; 8: 15–28.

27 Knudsen LM, Gaarsdal E, Jensen L et al. Evaluation of mobil-ized CD34! cell counts to guide timing and yield of large-scale collection by leukapheresis. J Hematother 1998; 7:45–52.

28 Weaver CH, Hazelton B, Birch R et al. An analysis ofengraftment kinetics as a function of the CD34 content of per-ipheral blood progenitor cell collections in 692 patients afterthe administration of myeloablative chemotherapy. Blood1995; 86: 3961–3969.

29 Knudsen LM, Gaarsdal E, Jensen L et al. Improved primingfor mobilization of and optimal timing for harvest of periph-eral blood stem cells. J Hematother 1996; 5: 399–406.

30 Bolwell BJ, Goormastic M, Andresen S et al. Platelet trans-fusion requirements during autologous peripheral blood pro-genitor cell transplantation correlate with the pretransplantplatelet count. Bone Marrow Transplant 1997; 20: 459–463.

31 Lowenthal RM, Faberes C, Marit G et al. Factors influencinghaemopoietic recovery following chemotherapy-mobilisedautologous peripheral blood progenitor cell transplantation forhaematological malignancies: a retrospective analysis of a 10-year single institution experience. Bone Marrow Transplant1998; 22: 763–770.


46932 Stewart DA, Guo D, Luider J et al. Factors predictingengraftment of autologous blood stem cells: CD34! subsetsinferior to the total CD34! cell dose. Bone Marrow Trans-plant 1999; 23: 1237–1243.

33 Reiffers J, Faberes C, Boiron JM et al. Peripheral blood pro-genitor cell transplantation in 118 patients with hematologicalmalignancies: analysis of factors affecting the rate ofengraftment. J Hematother 1994; 3: 185–191.

34 Nademanee A, Sniecinski I, Schmidt GM et al. High-dosetherapy followed by autologous peripheral-blood stem-celltransplantation for patients with Hodgkin’s disease and non-Hodgkin’s lymphoma using unprimed and granulocyte col-ony-stimulating factor-mobilized peripheral-blood stem cells.J Clin Oncol 1994; 12: 2176–2186.

35 Pettengell R, Morgenstern GR, Woll PJ et al. Peripheral bloodprogenitor cell transplantation in lymphoma and leukemiausing a single apheresis. Blood 1993; 82: 3770–3777.

36 Schwartzberg L, Birch R, Blanco R et al. Rapid and sustainedhematopoietic reconstitution by peripheral blood stem cellinfusion alone following high-dose chemotherapy. Bone Mar-row Transplant 1993; 11: 369–374.

37 Morton J, Morton A, Bird R et al. Predictors for optimal mobi-lization and subsequent engraftment of peripheral blood pro-genitor cells following intermediate dose cyclophosphamideand G-CSF. Leuk Res 1997; 21: 21–27.

38 Haas R, Witt B , Mohle R et al. Sustained long-term hemato-poiesis after myeloablative therapy with peripheral blood pro-genitor cell support. Blood 1995; 85: 3754–3761.

39 Steen R, Tjonnfjord GE, Gaudernack G et al. Differences inthe distribution of CD34 epitopes on normal haemopoieticprogenitor cells and leukaemic blast cells. Br J Haematol1996; 94: 597–605.

40 Sutherland DR, Keating A. The CD34 antigen: structure,biology, and potential clinical applications. J Hematother1992; 1: 115–129.

41 Civin CI, Almeida-Porada G, Lee MJ et al. Sustained, retrans-plantable, multilineage engraftment of highly purified adulthuman bone marrow stem cells in vivo. Blood 1996; 88:4102–4109.

42 Dercksen MW, Weimar IS, Richel DJ et al. The value of flowcytometric analysis of platelet glycoprotein expression ofCD34! cells measured under conditions that prevent P-sel-ectin-mediated binding of platelets. Blood 1995; 86: 3771–3782.

43 Dercksen MW, Rodenhuis S, Dirkson MK et al. Subsets ofCD34! cells and rapid hematopoietic recovery after periph-eral-blood stem-cell transplantation. J Clin Oncol 1995; 13:1922–1932.

44 Bender JG, Unverzagt K, Walker DE et al. Phenotypic analy-sis and characterization of CD34! cells from normal humanbone marrow, cord blood, peripheral blood, and mobilizedperipheral blood from patients undergoing autologous stemcell transplantation. Clin Immunol Immunopathol 1994; 70:10–18.

45 Tjonnfjord GE, Steen R, Evensen SA et al. Characterizationof CD34! peripheral blood cells from healthy adults mobil-ized by recombinant human granulocyte colony-stimulatingfactor. Blood 1994; 84: 2795–2801.

46 Baech J, Johnsen HE. Technical aspects and clinical impactof hematopoietic progenitor subset quantification. Stem Cells2000; 18: 76–86.

47 To LB, Haylock DN, Simmons PJ et al. The biology and clini-cal uses of blood stem cells. Blood 1997; 89: 2233–2258.

48 van der WE, Richel DJ, Holtkamp MJ et al. Bone marrowreconstitution after high-dose chemotherapy and autologousperipheral blood progenitor cell transplantation: effect of graftsize (see comments). Ann Oncol 1994; 5: 795–802.


49 Olivieri A, Offidani M, Montanari M et al. Factors affectinghemopoietic recovery after high-dose therapy and autologousperipheral blood progenitor cell transplantation: a single centerexperience. Haematologica 1998; 83: 329–337.

50 McQuaker I, Haynes A, Stainer C et al. Mobilisation of per-ipheral blood stem cells with IVE and G-CSF improvesCD34! cell yields and engraftment in patients with non-Hodgkin’s lymphomas and Hodgkin’s disease. Bone MarrowTransplant 1999; 24: 715–722.

51 Ketterer N, Salles G, Raba M et al. High CD34(!) cell countsdecrease hematologic toxicity of autologous peripheral bloodprogenitor cell transplantation. Blood 1998; 91: 3148–3155.

52 Bensinger W, Appelbaum F, Rowley S et al. Factors thatinfluence collection and engraftment of autologous peripheral-blood stem cells. J Clin Oncol 1995; 13: 2547–2555.

53 Scheid C, Draube A, Reiser M et al. Using at least 5 " 106/kgCD34! cells for autologous stem cell transplantation signifi-cantly reduces febrile complications and use of antibioticsafter transplantation. Bone Marrow Transplant 1999; 23:1177–1181.

54 Tricot G, Jagannath S, Vesole D et al. Peripheral blood stemcell transplants for multiple myeloma: identification of favor-able variables for rapid engraftment in 225 patients. Blood1995; 85: 588–596.

55 Watts MJ, Sullivan AM, Leverett D et al. Back-up bone mar-row is frequently ineffective in patients with poor peripheral-blood stem-cell mobilization. J Clin Oncol 1998; 16: 1554–1560.

56 Millar BC, Millar JL, Bell JB et al. Role of CD34! cells inengraftment after high-dose melphalan in multiple myelomapatients given peripheral blood stem cell rescue. Bone MarrowTransplant 1996; 18: 871–878.

57 Papadopoulos KP, Ayello J, Reiss RF et al. CD34! cell doserequirements for rapid engraftment in a sequential high-dosechemotherapy regimen of paclitaxel, melphalan, and cyclo-phosphamide, thiotepa, and carboplatin (CTCb) with PBPCsupport in metastatic breast cancer. J Hematother Stem CellRes 1999; 8: 357–363.

58 Haas R, Mohle R, Fruhauf S et al. Patient characteristics asso-ciated with successful mobilizing and autografting of periph-eral blood progenitor cells in malignant lymphoma. Blood1994; 83: 3787–3794.

59 Haynes A, Hunter A, McQuaker G et al. Engraftment charac-teristics of peripheral blood stem cells mobilised with cyclo-phosphamide and the delayed addition of G-CSF. Bone Mar-row Transplant 1995; 16: 359–363.

60 Schwella N, Beyer J, Schwaner I et al. Impact of preleukaph-eresis cell counts on collection results and correlation of pro-genitor-cell dose with engraftment after high-dose chemo-therapy in patients with germ cell cancer. J Clin Oncol 1996;14: 1114–1121.

61 Faucher C, Le Corroller AG, Chabannon C et al. Autologoustransplantation of blood stem cells mobilized with filgrastimalone in 93 patients with malignancies: the number of CD34!cells reinfused is the only factor predicting both granulocyteand platelet recovery. J Hematother 1996; 5: 663–670.

62 Menendez P, Redondo O, Rodriguez A et al. Comparisonbetween a lyse-and-then-wash method and a lyse-non-washtechnique for the enumeration of CD34! hematopoietic pro-genitor cells. Cytometry 1998; 34: 264–271.

63 Gratama JW, Orfao A, Barnett D et al. Flow cytometric enu-meration of CD34! hematopoietic stem and progenitor cells.European Working Group on Clinical Cell Analysis. Cytome-try 1998; 34: 128–142.

64 Sovalat H, Wunder E, Tienhaara A et al. Commentary: pros-pects for standardization of stem cell determination withinEurope. J Hematother 1993; 2: 293–296.


470


65 Verwer BJH, Ward DM. An automated classification algor-ithm for ProCOUNT flow cytometric acquisition and analysis.J Hematother 1997; 6: 169–173.

66 Read EJ, Kunitake ST, Carter CS et al. Enumeration ofCD34! hematopoietic progenitor cells in peripheral blood andleukapheresis products by microvolume fluorimetry: a com-parison with flow cytometry. J Hematother 1997; 6: 291–301.

67 Cabezudo E, Querol S, Cancelas JA et al. Comparison ofvolumetric capillary cytometry with standard flow cytometryfor routine enumeration of CD34! cells. Transfusion 1999;39: 864–872.

68 Johnsen HE, Hoffmann M, Gisseloe C et al. Recommendationfor assessing agreement between two methods of CD34 enu-meration. Bone Marrow Transplant 2001 (submitted).

69 Schiødt I, Knudsen LM, Jensen L et al. Flow cytometry com-parison of CD34 subsets in bone marrow and peripheral bloodafter priming with glycosylated or non-glycosylated rhG-CSF(letter). Bone Marrow Transplant 1998; 21: 1167–1169.

70 Larsson K, Bjorkstrand B, Ljungman P. Faster engraftmentbut no reduction in infectious complications after peripheralblood stem cell transplantation compared to autologous bonemarrow transplantation. Support Care Cancer 1998; 6: 378–383.

71 Estern Cooperative Oncology Group, ECOG. Common Tox-icity Criteria. www.ecog.dfci.harvard.edu. 2000.

72 Barnett D, Granger V, Storie I et al. Quality assessment ofCD34! stem cell enumeration: experience of the UnitedKingdom National External Quality Assessment Scheme (UKNEQAS) using a unique stable whole blood preparation. Br JHaematol 1998; 102: 553–565.

73 Barnett D, Janossy G, Lubenko A et al. Guidelines for theFlow Cytometric Enumeration of CD34! HaematopoieticStem Cells. Prepared by the CD34! Haematopoietic StemCell Working Party. General Haematology Task Force of theBritish Committee for Standards in Haematology. Clin LabHaematol 1999; 21: 301–308.

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