A European reference protocol for quality assessment and clinical validation of autologous haematopoietic blood progenitor and stem cell grafts

Abstract

Recently, the regulatory authorities have begun to show interest in haematopoietic stem cell products. On a professional rather than a regulatory basis, the International Society for Hematotherapy and Graft Engineering (ISHAGE) has established the Foundation for the Accreditation of Haematopoietic Cell Therapy (FACHT), which has drawn up guidelines for standards and accreditation of such activity. In Europe, the regulatory environment with regard to haematopoietic stem cell grafts, processing and storage are currently less stringent. However, in 1998 the European Joint Accreditation Committee Euro-ISHAGE/EBMT (JACIE) prepared a regulatory document ‘Standards for Blood and Marrow Progenitor Cell Collection, Processing and Transplantation’ which was approved by the EBMT General Assembly. The major objectives were to promote quality of medical and laboratory practice in haematopoietic progenitor cell transplantation. The standards extend and detail the pre-existing activity of EBMT centres including all phases of collection, processing and administration of these cells. This is the platform for the proposed reference protocol for CD34+ cell enumeration and clinical validation of quality assessment to ensure that appropriate standards of work and product quality are established and will be maintained. Bone Marrow Transplantation (2001) 27, 463–470.

Main

Current indications for high-dose therapy as adjuvant treatment are now being applied to newly diagnosed patients. This development has required evidence-based quality and safety assessment of haematopoietic stem cell grafts and introduced a new institution in medicine, the stem cell laboratory. In most cases this speciality has evolved from or within hematological research laboratories. However, the increased routine technologies applied in quality evaluation, ex vivo manipulation and safety assessment in stem cell handling, naturally places this activity in transfusion medicine in close collaboration with the clinic.

In 1998, the European Joint Accreditation Committee Euro-ISHAGE/EBMT (JACIE) prepared a regulatory document ‘Standards for Blood and Marrow Progenitor Cell Collection, Processing and Transplantation’,1 which was approved by the EBMT General Assembly. In the same year 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 the existing situation regarding quality assessment of stem cell grafts; (2) identify major problems in quality assessments and establish scientific protocols; (3) describe guidelines for the handling of auto- and allografts; (4) publish results of sub-committee studies; and (5) contribute a document to the EBMT operational manual.

The current situation regarding CD34+ cell enumeration has been analysed and published as an European Survey on flow cytometric determination of CD34-expressing cells.2 The problems in quality assessment were disclosed during the first sub-committee meeting held at the annual 1999 EBMT meeting. One major concern was paragraph D4.000 on quality management in the standard document stating: ‘D4.130 A nucleated cell count shall be performed for any component after collection and after any subsequent processing (if applicable). D4.131 CD34+ cell count shall be performed. D4.132 The target should be to transfuse a minimum of 2 × 106 CD34+ cells per kg body weight, but lower numbers may be acceptable in specific cases. (This does not apply to bone marrow or cord blood)’.1

The 1999 subcommittee meeting concluded that the numbers given in paragraph D4.132 were inappropriate as no convincing data exist from single- or multicentre studies to document a common protocol for CD34+ cell enumeration and strategy for clinical validation of numbers. This reflects a problem in preparing guidelines for quality assessments and is the background for this proposal of a European Reference Protocol on CD34+ cell enumeration by flow cytometry and a strategy for its validation by clinical end-points.

Laboratory analysis of progenitor and stem cells

First step in quality assessment was CD34 standardisation

Culture assays of colony-forming cells were originally performed, but due to the inconvenience of this method several laboratories focused on flow cytometric analysis of CD34+ cells34 in quality assessment and the first steps were taken in 1991 by Serke of Berlin, Siena of Milan and Fritsch of Vienna.5678 This development was promoted by the farseeing Mulhouse Group when in early 1992, they hosted the First European Workshop on Stem Cell Determination and Standardization. The resulting Milan/Mulhouse manual is still a valuable procedure reference in laboratory practice.910

The subsequent years have resulted in several publications about the standardisation of flow cytometry analysis111213141516171819202122232425 as well as reports on the clinical implication of the enumeration of CD34+ cells.24262728293031323334353637 The CD34 antigen is stage-specific and identifies cells in the early stages of haemopoietic differentiation. This population, therefore, contains progenitors committed to the myeloid, erythroid, megakaryocytic and lymphoid lineages, as well as primitive progenitors and stem cells capable of long-term reconstitution.38394041 Enumeration of CD34+ cells has been shown to be useful in the procedure of stem cell mobilisation and harvest from blood for transplantation and it seems informative for the prediction of fast or delayed three-lineage engraftment and blood cell recovery following high-dose therapy.5626272829

We do not yet know the minimal safe number of CD34+ cells needed for clinical engraftment of all lineages, as this may vary depending on the stem and progenitor cell subset composition in a given patient or autograft.4243444546 However, we do know that a graft content of more than 5–10 million CD34+ cells per kg body weight is safe, resulting in fast recovery of ANC and platelets before days 14 and 21, respectively, in a major fraction of patients and, most important, only have a minor risk for engraftment failures.26284748495051525354 In a survey of 1600 patients from nine published papers, including a minimum of 50 patients each, it is concluded that the overall median time to ANC and platelet recovery is 11 days (2–93) and 11 days (0–1000+), respectively. From 15 studies with information of reinfused low numbers (Table 1a and b), it is concluded that no definite lower level exists to document groups of patients at high risk for prolonged recovery, based on CD34 numbers below 1 million/kg,55 2 million/kg,265354555657 2.5 million/ kg,2849505152585960 3 million/kg61 or 5 million/kg.48 From such data, it is obvious that we will never obtain an exact number of CD34+ cells delineating an insufficient or safe graft. We have to reconsider these terms and change exact numbers into probabilities of obtaining clinical efficacy and avoiding toxicity evaluated by proper end-points (vide infra).

Table 1 Overall median number of CD34+ cells from nine studies of haematological recovery following autologous stem cell reinfusion
Table 2 Haematological recovery in subgroups of patients reinfused with varying low number of CD34+ cells

The final step in quality assessment is clinical validation

In general, new technologies, moving from the laboratory bench to the clinic, have to pass different stages before they are validated. In parallel with therapeutic studies passing phase I–IV, the four different phases believed to be necessary and informative in clinical validation of, for example, CD34+ cell enumeration as described in Table 2. In the first phase, the CD34 technique was established in the laboratory and analysed for specificity, sensitivity, reproducibility and accuracy.56789101617181920212223242526 The subsequent second phase, documented a likely clinical influence by single centres analysing retrospective material/data.27282930313233343637 The third phase prepared convincing single centre prospective evaluation5152535455565758 evolving into the most important phase four, a multicentre prospective evaluation based upon important clinical end-points (Table 2). Ideally, phases II–III document the usefulness, convincing one or more centres to participate in a phase IV validation trial, which however in this case has not yet been performed. In an attempt to establish a phase IV study within the EBMT we propose the European Reference Protocol for quality assessment by CD34+ cell enumeration with clinical validation based on end-points relevant for autograft-mediated supportive therapy (vide infra).

Table 3 The phases for clinical validation of a laboratory technique with special emphasis on CD34 enumeration

A European Reference Protocol on CD34+ cell enumeration (Table 3)

Table 4 The European Reference Protocol for CD34 enumeration in blood and leukapheresis products

Recently, several reports have presented protocols on flow cytometry enumeration and several companies have announced standard kits for absolute enumeration of CD34+ cells.111213141516171819202122232425 These protocols seem different at several levels depending on the designer and none has been proved superior to the others, if it fulfills ‘the state-of-the-art’ recommendation. The major conclusion from the work done over the last decade is that training diminishes the interlaboratory variations with only minor differences between first- and second-line strategies. The most important change in relation to the simple Milan/Mulhouse protocol is the recommendation of a ‘lyse no wash’ technique. This technique eliminates any potential cell loss caused by cell washing which has been shown to reduce the number of CD34+ events.62

One necessary step in such a strategy exemplified by this European protocol (Table 3) is to add the pan-CD45 antibody in order to be able to quantify leukocytes and discriminate these from erythroblasts and non-lysed erythrocytes in the live gate, ie in obtaining an accurate denominator for the CD34% calculation.13151763 The number of CD34+ events present in the low SSC region is still calculated on the basis of a minimum population in the range of 50 to 100 events positively stained by a CD34 class III antibody.56101564

The 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+ cells per volume can subsequently be calculated by multiplication of the CD34% and the leukocyte count obtained from a haematology analyzer (the dual platform technique). However, a more accurate determination may require a volumetric measurement added into the analysis which can be obtained, for example, by introducing a known number of reference particles per volume of sample65 or by scanning an exact volume666768 – the so-called single platform technique.63 These methods have the advantage of being independent of variation in leukocyte enumeration by the haematology analyser, but precision in pipetting the sample volume to get a correct absolute count is important.

CD34 class III antibodies with sufficient activity, independent of fluorochrome conjugation can be used if they have been appropriately titrated before use. The isotype control should also be selected after sufficient testing. Class I and II antibodies are not recommended.9173940

The most important step, however, may be choosing the red blood cell lysing reagent as this is not an ‘innocent by-stander’ in sample preparation. The different chemicals, particularly fixatives, may affect the cell and the staining intensity.

Introduction of such a reference protocol (Table 3) will allow each stem cell laboratory to choose their own methodology, including commercial kits available on the market. The consequence may be that it will be impossible to diminish interlaboratory variation in future analysis. On the other hand, it may be that various methods used in different laboratories in accordance with a reference protocol will produce interlaboratory variations, which are comparable by a strictly standardised method. Future bench workshops may 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 standardised analysis may be decreased or even eliminated by graft processing differences including thawing/freezing, ex vivo culture, purging, selection, etc. At the same time more important clinical questions are present regarding the prediction of ‘poor mobilisers’ and the prediction of ‘insufficient autografts’ with reduced efficacy and increased risk of side-effects following graft reinfusion (Table 4).

Table 5 Proposed objectives, end-points and grading for clinical validation of quality assessment of haematopoietic grafts

Laboratories wanting to shift from one method to another should compare the old method with a new one by the Bland and Altman method based on graphical techniques, simple calculations and assessment of repeatability, as recommended in a recent report from this committee.68

Clinical end-points and objectives in supportive therapy

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

Supportive reinfusion of haematopoietic stem and progenitor cells following marrow ablative therapy ultimately aims to re-establish haematopoiesis following an initial recovery of end-stage blood circulating cells to a level necessary for reducing the risk of side-effects, such as infections, bleeding 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 established for recovery of the immune system, ie B cells, T cells, NK cells and monocytes.

Consequently, engraftment has most often been evaluated by time to three-lineage recovery by calculating the time-dependent probability of reaching the above given levels following blood cell nadir after transplantation. A simple description of the observed data can be performed by means of Kaplan–Meier plots. The prognostic value of different factors as, for example, CD34+ cell numbers, subset numbers as well as other factors of proposed importance can be evaluated by log-rank test, for example. However, this strategy for analysis was established in the early days of allogeneic and autologous bone marrow transplantation and needs to be re-evaluated in actual practice. The use of blood progenitor grafts and growth factor administration may have changed the initial correlation between blood cell recovery and clinical events. Health, economic and life-quality considerations need to be included in assessment of supportive haematopoietic cell therapy. Of less importance may be the side-effects, eg haematological toxicity, as defined by time to three lineage recovery or engraftment. We propose a change to assessing the probabilities of obtaining primary, secondary or tertiary end-points. Such an assessment will allow us to handle each patient individually 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 their conclusions about quality assessment on surrogate markers and as suggested it seems to be time for a move towards evaluation on the basis of clinically relevant factors. Such data, although published from single centres, are not available from multicentre trials and have to be generated in a prospective manner.

It is worth mentioning that the introduction of peripheral blood autografting has not changed the risk of documented infections compared to the use of conventional bone marrow grafts, although a faster neutrophil recovery is substantially documented.6970 Furthermore, the risk of thrombocytopenic bleeding has neither been evaluated nor formally documented as being reduced by an increase of blood platelets from below to above 10, 20 or 50 × 109 platelets/l during recovery.30

It needs to be considered in future clinical trials whether: (1) Primary end-points should be events documenting efficacy of clinical importance, ie the risk of severe bleeding and infections including antibiotic administration and transfusion of blood components as well as time in hospital. (2) Secondary end-points should be an evaluation of toxicity in accordance with, for example, Common Toxicity Criteria (CTC),71 including a time-dependent grading of haematological toxicity. (3) Tertiary end-points should be the risk of regimen-related death or disease progression within 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 consideration actual clinical practice, as well as knowledge in biology. A time-dependent grading of efficacy is proposed with day 21 as the acceptable maximum time in hospital, which together with antibiotics, antifungal or transfusion therapy delineates three groups: a good outcome for patients discharged before day 22 with no transfusion therapy; a poor outcome for patients who stay in hospital more than 21 days on continuous therapy; and finally an intermediate group of patients who are discharged before day 22, but receive transfusions or interventional (not prophylactically) antibiotics/antifungal therapy.

Concerning toxicity, the proposed grading system is time-independent with outcome assessed according to the WHO recommendation for grading of organ toxicity as good if toxicity is grade 0–1, poor if grade 3–4 and as intermediate if grade 2–3, and can vary depending on the organ involved. By tradition haematological toxicity has to be time-dependent and an evaluation is proposed in Table 5.

Table 6 Proposed time-dependent grading of haematological toxicity (see Table 3)

Finally, evaluation of mortality and disease recurrence indicate good outcome if patients are alive with no disease recurrence up to day 90 and poor if the high-dose therapy is followed by death or relapse before day 90. An intermediate 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. Appropriate statistical methods would then be applied to give the best possible description of a correlation between the factor under consideration and the probabilities of specific end-points as for example described in Figure 1.

Figure 1
figure1

For patients undergoing haematopoietic stem cell transplantation, the Figure shows the estimated relative proportion of patients with the end-point in question (see Table 4) graded as ‘Good’, ‘Acceptable’ or ‘Poor’ and analysed for influence of the given risk factor, eg CD34 number. At any number of CD34+ cells transplantated one can read the risk for 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 decade is documented by the elimination of the risk of engraftment failure – not by the introduction of PBSPC grafting, but very likely due to improved quality assessment procedures. This has been achieved by hard work on a very simple technology allowing us to identify patients at risk of engraftment failure, due to either lack of mobilisation or obtaining an insufficient harvest.47

Future flow cytometry techniques should comply with the basic recommendations given in this report (Table 3) and be validated clinically by carefully planned prospective multicentre trials on efficacy and toxicity (Table 4). We would like to stress that this proposal does not intend to change or compete with guidelines for enumeration recently published.17637273 On the contrary, the European Reference Protocol attempts to cover all techniques providing the basis for starting prospective multicentre studies. Hopefully, when such data are available, the international community will reach a consensus on guidelines and reference protocols for supportive cell therapy in an attempt to make high-dose therapy a safe and cost-beneficial procedure. At that time we finally may be able to fulfil the paragraph D4.000 on Quality Management in the EBMT/ISHAGE standard.1 Much work has to be done in EBMT multicentre registration studies to obtain that goal.

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Serke, S., Johnsen, H. A European reference protocol for quality assessment and clinical validation of autologous haematopoietic blood progenitor and stem cell grafts. Bone Marrow Transplant 27, 463–470 (2001). https://doi.org/10.1038/sj.bmt.1702813

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Keywords

  • CD34
  • flow cytometry
  • standardization
  • reference protocol
  • haematopoietic stem cell transplantation
  • prediction of engraftment

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