JOURNAL OF HEMATOTHERAPY 5:261-270 (1996)Mary Ann Liebert, Inc.

BIRMA-K3, a New Monoclonal Antibody for CD34Immunophenotyping and Stem and Progenitor Cell Assay

HEINZ-GERT HÖFFKES,1 JENNIFER A. LOWE,2 RENÉ O. PEDERSEN,3GERNOLD SCHMIDKTE,4 and DOROTHY F. McDONALD2

ABSTRACT

A murine monoclonal antibody with specificity for the CD34 antigen has been produced and des-ignated BIRMA-K3. The antibody characterized as IgGl (kappa) has been shown to react with KG-la cells following treatment of the cells with glycoprotease enzyme, indicating reactivity with theclass III epitope of CD34. It was possible to show that class I and class II anti-CD34 antibodies were

not able to inhibit binding of BIRMA-K3. Investigation of FITC-labeled as well as PE-labeledBIRMA-K3 resulted in a clear cut-off staining of acute leukemias and CD34+ cell counts in pa-tients submitted to high-dose chemotherapy and stem cell transplantation. The results obtained cor-

relate strongly with those from HPCA-2, the Becton-Dickinson class III antibody.

INTRODUCTION

THE USE OF MONOCLONAL ANTIBODIES (mAb) againstthe CD34 antigen (1,2) permits flow cytometric

quantitation of the size of this cell pool, which appearsto be an important parameter in assessing the quality ofa hematopoietic graft (3). The determination of absoluteCD34 counts is believed to be of importance in optimiz-ing collection of mobilized CD34+ stem cells, and ab-solute CD34+ cell counts in peripheral blood correlatewith the numbers of CD34+ cells collected in apheresisproducts (4). CD34 is also an important antigen forleukemia and lymphoma immunophenotyping (5).

The analysis and quantitation of CD34+ cells will de-pend on the epitope recognized by the CD34 antibodyused. The CD34 antigen is composed of three differentepitopes with differential sensitivity to neuraminidaseand glycoprotease from Pasteurella haemolytica [6,7].The use of antibodies against class II and class III epi-topes of CD34 is recommended because the fluorescence

intensity of CD34+ cells in peripheral blood detected byclass I antibodies can be variable, depending on the levelof commitment of these cells [8]. Directly conjugated an-

tibodies are preferred, since they save time and result inreduced nonspecific background staining. Several anti-bodies are available conjugated to fluorescein isothio-cyanate (FITC) or phycoerythrin (PE). FITC labeling isadequate for leukemia as well as lymphoma typing [9],but for absolute CD34 counting, PE labeling is recom-mended because of the higher quantum yield of PE [8,10].

The aim of the present study was to characterize thenewly developed mAb BIRMA-K3, which is directedagainst the CD34 antigen. Experiments indicate thatBIRMA-K3 recognizes an epitope shared by HPCA-2,the Becton-Dickinson anti-CD34 antibody, namely, theclass III epitope. In addition, comparison of BIRMA-K3with other established monoclonal CD34 antibodies foron-line monitoring of absolute CD34 counts andleukemia immunophenotyping showed a strong correla-tion with HPCA-2.

'Division of Hematology/Oncology, Department of Medicine, Otto-von-Guericke University of Magdeburg, Germany.2National Blood Service, West Midland Centre, Birmingham, United Kingdom.3DAKO A/S, Glostrup, Denmark.4Division of Hematology, Department of Medicine, University of Essen, Germany.

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MATERIALS AND METHODS

Production of the monoclonal antibodyFemale Balb/c mice were immunized by i.p. injections

of the human acute myelogenous leukemia cell line KG-la (American Type Culture collection, Rockville, MD).The KG-la cells were grown in RPMI-1640 (Row Labs)supplemented with 10% fetal calf serum (FCS) and 2 mMglutamine. Cultured cells were washed and adjusted to100 X 106/ml in saline, and a 200 pi injection was givenon three occasions at 3 week intervals. Following a restperiod of 3 months, the mice were given a final boosterinjection of 20 X 106 cells 3 days before fusion. Spleencells were harvested from the immunized mice, and thesewere fused with cells of the X63 Ag8653 mouse myelomacell line by an established technique using polyethyleneglycol. Culture supernatants were screened for reactivitywith KG-la cells using affinity-purified FITC rabbitF(ab)2 antimouse immunoglobulin (F 0313, DAKO A/S,Glostrup, Denmark) as a fluorescent label, and tests were

analyzed using a FACScan (Becton-Dickinson,Heidelberg, Germany) flow cytometer and Lysis II soft-ware (Becton-Dickinson). Cells from positive wells were

cloned by limiting dilution. The clone chosen for studywas designated BIRMA-K3 (DAKO A/S, Glostrup,Denmark).

Characterization of the antibody BIRMA-K3

Reactivity with Established Cell Lines. The antibodyBIRMA-K3 was tested by an indirect immunofluores-cence technique for binding to 14 established cell lineschosen for their lack of reactivity with antibodies ofCD34 specificity. These cell lines were Burkitt lym-phoma lines Raji, Namalwa, and Daudi; B lymphoblas-toid cell lines LICR-H My and 04673; EBV B cell lineRPMI 8866; pre-B cell line Nairn 6; T cell lymphoblas-tic leukemia lines Jurkat, Molt 4, HSB2, and CEM;myeloma cell line RPMI 8266; promyelocytic leukemialine HL60; plasmocytoid cell line U266. Other cellstested were B cell lymphocytic leukemia cells, normalperipheral blood mononuclear cells, and buoyant anddense cells of tonsil origin.

Reactivity with Endothelium. The reactivity of anti-bodies to CD34 has been shown to be confined to im-mature hematopoietic cells and, in nonhematopoietic tis-sue, to endothelium. BIRMA-K3 antibody was tested forspecific reaction to endothelium in frozen tonsil sectionsusing an established immunohistofluorescence technique(Dr. G. Johnson, University of Birmingham).

Investigation of Blocking Effect of BIRMA-K3 on

Reactivity of Other mAb Directed Against the CD34Antigen. The following anti-CD34 antibodies were

tested: HPCA-1 unconjugated (11), HPCA-2 unconju-

gated and HPCA-2 (FTTC) (Becton-Dickinson), Q-BEND 10 unconjugated (12) and Q-BEND 10 (FITC)(Coulter/Immunotech, Krefeld/Hamburg, Germany),BIRMA-K3 unconjugated and BIRMA-K3(FITC) (DakoA/S). Cells from the cell line KG-la were transferredfrom the culture flasks into centrifuge tubes and cen-

trifuged at 300g for 5 min. The supernatant was aspirated,and the cell density was adjusted to 1 X 107/ml by adding0.01 M phosphate buffered saline (PBS). HPCA-1 (20pi) or Q-BEND 10(10 pi) or HPCA-2 (20 pi) or BIRMA-K3 (10 pi) undiluted or at 1:1024 dilution was added to1 X 106 cells. The cells were incubated at 4°C for 30 min.Then, 2 ml 0.01 M PBS was added and vortexed gently.After centrifugation at 300g for 5 min, the supernatantwas aspirated, leaving approximately 50 pA of fluid.Following treatment with primary antibody, directly con-

jugated anti-CD34 antibodies were added to KG-la cellsas follows: Q-BEND 10 (FITC) (10 pi) or HPCA-2(FITC) (20 pi) or BIRMA-K3 (FITC) (10 pi). Thesewere incubated at 4°C for 30 min in the dark. The cellswere washed once, and 300 pi of 0.01M PBS, 1%paraformaldehyde was added. The cells were analyzedon the FACScan using Lysis II software.

Determination of the Epitope Specificity ofBIRMA-K3.The CD34 antigen is known to express at least three epi-topes that currently permit discrimination of mAb toCD34 into three different classes. The epitopes for classesI and II mAb are sensitive to degradation by glycopro-teases, whereas the class III epitopes are enzyme resis-tant. For this experiment, KG-la cells, cultured as de-scribed previously, were washed, counted, and adjustedto a concentration of 5 X 106/ml. Aliquotsof200/¿1 were

taken, and 100 pi of PBS or the glycoprotease enzymeP. haemolytica (Cedarlane Laboratories, Ontario,Canada) at 2.4 mg/ml was added to these aliquots. Alltubes were incubated at 37°C for 30 min. After incuba-tion, all tubes were washed twice with ice-cold RPMIcontaining 10% FCS and 0.02% sodium azide, and thecells were left in a volume of approximately 50 pi.Unconjugated HPCA-1 5 pi (CD34 antibody of class I)(Becton-Dickinson) or 5 pi unconjugated BIRMA-K3was added to tubes containing either enzyme-treated cellsor untreated cells. Then, 10 pi of an irrelevant antibodywas added to cells as a negative control. Following 30min incubation at ambient temperature, cells were

washed in PBS/EDTA, and an FITC rabbit antimouse im-munglobulin second antibody (DAKO) was added.Incubation was carried out for 30 min at ambient tem-perature in the dark, after which the cells were washedonce in Isoton II (Coulter Electronics, Krefeld, Germany)before flow cytometric analysis on the FACScan. Sometreated and untreated cells were tested with the directlyconjugated class III anti-CD34 antibody, HPCA-2. In thisexperiment, cells were incubated with 20 pi antibody for30 min in the dark at 4°C. The cells were then washed

262

BIRMA-K3, A NEW ANTI-CD34

once with Isoton II before flow cytometric analysis. In a

separate experiment, the sensitivity of the CD34 antigento treatment with chymopapain was used to discriminatethe epitope specificity of BIRMA-K3 from that of theCD34 antibody Q-BEND 10, a class II antibody. KG-lacells were treated with chymopapain at a final concen-tration of 4 U/ml for 30 min at 37°C and then tested withboth antibodies in a similar manner to the previous ex-

periment.Immunoblotting. Information regarding the nature and

molecular weight of the epitope recognized by BIRMA-K3 was sought in immunoblotting experiments. SDSpolyacrylamide gel electrophoresis of a lysate of KG-lacells was carried out in parallel with a range of markersof known molecular weight (Sigma). A 7.5% running geland 3% stacking gel were used in a discontinuous buffersystem. Following electrophoresis, the proteins weretransferred onto 0.45 p nitrocellulose paper (Bio-Rad) ina standard blotting procedure using a transfer buffer con-

taining methanol. The portion of the blot carrying themolecular weight markers was cut off and stained withAmido black dye (BDH Poole, UK). The KG-la blot was

stained with BIRMA-K3, followed by a second antibody,peroxidase conjugated rabbit antimouse immunoglobu-

lins (DAKO). 33-Diaminobenzidine (D.A.B. Tablet Set,Sigma) was used as the peroxidase substrate to reveal thelocation of the protein band with which the BIRMA-K3antibody was reactive.

Reactivity with Clinical Samples. The reactivity ofBIRMA-K3 was investigated in three ways. First, the an-

tibody was used in an indirect flow cytometric techniquefor immunophenotyping samples of peripheral blood andbone marrow from 22 patients suffering from a variety ofacute leukemias and lymphomas, in parallel with HPCA-1 (data not shown). FITC-conjugated BIRMA-K3 (DAKODiagnostika, Hamburg, Germany) was also used for im-munophenotyping acute leukemias (n = 10), in parallelwith anti-CD34 HPCA-2 (PE) staining. Acute leukemiaswere investigated by routine multiparametric flowcytometry of CDw65(FITC)/CD33(PE)/CD13(PerCP),CD15(FITC)/CD14(PE)/CD16(PerCP),CD2(FITC)/CD14(PE)/CD64(PerCP),CD56(FITC)/CD7(PE)/CD19(PE-Cy5), and CD34(FITC)/CD38(PE)/HLA-DR(PerCP) triple staining in addition toconventional investigations according to the FAB classifi-cation (13).

Second, directly conjugated BIRMA-K3 (FITC andPE) was used to monitor CD34 counts in peripheral blood

FIG. 1. Staining of endothelium of blood vessels between the acini of mucous glands in a cryostat section of tonsil by the anti-CD34 antibody BIRMA-K3.

263

HÖFFKES ET AL.

and apheresis products of 50 patients suffering from low-grade and high-grade non-Hodgkin's lymphomas beforehigh-dose chemotherapy and stem cell transplantation.CD34-expressing hemopoietic cells were mobilized bycombined chemotherapy (ifosfamide, etoposide, arabi-noside-C, and dexamethasone) and GM-CSF administra-tion. CD34 counting was started when the leukocyte nadirwas passed, independent of the absolute leukocyte countper microliter. For CD34 monitoring, directly PE-con-jugated IMMU 133 (Immunotech, Hamburg, Germany,class I antibody), Q-Bend 10 (Immunotech, class II an-

tibody), and HPCA-2 (Becton-Dickinson, Heidelberg,Germany, class III antibody) were analyzed in parallelwith BIRMA-K3. According to multiparametric CD34analysis by Serke et al. (8,12), the samples were adjustedto a white blood cell count of about 10.000//Í.1 by dilut-ing into Dulbecco's phosphate-buffered saline (DPBS).Aliquots (100 ^tl) were pipetted into 4.5 ml polystyrenetubes. mAb were added at 10-20 pi per tube. Cells were

incubated with the antibodies for 15 min, and then redblood cells were lysed by adding ammonium chloridelysing reagent (Ortho, Raritan, NJ). The cells were pel-leted at 400g at 4°C for 5 min. The supernatant was as-

pirated, and cells were resuspended in DPBS and pel-leted again. Finally, cells were resuspended in DPBS andanalyzed by flow cytometry within 2 h. As an alterna-tive to ammonium chloride, OPTI lyse reagent(Immunotech) or FACS lyse reagent (Becton-Dickinson)was added after incubation with the mAb in some ex-

periments.Third, after informed consent, FITC-labeled and PE-

labeled BIRMA-K3, as well as PE-labeled HPCA-2, were

used for CD34 cell enumeration in a bone marrow aspi-rate of a young and healthy man being screened as a semi-compatible allograft bone marrow donor. The CD34staining was done as described previously.

RESULTS

BIRMA-K3 was shown to be an IgGl antibody byOuchterlony double diffusion in agar using isotype-spe-cific antibodies (mouse monoclonal antibody typing kit,The Binding Site, Birmingham, UK). Further analysisshowed that the light chain was kappa (DAKO).

CD34 blocking850,00

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FIG. 2. Blocking experiments with anti-CD34 antibodies using the KG-1 a cell line. HPCA-2 and BIRMA-K3 block each other'sbinding, whereas Q-BEND 10 binding was not influenced by HPCA-2 or BIRMA-K3. The blocking effect is abolished at highdilution (1:1024).

264

BIRMA-K3, A NEW ANTI-CD34

Reactivity with established cell lines

When tested for reactivity with 14 established cell linesother than KG-1 a, the antibody was reactive with no more

than 20% of cells in 11 of the cell lines. With the re-

maining 3 cell lines, reactivity was of the order of 50%,but the strength of reaction was very low and not con-

sidered significantly strong enough to be classed as pos-itive. The pattern of reactivity was consistent with an an-

tibody of anti-CD34 specificity. Reactivity with cellsfrom a patient with chronic B lymphocytic leukemia, nor-

mal peripheral blood mononuclear cells, and both buoy-ant and dense cells of tonsil origin was less than 20%(data not shown). This is also consistent with a CD34 an-

tibody.

Reactivity with endothelium

Cryostat sections of tonsil stained with BIRMA-K3 an-

tibody using an indirect immunofluorescence techniquerevealed extensive staining of endothelium (Fig. 1). Thisreactivity is compatible with that of an antibody of CD34specificity.

M2i

isotope control FITC

Marker Events % Gated % Total Mean Median Peak ChAllM1

85128055

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85.1280.55

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9.319.31

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BIRMA-K3 FITC

Marker Events % Gated % Total Mean Median Peak ChÄÜ 8597 100.00 85.97 302.45 305.05 352~M1 1016 11.82 10.16 8.72 6.61 6M2 7581 88.18 75.81 341.81 339.82 352

FIG. 3. FITC-conjugated BIRMA-K3 and isotype controlstaining in a case of acute myelogenous leukemia (FAB-M5a),showing a clear cut-off discrimination.

Investigation of blocking effect of BIRMA-K3 on

reactivity of other mAb directed against theCD34 antigen

Four CD34 antibodies were tested for cross-reactivity:HPCA-1 recognizing a class I epitope, Q-BEND 10 rec-

ognizing a class II epitope, HPCA-2 recognizing a classIII epitope, and BIRMA-K3. These experiments were

performed to test if antibodies against the different epi-topes were able to cross-inhibit binding. The results are

shown in Figure 2. The three leftmost bars represent themean fluorescence intensity (MFI) of HPCA-2/FITC,BIRMA-K3/FITC, and Q-BEND 10/FITC without prein-cubation with unconjugated antibodies. The next 12 barsshow three experiments in which preincubation withHPCA-1, Q-BEND 10, HPCA-2, and BIRMA-K3 was

carried out before subsequent incubation with HPCA-2/FITC, BIRMA-K3/FITC, and Q-BEND 10/FTTC, re-

spectively. Any inhibitory (blocking) effect of the pri-mary antibody on binding is displayed as a lowered MFIcompared with the control results (Fig. 2). HPCA-2 is in-hibited by both HPCA-2 and BIRMA-K3, and BIRMA-K3 is inhibited by BIRMA-K3 and HPCA-2, indicatingidentical specificity. Q-BEND 10 is inhibited only by Q-BEND 10. In all cases, the degree of inhibition was de-pendent on the concentration of the unconjugated anti-body and was abolished at a dilution of 1:1024, thusindicating that the inhibition was specific (1).

Reactivity with clinical samplesBIRMA-K3 and HPCA-2 gave equivalent staining with

samples of blood and BM from 22 patients with hémato-logie disorders in immunophenotyping studies. There was

good agreement in the numbers of cells marking positive,and no false positive reactivity was seen with BIRMA-K3(data not shown). In the triple staining experiments usingBIRMA-K3 for CD34(FITC)/CD38(PE)/HLA-DR(PerCP)staining with leukocytes of patients with acute myelogenousleukemia, the results (Figs. 3 and 4) illustrate a clear cut-offdiscrimination of the blasts characterized by intensive CD34staining. CD34 staining of different acute myelogenousleukemias as well as lymphoblastic leukemias showed no

difference between HPCA-2 (PE) and BIRMA-K3 (FITC)staining in all CD34-positive leukemias as defined byHPCA-2 staining. For comparison of BIRMA-K3 andHPCA-2 staining in normal BM aspirates, a single samplewas processed, resulting in a comparable staining pattern af-ter erythrocyte lysing (Ortho lysing reagent, Immunotech).The CD34 characterization of stem cells in a young andhealthy man showed comparable staining of FITC-labeledand PE-labeled BIRMA-K3 as well as PE-labeled HPCA-2(Fig. 5).

Comparison of different class II and class III directedCD34 antibodies and BIRMA-K3 in counting CD34+

265

HOFFKES ET AL.

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FIG. 5. FITC-conjugated and PE-conjugated BIRMA-K3 as well as PE-conjugated HPCA-2 staining in a bone marrow aspi-rate of a young and healthy man screened as a potential allograft donor.

FIG. 4. FITC-conjugated BIRMA-K3 staining in a case of acute myelogenous leukemia (FAB-M5a), showing a strong co-ex-

pression of CD34(FITC)/CD38(PE)/HLA-DR(PerCP).

267

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Table 1. Effect of Treatment of Ka-1a Cells with Glycoprotease Enzyme from Pasteurella haemolytica onReactivity of Three Monoclonal Antibodies to CD34

No. of cells reactive (%)Untreated cells Enzyme-treated cells CD34 epitope classification

HPCA-1 82 4 IHPCA-2 77 81 HIBIRMA-K3 77 81 DJ

cells from patients suffering from malignant lymphomasshowed a strong correlation between the HPCA-2 andBIRMA-K3 (Fig. 6). Both the IMMU 133 and Q-BEND10 showed a numeric lower CD34 count. FITC-conjugatedBIRMA-K3 was also seen to give a lower numeric CD34+count as compared with PE-conjugated BIRMA-K3, butthe differences were not significant.

Determination of epitope specificity of BIRMA-K3

The antibody was tested by an indirect fluorescencetechnique in parallel with the unlabeled class I antibodyHPCA-1 and with the directly labeled class III antibodyHPCA-2(FITC) against KG-1A cells. Some cell sampleshad been pretreated with glycoprotease enzyme, and oth-ers ware left untreated. All antibodies reacted equivalentlywith the untreated cell samples, but following treatmentwith glycoprotease, the reactivity of the cells with HPCA-1 was reduced considerably. In contrast, the reactivity ofthe treated cells with both HPCA-2 and BIRMA-K3 was

enhanced (Table 1). From these results, we infer that theBIRMA-K3 antibody is specific for the class III epitopeof CD34. Testing with KG-la cells pretreated with the en-

zyme chymopapain was carried out on a separate occa-

sion, when reactivity of BIRMA-K3 was compared withthat of the class II antibody Q-BEND 10 (data not shown).The reactivity of both antibodies with untreated cells was

equivalent, but following chymopapain treatment, the re-

activity of Q-BEND 10 was reduced, and that of BIRMA-K3 was unaffected, further indicating the specificity ofBIRMA-K3 for the enzyme-resistant class III epitope.

ImmunoblottingImmunoblotting experiments were carried out on three

occasions. On two occasions, weak staining of the nitro-cellulose was observed, suggesting a molecular weightof 105-120 kDa for the protein recognized by theBIRMA-K3 antibody. On the third occasion, no stainingwas observed at all. These results are consistent with con-

clusions drawn from testing in the Vth Leucocyte TypingWorkshop, where it was observed that class III epitopesare likely to be denaturation sensitive (14).

DISCUSSION

A murine mAb with specificity for the CD34 antigenhas been produced and designated BIRMA-K3. The an-

tibody characterized as IgGl (kappa) has been shown toreact with KG-la cells following treatment of the cellswith glycoprotease enzyme, indicating reactivity with theclass III epitope of CD34 (6). Varying results wereachieved in immunoblotting experiments using BIRMA-K3, suggesting that the epitope of 105-120 kDa is de-pendent on sialic acid residues removed under the re-

ducing conditions of the experiments. This again isindicative of class III specificity. The antibody is usefulfor immunophenotyping patients with leukemias as wellas lymphomas and for enumeration of hemopoietic stemand progenitor cells. Comparison of class I, class II, andclass III antibodies in the clinical part of the work re-vealed no significant differences between the absoluteCD34 counts using Q-BEND 10, HPCA-2, and BIRMA-K3 in patients with malignant lymphomas. It should benoted that HPCA-2 and BIRMA-K3 showed a strong cor-

relation, with Q-BEND 10 showing numerically lowerCD34 counts.

The blocking experiments confirm that class II andclass III antibodies recognize different epitopes and thatthe two class Ill-reactive antibodies, HPCA-2 andBIRMA-K3, show similar specificity. Because at presentthere are no FITC-conjugated CD34 class I antibodies, itwas not possible in these experiments to investigate di-rectly the inhibitory effect of class II and class III anti-bodies on class I antibodies. However, it was possible toshow that HPCA-1 (class I) was not able to inhibit bind-ing of BIRMA-K3 or of known class II and class III an-

tibodies.In conclusion, it was demonstrated that the new anti-

CD34 antibody BIRMA-K3 is directed against the classIII epitope of the CD34 antigen and can be used in thesame manner as HPCA-2 for CD34 immunophenotypingof leukemias and lymphomas as well as enumeration ofstem and progenitor cells. The antibody has been shownto perform well both in its unconjugated form and whenconjugated to FITC and PE, making it a very versatilereagent. BIRMA-K3 has been submitted to the Vlth

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HOFFKES ET AL.

International Workshop and Conference on LeucocyteDifferentiation Antigens (1996).

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14. Greaves, M.F., Titley, I., Colman, S.M., et al. CD34 clus-ter workshop report. In Leucocyte Typing V, eds.Schlossmann, S.F., Boumsell, L., Gilks, W., et al. OxfordUniversity Press, Oxford, New York, Tokyo, 1995, vol. 1,p. 841.

Address reprint requests to:Dr. Heinz-Gert Höffkes

Division of Hematology/OncologyDepartment of Medicine

Otto-von-Guericke University of MagdeburgLeipziger Strasse 44

39120 MagdeburgGermany

270