repere normal bm 2004

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Improving Lab Practice

Immunophenotypic Differentiation Patterns of NormalHematopoiesis in Human Bone Marrow:

Reference Patterns for Age-Related Changesand Disease-Induced Shifts

E.G. van Lochem,* V.H.J. van der Velden, H.K. Wind, J.G. te Marvelde,N.A.C. Westerdaal, and J.J.M. van Dongen

Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

Background: The abundance of monoclonal antibodies (mAb) and the routine use of quadruple stainingsin flow cytometry allow stepwise analysis of bone marrow (BM) samples that are suspected for abnormalhematopoiesis. A screening phase that precedes lineage-specific classification phases should be sufficient

to assess whether the BM has a normal or abnormal composition, as well as to identify the abnormaldifferentiation lineage.

Methods: For a quick and easy flow cytometric screening of BM samples, we selected six quadrupleimmunostainings that cover multiple differentiation stages of the B-cell, monocytic, granulocytic, and erythroidlineages: TdT/CD20/CD19/CD10 and CD45/CD34/CD19/CD22 for B cells, CD34/CD117/CD45/CD13.33 for precur-sor granulocytic and precursor monocytic cells (myelo/monoblasts), CD14/CD33/CD45/CD34 for monocytic cells,CD16/CD13/CD45/CD11b for granulocytic cells, and CD71/CD235a/CD45/CD117 for erythroid cells.

Results: The six quadruple immunostainings reveal specific staining patterns in normal BM, which allowthe recognition of various subpopulations of the respective lineages. These staining patterns can be used asa frame of reference for recognition of normal and abnormal BM development. Examples of normal(age-related) variations in these otherwise stable staining patterns are presented together with severalabnormal differentiation patterns.

Conclusions: Although alternative immunostainings can be used (e.g., including NK- and T-cell markers),we feel that the selected six stainings represent a comprehensive and easy screening phase for quick

identification of shifts in the composition of the studied differentiation lineages, reflecting age-relatedchanges or disease-induced BM abnormalities. 2004 Wiley-Liss, Inc.

Key terms: flow cytometry; normal bone marrow; staining patterns; immunophenotyping; expression profile

Flow cytometric analysis of bone marrow (BM) samplesis performed routinely for identification, frequency assess-ment, and further characterization of the various leuko-cyte subpopulations when abnormal hematopoiesis is sus-pected. The BM compartment is a complex tissuecontaining cells of multiple hematopoietic lineages with

various maturational stages per lineage. In healthy individ-uals, the BM precursor cells guarantee continuous produc-tion of the various hematopoietic differentiation lineages.The differentiation and maturation can be monitored bychanges in cytomorphology and immunophenotype. Es-pecially in the B-cell lineage, multiple stages have beendefined based on their immunophenotype (15). Also inthe monocytic, granulocytic, erythroid and thrombocyticlineages several differentiation stages can be identified byimmunophenotyping (6 8). Knowledge of the expres-sion (levels) of various lineage-specific, immature, and

mature markers in normal hematopoietic development

provides a frame of reference for recognition of abnormal

differentiation patterns.

Abnormal BM hematopoiesis may result from (1) anarrest during precursor B-cell differentiation; (2) BM re-

generation after immunosuppressive therapy or as a re-

sponse to infection; (3) dysplasia in one or more myeloidlineages; (4) increased turnover of hematopoietic cells

*Correspondence to: E.G. van Lochem, Department of Immunology,

Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein40, 3015 DR Rotterdam, The Netherlands.

E-mail: [email protected]

Received 18 August 2003; Accepted 29 October 2003Published online 14 June 2004 in Wiley InterScience (www.

interscience.wiley.com).DOI: 10.1002/cyto.b.20008

Cytometry Part B (Clinical Cytometry) 60B:113 (2004)

2004 Wiley-Liss, Inc.


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caused by peripheral cell depletion; or (5) unregulatedexpansion of neoplastic hematopoietic cells. Most ofthese abnormalities will present as major shifts in BMcomposition or as specific shifts in the relative distributionof subsets in one or more lineages. In the case of a B-celldifferentiation arrest, the absence of the more mature B

cells and the relative increase of precursor B cells will bethe most striking observation (5). In regenerating BM aftercytotoxic treatment, the immature precursor B cells willalso be overrepresented, but mature B cells will still bedetectable (9,10). In most BM-derived hematopoietic ma-lignancies, such as precursor B-acute lymphoblastic leuke-mia (ALL) and acute myeloid leukemia (AML), the rela-tively homogeneous leukemic cell population dominatesthe BM compartment with profound suppression of otherhematopoietic lineages. These leukemic cells frequentlyhave an aberrant immunophenotype not found in normalBM (1116). Knowledge of the normal immunopheno-typic differentiation patterns in BM facilitates recognitionof ALL and AML cells, even if they occur at low frequen-

cies (1720).Studies of normal BM hematopoiesis are improved bytechnological innovation of flow cytometry to the routineuse of four-color analyses. To generate significant datafrom these multi-parameter analyses, it is important toattune the composition of the monoclonal antibody(mAb) combinations to the part of hematopoiesis understudy: the relative distribution of the BM leukocytes overthe major lineages; immature precursor cells; more ma-ture differentiation stages; a specific lineage (B- or myeloidcells); or, a specific differentiation stage within a lineage.

Numerous leukocyte antigens have been studied exten-sively for their expression (levels) on various hematopoi-etic lineages and differentiation (11,21,22). It is not our

aim to review the complete expression profiles of allleukocyte markers or to describe all minor BM subpopu-lations. We focus on a limited set of six four-color immu-nostainings, which were selected for rapid screening of allmajor leukocyte (sub)populations in BM in order to easilyidentify shifts or aberrancies in normal BM hematopoiesis(i.e., B-cell, myeloid, and erythroid lineages). We did notinclude immunostainings for identification of the naturalkiller (NK)-/T-cell lineage, since NK-/T-cell differentiationdoes not take place in BM.

METHODOLOGYTechnical Considerations for the Design of the

Four-Color Immunostainings

The selection of the most informative mAb combina-tions should preferably be based on the properties ofantibodies, antigens, and fluorochromes. First, severalclones in the same cluster of differentiation (CD) recog-nize different epitopes or the same epitopes with differentaffinity (22). For example, CD15 mAb can recognize sia-lylated or nonsialylated epitopes, and CD34 antibodies(type I, II, and III mAb) recognize different epitopes with

variable sensitivity for enzymes (23). Second, aspecificFc-receptor (FcR) binding, causing an increased back-ground signal, can be minimized by selecting mAbs of

specific immunoglobulin subclasses or by using F(ab)2fragments, thereby avoiding interaction with FcRs (24).Third, most mAb are available with different fluoro-chrome-conjugates; the choice of fluorochrome-conjugateshould depend on the expression level of the antigen.Because of their strong fluorescence signal, PE- and APC-

conjugated mAb are the best choice for the detection ofweakly expressed antigens, as well as for antigens withvariable expression from weak to strong.

The mAb clones and fluorochrome-conjugates thatwere used for the six four-color immunostainings to assessthe normal BM differentiation patterns are summarized inTable 1. Our standard immunophenotyping procedureuses ammonium chloride-lysed whole BM samples to pre-

vent selective loss of cells and to conserve light scattercharacteristics (25). Cells were permeabilized for intracel-lular immunostaining of TdT by using FACSBrand lysingsolution (BD Biosciences; San Diego, CA) (26). Data wereacquired on a FACSCalibur (BD Biosciences) using CellQuest Pro Software and analysed using both Cell Quest

Pro and Paint a Gate Pro software.

Strategy for the Design of Four-Color Immunostainings

In this report we present a limited set of six immuno-stainings that unravel the normal differentiation patterns

within the four major hematopoietic lineages in BM. Be-cause T-cell development normally does not occur in BM,no T- or NK-cell-specific immunostainings were included.

In five of the six selected immunostainings, a lineage-specific marker is used to select the lineage of interest.The combination of CD19 or CD13, with side-scatter (SSC)

Table 1MAbs Used for the Six Selected Quadruple ImmunostainingsThat Cover the Development of the Four Major Hematopoietic

Lineages in Normal BM

CD Clone Fluorochromes Manufacturer

TdT FITC SuperTechsCD10 HI 10a APC BD BiosciencesCD19 4G7 PE BD Biosciences

SJ25C1 PerCP-Cy5.5 BD BiosciencesCD20 L27 PE BD Biosciences

L27 PerCP BD BiosciencesCD22 S-HCL-1 APC BD BiosciencesCD34 8G12 FITC BD Biosciences

8G12 PE BD Biosciences8G12 APC BD Biosciences

CD45 2D1 FITC BD Biosciences2D1 PerCP BD Biosciences

CD117 104D2 PE BD Biosciences104D2 APC BD Biosciences

CD11b D12 APC BD BiosciencesCD13 My7 RD1 Beckman Coulter

WM15 APC BD BiosciencesCD14 MOP9 FITC BD Biosciences

CD16 5D2 FITC BD BiosciencesCD33 906 RD1 Beckman-CoulterP67.6 APC BD Biosciences

CD71 LO1.1 FITC BD BiosciencesCD235a GPA PE Dako Cytomation

MAbs, monoclonal antibodies; BM, bone marrow.

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characteristics in a dot plot is useful to select and studythe immunophenotypic differentiation patterns within theB cell and granulocytic/monocytic lineage, respectively.

Apart from lineage-specific markers and immature or ma-ture markers that are restricted to a particular cell lineage,markers with a much broader expression pattern can be

useful in the study of normal differentiation patterns inBM. The pan-leukocyte marker CD45 gives additional in-formation about the major subpopulations, as the CD45antigen is differently expressed in various lineages anddifferentiation stages (6,27,28). CD45 expression is highon lymphocytes and monocytes, whereas granulocytes,precursor B cells, precursor granulocytic cells, and pro-erythroblasts are also CD45 positive, but at lower levels(29,30). In contrast, (more) mature erythroid cells aregenerally CD45 negative (6,31). Therefore, when com-bined with SSC characteristics, CD45 can help distinguishthe major leukocyte (sub)populations in BM. CD34 isanother non-lineage-restricted marker, expressed by theprecursor stages of the various cell lineages in BM (32,33).

The normal differentiation patterns within a specific lin-eage can be displayed by the application of dynamicmarkers, i.e., markers that are gradually up- or downregu-lated during differentiation. The immunophenotypicchanges are best illustrated when mAb directed againstmarkers with overlapping expression profiles are com-bined in one staining. For example, CD10 and CD20 (inB-cell development) and CD13 and CD16 (in myeloiddevelopment) are informative combinations (4,7). Ideally,the selected antibody combinations cover multiple con-secutive differentiation stages, resulting in dot plots thatprovide direct insight in the completeness and relativedistribution of the major subpopulations in the differenti-ation lineage under study.

The composition of the selected immunostainings wasbased on our extensive experience obtained during thelast decade with 9001,200 BM samples analysed each

year. All flow cytometric analyses of BM samples wereperformed routinely with three-color immunostainingsduring 19961999 and with four-color immunostainingsfrom 1999 until the present.

We selected a set of six four-color immunostainings forthe analysis of the B-cell, monocytic, granulocytic anderythroid cell lineages. Per lineage, the major subpopula-tions are described and illustrated in the respective plotsand differentiation schemes. Normal age-related variationsin the expression profiles are discussed followed by ex-amples of disease-induced shifts. We also discuss some

alternative markers or four-color immunostainings thatcan be used for the same purpose.

BONE MARROW DIFFERENTIATION PATTERNSB-Cell Differentiation

Normal B-cell differentiation. Early B-cell differenti-ation has been extensively studied in BM using flow cyto-metric immunophenotyping. Initially, most studies fo-cused on precursor B-acute lymphoblastic leukemia(precursor B-ALL) to unravel early B-cell development.Later, studies on the B-cell compartment in normal BM

further unraveled the consecutive expression of antigens(25,20). Refinements in multiparameter flow cytometryallowed the analysis of large numbers of B cells by whichminor B-cell populations could be identified (4,5,34). Thetwo selected quadruple immunostainings have proved ex-tremely useful for fast screening of the BM B-cell compart-

ment, enabling the identification of (pathological) abnor-malities in B-cell differentiation. Immunostaining 1: TdT/CD20/CD19/CD10.The

combination of CD10 and CD20 with a pan-B-cell marker(CD19) is a well-established combination for analysis ofB-cell differentiation (2,4). Within the CD19 B cells, atleast four sequential maturation stages can be discrimi-nated based on CD10 and CD20 expression (Fig. 1A:stages IIV). These four differentiation stages form a con-tinuous staining pattern in a CD10/CD20 plot (Fig. 1D) asa result of the stepwise loss of CD10 and the gradual gainof CD20 during maturation. Plasma cells, when present,reveal a minor discrepant population in this dot plot,being CD10CD20. As both CD10 and CD20 show a

wide range in expression levels during B-cell differentia-tion, APC- or PE-conjugates of these mAb are preferred fordiscrimination of the various subpopulations. Adding TdTto the CD10/CD20 combination of mAb enables a betterdiscrimination between the immature CD10bright cells thatare TdT and the more mature CD10 cells that areTdT(Fig. 1C). As detection of TdT concerns an intracel-lular staining, the addition of TdT delays the otherwise

very quick screening. In our opinion, however, the advan-tage of a better discrimination between the CD10 pre-cursor B-cell populations compensates the more complexintracellular staining. First, a low expression of TdT com-bined with a high CD10 expression is characteristic forprecursor B-ALL (20). Second, the ratio betweenTdTCD10 and TdTCD10 precursor B cells can beindicative for (drug-induced) regeneration of BM (9,10).

Immunostaining 2: CD45/CD34/CD19/CD22. Thesecond quadruple staining concerns the combination ofCD34 with CD22, and CD45 and CD19. Again, four majorB-cell subpopulations (Fig. 1A: stages IIV) can be discrim-inated within the CD19 B cells. The most immature Bcells from the first immunostaining (TdTCD10 cells)globally correspond to the CD34CD22CD45dim cells inthis immunostaining (Fig. 1E,F). The CD45 expressionincreases during maturation followed by an increasedCD22 expression (Fig. 1F). The mature CD10CD20 Bcells from the first immunostaining globally correspond to

the CD22bright

CD45bright

cells in the CD22/CD45 dot plot.When present, CD22CD19CD34 pro-B cells can beidentified as a minor population in addition to the earlierdescribed four stages of B-cell differentiation (5,25).

In a standardized setting, with a calibrated flow cytom-eter and using the same mAb clones, the staining patternsfrom both immunostainings as displayed in the respectiveplots are quite stable. Shifts in the relative sizes of thesubpopulations or a different location in the plot maytherefore indicate (ab)normal variations or aberrancies inthe B-cell development.

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Normal variations and abnormalities in B-cell dif-ferentiation. The expression profiles from the two se-

lected immunostainings are quite stable, but shifts in therelative sizes of the subpopulations can be found depen-dent on the age of the individual (4). In children theCD10 subpopulations predominate, while in adults theCD10CD20 B cells are more frequent. In the elderly(generally75 years), the mature B-cell population caneven occupy most of the BM B-cell compartment (Fig.2AC). Moreover, the CD10CD20 plasma cells repre-sent a substantial subpopulation of the CD19 B cells inthe BM of an older person (cyan colored). These age-related shifts should be regarded as normal.

Irrespective of age, shifts in the relative distribution of the(precursor) B-cell subpopulations can also be found in regen-

erating BM (Fig. 3AC). Precursor B-cell regeneration can beobserved after chemotherapy for acute leukemia or duringtemporary therapy stops. Differences in the precursor B-cellregeneration patterns are known to be related to the inten-sity of the preceding treatment (9,10). Following a high-intensity therapy block such as induction treatment of ALLpatients, the more immature TdTCD10 precursor B cellsdominate over the TdTCD10 precursor B cells during BMregeneration, whereas the ratio between the TdTCD10

and TdTCD10 precursor B cells is inversed when thepreceding therapy block has been less intense (9).

FIG. 1. Normal B-cell developmentin bone marrow (BM). A: In thisscheme for normal B-cell develop-ment in BM, differentiation stagesare depicted that can be recognizedwith the two selected four-color im-munostainings 1 and 2. The colors inthe differentiation scheme (A) glo-bally correspond to the colors in theunderlying dot plots (BF) and repre-sent different B-cell subpopulationsin childhood BM (4 year old healthychild). To identify B cells, a CD19gate was used in both immunostain-ings: TdT/CD20/CD19/CD10 (B-D)and CD45/CD34/CD19/CD22 (E,F).Within the CD19 B cells, at leastfour subpopulations can be identifiedwith both immunostainings.

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FIGURE 2

FIGURE 3

FIGURE 4

4

FIG. 2. Age-related shift in B-cell compartment of elderly individuals.In bone marrow (BM) of elderly individuals, the relative size of the(CD10) precursor B-cell population is decreased when compared to BMof young individuals (see Fig. 1). The CD19 B-cell population in theadult BM (A) is predominated by the more mature TdTCD10CD20 Bcells (B,C). The cyan dots represent the plasma cell population(CD19CD10CD20TdT), which can be a substantial subpopulationin the BM of elder individuals. Note the change in using CD19-PEinstead of CD19-PerCP, and CD20-PerCP instead of CD20-PE.

FIG. 3. Regenerating precursor B cells in bone marrow (BM) aftercytotoxic treatment. In BM from a patient treated for precursor B-acutelymphoblastic leukemia (BALL), massive regeneration of the precursorB-cell compartment is seen as soon as the therapy is (temporarily)

stopped after induction treatment. Analysis of CD19

B cells (A) showshigh numbers of (nonleukemic) CD10 precursor B cells in the BMsample, with relatively low numbers of more mature CD10CD20 Bcells (B,C).

FIG. 4. Alternative immunostainings for normal B-cell development inbone marrow (BM). Alternative immunostainings that show the differentstages in B-cell development are CyIg/SmIgM/CD20/CD10 and SmIg/SmIg /CD20/CD10. When gated on CD10 and CD20 small cells(lymphocyte scatter) (A,B), the stepwise gain of CyIg and gradual in-crease in SmIgM expression is observed (C) using alternative stainingCyIg/SmIgM/CD20/CD10: CyIgCD10CD20 precursor B cells dif-ferentiate to mature SmIgMCD10CD20 B lymphocytes. Using acomparable gating strategy, the differentiation into either Ig- or Igpositive B cells can be shown (D) using alternative staining SmIg/SmIg/CD20/CD10.



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Besides the shifts in the relative sizes of the subpopu-lations, shifts in expression levels of markers can beobserved. This should be considered with great suspi-cion. Precursor B-ALL frequently display aberrantmarker expression by which the malignant population

will occupy a so-called empty space of the dot plot

template: the cells are located in a space where nor-mally no cells are found (20). Examples include veryhigh expression of CD10 combined with low expres-sion or negativity for TdT or low expression or nega-tivity for CD45 combined with a relatively high CD22expression (20,35,36).

Other possible quadruple immunostainings for

studying B-cell differentiation. The two selected qua-druple immunostainings (i.e., TdT/CD20/CD19/CD10 andCD45/CD34/CD19/CD22) are efficient for first screening ofthe B-cell compartment in BM. They quickly reveal shifts orabnormal patterns in the B-cell differentiation profile, whichthen need to be further analyzed in detail. It is obvious thatthese two immunostainings are not the only possibilities forscreening of the B-cell compartment in BM.

In combination with CD10, CD20, and TdT, one canargue the use of CD22 instead of CD19, as a (minor)subpopulation of precursor B cells (pro-B cells) isknown to be CD19 negative (5). On the other hand,normal plasma cells are generally CD22 negative(37,38). Since both CD19 and CD22 B cells concernonly minor subpopulations of B cells, both markers areuseful as pan-B-cell markers for the major B-cell sub-populations. In the two B-cell immunostainings we pre-fer to use CD19 as the pan-B-cell marker for gating theB-cell lineage, because of its homogeneous expression.The expression of CD22 is much more heterogeneous,

varying from dim to bright positive, as is illustrated inFigure 1F. This heterogeneous expression and the ex-pression of CD22 on basophils (39) makes CD22-basedB-cell gating more complicated. In some cases, rela-tively high background staining of CD19 can be seen onmonocytes: this is caused by binding of the mAb to FcR.This background staining generally does not affect B-cell gating if a combination of CD19 positivity with lowSSC characteristics is used as a selection gate.

As indicated, the intracellular TdT staining may delaythe screening process. For reasons of efficiency, the im-munostainings CD45/CD20/CD19/CD10 or CD34/CD20/CD19/CD10 will be reasonable alternatives to discrimi-

nate the various immature B-cell subsets, taking intoaccount that the expression patterns of TdT, CD45 andCD34 are different (Fig. 1).

CD10 and CD20 can also be combined with manyother differentiation-stage specific markers like cyto-plasmic immunoglobulin chain (CyIg), surface mem-brane (Sm)IgM or SmIg light chain. These immunostain-i ng s w i ll g i ve e xte n de d i nf o rma ti o n a bou t t h ematurational stage of the various subpopulations (Fig.4), but will not identify additional major B-cell subpopu-lations in BM.

Monocytic Cell Differentiation

Normal monocytic differentiation. Immunostainingof cells of the monocytic lineage can be hampered by theaspecific binding of mAb via FcR on monocytes or byautofluorescence of the monocytes. For specific stainingof more mature monocytes, IgG2a mAb should be avoided

whenever possible because monocytes express high lev-els of the IgG2a binding FcRs (CD64) (24).

The most immature monocytic precursor cell (mono-blast) can immunophenotypically not be discriminatedfrom the most immature granulocytic precursor cell (my-eloblast) or from their common progenitor. Immunostain-ing 3 was selected to identify the myelo/monoblasts. Twoadditional differentiation stages can immunophenotypi-cally be identified with an immunostaining specific formonocytic differentiation (Fig. 5A; immunostaining 4).

Immunostaining 3. CD34/CD117/CD45/CD13.33.Both CD33 and CD13 are specific markers for the granu-locytic and monocytic lineage. As their expression level isheterogeneous during differentiation, a mixture of CD13

and CD33 mAb was used in this third immunostaining tooptimize the staining of all cells that belong to the gran-ulocytic and monocytic lineage.

For the identification of myelo/monoblasts, a combinationof CD45 expression and SSC pattern is useful in the gatingprocedure (Fig. 5B). A dim expression of CD45 is seen ongenerally all precursor cells in BM: myeloblasts, monoblast,precursor B cells, and erythroblasts. CD34 is expressed onprecursors-cells of granulocytic, monocytic and B-cell lin-eage, while CD13.33 and CD117 are expressed predomi-nantly on precursor cells of the granulocytic/monocytic lin-eage. CD117 is also expressed on the (CD34dimCD13.33)promyelocytes (Fig. 5C), on a minor subpopulation of pre-

cursor B cells and at very high levels on mast cells (4042).The latter populations is however hardly detectable in nor-mal BM. The CD34CD117CD45dimCD13.33 cells in thisimmunostaining represent the myelo/monoblasts, while themajority of CD34CD117CD45dimCD13.33 cells are pre-cursor B cells (Fig. 5B,D).

Immunostaining 4. CD14/CD33/CD45/CD34. Theprogression from monoblast to promonocyte is marked byan increase in CD33 expression and the disappearing ofCD34 expression, while the cells maintain intermediate lev-els of CD45 (Fig. 5E,G). The CD34CD33highCD45intermediate

promonocytes express low levels of CD15 on their surface.Subsequently, the CD45 expression is increased and the cellsgain CD14 (Fig. 5F). This CD14 expression marks the mono-

cyte stage. In contrast to the neutrophils, the monocytic cellsretain HLA-DR during their maturation.

Normal variations and abnormalities in mono-cytic development. Shifts in the relative distribution ofthe different immunophenotypically identified monocyticsubpopulations are rarely seen as a result of normal (age-related) variations. Monoblasts or promonocytes are over-represented in cases of monoblast-predominant AML(AML-M5a) or promonocyte-predominant AML (AML-M5b), respectively (Fig. 6). Consequently, the shifts in theimmunophenotypic profiles will be as prominent as the

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shifts in the relative frequency of the leukemic cell pop-ulation. Maturation asynchronisms such as coexpressionof CD34/CD11b or CD34/CD15; cross-lineage marker ex-pression such as CD19, CD2, or CD7; and aberrant inten-sities of expression may be correlated with the prognosis

of the disease in AML (16,17,43).In cases of myeloid dysplastic syndrome (MDS) shifts in

the frequency of monocytes (in normal BM, 18%) can beseen. Either monocytosis or a lack of monocytes candistinguish different types of MDS (44,45). For thosecases, immunostaining 4 (CD14/CD33/CD45/CD34) isuseful to quantify the CD14 CD45bright monocytes.

Other possible quadruple immunostainings forstudying monocytic differentiation. For evaluation ofmonocytic development, CD36, CD64, or CD4 (dimlyexpressed on monocytes) can be used in addition to

CD14. CD36 expression and dim CD4 expression oftenprecede CD14 expression and may indicate the mono-cytic differentiation of an otherwise CD34 AML. In addi-tion, CD68 is useful to identify the final stage of monocyticdifferentiation, i.e., macrophages. With their increased

FSC and SSC, macrophages can be distinguished fromtheir precursors. However, in BM generally no macro-phages are detectable.

As mentioned before, transient dim CD15 expression isseen on cells of the monocytic lineage preceding theCD14 expression and is only detectable with high affinityCD15 mAb (22,24). However, low expression of CD15 onCD34 precursor cells identifies not only commitment tomonocytic development, but also granulocytic precursorcells in transition from CD34CD15 blasts to CD34/CD15bright promyelocytes.(46).

FIG. 5. Normal monocytic develop-ment in bone marrow (BM). A: In thisscheme for normal monocytic devel-opment in BM, differentiation stagesare depicted that can be identified bythe two selected four-color immuno-stainings 3 and 4. The colors in thedifferentiation scheme globally corre-spond to the colors in the underlyingdot plots and represent different sub-populations. BD: Expression profilesof immunostaining 3 (CD34/CD117/CD45/CD13.33). A combination oflow CD45 expression and low SSC isused to identify the precursor cells inBM with immunostaining 3. TheCD34CD117CD45dimCD13.33

cells in this immunostaining repre-sent the myelo/monoblasts (reddots), the CD34CD117CD45dim

cells (blue dots) represent the pre-cursor B cells. The green dots repre-

sent the promyelocytes (see also Fig.7A), which can be identified with thisimmunostaining by the increasedSSC and high CD13.33 expression(D). Note that a mix of APC-conju-gated CD13 and CD33 mAb is usedto optimize the staining of all cellsthat belong to the granulocytic andmonocytic lineage. Normal mono-cytic development in BM is illus-trated with immunostaining 4 (CD14/CD33/CD45/CD34), when an SSClow

gate is combined with a CD45lowhigh

gate (E). Three differentiation stagescan be discriminated including themyelo/monoblast stage: CD34

CD33CD14 myelo/monoblasts,CD34/CD33highCD14 pro-mono-cytes and CD34CD33highCD14

monocytes (F and G). LWBM: ammo-niumchloride-lysed whole BM.



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Granulocytic Cell Differentiation

Normal granulocytic (neutrophil) differentiation.The granulocytic differentiation in BM generally concernsneutrophil differentiation, as eosinophils and basophils

comprise only minor subpopulations in normal BM. Asmentioned before, the most immature granulocytic pre-

cursor cell (myeloblast) cannot be discriminated immuno-

phenotypically from the most immature monocytic pre-

FIG. 6. Acute myeloid leukemia(AML) with monocytic differentia-tion. bone marrow (BM) of a patientwith an acute monocytic leukemia.Most cells in the BM are of monocyticlineage. Monoblasts, promonocytes,and monocytes can be recognized;the promonocytes are most predomi-nant. The dot plots illustrate the im-munophenotypic heterogeneity withinan acute leukemia.

FIG. 7. Normal granulocytic devel-opment in bone marrow (BM).A: Scheme for normal granulocyticdevelopment in BM, depicting differ-entiation stages that can be identi-fied by the two selected four-colorimmunostainings 3 and 5. The colors

in the differentiation scheme globallycorrespond to the colors in the under-lying dot plots and represent differ-ent subpopulations. The expressionprofiles of immunostaining 3 (CD34/CD117/CD45/CD13.33) are pre-sented in Fig. 5BD. Light scattercharacteristics in combination withthe intermediate CD45 expression isused to identify the granulocytic lin-eage (B), in immunostaining 5(CD16/CD13/CD45/CD11b). Fivedifferentiation stages are distinguish-able, based on the gradual increaseof CD11b (E,F) and CD16 expression(C,E,G) and the dynamic CD13 ex-pression (F,G) in combination withSSC characteristics (D). CD16 ex-pression is preceded by the CD11b

expression (E).

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cursor cell (monoblast). One of the two immunostainings we selected for the monocytic differentiation (immuno-staining 3) identifies both the monocytic and granulocyticprecursor cells, while the other (immunostaining 5) un-ravels the further granulocytic differentiation.

Immunostaining 3: CD34/CD117/CD45/CD13.33.

For screening of abnormalities in the immature granulo-cytic/monocytic precursor cells, we selected a quadruplestaining that covers the early precursors of both lineages:combination of the precursor markers CD34 and CD117

with CD45 and a mixture of CD13 and CD33 will revealeventual shifts in the relative size or immunophenotype ofthe myelo/monoblasts (Fig. 5BD). This immunostainingalso identifies promyelocytes as CD117CD34dim

CD13.33high cells with an increased SSC. Immunostaining 5: CD16/CD13/CD45/CD11b. The

second quadruple immunostaining for the granulocyticdifferentiation concerns the combination of CD45 andCD13 with CD16 and CD11b. An intermediate CD45 ex-pression discriminates the cells of the granulocytic lineage

from the CD45high

lymphocytes and monocytes. CD45expression, in combination with the granulocytic/mono-cytic lineage marker CD13 and light scatter characteris-tics, is useful to select and study the granulocytic differ-entiation patterns (45,47).

CD13 is a unique marker in the granulocytic differenti-ation as it is dynamically expressed during granulocyticdifferentiation. It is expressed at high levels on myelo-blasts and promyelocytes. CD13 is downregulated anddimly expressed on myelocytes and is gradually upregu-lated again as the granulocytic cells develop into seg-mented neutrophils (Fig. 7D). Combination of this dy-namic marker with CD11b and CD16 in oneimmunostaining reveals four sequential maturation stages

in the neutrophil development (Fig. 7A: stage IIV; Fig.7EG). CD11b and CD16 are initially expressed at lowlevels, but their expression increases during the develop-ment process, particularly in the last 2 stages (stages IVand V; Fig. 7E).

With the quadruple immunostaining 5 (CD16/CD13/CD45/CD11b), the myelo/monoblasts can be identified asCD16CD13CD45intermediateCD11b. They have an inter-mediate forward scatter (FSC) but still a low SSC andglobally correspond to the CD34CD117CD45intermediate

CD13.33 cells from immunostaining 3 (Fig. 7). When themyeloblasts progress to the stage of promyelocytes, adramatic increase in SSC is seen (Fig. 7B,D). From thismoment, the cells express CD15 and retain their high SSC.

In the subsequent maturation stages CD11b is acquired,followed by CD16 (Fig. 7EG). Note that the relativelyhigh fluorescence signal for CD16-fluoroscein isothiocya-nate (FITC) of promyelocytes and myelocytes in Figure7E,G has nothing to do with early CD16 expression but

with the high autofluorescence of these subpopulations inthe FL1 (FITC) channel.

Like B-cell development, the staining patterns of immu-nostaining 5 for normal granulocytic differentiation in BM,are quite stable with regard to the immunophenotypiccharacteristics and relative distribution of the various sub-

populations (provided that the same mAb clones areused).

Normal variations and abnormalities in neutro-

phil differentiation. The myeloid differentiation mainlyconcerns the maturation to neutrophilic granulocytes, asthe other end-stage granulocytes (i.e., the basophils and

eosinophils) comprise only minor cell populations in nor-mal BM. However, as a result of immune responses tospecific agents or in case of a chronic myeloid leukemia(CML), shifts can be observed in the relative distributionof the various end-stage granulocytes (48,49). In BM of apatient with a CML-accelerated phase, eosinophils andbasophils can be identified as prominent subpopulationsof the granulocytic lineage (Fig. 8). On the FSC/SSC dotplot, eosinophils show a high SSC and lower FSC com-pared to the neutrophils (Fig. 8A). Expression of CD16 oneosinophils is dim and also expression of CD11b andCD13 is somewhat lower compared to neutrophils (Fig.8C). In contrast, CD45 expression on eosinophils is con-sistently higher then on neutrophils (Fig. 8B). Basophilshave a much lower SSC compared to the other granulo-cytic subpopulations and are hard to distinguish fromlymphocytes and, to a lesser extend, monocytes based onlight scatter characteristics. An intermediate CD13 expres-sion on basophils (high on monocytes and absent onlymphocytes) and a lower CD45 expression (Fig. 8B), helpidentify these cells. Furthermore, basophils have a uniqueimmunophenotype, different from the other granulocyticcells. They express CD22, but are negative for other B-cellmarkers (39). An increase in the relative size of the baso-philic cell population can be seen in the chronic andaccelerated phase of CML (48).

Shifts in the relative distribution of the granulocytic

subpopulations and in the immunophenotypic character-istics are seen in myeloid malignancies like AML andmyeloid dysplastic syndrome (MDS) (50,51). An increaseof immature granulocytic precursor cells or a reduction in

well-differentiated granulocytes can be seen in both dis-eases. The two immunostainings were not selected for theidentification of all sorts of immunophenotypic aberran-cies that can be expected in patients with AML or MDS,but they are useful for a quick screening of BM for suchabnormalities. First, immunostaining 3 (CD34/CD117/CD45/CD13.33) will illustrate that most CD34 precursorcells in MDS or AML BM belong to the myelo/monocyticlineage, and not to the B-cell lineage, as they are CD117

and/or CD13.33

. In addition, the intermediate CD45expression in combination with SSC identifies increasednumbers of myelo/monoblast cells in both immunostain-ings. Second, morphological abnormalities like hypo- orhypergranulation will be reflected in reduced or increasedSSC values (Fig. 9). Third, in MDS an overexpression ofCD13 and CD33 and a reduced expression of CD11b andCD16 is frequently seen and consequently the CD13/CD16 and CD13/CD11b dot plots of immunostaining 5

will show an aberrant expression profile of the granulo-cytic cells (Fig. 9).



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Other possible quadruple immunostainings for

studying granulocytic differentiation. As mentionedbefore, both CD33 and CD13 are specific markers for thegranulocytic and monocytic lineage that can be expressedheterogeneously during differentiation. To optimize thestaining of all cells that belong to the granulocytic andmonocytic lineage, a mixture of CD13 and CD33 mAb canbe used. An aberrant cross-lineage expression of CD13and/or CD33 is seen in 4060% of precursor B-ALL pa-tients. CD65 can be used as an alternative pan-myeloidmarker to cover a large part of the granulocytic differen-tiation from myeloblasts, until end-stage granulocytes.

Although described as a pan-granulocytic marker, CD15expression is not confined to the granulocytic differentia-tion. In precursor cells committed to the monocytic differ-

entiation (like promonocytes), CD15 can be expressed atlow levels. Low-affinity mAb will only stain the granulocyticlineage, while high-affinity mAb (e.g., clone MM1; BD Bio-sciences) will also stain the immature monocytic cells. ThismAb is useful in combination with CD34 and monocyticmarkers to identify the monocytic precursor cells.

CD66b expression precedes CD16 expression compa-rable to CD11b, being present on (pro-) myelocytes. Con-

sequently, CD66b in combination with a pan-myeloid

marker, like CD13 or CD33, will also reveal differentstages in the granulocytic differentiation and is therefore

an alternative mAb for CD11b. We selected the combina-tion of CD11b with CD16 as CD11b distinguishes the early

differentiation stages in granulocytic differentiation very

well, whereas CD16 distinguishes the more mature differ-entiation stages very well. In addition to CD16 and

CD11b, other markers that are not restricted to the gran-ulocytic or monocytic lineage, like CD64 or CD35, will

also show variations in their expression levels during

granulocytic differentiation and are useful in combinationwith CD13 and/or CD33 (52).

Erythroid differentiation

Normal erythroid differentiation. To minimize

noise when measuring leukocytes, erythrocytes in BM sam-ples are generally lysed. The many commercially available

permeabilizing or lysing reagents and ammonium chloride(NH4Cl) preserve the lyse-resistant nucleated red cells of the

erythroid lineage for immunophenotypic analyses.

FIG. 8. Eosinophilic and basophilic granulocytes in bone marrow (BM) of a chronic myeloid leukemia (CML) patient. In BM of a patient withCML-accelerated phase, relatively high numbers of eosinophilic (yellow dots) and basophilic (red dots) granulocytes can be identified. Eosinophilicgranulocytes occupy a discrepant position in the forward scatter/side scatter (FSC/SSC) dot plot (A) and show a CD45 expression comparable tomonocytes (B; blue dots). Basophilic granulocytes are hard to distinguish from lymphocytes in a FSC/SSC dot plot (A; green dots). An intermediate CD45expression comparable to neutrophilic granulocytes however, discriminates the basophilic granulocytes (B). In the CD11b/CD13 dot plot, the basophilicand eosinophilic granulocytes show a slightly lower CD11b expression than the mature neutrophilic granulocytes ( c).

FIG. 9. Hypogranular granulocytes in bone marrow (BM) of a MDS patient. BM of an MDS patient displays various aberrancies in the granulocyticlineage. Hypogranularity of the granulocytes results in a reduced SSC ( A). Furthermore, the staining patterns in the CD11b/CD13 and CD13/CD16 dotplots are clearly changed with strongly reduced promyelocytic and myelocytic subsets (B,C).

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Immunostaining 6. CD71/CD235a/CD45/CD117.This selected quadruple immunostaining clearly identifiestwo sequential differentiation stages of nonlysed nucle-ated erythroid cells in BM samples after red cell lysis (Fig.10A). Cells of the erythroid lineage are generally identified

by the gradual loss of CD45 (6). Only the CD117

CD71

erythroid-precursors and pro-erythroblasts do expressCD45 dimly. During maturation, the expression of thetransferrin receptor CD71 slightly increases, where-afterCD235a (glycophorin A) is being expressed. The red cellsretain CD235a but lose CD71 together with loss of thenucleus (6,31).

Normal variations or abnormalities in erythroiddevelopment. The nucleated erythroid population canbe much more predominant in cases of a severe aplasticanemia or MDS, or during BM regeneration after drug-induced BM suppression. In regenerating BM of children

treated for ALL, the erythroid cell population can occupyup to 50% of the cells in the lysed BM sample (53). TheseCD45CD71CD235a cells include both the nucleatederythroid cells and the reticulocytes, which tend to bemore resistant to lysis when present in high numbers in

young children (Fig. 11AC). Although shifts in the relativefrequency of nucleated red cells can be seen in regenerating

FIG. 10. Normal erythroid development in bone mar-row (BM). A: In this scheme for normal erythroid devel-opment in BM, differentiation stages are depicted thatcan be identified by the selected four-color immuno-staining 6. The colors in the differentiation schemeglobally correspond to the colors in the underlying dotplots and represent different subpopulations. Whengated on SSClow in combination with CD45neg-low, onlythe erythroblasts are seen in normal BM (B,C). Pro-erythroblasts will form a minor population in normalBM and erythrocytes are lysed in the sample procedure.

FIG. 11. Erythroid development in bone marrow (BM) of a patient with regenerating BM. In regenerating BM of a patient treated for acute lymphoblasticleukemia (ALL), three differentiation stages can be discriminated in the erythroid development: CD117CD45lowCD71CD235a pro-erythroblasts (reddots), CD117CD45neg-lowCD71CD235a erythroblasts (green) and CD117CD45CD71CD235a nonlysed erythrocytes (blue dots) can bediscriminated.



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BM, severe anemia, megaloblastic anemia or brisk hemolyticanemia, or BM of very young children, immunophenotypicshifts as a result of aberrant marker expression are rare. Incontrast, in MDS patients aberrant expression of CD71and/or CD235a is frequently observed (50).

CONCLUDING REMARKS

We present six quadruple immunostainings that detect allmajor leukocyte populations in BM. All six quadruple immu-nostainings reveal remarkably stable immunostaining pat-terns and are therefore useful as a frame of reference forquick identification of shifts in normal BM development.These shifts can either be age-related or disease- or drug-induced. Age-related shifts in normal BM development gen-erally concern shifts in the relative distribution of the varioussubpopulations within the respective lineages. Although sev-eral subpopulations may become more predominant thanothers during aging, the shape of the immunophenotypicstaining pattern remains unaltered. Alterations in the relativedistribution of the subpopulations can also be seen in regen-

erating or reactive BM when erythroid, precursor B-cell ormyelo/monoblast populations are clearly overrepresented.In most cases of malignant hematopoiesis, such as MDS oracute leukemia, the aberrant immunophenotype often posi-tions the malignant cell population at a separate place in thedot plot. Such immunophenotypic shifts should thereforealways be considered as an abnormality in the hematopoie-sis. Further detailed flow cytometric analyses of the relevantcell lineage is then recommended.

In this report we do not include immunostainings foridentification of the NK-/T-cell lineage, since NK-/T-celldifferentiation does not take place in BM. The T-cell pop-ulation found in normal BM comprises mature blood de-rived T lymphocytes. T-cell immunostainings of normal

BM samples will therefore reveal nondynamic immuno-staining patterns with a homogeneous T-cell populationand no clear subpopulations other than CD4 and CD8

T lymphocytes. Adding a single T-cell marker to the sixselected quadruple stainings will not give additional infor-mation, as changes in the relative number of T cells in theBM will mainly reflect the quality of the aspirated BM.However, for screening BM involvement in immature ormature T-cell malignancies, we suggest the addition oftwo specific T-cell immunostainings: CD2/CD5/CD3/CD7and CD4/CD8/CD3/HLA-DR (19).

Reference values for the major leukocyte subpopulationsin BM are only partly available, but the dot plot templates ofthe six selected immunostainings can serve as a reference.

The normal immunophenotypic expression profiles helpidentify normal and abnormal variations in hematopoiesisand are therefore useful as first screening.

ACKNOWLEDGMENTS

The authors thank Marieke Comans-Bitter for preparingthe figures; the hematologists and pediatricians for sub-mitting patient samples to our laboratory; and IvonneBronmans, Anja de Jong, Jac Kuijpers-Entrup, Marja Smits,Dennis Tielemans, and Patricia Hoogeveen for their excel-lent technical support.

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