Klaus Steenberg Larsen

Louise C Andresen

Claus Beier

Sven Jonasson

Per Ambus Kristian R Albert

Karen Stevnbak

Marie Frost Arndal

Mette S Carter

Soren Christensen

Merete ; Selsted

Per Ambus

Karen S Andersen

Søren

Martin Holmstrup

Andreas Ibrom

Jane Kongstad

Leon Van Der Linden

Kristine Maraldo

Anders Michelsen

Teis N Mikkelsen

Kim Pilegaard

Anders Priemé

Helge Ro-Poulsen

Inger K Schmidt

Merete B Selsted

Reduced N cycling in response to drought, warming, and elevated CO2 in a Danish heathland: Synthesizing results of the CLIMAITE project

Keywords: Climate driver interactions, C/N ratio, multi-factor climate change experiment, nitrogen cycling, nitrogen mineralization, N2O, soil fauna Climate driver interactions, C/N ratio, multi-factor climate change experiment, 23 nitrogen cycling, nitrogen mineralization, N 2 O, soil fauna

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Field-scale experiments simulating realistic future climate scenarios are important tools for investigating the effects of current and future climate changes on ecosystem functioning and biogeochemical cycling. We exposed a semi-natural Danish heathland ecosystem to elevated atmospheric carbon dioxide (CO2), warming, and extended summer drought in all combinations. Here, we report on the short-term responses of the nitrogen (N) cycle after two years of treatments. Elevated CO2 significantly affected above-ground stoichiometry by increasing the carbon to nitrogen (C/N) ratios in the leaves of both co-dominant species (Calluna vulgaris and Deschampsia flexuosa), as well as the C/N ratios of Calluna flowers and by reducing the N concentration of Deschampsia litter. Below-ground, elevated CO2 had only minor effects, whereas warming increased N turnover, as indicated by increased rates of microbial NH4+-N consumption, gross mineralization, potential nitrification, denitrification and N2O emissions. Drought reduced below-ground gross N mineralization and decreased fauna N mass and N mineralization. Leaching was unaffected by treatments but was significantly higher across all treatments in the second year than in the much drier first year indicating that ecosystem N loss is highly sensitive to changes and variability in amount and timing of precipitation. Interactions between treatments were common and although some synergistic effects were observed, antagonism dominated the interactive responses in treatment combinations, i.e. responses were smaller in combinations than in single treatments. Nonetheless, increased C/N ratios of photosynthetic tissue, decreased litter N production, and decreased fauna N mineralization prevailed in the full treatment combination indicating reduced N turnover in the simulated future climate scenario, which could act to reduce the potential growth response of plants to elevated atmospheric CO2 concentration.

In conclusion, effects observed in single-factor studies should be evaluated with caution. Multi-factor climate experiments are needed for improving realistic estimation of the combined ecosystem responses to future climate changes.

62

Growth in many natural and semi-natural terrestrial ecosystems is limited by nitrogen 63 and characterized by strong competition for nitrogen between plants and the soil communities 64 of fungi and bacteria (Marion et al. 1989;Jonasson et al. 1996). Consequently, ecosystem N 65 availability is an important factor controlling the carbon uptake in terrestrial ecosystems and 66 therefore also plays a dominant role in controlling the feedback between the biosphere and the 67 atmosphere in a warmer and more CO 2 enriched world. The N cycle at the site is dominated by higher internal N turnover in comparison to the inputs 299 and losses by leaching and gaseous emission to the atmosphere (Fig. 1). Gross mineralization 300 (6.7 ± 0.96 g N m -2 y -1 ) is five times higher than bulk deposition (1.35 ± 0.04 g N m -2 y -1 ), an 301 order of magnitude higher than losses by leaching (0.80 ± 0.37 g N m -2 y -1 ) and two orders of 302 magnitude higher than gaseous losses in form of N 2 O (0.031 ± 0.006 g N m -2 y -1 ). other treatments causing the significant interactions for these three variables (Table 1).

361

The drought treatment reduced the N pools of enchytraeids and protozoans and their indicating an overall decrease in the N turnover in response to drought (Fig. 3b). In contrast,

362 NH 4 + -N mineralization (

365

warming stimulated microbial ammonium consumption in the top 5 cm of soil ( warming treatments was therefore an increase in N turnover (Fig. 3c).

370

While no direct main effects of elevated CO 2 were observed, it influenced the Across the 47 variables reported (Table 1) the drought treatment lead to the highest number of 379 significant main effects (11) followed by warming and CO 2 treatments (6 and 4, respectively).

380

Simultaneously there were a large number of interactions between the three treatments (14).

381

Interestingly, the analysis of the interactions revealed a dominance of antagonistic effects 382 (Fig. 4) with more than twice as many antagonistic effects as synergistic effects. Furthermore,

383

simple additive effects were less frequent than both antagonistic and synergistic effects. The N cycle at the experimental site is characterized by higher internal N turnover than bulk studies and found that three-way interactions were rare while two-way interactions were more 512 common. Generally, interactions were positive between elevated CO 2 and warming and 513 negative between elevated CO 2 and reduced precipitation with respect to net primary 514 production (NPP).

515

A general pattern of most published studies is the focus on a few important variables, 516 i.e. overall changes in NPP or biomasses or specific studies focusing on processes, e.g. See Table 1 for statistical significance of observed changes. ), C/N ratios and N concentrations (mg L -1

) in 2007. Data are means with SE for ambient (A) and means with SE normalized to the mean of the ambient treatment (all other treatments than A).

1)

Significant P values (P ≤ 0.05) from statistical analysis. Direction of main treatment effects is indicated with arrows. Significant interactions are indicated as antagonistic ( §), or synergistic ( ‡). Additive effects of multiple significant main effects are also indicated (+). 2) Significant single treatment effects as indicated by evaluation of main effects plus interaction(s) by analysis of DLSM (see text for further details). Direction of effects is indicated with arrows. n.s. = not significant.

  in response to drought, warming, and elevated CO2 in a Danish heathland: Synthesizing results of the CLIMAITE project after two years of treatments

  photosynthesis and growth(Luo et al. 2006; de Graaff et al. 2006), warming-induced

  N mineralization and immobilization were determined by the isotope pool-236 dilution approach applied in laboratory incubations. Soil cores within each plot were sampled 237 in September 2007 (0-5 and 5-10 cm soil depth, respectively), sieved and stored at 5 °C for 238 about one week until incubations were initiated. Fresh samples (10 g) were weighed in 250 ml 239 poly-ethylene bottles and mixed thoroughly with 400 µl of a 15 N-NH 4 Cl solution (5 atm % 240 15 N) to provide 5 µg N g -1 soil. One set of samples was analyzed immediately upon substrate 241 application, and a second set was analyzed after 7 days of incubation at 20 °C. Contents of 242 mineral N and the 15 N contents were determined upon extraction and micro-diffusion 243 (Sørensen & Jensen 1991). Gross daily rates of mineralization and immobilization were 244 calculated according to equations given by Kirkham & Bartholomew (1954). Upscaling to 245 done using the mean annual soil temperatures in 5 cm depth (10 °C) at the 246 experimental site and assuming a Q 10 of 2. soil cores taken in November 2007 were gently homogenized by hand 250 and major roots were removed. Soil samples were stored at 4 °C until analysis (less than 24 251 h). Potential nitrification was determined in 3 g soil samples by a modification of the method 252 described by Belser & Mays (1980) but without adding chlorate. Potential nitrification rates 253 were estimated from the increase in nitrite+nitrate concentrations. Potential denitrification 254 rates (Wolsing & Prieme 2004) in 10 g soil samples were estimated from linear regression of 255 increase in headspace N 2 O concentration during 180 min incubation. collected monthly from passive PVC soil water draining collectors below 259 the organic soil layer (approximately 5 cm depth) and polytetrafluoroethylene (PTFE) suction 260 cups with continuous sub-atmospheric pressure (Prenart Super Quarz, Frederikberg, DK) 261 below the main rooting zone (60 cm depth). Concentrations of NH 4 + -N, NO 3 --N, and total N 262 (by UV-persulphate oxidation) were analyzed on an Autoanalyzer 3 (Bran+Luebbe Gmbh, 263 Germany). DON was calculated as total N minus inorganic N. Precipitation was measured at 264 the site and the volume of percolating water was estimated by simulation with the CoupModel 265 model (Jansson & Moon 2001; Svensson et al. 2008) -a coupled ecosystem model with a 266 balance of abiotic and biotic processes in the soil-plant-atmosphere system. The basic 267 structure of the model is a depth profile of the soil, and calculations of water and heat flows 268 common soil properties. The model was parameterised using measured soil layer 269 properties and against time series of soil temperature and moisture content, using on site 270 measurements of radiation, precipitation and wind speed as driving data and a generalised 271 likelihood uncertainty evaluation approach. Percolation of water below 60 cm was calculated 272 as an output of the simulations and leaching of nitrogen at that depth was calculated as the 273 product of the sum of water percolation and the concentration measured in the soil water for 274 each water collection period (Schmidt et al. 2004). conducted using the multiple linear regression procedure PROC 278 MIXED of SAS (SAS Institute 2003). The statistical model included a random statement that 279 accounted for the experimental design (Random Block octagon octagon*D octagon*T). The 280 same model was used for all tested variables and included the three main factor effects (CO 2 , 281 D, and T) as well as all possible interactions (D*CO 2 , T*CO 2 , T*D, and T*D*CO 2 ). P-values 282 ≤ 0.05 were considered significant. Data were transformed when necessary to obtain 283 normality and homogeneity of variance. Differences of Least Squares Means (DLSM) were 284 used to interpret significant treatment interactions. During the DLSM evaluation, each 285 significant interaction was categorized as either antagonistic, i.e. the combined effect led to a 286 reduction of the observed individual effects or synergistic, i.e. the combined effect led to 287 either a) an amplification of the observed individual effect(s) or b) to a significant effect only 288 when treatments were combined. The DLSM evaluation also revealed if interactions were due

371

  responses to drought and warming primarily by reducing some of the significant individual 372 effects in the three-factor combination. Still, the drought-induced decrease of fauna-N and 373 fauna N mineralization as well as the warming-induced increased denitrification did not 374 interact significantly with the other treatments and consequently remained in the full 375

517&Figure 2 799

 2 Figure captions

  802

  Figure 3

  Figure 1

  Figure 2

  The above-ground biomass N of the dominant plant species Calluna and Deschampsia did not 323 respond to the climate manipulations (Table1). Root biomass N of Deschampsia was also 324 unaffected by the treatments, whereas root biomass N of Calluna increased in response to treatment had fewer effects on above-ground processes than the other treatments, showing 343 only an increase of N concentration in Deschampsia litter (Table1,P = 0.03, Fig 2c). Deschampsia litter N production are found in the full combination of treatments, i.e. in the 348 simulation of the projected future climate scenario. In contrast, the C/N ratio of mosses 349 increases in two of the three single treatments and N concentrations in Deschampsia litter

	319	precipitation in July) with extensive leaching compared to a dry summer in 2006 (60 mm
	320 344	precipitation in July) when summer leaching was absent.
	321 345	When all treatments were combined (Fig. 2d), the lack of interactions between single
	322 346	Plant responses to climate manipulations treatment effects for leaf level C/N ratios indicates that the effect is found also in treatment
	347	combinations. Consequently, increased C/N ratios of leaves as well as decreased
		F o r F o r
		R R
		e v e v
		i e i e
		w w
	312 313 358 359 360	O n l y O n l -N, NH 4 -+ -N and DON (Table 1, Fig. 3a) in y difference between bulk deposition and sum of total N leaching below 60 cm soil depth and leachate concentrations in 5 cm soil depth of NO 3 the single CO 2 treatment. These effects were reduced when elevated CO 2 was combined with
	314	N 2 O emission. While bulk deposition in 2006 (1.27 ± 0.07 g N m -2 y -1 ) was similar to the
	315 338	deposition in 2007 (1.35 ± 0.04 g N m -2 y -1 ), total N leaching across all treatments was
	316 339	significantly lower (P < 0.0001) in 2006 (0.13 ± 0.06 g N m -2 y -1 ) than in 2007 (0.56 ± 0.20 g Deschampsia leaves and also mosses (single treatment only) responded to drought
		N m -2 y -1 ) with no significant treatment effects in either 2006 (data not shown) or in 2007 with increased C/N ratios (Table 1, P = 0.002 and P ≤ 0.05, respectively) whereas Calluna
	318
	342

Negligible

303

N 2 O reductase activity was observed in the soil samples with N 2 O uptake rates less than 1 ng 304 N g -1 dw h -1 in all samples and N 2 production is therefore assumed to be negligible (data not 305 shown). Above ground, about 90 % of N is found in the two dominant species Calluna (~60 306 %) and Deschampsia (~30 %). Below ground, most N is stored in the SOM pool (~90 %) with 307 Deschampsia roots (~4 %), Calluna roots (~3 %) and soil microbial (~1.5 %) pools 308 dominating the remaining fraction. Soil fauna biomass was ~10 % of microbial biomass but 309 their mineralization of N is estimated to 5.0 ± 0.6 g NH 4 + -N m -2 y -1 , i.e. an amount of labile N much larger than the N input to the system by N bulk deposition. The system retained 0.53 g 311 N m -2 y -1 of bulk deposition in the ambient, non-manipulated plots in 2007 as estimated by

317

(Table

1

). The major cause of this interannual difference was a wet summer in 2007 (202 mm 325 drought (Table 1, P = 0.04) indicating a change in the root:shoot N allocation pattern. While 326 N in above-ground biomasses were unaffected by treatments, the C/N ratios of Calluna 327 flowers as well as leaves from both Calluna and Deschampsia increased in response to 328 elevated CO 2 (Table 1, P = 0.02, 0.04, and 0.01, respectively). The evaluation by DLSM of 329 the three-way interaction for the C/N ratio of mosses (Table 1, P = 0.003) showed that the 330 interaction was due to significantly higher C/N ratios in the single treatments of drought and 331 elevated CO 2 , while ratios were unchanged when drought and elevated CO 2 were combined 332 with each other as well as when they were combined with warming. The observed increases in 333 C/N ratios in new plant tissue of leaves and flowers were not seen in whole-plant C/N ratios 334 of Deschampsia or in the green tissue or wood fractions of Calluna. Yet, the N concentration 335 of Deschampsia litter decreased under elevated CO 2 (Table 1, P = 0.05), indicating that the 336 changes in C/N ratios starting in the leaves, are already beginning to cascade through the 337 system, after only two years of treatments (Fig. 2a). 340 leaves were unaffected (Table 1, Fig. 2b). Also, the pool of N in the annually produced 341 Deschampsia litter decreased in response to drought (Table 1, P = 0.01). The warming 350 either increase or decrease depending on treatment but these effects disappear when all 351 treatments are combined. 352 353 Responses of below-ground processes to climate manipulations 354 The below-ground N turnover was significantly affected by the climate manipulations, 355 primarily by the warming and drought treatments (Table 1, Fig. 3) showing changes 356 cascading through pools and processes. Elevated CO 2 as a main factor had no effects below 357 ground but the analysis of DLSM when evaluating the interactions revealed reductions in

Table 1

 1

	, P = 0.03 and 0.04, respectively), decreased gross

mineralization (Table 1, P = 0.03), and reduced the pool of NH 4 + -N (Table 1, P = 0.02), thus 364

Table 1

 1 ) as well as the gross mineralization rate (single treatment only, Table 1, P ≤ 0.05) and 367 nitrification rates (Table 1, P = 0.004), leading to both increased denitrification (Table 1, P = 368 0.02) and N 2 O emission (Table 1, P = 0.05). Overall, the below-ground response to the

	, P =

  likely to influence the overall competitive relationship, potentially disturbing the 408 balance of the equilibrium, especially if the rates of mortality of Calluna are increased. However, the increased C/N ratios observed in leaves, and for Calluna also 432 observed in flower biomass were after two years of treatments not large enough to affect 433 whole plant biomass C/N ratios, which were not affected by the treatments. Previous studiesNowak et al. 2004). A review byBarnard et al. (2005) of 25 multi-factor studies 506 concluded that interactions were generally rare. These 25 studies were dominated by 507 experimentally elevated CO 2 combined with increased N supply. The lack of interactions

	increase, are 409 The mineralization of NH 4 410 C/N ratios. 434 2010a). However, in contrast to our expectation, we observed very few direct effects of + -N by soil fauna is often neglected but contributes 457 elevated CO 2 on below-ground N processes. Still, the decreased concentrations of organic and 458 inorganic N leachate water at 5 cm soil depth imply that mineralization processes are affected 459 2004; 508
	411 435 460 509	significantly to the pool of plant available N in the soil (Osler & Sommerkorn 2007). In the have found increasing C/N ratios in other plant tissues than leaves (McGuire et al. 1995; or possibly that root N uptake has increased. In contrast to the lack of direct changes in could be due to the common trend that most ecosystems respond strongly to increased N
	388 412 436 461 510	present study, we calculated fauna N mineralization from estimated biomasses and literature Cotrufo et al. 1998), but the change often is much weaker than that observed for leaves response to elevated CO 2 , soil processes were more sensitive to the drought and warming supply, which hides weaker interactive responses. Luo et al. (2008) analyzed and modeled
	389 413 437 462 511	N deposition and losses by leaching and N 2 O production as generally seen in natural and values of the turnover times and yields of microfauna and mesofauna. Obviously, upscaling of (McGuire et al. 1995), indicating that the C/N effect of elevated CO 2 is closely linked to the treatments. Our study supports previous observations of increased below-ground N turnover interactions between elevated CO 2 , warming and changes in precipitation in 7 multi-factor
	390 414 463	semi-natural ecosystems. In comparison to our estimated annual gross mineralization rate (6.7 e.g. enumeration of bacterivorous protozoa and nematodes to gross nitrogen mineralization processes involved in the carbon sequestration in the photosynthetic apparatus (Körner 2000). in response to warming (Rustad et al. 2001; Schmidt et al. 2004). We observed increased
	391	± 0.96 g N m -2 y -1 ), Emmett et al. (2004) reported net mineralization rates of 3.8 to 6.6 g N m -2 y -1 from another Danish dry heathland and Aerts (1993) reported rates ranging from 4.4 to F o r should be evaluated with caution. Values for bacterivore biomass, turnover, and growth yield F o r F o r microbial NH 4 + -N consumption, gross mineralization, potential nitrification, and F o r F o r
		R R R R R
		e v e v e v e v e v
		i e i e i e i e i e
	422	w w w w w
	423 424 425 449 450 475 499	O n l y Elevated atmospheric CO 2 concentration is known to induce down-regulation of Responses to elevated CO 2 , warming and drought O n l y O n l O n l O n l therefore reluctant to put too much emphasis on this result and conclude overall that total y y y
	426 451 476 500	photosynthetic capacity, e.g. via reduced content of Rubisco in the leaves (Ainsworth & Long biomass N of both dominant species as well as mosses after two years of treatments were only this study, together with the negative effects of drought on top soil layer populations of and N 2 O emissions) were additive responses with opposing directions, which therefore acts to
	404 427 452 477 501	2005; Ainsworth & Rogers 2007) and reduction of leaf nitrogen content and increased C/N marginally affected by the applied climate drivers. enchytraeids (Maraldo et al. 2010) and other fauna groups and their decreased N reduce the observed effects in treatment combinations.
	405 453 478 502	should decrease due to progressive nitrogen limitation as the treatment exposure continues, ratios of leaves (Paterson et al. 1999; Lutze & Gifford 2000; van Heerwaarden et al. 2005; mineralization rates, indicate that the fauna response plays a significant role in the observed Previous studies have investigated interactions between elevated CO 2 and e.g. N Increased rhizodeposition is a common plant response to elevated CO 2 (Pendall et al. this, in combination with the P limitation for Deschampsia, would be beneficial to the slower 2004; Allard et al. 2006), and strong effects on below-ground C cycling have been observed decrease in N turnover in drought-exposed plots. Furthermore, while the warming effects supply (Lutze & Gifford 2000; Lutze et al. 2000; van Heerwaarden et al. 2005; Reich et al.
	407
	431 481 505
		20 21 22 24

392

12.6 g N m -2 y -1 under different plant species in wet and dry Dutch heathlands, where 393 atmospheric deposition is 2-3 times higher than in Denmark. Inter-annual variability in N 394 leaching was significant, while treatment differences after two years of treatments were not 395 significant. The observed difference in leaching between

2006 and 2007

shows that the 396 ecosystem N loss by leaching is highly dependent on the amount and timing of precipitation.

397

In a future climate with more precipitation distributed in fewer, but more severe rain events, N 398 leaching is likely to increase in this ecosystem. The inter-annual variability in leaching may 399 also indicate that the ecosystem is close to N saturation as also suggested from an adjacent 400 short-term experiment where N and phosphorus (P) addition resulted in only a modest 401 increase in fine root biomass of Deschampsia, and no effects on Calluna roots (Nielsen et al. 402 2009). Furthermore, Deschampsia responded more when both N and P were added indicating 403 that this species is currently more limited by P than N availability. If nitrogen availability 406 growing Calluna. However, other climate factors, i.e. water availability and temperature 415 on bacteria are all needed to perform the upscaling and very few references exist in the 416 literature on such values. This said, the value for fauna-related N mineralization in the 417 ambient treatment based on this calculation seems trustworthy based on the gross N 418 mineralization from the pool dilution assay of 6.7 g N m -2 y -1 . The assumption that most 419 bacterial mineralization is facilitated through predation and mineralization by the soil fauna 420 implies that N mineralization by bacteria is responsible for approximately 75% of the total 421 activity, the rest being due to fungi. 428 Finzi et al. 2006; Chen et al. 2007; Hovenden et al. 2008). At our experimental site, we 429 observed reduced photosynthetic capacity (Albert et al. 2010) and leaf nitrogen under 430 elevated CO 2 for the dominant species, Calluna and Deschampsia, as well as increased leaf 438 As hypothesized, however, N concentration in Deschampsia litter after two years of 439 treatments was also reduced by elevated CO 2 , possibly showing that the effect is beginning to 440 cascade from leaf level to other ecosystem N pools, although the effect disappears in the full 441 treatment combination. Together with the observed increased concentration of tannins 442 (Schmidt et al. 2007) in plant tissues, we anticipate that the changes in C/N ratios will affect 443 litter decomposition rates as the experimental treatments continue.

444

In contrast to our expectations, the above-and below-ground plant N pools were not 445 increased by elevated CO 2 . However, total root biomass N of Calluna increased unexpectedly 446 in response to drought. It is inherently difficult to estimate total roots from small soil samples 447 due to the large spatial heterogeneity in root distribution and, at the same time, collection of 448 larger samples is problematic due to the destruction of the experimental plots. We are 454 in response to elevated CO 2 in the current experiment, e.g. increased soil respiration rates 455

(Selsted et al. 2010

) and increased production of dissolved organic carbon

(Andresen et al. 464

denitrification from lab incubations of soils, as well as increased in situ N 2 O emissions. Soil 465 and litter incubations in situ also showed that warming promoted N cycling

(Andresen et al. 466 2010b)

. Previous field studies with artificial warming also showed no warming effect on N 2 O 467 emission rates

(Peterjohn et al. 1994; Mchale et al. 1998)

. However, we interpret our 468 observation of five variables that all responded in the same direction to warming as a strong 469 indicator that the warming effect on N turnover was real.

470

The below-ground N turnover was reduced by drought as observed by reduced gross 471 N mineralization and fauna N mineralization. Reduced N mineralization in response to 472 drought has previously been observed

(Emmett et al. 2004; Schmidt et al. 2004

; Sowerby et 473 al. 2008) but the negative effects of drought on N mineralization are often attributed directly 474 to reduced microbial activity. The importance of soil fauna to the N mineralization shown in 479 were generally reduced in combination with drought and elevated CO 2 , the negative effect of 480 drought on fauna N biomass and N mineralization was not reduced by interactions.

ecosystem is more robust when more than one factor is changed. For instance, the C/N ratio 495 of mosses increased in both single treatments of drought and elevated CO 2 but was unaffected 496 when treatments were combined. Similarly, the increased gross mineralization rate observed 497 in the single warming treatment was reduced especially in combination with elevated CO 2 .

498 Furthermore, two of the three additive effects observed (Deschampsia litter N concentration 503 2006), warming

(Peltola et al. 2002; Tingey et al. 2003; Hovenden et al. 2008; Dijkstra et al. 504

2010), biodiversity

(Reich et al. 2001; Niklaus et al. 2001

) and water relations (Morgan et al.

Table 1 .

 1 Table 1 for statistical significance of observed changes. Squares Means (DLSM) were categorized as antagonistic, i.e. combination leads to reduction 814 of effects, or as synergistic, i.e. combination leads to amplification of single effects or 815 significant effect is only observed in combination. Number of observed simple additive 816 effects is also shown, i.e. cases where two significant individual effects were observed 817 without significant interaction. Note that the sum of antagonistic and synergistic effects are 15 818 because one three-way interaction (potential nitrification -see Table 1) was caused by both Pools and fluxes of N (g N m

	809

(Festuca pratensis) and Barley (Hordeum vulgare).Biology and Fertility of Soils, 3,[19][20][21][22][23][24][25]

Acknowledgements

Consequently, the effect prevailed in the full combination of treatments, indicating that N 482 mineralization will be reduced in the full future climate scenario. Therefore, despite the fact 483 that this ecosystem currently show no clear sign of N limitation of plant growth, we cannot 484 rule out the possibility that progressive nitrogen limitation will play a role in the longer term,


Roos J Leguit
email: rleguit2@umcutrecht.nl

Jan G Van Den Tweel

THE PATHOLOGY OF BONE MARROW FAILURE

Keywords: bone marrow, histopathology, myelodysplastic syndromes, inherited bone marrow failure syndromes, trephine biopsy bone marrow, histopathology, myelodysplastic syndromes, inherited bone marrow failure syndromes, trephine biopsy

whether they are published or not. The documents may come

Introduction

An important indication for bone marrow investigation is the presence of bone marrow failure, which manifests itself as (pan)cytopenia. The causes of cytopenia are varied and differ considerable between childhood and adulthood as inherited bone marrow failure syndromes are important causes of bone marrow failure in the paediatric age group but play only a minor role in adulthood. The inherited bone marrow failure syndromes are frequently, but not always associated with physical abnormalities and sometimes the haematological abnormalities precede the onset of other abnormalities. Furthermore, some of these inherited syndromes may not be recognized until adulthood, [START_REF] Balaban | Diamond-Blackfan syndrome in adult patients[END_REF][START_REF] Lipton | Improving clinical care and elucidating the pathophysiology of Diamond Blackfan anemia: an update from the Diamond Blackfan Anemia Registry[END_REF] and most of them are associated with an increased risk of developing myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML). Some syndromes, especially Fanconi anaemia and the Shwachman-Diamond syndrome, also have a tendency to progress to aplastic anaemia. The bone marrow in these inherited bone marrow failure syndromes is often hypocellular and may show some dysplastic changes making distinction from refractory cytopenia, a low-grade form of MDS, difficult or impossible at times.

In adults, common forms of anaemia are iron deficiency anaemia, megaloblastic anaemia, anaemia of chronic disease and haemolytic anaemia. Thrombocytopenia develops due to excessive use, excessive peripheral destruction or due to impaired production of platelets in the bone marrow. Neutropenia in adults is mainly autoimmune, drug related or associated with infections. In most cases of adult single cell cytopenia, a bone marrow trephine biopsy is not necessary for the diagnosis. However, in case of cytopenia of unknown origin or in case of pancytopenia, a bone marrow biopsy is often performed, amongst others to exclude or confirm the diagnosis of MDS. Since dysplasia of one or more cell lines can be seen not only in MDS but also in a wide variety of other situations as illustrated in this review, correlation with clinical parameters, the bone marrow aspirate, the peripheral blood smear and cytogenetics is essential in the assessment of bone marrow disorders. This review gives a comprehensive overview of the histopathology of bone marrow failure in children and adults. The causes of bone marrow failure are classified according to the main presenting haematological abnormality (i.e. anaemia, neutropenia, thrombocytopenia or pancytopenia). HIV can cause significant dyshaematopoiesis simulating MDS and will be discussed under the heading of pancytopenia. Cytopenias associated with inherited will not be discussed in this review, neither will infections like kala azar, enteric fever, malaria, disseminated tuberculosis and leishmania, which are common causes of pancytopenia in non-Western countries. [START_REF] Gupta | A study of clinico-haematological profiles of pancytopenia in children[END_REF][START_REF] Memon | Etiological spectrum of pancytopenia based on bone marrow examination in children[END_REF] Anaemia Anaemia is a frequently occurring isolated disorder. In the majority of cases a bone marrow biopsy is redundant, since it does not add to the diagnosis, e.g. in patients with proven iron or Vitamin B12 deficiency. Nevertheless, there are situations in which a biopsy is performed in order to make a diagnosis or to exclude other pathologies.

Pure Red Cell Aplasia

Pure red cell aplasia (or erythroblastopenia) is a rare disease characterized by a moderate to severe normocytic anaemia, a profound reticulocytopenia and absence of erythroblasts in the bone marrow. It can be divided into three groups: a congential form called Diamond-Blackfan anaemia (DBA), an acquired acute self-limiting form predominantly seen in childhood called transient erythroblastopenia of childhood (TEC), and an acquired chronic form that occurs more frequently in adults. The inherited form, DBA, is associated with physical abnormalities like craniofacial anomalies, radial ray abnormalities, renal defects and cardiac defects in almost 50% of patients. [START_REF] Lipton | Improving clinical care and elucidating the pathophysiology of Diamond Blackfan anemia: an update from the Diamond Blackfan Anemia Registry[END_REF] This disorder typically presents in the first year of life, although it may be first recognized in adulthood. [START_REF] Balaban | Diamond-Blackfan syndrome in adult patients[END_REF][START_REF] Lipton | Improving clinical care and elucidating the pathophysiology of Diamond Blackfan anemia: an update from the Diamond Blackfan Anemia Registry[END_REF] In addition to anaemia, the development of neutropenia and/or thrombocytopenia has been described during the course of the disease. [START_REF] Giri | Clinical and laboratory evidence for a trilineage haematopoietic defect in patients with refractory Diamond-Blackfan anaemia[END_REF] The genes mutated in DBA that have been identified so far, encode ribosomal proteins. [START_REF] Ellis | Diamond Blackfan anemia: a disorder of red blood cell development[END_REF][START_REF] Flygare | Diamond-Blackfan anemia: erythropoiesis lost in translation[END_REF] Less than 2 percent of DBA patients will develop haematopoietic or non-haematopoietic malignancies or MDS. [START_REF] Lipton | Improving clinical care and elucidating the pathophysiology of Diamond Blackfan anemia: an update from the Diamond Blackfan Anemia Registry[END_REF] Transient erythroblastopenia of childhood (TEC) occurs predominantly in young children with a mean age of 23 months. [START_REF] Gerrits | Transient erythroblastopenia of childhood. A review of 22 cases[END_REF] Neutropenia has been described in up to 65% of cases. [START_REF] Cherrick | Transient erythroblastopenia of childhood. Prospective study of fifty patients[END_REF] The aetiology is still unknown, but infections by human herpes virus 6 [START_REF] Penchansky | Transient erythroblastopenia of childhood associated with human herpesvirus type 6, variant B[END_REF] and parvovirus B19 [START_REF] Prassouli | Classic transient erythroblastopenia of childhood with human parvovirus B19 genome detection in the blood and bone marrow[END_REF][START_REF] Geetha | Pure red cell aplasia caused by Parvovirus B19 infection in solid organ transplant recipients: a case report and review of literature[END_REF] have been demonstrated in some cases although these viruses could not be found in other studies. [START_REF] Skeppner | Transient erythroblastopenia of childhood: prospective study of 10 patients with special reference to viral infections[END_REF] Genetic factors may also play a role, as several cases of TEC occurring in siblings F o r P e e r R e v i e w 5 and twins have been described. [START_REF] Shaw | Transient erythroblastopenia of childhood in siblings: case report and review of the literature[END_REF][START_REF] Skeppner | Transient red cell aplasia in siblings: a common environmental or a common hereditary factor?[END_REF] TEC is always self-limiting and spontaneous recovery usually occurs within 1 to 2 months.

The acquired chronic form of pure red cell aplasia can be primary or secondary to various underlying factors like infections, haematologic malignancies, solid tumours, collagen vascular diseases or drugs. [START_REF] Djaldetti | Pure red cell aplasia--a rare disease with multiple causes[END_REF] Large granular lymphocyte leukaemia is the most common underlying haematologic malignancy. [START_REF] Sawada | Long-term outcome of patients with acquired primary idiopathic pure red cell aplasia receiving cyclosporine A. A nationwide cohort study in Japan for the PRCA Collaborative Study Group[END_REF] In up to 22% of patients with pure red cell aplasia, an associated thymoma is found.

The bone marrow in pure red cell aplasia is either normocellular or hypocellular and typically shows selective decrease or absence of red cell precursors beyond the proerythroblast stage (Figure 1). However, during the recovery phase of the disease, increased erythropoetic activity can be observed. [START_REF] Dickerman | Transient erythroblastopenia of childhood presenting with reticulocytosis and erythroid hyperplasia in the bone marrow[END_REF] The maturation of the granulocytic and megakaryocytic series is normal and iron stores are often increased. [START_REF] Balaban | Diamond-Blackfan syndrome in adult patients[END_REF][START_REF] Burkhardt | Bone biopsy in haematological disorders[END_REF] There is frequently an increase in haematogones (benign lymphoid precursors), which might lead to an erroneous diagnosis of acute lymphoblastic leukaemia. [START_REF] Gerrits | Transient erythroblastopenia of childhood. A review of 22 cases[END_REF][START_REF] Foot | Transient erythroblastopenia of childhood with CD10, TdT, and cytoplasmic mu lymphocyte positivity in bone marrow[END_REF] In parvovirus infections, enlarged proerythroblasts with intranuclear inclusions may be seen and the dyserythropoiesis might be so marked as to simulate congenital dyserythropoietic anaemia. [START_REF] Carpenter | Acute parvovirus B19 infection mimicking congenital dyserythropoietic anemia[END_REF] Immunohistochemical stainings can be used to identify the parvovirus antigens.

Sideroblastic anaemia

Sideroblastic anaemia includes a heterogeneous group of rare conditions, characterized by the presence of ring sideroblasts in the bone marrow. Abnormal haem synthesis causes mitochondrial iron overload in erythroblasts. Several inherited forms have been described

showing different modes of inheritance, a broad clinical spectrum and an initial clinical presentation ranging from foetal life to late childhood. The most frequent form is X-linked sideroblastic anaemia caused by mutations of delta-aminolevulinic acid synthase 2 (ALAS2), an enzyme involved in haem synthesis in erythroid precursors. Acquired forms of sideroblastic anaemia can be primary, as in refractory anaemia with ring sideroblasts (RARS), secondary due to the toxic effects of alcohol, lead and drugs, or due to copper deficiency. [START_REF] Sheftel | Mitochondrial iron metabolism and sideroblastic anemia[END_REF][START_REF] Gregg | Copper deficiency masquerading as myelodysplastic syndrome[END_REF][START_REF] Halfdanarson | Hematological manifestations of copper deficiency: a retrospective review[END_REF] Once considered a morphological hallmark of sideroblastic anaemia, ring sideroblasts can also be seen in other conditions. The diagnosis of sideroblastic anaemia is usually made on the peripheral blood smear and the bone marrow aspirate, where dysmorphic erythrocytes, ring sideroblasts and erythroid hyperplasia are seen. Bone marrow trephine biopsies show some degree of erythroid hyperplasia with increased iron stores. Ring sideroblasts can be easily seen in plasticembedded material and smears. Decalcified paraffin-embedded biopsies might sometimes give false negative results. Bone marrow trephine biopsies are most useful in distinguishing sideroblastic anaemia from MDS, the latter showing also dysplastic changes in the granulocytic and megakaryocytic lineages.

Congenital dyserythropoietic anaemia

Congenital dyserythropoietic anaemia (CDA) comprises a group of rare inherited disorders of erythropoiesis usually characterized by a mild to moderate anaemia, ineffective erythropoiesis and frequently striking dyserythropoietic changes in the red cell precursors. The group of CDA was originally classified in three types (I,II, III), but variants have been reported and classified as type IV-VII. [START_REF] Wickramasinghe | Advances in the understanding of the congenital dyserythropoietic anaemias[END_REF] In CDA II, the most frequent form, there is typically a positive acidified serum test (Ham test), thereby mimicking paroxysmal nocturnal haemoglobinuria.

The mode of inheritance can be autosomal recessive or autosomal dominant. CDA I in particular is associated with dysmorphic features which mainly affect the distal extremities. [START_REF] Wickramasinghe | Dyserythropoiesis and congenital dyserythropoietic anaemias[END_REF] Thrombocytopenia has been described in patients with CDA II. [START_REF] Iolascon | Genetic heterogeneity of congenital dyserythropoietic anemia type II[END_REF] The characteristic dyserythropoietic features of CDA can be best observed in the bone marrow aspirate, a bone marrow trephine biopsy usually does not add to the diagnosis. The bone marrow trephine will confirm the often marked dyserythropoiesis and erythroid hyperplasia. [START_REF] Wickramasinghe | Advances in the understanding of the congenital dyserythropoietic anaemias[END_REF][START_REF] Holla | Congenital dyserythropoietic anemia type I[END_REF] Megaloblastic changes are seen in most types, [START_REF] Wickramasinghe | Advances in the understanding of the congenital dyserythropoietic anaemias[END_REF][START_REF] Holla | Congenital dyserythropoietic anemia type I[END_REF] and iron stores are often increased. [START_REF] Holla | Congenital dyserythropoietic anemia type I[END_REF] CDA I is characterized by megaloblastic changes with internuclear chromatin bridges between erythroblasts. CDA II typically has large portions (10-40%) of binucleated and multinucleated erythroblasts, shows increased karyorrhexis, and may contain pseudo-Gaucher cells. [START_REF] Van | Gaucher-like cells and congenital dyserythropoietic anaemia, type II (HEMPAS)[END_REF] The most distinctive feature of CDA III is the high proportion of giant multinucleated erythroblasts with up to 12 nuclei. [START_REF] Wickramasinghe | Advances in the understanding of the congenital dyserythropoietic anaemias[END_REF] Although typical for CDA III, these giant multinucleated erythroblasts can also be seen in MDS and erythroleukaemia. Non-erythroid haematopoietic cell lineages are usually normal in CDA, but in patients with thrombocytopenia a severe reduction of megakaryocytes has been described. [START_REF] Iolascon | Genetic heterogeneity of congenital dyserythropoietic anemia type II[END_REF] CDA must be differentiated from other inherited and acquired disorders showing dyserythropoiesis, such as thalassaemia, sideroblastic anaemia, vitamin B12 or folate deficiency, iron deficiency, excess infections like malaria and kala azar, or liver disease. [START_REF] Wickramasinghe | Dyserythropoiesis and congenital dyserythropoietic anaemias[END_REF] As some cases of CDA may present rather late in life they can be misdiagnosed as MDS. The latter, however, will show dysplastic changes in other cell lineages as well.

Haemolytic anaemia

Haemolytic anaemia is caused by increased destruction of red cells and can be inherited or acquired. Inherited haemolytic anaemia is either due to red cell membrane defects like in hereditary spherocytosis, due to haemoglobin abnormalities as in thalassaemia and sickle cell anaemia, or due to metabolic disorders of the red cell, like in G6PD deficiency. In thalassaemia, there is additionally ineffective erythropoiesis.

Acquired haemolytic anaemia can be immune-related or non-immune. Haemolytic disease of the newborn (erythroblastosis foetalis) is the result of the immune destruction of foetal red cells by alloantibodies transmitted through the placenta from maternal blood. Evans syndrome is a childhood disorder caused by autoantibodies against red blood cells and platelets, resulting in haemolytic anaemia and thrombocytopenia. In paroxysmal nocturnal haemoglobinuria (PNH), haemolytic anaemia develops due to an acquired stem cell abnormality resulting in alterations in the cell membrane composition and is discussed separately below.

Regardless of the cause of haemolytic anaemia, the bone marrow shows hypercellularity with substantial erythroid hyperplasia, sometimes with a left shift and variable dyserythropoiesis.

Iron stores are increased, sometimes extensively. In addition, autoimmune haemolytic anaemia often shows small lymphoid infiltrates. In thalassaemia major, the erythroid hyperplasia is extensive with complete replacement of the fat cells and marked dyserythropoiesis, sometimes with megaloblastic changes. [START_REF] Block | Histopathologic studies in beta-thalassemia minor and intermedia[END_REF] Macrophages are often increased in number and can have the form of pseudo-Gaucher cells [START_REF] Sharma | Pseudo-Gaucher cells in Hb E disease and thalassemia intermedia[END_REF][START_REF] Zaino | Gaucher's cells in thalassemia[END_REF] or sea-blue histiocytes. A variable degree of granulocytic and megakaryocytic hyperplasia has been described in thalassaemia, and increased karyorrhexis is indicative of the ineffective haematopoiesis. [START_REF] Block | Histopathologic studies in beta-thalassemia minor and intermedia[END_REF] The dyserythropoiesis can be so marked that it can mimic CDA.

In sickle cell disease, sickle cells may be seen within bone marrow sinusoids during episodes of sickling. However, sickle cells can also be found in patients with the sickle cell trait.

During a sickle cell crisis, infarction of bone and bone marrow may be present, while fibrotic

Paroxysmal nocturnal haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH) is an uncommon acquired haematological disorder characterized by intravascular haemolysis, nocturnal haemoglobinuria, thrombotic events, serious infections, and bone marrow failure. It results from a non-malignant clonal expansion of one or more haematopoietic stem cells that have acquired mutations in an Xlinked gene, PIGA, causing affected cells and their progeny to be deficient in the complement regulator glycosylphosphatidylinositol anchored proteins (GPI-AP). This deficiency causes increased susceptibility of red cells to complement, resulting in intravascular haemolysis. [START_REF] Miyata | The cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis[END_REF] In addition to anaemia, neutropenia and thrombocytopenia are common features. [START_REF] Socie | Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors[END_REF] The mean age of presentation is 33 years with a broad range (6-82 years). [START_REF] Socie | Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors[END_REF] There is a close relationship between PNH and aplastic anaemia (AA). Thirty percent of PNH patients have a previous history of AA. [START_REF] Socie | Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors[END_REF] On the other hand, PNH clones (often repeatedly recurring) are observed in a high percentage of patients with AA. [START_REF] Wanachiwanawin | A cohort study of the nature of paroxysmal nocturnal hemoglobinuria clones and PIG-A mutations in patients with aplastic anemia[END_REF] Classic PNH requires features of intravascular haemolysis. Patients with AA and small to moderate PNH clones but without signs of haemolysis are often referred to as having an AA/PNH overlap syndrome.

Bone marrow cellularity in classical PNH ranges from normocellular to hypercellular with marked erythroid hyperplasia. Hypocellular bone marrow with relative red-cell hyperplasia is seen in cases of AA/PNH overlap syndrome. Some degree of dyserythropoiesis may be present and distinction from low grade MDS, especially in children, can often not be made on morphological grounds alone.

Iron deficiency anaemia

Iron deficiency anaemia develops due to shortages of iron stores and typically is microcytic and hypochromic. Thrombocytosis and, in severe iron deficiency, thrombocytopenia has been reported. [START_REF] Perlman | Thrombocytopenia in children with severe iron deficiency[END_REF][START_REF] Kuku | Platelet counts in adults with iron deficiency anemia[END_REF] Although this disease is usually acquired and can be attributed to chronic blood loss or inadequate dietary intake, an inherited autosomal recessive disorder has recently been described, causing an iron-refractory iron deficiency anaemia (IRIDA). [START_REF] Finberg | Mutations in TMPRSS6 cause ironrefractory iron deficiency anemia (IRIDA)[END_REF][START_REF] Guillem | Two nonsense mutations in the TMPRSS6 gene in a patient with microcytic anemia and iron deficiency[END_REF][START_REF] Melis | A mutation in the TMPRSS6 gene, encoding a transmembrane serine protease that suppresses hepcidin production, in familial iron deficiency anemia refractory to oral iron[END_REF] IRIDA is unresponsive to oral iron therapy and is partially responsive to parenteral iron therapy. It is caused by mutations in the TMPRSS6 gene which encodes a transmembrane serine protease produced in the liver that controls the expression of hepcidin, a hormone regulating systemic iron. [START_REF] Finberg | Mutations in TMPRSS6 cause ironrefractory iron deficiency anemia (IRIDA)[END_REF][START_REF] Guillem | Two nonsense mutations in the TMPRSS6 gene in a patient with microcytic anemia and iron deficiency[END_REF][START_REF] Melis | A mutation in the TMPRSS6 gene, encoding a transmembrane serine protease that suppresses hepcidin production, in familial iron deficiency anemia refractory to oral iron[END_REF] The bone marrow in iron deficiency anaemia usually shows a mild to moderate erythroid hyperplasia with a predominance of intermediate normoblasts. However, when the anaemia becomes more chronic, erythropoiesis can be decreased. Dyserythropoiesis, correlated with the severity of iron deficiency, can be seen as nuclear irregularities, increased karyorrhexis and erythroblast multinuclearity. [START_REF] Hill | Iron deficiency and dyserythropoiesis[END_REF] In case of additional thrombocytopenia, megakaryocytes are reported to be decreased, adequate or increased. [START_REF] Perlman | Thrombocytopenia in children with severe iron deficiency[END_REF] The bone marrow reticulum cells are characteristically depleted of iron. In case of IRIDA, however, the bone marrow may show detectable iron after intravenous iron therapy, while serum iron concentrations remain low. [START_REF] Guillem | Two nonsense mutations in the TMPRSS6 gene in a patient with microcytic anemia and iron deficiency[END_REF] Sometimes, decalcification needed for paraffin-embedded trephine biopsies may leach out some or all of the iron, so that a positive iron staining can rule out iron deficiency anaemia, but a diagnosis of iron deficiency cannot always be made on decalcified specimens.

Anaemia of chronic disease / anaemia of chronic inflammation

Anaemia of chronic disease (ACD) is a common cause of anaemia in adults and is primarily found in patients with diseases causing immune activation like chronic infections, autoimmune disorders, malignancies, chronic organ rejection and chronic kidney diseases. [START_REF] Weiss | Anemia of chronic disease[END_REF] In autoimmune diseases, ACD is the main mechanism of anaemia, although this can also be caused by other mechanisms such as iron deficiency or drug effects. [START_REF] Voulgarelis | Anaemia in systemic lupus erythematosus: aetiological profile and the role of erythropoietin[END_REF] In addition, autoimmune disorders can be associated with pure red cell aplasia, [START_REF] Mavragani | Pure red cell aplasia in a Sjogren's syndrome/lupus erythematosus overlap patient[END_REF][START_REF] Hara | Combined pure red cell aplasia and autoimmune hemolytic anemia in systemic lupus erythematosus with anti-erythropoietin autoantibodies[END_REF] aplastic anaemia, [START_REF] Das | Remission of aplastic anemia induced by treatment for Graves disease in a pediatric patient[END_REF] haemophagocytic syndrome [START_REF] Fukaya | Clinical features of haemophagocytic syndrome in patients with systemic autoimmune diseases: analysis of 30 cases[END_REF] and autoimmune myelofibrosis. [START_REF] Bass | Pathology of autoimmune myelofibrosis. A report of three cases and a review of the literature[END_REF] Although anaemia in chronic kidney diseases can be the result of inflammatory changes and ACD, it could also merely be due to a poor production of erythropoietin.

In ACD, T-cells and monocytes are activated resulting in the production of cytokines and eventually in disturbances in iron homeostasis, impaired proliferation of erythroid progenitor cells and an inadequate erythropoietin response. [START_REF] Weiss | Anemia of chronic disease[END_REF] Hepcidin, the liver hormone regulating iron metabolism, plays an important role in ACD. [START_REF] Weiss | Anemia of chronic disease[END_REF][START_REF] Munoz | An update on iron physiology[END_REF][START_REF] Nemeth | Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein[END_REF][START_REF] Nemeth | IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin[END_REF] The anaemia is typically normocytic and normochromic.

The bone marrow in anaemia of chronic disease can be hypocellular, normocellular or hypercellular. [START_REF] Voulgarelis | Bone marrow histological findings in systemic lupus erythematosus with hematologic abnormalities: a clinicopathological study[END_REF][START_REF] Yetgin | Myelodysplastic features in juvenile rheumatoid arthritis[END_REF] Most often there is erythroid hypoplasia, though the erythropoiesis can be normal or even increased. [START_REF] Voulgarelis | Bone marrow histological findings in systemic lupus erythematosus with hematologic abnormalities: a clinicopathological study[END_REF][START_REF] Yetgin | Myelodysplastic features in juvenile rheumatoid arthritis[END_REF][START_REF] Kirel | Anaemia in juvenile chronic arthritis[END_REF] The myeloid and megakaryocytic series are frequently increased, but can be normal or decreased. [START_REF] Voulgarelis | Bone marrow histological findings in systemic lupus erythematosus with hematologic abnormalities: a clinicopathological study[END_REF][START_REF] Yetgin | Myelodysplastic features in juvenile rheumatoid arthritis[END_REF] Dysplastic changes in all cell lineages are present in the vast majority of cases. [START_REF] Voulgarelis | Bone marrow histological findings in systemic lupus erythematosus with hematologic abnormalities: a clinicopathological study[END_REF][START_REF] Yetgin | Myelodysplastic features in juvenile rheumatoid arthritis[END_REF][START_REF] Kirel | Anaemia in juvenile chronic arthritis[END_REF][START_REF] Oka | Reversible bone marrow dysplasia in patients with systemic lupus erythematosus[END_REF] Although some authors claim that there is no correlation between myelodysplastic changes in the bone marrow and disease activity, [START_REF] Yetgin | Myelodysplastic features in juvenile rheumatoid arthritis[END_REF] others do and even describe disappearance of the dysplasia with remission of the autoimmune disorder. [START_REF] Oka | Reversible bone marrow dysplasia in patients with systemic lupus erythematosus[END_REF] Iron stores are normal or increased unless a coexisting iron deficiency anaemia is present. Other bone marrow findings include architectural disturbances with paratrabecular localization of erythroid and megakaryocytic precursors, increase in reticulin fibres and occasionally collagen fibrosis with osteosclerosis, necrosis, dilatation of sinuses and the presence of lymphoid aggregates. [START_REF] Voulgarelis | Bone marrow histological findings in systemic lupus erythematosus with hematologic abnormalities: a clinicopathological study[END_REF] Furthermore, an increase in polytypic plasma cells of up to 33% has been described in autoimmune disorders. [START_REF] Kirel | Anaemia in juvenile chronic arthritis[END_REF][START_REF] Lee | A case of reactive plasmacytosis mimicking multiple myeloma in a patient with primary Sjogren's syndrome[END_REF][START_REF] Tanvetyanon | Severe anemia and marrow plasmacytosis as presentation of Sjogren's syndrome[END_REF]

Megaloblastic anaemia

Megaloblastic anaemia comprises a heterogeneous group of disorders characterized by macrocytaemia in the blood smear occurring due to impaired DNA synthesis. The mildest form shows only macrocytosis without anaemia. Severe cases may also show leucopenia and/or thrombocytopenia, since DNA synthesis is affected in all haematopoietic cell lineages.

The most common cause of megaloblastic anaemia is vitamin B12 or folate deficiency.

Vitamin B12-and folate-independent causes of megaloblastic anaemia are inherited or acquired abnormalities of nucleic acid synthesis (mostly due to drugs), complex mechanisms or unknown mechanisms as in MDS, AML and CDA. [START_REF] Wickramasinghe | Diagnosis of megaloblastic anaemias[END_REF] Pernicious anaemia is a specific form of megaloblastic anaemia due to vitamin B12 deficiency in the setting of autoimmune atrophic gastritis.

In megaloblastic anaemia due to vitamin B12 or folate deficiency, the bone marrow is usually markedly hypercellular with striking erythroid hyperplasia and numerous megaloblasts, although the M/E ratio may be increased. The picture can sometimes simulate acute myeloid leukaemia. In non-anaemic cases, erythropoiesis may be normoblastic or only mildly megaloblastic. [START_REF] Wickramasinghe | Diagnosis of megaloblastic anaemias[END_REF] Granulocytic precursors can also be increased and often show nuclear/cytoplasmic asynchrony in the form of giant metamyelocytes and giant bands. There can be a reduction of megakaryocytes, which may be abnormally large and hyperlobulated even with bizarre multilobated nuclei. Iron stores may vary between increased and decreased.

After start of therapy, conversion to normoblastic haematopoiesis begins within 12 hours and is usually complete within 2-3 days.

Neutropenia

Severe neutropenia is defined as an absolute blood neutrophil count < 500/µL. The acute form is most often caused by drugs or acute viral infections, [START_REF] Boxer | Neutropenia: causes and consequences[END_REF] usually lasts only a few days or weeks and is generally not biopsied. The chronic form can be due to a variety of underlying disorders that may be either congenital or acquired. [START_REF] Dale | The Severe Chronic Neutropenia International Registry: 10-Year Follow-up Report[END_REF]

Shwachman-Diamond syndrome

Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive inherited disorder characterized by insufficiency of the exocrine pancreas (replacement by fat), metaphyseal chondrodysplasia, growth retardation and other physical abnormalities. Neutropenia, chronic or intermittent, is the most important haematological abnormality and is observed in 88-100% of patients, but anaemia and/or thrombocytopenia also frequently occur. [START_REF] Aggett | Shwachman's syndrome. A review of 21 cases[END_REF][START_REF] Dror | Clonal evolution in marrows of patients with Shwachman-Diamond syndrome: a prospective 5-year follow-up study[END_REF][START_REF] Ginzberg | Shwachman syndrome: phenotypic manifestations of sibling sets and isolated cases in a large patient cohort are similar[END_REF][START_REF] Mack | Shwachman syndrome: exocrine pancreatic dysfunction and variable phenotypic expression[END_REF][START_REF] Smith | Haematological abnormalities in Shwachman-Diamond syndrome[END_REF] Recurrent infections, sometimes fatal, are common. Approximately 90% of patients meeting the clinical criteria of SDS have mutations in the SBDS gene on chromosome 7. [START_REF] Boocock | Mutations in SBDS are associated with Shwachman-Diamond syndrome[END_REF] The disease has a propensity to develop into aplastic anaemia, MDS or AML, although clonal chromosome anomalies may persist for a long time without evolving to MDS or AML, and may even regress. [START_REF] Dror | Clonal evolution in marrows of patients with Shwachman-Diamond syndrome: a prospective 5-year follow-up study[END_REF][START_REF] Maserati | The route to development of myelodysplastic syndrome/acute myeloid leukaemia in Shwachman-Diamond syndrome: the role of ageing, karyotype instability, and acquired chromosome anomalies[END_REF] Although the marrow dysplasia and cytogenetic abnormalities are nearly equally distributed among the sexes, progression to AML occurs predominantly in males. [START_REF] Ginzberg | Shwachman syndrome: phenotypic manifestations of sibling sets and isolated cases in a large patient cohort are similar[END_REF][START_REF] Maserati | The route to development of myelodysplastic syndrome/acute myeloid leukaemia in Shwachman-Diamond syndrome: the role of ageing, karyotype instability, and acquired chromosome anomalies[END_REF] The bone marrow cellularity in SDS is most often decreased, but may be normal or even increased. [START_REF] Ginzberg | Shwachman syndrome: phenotypic manifestations of sibling sets and isolated cases in a large patient cohort are similar[END_REF][START_REF] Smith | Haematological abnormalities in Shwachman-Diamond syndrome[END_REF] Maturation arrest or absence or myeloid precursors is seen in only a minority of patients (Figure 2). [START_REF] Aggett | Shwachman's syndrome. A review of 21 cases[END_REF][START_REF] Ginzberg | Shwachman syndrome: phenotypic manifestations of sibling sets and isolated cases in a large patient cohort are similar[END_REF][START_REF] Smith | Haematological abnormalities in Shwachman-Diamond syndrome[END_REF] Normal bone marrow aspirates have been described, even during episodes of neutropenia, but this may be due to patchy distribution of the hypoplasia. [START_REF] Aggett | Shwachman's syndrome. A review of 21 cases[END_REF][START_REF] Ginzberg | Shwachman syndrome: phenotypic manifestations of sibling sets and isolated cases in a large patient cohort are similar[END_REF] Mild dysplastic changes in granulopoiesis, erythropoiesis or megakaryopoiesis as assessed on aspirates, are common and can be transient. [START_REF] Dror | Clonal evolution in marrows of patients with Shwachman-Diamond syndrome: a prospective 5-year follow-up study[END_REF][START_REF] Ginzberg | Shwachman syndrome: phenotypic manifestations of sibling sets and isolated cases in a large patient cohort are similar[END_REF][START_REF] Smith | Haematological abnormalities in Shwachman-Diamond syndrome[END_REF] These dysplastic changes are considered to be part of the syndrome and not indicative of MDS. [START_REF] Dror | Clonal evolution in marrows of patients with Shwachman-Diamond syndrome: a prospective 5-year follow-up study[END_REF] When pancytopenia develops, marrow changes consistent with aplastic anaemia are seen. Furthermore, bone abnormalities consistent with low turnover osteoporosis frequently occur. There often is a marked reduction in trabecular bone volume, in the amount of osteoid (with normal mineralization) and in numbers of osteoblasts and osteoclasts. [START_REF] Toiviainen-Salo | Shwachman-Diamond syndrome is associated with low-turnover osteoporosis[END_REF] The osteoporosis is thought to be an integral feature of SDS that might be directly related to the bone marrow dysfunction and neutropenia.

Severe congenital neutropenia and Kostmann syndrome

Severe congenital neutropenia (SCN) is a form of chronic neutropenia. It was originally described in 1956 by Kostmann as an autosomal recessive inherited marrow failure syndrome involving only the neutrophilic lineage. [START_REF] Kostmann | Infantile genetic agranulocytosis; agranulocytosis infantilis hereditaria[END_REF] Subsequently it was found to consist of a heterogeneous group of disorders with either an autosomal dominant or autosomal recessive pattern of inheritance, or sometimes occurring sporadically. Several molecular defects have been identified in SCN. The majority of patients have an autosomal dominant trait mainly due to mutations in ELA2, the gene encoding neutrophil elastase. [START_REF] Horwitz | Neutrophil elastase in cyclic and severe congenital neutropenia[END_REF][START_REF] Dale | Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia[END_REF] The autosomal recessive form (Kostmann syndrome) accounts for about 30% of cases and is mostly due to mutations in the HAX1 gene. [START_REF] Klein | HAX1 deficiency causes autosomal recessive severe congenital neutropenia (Kostmann disease)[END_REF] In others, the underlying molecular cause is still unknown. SCN usually becomes symptomatic within the first 6 months of life, presenting with severe pyogenic infections. The total white cell count may be close to normal due to increased numbers of eosinophils and monocytes. [START_REF] Dale | Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia[END_REF][START_REF] Skokowa | Severe congenital neutropenia: inheritance and pathophysiology[END_REF] There can be a mild anaemia and thrombocytosis [START_REF] Skokowa | Severe congenital neutropenia: inheritance and pathophysiology[END_REF] and there is an increased risk of transformation to MDS, [START_REF] Hasle | A pediatric approach to the WHO classification of myelodysplastic and myeloproliferative diseases[END_REF] or AML. [START_REF] Rosenberg | The incidence of leukemia and mortality from sepsis in patients with severe congenital neutropenia receiving long-term G-CSF therapy[END_REF] Rosenberg et al studied 374 patients with SCN and found that the cumulative incidence for MDS/AML was 21% after 10 years of G-CSF therapy and 36% after 12 years. [START_REF] Rosenberg | The incidence of leukemia and mortality from sepsis in patients with severe congenital neutropenia receiving long-term G-CSF therapy[END_REF] In SCN, bone marrow cellularity is normal or slightly decreased, with a characteristic absence of fully developed granulocytes which occurs due to a maturation arrest at the promyelocyte or myelocyte stage (Figure 3). [START_REF] Dale | Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia[END_REF][START_REF] Skokowa | Severe congenital neutropenia: inheritance and pathophysiology[END_REF][START_REF] Boztug | A syndrome with congenital neutropenia and mutations in G6PC3[END_REF] During infections however, there can be a transient increase of mature neutrophils whereas maturation of other haematopoietic lineages is normal.

Marrow monocytosis and eosinophilia are common. [START_REF] Skokowa | Severe congenital neutropenia: inheritance and pathophysiology[END_REF] In cases where neutropenia results from infections, drugs, allo-or autoimmunity, metabolic disorders or immunodeficiency syndromes, the bone marrow shows complete maturation of the granulocytic series, or blockage only at the band stage.

Cyclic neutropenia

Cyclic neutropenia, also a form of chronic neutropenia, is characterized by regular periods in which the number of granulocytes fluctuates from normal to (severely) neutropenic, lasting for between 19 and more than 21 days. [START_REF] Haurie | Cyclical neutropenia and other periodic hematological disorders: a review of mechanisms and mathematical models[END_REF] In addition, there is an inverse oscillation of the platelets, often of the monocytes and eosinophils and occasionally of the reticulocytes, which all oscillate from normal to high levels. [START_REF] Haurie | Cyclical neutropenia and other periodic hematological disorders: a review of mechanisms and mathematical models[END_REF] Cyclic neutropenia occurs sporadically or as a result of autosomal dominant inheritance. As in severe congenital neutropenia, ELA2 mutations are 13 the most common cause for both sporadic and autosomal dominant cyclic neutropenia. [START_REF] Dale | Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia[END_REF][START_REF] Horwitz | Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis[END_REF] There is insufficient evidence for an increased risk of MDS and AML in cyclic neutropenia. 79- 81 In cyclic neutropenia, maturation arrest or absence of granulocyte precursors in the otherwise unremarkable bone marrow precedes peripheral blood neutropenia, and is followed by a return to normal maturation before the improvement of peripheral blood counts. [START_REF] Donadieu | Analysis of risk factors for myelodysplasias, leukemias and death from infection among patients with congenital neutropenia. Experience of the French Severe Chronic Neutropenia Study Group[END_REF][START_REF] Birgens | Reversible adult-onset cyclic haematopoiesis with a cycle length of 100 days[END_REF][START_REF] Quesenberry | Cyclic hematopoiesis: disorders of primitive hematopoietic stem cells[END_REF] Immune-related neutropenia Immune-related neutropenia can be divided in autoimmune, alloimmune and drug-related neutropenia. Autoimmune neutropenia can occur as an isolated phenomenon, or as one of the manifestations of an autoimmune disease such as systemic lupus erythematosus. Alloimmune neutropenia can occur after allogeneic stem cell transplantation. Although drug associated neutropenia can be due to antineutrophil antibodies, the main mechanism of most druginduced neutropenias is non-immune.

The bone marrow in autoimmune neutropenia usually shows normal or hyperplastic granulopoiesis with a reduced proportion of mature neutrophils, sometimes with a maturation arrest at the promyelocyte stage. [START_REF] Laporte | Auto-immune neutropenia associated with metformin in a patient with splenic marginal zone lymphoma[END_REF] Occasionally, lipidladen macrophages or sea-blue histiocytes are seen. [START_REF] Papadaki | Severe autoimmune neutropenia associated with bone marrow sea-blue histiocytosis[END_REF] In autoimmune neutropenia of infancy, phagocytosis of neutrophils by bone marrow macrophages has been described. [START_REF] Shimizu | Intramedullary neutrophil phagocytosis by histiocytes in autoimmune neutropenia of infancy[END_REF] In drug-induced autoimmune neutropenia, the marrow can be normo-or hypocellular with marked hypoplasia and left-shifting of the myeloid series with unremarkable erythropoiesis and megakaryopoiesis, sometimes in the presence of lymphohistiocytic aggregates, small noncaseating granulomas and lipogranulomas. [START_REF] Akhtari | Neutropenia in a patient treated with ipilimumab (anti-CTLA-4 antibody)[END_REF]

Non-immune neutropenia

Chronic neutropenia not related to an immune phenomenon can be idiopathic or associated with infections (notably typhoid fever), metabolic disorders, immunodeficiency and drugs. In the chronic idiopathic form, impaired granulopoiesis might be due to overproduction of cytokines by immune cells within the bone marrow microenvironment, that may exert an inhibitory effect on myelopoiesis by inducing Fas-mediated apoptosis in the granulocyte progenitors. [START_REF] Papadaki | Impaired granulocytopoiesis in patients with chronic idiopathic neutropenia is associated with increased apoptosis of bone marrow myeloid progenitor cells[END_REF] It typically has a low incidence of infection and usually has a benign outcome. The bone marrow features in chronic idiopathic neutropenia are well described by Papadaki et al. [START_REF] Papadaki | Morphologically defined myeloid cell compartments, lymphocyte subpopulations, and histological findings of bone marrow in patients with nonimmune chronic idiopathic neutropenia of adults[END_REF] The bone marrow trephine biopsy is usually normocellular, but can be hypocellular or hypercellular. In nearly half the cases, the granulocytic series shows left shifting, which is most profound in patients with severe neutropenia. Granulopoiesis is hyperplastic in a minority of cases. Erythropoiesis can be slightly hyperplastic with occasional megaloblastic features. Megakaryopoiesis is usually normal, but can sporadically be increased or contain micromegakaryocytes. An increased proportion of plasma cells is seen in half of cases and is inversely correlated to the number of circulating neutrophils. Non-paratrabecular lymphoid aggregates without germinal centres are frequently found and there can be an increase in the numbers of dispersed B-lymphocytes. Eosinophils and histiocytes as well as reticulin fibers can be slightly increased.

Thrombocytopenia

Inherited forms of thrombocytopenia are rare; two of them will be discussed. Acquired forms of thrombocytopenia can be seen in both children and adults, and often occur in combination with other cytopenias such as aplastic anaemia or MDS. Several acquired forms of isolated thrombocytopenia are discussed below.

Congenital amegakaryocytic thrombocytopenia

Congenital amegakaryocytic thrombocytopenia (CAMT) is characterized by severe thrombocytopenia due to a lack of megakaryocytes. It presents at birth with haemorrhages of the skin or gastrointestinal tract mucosa without the physical anomalies typical of other inherited bone marrow failure syndromes. In most patients with CAMT, mutations in the cmpl gene coding for the thrombopoietin (TPO) receptor are present. [START_REF] Ballmaier | c-mpl mutations are the cause of congenital amegakaryocytic thrombocytopenia[END_REF] A very high proportion of patients subsequently develop multilineage bone marrow failure/aplastic anaemia in the first or second decade of life, which also seems to be the result of the c-mpl mutations. [START_REF] Ballmaier | Thrombopoietin is essential for the maintenance of normal hematopoiesis in humans: development of aplastic anemia in patients with congenital amegakaryocytic thrombocytopenia[END_REF] At the time of diagnosis, the bone marrow trephine usually shows normal cellularity with absent or low numbers of megakaryocytes but unremarkable erythropoiesis and granulopoiesis (Figure 4). [START_REF] Ballmaier | Thrombopoietin is essential for the maintenance of normal hematopoiesis in humans: development of aplastic anemia in patients with congenital amegakaryocytic thrombocytopenia[END_REF] When present, the megakaryocytes are usually small and immature. [START_REF] Ballmaier | Advances in the understanding of congenital amegakaryocytic thrombocytopenia[END_REF] Although a few CAMT patients have been described with normal numbers of megakaryocytes in the initial bone marrow samples, serial bone marrow samples in these patients show progression to amegakaryocytosis. [START_REF] Rose | Congenital amegakaryocytic thrombocytopenia: the diagnostic importance of combining pathology with molecular genetics[END_REF] When aplastic anaemia develops, it is morphologically indistinguishable from aplastic anaemia due to other causes.

Thrombocytopenia with absent radii

Thrombocytopenia with absent radii (TAR) is a rare inherited disorder that usually has an autosomal recessive mode of inheritance. It is characterized by a usually severe thrombocytopenia at birth and bilaterally absent radii. In contrast to Fanconi anaemia, thumbs are present. Skeletal abnormalities of the lower extremities as well as cardiac, renal and facial anomalies can be present. About half of patients have cow's milk intolerance. Other characteristic findings are periodic leukaemoid reactions and eosinophilia. [START_REF] Hall | Thrombocytopenia with absent radius (TAR)[END_REF] Platelet numbers increase with age and can reach near-normal levels in adulthood. Anaemia may develop secondary to extensive bleeding. The genetic basis of this syndrome is still unclear. [START_REF] Bonsi | Thrombocytopenia with absent radii (TAR) syndrome: from hemopoietic progenitor to mesenchymal stromal cell disease?[END_REF] The bone marrow in TAR is normo-or hypercellular with absent or decreased megakaryocytes, which, if present, are small, hypolobulated and immature. [START_REF] Hedberg | Thrombocytopenia with absent radii. A review of 100 cases[END_REF] When platelet counts increase, the number of megakaryocytes also increases and more mature forms are seen. Erythropoiesis is normal or hyperplastic secondary to the bleeding episodes, but can occasionally be decreased. [START_REF] Hedberg | Thrombocytopenia with absent radii. A review of 100 cases[END_REF] Granulopoiesis is normal or increased and can be left shifted, especially during a leukaemoid reaction. Eosinophilia is common.

Immune-related thrombocytopenia

Immune thrombocytopenic purpura (ITP) is usually the result of autoantibodies causing increased removal of platelets by the spleen and liver, although ineffective platelet production also seems to play a role. [START_REF] Cines | Immune thrombocytopenic purpura[END_REF] ITP is classified as primary (idiopathic) versus secondary or as acute (usually in childhood) versus chronic (usually in adult women). [START_REF] Cines | Immune thrombocytopenic purpura[END_REF] Secondary ITP can be associated with other autoimmune diseases, lymphoproliferative disorders, viral infections and drugs.

Alloantibodies can cause thrombocytopenia after blood transfusion and neonatally when maternal antibodies are transmitted through the placenta.

In ITP, the bone marrow is normocellular with increased or normal numbers of megakaryocytes and unremarkable erythropoiesis and granulopoiesis. [START_REF] Jubelirer | The role of the bone marrow examination in the diagnosis of immune thrombocytopenic purpura: case series and literature review[END_REF][START_REF] Muhury | Megakaryocytic alterations in thrombocytopenia: a bone marrow aspiration study[END_REF] [START_REF] Jubelirer | The role of the bone marrow examination in the diagnosis of immune thrombocytopenic purpura: case series and literature review[END_REF] Immature megakaryocytes with hypolobated nuclei are typical for ITP, although dysplastic megakaryocytes, naked megakaryocyte nuclei and, to a lesser degree, micromegakaryoctyes are also frequently found in ITP bone marrow aspirates. [START_REF] Muhury | Megakaryocytic alterations in thrombocytopenia: a bone marrow aspiration study[END_REF] Secondary ITP may in addition show features of the associated disorder. When thrombocytopenia is due to direct megakaryocytic damage by allo-or drug-related antibodies, their number is usually reduced. TPO receptor agonists used in the treatment in ITP may induce reversible and dose-dependent bone marrow changes such as megakaryocyte hyperplasia and increased reticulin fibrosis. 100

Non-immune thrombocytopenia

Non-immune thrombocytopenia can be due to impaired production of platelets in the bone marrow. Besides the above mentioned inherited amegakaryocytic syndromes, other (very rare) syndromes with congenital thrombocytopenia are known. An acquired syndrome called amegakaryocytic thrombocytic purpura (ATP) and its cyclic variant have been described in adults, being rare disorders characterized by severe thrombocytopenia with absence or marked reduction of megakaryocytes in the bone marrow. 101,102 The aetiology seems to be multifactorial. 101,102 Ineffective production of platelets is also the cause of thrombocytopenia in MDS, which is a clonal haematopoietic stem cell disorder.

Non-immune thrombocytopenia can furthermore be the result of increased utilization or destruction of platelets outside the bone marrow as seen in thrombocytic thrombocytopenic purpura (TTP), diffuse intravascular coagulation (DIC), haemolytic uremic syndrome (HUS) and certain infections.

In the amegakaryocytic thrombocytopenia syndromes, megakaryocytes are absent or markedly reduced with only small, immature forms. Very few abnormalities are present in the other haematopoietic cell lineages. In cyclic ATP, megakaryocytes are markedly reduced during platelet nadirs and atypical micromegakaryocytes can be seen. 102 When platelet counts are increasing, the number of megakaryocytes becomes normal or increased and their morphology turns to normal. 102 In MDS, megakaryocytes are dysplastic and dysplasia can be seen in other lineages too. When thrombocytopenia has developed acutely due to increased utilization or destruction of platelets, the bone marrow is morphologically normal. With time, however, there will be an in increase in the number of megakaryocytes together with a reduction in their average size.

Fanconi anaemia

Fanconi anaemia (FA) is an autosomal recessive or, rarely, X-linked recessive bone marrow failure syndrome with characteristically an abnormal high frequency of spontaneous chromosomal breakages and hypersensitivity to DNA cross-linking agents such as diepoxybutane and mitomycin-C. FA shows considerable genetic heterogeneity, with 13 subtypes currently recognized. [103][104][105] It is associated with a wide range of congenital abnormalities including generalized hyperpigmentation of the skin, microphthalmia, radial ray anomalies, genitourinary abnormalities and short stature. However, in about 30% of FA patients no physical abnormalities are found. 106 Haematological abnormalities generally develop during the first decade, usually starting with thrombocytopenia and/or anaemia, eventually progressing to pancytopenia in >90% of patients. 105 There is a strikingly increased risk of MDS, AML and solid tumours, particularly squamous cell carcinomas of the head and neck, anogenital regions and skin. 107 The development of MDS is highly associated with the presence of clonal chromosomal abnormalities, mostly involving chromosomes 1, 3 and 7. 108 The first presentation of FA may be in adulthood with marrow failure or a malignancy. 109 In early stages, the bone marrow of FA patients might still be normocellular but with disease progression, it becomes increasingly hypocellular and eventually a histological picture indistinguishable from other forms of aplastic anaemia will ensue. Megakaryocytes are often decreased in number. The erythroid lineage usually shows hyperplasia with erythroblastic islands, megaloblastic changes and dysplasia. 108,110,111 As dysplasia limited to the erythroid lineage is frequently found in FA, one must be very hesitant to regard these findings as MDS (Figure 5). Chioc et al. described the morphological and cytogenetic characteristics of FArelated MDS. 108 An increase in blasts and the presence of dysgranulopoiesis are the most

Dyskeratosis congenita

Dyskeratosis congenita (DC) is typically characterized by the mucocutaneous triad of nail dystrophy, mucosal leucoplakia and abnormal skin pigmentations, although a variety of other abnormalities have been reported. The classical features usually appear during childhood. 112 The pattern of inheritance may be X-linked recessive, autosomal dominant or autosomal recessive. The mutations that have been identified thus far interfere with telomerase activity, although the genetic basis of disease is still unknown in the majority of patients. 113,114 DC has an increased risk of MDS and acute leukaemia as well as of solid tumours, especially squamous cell carcinomas. Most patients (>90%) progress to aplastic anaemia with bone marrow failure being the major cause of mortality. 112 The onset of cytopenia can precede the appearance of the characteristic mucocutaneous triad.

The bone marrow findings in DC range from normal to variable stages of aplasia depending on the stage of the disease and eventually will be indistinguishable from aplastic anaemia due to other causes.

Aplastic anaemia

Aplastic anaemia (AA) is characterized by pancytopenia and an acellular or markedly hypocellular bone marrow. AA can be idiopathic (~70%), inherited (~15-20%) or secondary to radiation, drugs, viruses or immune disorders (~10-15%). 105 Idiopathic and secondary AA are usually immune mediated by autoreactive lymphocytes mediating destruction of haematopoietic stem cells. 115 The clinical course of AA is highly variable with some patients having only mild symptoms, needing little or no therapy, and others presenting with lifethreatening pancytopenias. 115 Patients with AA are at risk for the development of clonal disorders, e.g. PNH clones. [START_REF] Wanachiwanawin | A cohort study of the nature of paroxysmal nocturnal hemoglobinuria clones and PIG-A mutations in patients with aplastic anemia[END_REF]116 In addition, the development of MDS [117][118][119] and AML is related to emerging clonal chromosomal abnormalities, mostly monosomy 7. It is important to identify patients with AA in the setting of one of the inherited bone marrow failure syndromes as these rarely respond to immunosuppressive therapy. Since the histopathology of idiopathic, inherited and secondary AA is similar, the distinction can only be made by ancillary tests. A trephine biopsy is essential for diagnosing AA and whatever the underlying cause, bone marrow morphology is similar. There is a marked reduction in haematopoietic cells with replacement by fat cells and a variable inflammatory infiltrate of plasma cells, mast cells, interspersed or aggregated lymphocytes, macrophages and sometimes eosinophils (Figure 6). This reactive infiltrate might be so extensive that at first glance the marrow may even seem hypercellular. In addition, there may be oedema and/or haemorrhages, 120, 121 necrosis or cellular debris. Macrophage iron is often increased; the amount of reticulin is normal or only slightly increased. Some cases may contain so-called hot spots: hypercellular areas consisting mainly of erythroid precursors which are often left-shifted and synchronized at the same stage of development. 121 In adults, clusters of more than 20 erythroid cells and the presence of leftshifted and/or synchronized erythropoiesis in an otherwise empty marrow are considered part of AA. In children however, these findings should be regarded as evidence for refractory cytopenia of childhood, a low-grade form of paediatric MDS, according to the WHO classification 2008. 122 In addition, aplasia and dyserythropoiesis are frequently found in Fanconi anaemia so this disorder should always be excluded by ancillary chromosomal breakage tests before diagnosing AA or MDS in young persons. Furthermore, the presence of dyserythropoiesis in an aplastic marrow should also raise the question of PNH-associated AA in both children and adults.

It is always important to differentiate AA from hypocellular MDS (h-MDS). In AA, cell atypia is found only in erythroblasts and not in other cell lineages. 123 While megakaryocytes are absent or reduced in numbers but otherwise normal in AA, 121 they are present and atypical in the majority of cases of h-MDS. 123 The presence of reticulin fibrosis further favours h-MDS over AA. 124 The amount of CD34 positive blasts is often low in AA, whereas it is normal or increased in h-MDS. 125,126 In hypocellular AML, another important differential diagnosis of AA, the amount of blasts is per definition increased (≥ 20% in adults and ≥ 30% in children). Their identification by immunostains is therefore crucial.

Finally, it should be kept in mind that subcortical bone and sites other than central axial bone marrow normally contain little haematopoiesis in adult patients, so that trephine biopsy specimens taken from these areas are not suitable for diagnosing AA.

Myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal haematopoietic stem cell disorders characterized by ineffective haematopoiesis resulting in peripheral cytopenia, and a propensity to progress to AML. MDS mainly occur in the elderly, but occasionally can be seen in childhood as well. 127,128 In adults, low-grade MDS usually present with anaemia whereas in childhood they more often present with thrombocytopenia or leukopenia. 129 Two thirds of cases of paediatric MDS are considered primary, whereas one third of cases has an association with an inherited/constitutional disorder or follows chemotherapy. The most recent WHO classification recognizes several subgroups, mainly based on morphology, blast count and cytogenetic abnormalities. 130 The threshold for diagnosing AML is an increase in myeloblast in peripheral blood or bone marrow of ≥ 20% in adults and of ≥ 30% in children.

However, MDS secondary to chemotherapy or radiation therapy should be classified together with therapy-related AML as 'therapy-related myeloid neoplasm' regardless the blast count.

Furthermore, MDS associated with Down syndrome, which accounts for 20-25% of cases of MDS occurring in childhood, 131 is considered to be a unique biological entity and is classified together with AML as 'Down syndrome-related myeloid leukaemia' irrespective of the blast count.

The bone marrow of patients with MDS usually is hypercellular but hypocellularity can be seen, especially in children. 132 Dyshaematopoiesis is the hallmark of the disease and the bone marrow architecture is usually disturbed (Figure 7). There is often erythroid hyperplasia and dyserythropoies is manifested by left-shifting, synchronization of the erythroid precursors within the erythroid islands, nuclear abnormalities and sometimes presence of megaloblasts.

Dysgranulopoiesis is characterized by decreased maturation of the myeloid precursors and clusters of immature myeloid precursors abnormally located in the centre of the bone marrow fields (so-called ALIPs) which are typically seen in high-grade MDS. In trephine bone marrow biopsies, dysplastic features are most prominent in the megakaryocytes, which typically show hypolobulation, wide separation of the nuclear lobes, rounded nuclei and micromegakaryocytes. Iron stores are increased due to ineffective haematopoiesis or transfusions and significant fibrosis can be seen in approximately 10% of cases. Other common findings include the presence of oedema, increased number of microvessels, plasmacytosis, increased mast cells, macrophages with increased cellular debris, lymphocytosis and lymphoid follicels. 133 The dysplastic features of hypocellular MDS are similar to the normo-and hypercellular forms. 133 Distinction of hypocellular MDS form AA is important and is discussed under AA (see above). Since dysplastic changes are a feature of HIV infection itself, or can be induced by antiretroviral therapy distinction from MDS can be challenging. Furthermore, superimposed infections can complicate the picture.

The bone marrow in patients with HIV can be either hypercellular, normocellular or hypocellular. 134 The bone marrow architecture is often disturbed and dysplastic changes are almost always present which can be similar to those of MDS. However, dyserythropoiesis is usually less severe in HIV than in MDS and occurs mainly in patients treated with HAART therapy with megaloblastic changes being particularly associated with AZT therapy. 135,136 In contrast to MDS, in which erythropoiesis is usually hyperplastic, the myeloid/erythroid ratio in HIV is usually normal and initially there may even be neutrophilic and megakaryocytic hyperplasia in HIV. 136 An increase in blasts can be seen in MDS, but never in HIV. In addition, bone marrow trephine biopsies in HIV patients often show eosinophilia, lymphohistiocytic infiltrates and reactive plasmacytosis. 137 Granulomas are encountered in approximately 15% of bone marrow trephines and might by merely due to the HIV infection but a thorough search for other infections is required. HIV associated stromal changes include edema, gelatinous transformation and increased reticulin fibers. 136,137 Dense collagen fibrosis, however, is not a feature of HIV. 136 In all instances one should carefully search for opportunistic infections, especially mycobacterial, fungal and protozoal infections.

Lastly, there can be bone marrow involvement by lymphomas, Kaposi sarcoma and Castleman disease, resulting in bone marrow failure.

Concluding remarks:

Dyshaematopoiesis is the hallmark of MDS, but as illustrates in this review, dysplasia of one or more cell lines can be seen in a wide variety of other situations such as in inherited bone marrow failure syndromes, PNH, infections such as HIV and parvovirus B19, drugs and autoimmune diseases. Therefore, correlation with clinical parameters, the bone marrow aspirate, the peripheral blood smear and cytogenetics is essential in the assessment of bone marrow disorders. The bone marrow is normocellular but consist mainly of granulopoisis and megakarypoiesis.

Maturing erythropoiesis is not seen. Iron stores are increased due to blood transfusions.

  due to marrow infiltration by leukaemia and solid tumours

  MDS, AML, paroxysmal nocturnal haemoglobinuria, aplastic anaemia, AIDS,

  of new bone formation and foamy macrophages are signs of previous infarctions.

68

 68

Pancytopenia

  Pancytopenia can result from a wide variety disorders such as aplastic anaemia, MDS, acute leukemia, infections (most often HIV), familial haemophagocytic syndrome, severe folate or Vit.B12 deficiency, autoimmune disorders such as systemic lupus erythematosus, PNH, inherited bone marrow failure syndromes, hypersplenism or drugs. Some of these disorders more often present as a single cell cytopenia and have already been discussed, others are discussed below.

  diagnosing FA-related MDS, and are highly correlated to the presence of clonal abnormalities, followed by dysmegakaryopoiesis.108

  Furthermore, it is important to differentiate AA from hypocellular MDS and hypocellular AML.

21 HIV

 21 Patients with HIV infection can suffer from various cytopenias and late in the course of the disease, pancytopenia with marked lymphopenia may ensue. A bone marrow biopsy may be performed in search of infection, for staging of malignant lymphoma or to rule out MDS.

33 Figure 1 :

 331 Figure 1: Pure red cell aplasia

Figure 1 :

 1 Figure 1: Pure red cell aplasia in an 18 year old male with Hodgkin lymphoma in remission.

Figure 2 :

 2 Figure 2: Shwachman-Diamond syndrome. Case of an 8 year old boy with a hypocellular bone marrow, dispersed erythropoiesis and mainly immature granulocyte precursors.Megakaryocytes are present in normal numbers. This patient later developed pancytopenia.

Figure 3 :

 3 Figure 3: Severe congenital neutropenia. Normocellular bone marrow in an 8 year old child, showing normal erythropoiesis and megakaryopoiesis. Neutrophil maturation is virtually absent. Eosinophils are increased.

Figure 4 :

 4 Figure 4: Congenital amegakaryocytic thrombocytopenia. Normocellular marrow with maturing granulopoiesis, slightly hyperplastic erythropoiesis and absence of normal megakaryocytes.

Figure 5 :

 5 Figure 5: Fanconi anaemia in a 4 year old boy. Hypocellular bone marrow with a focus of early erythroblasts. This dyserythropoiesis is typical of FA and should not be confused with MDS.

Figure 6 :

 6 Figure 6: Aplastic anaemia in a 14 year old girl. Markedly hypocellular bone marrow devoid of hematopoietic cells with only scattered lymphocytes, plasma cells and mast cells.

Figure 7 :

 7 Figure 7: Myelodysplastic syndrome in a 65 years old female. Hypercellular bone marrow with a disturbed architecture. Erythropoiesis is scattered and immature forms and nuclear irregularities are seen. There is decreased maturation of the myeloid precursors and immature myeloid precursors are abnormally located in the centre of the bone marrow fields. Megakaryocytes are small and atypical showing hypolobulation.

Published on behalf of the British Division of the International Academy of Pathology Histopathology