Prof Cyril Fisher
email: cyrilfisher@gmail.com

MD Dsc Frcpath

Keywords: ; royal marsden hospital, histopathology soft tissue tumours, immunohistochemistry, sarcoma, diagnosis Soft Tissue Tumour Immunohistochemistry Immunohistochemistry, sarcoma, diagnosis, soft tissue tumour

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Introduction

Soft tissue tumours comprise numerous different tumour types that are classified according to the type of mesenchymal tissue which they resemble. The main object of immunohistochemistry in soft tissue neoplasms, and especially sarcomas, is to identify differentiation in the neoplastic cells. This is straightforward for those tumours that correspond to normal tissue types, but less so for those sarcomas characterized by specific chromosomal translocations since they represent novel lineages. In some cases, specific antigens are expressed; however, an initial panel of antibodies is often required in order to establish the broad lineage, with a subsequent, more focused panel to allow precise classification.

Tumours with adipocytic or osteochondroid differentiation or vascular space formation can often be recognized from the light microscopic appearances. Many soft tissue neoplasms, however, show spindle cell, epithelioid cell, small round cell or pleomorphic morphology on initial examination and require further characterisation by immunohistochemistry, electron microscopy (although this now has a limited role [START_REF] Fisher | The comparative roles of electron microscopy and immunohistochemistry in the diagnosis of soft tissue tumours[END_REF] ), and genetic analysis. This article reviews selected applications of immunohistochemistry in diagnosis in each of the principal morphological groups, concentrating on areas of most use in daily practice. It is important to remember that immunohistochemical evaluation must be employed with the clinical picture, the morphology, and, when necessary, other ancillary techniques such as molecular genetics and cytogenetics. The differential diagnosis of the principal benign soft tissue tumours is straightforward when the lineage is obvious. Difficulty is sometimes encountered, especially in core biopsies, in assessing malignancy, the morphological criteria for which vary according to the tumour type. In specific situations, immunohistochemistry is occasionally of assistance in distinguishing benign from malignant lesions. For example, diffuse S100 protein positivity in a spindled nerve sheath tumour is more suggestive of schwannoma than malignant peripheral nerve sheath tumour (MPNST), especially if there are portions of capsule in the biopsy. Recently, it has been suggested that a rare CD34-positive cellular fibrous histiocytoma can be distinguished from the commonly CD34-positive dermatofibrosarcoma by the expression of podoplanin (D2-40) in the former but not the latter. [START_REF] Bandarchi | D2-40, a novel immunohistochemical marker in differentiating dermatofibroma from dermatofibrosarcoma protuberans[END_REF] Combinations of CD34 with other antibodies can be useful in selected circumstances (Table 1). Cell cycle-related and proliferation markers are of little use in individual cases. The proliferation index with Ki67 can be indicative of malignancy when high, but it can also be high in reactive lesions such as nodular fasciitis.

Spindle Cell Tumours

BENIGN SPINDLE CELL NEOPLASMS

INTERMEDIATE SPINDLE CELL NEOPLASMS

Fibromatosis is a non-metastasizing lesion which is a consideration in the differential diagnostic of many types of spindle cell sarcoma. The lesional cells are myofibroblasts which are focally positive for smooth muscle actin (SMA) and occasionally for desmin in scattered cells. S100 protein can be focally positive but CD34 is negative. Nuclear immunoreactivity for beta-catenin is found in variable numbers of nuclei in about 80% of deep fibromatoses, but only 5% of superficial ones. [START_REF] Ng | Nuclear beta-catenin in mesenchymal tumors[END_REF][START_REF] Bhattacharya | Nuclear beta-catenin expression distinguishes deep fibromatosis from other benign and malignant fibroblastic and myofibroblastic lesions[END_REF] Non-specific paranuclear staining is common in other myofibroblastic lesions and must not be misinterpreted. Nuclear Solitary fibrous tumour is diffusely positive for CD34 in 95% of cases, as well as for bcl-2, CD99 and beta-catenin. [START_REF] Ng | Nuclear beta-catenin in mesenchymal tumors[END_REF][START_REF] Renshaw | CD34 and AE1/AE3. Diagnostic discriminants in the distinction of solitary fibrous tumor of the pleura from sarcomatoid mesothelioma[END_REF][START_REF] Chan | Solitary fibrous tumour--everywhere, and a diagnosis in vogue[END_REF][START_REF] Chilosi | bcl-2 expression in pleural and extrapleural solitary fibrous tumours[END_REF] Occasional cells expressing cytokeratin (CK), 9 epithelial membrane antigen (EMA), or S100 protein can be seen, which does not alter the diagnosis. Malignant solitary fibrous tumour, manifesting as spindle or polygonal cell sarcoma contiguous with (or recurrent at the site of a previous) typical solitary fibrous tumour, usually retains CD34 expression diffusely or focally. 10,11,12 Angiomatoid fibrous histiocytoma expresses desmin in about 50% of cases, but myogenin is negative. Other antigens that are sometimes positive are h-caldesmon, SMA (but the morphology is unlike that of smooth muscle tumours), CD99 and EMA. 13 This tumour has three translocations resulting in the fusion genes EWSR1-CREB1 (the commonest), EWSR1-ATF1, or FUS-ATF1. 14 The first two are shared with clear cell sarcoma (see below).

MALIGNANT SPINDLE CELL NEOPLASMS

These include synovial sarcoma, malignant peripheral nerve sheath tumour, leiomyosarcoma, myofibrosarcoma and fibrosarcoma, and spindle cell variants of rhabdomyosarcoma and angiosarcoma. Non-mesenchymal tumours such as sarcomatoid Synovial sarcoma is diagnostically obvious when biphasic. The monophasic form is a spindle cell sarcoma composed of closely packed uniform cells with overlapping nuclei and scanty cytoplasm, while poorly differentiated synovial sarcoma has polygonal or small round cells with high proliferation index. 15 All types of synovial sarcoma exhibit epithelial differentiation with focal positivity for cytokeratins (including CK7 and CK19) and for EMA in most cases. Diffuse expression of bcl-2 and focal positivity for CD99, S100 protein, CD56 and calponin are also found in some, and other non-specific markers of occasional diagnostic value include beta-catenin (nuclear) [START_REF] Ng | Nuclear beta-catenin in mesenchymal tumors[END_REF] and calretinin (nuclear). 16 CD34 is almost always negative in synovial sarcoma 17 which is useful in excluding solitary fibrous tumour, since the immunophenotype of these two tumours can otherwise overlap. TLE1, an antibody derived from gene expression profiling studies, is emerging as a highly sensitive (nuclear) marker for synovial sarcoma of all morphologic types. [START_REF] Kosemehmetoglu | TLE1 expression is not specific for synovial sarcoma: a whole section study of 163 soft tissue and bone neoplasms[END_REF][START_REF] Jagdis | Prospective evaluation of TLE1 as a diagnostic immunohistochemical marker in synovial sarcoma[END_REF] It is also not wholly specific, since occasional examples of nerve sheath tumours can display positivity. However, its sensitivity means that it is useful in excluding the diagnosis of synovial sarcoma when the result is negative. Reduced expression of INI1 (see below) has been described in some synovial sarcomas. [START_REF] Kohashi | Reduced expression of SMARCB1/INI1 protein in synovial sarcoma[END_REF] Synovial sarcoma is characterized by a specific chromosomal translocation t(X;18)(p11.2;q11.2), which results in fusion of the SS18 (SYT) gene from chromosome 18 with one of several variants of the SSX gene on the X chromosome. [START_REF] Ladanyi | Impact of SYT-SSX fusion type on the clinical behavior of synovial sarcoma: a multi-institutional retrospective study of 243 patients[END_REF] Recently, an antibody to the SS18 gene product has been shown to be sensitive for identification of synovial sarcoma but it is not specific and has not found general application. Malignant peripheral nerve sheath tumours (MPNST) have focal nuclear positivity for S100 protein in up to 67% of cases, which correlates with ultrastructural evidence of Schwann cell differentiation. 23 There are no other 'specific' indicators of nerve sheath lineage (although electron microscopy can be contributory in S100 proteinnegative examples 23 ) but many MPNST are focally positive for CD34, and a variable proportion of cases express GFAP, myelin basic protein, CD57 and nestin. Cytokeratins are occasionally positive but the presence of CK7 or CK19 is more indicative of synovial sarcoma than of MPNST. [START_REF] Smith | Usefulness of cytokeratin subsets for distinguishing monophasic synovial sarcoma from malignant peripheral nerve sheath tumor[END_REF] Immunoreactivity for NSE or PGP9.5 is sometimes found in MPNST but these markers are non-specific and of no value in diagnosis. 25 EMA positivity is occasionally seen in MPNST with Schwannian differentiation, but is most useful in diagnosis of benign perineurioma [START_REF] Hornick | Soft tissue perineurioma: clinicopathologic analysis of 81 cases including those with atypical histologic features[END_REF] and its rare malignant counterpart, [START_REF] Hirose | Perineurial malignant peripheral nerve sheath tumor (MPNST). A clinicopathologic, immunohistochemical, and ultrastructural study of seven cases[END_REF] which usually lack S100 protein. Another useful marker of perineurial cell differentiation is claudin-1 which, as a tight junction-associated protein, [START_REF] Billings | Aberrant expression of tight junction-related proteins ZO-1, claudin-1 and occludin in synovial sarcoma: an immunohistochemical study with ultrastructural correlation[END_REF] has a granular distribution in normal perineurial cells and in perineurioma. [START_REF] Folpe | Expression of claudin-1, a recently described tight junction-associated protein, distinguishes soft tissue perineurioma from potential mimics[END_REF] Claudin-1 has also been reported as positive in low-grade fibromyxoid sarcoma [START_REF] Thway | Claudin-1 is expressed in perineurioma-like low-grade fibromyxoid sarcoma[END_REF] which can resemble perineurioma, especially in cutaneous lesions, so that genetic analysis (see below) can be required for diagnosis.

Leiomyosarcoma is most often morphologically distinctive, with its non-tapered cells with eosinophilic cytoplasm and squared-off nuclei, arranged in cellular fascicles in a distinctive rectilinear pattern. Immunohistochemically, desmin, SMA, muscle specific actin (MSA), h-caldesmon, calponin and smooth muscle myosin (SMM) are expressed in the majority of leiomyosarcomas. Some examples co-express cytokeratins (frequently with dot pattern), EMA and S100 protein but usually only when the muscle-specific expression is variable but generally negative in leiomyosarcoma, and CD117 is absent from smooth muscle tumours. A spindle cell neoplasm expressing SMA but not desmin or h-caldesmon is unlikely to be a smooth muscle tumour and is more likely to be myofibroblastic. [START_REF] Ceballos | Is anti-h-caldesmon useful for distinguishing smooth muscle and myofibroblastic tumors? An immunohistochemical study[END_REF] Myofibrosarcomas are spindle cell or pleomorphic sarcomas which display myofibroblastic differentiation. 32 Low grade myofibrosarcomas are composed of tapered cells containing ovoid nuclei and punctate nucleoli, arranged in sheets and fascicles. 33 They are positive for SMA (with a 'tram-track' subplasmalemmal accentuation), and usually express calponin and less commonly desmin. H-caldesmon and SMM are negative, distinguishing myofibrosarcoma from leiomyosarcoma. 34 CD34 and S100 protein are also negative; the combination of CD34 and desmin immunoreactivity is found in mammary-type myofibroblastomas, 35 but their ultrastructure is more like that of smooth muscle cells than of true myofibroblasts. 36 Inflammatory myofibroblastic tumour (IMT) is a low-grade myofibroblastic malignancy with fasciitis-like, fascicular and sclerosing histological patterns. 37 In addition to the usual myofibroblastic markers, about half of IMT, especially those in viscera and in children, express ALK, and recent evidence suggests that lack of ALK is prognostically adverse. 38 In IMT, the ALK gene is rearranged with one of six partner genes, most of which result in cytoplasmic staining on immunohistochemistry, except for ALK-RANBP2. The latter gives a distinctive nuclear membranous distribution as RANBP2 binding proteins are localised at the nuclear pore complex. 39 Adult fibrosarcoma is now a rare tumour which is by definition negative for all lineage markers (fibrosarcomas express vimentin but this is a wholly non-specific marker which need not usually be included in diagnostic panels). However, specific subtypes of fibrosarcoma are positive for CD34, including those arising in dermatofibrosarcoma, about half of myxofibrosarcomas, and a smaller proportion of myxoinflammatory fibroblastic sarcomas. 40 Some superficial fibrosarcomas that are strongly CD34 positive but lack an apparent dermatofibrosarcomatous component have been shown to have the same COL1A1-PDGFRB fusion gene transcripts as dermatofibrosarcoma. 41 In a CD34negative fibrosarcoma, an occasional SMA-positive cell can sometimes be seen which is attributable to focal myofibroblastic differentiation, but diffuse or multifocal SMA staining indicates categorization as myofibrosarcoma. 32 Low grade fibromyxoid sarcoma occasionally has SMA or CD34 positivity (and, as previously noted, can express EMA and claudin-1 [START_REF] Thway | Claudin-1 is expressed in perineurioma-like low-grade fibromyxoid sarcoma[END_REF] ), but the diagnosis is best confirmed by genetic demonstration of the t(7;16)(q33;p11) or FUS-CREB3L2 (or rarely FUS-CREB3L1) fusion gene, [42][START_REF] Mertens | Clinicopathologic and molecular genetic characterization of low-grade fibromyxoid sarcoma, and cloning of a novel FUS/CREB3L1 fusion gene[END_REF][START_REF] Matsuyama | Molecular detection of FUS-CREB3L2 fusion transcripts in low-grade fibromyxoid sarcoma using formalin-fixed, paraffin-embedded tissue specimens[END_REF] which has also been reported in some examples of sclerosing epithelioid fibrosarcoma. [START_REF] Guillou | Translocation-positive low-grade fibromyxoid sarcoma: clinicopathologic and molecular analysis of a series expanding the morphologic spectrum and suggesting potential relationship to sclerosing epithelioid fibrosarcoma: a study from the French Sarcoma Group[END_REF] Spindle cell rhabdomyosarcoma of juvenile (adolescent) [START_REF] Cavazzana | Spindle cell rhabdomyosarcoma. A prognostically favorable variant of rhabdomyosarcoma[END_REF][START_REF] Leuschner | Spindle cell rhabdomyosarcoma: histologic variant of embryonal rhabdomyosarcoma with association to favorable prognosis[END_REF] or adult type [START_REF] Nascimento | Spindle cell rhabdomyosarcoma in adults[END_REF][START_REF] Mentzel | Spindle cell rhabdomyosarcoma in adults: clinicopathological and immunohistochemical analysis of seven new cases[END_REF][START_REF] Stock | Adult-type rhabdomyosarcoma: analysis of 57 cases with clinicopathologic description, identification of 3 morphologic patterns and prognosis[END_REF] is diffusely positive for desmin, and at least focally so in nuclei for myogenin and MyoD1.

The latter is less sensitive, but in the rare sclerosing pseudovascular rhabdomyosarcoma, MyoD1 is more widely expressed than myogenin. [START_REF] Folpe | Sclerosing rhabdomyosarcoma in adults: report of four cases of a hyalinizing, matrix-rich variant of rhabdomyosarcoma that may be confused with osteosarcoma, chondrosarcoma, or angiosarcoma[END_REF] It should be noted that these are nuclear antigens and that cytoplasmic staining for either is non-specific and does not denote rhabdomyoblastic differentiation.

Myoglobin, a previously widely used Spindle cell angiosarcoma is an uncommon soft tissue sarcoma of skin or soft tissue in which diagnostic vasoformative areas can be present only focally and therefore absent from a core biopsy. The tumour can be identified by its expression of CD31, and less commonly of CD34 and other endothelial markers (see discussion of epithelioid endothelial neoplasms below). CD117 positivity has also been reported in angiosarcoma [START_REF] Miettinen | KIT (CD117): a review on expression in normal and neoplastic tissues, and mutations and their clinicopathologic correlation[END_REF] and in Kaposi sarcoma (KS). The latter, however, demonstrates highly specific immunoreactivity for HHV8 (KSHV) in the nuclei of its spindled and endothelial cells, which is not found in other endothelial neoplasms. [START_REF] Boshoff | Kaposi's sarcoma-associated herpesvirus infects endothelial and spindle cells[END_REF] Podoplanin (D2-40) is a lymphatic endothelial marker that has a membranous staining pattern in Kaposi sarcoma, but also in some angiosarcomas and gastrointestinal stromal tumours (which also display CD34 and CD117, as in angiosarcoma). [START_REF] Ordonez | Podoplanin: a novel diagnostic immunohistochemical marker[END_REF] Antibodies to vascular endothelial growth factor and its receptor (such as VEGF-C and VEGFR-3) are also immunoreactive in KS. [START_REF] Dupin | Distribution of human herpesvirus-8 latently infected cells in Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma[END_REF][START_REF] Folpe | Vascular endothelial growth factor receptor-3 (VEGFR-3): a marker of vascular tumors with presumed lymphatic 28 differentiation, including Kaposi's sarcoma, kaposiform and Dabska-type hemangioendotheliomas, and a subset of angiosarcomas[END_REF] Follicular dendritic cell sarcoma (FDCS), which sometimes arises in soft tissue sites, [START_REF] Chan | Follicular dendritic cell sarcoma. Clinicopathologic analysis of 17 cases suggesting a malignant potential higher than currently recognized[END_REF][START_REF] Fisher | Myxoid variant of follicular dendritic cell sarcoma arising in the breast[END_REF][START_REF] Biddle | Extranodal follicular dendritic cell sarcoma of the head and neck region: three new cases, with a review of the literature[END_REF] is characterised by fascicles and whorls of cells in which the nuclei typically display prominent membranes and a speckled chromatin pattern. Immunoreactivity for CD21, CD35 (the two are more sensitive when used together as a cocktail [START_REF] Chan | Follicular dendritic cell sarcoma. Clinicopathologic analysis of 17 cases suggesting a malignant potential higher than currently recognized[END_REF] ) and CD23 is diagnostic, and other markers reportedly helpful in identifying FDCS include fascin, clusterin [START_REF] Grogg | A survey of clusterin and fascin expression in sarcomas and spindle cell neoplasms: strong clusterin immunostaining is highly specific for follicular dendritic cell tumor[END_REF] and D2-40. [START_REF] Yu | Podoplanin (D2-40) is a novel marker for follicular dendritic cell tumors[END_REF] Many examples are also positive for S100 protein and EMA. [START_REF] Chan | Proliferative lesions of follicular dendritic cells: an overview, including a detailed account of follicular dendritic cell sarcoma, a neoplasm with many faces and uncommon etiologic associations[END_REF] Sarcomatoid carcinoma can express cytokeratins focally, especially those of high molecular weight, but can also be negative. 64 The diagnosis can be facilitated by finding Spindle cell carcinoma can also express SMA, but desmin, h-caldesmon, CD34 and S100 protein are absent. SMA-positive/CK-negative sarcomatoid carcinoma, which is usually a pleomorphic neoplasm, can be difficult to distinguish from pleomorphic myofibrosarcoma without knowledge of the history and the location of the tumour.

Gastrointestinal stromal tumours (GIST) can arise not only in the gastrointestinal

tract but also in extra-gastrointestinal locations, especially omentum, mesentery and retroperitoneum. The two predominant patterns are spindled or epithelioid, which have a similar immunophenotype. GIST typically express CD117, the product of the KIT gene, which encodes a receptor tyrosine kinase, and which in GIST shows a variety of prognosis-related mutations. [START_REF] Miettinen | Gastrointestinal stromal tumors: review on morphology, molecular pathology, prognosis, and differential diagnosis[END_REF][START_REF] Andersson | Gastrointestinal stromal tumors with KIT exon 11 deletions are associated with poor prognosis[END_REF][START_REF] Lasota | KIT and PDGFRA mutations in gastrointestinal stromal tumors (GISTs)[END_REF] A small number of GIST are CD117-negative, and some of these have mutations in the gene which encodes platelet-derived growth factor receptor A (PDGFRA). [START_REF] Lasota | KIT and PDGFRA mutations in gastrointestinal stromal tumors (GISTs)[END_REF] These are often epithelioid gastric GIST. The antibody DOG1, identified by gene expression profiling, has high sensitivity and specificity for GIST and can identify most KIT-negative tumours. [START_REF] Espinosa | A novel monoclonal antibody against DOG1 is a sensitive and specific marker for gastrointestinal stromal tumors[END_REF][START_REF] West | The novel marker, DOG1, is expressed ubiquitously in gastrointestinal stromal tumors irrespective of KIT or PDGFRA mutation status[END_REF] Other antigens found in GIST include CD34 in 50-100% of tumours (varying with site [START_REF] Miettinen | Immunohistochemical spectrum of GISTs at different sites and their differential diagnosis with a reference to CD117 (KIT)[END_REF] ), SMA (varying inversely with CD34 expression), h-caldesmon, bcl-2 and, less commonly, desmin or S100 protein. Betacatenin is negative in nuclei, in contrast to fibromatosis. 5

Epithelioid and clear cell tumours

Epithelioid soft tissue neoplasms include epithelioid sarcoma, and epithelioid variants of MPNST, leiomyosarcoma, and fibrosarcoma, as well as clear sarcoma, alveolar soft part sarcoma, PEComa (Table 2), and melanoma and carcinoma when presenting in soft tissue locations. Epithelioid sarcoma (EpS), of both classical and proximal types, is positive for the epithelial markers cytokeratins and EMA and, in about 50% of cases for CD34. [START_REF] Fisher | Epithelioid sarcoma of Enzinger[END_REF] This is particularly useful in making the distinction from primary or metastatic carcinomas, which are CD34 negative. In addition, CK5/6, a useful marker for carcinomas especially those with squamous differentiation, is detectable in only a small proportion of epithelioid sarcomas. 72 EpS generally lacks desmin, S100 protein and FLI-1, [START_REF] Folpe | Expression of Fli-1, a nuclear transcription factor, distinguishes vascular neoplasms from potential mimics[END_REF] which help in diagnosis from rhabdomyosarcoma, melanoma, and epithelioid angiosarcoma respectively. Antibodies to INI1 are negative in about 90% of EpS, in malignant rhabdoid tumours (MRT), in about half the cases of epithelioid MPNST, and in some extraskeletal myxoid chondrosarcomas with rhabdoid cytomorphology. [START_REF] Modena | SMARCB1/INI1 tumor suppressor gene is frequently inactivated in epithelioid sarcomas[END_REF][START_REF] Orrock | INI1 and GLUT-1 expression in epithelioid sarcoma and its cutaneous neoplastic and nonneoplastic mimics[END_REF][START_REF] Hornick | Loss of INI1 expression is characteristic of both conventional and proximal-type epithelioid sarcoma[END_REF] This relates to the location of the SMARC/INI gene on chromosome 22q, which is mutated or deleted in MRT and EpS. [START_REF] Modena | SMARCB1/INI1 tumor suppressor gene is frequently inactivated in epithelioid sarcomas[END_REF] Epithelioid MPNST differs from its spindle cell counterpart in displaying diffuse S100 protein expression in most cases. [START_REF] Laskin | Epithelioid variant of malignant peripheral nerve sheath tumor (malignant epithelioid schwannoma)[END_REF] [START_REF] Hornick | Loss of INI1 expression is characteristic of both conventional and proximal-type epithelioid sarcoma[END_REF] and D2-40 is positive in some cases. [START_REF] Ordonez | Podoplanin: a novel diagnostic immunohistochemical marker[END_REF] In addition, epithelioid angiosarcoma is frequently cytokeratin-positive, while usually lacking EMA. [START_REF] Gray | Cytokeratin expression in epithelioid vascular neoplasms[END_REF][START_REF] Meis-Kindblom | Angiosarcoma of soft tissue: a study of 80 cases[END_REF] Epithelioid sarcoma, especially the proximal variant, can be morphologically similar to epithelioid angiosarcoma and can also co-express CD34 and cytokeratins, but lacks CD31, FVIIIRAg, FLI-1, and INI1. Epithelioid sarcoma-like haemangioendothelioma is a very rare low-grade spindle cell neoplasm that displays positivity for cytokeratins, CD31, and FL1-1, but lacks CD34 (Table 1). [START_REF] Billings | Epithelioid sarcoma-like hemangioendothelioma[END_REF]81 PEComa is the term used for a group of clinically disparate tumours composed of supposed perivascular epithelioid cells -spindle or polygonal cells with clear or granular cytoplasm and uniform nuclei, which are arranged in nests around sinusoidal vessels. [START_REF] Pea | Perivascular epithelioid cell[END_REF][START_REF] Folpe | Perivascular epithelioid cell neoplasms: pathology and pathogenesis[END_REF] Members of the PEComa 'family' include angiomyolipoma, lymphangioleiomyomatosis, clear cell 'sugar' tumour of lung and other sites, and clear cell myomelanocytic tumour.

PEComas co-express SMA and the melanocytic antigens HMB-45, melan-A, and microophthalmia transcription factor (MITF). It has become recognized that PEComas can also focally express S100 protein, desmin and h-caldesmon as well as TFE3 in some cases. [START_REF] Folpe | Perivascular epithelioid cell neoplasms of soft tissue and gynecologic origin: a clinicopathologic study of 26 cases and review of the literature[END_REF] Most PEComas are benign but malignant examples are increasingly reported. [START_REF] Folpe | Perivascular epithelioid cell neoplasms of soft tissue and gynecologic origin: a clinicopathologic study of 26 cases and review of the literature[END_REF][START_REF] Harris | Malignant perivascular epithelioid cell tumour ("PEComa") of soft tissue: a unique case[END_REF] The principal differential diagnosis ( [START_REF] Meis-Kindblom | Clear cell sarcoma of tendons and aponeuroses: a historical perspective and tribute to the man behind the entity[END_REF] The morphologically similar clear cell sarcoma of the gastrointestinal tract with osteoclastlike giant cells is also S100 protein positive but lacks melan-A and HMB45 expression, and usually has t(2;22)(q33;q12) with EWS-CREB1 fusion. [START_REF] Kosemehmetoglu | Clear cell sarcoma of tendons and aponeuroses, and osteoclast-rich tumour of the gastrointestinal tract with features resembling clear cell sarcoma of soft parts: a review and update[END_REF] However, examples of both fusions have been described at both sites. [START_REF] Wang | Detection and characterization of EWSR1/ATF1 and EWSR1/CREB1 chimeric transcripts in clear cell sarcoma (melanoma of soft parts)[END_REF] The same fusions are also found in angiomatoid fibrous histiocytoma, [START_REF] Rossi | EWSR1-CREB1 and EWSR1-ATF1 fusion genes in angiomatoid fibrous histiocytoma[END_REF] representing one of several examples of promiscuity among gene fusions. 90 Alveolar soft part sarcoma has long been an enigmatic tumour in which the occasional report of immunoreactivity for desmin and, for a time, MyoD1, led to the assumption that the tumour showed skeletal muscle differentiation. This tumour, however, is now known to be a translocation-associated sarcoma characterized by t(X;17)(p11;q25) with fusion of TFE3 and ASPL genes. [START_REF] Ladanyi | The der(17)t(X;17)(p11;q25) of human alveolar soft part sarcoma fuses the TFE3 transcription factor gene to ASPL, a novel gene at 17q25[END_REF] and CDK4 amplification, detectable by fluorescence in situ hybridization (FISH) and also by immunohistochemistry. [START_REF] Binh | MDM2 and CDK4 immunostainings are useful adjuncts in diagnosing well-differentiated and dedifferentiated liposarcoma subtypes: a comparative analysis of 559 soft tissue neoplasms with genetic data[END_REF] Most retroperitoneal (and some extremity 100 ) undifferentiated pleomorphic sarcomas (MFH) and myxofibrosarcomas express these markers (in nuclei) and are now considered to represent dedifferentiated liposarcomas even when no well-differentiated component remains. [100][101][102][103] In addition, dedifferentiated liposarcoma can express actin, desmin and CD34 focally in the pleomorphic areas, 101 which should not be misinterpreted as leiomyosarcoma.

Sarcomatoid carcinoma can appear as a pleomorphic neoplasm. This usually expresses cytokeratins in a multifocal or diffuse distribution that is best demonstrated with a broad spectrum antibody or 'cytokeratin cocktail'. Even very focal cytokeratin expression should prompt consideration of a primary carcinoma, and it can be accepted as evidence of epithelial differentiation in an organ-based (visceral) pleomorphic neoplasm.

It should be remembered, however, that some undifferentiated pleomorphic sarcomas in soft tissue have (aberrant) expression of cytokeratin, usually in a few scattered cells. 104, 105 Also, as previously noted, spindled carcinomas can acquire myofibroblastic differentiation and thereby be focally positive for SMA (but not desmin or h-caldesmon).

As always, the final diagnosis must summate all data including clinical and radiological findings. neuroectodermal tumour (ES), desmoplastic small round cell tumour (DSRCT), alveolar rhabdomyosarcoma (ARMS), neuroblastoma (NB), and poorly differentiated synovial sarcoma (PDSS). Most of these occur predominantly in childhood and adolescence, but are also seen sporadically in adults. 106 Several of these sarcoma subtypes have specific, translocations and consequent fusion genes that allow precise diagnosis, and in some instances provide prognostic information, using FISH or PCR-based methods. 90, 107 A molecular pathology service is not always readily available, but the immunophenotypes of these small round cell sarcomas differ sufficiently to enable diagnosis in many cases.

Small round cell tumours

Lymphomas, especially of lymphoblastic type, must also be considered in the differential diagnosis, and TdT should be part of the diagnostic panel in the appropriate clinical setting. Similarly leukaemic deposits, and small cell carcinomas might need exclusion by immunostaining for myeloid markers or cytokeratins, neuroendocrine antigens and TTF1 respectively.

Ewing sarcoma is a translocation-associated tumour, which can arise in bone or extraskeletal sites; examples with evidence of neural differentiation are termed PNET. 90 The large majority of ES display diffuse membranous staining for CD99, and some express neurofilament proteins and NSE. About 70% show nuclear immunoreactivity for FLI-1. 108 A small number are also positive for S100 protein or display dot positivity with broad spectrum anticytokeratin antibodies and membranous staining for EMA. CD56 is usually negative, unlike in small cell neuroendocrine carcinoma and PDSS. 109 The latter also expresses TLE1, but some cases have a similar immunophenotype to ES and although there are subtle morphological differences (ES tends to be more uniform and

  positivity can also be found in about 40% of solitary fibrous tumours, synovial sarcomas and endometrial stromal sarcomas, as well as in hypertrophic scars. It is absent from gastrointestinal stromal tumour, leiomyosarcoma, inflammatory myofibroblastic tumour and MPNST, which is of use in the differential diagnosis of intraabdominal spindle cell neoplasms.[START_REF] Montgomery | Beta-catenin immunohistochemistry separates mesenteric fibromatosis from gastrointestinal stromal tumor and sclerosing mesenteritis[END_REF] Conversely, fibromatosis is negative for hcaldesmon, ALK, CD34, CD117 and DOG1.

  and follicular dendritic cell sarcoma can present in soft tissue locations and thereby enter the differential diagnosis.

22

 22

  present. CD99 can also be seen in a paranuclear dot pattern. CD34

  skeletal muscle differentiation, is of low sensitivity and specificity and is no longer recommended for routine use.

  differentiation or surface dysplasia, or a nested reticulin pattern.

  Absence of INI1 is extremely useful for diagnosis of these tumours since all other epithelioid or polygonal cell tumours in the differential diagnosis display nuclear positivity for this marker. MRT can be distinguished from EpS by absence of CD34, although clinical data are required to distinguish MRT from CD34negative EpS.

  soft tissue neoplasms includes Ewing sarcoma/primitive

Table 2

 2

	) is with clear sarcoma of tendons and

  Banerjee SS, Shenjere P, Fisher C. The myofibroblast and its tumours. J Fletcher CD. Mammary-type myofibroblastoma of soft tissue: a tumor closely related to spindle cell lipoma. Am J Surg Pathol 2001;25;1022-1029.36.Eyden BP, Shanks JH, Ioachim E, Ali HH, Christensen L, Howat AJ. Myofibroblastoma of breast: evidence favoring smooth-muscle rather than myofibroblastic differentiation. Ultrastruct Pathol 1999;23;249-257. 37. Gleason BC, Hornick JL. Inflammatory myofibroblastic tumours: where are we now? J Clin Pathol 2008;61;428-437. 38. Coffin CM, Hornick JL, Fletcher CD. Inflammatory myofibroblastic tumor: comparison of clinicopathologic, histologic, and immunohistochemical features including ALK expression in atypical and aggressive cases. Am J Surg Pathol 2007;31;509-520. 39. Wilken N, Senecal JL, Scheer U, Dabauvalle MC. Localization of the Ran-GTP binding protein RanBP2 at the cytoplasmic side of the nuclear pore complex. Eur J Cell Kindblom JM, Kindblom LG. Acral myxoinflammatory fibroblastic sarcoma: a low-grade tumor of the hands and feet. Am J Surg Pathol 1998;22;911-924. Hasegawa T, Hirose T, Ayala AG et al. Adult neuroblastoma of the retroperitoneum and abdomen: clinicopathologic distinction from primitive neuroectodermal tumor. Am J Surg Pathol 2001;25;918-924. 122. Chibon F, Mariani O, Derre J et al. A subgroup of malignant fibrous histiocytomas is associated with genetic changes similar to those of well-differentiated liposarcomas. Cancer Genet Cytogenet 2002;139;24-29. 123. Oikawa K, Ishida T, Imamura T et al. Generation of the novel monoclonal antibody against TLS/EWS-CHOP chimeric oncoproteins that is applicable to one of the most sensitive assays for myxoid and round cell liposarcomas. Am J Surg Pathol 2006;30;351-356.

	lacks intercellular reticulin), genetic analysis may be required to make the diagnosis. The Immunohistochemistry with an antibody to PAX5 can detect all translocation-positive MPNST, myxofibrosarcomas and embryonal rhabdomyosarcomas are immunoreactive 9. McGuire LJ, Chan HS, Pang J. Solitary fibrous tumor of pleura: expression of 32. Fisher C. Myofibrosarcoma. Virchows Arch 2004;445;215-223. 121.
	morphologic spectrum of ES is expanding due to increased recognition of atypical variants aided, in part, by molecular studies. 110, 111 Although there is a wide range of cases of either type. 119 Neuroblastoma can be recognized not only by its clinical context (the vast with both 99 but, apart from morphological considerations, the diagnosis of DDL is supported by positivity for these two markers in the differentiated adipose tissue adjacent cytokeratins. Pathology 1990;22;232-234. 10. Nielsen GP, O'Connell JX, Dickersin GR, Rosenberg AE. Solitary fibrous tumor 33. Eyden B, Clin Pathol 2009;62;236-249.
	available antibodies at least three of a panel composed of CD99, FLI-1, HNK1, and majority are in patients <4 years old and arise in adrenal or other sympathetic nervous to the pleomorphic neoplasm. of soft tissue: a report of 15 cases, including 5 malignant examples with light 34. Fisher C. Myofibroblastic malignancies. Adv Anat Pathol 2004;11;190-201.
	F o r P e e r R e v i e w caveolin-1 reportedly show immunoreactivity in all cases with caveolin-1 being positive in CD99-negative cases. 111 Desmoplastic small round cell tumour was originally described in the abdomen and pelvis in adolescent males, and is now recognized in other locations. It shows presumed primitive mesothelial differentiation, and has a characteristic t(11;22)(p13;q12) rearrangement with formation of an EWS-WT1 fusion gene. Immunohistochemically, DSRCT shows nuclear staining with antibodies to the C-terminal end of WT1 as well as multilineage differentiation with dot positivity for desmin, cytokeratins (CK20 and CK5/6 are negative), and neural markers including chromogranin A, synaptophysin, neurofilaments, and CD56. 112, 113 However, not all lineage markers are expressed in every case. 114 DSRCT is usually negative for CD99 but 10-20% (and especially those with variant translocations) can be positive which does not therefore exclude the diagnosis. 115, 116 Alveolar rhabdomyosarcoma, as with other rhabdomyosarcoma subtypes, has diffuse cytoplasmic staining for desmin. Myogenin is expressed in a majority of tumour cell nuclei and is more sensitive than MyoD1. 117 CD56 is also positive as in other types F o r P e e r R e v i e w system locations), but also by its expression of NB84, NSE, CD56 and neurofilament proteins. 120 Antibodies to these markers, which can be used as a panel, are particularly useful in detection of residual disease in bone marrow specimens taken after chemotherapy. Neuroblastoma is negative for cytokeratins, desmin and CD99, which can be particularly helpful in diagnosis of rare examples in adults. 121 Adipose tumours The distinction between lipoma and atypical lipomatous tumour (ALT)/well differentiated liposarcoma can be difficult since it depends on focal morphologic subtleties which are not always represented in the samples taken. The diagnosis is facilitated by the demonstration of immunohistochemical positivity for products of the murine double minute type 2 (MDM2) and cyclin-dependent kinase 4 (CDK4) genes which are overexpressed in ALT as a result of amplification in the 12q14-15 chromosomal region. All ALT are immunoreactive in nuclei for MDM2 (often focally) and 91% for CDK4 (more diffusely). 99 These have application not only in the differential diagnosis of lipoma and ALT, but also in determining whether the fatty tissue at the F o r P e e r R e v i e w Conclusion Existing antibody panels, when correctly selected, have a major role in diagnosis of soft tissue sarcomas. Diagnostically useful new antibodies, such as DOG1 and TLE1, are emerging from gene profiling studies, and in addition to the usual reagents, antibodies to fusion gene products might theoretically be of value in detecting translocation sarcomas. For example, antibodies raised against TLS/EWS-CHOP chimeric oncoproteins have shown high specificity and sensitivity for myxoid and round cell liposarcoma. 123 These approaches can be expected to lead to improved diagnosis of soft tissue tumours and perhaps to more efficient use of immunohistochemistry with limited and therefore less expensive panels. F o r P e e r R e v i e w F o r P e e r R e v i e w microscopic, immunohistochemical, and ultrastructural data. Mod Pathol 1997;10;1028-1037. 11. Vallat-Decouvelaere AV, Dry SM, Fletcher CD. Atypical and malignant solitary fibrous tumors in extrathoracic locations: evidence of their comparability to intra-thoracic tumors. Am J Surg Pathol 1998;22;1501-1511. 12. Fisher C. Low-grade sarcomas with CD34-positive fibroblasts and low-grade myofibroblastic sarcomas. Ultrastruct Pathol 2004;28;291-305. 13. Thway K. Angiomatoid fibrous histiocytoma: a review with recent genetic findings. Arch Pathol Lab Med 2008;132;273-277. 14. Antonescu CR, Dal Cin P, Nafa K et al. EWSR1-CREB1 is the predominant gene fusion in angiomatoid fibrous histiocytoma. Genes Chromosomes Cancer 2007;46;1051-1060. 15. Fisher C. Synovial sarcoma. Ann Diagn Pathol 1998;2;401-421. 16. Miettinen M, Limon J, Niezabitowski A, Lasota J. Calretinin and other mesothelioma markers in synovial sarcoma: analysis of antigenic similarities and differences with malignant mesothelioma. Am J Surg Pathol 2001;25;610-617. F o r P e e r R e v i e w F o r P e e r R e v i e w F o r P e e r R e v i e w 35. F o r P e e r F o r P e e r F o r P e e r F o r P e e r F o r P e e r F o r P e e r F o r P e e r F o r P e e r F o r P e e r F o r P e e r F o r P e e r McMenamin ME, Biol 1995;68;211-219. 40. Sheng WQ, Hashimoto H, Okamoto S et al. Expression of COL1A1-PDGFB Meis-41. R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w
	of rhabdomyosarcoma. 70-90% of ARMS have either t(2;13)(q35;q14) creating a PAX3-margin of excision of ALT is neoplastic or not. As mentioned above, in dedifferentiated 17. Pelmus M, Guillou L, Hostein I, Sierankowski G, Lussan C, Coindre JM. fusion transcripts in superficial adult fibrosarcoma suggests a close relationship to
	FOXO1 fusion gene or t(1;13)(p36;q14) with resulting PAX7-FOXO1 fusion. 118 liposarcoma (DDL), which is genetically related to ALT, CDK4 and MDM2 are similarly Monophasic fibrous and poorly differentiated synovial sarcoma: immunohistochemical dermatofibrosarcoma protuberans. J Pathol 2001;194;88-94.
	overexpressed. 102, 122 Their specificity in DDL is reduced since a small proportion of
	18 19 20 22 25 36

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

Acknowledgment

I acknowledge NHS funding to the NIHR Biomedical Research Centre.


De Tute

Ruth M De Tute

A REVIEW OF FLOW CYTOMETRY AND ITS USE IN THE DIAGNOSIS AND MANAGEMENT OF MATURE LYMPHOID MALIGNANCIES

Keywords: Flow Cytometry, Lymphoma, Lymphoid Neoplasm, Diagnosis

As well as being effective in determining presence of disease at presentation and assist with classification, the group concluded that the technique is also effective for staging of a newly diagnosed malignancy, minimal residual disease monitoring and investigation of relapse or progression.

The principles of flow cytometry

Flow cytometry is a technique which allows analysis of the physical properties and patterns of antigen expression in suspensions of intact cells. Preparation of the suspended cells usually involves lysis of any erythrocytes present and staining of the remaining leucocytes with antibodies conjugated with fluorescent dyes (fluorochromes). Once the sample has been incubated, washing is necessary to remove any excess antibody and the cells are then re-suspended prior to acquisition on a flow cytometer. As the fluid is introduced to the flow cytometer the fluidics system of the analyser employs a technique called hydrodynamic focusing to organise cells into a single cell stream. This stream then passes through a number of laser beams of differing wavelengths (this can range from a single laser to four or more depending on the cytometer). As each cell passes a laser beam it disrupts the path of the laser light, scattering it in many directions. This scatter is measured by a number of detectors to generate the parameters of forward scatter (FSC) and side scatter (SSC). These parameters provide information about the size and complexity of cells with, in general terms, FSC being proportional to cell size and SSC relating to the internal complexity or granularity of a cell. The lasers also cause excitation of any fluorochromes which are attached to antibodies bound to the cells and the resulting fluorescence emission is measured using a series of fluorescence detectors and optical filters. Emission spectra of some fluorochromes overlap considerably so mathematical adjustment of the data, termed compensation, is applied to allow for this. Selection of

Flow cytometric analysis

Each measurement taken by a detector is termed an event and each event equates to a single cell. A detector gives every event above the threshold of detection a numerical value so that by the end of acquisition each cell has a set of numerical values representing all the different parameters that have been measured. Combining of regions is accomplished using the Boolean logic operators AND, OR and NOT (Figure 2 and Figure 3). Once a gate is applied it is possible to examine those cells or events alone to establish a detailed immunophenotypic picture of the population.

Flow cytometric approach to the classification of B-cell neoplasms

The analysis of suspected B-cell neoplasms by flow cytometry proceeds through four well defined steps. The B-cell population is identified and gating established; the stage of B-cell development represented in the sample is assessed; the clonality of the B-cell population is identified and finally a detailed composite phenotype is produced which includes the identification of tumour specific markers.

i) Identification of cell lineage

The first B-cell antigen to be described was B1 (now known as CD20) and it was first used to characterise malignant cells from lymphoma and leukaemia patients in 1981 ( 7 ). Since then many more antigens have been identified which are expressed exclusively on B-cells, including CD19, CD22, CD79a, CD79b and other components of the B-cell receptor complex. The most commonly used marker for flow cytometric B-cell gating is CD19 as this marker is present at all stages in the life-cycle of a Bcell, from early progenitors through to terminally differentiated plasma cells. An example of gating with CD19 can be seen in Figure 4. Some B-cell lymphomas show down-regulation of CD19 and this can make detection of these cell populations problematic using CD19 alone, especially in the context of a background of normal B-cells. Low levels of CD19 expression are most commonly seen in follicular lymphoma but can be seen occasionally in most types of B-cell neoplasm ( 8 ). To overcome the gating issues caused by decreased expression it is advisable to use combinations of gating markers, for example CD19 and CD20, as shown in Figure 5. As with gating , the assessment of developmental stage can be enhanced by using markers in combination. A good example is assessment of CD10 in combination with CD20. Unlike progenitors, neoplastic germinal centre-derived B-cells have strong CD20 expression and so the two populations can be distinguished if the correct antigens are examined together. The use of CD20 and CD10 in combination to demonstrate a normal regenerative B-cell population is seen in Figure 6.

iii) Presence of clonal populations

In the vast majority of presenting B-cell neoplasms there will be a large population of monoclonal cells expressing a single kappa or lambda light chain. Light chain expression is straight forward to assess using flow cytometry once circulating immunoglobulin has been removed by washing. A monoclonal population will result in perturbation of the balance of cells expressing kappa and lambda light chains, resulting in a kappa:lambda ratio outside the reference range. Examples of monoclonal populations can be seen in Figure 7. Light chain restriction, however, is Improvements in the sensitivity and specificity of flow cytometric assays have made it possible to detect small populations of neoplastic cells. In samples taken following treatment or in staging marrow aspirates the neoplastic cells may be mixed with normal progenitor and mature polyclonal B-cells. In these instances reliance on assessment of the kappa:lambda ratio is not sufficient as there may not be a large enough number of abnormal cells present to take the ratio outside the normal range.

An example of an occult monoclonal population can be seen in Figure 8. In these cases it is essential to have detailed knowledge about the immunophenotype of the neoplastic cells so they can be distinguished from normal B-cells without the use of clonality. to distinguish these neoplastic cells from all other populations based on differences in antigen expression in both presentation and follow up specimens ( 10 ).

For some antigens, these differences will be positivity compared to negativity. For others, it is intensity of expression that is altered and the quantitative nature of flow cytometry data can be a real advantage over other phenotyping techniques where intensity is much harder to assess.

A detailed immunophenotype is not only diagnostically important but also generates information which can be prognostically important. Expression of certain antigens, for example CD38 in CLL ( 11), have prognostic implications. Antigens within the extended phenotype will also influence decisions regarding treatment with antibodybased regimens such as Alemtuzamab (anti-CD52) or Rituximab (anti-CD20).

Treatment itself can affect the level of expression of certain markers, for example Bcells in a patient that has been treated with Rituximab will show significantly weaker CD20 expression than prior to treatment ( 12). This is important to consider in the construction of panels that may be used post-treatment. Any markers to be used in an MRD setting, especially those that will be relied upon for gating, should ideally be 'therapy proof'.

Flow cytometric characteristics of individual B-cell malignancies

The immunophenotypes of some of the most commonly seen mature B-cell neoplasms can be seen in Table 1. CLL is perhaps the most immunophenotypically well-defined of all the mature B-cell lymphoproliferative disorders. Due to the high levels of circulating disease present in most cases, access to material for flow cytometric analysis is straight-forward and in many instances a diagnosis can be made on the basis of a peripheral blood sample alone. The immunophenotype of a typical case of CLL can be seen in Figure 9. There are also a number of antigens which have been identified as prognostic factors in CLL, including CD38, ZAP70 and CD49d ( Flow cytometry plays a crucial role in directing FISH or molecular testing, especially for CCND1/IGH@ or t (11;14) in mantle cell lymphoma. Particularly in peripheral blood samples, it is critical that atypical cases are not mis-classified as CLL or as a CD5-negative BLPD without proceeding to molecular analysis. Recently, CD200 has been identified as an antigen that can be used to distinguish between MCL and CLL.

Although atypical cases exist, in general the neoplastic cells in MCL are CD200 negative and those in CLL are CD200 positive ( 23), as shown in Figure 12. As well as analysing antigens on the cell surface, it is also possible to detect intra cellular proteins by flow cytometry. BCL2 expression can distinguish follicular lymphoma cells from other CD10+ events, namely normal progenitors and nonmalignant germinal centre cells ( 25 ). It is therefore useful for differentiating between case of neoplasia and reactive hyperplasia ( 26). An example is depicted in Figure 13.

Some other B-cell lymphomas still remain challenging for flow cytometrists.

particularly where there is no single disease specific phenotype which characterises the entity and there is great heterogeneity between cases, This is especially the case for diffuse large B-cell lymphoma (DLBCL). In the last few years, gene expression profiling has generated large amounts of data about the neoplastic cells of many haematological malignancies, some of which has been applied to improve diagnostic

Flow cytometric approach to the classification of T-cell and NK-cell neoplasms.

The analysis of suspected T-cell and NK cell derived tumour follows the same steps which have been described above for B-cell malignancies.

iv) Detailed immunophenotype

The large number of normal sub-populations present in the T-cell and NK cell lineages makes identification of phenotypically abnormal population more challenging than for the B-cell lineage. Even so, there are disease specific phenotypes established for some disease entities and these can be used to classify abnormal populations.

Treatment with therapeutic antibodies is also a possibility for some T-cell neoplasms and assessment of expression of CD52 (which is needed for treatment with Alemtuzamab) and CD25 (against which Daclizumab is directed) is an important part of the extended phenotyping process.

Flow cytometric characteristics of individual T-cell malignancies

The situation described for the B-cell neoplasms is similar in the T-cell lineage disorders. There are a proportion of entities which are immunophenotypically well- This can generate sampling issues and reduce the number of unfixed specimens received that are suitable for flow cytometric analysis. Down-regulation of T-cell markers is common in T-cell malignancies and this can make lineage identification difficult, with cells having a "null" phenotype ( 37 ). In other cases, for example Anaplastic Large Cell Lymphoma (ALCL), the neoplastic cells are large and can be low in number, meaning adaptation of gating strategies is necessary ( 38 ).

F

Advantages and disadvantages of flow cytometry as a technique

Analysis of flow cytometric data is an objective process involving both qualitative and quantitative measurements. Results are not influenced by the same levels of subjectivity as are present in morphological evaluation. Cells are defined by their pattern of antigen expression and in most cases lineage identification is straight forward. The multiparametric nature of flow cytometry data allows numerous antigens to be assessed simultaneously, making it possible to examine antigen expression and co-expression on specific populations. Large numbers of cells can be analysed very rapidly, with most laboratories routinely collecting data on a minimum of 100,000 cells at presentation and between 500,000 and 1,000,000 cells in an MRD setting. Results can be delivered more quickly than histological analysis as samples do not need to be subjected to the lengthy fixing and processing stages necessary for cytomorphological and immunohistochemical analysis.

The data generated by flow cytometry is only as reliable as the sample from which it results. For tissue biopsies, especially those which are core or fine-needle in origin, and bone marrow aspirates, lack of neoplastic cells in the sample does not necessarily constitute absence of disease ( 39 ). Many neoplastic nodes or masses can show lack of uniform involvement and inaccurate sampling can lead to a biopsy which is devoid of malignant cells but also unrepresentative of the total mass. Accuracy can be improved by performing biopsies with CT guidance but it is still important to consider that a normal result on a minimally sized biopsy does not exclude a diagnosis of lymphoma.

In bone marrow aspirate samples, lymphoma cells can aggregate in clusters which adhere to the trabeculae and are therefore not sampled when an aspirate is taken. The quality of the aspirate sample can also be affected by dilution of the collection with peripheral blood. In both these cases the samples will not be representative of the situation in vivo. diagnosis cannot be reached then a surgical biopsy will be inevitable causing a delay in treatment which may, in some cases, affect patient outcome. Flow cytometric data can greatly assist in reaching a diagnostic conclusion in these cases ( 41 , [START_REF] Meda | Diagnosis and Subclassification of Primary and Recurrent Lymphoma[END_REF] ) with studies showing the sensitivity of flow cytometry in the context of FNAs been equal

to or greater than that of PCR ( 43). Knowledge regarding cellular composition of the sample, presence of monoclonal populations and presence of phenotypic abnormalities will facilitate the most efficient use of the limited cells or tissue samples which are available by directing further testing. The integration of flow cytometric analysis of cellular aspirate and histological evaluation of a needle-core biopsy is a promising approach to improving the applicability of minimally invasive diagnostic techniques.

. The need for fresh tissue or cells restricts the number of biopsies that can be analysed by flow cytometry. In many centres it is not routine practice to acquire unfixed tissue even when lymphoma is suspected clinically. This is despite the advantages of having access to flow cytometric data, improved molecular analysis and, in some cases, improved fixation and processing of routine histological blocks. Hopefully, this will be improved by the centralisation of diagnostic services for haematological malignancies which will provide wider access to the full range of diagnostic techniques.

Improvements in the sensitivity and specificity of flow cytometric assays have made it possible to detect small populations of neoplastic cells in a background of normal mature and progenitor cells. Several groups have shown the presence of occult monoclonal B-cell populations in peripheral blood samples from normal individuals ( 44 , [START_REF] Nieto | Increased frequency (12%) of circulating chronic lymphocytic leukemia-like B-cell clones in healthy subjects using a highly sensitive multicolor flow cytometry approach[END_REF] ). Flow cytometric analysis has also been shown to be more sensitive than histological assessment in the evaluation of bone marrow samples referred for staging of a known mature B-cell neoplasm ( 16 , [START_REF] Talaulikar | Staging bone marrow in diffuse large B-cell lymphoma: the role of ancillary investigations[END_REF] ). The clinical implications of finding these small clonal populations are still unclear. The presence of bone marrow involvement elevates the disease stage that is assigned to the patient and this has a direct impact on treatment decisions. Further work needs to be carried out to assess the impact which very low levels of bone involvement have on patient outcome, so that new guidelines can be developed for staging in these situations. acknowledging that no intervention may be required in many of these cases.

The future of flow cytometry in the analysis of mature lymphoid malignancies

The future of flow cytometry will undoubtedly bring cytometers with a greater numbers of lasers and parameters and this will be accompanied by new choices of fluorochromes. As our knowledge of the haematopoietic system and the cells within it continue to increase, there will also be new antigens to be investigated for their utility in the development of more disease-specific phenotypes. The vast majority of antibodies currently available are directed against antigens expressed on the cell surface but as techniques improve there will be an increase in reagents to examine the internal proteins.

As the complexity of flow cytometry increases, it will become more important for diagnostic and MRD approaches to be standardised. The Bethesda Consensus ( 4 , [START_REF] Greig | Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: recommendations for training and education to perform clinical flow cytometry[END_REF] produced recommendations on the use of reagents and the reporting of immunophenotyping results but there is a growing need for standardisation of antibody panels. The Euroflow Consortium is a group of diagnostic laboratories and biotechnology companies from across Europe ( 50 ). The Euroflow project aims to develop specific flow cytometric tests for the diagnosis and monitoring of haematological malignancies. A standardised method for testing of potentially neoplastic samples will increase reliability and reproducibility of diagnostic assays.

  morphological assessment for diagnosis, other classifications have been defined by the presence of a specific genetic abnormality or a specific immunophenotype. Flow cytometric immunophenotyping is central to the diagnostic strategy laid out for many of the mature lymphoid neoplasms. The International Consensus Recommendations on the flow cytometric immunophenotypic analysis of haematolymphoid neoplasia (Bethesda, 2006) suggests that flow cytometric immunophenotyping is an important part of the investigation in a range of clinical situations, including organomegaly, tissue masses, lymphocytosis and cytopenias ( 4 ).

  be made carefully to minimise the need for compensation correction. The emission data collected indicates presence or absence of antibodybound fluorochromes. This generates information about the antigens present on the cell to produce a composite immunophenotype.

  Figure 1.

  Flow cytometry data is usually analysed by the application of regions and gates, allowing identification of a population of interest and exclusion of other events in further analysis. Gates can be a single region or a combination of multiple regions.

Figure 1 .Figure 2 .

 12 Figure 1. Graphical representation of flow cytometric data. A is a single parameter histogram for CD3 expression in a normal peripheral blood sample. B and C show the same data in double parameter plots; B demonstrates CD3 expression as compared to side scatter and C shows CD3 vs CD8 expression.

  Application of flow cytometryEarly flow cytometers were used primarily for the enumeration of cells and, indeed, the same technology is still used in modern haematology full blood count analysers.Classification of cells became possible following the development of the monoclonal antibody ( 5 ). By the early 1980's a large number of monoclonal antibodies to human leucocyte antigens had been produced and this revolutionised the field of flow cytometry, allowing flow cytometric immunophenotyping to be used routinely. The last decade has seen major advances in flow cytometric instrumentation and also a substantial increase in the availability of antibodies and fluorochromes. Once confined to research institutions, cytometers with 8 (or more) colours are now available as clinical instruments for use in diagnostic laboratories. It is now possible to routinely assess 10 (or more) parameters simultaneously ( 6 ). The greater the

Figure 3 .

 3 Figure 3. Demonstration of the use of combined regions to interrogate flow cytometric data. A shows a CD3 vs SSC plot for a normal PB sample. A region (P1) has been drawn around the CD3+ events. In B only cells which fall within P1 are shown and a number of subpopulations can be seen. Two further regions have been drawn which apply only to cells in P1: P2 has been created around the CD4+ CD8-events and P3 has been drawn around the CD8+CD4-events. P2 and P3 can then be combined in numerous ways to examine the different sub-populations present. C, D and E show how applying a gate using each of the AND, OR and NOT operators can alter the events which are shown.

Figure 4 .

 4 Figure 4. Plot A shows B-cells identified by gating the CD19+ events. CD19 is expressed at all stages of B-cell development from progenitor to plasma cell. Plot B shows that the gated cells do indeed consist of a mixture of Kappa positive and Lambda positive mature B-cells and surface immunoglobulin negative B-cell progenitors.

  Once the B-cell population has been gated as described above, it is possible to obtain information about the stage of cellular development by assessing antigens which have variable expression throughout the life-cycle of the B-cell. One of the most important markers in this respect is CD10. Progenitor B-cells are CD10 positive and express intracellular CD79a/b. They lack CD20 and have no surface immunoglobulin. As the cells mature, expression of CD10 decreases and CD20 increases and the cells gain surface immunoglobulin. Knowledge about the normal differentiation pathways of cells can be used to identify cells which fall outside of the normal pattern.

A

Figure 5 .

 5 Figure 5. Demonstration of the gating problems encountered when neoplastic B-cells have weak CD19 expression. Row A shows B-cell gating using CD19 alone. The B-cells comprise 1.9% of total leucocytes and have a slightly skewed kappa:lambda ratio. In Row B, the same sample is gated using CD19 and CD20 in combination. The B-cell proportion increases to 3.8% and there is a clearer lambda positive monoclonal component visible. The final CD19 vs SSC plot shows the B-cells in blue and illustrates the number of cells excluded from the initial gate when CD19 was used alone.

Figure 6 .

 6 Figure 6. Using CD10 and CD20 to assess developmental stage of B-cells. The plots in Row A show regenerating B-cells in the bone marrow of a patient following therapy. There are large numbers of progenitor B-cells present which are CD10+CD20-. As the B-cells mature there is a gain of CD20 expression and a loss of CD10 positivity. This pathway of maturation is depicted by the blue arrow in the second plot.The plots in Row B show normal B-cells in a reactive lymph node biopsy. The majority of the cells have no CD10 expression and are CD20+. A small population of CD10+ events can be seen but these are not progenitors as they have strong CD20 expression. These are germinal centre B-cells. The blue arrow demonstrates how mature B-cells gain and then lose CD10 expression as they pass through the germinal centre and then exit again.

  of clonality and there are examples in the literature of nonclonal reactive B-cell populations which exhibit light chain restriction ( 9 ).

Figure 7 .Figure 8 .

 78 Figure 7. Monoclonal populations in samples from presentation lymphoma cases The sample represented in Plot A has a kappa positive neoplasm and no normal B-cells remain. In Plot B, although there are some normal polyclonal cells still present, there is still an obvious lambda positive population.

  Once a population of interest has been successfully gated it is possible to examine the immunophenotype of those cells in detail. Each sub-population of normal cells has a specific immunophenotype and abnormal phenotypes characterise many of common disease entities. If a disease-specific phenotype has been identified then it is possible

  As treatment regimens for CLL have become more effective it has become more important to be able to detect minimal residual disease (MRD) following therapy. It has been shown that presence of MRD predicts a poorer outcome(15) and that flow cytometry can be more effective in this setting than conventional PCR(16). There is now an internationally standardised approach to assessing MRD by flow and the assay can detect CLL cells when they represent as few as 0.01% of total leucocytes ( 17 ).

Figure 10

 10 Figure 10 demonstrates how the different B-cell populations present in a posttreatment bone marrow sample can be discriminated from each other.

Figure 10 .

 10 Figure 10. MRD analysis in CLL The plots above show analysis of a bone marrow with a minimal CLL population and demonstrate how the use of a few carefully selected antigens can discriminate between CLL cells (red), normal mature B-cells (green) and B-cell progenitors (blue). B-cells have been gated in a CD19 vs SSC plot and are 1.6% of total leucocytes. CLL cells represent 0.32% of total leucocytes.

Figure 9 .

 9 Figure 9. Typical immunophenotype of CLL.The plots show a BMA sample from a patient at presentation. B-cells are gated using CD19 and the subsequent plots who that the cells have a typical CLL phenotype.

Figure 11 .

 11 Figure 11. Immunophenotype of typical HCLThe above plots show analysis of a peripheral blood sample from a patient with a large population of neoplastic B-cells. These have a typical phenotype for HCL with strong CD103, CD25, CD11c and CD22 expression; note the larger than usual SSC and FSC which is caused by the projections on the cell surface rather than the size of the cell.

Figure 12 .Figure 13 .

 1213 Figure 12. Use of CD200 to discriminate CLL from MCL Plot A shows gated B-cells from a BMA sample involved with CLL. The cells demonstrate expression of CD23 and CD200. Plot B displays B-cells from a patient with MCL. The cells have a typical MCL phenotype and are negative for both markers. CD200 is extremely useful in cases of MCL which exhibit atypical CD23 expression.

  i) Cell lineage There are numerous antigens which indicate T-cell (CD2, CD3, CD5 and CD7) or NK-cell (CD2, CD16, CD56) lineage. Surface expression of CD3 occurs during T-cell maturation, with lymphoblasts showing only cytoplasmic CD3 positivity. CD2, CD5 and CD7 expression can be seen at all stages of development, although minor subpopulations which lack expression may exist in samples from healthy individuals. Unfortunately, the markers that are commonly relied upon for identification of T-cell lineage in normal cells often show weak or completely absent expression in neoplastic populations. As with the B-cell neoplasms, utilisation of multiple antigens for gating can improve identification but populations which are negative for several T-celllineage-related antigens will still pose a problem and are most likely to be misclassified. ii) Developmental stage Mature T-cells are defined by the presence of surface CD3 expression. T-cell lymphoblasts are negative for surface CD3 and positive for cytoplasmic CD3 and also express CD1a. Following TCR gene rearrangement, the cells become surface CD3 positive and exit from the thymus into the peripheral circulation as naïve T-cells.There are a vast number of mature T-cell sub-populations which can be present in a normal sample, including regulatory, effector and memory populations. Mature Tcells are often split crudely into helper (CD4+) and cytotoxic (CD8+) components and large variations in the ratio of these populations can be seen in normal and reactive not definitive of the presence of a malignant population, skewing of the CD4;CD8 ratio warrants further investigation.iii) Presence of clonal populationsPresence of a clonal T-cell population results in expansion of a group of cells with a restricted phenotype. TCR repertoire can be assessed by flow cytometry using antibodies which recognise products of V and D domains following VDJ recombination. The most commonly used is V-β analysis and, although the gold standard for this analysis is PCR, flow cytometric evaluation has been shown to be an efficient substitute in both peripheral blood and lymph node specimens ( 33 ,[START_REF] Salameire | Efficient characterization of the TCR repertoire in lymph nodes by flow cytometry[END_REF] ). In a normal sample there is heterogeneous expression of V-β subtypes, resulting in a population of T-cells expressing a large variety of V-β family members. A clonal Tcell population will have a far more homogeneous pattern of expression. As with light chain restriction in B-cell populations, non-malignant reactive populations can appear clonal and oligoclonality is seen in healthy individuals as age increases (35). Samples which contain small numbers of T-cells, such as those from aplastic patients, can falsely appear to be clonally restricted. Clonal populations can also be hidden amongst a background of normal polyclonal cells so results must be interpreted carefully.

Table 2 .

 2 Examples of immunophenotypes for some of the most commonly seen mature T-cell neoplasms.

Figure 14 .

 14 Figure 14. Typical phenotype of T-PLL. T-cells have been gated using CD3 vs SSC and then a secondary gate (not shown) has been used to select the T-PLL cells based on CD4 positivity.

Figure 15 .

 15 Figure 15. Typical phenotype of T-cell large granular lymphocytic leukaemia Cells gated using CD3 are CD8+, CD4-, TCRαβ+, CD57+ and CD16+.

Figure 16 .

 16 Figure 16. Typical phenotype of the neoplastic cells in Sezary syndrome CD3+ gated cells show the characteristic CD4+, CD8-, CD2+, CD7-, CD26-phenotype

Increasingly, centres are

  moving away from full excisional biopsies of lymph nodes, favouring instead less invasive procedures such as fine needle aspiration (FNA) or needle core biopsy ( 40 ). Although these procedures are favourable for the patient in terms of discomfort and recovery time, the resulting sample offers far fewer cells and greatly increases the diagnostic challenges. If the paucity of the sample is such that a

Flow cytometric acquisition generates

  large amounts of data that needs to be analysed by highly experienced operators. These individuals are trained to recognise subtle changes in antigen expression to identify abnormal cell populations ( 49 ). Infinicyt (Cytognos, Salamanca) is a newly developed software program for flow cytometric analysis which has been created as part of the Euroflow project and approaches evaluation of data in a completely new way ( 51 ). One of the aims of this development program which will allow automated analysis of data by comparing test data with a database of normal and malignant samples ( 52 ). This would allow high levels of reproducibility in flow cytometry analysis.Recently of a new flow technology has been introduced, (ImageStream Amnis Corporation, Seattle), which combines flow cytometric fluorescence measurements with digital images. This allows simultaneous assessment of surface antigens and morphology ( 53 ). This multispectral imaging technique has been successfully used to assess cells of the haematopoietic system ( 54 ) and offers interesting possibilities for the investigation of mature lymphoid malignancies.Concluding remarksOver the last decade, flow cytometry has been established as a critical tool in the diagnosis of haematological malignancies. This review has summarised the contribution the technique currently makes to the diagnosis and management of mature lymphoid neoplasms. Recent improvements in technology are beginning to yield assays with much higher levels of disease specificity and sensitivity which can be applied in routine diagnostic laboratories. Many types of lymphoma can now be correctly classified by flow cytometric analysis independent of other diagnostic modalities which allows much more robust integrated diagnostic protocols to be developed. The development of highly sensitive techniques will play an increasing role in disease monitoring through and beyond treatment. Flow cytometry now has the potential to improve substantially the diagnosis and monitoring of patients with lymphoma. To realise this potential fully will require a change in the way specimens are acquired and in the organisation of specialist haematopathology service. anaplastic large cell lymphoma. Arch. Pathol. , C.E. et al. Generation of flow cytometry data files with a potentially infinite number of dimensions. Cytometry A 73, 834-846 (2008). 52. Pedreira, C.E. et al. A probabilistic approach for the evaluation of minimal residual disease by multiparameter flow cytometry in leukemic B-cell chronic lymphoproliferative disorders. Cytometry A 73A, 1141-1150 (2008). 53. Zuba-Surma, E.K., Kucia, M., Abdel-Latif, A., Lillard, J.W. & Ratajczak, M.Z. The ImageStream System: a key step to a new era in imaging. Folia Histochem. Cytobiol 45, 279-290 (2007). 54. McGrath, K.E., Bushnell, T.P. & Palis, J. Multispectral imaging of hematopoietic cells: where flow meets morphology. J. Immunol. Methods 336, 91-97 (2008).

Table 1 .

 1 ). Examples of immunophenotypes for some of the most commonly seen

mature B-cell neoplasms.

  defined and these are shown in Table2. These entities tend to be those with high levels of circulating disease which means that large numbers of cells are usually available for analysis in a peripheral blood sample. Examples of flow cytometry plots for T-cell Prolymphocytic Leukaemia (T-PLL), Large Granular Lymphocyte

	Leukaemia (LGL) and Sezary syndrome (SS) can be seen in Figures 14, 15 and 16
	respectively. Again, a well-defined phenotype can be used to develop a successful
	MRD assay.
	Disease entity	Typical phenotype	Atypical expression
	T-cell Prolymphocytic	CD3+, CD2+, CD7+, CD5+,	Atypical cases can show a
	Leukaemia (T-PLL)	CD1a-, CD52++, CD4+, CD8-	distinct populations which
			co-expresses CD4 and
	o r	CD8, a characteristic not seen in other T-cell
	malignancies. There are also rare case which are CD8+, CD4-Atypical cases include examples which are CD4+, TCRαβ+ and also CD8+, TCRγδ expressing variants. Atypical cases include i TCRαβ, CD5wk/-, CD7wk/-, CD57+, P e e CD3+, CD8+, r CD16+ R e v Sezary syndrome (SS) CD3+, CD2+, CD5+, CD4+, Large Granular Lymphocyte Leukaemia (LGL) e down-regulation of T-cell CD7-, CD26-, TCRαβ antigens other than CD7, most commonly CD2 and w also rare examples with
			CD8 expression. Until
			recently, lack of CD26
			expression was thought to
			be a feature of all Sezary
			cells but current evidence
			suggests that only 60% of
			SS cases show CD26
			negativity and that
			expression is also lacking
			in a third of mycosis
			fungoides cases ( 36 ).

  individuals, requires further clarification but recent data suggest that the rate of progression may be relatively low. ( 47 ,[START_REF] Rawstron | Clinical and diagnostic implications of monoclonal B-cell lymphocytosis[END_REF] ). As flow cytometric technology continues to improve, it will become possible to find smaller populations of cells in all types of samples. It is critical that these results are considered in the clinical context to derive the maximum value from them and to ensure the best care for the patient,

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The clinical significance of the presence of occult monoclonal populations, which are increasingly detected in healthy

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


James Campbell
email: jim.campbell@integrare.es

Judith A Oulton

Barbara Mcpake

Jim Buchan

Increasing access to 'free' health services: are health workers not a missing link?

come

Increasing access to 'free' health services: are health workers not a missing link?

In September 2009 in front of a global audience in New York, leaders from Burundi, Ghana, Liberia, Malawi, Nepal and Sierra Leone made public commitments to increase access to free health services in their respective countries, particularly for pregnant women and children younger than 5 years [START_REF]Outcome document of the meeting of the Taskforce on Innovative International Financing for Health Systems[END_REF].

The commitments reflected, in part, a desire to: 'accelerate action towards universal access to primary health care' as laid out in the World Health Assembly Resolution of May 2009 [START_REF][END_REF];

specifically address poor maternal and child health outcomes by focusing on women and children, and in turn; enhance progress against the health-related Millennium Development Goals.

A common theme within the country commitments is the removal of financial barriers to access health services: in particular, user-fees at the point of service. This is not a new approach and has been implemented in both high-and low-income countries; albeit with varying degrees of success.

In a 1945 address to Congress on the health of the Nation, Harry S. Truman recognised that "the principal reason why people do not receive the care they need is that they cannot afford to pay for it on an individual basis at the time they need it". His resulting policy highlighted "that financial barriers in the way of attaining health shall be removed; that the health of all its citizens deserves the help of all the Nation" [START_REF] Truman | Special Message to the Congress Recommending a Comprehensive Health Program[END_REF]. The logic from 1945 is as relevant today.

Published evidence, including reports from the World Health Organisation (WHO) [START_REF] Who | Women and health : today's evidence tomorrow's agenda[END_REF], the WHO Commission on Social Determinants for Health (CSDH) [START_REF]Closing the gap in a generation: health equity through action on the social determinants of health[END_REF], and articles in The Lancet [START_REF] Yates | Universal health care and the removal of user fees[END_REF] and in the Bulletin of the World Health Organisation [START_REF] Lagarde | The impact of user fees on health service utilization in low-and middleincome countries: how strong is the evidence?[END_REF], indicates that user-fees are one of the most regressive forms of health financing and a barrier to reducing maternal and child mortality.

"The policy imposition of user fees for health care in low-and middle-income countries has led to an overall reduction in utilization and worsening health outcomes. Upwards of 100 million people are pushed into poverty each year through catastrophic household health costs. This is unacceptable" CSDH, 2008.

User -fee removal is generally considered a positive policy objective and should enable many individuals in lower-income quintiles to enjoy new and improved access to health services.

With such notable publications and health journals disseminating the case for removal of user-fees it is easy to grasp the political appeal to announce increased access to 'free' health services: there are indications that other countries will make similar commitments in 2010. But, whilst demand-side interventions are logical, and a focus on women and children absolutely essential [4] [8], there remain some concerns that the supply-side characteristics of a national health system, especially the dynamics of the health workforce, are a missing link to achieve the policy goals of universal health care. What is the net benefit of increasing access to 'free' health services if there is no qualified health worker available to provide care, or where you may queue all day only to be afforded an ineffectual consultation which undermines respect, trust, privacy and confidentiality? Such are the realities in many low-income countries, particularly in rural and remote areas, where health workers are in drastically short supply, and often over-burdened and/or under-resourced.

F
The potential for trade-off between access to a health facility and access to quality health care raises a number of issues. 'Access' and 'coverage', with or without a 'universal' prefix, are not meaningful concepts without some reference to quality and safety: increasing access does not necessarily correlate with improved care. Rather the realities of budget limits in low-income countries, even after donor resources are taken into account, suggest that universal access to a free public service may only be achieved at a basic level of quality. Many countries have identified an 'essential' or 'basic' package of care to which universal access is targeted in recognition of this. Yet even these restricted packages may require additional financial allocations if they are to be genuinely 'free'.

Where these issues are at play the trade-off between coverage and quality as countries seek to 'scale-up' and achieve universal coverage has critical implications for outcomes across the breadth of the health system [START_REF] Mangham | Scaling up in international health: what are the key issues?[END_REF].

Evidence suggests that health service utilisation rates tend to increase dramatically once financial barriers are removed [START_REF] Who | Women and health : today's evidence tomorrow's agenda[END_REF] [10] [START_REF] Gilson | Removing user fees for primary care in Africa: the need for careful action[END_REF] [12] [START_REF] Pearson | Issues Paper: The Case for Abolition of User Fees for Primary Health Services[END_REF]. The existing stock, competencies and distribution of the health workforce may not be able to respond to increased demand for services whilst maintaining and improving the quality of the services it provides. This is particularly relevant in the 6 countries that made public commitments to free health care: each is faced with critical shortages in their national health workforce. In a standardised, comparative survey conducted by WHO for the 2006 World health report each country was categorised as a 'crisis' country when benchmarked against the threshold of a minimum of 2.3 Doctors, Nurses and Midwives per 1,000 population in order to deliver essential services [START_REF] Who | [END_REF] (see FIGURE 1).

Whilst the science employed by WHO to arrive at the 2. (2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)'. GHWA has the laudable Vision that 'All people everywhere will have access to a skilled, motivated and supported health worker, within a robust health system'. WHO hosts the GHWA and is demonstrating its leadership role; in both the 2006 WHR and its 2010-2015 HRH strategy [START_REF]Strategy of WHO Human Resources for Health Team[END_REF] it has called for and committed to increasing strategic intelligence on the national health workforce. A revised vision "to ensure all people have equitable access to an adequately trained, competent, motivated and supported health workforce so they attain the highest possible level of health" is included in the latest WHO strategy. However, if both GHWA and WHO wish to enhance their respective performance and accountability frameworks in relation to their vision statements it will require new thinking that moves away from a workforce density focus to consider skills, distribution and performance. This will require innovation and action on the metrics and science to measure and evaluate whether countries are making progress in increasing "equitable access", especially for women and children, to a "skilled" or "competent" health workforce.

One option for the 6 countries to promote national objectives whilst simultaneously generating knowledge for other WHO Member States is to include particular emphasis on strategic intelligence of the health workforce. As the countries implement their 2009 policy commitments it will be beneficial to conduct prospective evaluation of the risks and supporting measures needed in removing user-fees, with an understanding of the supply/demand dynamics within the individual health labour markets and the need to create a baseline from which to monitor trends [START_REF] Mcpake | Freeing up healthcare: A guide to removing user fees, Save the Children[END_REF]. This will include determining the geographical and socio-economic dynamics of 'access to a .... health worker' as a proxy for 'access to health services'.

'Workforce Surveillance' is an evolving conceptual and scientific approach to support such strategic intelligence. Its current working definition is: "the routine, dynamic and systematic collection and analysis of HRH data for the provision of rigorous evidence and information which leads to action" and Prevention (CDC) to review the dynamics of the American health workforce [START_REF] Gotway Crawford | Perspectives on Public Health Workforce Research[END_REF].

A surveillance exercise in each country could validate a Concentration Index (CI) of the health workforce (by cadre and stratified by sex) to provide additional evidence for data-driven decisionmaking. The CSDH recommends a CI as a health equity surveillance measure [START_REF]Closing the gap in a generation: health equity through action on the social determinants of health[END_REF]; a WHO Expert Group has encouraged its adoption in the design, implementation and evaluation of initiatives to promote rural deployment and retention [START_REF] Huicho | Increasing access to health workers in underserved areas: A conceptual framework for measuring results[END_REF], and; the World Bank has recently adapted CI to review the geographical imbalances in health workforce distribution in seven African countries [START_REF] Benin | Reducing Geographical Imbalances of Health Worker Distribution: What works, what does not and why? A Labour Market Perspective of Urban-Rural Health Disparities, Policy Solution and Experiences in Sub-Saharan Africa[END_REF].

This would identify actual and potential gaps in the workforce and its impact on service provision, especially in underserved areas, and enable new policy discussions on the priority engagement, deployment and training of health workers to achieve maximum benefit from the available or planned financial resources.

"Documenting and mapping exclusion from various essential services can be a useful tool for planning and can also serve as a baseline against which to measure progress in coverage"

WHO, Women's Health (2009).

Nepal, for instance, has very recent evidence (December 2009) of how staff shortages, gender, knowledge and competencies continue to be contributing factors to maternal mortality and morbidity [START_REF] Suvedi | Nepal Maternal Mortality and Morbidity Study 2008/2009: Summary of Preliminary Findings[END_REF].

"The well known issues of staff availability and transfer continue to negatively affect the provision of skilled care, particularly Comprehensive Emergency Obstetric Care , at facilities built and equipped to provide this level of service, highlighting the vulnerability of systems"

Nepal -Maternal Mortality and Morbidity Survey.

Whilst there is expectation that Nepal's Aama programme to remove user-fees will reduce barriers to access there is recognition that it will not immediately alter health workforce dynamics. Active workforce surveillance to routinely capture stock, competencies and distribution, including a regularly updated CI, combined with improved time-to-event analyses of workforce trends (i.e. on stability and retention rates and their underlying causes) would add new insights to address this. If these insights were subsequently correlated with findings from the maternal mortality survey, it this failing: "It is not enough, however, that we have them....They should be located where their services are needed" [START_REF] Truman | Special Message to the Congress Recommending a Comprehensive Health Program[END_REF]. Like many since, he recognised that promoting free health care through the removal of user-fees without addressing health workforce distribution is unlikely to improve access to quality care services.

If we follow the premise that demand for health services is in part related to their perceived quality, then actions must be taken to strengthen the supply-side elements. And if removal of user-fees is targeted to those individuals who have been economically disadvantaged in the past, especially women and children, then the priority must be to strengthen the health workforce in the areas in which they live. This requires new strategic intelligence to prioritise HRH interventions, especially in rural and remote areas: workforce surveillance to enable prospective evaluation , evidence and action is one suggested approach.

[

  18]. Moving beyond an observational nature it seeks to capitalise on existing HRH metrics, monitoring and evaluation; supplementing this with context-specific real-time intelligence on health workforce trends from all available sources (including the public sector, professional associations and communities), and; employing inter-disciplinary and multi-disciplinary research methodologies to generate new insights. Whilst research and application is primarily in the context of strengthening HRH in low-and middle-income countries[START_REF] Campbell | Taking forward action on Human Resources for Health in Ethiopia, Kenya, Mozambique and Zambia: Synthesis and measures of success[END_REF] , workforce surveillance is also Office of Workforce and Career Development at the Centres for Disease Control

Deleted: [ 5 ][ 21 ]

 521 clearer pathway on implementing part 4 of the Consensus on Maternal, Newborn and Child Health (MNCH) -'to deploy skilled and motivated health workers in the right place at the right time' [24]. As the 6 countries implement their respective programmes of 'free' health care, with support from various international development partners (including a new Centre for Progressive Health Financing launched in March 2010 by the UK Department for International Development [25]) , we return again to Harry S. Truman when he spoke 65 years ago. One of the key elements he recognised was that access to care, irrespective of financial barriers, was multi-faceted. He argued that health workers -doctors, dentists, public health and hospital administrators, nurses and other experts -are 'one of the most important requirements for adequate health services'. At the time Truman spoke some rural districts in America had zero Doctors per 1,000 population. His commentary recognised

Figure 1 .

 1 Figures:

  -it has nonetheless acted as a catalyst to afford Human Resources for Health (HRH) a prominence in global health discourses. This includes the establishment of the Global Health Workforce Alliance (GHWA) in 2006 to support a 'Decade of Action on Human Resources for Health

	PERSPECTIVE -International Journal of Clinical Practice
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3/1,000 'crisis' threshold remains open to debate -for instance some countries with a health workforce density less than this are demonstrating progress against the health MDGs (i.e. Bhutan, China, El Salvador, Morocco, Peru) Deleted: ..