- Email: [email protected]
Synovial fluid analysis in the diagnosis of joint disease
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
Synovial fluid analysis in the diagnosis of joint disease
arthropathy where the prognosis is inversely related to delay in diagnosis.1
The properties of synovial fluid SF consists of a transudate of plasma from synovial blood vessels, supplemented with high molecular weight lipid and saccharide-rich molecules,2 produced by one of the two main types of synovial cells (type B synoviocytes derived from synovial fibroblasts). Type A synoviocytes are tissue macrophage-derived phagocytes that remove debris from the synovial fluid. SF differs from all other body fluids in that the synovium and cartilage, which are the tissues it contacts, do not have an intact surface cellular layer seated on a basement membrane. This means that the matrix of both tissues is in direct contact with and through SF allowing an homogenous biological environment to develop within the joint. Because of this it is probably better to regard SF as a semi-liquid, avascular, hypocellular, connective tissue rather than a true body fluid such as is seen in other situations (e.g. pericardial effusion). Cytological analysis of SF differs in three important ways from other body fluids. Firstly synovial joints are only very rarely affected by primary or secondary malignancies. Secondly the term “cytological analysis” of SF would be better described as “microscopy” of SF, as many of the diagnostic features found are not cellular but are particles such as cartilage, crystals and prosthetic wear particles. Thirdly the greatest diagnostic information comes from the recognition of individual cell types and their quantification. Normal SF: Is a supplemented ultra-filtrate of plasma Contains no clotting factors Is viscous Is paucicellular (<500 cells/mm3) Contains no particles Any or all of these may change with joint disease. High molecular weight clotting factors are introduced into the joint as a result of vascular leakage following trauma or in inflammation. This results in the need to anticoagulate specimens. The choice of anticoagulant can have profound influence on the final diagnosis. Anticoagulants such as EDTA will, because of their sequestering properties, dissolve complex calcium phosphates such as seen in bone-derived hydroxyapatite and calcium pyrophosphate making the diagnosis of osteoarthritis and pseudogout difficult, if not impossible. Other anticoagulants, such as sodium heparin, can crystalise out leading to the false diagnosis of crystal arthritis. Lithium heparin, by contrast, has none of these faults and is the anticoagulant of choice. As, at the time of aspiration it is impossible to predict which SF will clot or contain crystals, all SF samples should be anticoagulated with lithium heparin. The viscosity of the SF is dependent on the concentration and size of the proteoglycans it contains. Normal SF and that from non-inflammatory arthropathies is very viscous. By contrast, in inflammatory arthropathies, inflammatory mediators and/or products cause abnormal synthesis or breakdown of existing proteoglycans, reducing viscosity.
Paul Hermansen Tony Freemont
Abstract Normal synovial fluid consists of a transudate of plasma from synovial blood vessels supplemented with high molecular weight lipid and saccharide-rich molecules. This produces a paucicellular, viscous fluid, which behaves like a tissue. In primary (e.g. rheumatoid disease) and secondary (e.g. septic and crystal-induced) inflammatory arthropathies, changes occur to the cell numbers and cell type in the fluid forming the basis of a diagnostic test. The diagnostic value is enhanced by the appearance of endogenous and exogenous particles, particularly those associated with degenerative, crystal-induced, and prosthesis-associated arthropathies (e.g. fibrin, cartilage, crystals, metal and plastic). Cells and particles can be characterised under the microscope leading to a simple, inexpensive, “cytological” test for every type of joint disease.
Keywords arthritis; crystals; microscopy; polarisation; synovial fluid
Why perform synovial fluid microscopy? Histopathologists are frequently called upon to diagnose inflammatory or non-inflammatory arthropathies based on the examination of a synovial biopsy. Even experienced histopathologists can have difficulty in distinguishing inflammatory from non-inflammatory arthropathies since the latter frequently have a lymphocyte infiltrate in the synovium. Even if they can be distinguished it is usually impossible to make specific diagnoses as there are few differences that can be detected histologically between disorders in the same broad group. By contrast, synovial fluid (SF) microscopy is of greatest value in these disorders, supporting clinicians in making early and accurate diagnoses of a spectrum of inflammatory and noninflammatory arthropathies often before the full blown syndrome develops. SF microscopy, on as little as a 0.5 ml sample, permits the rapid diagnosis of joint disease (the full test takes between 10 minutes for crystal arthropathies and 2 hours for full cytological analysis), particularly disorders such as sepsis and crystal related
Paul Hermansen MSc FIBMS is Consultant BMS in the Cytology Department, Central Manchester University NHS Foundation Trust, Manchester, UK. Conflicts of interest: none declared. Tony Freemont BSc MD FRCP FRCPath is Professor of Osteoarticular Pathology, University of Manchester, Director of the Manchester MRC/EPSRC Molecular Pathology Node, Manchester NIHR Biomedical Research Centre, Manchester, UK. Conflicts of interest: none declared.
DIAGNOSTIC HISTOPATHOLOGY --:-
1
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
The traditional method of counting cells is to use a graduated pipette to dilute the SF by a known amount with normal saline, to which crystal violet has been added as a supravital stain. This cell suspension is then introduced into a haemocytometer counting chamber where the cells are manually counted on the microscope. With the advent of newer technologies for cell counting, assessing cell number has now changed. The nature of the proteoglycans makes microbore based cell counting techniques tricky but computerised chamber counting methodologies are highly practical and accurate. These automated methods, although in themselves more costly in terms of initial investment and consumables, can make SF analysis quicker and more accurate than the manual method, and, by reducing the amount of biomedical scientist time required, reduce the overall cost of the test. The number of nucleated cells contained in the SF is the primary indicator of inflammation. Where the cell count is less than 500/mm3 the patient can be assumed to have a noninflammatory arthropathy, whereas a cell count greater than 1000/mm3 indicates an inflammatory arthropathy. If between these figures, the percentage of neutrophils in a differential count distinguishes inflammatory from noninflammatory arthropathies, where more than 50% of the nucleated cells in the sample being polymorphs indicates inflammation.
Viscosity of SF can be assessed by mixing SF and a 2% solution of acetic acid leading to the formation of a white precipitate, produced by aggregation of proteins and hyaluronans (the mucin clot test). The nature and amount of precipitate varies from good to poor and reflects the quality and quantity of the protein/hyaluronan complex, such that in inflammatory joint diseases and haemarthrosis there is poor clot formation. Noninflammatory arthropathies exhibit a good mucin clot. The mucin clot test is not normally performed on routine specimens, but it is very useful in identifying surgical joint washouts or dilution with local anaesthetic, where, in the presence of a low cell count no clot will form. This is an important observation as dilution of SF with water will affect cell counts and remove monosodium urate crystals, adversely affecting diagnostics.
Why examine synovial fluid? Basic examination of SF will result in identifying combinations of cellular and non-cellular biomarkers, which can have diagnostic, prognostic and theranostic (following responses to treatment) value, in primary, secondary and tertiary care, where SF analysis can be used to influence the choice of care pathway, and assess treatment options. This valuable role for SF analysis is made possible because the absence of a cell or basement membrane barrier between synovium, cartilage and SF means that pathological changes in the tissues surrounding the joint are reflected in the volume, cellular composition and particulate load within the SF. SF is examined “fresh” requiring careful handling in the laboratory and a need to minimise cell and crystal loss, which can dramatically adversely affect diagnosis, during transport to the laboratory. Storage at 4 C is well tolerated by the SF and prolongs transport times, but even so, adequate diagnosis that even cooled samples must reach the laboratory within 48 hours of aspiration.3
Wet preparation
In order that no part of the analysis is omitted there is a sequential examination of SF specimens arriving in the laboratory. It follows four steps: Gross analysis Nucleated cell count The “wet prep” Preparation and examination of a stained cell monolayer Gross analysis relies on the visual inspection of the SF to determine colour, clarity and viscosity. Colour will change, particularly following haemorrhage such as that seen in trauma, anticoagulant use and in primary disorders of joints such as pigmented villonodular synovitis. Clarity of SF is variable and a cloudy or opaque appearance generally indicates an increase in cellular concentration, crystal content or the presence of lipid, microscopic clarification is then necessary.
The “wet prep” is an important part of the examination of SF and can often be relied upon to give a definitive diagnosis. Two preparations are made each serving a different purpose. Firstly using a glass Pasteur pipette for clarity, an aliquot of SF is removed from the container and by slowly returning the SF to the container visible particles of fibrin, cartilage or crystal aggregates can be seen in the thin part of the pipette and placed onto a microscope slide, this we call the “thick preparation”. It can be of great help if the viewing background provides contrast, for example fragments of cartilage are most easily seen against a dark background whereas prosthetic debris is most easily seen against a pale background, white laboratory coats and dark laboratory bench surfaces usually provide this contrast. It is of great importance that any pieces of fibrin clot are found, as frequently identification of crystal arthropathies with a low crystal burden relies on finding crystals trapped in the fibrin clot. A second, much thinner preparation, is made avoiding particulate matter and using only a few microlitres of SF. Coverslipping this preparation flattens the cells and allow intracytoplasmic inclusions to be identified. Large inclusions with specific refractile properties characterise a functional group of cells called ragocytes. Ragocytes have diagnostic significance (see below). “Wet preps” can be preserved for 24 hours by painting around the edge of the coverslip with nail varnish to minimise evaporation. This also helps provide additional safety features, as the SF is a potential infection hazard.
Total nucleated cell count
Thick preparation examination
A total nucleated cell count is performed in order that the degree of inflammation can be assessed and so that an optimally diluted cell suspension can be achieved for the subsequent cytocentrifuge preparation.
Crystals When screening for crystals it is of the greatest importance that aggregates of fibrin and other particles are included in the slide preparation, as it is these micro clots that will often contain the
Basic approach to synovial fluid analysis
DIAGNOSTIC HISTOPATHOLOGY --:-
2
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
signifies gout but in a quiescent or latent form at the time of aspiration. Calcium pyrophosphate dihydrate crystals (CPPD) are of a much “weaker” birefringence than the mono sodium urate crystals and appear as short rods rhomboids or cubes. As described above they have an opposite sign to urate, thus, when aligned along the fast wave of the plate they are yellow. In common with monosodium urate crystals they are often seen within cells. Pseudogout is diagnosed when the nucleated cell count exceeds 1000 cells/mm3 CPPD in a non-inflammatory setting (nucleated cell count <1000 cells/mm3) could indicate latent pseudogout but more commonly osteoarthritis or chondrocalcinosis. Pseudogout is a disease of old age. If seen in a relatively young patient, particularly a male below 50 years of age, a secondary underlying diagnosis of a systemic metabolic disease such as hypothyroidism, haemochromatosis or hypomagnesaemia should be considered. Both mono sodium urate and calcium pyrophosphate can occasionally be found in the same fluid indicating a mixed crystal arthropathy5 Figure 1. Apatite as a true crystalline form of hydroxyapatite is somewhat of a rarity in SF, only occurring as spherulites where it is then diagnostic of the peri-articular calcification known as Milwaukie shoulder.6 Although originally described in the shoulder this condition has been noted in many other joints. When apatite is seen in SF it is normally in the form of bone dust or bone debris as a result of the mechanical removal of bone or calcified cartilage, following cartilage loss, typically as a result of osteoarthritis Figure 2. If seen in an inflammatory setting destructive, erosive arthropathies such as rheumatoid or psoriatic arthritis, as well as secondary osteoarthritis, should be considered. Apatite is also commonly seen in joints in which there is loosening of joint prostheses.
crystals even though the surrounding fluid and cells may not. A recent review4 shows that there is poor consistency in crystal identification and characterisation between individuals and laboratories unless adequate training of personnel is first carried out, but we find the simple expediency of including clots in the “wet prep” reduces this problem. A list of the characteristics of the main crystal types can be seen below in Box 1. Mono sodium urate crystals (MSU) are seen in microscopic preparations as thin rod/needle shaped crystals or even collections of crystals radiating from a central point, which are described as “beach balls”. When examined by polarising microscopy they are highly birefringence, meaning they are bright white against the black background of the fully crossed polarising filters. Additional information on the nature of the crystal can be gained by adding a quarter wave retardation plate to the polarising system. This plate retards the polarised light by one quarter of a wavelength which produces a magenta coloured background. The slow wave of the plate is often marked by a double headed arrow frequently accompanied by the Greek symbol “ɤ”. In most cases the slow wave of the plate is perpendicular to the long axis of the plate holder. If the longitudinal axis of the crystal is aligned parallel to the long axis of the retardation plate and the crystal appears blue the crystal is said to have a positive sign. If the same crystal were rotated through 90 aligning the longitudinal axis along the slow wave of the plate the crystal would be yellow. This colour is characteristic of mono sodium urate crystals. With the light path setup in this way crystals of calcium pyrophosphate (the crystals seen in pseudogout) are blue (ie the opposite of urate crystals). Unfortunately some microscopes are configured such that the image is reversed, so that urate crystals would be blue and pyrophosphate yellow at the image seen through the eyepieces has been reversed. It is therefore critically important that each individual microscope is “calibrated”with a known urate specimen. Often urate crystals have been phagocytosed becoming intracellular. When found in an inflammatory setting as judged by a WBC count of >1000/mm3, the presence of urate crystals is diagnostic of acute gout. If found on a background of a noninflammatory arthropathy, the presence of urate crystals still
Characteristics of the main crystal types C
C
C
C
C
C
Mono sodium urate e highly birefringent, needle shaped, negative sign, water soluble Calcium Pyrophosphate e low birefringence, rhomboid/rectangular shape, positive sign, water insoluble, alizarin positive Apatite/bone e non-birefringent, amorphous granules or spherulites, alizarin positive Lipid/fatty acid e intra-lipid rosettes or sheaves, birefringent, hydrocarbon soluble Cholesterol e low birefringence, plates or crescents, hydrocarbon soluble Haematoidin e orange, highly birefringent, rhomboid form intense orange, fern form green
Figure 1 Mixed crystal inflammatory arthropathy containing both calcium pyrophosphate and mono sodium urate crystals. In the centre of the image are two parallel crystals, one showing a negative sign of birefringence, (yellow) and the other (blue) having the opposite, positive sign. The yellow urate crystal is needle shaped which contrasts with the stumpy rod shape of the calcium pyrophosphate.
Box 1
DIAGNOSTIC HISTOPATHOLOGY --:-
3
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
Figure 3 Urate “beach ball” composed of a cluster of mono sodium urate crystals that seem to radiate from a central point viewed in compensating, polarising microscopy.
Figure 2 Two large pieces of bone viewed in “pseudo phase”characteristic of bone fragments seen in osteoarthritis. Unstained.
Apatite crystalloids have low or zero birefringence. Although not difficult to visualise microscopically a simple chemical test can be used to improve detection. A drop of SF, preferably including a piece of fibrin clot, is placed on the slide and a solution of 2% alizarin red S dye in 4% acetic acid added and a coverslip applied. This simple chemical reaction produces an easily visualized, bright red, highly birefringent compound colocalising with the apatite.7 This test is specific and sensitive to any complex calcium phosphate, and therefore CPPD crystals will also react. Lipids can often be seen in SF as droplets of fat either within or (more commonly) outside cells. Some lipids occur in crystalline forms such as the plates of cholesterol or the droplets of cholesterol ester exhibiting the classic “Maltese cross” birefringence. One of the pitfalls that can cause confusion in the analysis of SF is the failure to recognize the difference between urate crystals and the presence of fatty acid crystals. Urate crystals either singly or as “beach balls” are never within a lipid droplet, whereas fatty acid crystals, which are also highly positively birefringent, and which can also occur as single needle shaped crystal or “beach balls”, can only occur within lipid droplets (Figures 3 and 4). Contamination of the SF by depot steroid crystals can be problematic and causes difficulties during crystal examination where they are seen as highly birefringent crystals that have no discernible optical sign. There are many other rarer crystals seen in SF which can be discounted in day to day practice, but one that can give excellent, reliable, clinical information is hematoidin. This self-coloured orange/brown crystal that occurs in either rhomboidal or fern like form, is highly birefringent, the rhomboidal form being bright orange and the fern form bright green in polarised light. In a non-inflammatory SF the significance is that of a chronic haemarthrosis, whereas, much more importantly, when associated with an inflammatory SF they are diagnostic of septic arthritis. Haematoidin is most often present in untreated sepsis of at least 1 week’s duration Figure 5. Although formed from the breakdown
DIAGNOSTIC HISTOPATHOLOGY --:-
Figure 4 Intra-lipid droplet fatty acid crystal cluster mimicking a urate “beach ball” viewing conditions the same as Figure 3.
Figure 5 A cluster of brown haematoidin crystals. The insert in the bottom right if the image shows intra-cytoplasmic Gram-negative bacteria, these were only found after a prolonged search initiated by the finding of the haematoidin crystal cluster. Gram stain.
4
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
of blood, they have no iron content and are similar to bilirubin in their chemical composition.
traumatic cruciate ligament damage, which will result in joint instability. Synovial villi are most often introduced into the joint during the aspiration of the joint fluid and can be considered an “artefactual biopsy”. Only when the synovial villus changes its external morphology can its presence indicate a specific disease. In osteoarthritis a characteristic fern or leaf like formation of the synovial villus is found Figure 7. SF analysis is increasingly being requested by orthopaedic surgeons in the assessment of patients with a failing prosthetic joint replacement where careful microscopic analysis can identify individual prosthetic components (e.g. plastic,8 metal,9 cement etc.) (Figure 8).
Debris The inside of the synovial joint is surrounded by synovial tissue and cartilage, in addition, the knee joint contains the fibrocartilaginous menisci and the ligamentous tissue of the cruciate ligaments. In disease, particularly in acute or low grade recurrent trauma, any of these tissues may fragment and the fragments be seen by SF microscopy. However the absence of these tissue fragments in SF does not mean they are not present in the joint as the particles in the joint may be too large to be aspirated using a conventional needle. In general the larger the bore of the needle, bearing in mind patient compliance, the more likely is the detection of diagnostic particles. Diagnostically, one of the most useful types of debris is fragmented cartilage containing clusters of chondrocytes. Only in osteoarthritis does articular cartilage undergo chondrocyte proliferation, so this feature is diagnostic of osteoarthritis. Chondrocyte proliferation is associated with cartilage fibrillation and so another feature of osteoarthritis is the presence of fragments of fibrillated cartilage, fragments of which in SF are “striped” in polarising light an appearance we describe as resembling a “tigers tail”. Figure 6. Frequently tigers tails or cartilage showing chondrocyte proliferation are found in combination with CPPD or apatite, and when seen together are absolutely diagnostic of osteoarthritis. Occasionally, fragments of cartilage are introduced into the joint where they start to proliferate, the SF acting like a tissue culture medium. This phenomenon is most often seen in the younger patient with chondromalacia patellae, particularly in a patient with a sporting background. The fragments of cartilage proliferate, forming cartilaginous loose bodies; any microscopic cartilaginous loose bodies identified should indicate an arthroscopy as larger cartilage nodules are often present. Particles of ligament are found most often in cases of rotational trauma to the knee joint where they are associated with a low cell count and a haemarthrosis. In an inflammatory disease such as rheumatoid disease, they are an indicator of non-
Ragocytes The thin “wet preparation” is used to assess the presence of ragocytes which were originally described as refractive intracytoplasmic spheres, which vary from black to green, depending
Figure 7 Synovial villus from a case of osteoarthritis with the leaf or fern like configuration. Unstained.
Figure 8 Prosthetic derived UHMW polyethylene seen as finely divided, refractile shards with a piece of bone (far right) this combination is diagnostic of a loosening and wear of the prosthesis. Unstained.
Figure 6 A “tiger’s tail” of fibrillated cartilage showing the collagen fibrillation in full crossed polarising filters. Unstained.
DIAGNOSTIC HISTOPATHOLOGY --:-
5
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
on the focal plane of the microscope.10 Pseudo-phase, where the condenser iris of the microscope is at its minimal aperture, gives the best microscope setting for the detection of ragocytes Figure 9. Ragocytes were originally thought of as a marker of rheumatoid disease, but due to improvements in therapy they are now not often seen in this condition. Ragocytes are counted and their number expressed as a percentage of nucleated cells seen in the “wet preparation”. If this percentage is between 70% and 90%, the diagnosis is most probably rheumatoid disease, but when greater than 90%, septic arthritis is the most likely diagnosis.
Septic arthritis Sepsis in a joint can be a life threatening situation, either because the bacteria are disseminating into the circulation from an infected joint following joint penetration (accidental or surgical), or the joint itself is infected by the haematological route. Either way both can be associated with potentially fatal septicaemia. Careful microscopic examination of SF cytocentrifuge preparations allows micro-organisms to be identified in approximately 87% of instances of clinical infective arthritis. Most cases of infective arthritis are caused by Gram positive bacteria and organisms making them detectable in Gram stained preparations. Unfortunately, significant numbers of cases are partially treated with antibiotics before aspiration and rendering previously Gram positive organisms Gram negative. It is therefore essential in this clinical setting to examine the normally stained slide for evidence of organisms. Figure 10. Sometimes no organisms can be identified, but the presence of a very high cell count (>30,000 cells/mm3), particularly if crystals and reactive arthritis (see below) have been ruled out, or haematoidin crystals are present should raise the real possibility of a septic arthritis. Another feature we have found useful for raising a high suspicion of septic arthritis is the presence of “galaxy cartilage”, an appearance of cartilage following partial breakdown by bacterial proteolytic enzymes. Figure 11. Most bacterial infections result in a raised neutrophil response. However mycobacterial infections may be associated with lymphocyte-rich fluids. Periprosthetic infections are a special type of infective arthritis associated with a joint replacement. Here, a florid cell response is never seen. However, there is now a body of work that says if the SF cell count exceeds 1700/mm3 and more than 60 % of the nucleated cells are polymorphs that patient has a periprosthetic infection.11 When first made this observation changed joint revision surgery as it meant the diagnosis of periprosthetic infection can be made before, rather than during, the operation. Care must also be taken in interpreting SF samples from patients with pre-existing inflammatory arthropathies, particularly
Monolayer preparation In order to identify and perform a differential cell count it is necessary to stain the cells. Traditionally this has been done by making a smear, as would be done routinely in haematology laboratories. Unfortunately SF contains much more protein and hyaluronan than blood, and if a smear is prepared, the background staining obscures cellular detail. To overcome this, a monolayer of cells is produced by cytocentrifugation. The recommended method is to dilute the SF to a cell count of 400 cells/ mm3 with sterile saline. 100 ml of the suspension is then loaded into the cytocentrifuge chamber with the filter paper and slide, and spun at 800 rpm for 30 minute. This produces a monolayer of cells on the slide. Following air drying the specimen is methanol fixed for a minimum of 5 minute. The dilution of the fluid serves two purposes, firstly a standard number of cells are available for microscopic examination and secondly the hyaluronan that would stain and obscure the cells are removed giving a much clearer picture with little or no background staining. Following fixation the cell monolayer is stained by the Jenner Giemsa technique, but any cytological stain can be used, depending on personal preference or when a specific objective such as the diagnosis of sepsis is required, where a Gram stain would be necessary.
Figure 9 Intra-cytoplasmic ragocytes granules seen in “pseudo-phase illumination”. When the focus of the microscope is varied slightly the inclusions appear to change from black to green, whereas lipid droplets just go in and out of focus. Many inclusions, particularly if over 90% of the nucleated cells strongly suggest sepsis. Unstained.
DIAGNOSTIC HISTOPATHOLOGY --:-
Figure 10 Intra-cytoplasmic, blue pairs of cocci. Although the bodies are readily identified as bacteria, a Gram stain would be necessary to gain further information as to their grouping. Jenner Giemsa stained.
6
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
Figure 11 Galaxy cartilage containing four chondrocytes at its centre. The typical effect of enzymatic degradation of the cartilage is seen where there is a separation of the cartilage into individual collagen fibrils giving the “hairy” appearance of the surface. Unstained.
Figure 12 Cytophagocytic macrophages. The centre cell containing a freshly phagocytosed apoptotic neutrophil, other large macrophages are in various stages of digesting apoptotic neutrophils. Jenner Giemsa stained.
making up the overwhelming majority of the cells within diseased joints, these 3 cell types (polymorphs, lymphocytes and macrophages) represent only a small proportion of the cell types that can be identified regularly within diseased joints. Most of these cell types are identifiable on morphological grounds in conventional Jenner Giemsa stained cytocentrifuge preparations. The following are the most commonly identified cells in SF.
rheumatoid disease. These patients are both at a greater risk of developing a superimposed infective arthritis and of having a disease that may mask the presence of infection. Cells For reasons that are not clearly understood, primary and metastatic neoplastic disease is exceptionally rare in joints. Occasionally, leukaemic cells may be found in SF, but there are only a handful of cases of other neoplastic processes involving joints. Malignant cells are therefore so rare as to be disregarded in everyday practice. The cells most commonly encountered in synovial flid are either cells derived from connective tissues (chondrocytes, fibroblasts) or cells one would usually associate with inflammation (neutrophils, macrophages, lymphocytes). Patterns of “inflammatory” cell types and/or the presence of certain types of cytoplasmic inclusion within the cells can be used to identify individual diseases or more commonly a disease group. The cells that are found in SF are therefore a reflection of the two major groups of joint diseases, namely the inflammatory arthropathies such as septic arthritis, gout, seronegative spondylarthropathies and rheumatoid disease, and the noninflammatory arthropathies resulting from trauma or due to osteoarthritis. For most of the non-inflammatory arthropathies a differential count of the cells within the fluid is unhelpful and unnecessary and is therefore not routinely performed. In inflammatory arthropathies, by contrast, identifying certain types of inflammatory cell or the proportion of the three most common cells, polymorphs, macrophages and lymphocytes, is the mainstay of distinguishing different types of inflammatory arthritis. In very general terms, in inflammatory arthropathies polymorphs dominate the cytological picture, with other cells such a lymphocytes and macrophages occurring in varying proportions in various arthropathies. This is often completely different to the proportions of these cell types within the synovium. Although
DIAGNOSTIC HISTOPATHOLOGY --:-
Neutrophil polymorphs: these cells are recognized by their characteristic nuclear morphology and eosinophilic cytoplasm in Jenner Giemsa stained cytocentrifuge preparations. They are the predominant cells in inflammatory arthropathies and in intraarticular haemorrhage, the former as a consequence of specific traffic into the SF and the latter because they are the most abundant nucleated cell in blood. In septic arthritis and acute crystal arthritis they frequently amount to more than 95% of the total. In the absence of crystals, finding >95% of the nucleated
Figure 13 The lower of the central group of three cells containing a classical “LE” cell and to the upper left of the group another neutrophil but containing “small LE” inclusions. Jenner Giemsa stained.
7
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
cells as polymorphs and a total nucleated cell count >30,000/ mm3 is practically diagnostic of septic arthritis, even when organisms cannot be identified.
Synoviocytes It is perhaps unexpected, that the cells universally in contact with SF are one of the least common cells found in SF. This is perhaps a reflection of the way these cells adhere to the underlying synovial matrix. Both types of synoviocyte are seen in SF. Type A synoviocytes, which are functionally activated macrophages and type B synoviocytes which are functionally connective tissue cells involved in the production of specialised matrix molecules have distinct and distinctive morphologies. Type A synoviocytes are large cells (>20 mm) which generally have a vacuolated cytoplasm and a small nucleus that occupies less that 20% of the cell. It is most commonly seen in osteoarthritis. The type B synoviocyte is smaller (approx. 20 mm) and has a somewhat basophilic stippled cytoplasm, with a faintly cytoplasmic frill. The nucleus of the type B synoviocyte occupies 25% e50% of the cell. They are most commonly seen in seronegative arthropathies where they generally form a small percentage of the total number of cells.
Lymphocytes This cell can be of the typical small type, although biological factors in SF can frequently transform the lymphocyte into a larger activated forms or even cells resembling lymphoblasts. In the circulation these changes may be interpreted as leukaemic in origin but within SF they are “normal” in certain types of inflammatory arthropathy and do not represent a malignancy. Typically small lymphocytes are described as up to 12 mm in diameter with a nuclear/cytoplasmic ratio greater than 9:1. They predominate in approximately 10% of all cases in inflammatory arthritis, and in rheumatoid disease they indicate a better longterm prognosis for the joint than when neutrophils predominate. When seen in the company of LE cells (see below) they strongly suggest the diagnosis of systemic lupus erythematosus. Lymphocytes up to 30 mm with a nuclear/cytoplasmic ratio of about 1:1 indicate lymphocyte transformation and activation. All lymphocytes, other than small lymphocytes, have a peripheral cytoplasmic blue colouration often containing vacuoles when Jenner Giemsa stained.
Cytophagocytic mononuclear cells (CPM) This is a functional cell type. It refers to a macrophage that has phagocytosed apoptotic polymorphs Figure 12. The cells are normally seen whenever apoptosis is occurring as phagocytosis by macrophages is the usual way neutrophils are removed from the joint. They are, however, most abundant in the seronegative spondylarthropathies and as such are seen on an inflammatory background, either with polymorph or lymphocyte predominance. The seronegative spondylarthropathies are a group of diseases which includes the peripheral arthritis associated with psoriasis, inflammatory bowel disease, Behcet’s disease, ankylosing spondylitis, many types of juvenile idiopathic arthritis and reactive arthritis (an oligoarthropathy occurring in association with extra-articular infection, notably of the gastrointestinal and genitourinary tracts). If more than 1% of all large mononuclear
Macrophages These form one of the three morphologically distinct categories of large mononuclear cells encountered in SF, the others being transformed lymphocytes and synoviocytes. They are common in all types of arthritis and are frequently the most common cell found in non-inflammatory arthropathies. Where the total nucleated cell count is high and the cellular pattern is predominantly cells of the macrophage lineage, then a true viral arthritis, i.e. one in which the virus is present in the joint should be suspected. In non-inflammatory arthropathies, macrophages in combination with eosinophils indicate a resolving haemarthrosis.
Diagnosis of non-inflammatory synovial fluid Nucleated cell count < 500
Are monosodium urate crystals present?
Is prosthetic debris present?
Quiescent gout
Is apatite present?
Prosthetic wear
Wear and loosening Is/are – cartilage, apatite, pyrophosphate crystals present? Is frank haemorrhage present lipid or meniscus or ligament?
Osteoarthritis
Intra-articular trauma No specific features Non-inflammatory arthropathy
Figure 14
DIAGNOSTIC HISTOPATHOLOGY --:-
8
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
cells are CPM, a confident diagnosis of a seronegative spondylarthropathy can be made. CPMs are not found in SF from rheumatoid disease patients except when the patient has an extra-articular infection, particularly of the lung or bowel. Neither are they seen in septic arthritis, unless the patient has AIDS.
Eosinophils Eosinophils are most often seen following intra-articular haemorrhage or arthrography, as well as in the allergic reaction to injected medications such as artificial SF. LE cells Phagocytes containing a cytoplasmic inclusion of nuclear material are not uncommon and do not have the same significance in SF as they do in blood. However, they should always raise the possibility of systemic lupus erythematosus when found in a fluid rich in lymphocytes Figure 13.
Mast cells Although mast cells can be found in most arthropathies they are seen most commonly in inflammatory arthritis in patients with a seronegative spondylarthropathy and in noninflammatory arthropathies associated with trauma.
Concluding remarks Cells in mitosis Neoplastic infiltration of joints is very rare. Mitotic figures are relatively common by comparison and, no matter how bizarre they appear, are usually of little diagnostic or prognostic significance.
By examining the type and number of cells, crystals and debris it is possible to give either a specific single diagnosis (e.g. gout, osteoarthritis, rheumatoid arthritis) or where this is not possible, to give a more generalised conclusion varying from relatively
Diagnosis of inflammatory synovial fluid Nucleated cell count 1000
Are monosodium urate crystals present?
Is prosthetic joint in-situ and WBC count 5000?
Acute gout
Assume sepsis unless an inflammatory arthropathy can be proven
Are calcium pyrophosphate crystals present? Pseudogout Both?
Do ragocytes exceed 60%?
Mixed crystal arthropathy
Rheumatoid disease Do they exceed 90%? Septic arthritis
Are CPM present? Sero-negative spondylarthropathy If polymorph 60%
The percentage of neutrophils 50%
Reactive arthropathy
Sero-negative type of primary inflammatory arthropathy
WBC count 5,000 and hematoidin crystals present and/or galaxy cartilage present in the absence of CPPD crystals
If haemarthrosis is the WBC count 3,000? Haemarthrosis superimposed on inflammatory arthropathy
Septic arthritis
No significant features
Lymphocytes 60% and LE cells present
Primary inflammatory arthropathy
Systemic lupus erythematosus WBC count 5,000 and small lymphocytes 70% Tuberculosis
Figure 15
DIAGNOSTIC HISTOPATHOLOGY --:-
9
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
specific diagnoses such as “seronegative spondylarthropathy” to the most general of diagnoses such as “inflammatory” arthritis. Figures 14 and 15 below are simple algorithms that form the basis of much of diagnostic SF microscopy. SF microscopy is of greatest value in distinguishing inflammatory from non-inflammatory arthropathies and in defining specific disorders within these two groups. It is also important in the diagnosis of early inflammatory disease where it might be possible, on the basis of cytology, to identify a specific arthropathy before the clinical syndrome develops. In these cases accurate early diagnosis often allows the implementation of specific therapies before irreversible joint damage has occurred. The identification of non-cellular particles particularly crystals, endogenous joint debris and prosthetic joint debris has great significance, all of which will greatly influence clinical therapy and surgical intervention. Finally, it permits the very rapid diagnosis of acute joint disease, particularly in the difficult clinical differentiation of septic arthritis and acute crystal arthropathies where a misdiagnosed septic arthritis has an increased mortality and morbidity for the patient. The simple observations described above are based on conventionally illuminated and sometimes stained preparations examined microscopically. These can simply represent an increase in the cytopathologists workload but one of considerable importance to Rheumatologists, Orthopaedic Surgeons, Accident and Emergency Physicians and General Practitioners. Of course the greatest benefit is to the patient who receives an accurate and speedy diagnosis of their joint problem. A
3 4
5
6
7
8
9
10
11 REFERENCES 1 Swan A, Amer H, Dieppe P. The value of synovial fluid assays in the diagnosis of joint disease: a literature survey. Ann Rheum Dis 2002 Jun; 61: 493e8. 2 Barton K, Ludwig T, Achari Y, Shrive N, Frank C, Schmidt T. Characterization of proteoglycan 4 and hyaluronan composition
DIAGNOSTIC HISTOPATHOLOGY --:-
10
and lubrication function of ovine synovial fluid following knee surgery. J Orthop Res 2013 Oct; 31: 1549e54. Jones ST, Denton J, Holt PJ, Freemont AJ. Refrigeration preserves synovial fluid cytology. Ann Rheum Dis 1993 May; 52: 384. Lumbreras B, Pascual E, Frasquet J, Gonz_alez-Salinas J, Rodríguez E, Hern_andez-Aguado I. Analysis for crystals in synovial fluid: training of the analysts results in high consistency. Ann Rheum Dis 2005 Apr; 64: 612e5. Heselden EL, Freemont AJ. Synovial fluid findings and demographic analysis of patients with coexistent intra-articular monosodium urate and calcium pyrophosphate crystals. J Clin Rheumatol 2016; 22: 68e70. Paul H, Reginato AJ, Schumacher HR. Alizarin red S staining as a screening test to detect calcium compounds in synovial fluid. Arthritis Rheum 1983 Feb; 26: 191e200. Peterson C, Benjamin JB, Szivek JA, Anderson PL, Shriki J, Wong M. Polyethylene particle morphology in synovial fluid of failed knee arthroplasty. Clin Orthop Relat Res 1999 Feb; 359: 167e75. Khan WS, Agarwal M, Malik AA, Cox AG, Denton J, Holt EM. Chromium, cobalt and titanium metallosis involving a Nottingham shoulder replacement. J Bone Jt Surg Br 2008 Apr; 90: 502e5. Hollander JL, McCarty Jr DJ, Rawson AJ. The “R.A. cell”, “ragocyte”, or “inclusion body cell”. Bull Rheum Dis 1965 Sep; 16: 382e3 [No abstract available]. Trampuz A, Hanssen AD, Osmon DR, Mandrekar J, Steckelberg JM, Patel R. Synovial fluid leukocyte count and differential for the diagnosis of prosthetic knee infection. Am J Med 2004 Oct 15; 117: 556e62. Jones ST, Denton J, Holt PJ, Freemont AJ. Possible clearance of effete polymorphonuclear leucocytes from synovial fluid by cytophagocytic mononuclear cells: implications for pathogenesis and chronicity in inflammatory arthritis. Ann Rheum Dis 1993 Feb; 52: 121e6.
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
Synovial fluid analysis in the diagnosis of joint disease
arthropathy where the prognosis is inversely related to delay in diagnosis.1
The properties of synovial fluid SF consists of a transudate of plasma from synovial blood vessels, supplemented with high molecular weight lipid and saccharide-rich molecules,2 produced by one of the two main types of synovial cells (type B synoviocytes derived from synovial fibroblasts). Type A synoviocytes are tissue macrophage-derived phagocytes that remove debris from the synovial fluid. SF differs from all other body fluids in that the synovium and cartilage, which are the tissues it contacts, do not have an intact surface cellular layer seated on a basement membrane. This means that the matrix of both tissues is in direct contact with and through SF allowing an homogenous biological environment to develop within the joint. Because of this it is probably better to regard SF as a semi-liquid, avascular, hypocellular, connective tissue rather than a true body fluid such as is seen in other situations (e.g. pericardial effusion). Cytological analysis of SF differs in three important ways from other body fluids. Firstly synovial joints are only very rarely affected by primary or secondary malignancies. Secondly the term “cytological analysis” of SF would be better described as “microscopy” of SF, as many of the diagnostic features found are not cellular but are particles such as cartilage, crystals and prosthetic wear particles. Thirdly the greatest diagnostic information comes from the recognition of individual cell types and their quantification. Normal SF: Is a supplemented ultra-filtrate of plasma Contains no clotting factors Is viscous Is paucicellular (<500 cells/mm3) Contains no particles Any or all of these may change with joint disease. High molecular weight clotting factors are introduced into the joint as a result of vascular leakage following trauma or in inflammation. This results in the need to anticoagulate specimens. The choice of anticoagulant can have profound influence on the final diagnosis. Anticoagulants such as EDTA will, because of their sequestering properties, dissolve complex calcium phosphates such as seen in bone-derived hydroxyapatite and calcium pyrophosphate making the diagnosis of osteoarthritis and pseudogout difficult, if not impossible. Other anticoagulants, such as sodium heparin, can crystalise out leading to the false diagnosis of crystal arthritis. Lithium heparin, by contrast, has none of these faults and is the anticoagulant of choice. As, at the time of aspiration it is impossible to predict which SF will clot or contain crystals, all SF samples should be anticoagulated with lithium heparin. The viscosity of the SF is dependent on the concentration and size of the proteoglycans it contains. Normal SF and that from non-inflammatory arthropathies is very viscous. By contrast, in inflammatory arthropathies, inflammatory mediators and/or products cause abnormal synthesis or breakdown of existing proteoglycans, reducing viscosity.
Paul Hermansen Tony Freemont
Abstract Normal synovial fluid consists of a transudate of plasma from synovial blood vessels supplemented with high molecular weight lipid and saccharide-rich molecules. This produces a paucicellular, viscous fluid, which behaves like a tissue. In primary (e.g. rheumatoid disease) and secondary (e.g. septic and crystal-induced) inflammatory arthropathies, changes occur to the cell numbers and cell type in the fluid forming the basis of a diagnostic test. The diagnostic value is enhanced by the appearance of endogenous and exogenous particles, particularly those associated with degenerative, crystal-induced, and prosthesis-associated arthropathies (e.g. fibrin, cartilage, crystals, metal and plastic). Cells and particles can be characterised under the microscope leading to a simple, inexpensive, “cytological” test for every type of joint disease.
Keywords arthritis; crystals; microscopy; polarisation; synovial fluid
Why perform synovial fluid microscopy? Histopathologists are frequently called upon to diagnose inflammatory or non-inflammatory arthropathies based on the examination of a synovial biopsy. Even experienced histopathologists can have difficulty in distinguishing inflammatory from non-inflammatory arthropathies since the latter frequently have a lymphocyte infiltrate in the synovium. Even if they can be distinguished it is usually impossible to make specific diagnoses as there are few differences that can be detected histologically between disorders in the same broad group. By contrast, synovial fluid (SF) microscopy is of greatest value in these disorders, supporting clinicians in making early and accurate diagnoses of a spectrum of inflammatory and noninflammatory arthropathies often before the full blown syndrome develops. SF microscopy, on as little as a 0.5 ml sample, permits the rapid diagnosis of joint disease (the full test takes between 10 minutes for crystal arthropathies and 2 hours for full cytological analysis), particularly disorders such as sepsis and crystal related
Paul Hermansen MSc FIBMS is Consultant BMS in the Cytology Department, Central Manchester University NHS Foundation Trust, Manchester, UK. Conflicts of interest: none declared. Tony Freemont BSc MD FRCP FRCPath is Professor of Osteoarticular Pathology, University of Manchester, Director of the Manchester MRC/EPSRC Molecular Pathology Node, Manchester NIHR Biomedical Research Centre, Manchester, UK. Conflicts of interest: none declared.
DIAGNOSTIC HISTOPATHOLOGY --:-
1
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
The traditional method of counting cells is to use a graduated pipette to dilute the SF by a known amount with normal saline, to which crystal violet has been added as a supravital stain. This cell suspension is then introduced into a haemocytometer counting chamber where the cells are manually counted on the microscope. With the advent of newer technologies for cell counting, assessing cell number has now changed. The nature of the proteoglycans makes microbore based cell counting techniques tricky but computerised chamber counting methodologies are highly practical and accurate. These automated methods, although in themselves more costly in terms of initial investment and consumables, can make SF analysis quicker and more accurate than the manual method, and, by reducing the amount of biomedical scientist time required, reduce the overall cost of the test. The number of nucleated cells contained in the SF is the primary indicator of inflammation. Where the cell count is less than 500/mm3 the patient can be assumed to have a noninflammatory arthropathy, whereas a cell count greater than 1000/mm3 indicates an inflammatory arthropathy. If between these figures, the percentage of neutrophils in a differential count distinguishes inflammatory from noninflammatory arthropathies, where more than 50% of the nucleated cells in the sample being polymorphs indicates inflammation.
Viscosity of SF can be assessed by mixing SF and a 2% solution of acetic acid leading to the formation of a white precipitate, produced by aggregation of proteins and hyaluronans (the mucin clot test). The nature and amount of precipitate varies from good to poor and reflects the quality and quantity of the protein/hyaluronan complex, such that in inflammatory joint diseases and haemarthrosis there is poor clot formation. Noninflammatory arthropathies exhibit a good mucin clot. The mucin clot test is not normally performed on routine specimens, but it is very useful in identifying surgical joint washouts or dilution with local anaesthetic, where, in the presence of a low cell count no clot will form. This is an important observation as dilution of SF with water will affect cell counts and remove monosodium urate crystals, adversely affecting diagnostics.
Why examine synovial fluid? Basic examination of SF will result in identifying combinations of cellular and non-cellular biomarkers, which can have diagnostic, prognostic and theranostic (following responses to treatment) value, in primary, secondary and tertiary care, where SF analysis can be used to influence the choice of care pathway, and assess treatment options. This valuable role for SF analysis is made possible because the absence of a cell or basement membrane barrier between synovium, cartilage and SF means that pathological changes in the tissues surrounding the joint are reflected in the volume, cellular composition and particulate load within the SF. SF is examined “fresh” requiring careful handling in the laboratory and a need to minimise cell and crystal loss, which can dramatically adversely affect diagnosis, during transport to the laboratory. Storage at 4 C is well tolerated by the SF and prolongs transport times, but even so, adequate diagnosis that even cooled samples must reach the laboratory within 48 hours of aspiration.3
Wet preparation
In order that no part of the analysis is omitted there is a sequential examination of SF specimens arriving in the laboratory. It follows four steps: Gross analysis Nucleated cell count The “wet prep” Preparation and examination of a stained cell monolayer Gross analysis relies on the visual inspection of the SF to determine colour, clarity and viscosity. Colour will change, particularly following haemorrhage such as that seen in trauma, anticoagulant use and in primary disorders of joints such as pigmented villonodular synovitis. Clarity of SF is variable and a cloudy or opaque appearance generally indicates an increase in cellular concentration, crystal content or the presence of lipid, microscopic clarification is then necessary.
The “wet prep” is an important part of the examination of SF and can often be relied upon to give a definitive diagnosis. Two preparations are made each serving a different purpose. Firstly using a glass Pasteur pipette for clarity, an aliquot of SF is removed from the container and by slowly returning the SF to the container visible particles of fibrin, cartilage or crystal aggregates can be seen in the thin part of the pipette and placed onto a microscope slide, this we call the “thick preparation”. It can be of great help if the viewing background provides contrast, for example fragments of cartilage are most easily seen against a dark background whereas prosthetic debris is most easily seen against a pale background, white laboratory coats and dark laboratory bench surfaces usually provide this contrast. It is of great importance that any pieces of fibrin clot are found, as frequently identification of crystal arthropathies with a low crystal burden relies on finding crystals trapped in the fibrin clot. A second, much thinner preparation, is made avoiding particulate matter and using only a few microlitres of SF. Coverslipping this preparation flattens the cells and allow intracytoplasmic inclusions to be identified. Large inclusions with specific refractile properties characterise a functional group of cells called ragocytes. Ragocytes have diagnostic significance (see below). “Wet preps” can be preserved for 24 hours by painting around the edge of the coverslip with nail varnish to minimise evaporation. This also helps provide additional safety features, as the SF is a potential infection hazard.
Total nucleated cell count
Thick preparation examination
A total nucleated cell count is performed in order that the degree of inflammation can be assessed and so that an optimally diluted cell suspension can be achieved for the subsequent cytocentrifuge preparation.
Crystals When screening for crystals it is of the greatest importance that aggregates of fibrin and other particles are included in the slide preparation, as it is these micro clots that will often contain the
Basic approach to synovial fluid analysis
DIAGNOSTIC HISTOPATHOLOGY --:-
2
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
signifies gout but in a quiescent or latent form at the time of aspiration. Calcium pyrophosphate dihydrate crystals (CPPD) are of a much “weaker” birefringence than the mono sodium urate crystals and appear as short rods rhomboids or cubes. As described above they have an opposite sign to urate, thus, when aligned along the fast wave of the plate they are yellow. In common with monosodium urate crystals they are often seen within cells. Pseudogout is diagnosed when the nucleated cell count exceeds 1000 cells/mm3 CPPD in a non-inflammatory setting (nucleated cell count <1000 cells/mm3) could indicate latent pseudogout but more commonly osteoarthritis or chondrocalcinosis. Pseudogout is a disease of old age. If seen in a relatively young patient, particularly a male below 50 years of age, a secondary underlying diagnosis of a systemic metabolic disease such as hypothyroidism, haemochromatosis or hypomagnesaemia should be considered. Both mono sodium urate and calcium pyrophosphate can occasionally be found in the same fluid indicating a mixed crystal arthropathy5 Figure 1. Apatite as a true crystalline form of hydroxyapatite is somewhat of a rarity in SF, only occurring as spherulites where it is then diagnostic of the peri-articular calcification known as Milwaukie shoulder.6 Although originally described in the shoulder this condition has been noted in many other joints. When apatite is seen in SF it is normally in the form of bone dust or bone debris as a result of the mechanical removal of bone or calcified cartilage, following cartilage loss, typically as a result of osteoarthritis Figure 2. If seen in an inflammatory setting destructive, erosive arthropathies such as rheumatoid or psoriatic arthritis, as well as secondary osteoarthritis, should be considered. Apatite is also commonly seen in joints in which there is loosening of joint prostheses.
crystals even though the surrounding fluid and cells may not. A recent review4 shows that there is poor consistency in crystal identification and characterisation between individuals and laboratories unless adequate training of personnel is first carried out, but we find the simple expediency of including clots in the “wet prep” reduces this problem. A list of the characteristics of the main crystal types can be seen below in Box 1. Mono sodium urate crystals (MSU) are seen in microscopic preparations as thin rod/needle shaped crystals or even collections of crystals radiating from a central point, which are described as “beach balls”. When examined by polarising microscopy they are highly birefringence, meaning they are bright white against the black background of the fully crossed polarising filters. Additional information on the nature of the crystal can be gained by adding a quarter wave retardation plate to the polarising system. This plate retards the polarised light by one quarter of a wavelength which produces a magenta coloured background. The slow wave of the plate is often marked by a double headed arrow frequently accompanied by the Greek symbol “ɤ”. In most cases the slow wave of the plate is perpendicular to the long axis of the plate holder. If the longitudinal axis of the crystal is aligned parallel to the long axis of the retardation plate and the crystal appears blue the crystal is said to have a positive sign. If the same crystal were rotated through 90 aligning the longitudinal axis along the slow wave of the plate the crystal would be yellow. This colour is characteristic of mono sodium urate crystals. With the light path setup in this way crystals of calcium pyrophosphate (the crystals seen in pseudogout) are blue (ie the opposite of urate crystals). Unfortunately some microscopes are configured such that the image is reversed, so that urate crystals would be blue and pyrophosphate yellow at the image seen through the eyepieces has been reversed. It is therefore critically important that each individual microscope is “calibrated”with a known urate specimen. Often urate crystals have been phagocytosed becoming intracellular. When found in an inflammatory setting as judged by a WBC count of >1000/mm3, the presence of urate crystals is diagnostic of acute gout. If found on a background of a noninflammatory arthropathy, the presence of urate crystals still
Characteristics of the main crystal types C
C
C
C
C
C
Mono sodium urate e highly birefringent, needle shaped, negative sign, water soluble Calcium Pyrophosphate e low birefringence, rhomboid/rectangular shape, positive sign, water insoluble, alizarin positive Apatite/bone e non-birefringent, amorphous granules or spherulites, alizarin positive Lipid/fatty acid e intra-lipid rosettes or sheaves, birefringent, hydrocarbon soluble Cholesterol e low birefringence, plates or crescents, hydrocarbon soluble Haematoidin e orange, highly birefringent, rhomboid form intense orange, fern form green
Figure 1 Mixed crystal inflammatory arthropathy containing both calcium pyrophosphate and mono sodium urate crystals. In the centre of the image are two parallel crystals, one showing a negative sign of birefringence, (yellow) and the other (blue) having the opposite, positive sign. The yellow urate crystal is needle shaped which contrasts with the stumpy rod shape of the calcium pyrophosphate.
Box 1
DIAGNOSTIC HISTOPATHOLOGY --:-
3
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
Figure 3 Urate “beach ball” composed of a cluster of mono sodium urate crystals that seem to radiate from a central point viewed in compensating, polarising microscopy.
Figure 2 Two large pieces of bone viewed in “pseudo phase”characteristic of bone fragments seen in osteoarthritis. Unstained.
Apatite crystalloids have low or zero birefringence. Although not difficult to visualise microscopically a simple chemical test can be used to improve detection. A drop of SF, preferably including a piece of fibrin clot, is placed on the slide and a solution of 2% alizarin red S dye in 4% acetic acid added and a coverslip applied. This simple chemical reaction produces an easily visualized, bright red, highly birefringent compound colocalising with the apatite.7 This test is specific and sensitive to any complex calcium phosphate, and therefore CPPD crystals will also react. Lipids can often be seen in SF as droplets of fat either within or (more commonly) outside cells. Some lipids occur in crystalline forms such as the plates of cholesterol or the droplets of cholesterol ester exhibiting the classic “Maltese cross” birefringence. One of the pitfalls that can cause confusion in the analysis of SF is the failure to recognize the difference between urate crystals and the presence of fatty acid crystals. Urate crystals either singly or as “beach balls” are never within a lipid droplet, whereas fatty acid crystals, which are also highly positively birefringent, and which can also occur as single needle shaped crystal or “beach balls”, can only occur within lipid droplets (Figures 3 and 4). Contamination of the SF by depot steroid crystals can be problematic and causes difficulties during crystal examination where they are seen as highly birefringent crystals that have no discernible optical sign. There are many other rarer crystals seen in SF which can be discounted in day to day practice, but one that can give excellent, reliable, clinical information is hematoidin. This self-coloured orange/brown crystal that occurs in either rhomboidal or fern like form, is highly birefringent, the rhomboidal form being bright orange and the fern form bright green in polarised light. In a non-inflammatory SF the significance is that of a chronic haemarthrosis, whereas, much more importantly, when associated with an inflammatory SF they are diagnostic of septic arthritis. Haematoidin is most often present in untreated sepsis of at least 1 week’s duration Figure 5. Although formed from the breakdown
DIAGNOSTIC HISTOPATHOLOGY --:-
Figure 4 Intra-lipid droplet fatty acid crystal cluster mimicking a urate “beach ball” viewing conditions the same as Figure 3.
Figure 5 A cluster of brown haematoidin crystals. The insert in the bottom right if the image shows intra-cytoplasmic Gram-negative bacteria, these were only found after a prolonged search initiated by the finding of the haematoidin crystal cluster. Gram stain.
4
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
of blood, they have no iron content and are similar to bilirubin in their chemical composition.
traumatic cruciate ligament damage, which will result in joint instability. Synovial villi are most often introduced into the joint during the aspiration of the joint fluid and can be considered an “artefactual biopsy”. Only when the synovial villus changes its external morphology can its presence indicate a specific disease. In osteoarthritis a characteristic fern or leaf like formation of the synovial villus is found Figure 7. SF analysis is increasingly being requested by orthopaedic surgeons in the assessment of patients with a failing prosthetic joint replacement where careful microscopic analysis can identify individual prosthetic components (e.g. plastic,8 metal,9 cement etc.) (Figure 8).
Debris The inside of the synovial joint is surrounded by synovial tissue and cartilage, in addition, the knee joint contains the fibrocartilaginous menisci and the ligamentous tissue of the cruciate ligaments. In disease, particularly in acute or low grade recurrent trauma, any of these tissues may fragment and the fragments be seen by SF microscopy. However the absence of these tissue fragments in SF does not mean they are not present in the joint as the particles in the joint may be too large to be aspirated using a conventional needle. In general the larger the bore of the needle, bearing in mind patient compliance, the more likely is the detection of diagnostic particles. Diagnostically, one of the most useful types of debris is fragmented cartilage containing clusters of chondrocytes. Only in osteoarthritis does articular cartilage undergo chondrocyte proliferation, so this feature is diagnostic of osteoarthritis. Chondrocyte proliferation is associated with cartilage fibrillation and so another feature of osteoarthritis is the presence of fragments of fibrillated cartilage, fragments of which in SF are “striped” in polarising light an appearance we describe as resembling a “tigers tail”. Figure 6. Frequently tigers tails or cartilage showing chondrocyte proliferation are found in combination with CPPD or apatite, and when seen together are absolutely diagnostic of osteoarthritis. Occasionally, fragments of cartilage are introduced into the joint where they start to proliferate, the SF acting like a tissue culture medium. This phenomenon is most often seen in the younger patient with chondromalacia patellae, particularly in a patient with a sporting background. The fragments of cartilage proliferate, forming cartilaginous loose bodies; any microscopic cartilaginous loose bodies identified should indicate an arthroscopy as larger cartilage nodules are often present. Particles of ligament are found most often in cases of rotational trauma to the knee joint where they are associated with a low cell count and a haemarthrosis. In an inflammatory disease such as rheumatoid disease, they are an indicator of non-
Ragocytes The thin “wet preparation” is used to assess the presence of ragocytes which were originally described as refractive intracytoplasmic spheres, which vary from black to green, depending
Figure 7 Synovial villus from a case of osteoarthritis with the leaf or fern like configuration. Unstained.
Figure 8 Prosthetic derived UHMW polyethylene seen as finely divided, refractile shards with a piece of bone (far right) this combination is diagnostic of a loosening and wear of the prosthesis. Unstained.
Figure 6 A “tiger’s tail” of fibrillated cartilage showing the collagen fibrillation in full crossed polarising filters. Unstained.
DIAGNOSTIC HISTOPATHOLOGY --:-
5
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
on the focal plane of the microscope.10 Pseudo-phase, where the condenser iris of the microscope is at its minimal aperture, gives the best microscope setting for the detection of ragocytes Figure 9. Ragocytes were originally thought of as a marker of rheumatoid disease, but due to improvements in therapy they are now not often seen in this condition. Ragocytes are counted and their number expressed as a percentage of nucleated cells seen in the “wet preparation”. If this percentage is between 70% and 90%, the diagnosis is most probably rheumatoid disease, but when greater than 90%, septic arthritis is the most likely diagnosis.
Septic arthritis Sepsis in a joint can be a life threatening situation, either because the bacteria are disseminating into the circulation from an infected joint following joint penetration (accidental or surgical), or the joint itself is infected by the haematological route. Either way both can be associated with potentially fatal septicaemia. Careful microscopic examination of SF cytocentrifuge preparations allows micro-organisms to be identified in approximately 87% of instances of clinical infective arthritis. Most cases of infective arthritis are caused by Gram positive bacteria and organisms making them detectable in Gram stained preparations. Unfortunately, significant numbers of cases are partially treated with antibiotics before aspiration and rendering previously Gram positive organisms Gram negative. It is therefore essential in this clinical setting to examine the normally stained slide for evidence of organisms. Figure 10. Sometimes no organisms can be identified, but the presence of a very high cell count (>30,000 cells/mm3), particularly if crystals and reactive arthritis (see below) have been ruled out, or haematoidin crystals are present should raise the real possibility of a septic arthritis. Another feature we have found useful for raising a high suspicion of septic arthritis is the presence of “galaxy cartilage”, an appearance of cartilage following partial breakdown by bacterial proteolytic enzymes. Figure 11. Most bacterial infections result in a raised neutrophil response. However mycobacterial infections may be associated with lymphocyte-rich fluids. Periprosthetic infections are a special type of infective arthritis associated with a joint replacement. Here, a florid cell response is never seen. However, there is now a body of work that says if the SF cell count exceeds 1700/mm3 and more than 60 % of the nucleated cells are polymorphs that patient has a periprosthetic infection.11 When first made this observation changed joint revision surgery as it meant the diagnosis of periprosthetic infection can be made before, rather than during, the operation. Care must also be taken in interpreting SF samples from patients with pre-existing inflammatory arthropathies, particularly
Monolayer preparation In order to identify and perform a differential cell count it is necessary to stain the cells. Traditionally this has been done by making a smear, as would be done routinely in haematology laboratories. Unfortunately SF contains much more protein and hyaluronan than blood, and if a smear is prepared, the background staining obscures cellular detail. To overcome this, a monolayer of cells is produced by cytocentrifugation. The recommended method is to dilute the SF to a cell count of 400 cells/ mm3 with sterile saline. 100 ml of the suspension is then loaded into the cytocentrifuge chamber with the filter paper and slide, and spun at 800 rpm for 30 minute. This produces a monolayer of cells on the slide. Following air drying the specimen is methanol fixed for a minimum of 5 minute. The dilution of the fluid serves two purposes, firstly a standard number of cells are available for microscopic examination and secondly the hyaluronan that would stain and obscure the cells are removed giving a much clearer picture with little or no background staining. Following fixation the cell monolayer is stained by the Jenner Giemsa technique, but any cytological stain can be used, depending on personal preference or when a specific objective such as the diagnosis of sepsis is required, where a Gram stain would be necessary.
Figure 9 Intra-cytoplasmic ragocytes granules seen in “pseudo-phase illumination”. When the focus of the microscope is varied slightly the inclusions appear to change from black to green, whereas lipid droplets just go in and out of focus. Many inclusions, particularly if over 90% of the nucleated cells strongly suggest sepsis. Unstained.
DIAGNOSTIC HISTOPATHOLOGY --:-
Figure 10 Intra-cytoplasmic, blue pairs of cocci. Although the bodies are readily identified as bacteria, a Gram stain would be necessary to gain further information as to their grouping. Jenner Giemsa stained.
6
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
Figure 11 Galaxy cartilage containing four chondrocytes at its centre. The typical effect of enzymatic degradation of the cartilage is seen where there is a separation of the cartilage into individual collagen fibrils giving the “hairy” appearance of the surface. Unstained.
Figure 12 Cytophagocytic macrophages. The centre cell containing a freshly phagocytosed apoptotic neutrophil, other large macrophages are in various stages of digesting apoptotic neutrophils. Jenner Giemsa stained.
making up the overwhelming majority of the cells within diseased joints, these 3 cell types (polymorphs, lymphocytes and macrophages) represent only a small proportion of the cell types that can be identified regularly within diseased joints. Most of these cell types are identifiable on morphological grounds in conventional Jenner Giemsa stained cytocentrifuge preparations. The following are the most commonly identified cells in SF.
rheumatoid disease. These patients are both at a greater risk of developing a superimposed infective arthritis and of having a disease that may mask the presence of infection. Cells For reasons that are not clearly understood, primary and metastatic neoplastic disease is exceptionally rare in joints. Occasionally, leukaemic cells may be found in SF, but there are only a handful of cases of other neoplastic processes involving joints. Malignant cells are therefore so rare as to be disregarded in everyday practice. The cells most commonly encountered in synovial flid are either cells derived from connective tissues (chondrocytes, fibroblasts) or cells one would usually associate with inflammation (neutrophils, macrophages, lymphocytes). Patterns of “inflammatory” cell types and/or the presence of certain types of cytoplasmic inclusion within the cells can be used to identify individual diseases or more commonly a disease group. The cells that are found in SF are therefore a reflection of the two major groups of joint diseases, namely the inflammatory arthropathies such as septic arthritis, gout, seronegative spondylarthropathies and rheumatoid disease, and the noninflammatory arthropathies resulting from trauma or due to osteoarthritis. For most of the non-inflammatory arthropathies a differential count of the cells within the fluid is unhelpful and unnecessary and is therefore not routinely performed. In inflammatory arthropathies, by contrast, identifying certain types of inflammatory cell or the proportion of the three most common cells, polymorphs, macrophages and lymphocytes, is the mainstay of distinguishing different types of inflammatory arthritis. In very general terms, in inflammatory arthropathies polymorphs dominate the cytological picture, with other cells such a lymphocytes and macrophages occurring in varying proportions in various arthropathies. This is often completely different to the proportions of these cell types within the synovium. Although
DIAGNOSTIC HISTOPATHOLOGY --:-
Neutrophil polymorphs: these cells are recognized by their characteristic nuclear morphology and eosinophilic cytoplasm in Jenner Giemsa stained cytocentrifuge preparations. They are the predominant cells in inflammatory arthropathies and in intraarticular haemorrhage, the former as a consequence of specific traffic into the SF and the latter because they are the most abundant nucleated cell in blood. In septic arthritis and acute crystal arthritis they frequently amount to more than 95% of the total. In the absence of crystals, finding >95% of the nucleated
Figure 13 The lower of the central group of three cells containing a classical “LE” cell and to the upper left of the group another neutrophil but containing “small LE” inclusions. Jenner Giemsa stained.
7
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
cells as polymorphs and a total nucleated cell count >30,000/ mm3 is practically diagnostic of septic arthritis, even when organisms cannot be identified.
Synoviocytes It is perhaps unexpected, that the cells universally in contact with SF are one of the least common cells found in SF. This is perhaps a reflection of the way these cells adhere to the underlying synovial matrix. Both types of synoviocyte are seen in SF. Type A synoviocytes, which are functionally activated macrophages and type B synoviocytes which are functionally connective tissue cells involved in the production of specialised matrix molecules have distinct and distinctive morphologies. Type A synoviocytes are large cells (>20 mm) which generally have a vacuolated cytoplasm and a small nucleus that occupies less that 20% of the cell. It is most commonly seen in osteoarthritis. The type B synoviocyte is smaller (approx. 20 mm) and has a somewhat basophilic stippled cytoplasm, with a faintly cytoplasmic frill. The nucleus of the type B synoviocyte occupies 25% e50% of the cell. They are most commonly seen in seronegative arthropathies where they generally form a small percentage of the total number of cells.
Lymphocytes This cell can be of the typical small type, although biological factors in SF can frequently transform the lymphocyte into a larger activated forms or even cells resembling lymphoblasts. In the circulation these changes may be interpreted as leukaemic in origin but within SF they are “normal” in certain types of inflammatory arthropathy and do not represent a malignancy. Typically small lymphocytes are described as up to 12 mm in diameter with a nuclear/cytoplasmic ratio greater than 9:1. They predominate in approximately 10% of all cases in inflammatory arthritis, and in rheumatoid disease they indicate a better longterm prognosis for the joint than when neutrophils predominate. When seen in the company of LE cells (see below) they strongly suggest the diagnosis of systemic lupus erythematosus. Lymphocytes up to 30 mm with a nuclear/cytoplasmic ratio of about 1:1 indicate lymphocyte transformation and activation. All lymphocytes, other than small lymphocytes, have a peripheral cytoplasmic blue colouration often containing vacuoles when Jenner Giemsa stained.
Cytophagocytic mononuclear cells (CPM) This is a functional cell type. It refers to a macrophage that has phagocytosed apoptotic polymorphs Figure 12. The cells are normally seen whenever apoptosis is occurring as phagocytosis by macrophages is the usual way neutrophils are removed from the joint. They are, however, most abundant in the seronegative spondylarthropathies and as such are seen on an inflammatory background, either with polymorph or lymphocyte predominance. The seronegative spondylarthropathies are a group of diseases which includes the peripheral arthritis associated with psoriasis, inflammatory bowel disease, Behcet’s disease, ankylosing spondylitis, many types of juvenile idiopathic arthritis and reactive arthritis (an oligoarthropathy occurring in association with extra-articular infection, notably of the gastrointestinal and genitourinary tracts). If more than 1% of all large mononuclear
Macrophages These form one of the three morphologically distinct categories of large mononuclear cells encountered in SF, the others being transformed lymphocytes and synoviocytes. They are common in all types of arthritis and are frequently the most common cell found in non-inflammatory arthropathies. Where the total nucleated cell count is high and the cellular pattern is predominantly cells of the macrophage lineage, then a true viral arthritis, i.e. one in which the virus is present in the joint should be suspected. In non-inflammatory arthropathies, macrophages in combination with eosinophils indicate a resolving haemarthrosis.
Diagnosis of non-inflammatory synovial fluid Nucleated cell count < 500
Are monosodium urate crystals present?
Is prosthetic debris present?
Quiescent gout
Is apatite present?
Prosthetic wear
Wear and loosening Is/are – cartilage, apatite, pyrophosphate crystals present? Is frank haemorrhage present lipid or meniscus or ligament?
Osteoarthritis
Intra-articular trauma No specific features Non-inflammatory arthropathy
Figure 14
DIAGNOSTIC HISTOPATHOLOGY --:-
8
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
cells are CPM, a confident diagnosis of a seronegative spondylarthropathy can be made. CPMs are not found in SF from rheumatoid disease patients except when the patient has an extra-articular infection, particularly of the lung or bowel. Neither are they seen in septic arthritis, unless the patient has AIDS.
Eosinophils Eosinophils are most often seen following intra-articular haemorrhage or arthrography, as well as in the allergic reaction to injected medications such as artificial SF. LE cells Phagocytes containing a cytoplasmic inclusion of nuclear material are not uncommon and do not have the same significance in SF as they do in blood. However, they should always raise the possibility of systemic lupus erythematosus when found in a fluid rich in lymphocytes Figure 13.
Mast cells Although mast cells can be found in most arthropathies they are seen most commonly in inflammatory arthritis in patients with a seronegative spondylarthropathy and in noninflammatory arthropathies associated with trauma.
Concluding remarks Cells in mitosis Neoplastic infiltration of joints is very rare. Mitotic figures are relatively common by comparison and, no matter how bizarre they appear, are usually of little diagnostic or prognostic significance.
By examining the type and number of cells, crystals and debris it is possible to give either a specific single diagnosis (e.g. gout, osteoarthritis, rheumatoid arthritis) or where this is not possible, to give a more generalised conclusion varying from relatively
Diagnosis of inflammatory synovial fluid Nucleated cell count 1000
Are monosodium urate crystals present?
Is prosthetic joint in-situ and WBC count 5000?
Acute gout
Assume sepsis unless an inflammatory arthropathy can be proven
Are calcium pyrophosphate crystals present? Pseudogout Both?
Do ragocytes exceed 60%?
Mixed crystal arthropathy
Rheumatoid disease Do they exceed 90%? Septic arthritis
Are CPM present? Sero-negative spondylarthropathy If polymorph 60%
The percentage of neutrophils 50%
Reactive arthropathy
Sero-negative type of primary inflammatory arthropathy
WBC count 5,000 and hematoidin crystals present and/or galaxy cartilage present in the absence of CPPD crystals
If haemarthrosis is the WBC count 3,000? Haemarthrosis superimposed on inflammatory arthropathy
Septic arthritis
No significant features
Lymphocytes 60% and LE cells present
Primary inflammatory arthropathy
Systemic lupus erythematosus WBC count 5,000 and small lymphocytes 70% Tuberculosis
Figure 15
DIAGNOSTIC HISTOPATHOLOGY --:-
9
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002
MINI-SYMPOSIUM: OSTEOARTICULAR PATHOLOGY
specific diagnoses such as “seronegative spondylarthropathy” to the most general of diagnoses such as “inflammatory” arthritis. Figures 14 and 15 below are simple algorithms that form the basis of much of diagnostic SF microscopy. SF microscopy is of greatest value in distinguishing inflammatory from non-inflammatory arthropathies and in defining specific disorders within these two groups. It is also important in the diagnosis of early inflammatory disease where it might be possible, on the basis of cytology, to identify a specific arthropathy before the clinical syndrome develops. In these cases accurate early diagnosis often allows the implementation of specific therapies before irreversible joint damage has occurred. The identification of non-cellular particles particularly crystals, endogenous joint debris and prosthetic joint debris has great significance, all of which will greatly influence clinical therapy and surgical intervention. Finally, it permits the very rapid diagnosis of acute joint disease, particularly in the difficult clinical differentiation of septic arthritis and acute crystal arthropathies where a misdiagnosed septic arthritis has an increased mortality and morbidity for the patient. The simple observations described above are based on conventionally illuminated and sometimes stained preparations examined microscopically. These can simply represent an increase in the cytopathologists workload but one of considerable importance to Rheumatologists, Orthopaedic Surgeons, Accident and Emergency Physicians and General Practitioners. Of course the greatest benefit is to the patient who receives an accurate and speedy diagnosis of their joint problem. A
3 4
5
6
7
8
9
10
11 REFERENCES 1 Swan A, Amer H, Dieppe P. The value of synovial fluid assays in the diagnosis of joint disease: a literature survey. Ann Rheum Dis 2002 Jun; 61: 493e8. 2 Barton K, Ludwig T, Achari Y, Shrive N, Frank C, Schmidt T. Characterization of proteoglycan 4 and hyaluronan composition
DIAGNOSTIC HISTOPATHOLOGY --:-
10
and lubrication function of ovine synovial fluid following knee surgery. J Orthop Res 2013 Oct; 31: 1549e54. Jones ST, Denton J, Holt PJ, Freemont AJ. Refrigeration preserves synovial fluid cytology. Ann Rheum Dis 1993 May; 52: 384. Lumbreras B, Pascual E, Frasquet J, Gonz_alez-Salinas J, Rodríguez E, Hern_andez-Aguado I. Analysis for crystals in synovial fluid: training of the analysts results in high consistency. Ann Rheum Dis 2005 Apr; 64: 612e5. Heselden EL, Freemont AJ. Synovial fluid findings and demographic analysis of patients with coexistent intra-articular monosodium urate and calcium pyrophosphate crystals. J Clin Rheumatol 2016; 22: 68e70. Paul H, Reginato AJ, Schumacher HR. Alizarin red S staining as a screening test to detect calcium compounds in synovial fluid. Arthritis Rheum 1983 Feb; 26: 191e200. Peterson C, Benjamin JB, Szivek JA, Anderson PL, Shriki J, Wong M. Polyethylene particle morphology in synovial fluid of failed knee arthroplasty. Clin Orthop Relat Res 1999 Feb; 359: 167e75. Khan WS, Agarwal M, Malik AA, Cox AG, Denton J, Holt EM. Chromium, cobalt and titanium metallosis involving a Nottingham shoulder replacement. J Bone Jt Surg Br 2008 Apr; 90: 502e5. Hollander JL, McCarty Jr DJ, Rawson AJ. The “R.A. cell”, “ragocyte”, or “inclusion body cell”. Bull Rheum Dis 1965 Sep; 16: 382e3 [No abstract available]. Trampuz A, Hanssen AD, Osmon DR, Mandrekar J, Steckelberg JM, Patel R. Synovial fluid leukocyte count and differential for the diagnosis of prosthetic knee infection. Am J Med 2004 Oct 15; 117: 556e62. Jones ST, Denton J, Holt PJ, Freemont AJ. Possible clearance of effete polymorphonuclear leucocytes from synovial fluid by cytophagocytic mononuclear cells: implications for pathogenesis and chronicity in inflammatory arthritis. Ann Rheum Dis 1993 Feb; 52: 121e6.
Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Hermansen P, Freemont T, Synovial fluid analysis in the diagnosis of joint disease, Diagnostic Histopathology (2017), http://dx.doi.org/10.1016/j.mpdhp.2017.04.002