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Table 35.2

and related interpretative criteria

Macroscopic examination Cytometric examination

- Transudate=clear from pale to straw yellow

- Exudate=from pale to straw yellow with a different degree of turbidity, sometimes milky; if the latescence persists after centrifugation, chylothorax occurs

Macroscopic and cytometric analysis of the pleural uid

The total cellularity is usually between 1395 and 3794×10 between 64% and 80%, lymphocytes between 18% and 36%, neutrophils between 0% and 1% and mesothelial cells between 0% and 2% In pleural effusions:

- Total cellularity <1000×106/L is indicative of an effusion of probable transudative origin

- Total cellularity ≥1000×106/L is indicative of an effusion of probable exudative origin. In this case a count of:

- Neutrophils ≥50% is indicative

of probable bacterial pneumonia, pulmonary infarction, pancreatitis, etc.

- Lymphocytes ≥50% is indicative

of probable tuberculous or viral infection, chylothorax, neoplasm, rheumatoid pleurisy, etc.

- Eosinophils ≥10% is indicative

of probable pneumothorax, trauma, pulmonary infarction, parasitic or fungal infections, drug reactions, rheumatic disorders, Hodgkin’s lymphoma,

and idiopathic eosinophilia Comment. About 80% of transudates have a cellularity <1000×10 while cellularity >10,000×10 usually associated with parapneumonic effusions. Finally, about 10% of transudates have a neutrophil count ≥50% and about 30% have a lymphocyte count ≥50%. Regardless of the number of cells found, it is the qualitative evaluation that allows their characterization in a neoplastic sense

/L, macrophages

/L cells,

/L are

In the case of neoplastic or chylous effusion, the uid may be milky. Table35.3 describes the differential characteristics of chylous and pseudochylous effusion.

Normally, the volume of uid in the pleural cavity ranges between 4.1 and 12.7 mL, the total cellularity is between 1395 and 3794 × 106/L, macrophages between 64% and 80%, lymphocytes between 18% and 36%, neutrophils between 0% and 1%, and mesothelial cells between 0% and 2%.

In pleural effusions of exudative origin, on the other hand, the cell count is greater than 1000×106/L.In this condition, neutrophilia greater than 50% is indicative of an inamma-

tory process of bacterial etiology (e.g., parapneumonic effusion). A lymphocytosis higher than 50% is associated with tubercular infection, chylous effusions due to mechanical damage of the lymphatic system, and lymphoproliferative diseases. Lymphocyte characterization is especially useful in exudative effusions; in these cases, immuno-phenotyping can distinguish between benign and malignant lymphoproliferative diseases.

Eosinophilia (>10%) occurs in conditions as diverse as pneumothorax, pulmonary embolism, traumatic hemothorax, and Churg-Strauss syndrome.

The count of red blood cells distinguishes between serous effusion and hemothorax. The latter may be secondary to trauma, pulmonary embolism, and neoplasia.

Total cellularity is useful in the differential diagnosis between exudates and transudates: in the latter, approximately 80% have a cellularity of less than 1000×106/L cells; cellularities greater than 1000×106/L are usually associated with parapneumonic effusions.

Biochemistry

Light’s criteria include assaying total protein and LDH on pleural uid and serum to calculate the ratio. Table35.1 sum- marizes the biochemical criteria for identifying an exudate with greater accuracy than using total protein alone.

The pH has a high positive prognostic value in nonpneumococcal pneumonia. The presence of creatinine or urea indicatesthe presence of urine in pleural uid. C-reactive protein (CRP) of pleural uid may be more sensitive than blood protein in diagnosing non-pneumococcal pneumonia.

The usefulness of assaying certain tumor-associated biological markers in pleural uid is controversial. For example, the literature reports the case of a patient with a marked increase in prostate-specic antigen (PSA), which proved to be decisive in establishing the diagnosis of metastatic prostate cancer. The patient presented with severe anemia, peripheral edema, and pleural and pericardial effusions, but no neoplastic cells.

A milky pleural uid suggests the presence of a chylothorax, which is always due to obstruction of the thoracic duct for mainly neoplastic or traumatic reasons. However, it is useful to distinguish this situation from a pseudochylothorax, which is usually due to rheumatoid pleurisy, tuberculosis, or myxedema. In these cases, triglyceride and chylomicron detection may be helpful; table35.3 summarizes the criteria for the differential diagnosis between the two situations.

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Table 35.3

effusions and related interpretative criteria

Characteristic Chylous Pseudochylous Onset Rapid Gradual Appearance Milky or yellow

Microscopic exam Lymphocytosis Mixed cellularity and

Triglycerides Electrophoresis of

lipoproteins

Main characteristics of chylous and pseudochylous pleural

tending to blood color

≥110mg/dL Chylomicrons present Chylomicrons absent

Milky or grayish or metallic

cholesterol crystals <50mg/dL

Pericardial Fluid

In the healthy adult, pericardial uid ranges from 10 to 50mL, produced similarlyas in the pleura.

Pericardial effusions are mainly due to phlogistic and/or neoplastic processes that compromise the integrity of the permeability of endothelial and mesothelial cells, and induce obstruction of the lymphatic pathways of the affected area.

Pericardial uid is pale yellow and clear. Large effusions (>350mL) are due to neoplastic inltration (lymphoproliferative neoplasms or metastatic processes), uremia or microbial agents (bacterial, tubercular, or fungal infections), inammatory processes, autoimmune diseases, cardiac infarction with mediastinal damage, and iatrogenic causes. The most frequent cause of idiopathic pericardial effusion is enterovirus infections. Patients with HIV infection often present with asymptomatic pericardial effusions that increase as the infectious disease progresses.

Pericardial uid may be hematic due to incorrect sampling maneuver (with penetration of the heart chambers) or in the case of a hemorrhagic effusion, with correct sampling. In the latter case, the hematocrit or the number of red blood cells is lower than that of the blood. Moreover, in the rst case the sample coagulates, in the second case this is very rare.

Post-pericardiotomy syndrome may occur in subjects undergoing cardiac surgery, who are still in hospital but sometimes already at home. It is characterized by fever, thoracic- pleural pain, and other signs of pleural, pericardial, and, less frequently, pulmonary inammation. In more than 80% of cases, a pleural exudate develops, which is serous to frankly hematic, with pH>7.4 and normal glucose levels. There are no specic laboratory tests except for detection of anti-myocardial antibodies and, serum C3, which may be decreased.

Table 35.4 shows the main laboratory tests performed in pericardial uid.

Table 35.4

cardial uid and related interpretative criteria

Macroscopic examination Cytometric examination

- Transudate=clear from pale to straw yellow

- Exudate=from pale to straw yellow with different degree of turbidity

Brief description of the main laboratory tests on the peri-

Clinical biochemistry

Normally they are present from a minimum

10×10

/L cells up to a maximum of 1900– 2210×10 In effusions of an exudative nature, the average cellularity is generally ≥14.116×10 In effusions of probable neoplastic origin, the average value is 3600×10 Comment. In the above cases, the standard deviations detected are very large and sometimes overlap in the different categories of pericardial effusions Differential cell count has the following meaning: Neutrophils >70% are

Mononuclear cells/

Comment. Regardless of the number of cells found, the qualitative evaluation allows their neoplastic characterization

/L cells

indicative of bacterial infection or rheumatic effusion

monocytes >75% are indicative of probable monocytosis secondary to hypothyroidism or malignant effusion

Diagnostic cutoff for exudate:

- Total protein >3g/dL and liquid/serum protein ratio >0.5

- LDH >200U/L

- Liquid/serum LDH ratio >0.6

Glucose <40mg/ dL is frequently found in bacterial, tuberculous, rheumatic, or neoplastic effusions. To date, there is insufcient evidence to indicate an appropriate use of other analytes in routine practice

Macroscopic Evaluation andCellular Analysis

Pericardial uid for diagnostic purposes is obtained by pericardiotomy or pericardiocentesis.

Normal pericardial uid or uid of exudative origin is generally clear yellow, whereas the uid is turbidin exudative effusions, such as neoplastic or infected effusions. Finally, as described above, pericardial uid may sometimeshave a hemorrhagic appearance.

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The mean value of cellularity can range from a minimum of 10×106/L to a maximum of 1900–2210×106/L cells. In contrast, the mean value ranges from 3600 × 106/L up to 14,116×106/L cellsin pericardial effusions of probable neoplastic origin.

The effusions of bacterial or rheumatic etiology are generally characterized by the presence of about 70% of neutrophil granulocytes, while those secondary to hypothyroidism and/or neoplastic nature are characterized by 75% or more of monocytes or other mononuclear cells. Distinguishing activated mesothelial cells from neoplastic cells often requires further investigation, in addition to purely morphological ones.

Biochemistry

Light’s criteria for distinguishing an exudate from a transudate also apply to pericardial uid. However, the literature data do not show adequate specicity and sensitivity and, even if applied, have not been validated for this type of uid; further studies, possibly multicentric and with large numbers, are necessary (Table35.4). Glucose, total protein, pH, lipids, lactate dehydrogenase (LDH), and adenosine deaminase (ADA) should be assayed on this uid.

A glucose concentration <40mg/dL is frequent in bacterial, tubercular, rheumatic, or neoplastic effusions. Higher glucose values have less diagnostic value.

A protein concentration <3.0 g/dL has a sensitivity of 97% for an exudate, but a much lower specicity (around 22%), resulting in poor utility.

The pH of pericardial uid can be markedly decreased (<7.1) in rheumatic and purulent pericarditis. Pathologies such as neoplasms, uremia, tuberculosis, and idiopathic forms show a moderate decrease in pH (7.2–7.3).

Triglyceride and cholesterol assays may be useful in distinguishing between chylous and pseudochylous effusions, along with lipoprotein electrophoresis, which is useful in checking for the presence of chylomicrons (Table35.3). In pericardial uid, LDH >200U/L (i.e., pericardial uid LDH/ plasma LDH ratio >0.6) is characteristic of an exudate.

The ADA increases signicantly in tubercular pericarditis, at a cutoff of 30U/L the sensitivity exceeds 90%, while the specicity does not reach 70%. By raising the cutoff to 40U/L, both sensitivity and specicity exceed 90%.

Ascitic Fluid or Peritoneal Eusion

The pathological accumulation of fluid in the peritoneal cavity is called ascites. The most common causes of ascites include cirrhosis of the liver, certain neoplasms, congestive heart failure, nephrotic syndrome, and pancreatic

disease. The mechanism of ascitic fluid accumulation is controversial. Portal venous hypertension pushes fluid out of the vascular space, causing hepatic and interstitial edema. Both the liver and the intestine may, therefore, be the source of such accumulation. After the fluid enters the abdomen, it is reabsorbed by the lymphatic vessels. If the reabsorption capacity of the lymphatic system is exceeded, the fluid moves to the peritoneal space, causing ascites. History and physical examination provide clues to its possible etiology. Radiological and ultrasonographic evaluation can help detect small amounts of peritoneal fluid, andestablish the possible etiology of ascites. Table35.5 shows the main laboratory tests performed on this fluid.

Macroscopic Evaluation andCellular Analysis

The ascitic uid sample is usually obtained by paracentesis or drainage following paracentesis.

The appearance of the ascitic uid is typical of cavitary uid, while in case of jaundice it is generally clear and of an intense yellow color.

Table 35.5 Main laboratory test on ascitic uid and related interpretative criteria

Macroscopic examination

- Transudate=clear yellow

- Exudate=cloudy of variable color:

- Yellow-green

(bile contamination)

- Iridescent green

(acute pancreatitis)

- Milky (chylous

or pseudochylosis infections or

effusions) In chylous effusions the milky appearance persists even after centrifugation

Cytometric examination Clinical biochemistry

The diagnostic cutoffs are

- Neutrophils >250×106/L, indicative of probable non perforated bacterial peritonitis

- Total cellularity >1000×10 with prevalence of lymphocytes, indicative of probable tuberculous peritonitis

- Eosinophils >10%, indicative of probable chronic inammation

Comment. Regardless of the number of cells, the qualitative evaluation allows their neoplastic characterization

The albumin gradient values:

- ≥11g/L or 1.1g/ dLis indicative of portal hypertension ascites. This condition may include cirrhosis, fulminant hepatitis, vein-occlusive

disease, hepatic vein obstruction (e.g.,

Budd-Chiari syndrome), congestive heart failure

- <11g/L or 1.1g/ dLis indicative of ascites from alterations of the peritoneum. This condition may include primary or secondary peritoneal carcinomatosis, tuberculous peritonitis, parasitic or fungal infection, pancreatitis

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The reference range for cellularity of sterile ascitic uid is 0 to 562×106/L.In sterile ascitic uid, neutrophils have an average value of 27%. The absolute neutrophil count ranges from 0 to 2532×106/L with an average of 82×106/L cells.

To date, it is assumed that, in a peritoneal effusion, a neutrophil count <250×106/L cells is diagnostic of sterile ascites, whereas a value ≥250×106/L is diagnostic of spontaneous bacterial peritonitis. A total nucleated element count >1000 × 106/L cells, with lymphocytes predominating, is diagnostic of tuberculous peritonitis.

The count of nucleated elements,especially leukocytes, and their differentiation, has a substantial diagnostic value in the evaluation of various inammatory conditions and is useful in the differential diagnosis and, therefore, in directing toward the appropriate therapy.

Biochemistry

The determination of the albumin gradient, i.e., the difference between the serum and ascitic uidalbumin allows to differentiatethe etiology of the effusion (Table35.6).

Fluids of biliary origin may be found in the peritoneal cavity during surgery on the biliary excretory tract or liver transplantation. A high bilirubinconcentration in the abdominal/ascitic uid is practically conclusive for damage to the biliary excretory tract, which almost always requires surgical correction.

Damage to the exocrine pancreas, from surgical maneuvers or medical causes, may result in pancreatic enzymes leaking into the peritoneum. The assay of pancreatic amylase, or lipase, in the uid of peritoneal origin can conrm or exclude pancreatic damage, along with the presence or absence of an acute abdomen. If the same enzymes are also elevated in pleural uid from the left lung, it can be concluded that pancreatic damage has also involved this organ.

Renal damage of various etiology (neoplastic, brosis, stone, surgical, traumatic) may result in urine leaking into the pleural and/or peritoneal cavity. Lithotripsy may involve uroperitoneum, as well as an unsuccessful external urinary diversion. Conrmation that the uid in the two cavities is urine is obtained by measuring creatinine (2–10 mg/dL) and urea, which are signicantly present. The creatinine to serum creatinine ratio will be greater than one. Finally, if uid is inadvertently aspirated directly from the bladder, the urea/creatinine ratio should be higher than if the uid were peritoneal due to the greater and faster reabsorption of urea itself.

The alkaline phosphatase (ALP) assay is useful in diagnosing damage to the abdominal hollow organs, making laparoscopy unnecessary. Values greater than 10 U/L have sensitivity and specicity greater than 95%. High ALP values are also diagnostic to differentiate a primary peritonitis from one secondary to intestinal perforation. In the latter,

Table 35.6 Clinical utility of some analytes in cavity liquids

Pleural effusion Ascites or peritoneal effusion

Analyte Light’s criteria X X X LDH X X O Proteins X X O Cholesterol X X O Bilirubin X X X Enzymes Anti-trypsin O X O ADA X O O Albumin gradient X X O Protein gradient X Amylase X X X PH X O O Glucose X X X CEA X X X CA19.9 X X O CA125 X X O CYFRA21-1 O O O CA15.3 X X O α-Fetoprotein

X indicates that the diagnostic usefulness has been conrmed in several studies, O indicates that there is no robust evidence to really dene usefulness in clinical practice, ADA adenosine deaminase, CEA carcinoembryonic antigen, CA19.9 carbohydrate antigen 19.9, CA125 carbohydrate antigen 125, CA15.3 carbohydrate antigen 15.3

Other enzymes besides LDH: amylase, ADA

Useful Useless Of limited utility Useful Useless Of limited utility Useful Useless Of limited utility

X X X

O X O

Pericardial effusion

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ALP exceeds 240U/L, a value that is almost never observed in a primary form of the disease.

LDH is often elevated in neoplastic effusions. A peritoneal LDH/serum LDH ratio >0.6 has a sensitivity greater than 80%.

Combined measurement of peritoneal LDH and cholesterol discriminates peritoneal carcinomatosis from hepatocarcinoma ascites.

In ovarian carcinomas, peritoneal uid LDH is more useful in distinguishing malignant from benign forms than the serum value of the enzyme.

Finally, in spontaneous bacterial peritonitis, LDH allows a correct diagnosis in three out of four cases by using the 0.4 cutoff of the peritoneal LDH/serum LDH ratio.

The ADA distinguishes well, in endemic areas, a tuberculous peritonitis from those of different etiology, with sensitivity and specicity higher than 90%.

Synovial Fluid

Pathophysiology

Synovial uid isin varying amounts in the joint space and consists of hyaluronic acid, glucose, proteins, lipids, electrolytes, enzymes, and cells. Its main functions are hydration and lubrication of the joint; it also provides a means for the passage of nutrients to the cartilage tissue. Synovial uid is produced by ultraltration of peripheral blood through the synovium and the synthesis of hyaluronic acid by synovial cells.

Normal synovial uid has the following characteristics: high viscosity, clarity and an opaque yellow color, low erythrocyte and leukocyte cellularity, with a predominance of lymphocytes and monocytes or macrophages; the neutrophil count is less than 10%. Normal synovial uid is free of crystals and brinogen,and is sterile. Synovial uid and its characteristics vary in different clinical conditions; its analysis can provide the clinician (orthopedist or internist) with helpful information to clarify the etiology of a joint pathology or its evolution.

The search for crystals in synovial uid is helpful in differential diagnostics; both the qualitative nature of the crystals and their semi-quantitative evaluation (rare, a few, or numerous) are of considerable clinical interest. The main constituents are monosodium urate (acute or chronic gout); calcium pyrophosphate (pseudogout or chondrocalcinosis); apatite (pre-articular calcication); and cholesterol crystals can also be found in joint effusions of subjects with chronic rheumatism.

The cell count in synovial uid takes on different characteristics depending on the pathology involving the joint.

Bacterial Arthritis

Synovial uid has a purulent appearance, and the cellular component consists predominantly of neutrophils, even in concentrations greater than 100,000×106/L cells. In Reiter’s syndrome (often associated with Chlamydia trachomatis infection), synovial uid has a cellularity of approximately 40,000 × 106 cells/L, with 80% neutrophils. Occasionally, Reiter cells (macrophages with basophilic cytoplasmic inclusions with panoptic staining) are also present.

Rheumatoid Arthritis

The average cell count in this disease is 30,000×106/L cells, which varies according to the disease phase. During the acute phase, lymphocytes and macrophages prevail, which are not pathognomonic ofrheumatoid arthritis because they can also be found in other joint diseases.

Acute Joint Rheumatism

Synovial uid is rich in brin, with lymphocytes, histiocytes, and sometimes synoviocytes predominating. The average cell count is 10,000×106/L cells.

Systemic Lupus Erythematosus

Synovial uid is straw-colored, and the predominant cells are lymphocytes, in variable numbers. The characteristic cell is Hargraves’ cell, or LE: a neutrophil twice or thrice the normal size withan eccentric nucleus surrounding an inclusion that appears red on panoptic staining. The average cell count is 12,000×106/L cells.

Macroscopic Evaluation, Crystal Detection, andCellular Analysis

Synovial uid is collectedthrough arthrocentesis in quantities of 3–5mL.

Normal synovial uid is generally light yellow, transparent, and viscous. In cases of rheumatic or infected effusions, it may be turbid; sometimes it may have a hemorrhagic appearance.

Gout

In the acute phase, the synovial uid may appear cloudy to milky. Fresh urate crystals, with the appearance of needles or rods, can be observed either free or in the cytoplasm of neutrophils. The average cell count is 15,000×106/L cells, and neutrophils are the predominant cell population, followed by synoviocytes.

Pseudogout or Chondrocalcinosis

The synovial uid is similar in cellular composition and appearance to that of gout, but the crystals are of calcium

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pyrophosphate, either in free form or phagocytosed. They are distinguishable by their rhomboidal shape and inhomogeneous size compared to urate crystals.

Traumatic Arthropathy

Synovial uid is hemorrhagic or xanthochromic in color, with poor cellularity (average cell count <2000 × 106/L cells), predominantly neutrophils, sometimes also tissue cells such as chondrocytes or bone chips and connective tissue.

Villonodular Synovitis

It is a rare benign tumor of the synovial membrane, in which the synovial uid is serohemorrhagic or brown, with few cellular elements (<3000 × 106/L cells), mainly histiocytes with, within them, hemosiderin, and sometimesplurinucleated giant cells.

Primary or Secondary Neoplastic Arthritis

The uid is serohemorrhagic with poor cellularity (mean cell count <2000×106/L), possible presence of “foreign” cellular elements typical of the histotype of the primary tumor.

Prosthetic Infections

In orthopedics, cytometric analysis of synovial uid is essential for the differential diagnosis between periprosthetic infections and other non-infectious diseases.

Many studies propose different decision thresholds, both for the leukocyte count and for the relative value of neutrophils and/or polymorphonucleates; unfortunately, the decision cutoffs are often not concordant, and this limitation has repercussions in clinical practice.

Pathological synovial uid can be divided into four categories, as shown in Table35.7.

may be helpful in selected cases. Table35.8 shows the reference values of the main measurands in synovial uid.

Proteins are generally >3.0g/dL in any type of inammatory process, thus they havelittle diagnostic or prognostic usufulness.

Cholesterol andTriglycerides

The concentration of lipoproteins in synovial uid is, on average, 40% lower than in plasma. In inammatory processes and in crystal-induced arthritis (rheumatoid arthritis, SLE, gout), their levels increase signicantly. Lipid effusions have been roughly divided into three groups: cholesterolrich, lipid droplets, and chylous.

In synovial effusions, cholesterol can reach concentrations as high as 2600mg/dL.

Chylous synovial effusions may be observed, rarely, in association with rheumatoid arthritis, SLE, trauma, lariasis, and pancreatitis (pancreatic-arthritic syndrome).

Glucose

The glucose level should be interpreted against the blood glucose level. Transient postprandial hyperglycemia is not reected until several hours later in a physiologic increase in synovial uid glucose. At equilibrium, normal synovial glucose is 10mg/dL lower than plasma glucose, or even less. In general, in non-inammatory or hemorrhagic joint diseases (osteoarthritis, villonodular synovitis pigmentosa, trauma, hemangioma, etc.), glucose may be, respectively, 10 and 20 mg/dL less than in plasma. In inammatory diseases, instead, the distance between plasma glucose and joint glucose can vary from 0 to 40mg/dL, while in infectious synovial forms, glucose can be 20–100mg/dL less than in plasma; to a lesser extent (from 0 to 80mg/dL), this is observed in crystal-induced forms.

Biochemistry

Although not routinely measured on synovial uid, some measurands, such as glucose, uric acid, lactic acid, lipids (cholesterol and triglycerides), proteins, and some enzymes

Table 35.7 Main laboratory tests on the synovial uid and related interpretative criteria

Macroscopic exam

Cytometric exam

Appearance Transparent Transparent Transparent/opaque Opaque Opaque Color Light to

Leukocytes (×10

Polymorphonuclear (%) Red blood cells Absent Absent Absent Present Present

Lactic Acid

The upperreference limit of the lactic acid is 25–30mg/dL.

There is ample evidence that synovial uid lactic acid is generally increased in monoarticular septic arthritis compared to non-septic forms.

In septic arthritis, lactic acid can reach concentrations of 1170mg/dL, 40 times the value of the upper reference inter-

Normal

paleyellow

/L) 0–150 <3000 3000–75.000 50,000–

<25 <30 >50 >90 <50

Group 1– Not inammatory

Xanthochromic Xanthochromic/

Group 2– Inammatory

white/blood

Group 3– Infectious

White Red/brown or

200,000

Group 4– Hemorrhagic

xanthromic 50–10,000

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Table 35.8

uid

Cholesterol Triglycerides Glucose (plasma-synovial) Lactic acid Proteins Uric acid (males) Uric acid (females) <6.0mg/dL Hyaluronic acid 3–4g/L

Reference values for the main biomarkers in the synovial

Reference range

≤65% of the corresponding plasma ≤40% of the corresponding plasma ≤10.0mg/dL ≤30.0mg/dL ≤3.0g/dL ≤8.0mg/dL

val, contrary to what happens in degenerative forms. However, it has been observed that lactic acid does not always increase in bacterial forms; an example is Neisseria gonorrhoeae form. This bacterium does not induce an increase in synovial lactic acid. In general, a lactic acid >110mg/dL indicatesa septic form.

Uric Acid

The denitive diagnosis of gout is based on microscopic observation of monosodium urate crystals in synovial uid.

Uric acid assay on synovial uid proves to be a valuable aid in the denitive diagnosis of gout. The decision limits for the diagnosis are moderately controversial. For some authors, there is a substantial equivalence between the serumconcentration of uric acid compared to the synovial uid; for others, instead, its values in gout are higher than in the serum, thus constituting a situation without interpretative uncertainties. Probably both observations are true, due to a dynamic balance between the constitution and dissolution of crystals that would be reected on the concentration of uric acid in this uid.

Enzymes

Numerous enzymes have been studied in synovial uid disorders: LDH, aspartate aminotransferase (AST), ALP, γ-glutamyltransferase, ADA, neuraminidase (lysozyme), cytidine deaminase, and several others. Their presumed clinical utility has not always been conrmed in practice. Among the enzymes of proven diagnostic help, LDH certainly stands out. Values between 400 and 700U/L suggest a moderatemedium inammatory state, while values >700U/L strongly indicates severe inammatory state.

C-Reactive Protein (CRP)

When the inammatory phenomenon is very circumscribed, in particular clinical pictures, assaying the CRP in a biological uid can be of considerable diagnostic and prognostic help as in the case of joint infections following implantation of prostheses, representing a more powerful tool than the same imaging.

Cerebrospinal Fluid

The cerebrospinal uid (CSF), part of the central nervous system (CNS), is clear and colorless (“rock water”); it contains numerous solutes, a very low concentration of proteins, and is isosmotic with plasma, due to a higher concentration of chlorides. The concentration of proteins, under physiological conditions, is 20–45 mg/dL. Its total volume is 140mL, although about 500 mL is produced in the course of a day.

CSF is produced in the ventricles by the choroidal plexuses, vascular formations of pial origin, projecting into the ventricles. The inner part of the ventricles, as well as the central canal of the spinal cord, is lined by a thin epithelial membrane called the ependyma. The choroid plexuses are equipped with unidirectional transport systems of ions from the periphery to the CSF (this involves by osmosis a movement of water) and, by dedicated mechanisms, glucose, and amino acids. Finally, the choroid plexuses synthesize the proteins of the CSF, such as transthyretin and asialotransferrin. The CSF passes from the ventricles to the subarachnoid spaces, where Pacchioni’s granulations reabsorb itand ends up in the venous system and, according to recent studies, also in the lymphatic system.

The blood-liquor barrier (BLB) allows the passage of plasmaticmolecules, especially proteins, into the CSF and should not be confused with the blood-brain barrier (BBB), which is, instead, impermeable, like a cell membrane, due to the presence of tight junctions that “weld” endothelial cells together.

CSF is enriched with solutes. Indeed,albumin, a protein used as a model for the passage from plasma to CSF because it is exclusively synthesized by the liver, has a concentration

2.5 times higher in the lumbar CSF than in the ventricular one.

CSF also has a cellular component (lymphocytes and monocytes) due to previously unknown connections between the meninges and the lymphatic vessels.

Circulating in a rigid bone structure, in case of increased production or reduced reabsorption, the amount of CSF cannot increase much, but its pressure increases, creating an endocranial hypertension that pathologically compresses the CNS.The CNS is reduced in volume and the CSF volume increases, a phenomenon called hypertensive hydrocephalus. Normotensive hydrocephalus occurs when CSF volume increasesdue to a primitive reduction of the cerebral mass, generally due to atrophic phenomena.

CSF is collected by lumbar puncture, usually performed by the neurologist through the intervertebral space between L4 and L5 or L5 and S1, where there is no spinal cord. This maneuver frequently results in blood contamination due to accidental rupture of capillaries in the needlepath. For every

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600 redblood cells present in thesampledue to collection, there is one additional leukocyte in the CSF, and for every 1000 red blood cells, there is a 1 mg/dL increase in total proteins.

To discriminate between the presence of blood in the sample due to traumatic lumbar puncture and blood from subarachnoid hemorrhage, the CSF should be collected, whatever the quantity taken, in three tubes after having numbered them: in case of blood appearance from puncture, this will tend to disappear passing from the rst to the third tube.

CSF is currently studied in the suspicion of the following diseases:

• Inammatory meningitis on an infectious basis, or due to

carcinomatosis bymetastatic localization of tumors out-

side or within CNS (not to be confused with meningio-

mas, which are primary tumors of the meninges).

• Encephalitis, which can be infectious, inammatory,auto-

immune, often as a result of previous infection of the CNS

or degenerative type, either on a genetic basis or acquired

sporadic.

• Polyradiculoneuritis, inammation of spinal and cranial

nerve roots, acute or chronic.

• Myelitis, diseases conned to the spinal cord.

• Acute hemorrhagic phenomena, such as subarachnoid

hemorrhages.

tient (QAlb). The reference values of total protein and QAlb change with age: they are higher in infants, lower in children, and rise with age. It was determined that the reference value (RV) of QAlb, for subjects aged 11–80years, should be calculated using the formula: QAlb=age/25+8.

The QAlb is a dimensionless gure, which does not require standardization of the albumin assay. The dosage of proteins of brain origin is expressed with absolute values, generally in mass concentration, with thresholds discriminating sick from healthy.

The dosage of intrathecal synthesis proteins is fundamental, because they are pathognomonic of CNS pathology. Electrophoretic fractionation is very useful, especially with isoelectrophoresis (IEF), as in the case of IgG of intrathecal synthesis, for which the patterns have long been encoded. The formulas quantifying the intrathecal fraction, the more complex ones, such as the Reiber equation, take into account the greater passage of specic proteins as the barrier damage increases and are to be preferred because they are more specic.

Analyses can be of the following types:

• Macroscopic analysis, usually performed by the neurolo-

gist at the time of the lumbar puncture

• Cytometric and cytological examination to count and

identify the CSF cells

• Biochemical examination (metabolites, proteins, etc.)

The molecules reach the CSF by simple passage through the blood-liquor barrier, or by secretion from the choroid plexuses, or by drainage from the central nervous tissue. Exit, not at all selective, occurs from the arachnoid granulations and most likely from the lymphatic structures of the meninges, as recently proposed.

CSF proteins fall into three categories according to their origin:

1. Plasma, by simple passage from the plasma to the CSF

through the BLB, in an amount dependent on the state of the barrier.

2. Brain, only from the CNS; they increase in case of tissue

damage (e.g., tau proteins and phosphorylated tau in Alzheimer’s disease).

3. Mixed (plasma-cerebral).

In CSF diagnostics, the use of proteins has a threepronged approach: assaying proteins as markers of barrier status, assaying proteins of brain origin, and assaying proteins of intrathecal synthesis.

The total protein assay is the most widely used test to diagnose barrier damage.As damage increases, its selectivity decreases. However, the test cannot detect simultaneous or exclusive increases in brain-derived proteins. The recommended method for assessing barrier damage is to assay albumin, either in CSF or serum, calculate the ratio, and multiply the result by 1000. This gives the albumin quo-

Macroscopic Evaluation andCellular Analysis

Under pathological conditions, the CSF loses its transparent and clear appearance, becoming increasingly turbid. The cornerstone of cellular analysis is not only cytometric analysis, by cell counting and differential counting of lymphocytes, monocytes, and granulocytes, but also qualitative evaluation by cytological examination for the search and reporting of particularly voluminous cells, probably of neoplastic origin, especially if in clusters. The cytometric examination is fundamental for diagnosing meningitis and allowing to dene thetype. Bacterial meningitis are characterized by a high number of neutrophil granulocytes (typically greater than 1000× 106/L), while viral meningitis are characterized by a lower number of mononuclear, typically lymphocytes.

In the resolving stages of subarachnoid hemorrhages, siderophages may also be present: macrophages with iron inclusions.

Biochemistry

Glucose is used to estimate the extent of the presence of bacteria and cells; it should be reported as CSFto serum glucose ratio; reference value >0.4–0.5.

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Lactates, a product of anaerobic glucose metabolism, increase under all conditions in which glucose is metabolized in the CSF.Since they are not affected by plasma concentration, they are reported in mmol/L; reference value <2.8.

Total protein is a crude indicator of blood-liquor barrier damage.

Immunoglobulin G, when intrathecally synthesized, has long been used, and still is, to make the diagnosis of multiple sclerosis (MS).

M-immunoglobulins have been dened as indicators of the type of CNS diseaseand not markers of recent infections: However, epidemiological studies and case reports have shown that they appear rst and alone in acute infections.

Immunoglobulin A is a marker of purulent meningitis and neurotuberculosis.

Immunoglobulin free light chains are candidates to become the best indicator of immune activation in the CNS.

Specic antibodies should be investigated using criteria that allow their intrathecal synthesis fraction to be identied exclusively by calculating the Antibody Index (AI): the numerator is the quotient of the specic antibody and the denominator is the quotient of the total immunoglobulins of the same class. The search for specic liquid antibodies to infectious agents, especially viral, is also extremely useful in the era of molecular diagnostics. A particular application of the search for specic IgG antibody response is the so-called MRZ (measles, rubella, zoster) reaction searchto discriminate MS patients from patients with viral infection,oraffected by other CNS inammatory diseases.

The search for tumor markers in the CSF is very useful for diagnosing meningeal carcinomatosis together with the search for tumor cells. CEA and mucin markers such as CA15.3, CA125, and CA19.9 are used. β-hCG is a specic marker of cerebral germinoma, but it may be present in highly undifferentiated tumors.

Autoantibodies against the CNS are responsible for well- dened neurological diseases. They are mainly to be sought in serum. Intrathecal synthesis can be demonstrated in the CSF, but the synthesis is not of particular clinical signicance. Only anti-MNADr autoantibodies are to be sought in the CSF, because they are often higher in the CSF than in the serum, where sometimes they may be absent.

Biomarkers of Alzheimer’s disease (AD), a neurodegenerative disease accounting for 50–60% of clinically diagnosed forms of dementia, are exclusively in the CSF.They include β-amyloid protein, resulting from the proteolysis of an amyloid precursor and constituting amyloid plaques; total tau protein (T-tau), associated with microtubules and mainly located in the axon, which has higher values in AD patients; phosphorylated tau protein (p-tau), a hyperphosphorylated form of tau protein and expression of neurobrillary degeneration, which has higher values in AD patients.

Creutzfeldt-Jakob disease, the best known prion disease, does not have a specic biomarker. For its diagnosis, the protein 14-3-3, not related to the prion, but typical of extensive neuronal necrosis, is used.

Markers of subarachnoid hemorrhage are indicative of a rupture of a vessel in the subarachnoid space, representing an acute and dramatic event with low probability of survival for the patient. The reference examination is computed tomography of the brain, which demonstrates the presence of a hemorrhagic infarction. The CSF examination is performed only in doubtful cases, 2% of the total, especially in the post-acute phase, in search of hemoglobin and bilirubin, the main product of hemoglobin metabolism.

The markers of CSF, presence of CSF outside its physiological sites, indicate an abnormal communication of the cerebral spaces with the external environment, with a strong risk of infection. The most frequent form is rhinoliquorrhoea, a non-acute episode, often post-traumatic, of otorhinolaryngological scope. Post-neurosurgical CSF may represent an urgent complication of a recent neurosurgical procedure. The search for asialotransferrin, isoform of transferrin produced in the CNS, is the reference test, but the β-trace protein (β-TP), produced in the CNS, present in the CSF in concentrations much higher than in plasma, and rapidly measured on automatic analyzers is now the test of choice for these urgent requests.

CSF chlorides, which are higher in CSF than in plasma, decrease in chronic meningitis, such as tuberculosis.

CSF LDH is the only enzyme currently assayed, because it is produced by CSF cells. It is an absolutely non-specic marker of inammation, if its isoenzymes are not assayed. Thus, it has a high negative predictive value. Physiological values are <21 U/L.