chapter 3. body fluid analysis: serous fluid

body fluid

1. Chapter three Serous Fluid analysis

2. Chapter outline Introduction to Serous fluid analysis Routine laboratory assays Collection of sample Gross appearance Cell counts Chemical analysis Morphological Examination Microbiological Examination Serological Examination

3. Learning Objectives Upon completion of this chapter the student will be able to: Describe the sample collection and examination of Serous fluid. Describe the function of the Serous fluid analysis . Discuss difference between exudates and transudes Explain the normal values of chemical analysts of Serous fluid

4. Learning Objectives 5.Differentiate between a hemothorax and a hemorrhagic exudate . 6 Differentiate between a chylous and a pseudochylous exudate . 7 State the significance of increased neutrophils, lymphocytes, eosinophils , and plasma cells in pleural fluid. 8 Describe the morphologic characteristics of Serous Fluid

5. Introduction The closed cavities of the body—namely, the pleural, pericardial, and peritoneal cavities—are each lined by two membranes referred to as the serous membranes. One membrane lines the cavity wall (parietal membrane), and the other covers the organs within the cavity (visceral membrane). The fluid between the membranes is called serous fluid, and it provides lubrication between the parietal and visceral membranes.

6. Introduction An effusion is fluid which collects in a body cavity or joint. Fluid which collects due to an inflammatory process is referred to as an exudate and that which forms due to a non-inflammatory condition is referred to as a transudate. When the effusion is an exudate , it is important to investigate whether the inflammatory process is an infective one (septic) or caused by a non-infective process, e.g. malignancy. When the fluid is a transudate, no further microbiological testing is required.

7. Formation Serous fluids are formed as ultrafiltrates of plasma, with no additional material contributed by the mesothelial cells that line the membranes. Production and reabsorption are subject to hydrostatic and colloidal ( oncotic ) pressures from the capillaries that serve the cavities and the capillary permeability.

8. Pathologic Causes of Effusions 1. Increased capillary hydrostatic pressure Congestive heart failure Salt and fluid retention 2. Decreased oncotic pressure Nephrotic syndrome Hepatic cirrhosis Malnutrition Protein-losing enteropathy 3. Increased capillary permeability Microbial infections Membrane inflammations Malignancy 4. Lymphatic obstruction Malignant tumors, lymphomas Infection and inflammation Thoracic duct injury

9. Pleural Fluid

10. PLEURAL EFFUSION The pleural space lies between the lung and chest wall and normally contains a very thin layer of fluid, which serves as a coupling system. A pleural effusion is present when there is an excess quantity of fluid in the pleural space. Pleural effusions may be of either transudative or exudative origin. In addition the pleural fluid cholesterol and fluid:serum cholesterol ratio and the pleural fluid:serum total bilirubin ratio will done

11. Pleural Fluid The pleural cavity normally contains a small amount of fluid that facilitates movement of the visceral and parietal pleura each other. A plasma filtrate derived from capillaries of the parietal pleura. Produced continuously at a rate dependent on capillary hydrostatic pressure, plasma oncotic pressure, and capillary permeability. Reabsorbed through the lymphatic and venules of the visceral pleura.

12. Pleural Fluid cont’d… Effusion: an accumulation of fluid, which results from an imbalance of fluid production and reabsorption. Serous effusion: fluid accumulation in the pleural, pericardial, and peritoneal cavities Pleural fluid accumulates when pleural fluid formation exceeds pleural fluid absorption. Normally, fluid enters the pleural space from the capillaries in the parietal pleura and is removed via the lymphatics situated in the parietal pleura.

13. Pleural Fluid cont’d… Fluid can also enter the pleural space from the interstitial spaces of the lung via the visceral pleura or from the peritoneal cavity via small holes in the diaphragm. The lymphatics have the capacity to absorb 20 times more fluid than is normally formed. Accordingly, a pleural effusion may develop when there is excess pleural fluid formation or when there is decreased fluid removal by the lymphatics .

14. The normal formation and absorption of pleural fluid.

15. Diagnostic Approach When a patient is found to have a pleural effusion, an effort should be made to determine the cause The first step is to determine whether the effusion is a transudate or an exudate . A transudative pleural effusion occurs when systemic factors that in fluence the formation and absorption of pleural fluid are altered.

16. Diagnostic Approach Transudative and exudative pleural effusions are distinguished by measuring the lactate dehydrogenase (LDH) and protein levels in the pleural fluid. Exudative pleural effusions meet at least one of the following criteria, whereas transudative pleural effusions meet none: 1. pleural fluid protein/serum protein 0.5 2. pleural fluid LDH/serum LDH 0.6 3. pleural fluid LDH more than two-thirds normal upper limit for serum

17. Pleural Fluid cont’d… Specimen Collection The physician is expected to draw approprate amount of sample from the patient Should be labeled with all information like above one Should be handled appropriately during delivery to the laboratory Avoid in appropriate testing Except for an EDTA tube for total and differential cells counts, the specimen should be collected in heparinized tubes to avoid clotting

18. Specimen Collection cont’d… Aliquots for aerobic and anaerobic bacterial cultures are best inoculated into blood culture media at the bedside. If malignancy, fungal infection, or mycobacterial infection is suspected, all remaining fluid (100 mL or more) should be submitted to maximize yield of stains and culture. fresh specimens for cytology may be stored up to 48 hours in the refrigerator with satisfactory results initial classification of a pleural fluid as a transudate or an exudate greatly simplifies the process of arriving at a correct final diagnosis

19. Transudates and Exudates cont’d…. Transudates : are usually bilateral owing to systemic conditions leading to increased capillary hydrostatic pressure or decreased plasma oncotic pressure Non-inflammatory condition No need further investigation

20. Transudates :cont… increased hydrostatic pressure or decreased plasma oncotic pressure Congestive heart failure Hepatic cirrhosis Hypoproteinemia (e.g., nephritic syndrome)

21. Exudates: Exudates are more often unilateral, associated with localized disorders that increase vascular permeability or interfere with lymphatic resorption Because of any inflammatory condition

22. Exudates: cont… Exudates: increased capillary permeability or decreased lymphatic resorption Infections  Bacterial pneumonia  Tuberculosis granulomatous diseases (e.g., sarcoidosis, histoplasmosis, etc.)  Viral or mycoplasma pneumonia Neoplasms Bronchogenic carcinoma  Lymphoma  Pulmonary infarct (may be associated with hemorrhagic effusion)

23. Transudate and Exudates cont’d… Classical teaching stressed that exudates and transudates can be distinguished on the basis of total protein concentrations above (exudates) or below ( transudates ) 3.0 g/ dL . However, using total protein alone misclassifies both exudates and transudates by about 30%

24. Recommended Tests The evaluation of serous body fluids (pleural, pericardial, peritoneal) is directed first toward differentiating transudative from exudative effusions.

25. Gross Examination Transudates are typically clear, pale yellow to straw-colored, odorless, and do not clot. Approximately 15% of transudates are blood tinged. A bloody pleural effusion ( hematocrit > 1%) suggests trauma, malignancy, or pulmonary infarction A traumatic tap is suggested by uneven blood distribution, fluid clearing with continued aspiration, or formation of small blood clots.

26. Exudates may grossly resemble transudates , but most show variable degrees of cloudiness or turbidity, and often clot if not heparinized . A fecalent odor may be detected in anaerobic infections. Turbid, milky, and/or bloody specimens should be centrifuged and the supernatant examined. If the supernatant is clear, the turbidity is most likely due to cellular elements or debris. If the turbidity persists after centrifugation, a chylous or pseudochylous effusion is likely Gross Examination

27.  Characteristic Features of Chylous and Pseudochylous Effusions Feature Chylous Pseudochylous Onset Sudden Gradual Appearance Milky-white, or yellow to bloody Milky or greenish, metallic sheen Microscopic examination Lymphocytosis Mixed cellular reaction, cholesterol crystals Triglycerides [*][†] ≥ 110 mg/dL (≥ 1.24 mmol/L) < 50 mg/dL (< 0.56 mol/L) Lipoprotein electrophoresis Chylomicrons present Chylomicrons absent

28. Microscopic Examination Cell Counts. Leukocyte counts are unreliable in separating transudates (< 1000/ μL ) from exudates (> 1000/ μL ). Although red cell counts above 100 000/ μL are highly suggestive of malignancy, trauma, or pulmonary infarction, they have little practical value. Differential Leukocyte Count and Cytology . Examination on a stained smear, prepared by cytocentrifugation and an air-dried Romanowski's stain. Indeed, examination by the hematology laboratory can be highly effective in the detection of malignant cells

29. Cell Counts. Similar to other body fluids, an increase in pleural fluid neutrophils is indicative of a bacterial infection, such as pneumonia. Neutrophils are also increased in effusions resulting from pancreatitis and pulmonary infarction.

30. Mesothelial cells are common in pleural fluids from inflammatory processes

31. well  -differentiated breast carcinoma cells in pleural fluid

32.   Undifferentiated oat cell carcinoma of lung showing typical molding of nuclei

33. Normal pleural fluid mesothelial cells, lymphocytes, and monocytes (250).

34. Neutrophilia (> 50%) Bacterial pneumonia (Para pneumonic effusion) Pulmonary infarction Pancreatitis Subphrenic abscess Early tuberculosis Transudates (over 10%)

35. Lymphocytosis (> 50%) Tuberculosis (mesothelial cells are rare) Viral infection Malignancy True chylothorax Rheumatoid pleuritis Systemic lupus erythematosus Uremic effusions Transudates (approximately 30%)

36. Eosinophilia (> 10%) Pneumothorax (air in pleural space) Trauma Pulmonary infarction Congestive heart failure Infection (especially parasitic, fungal) Hypersensitivity syndromes Drug reaction Rheumatologic diseases Hodgkin's disease Idiopathic

37. Chemistry Tests chemical tests performed on pleural fluid are glucose, pH, adenosine deaminase (ADA), and amylase. Triglyceride levels may also be measured to confirm a chylous effusion. Decreased glucose levels are seen with tuberculosis, rheumatoid inflammation, and purulent infections. As an ultrafiltrate of plasma, pleural fluid glucose levels parallel plasma levels with values less than 60 mg/ dL considered decreased. Fluid values should be compared with plasma values. Pleural fluid lactate levels are elevated in bacterial infections and can be considered in addition to the glucose level.

38. Chemical Analysis Protein. The measurement of pleural fluid total protein or albumin has little clinical value except when combined with other parameters to differentiate exudates from transudates . Protein electrophoresis shows a pattern similar to serum except for a higher proportion of albumin; it has little value for differential diagnosis

39. Glucose. The glucose level of normal pleural fluid, transudates , and most exudates is similar to serum levels. Decreased pleural fluid glucose, accepted as a level below 60 mg/ dL (3.33 mmol /L) or a pleural fluid/serum glucose ratio less than 0.5, is most consistent and dramatic in rheumatoid pleuritis and grossly purulent parapneumonic exudates ( Sahn , 1982 ). Low pleural fluid glucose may also be present in malignancy, tuberculosis, nonpurulent bacterial infections, lupus pleuritis , and esophageal rupture.

40. Lactate. Pleural fluid lactate levels can be a useful adjunct in the rapid diagnosis of infectious pleuritis. Levels are significantly higher in bacterial and tuberculous pleural infections than in other pleural effusions. Moderate elevations are generally observed in malignant effusions ( Brook, 1980 ). Values greater than 90 mg/dL (10 mmol/L) have a positive predictive value for infectious pleuritis of 94% and a negative predictive value of 100% ( Gastrin,

41. Enzymatic tests Amylase elevations above the serum level (usually 1.5-2.0 or more times greater) indicate the presence of pancreatitis, esophageal rupture, or malignant effusion Elevated amylase derived from esophageal rupture or malignancy is the salivary isoform , which differentiates it from pancreatic amylase Pleural fluid lactate dehydrogenase (LD) levels rise in proportion to the degree of inflammation. In addition to its use in separating exudates from transudates , declining LD levels during the course of an effusion indicate that the inflammatory process is resolving.

42. Interferon-gamma (INF-gamma). Pleural fluid INF-gamma levels are significantly increased in pleural fluid of patients with tuberculous pleuritis. The sensitivity of levels 3.7 IU/L or greater is 99% and the specificity is 98%. The test sensitivity does not differ in HIV-positive and HIV-negative patients. Only about 20% of patients with effusions due to hematologic malignancies have INF-gamma levels slightly above 3.7 IU/L

43. pH . Pleural fluid pH measurement has the highest diagnostic accuracy in assessing the prognosis of parapneumonic (pneumonia-related) effusions. A parapneumonic exudate with a pH greater than 7.30 generally resolves with medical therapy alone. A pH less than 7.20 indicates a complicated parapneumonic effusion ( loculated or associated with empyema ) requiring surgical drainage.

44. Immunologic Studies Approximately 5% of patients with rheumatoid arthritis (RA) and 50% with systemic lupus erythematosus (SLE) develop pleural effusions sometime during the course of their disease. Rheumatoid factor ( RF) is commonly present in pleural effusions associated with seropositive RA. Although a pleural fluid titer of 1:320 or greater in a patient with known RA is reasonable evidence of rheumatic pleuritis Antinuclear antibody (ANA) titers may be useful in the diagnosis of effusions due to lupus pleuritis

45. Microbiological Examination Bacteria most commonly associated with parapneumonic effusions are Staphylococcus aureus, Streptococcus pneumoniae, beta-hemolytic group A streptococci gamma-streptococci some Gram-negative bacilli. patients with suspected M. tuberculosis , direct staining of tuberculous effusions for acid-fast bacteria has a sensitivity of 20-30 %

46. Pericardial Fluid

47. Pericardial Fluid Normally, only a small amount (10 to 50 mL ) of fluid is found between the pericardial serous membranes. Pericardial effusions are primarily the result of changes in the permeability of the membranes due to infection ( pericarditis ), malignancy, and trauma-producing exudates. Metabolic disorders such as uremia, hypothyroidism, and autoimmune disorders are the primary causes of transudates . The presence of an effusion is suspected when cardiac compression ( tamponade ) is noted during the physician’s examination.

48. Pericardial Fluid 10-50 mL of fluid is normally present in the pericardial space produced by a transudative process similar to pleural fluid. Pericardial effusion caused by : viral infection enterovirus being the most common. bacterial, tuberculous or fungal infections autoimmune disorders, renal failure, myocardial infarction. Many of the recommended laboratory tests described for pleural fluid also pertain to pericardial effusions

49. Pericardial Fluid cont’d… Specimen Collection Performed by experienced professional should be delivered within 1 hours if delay is mandatory the approprate preservative for the desired test should be added as soon as it reached the lab gross examination should be performed

50. Pericardial Fluid cont’d… Gross Examination Normal pericardial fluid : pale yellow and clear. Large effusions (> 350 mL ) are most often caused by malignancy or uremia, or are idiopathic. Turbid effusions :Infection or malignancy clear and straw-colored: effusions due to uremia

51. Appearance Normal and transudate pericardial fluid appears clear and pale yellow. Effusions resulting from infection and malignancy are turbid, and malignant effusions are frequently blood streaked. Grossly bloody effusions are associated with accidental cardiac puncture and misuse of anticoagulant medications. Milky fluids representing chylous and pseudochylous effusions may also be present.

52. Microscopic Examination The hematocrit and red cell count document the presence of a hemorrhagic effusion Total leukocyte counts over 10 000/μL suggest bacterial, tuberculous, or malignant pericarditis. Although formal leukocyte differentials add little diagnostic information, a stained smear should always be examined.

53. Chemical Analysis Protein . A value greater than 3.0 g/ dL has a sensitivity of 97% for exudative effusions Glucose . Pericardial glucose levels less than 60 mg/ dL have a diagnostic accuracy of only 36% in identifying pericardial exudates pH . Pericardial fluid pH may be markedly decreased (< 7.10) in rheumatic or purulent pericarditis

54. Microbiological Examination The sensitivity of the Gram stain and culture for bacterial pericarditis is similar to other serous body fluids Diagnosis of a specific etiologic agent in viral pericarditis is difficult because the viruses (e.g., Coxsackieviruses , influenza virus, mumps) are rarely isolated from pericardial fluid. Obtaining acute and convalescent sera for antibody response to suspected viral pathogens may help support the diagnosis. The sensitivity of acid-fast stains and culture for tuberculous pericarditis is about 50% PCR is a sensitive technique

55. Peritoneal Fluid

56. Peritoneal Fluid Accumulation of fluid between the peritoneal membranes is called ascites , and the fluid is commonly referred to as ascitic fluid rather than peritoneal fluid. In addition to the causes of transudative effusions, hepatic disorders such as cirrhosis are frequent causes of ascitic transudates . Bacterial infections (peritonitis)—often as a result of intestinal perforation or a ruptured appendix—and malignancy are the most frequent causes of exudative fluids

57. Peritoneal Fluid Ascites is the pathologic accumulation of excess fluid in the peritoneal cavity. Up to 50 mL of fluid is normally present in this mesothelial-lined space. As with pleural and pericardial fluids, it is produced as an ultrafiltrate of plasma dependent on vascular permeability, and hydrostatic and oncotic Starling forces.

58. Peritoneal Fluid cont’d… Specimen Collection Done by clilinicians Collected in in sterile test tube Delivered to the lab within 1 hour The test performed in appropriate technique Sufficient sample is needed( A minimum of 30 m) Sample of cell count should be placed in an EDTA- anticoagulated venipuncture tube. Culture specimens should include blood culture bottles that have been inoculated at the bedside with ascetic fluid (10 mL per culture bottle).

59.  Recommended Tests in Peritoneal Effusion Useful in most patients Gross examination Cytology Stains and culture for microorganisms Serum– ascites albumin concentration gradient Useful in selected disorders Total leukocyte and differential cell counts RBC count ( lavage ) Bilirubin Creatinine/urea nitrogen Enzymes (ADA, ALP, amylase, LD, telomerase) Lactate Cholesterol (malignant ascites )

60. Peritoneal Fluid cont’d… Gross Examination Milky fluid that does not clear with centrifugation suggests a chylous or pseudochylous effusion. True chylous peritoneal effusions are significantly less common than chylous pleural fluids. Caused by disruption or blockage of lymphatic flow by trauma, lymphoma, carcinoma, tuberculosis or other granulomatous diseases , hepatic cirrhosis, adhesions, or parasitic infestation.

61. Peritoneal Fluid cont’d… Microscopic Examination The total leukocyte count is useful in distinguishing ascites due to uncomplicated cirrhosis from spontaneous bacterial peritonitis (SBP), which is caused by migration of bacteria from the intestine into the ascetic fluid. Approximately 90% of patients with SBP will have leukocyte counts greater than 500/μL, over 50% of which are neutrophils

62.   Neutrophils in a patient with bacterial peritonitis

63. Peritoneal Fluid cont’d… Eosinophilia most commonly > 10% associated with the chronic inflammatory process with chronic peritoneal dialysis. it is also reported in congestive heart failure, vasculitis, lymphoma, and ruptured hydatid cyst.

64. Peritoneal Fluid cont’d… Chemical Analysis Protein. Spontaneous bacterial peritonitis is commonly associated with low total protein (< 3.0 g/dL) and a high serum–ascites albumin gradient (> 1.1 g/dL), making total protein measurements of little value in this disorder. Glucose. peritoneal fluid glucose levels of 50 mg/dL or less are present in 30-60% of cases of tuberculous peritonitis and about 50% of patients with abdominal carcinomatosis

65. Peritoneal Fluid cont’d… Microbiological Examination The bacteria in SBP are most often normal intestinal flora and over 92% are monomicrobial. The Gram stain has a sensitivity of 25% in SBP and routine cultures are positive in only about 50% of cases Ascetic fluid total neutrophils count is the preferred method for the diagnosis of SBP