role of pleural fluid analysis in pulmonary pathology.pptx

Fluid analysis

1. ROLE OF PLEURAL FLUID ANALYSIS IN PULMONARY PATHOLOGY Dr.M.P.Sreelekha PG-III

2. INTRODUCTION: Pleural cavity is a potential space between chest wall and lung lined on either side by parietal and visceral pleura. Contains fluid (<10 ml) in each cavity. Pleural fluid is an ultra-filtrate of plasma and lubricates the two opposing pleural layers. Formation: capillaries in parietal pleura. (depending on permeability, hydrostatic pressure, plasma colloid oncotic pressure) Resorption: through the lymphatics and venules of the visceral pleura

3. It is clear and straw or pale yellow in colour. Composition is similar to plasma except for low protein (<1.5gm/dl).

4. PLEURAL EFFUSION: Accumulation of excess fluid in pleural cavity is called as pleural effusion Irrespective of their cellular composition, all effusions are pathological. Pleural cavities can hold up to 3 L of fluid in each cavity The causative factors include increased vascular permeability, vasodilatation, blockage of lymphatics, breakdown of small blood vessels and haemodynamic imbalance of the microcirculation Reactive effusions are more common than malignant effusions.

5. Specimen collection and storage: Freshly collected fluid, up to 1000 mL, should be transported to the laboratory. Anticoagulants: EDTA (for cell counts), heparin ( 3 U/mL of fluid) (prevents clotting) Fixatives should be avoided for collecting and transporting effusion specimens Storage: up to 48 hours when refrigerated between 2-8°c

6. Bacterial culture should be carried out bedside If malignancy, fungal infection, or mycobacterial infection is suspected, all fluid (100 mL or more) should be submitted to maximize yield of stains and culture

7. Cytopreparation For semiquantitative evaluation of cellularity, a direct smear of the unconcentrated effusion specimen-stained with Diff- Quik is recommended. Concentrated sediment is processed for Diff- Quik , PAP, and haematoxylin and eosin (H & E)-stained smears and cell block sections. Cell blocks are more effective for IHC, special stains such as mucicarmine, PAS in suspected cases of malignancies. Methods used: wet fixed smears, air dried smears, cytospin smears, LBC smears

8. Initial classification of a pleural fluid as a transudate or an exudate greatly simplifies the process of arriving at a correct final diagnosis. It determines whether further testing is needed. Transudates generally require no further work-up. Treated by treating systemic illness.

9. Other criteria for exudates:

10. It is now well accepted that test combinations increase sensitivity and improve accuracy.

11. EXUDATES WORK UP Gross examination Examination of Romanowski-stained smear (malignant cells, LE cells) and cell counts Stains and cultures for microorganisms Cytology Chemical analysis

12. Gross examination Colour of pleural fluid Analysis Orange to deep red Malignant effusions Light brown Chronic haemorrhagic effusions ( hemosiderophages ) Chocolate brown Melanoma cells Thick greyish white to yellow sediment Hypercellular effusions Milky white with top lipid layer Chylous effusion viscous Diffuse malignant mesothelioma

13. BLOODY: Trauma, Malignancy, Pulmonary infarction. Haemothorax: if haematocrit of pleural fluid is >50% of blood haematocrit. TURBID/MILKY/BLOODY ON CENTRIFUGATION SUPERNATANT CLEAR TURBIDITY PERSISTS CELLULAR ELEMENTS/ DEBRI CHYLOUS EFFUSION PSUEDO CHYLOUS EFFUSION

14. CHYLOUS PSEUDOCHYLOUS CAUSE: Produced by leakage from the thoracic duct from obstruction by lymphoma, carcinoma, or traumatic disruption Accumulate gradually through the breakdown of cellular lipids in longstanding effusions such as rheumatoid pleuritis, tuberculosis, or myxedema 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

15. MICROSCOPY (exudate) TOTAL COUNT >1000/ microlit Performed on a stained smear, preferably prepared by cytocentrifugation and an air-dried Romanowski's stain. Filtration or automated concentration methods with Papanicolaou stain may also be used. Both smears and blocks are to be prepared if malignancy is suspected.

16. MICROSCOPY INFLAMMATORY CELLS MESOTHELIAL CELLS MALIGNANT CELLS NEUTROPHILIA (>50%) LYMPHOCYTOSIS (>50%) EOSINOPHILS (>10%) Bacterial pneumonia Pulmonary infarction Pancreatitis Subphrenic abscess Early tuberculosis Tuberculosis Viral infection Malignancy Chylothorax Rheumatoid pleuritis SLE Uremic effusions Pneumothorax Trauma Pulmonary infarction CHF Infection -parasitic, fungal Drug reaction Rheumatologic diseases Hodgkin's disease Idiopathic Inflammation RARE IN: TB pleurisy Empyema Rheumathoid plueritis pleurodesis MORPHOLOGY: Romanowsky/PAP stain IHC FLOW CYTOMETRY

17. Purulent Effusions: >90% neutrophils (degenerating) 5-10% lymphocytes and macrophages Mesothelial cells are rare in purulent effusions and if present show features of degeneration with vacuolated cytoplasm Characteristic background staining due to cellular debris*

18. Sterile Acute Inflammatory Effusions Pneumonia, influenza, pulmonary collapse, and in the "sympathetic" effusion secondary to subdiaphragmatic abscess. Pleomorphic cellular exudate including neutrophils, lymphocytes, macrophages, mesothelial cells, eosinophils and basophils, usually in that order of frequency.

19. Pulmonary Infarct Show numerous degenerating mesothelial cells Blood stained Effusion related to SLE: LE cell if identified is diagnostic Shows variable cell morphology High chances of finding LE cell if neutrophils are more.

20. Tuberculous effusions: Mesothelial cells scarce Lymphocyte predominant Neutrophils 10-40% if effusion < 14 days Pus cells – tuberculous empyema Lymphocyte rich with scarce mesothelial cells: Tuberculous effusion Malignant effusion Postpnemonic effusion

21. Malignant effusions: Two types: Indirect : inflammatory effusion secondary to obstruction and collapse of lobe. Ex: Bronchial carcinoma Direct : Due to metastatic involvement of pleura. Features suggesting malignant (direct) effusions: Heavy bloodstaining High proportion of lymphocytes Abundance of mesothelial cells

22. ‘Two cell population approach ’ for making a final interpretation of malignancy in effusion specimens is simple and effective. Mesothelial cells in effusions show a wide morphological spectrum, all cells are seemingly of one type and demonstrate a subtle morphological continuum. Malignant effusions with metastatic tumour cells usually show a morphologically alien population (easily made out in diff quick )

23. MESOTHELIOMA: More than 80% of all patients with malignant mesothelioma develop body cavity effusion at an early stage of the disease. Malignant cells are similar to mesothelial cells with large size, multinucleation Arranged as large aggregates and cell balls forming hollow spheres, papillary clusters Canabalism Nuclear irregularities and uneven vacuolations absent ( adenoca ) Imaging findings such as pleural-based mass and relevant clinical findings such as relapsed effusions must be taken into consideration, and support a cytologic diagnosis of malignant mesothelioma

24. E-Cadherin: Malignant mesotheliomas Positive; Reactive mesothelial cells negative

25. ADENOCARCINOMA Most common malignancy in malignant effusion. ( mets ) Shows 2 cell population- reactive mesothelial cells and malignant cells Cellular pattern: Predominantly appear as clusters : The clusters of malignant cells are tight and compact, usually with smooth borders The cells mold , each taking the shape of the neighboring cell, a “ cell within cell ” pattern may be seen Cellular morphology: Pleomorphism, multinucleation, high nucleo -cytoplasmic ratio, hyperchromasia , abnormal nucleoli, and clumped, irregular chromatin.

26. Immunocytochemistry panel for adenoca and mesotheliomas Adenocarcinomas - the most common pattern—EMA positive, CEA positive, Ber-EP4 positive, B72.3 positive, and LeuM1 positive Malignant mesotheliomas, the most common patterns - EMA positive, CEA negative, Ber-EP4 negative, B72.3 negative, and LeuM1 negative

27. Squamous cell carcinoma lung: Rarely showed in pleural effusions Mature squamous cells if seen are due to: 1. Introduction of microscopic fragments of skin during puncture. 2. Perforation of oesophagus, stomach or bowel, so that swallowed buccal cells gain access to the pleural cavity

28. Small cell carcinoma lung: Commonest type of malignant cell in effusions due to primary carcinoma of the lung

29. Chemical analysis: Glucose: Decreased (<60mg/dl or p.f glucose: serum glucose - <0.5) seen in: Rheumathoid pleuritis Grossly purulent parapnemonic effusions Malignancies Tuberculous effusions Lupus pleuritis Esophageal rupture

30. Pleural fluid lactate: Significantly increased: (>90mg/dl) – Tuberculous effusion, pneumonic effusions Moderately increased : Malignancy Lactate dehydrogenase (LD) Proportional to degree of inflammation It is use in separating exudates from transudates. Declining LD levels during the course of an effusion indicate that the inflammatory process is resolving

31. Enzymes: Amylase: (1.5 – 2 times serum) Seen in pancreatitis, esophageal rupture and malignant effusions. ADA: adenosine deaminase (>50IU/L) – TB Pleuritis PH : >7.3 – pleural effusion in pneumonia resolves with medical treatment <7.2 – complicated paranemonic effusion – requires surgical drainage 7.2-7.3 with glucose <60 mg/dl – impending empyema <6 – oesophageal rupture

32. CRP: done as index of disease activity and measure response to therapy. >90mg/dl – para pneumonic infections 26mg/dl – tuberculous effusion 23mg/dl – malignant effusions 5. Tuberculostearic acid (75% sensitive) – Active tuberculosis

33. Immunologic studies: Rheumathoid factor: (>1:320) – rheumatic pleuritis (non specific) ANA (anti nuclear antibody): > 1:160 – Lupus pleuritis Microbiological examination: Grams stain Anaerobic culture Aerobic culture If TB suspected – ZN stain, Granulomas in peripheral smear, Culture

34. Likelihood of isolation of organisms is increased if pleural fluid is inoculated in blood culture bottles at the bedside. Ziehl-Neelsen stained smear is positive in <20% of tuberculous pleural effusions and culture in <40% of cases. If tuberculosis is suspected and culture is negative, polymerase chain reaction for mycobacterial DNA or Increased level of adenosine deaminase (released in pleural fluid from activated lymphocytes) can establish the diagnosis

35. SUMMARY:

36. References: McPherson & Pincus: Henry's Clinical Diagnosis and Management by Laboratory Methods, 21st edition;2006. Winifred grey, Gabrijela kocjan : Diagnostic cytopathology, 3 rd edition; 2010 Shirish M Kawthalkar : Essentials of clinical pathology, 1 st edition;2010 Leopold G Koss: Koss diagnostic cytology and its histologic basis, 5 th edition; volume 2; 2006 José M. Porcel , Richard W. Light. Diagnostic Approach to Pleural Effusion in Adults. Am Fam Physician. 2006 Apr 1;73(7):1211-1220. Dixit R, Agarwal K C, Gokhroo A, Patil CB, Meena M, Shah NS, Arora P. Diagnosis and management options in malignant pleural effusions. Lung India 2017;34:160-6 A. I. SPRIGGS, The cytology of effusions pleural, pericardial and peritoneal and of cerebrospinal fluid, 2 nd edition; 1968 Parvin ganzei azer : Effusion cytology A practical guide to cancer diagnosis, 2011