1. CLINICAL LABORATORY DIAGNOSTICS OF PATHOLOGICAL PROCESSESS IN LUNGS Marushchak Maria

2. Obtaining a sputum sample Mouth should be free of foreign objects Mouth should be free of foreign objects Remove food, gum, or Tobacco Remove food, gum, or TobaccoTobacco Remove dentures Remove dentures Early morning specimen is best Early morning specimen is best Induce sputum if necessary Induce sputum if necessary Nebulized hypertonic saline or distilled water Nebulized hypertonic saline or distilled water Chest percussion Chest percussion Postural drainage Postural drainage Cough into sterile specimen cup Cough into sterile specimen cup Cough

3. Special Circumstances Tuberculosis suspected Tuberculosis suspected Tuberculosis Sputum collected in negative pressure room Sputum collected in negative pressure room Early morning gastric aspirate Early morning gastric aspirate Bronchoscopy with bronchial lavage Bronchoscopy with bronchial lavage Anaerobic culture specimen Anaerobic culture specimen Transtracheal aspiration Transtracheal aspiration Thoracentesis Thoracentesis Thoracentesis Direct lung puncture Direct lung puncture Viral Culture Specimens Viral Culture Specimens Patient gargles and expectorates with nutrient broth Patient gargles and expectorates with nutrient broth Nasopharyngeal swab transported in viral medium Nasopharyngeal swab transported in viral medium

4. Preparation of Sputum for Lab Fixation of sputum for cytology (prevents air drying) Fixation of sputum for cytology (prevents air drying) Patient expectorates into jar of 70% Ethanol Patient expectorates into jar of 70% EthanolEthanol Spread fresh sputum on slide and spray pap fixative Spread fresh sputum on slide and spray pap fixative Culture specimen transport to lab Culture specimen transport to lab Sputum Gram Stain assesses sample for adequacy Sputum Gram Stain assesses sample for adequacy Sputum Gram Stain Sputum Gram Stain Anaerobic cultures transported in air tight container Anaerobic cultures transported in air tight container Transport to lab for immediate plating Transport to lab for immediate plating Aerobic culture specimen Aerobic culture specimen Bring to lab as quickly as possible Bring to lab as quickly as possible Refrigerate specimen if transport delayed Refrigerate specimen if transport delayed Consider washing specimen of oral flora Consider washing specimen of oral flora Rinse several times with saline Rinse several times with saline Discard supernatant (non-viscous Saliva) Discard supernatant (non-viscous Saliva)Saliva Tuberculosis culture Tuberculosis culture Tuberculosis May be stored at room temperature for up to 48 hour May be stored at room temperature for up to 48 hour

5. Diagnosis #1: Necrotizing granulomas, cervical lymph node, acid-fast organisms present. Non-necrotizing granulomas, pleura, no acid-fast organisms seen. Cervical lymph node: A low-power view of the cervical lymph node shows necrotic nodules surrounded by a layer of pale histiocytes. Giant cells and non-necrotizing granulomas were absent. An acid-fast stain revealed beaded bacilli consistent with mycobacteria. Cervical lymph node: A low-power view of the cervical lymph node shows necrotic nodules surrounded by a layer of pale histiocytes. Giant cells and non-necrotizing granulomas were absent. An acid-fast stain revealed beaded bacilli consistent with mycobacteria.

6. Pleural biopsy: Non- necrotizing granulomas with central, epithelioid cells and surrounding lymphoid cells are present. Giant cells are absent. No acid-fast organisms were found.

7. Sputum Gross Exam

8. Estimate daily volume of Sputum Small amounts Small amounts Lung Abscess Lung Abscess Lung Abscess Lung Abscess Pneumonia Pneumonia Pneumonia Tuberculosis Tuberculosis Tuberculosis Copious amounts (>200 cc/day) Copious amounts (>200 cc/day) Bronchiectasis Bronchiectasis Bronchiectasis Bronchopleural Fistula Bronchopleural Fistula Bronchopleural Fistula Bronchopleural Fistula

9. Sputum Color Bloody Sputum (Hemoptysis) Bloody Sputum (Hemoptysis) Bloody SputumHemoptysis Bloody SputumHemoptysis Rusty Sputum (Prune-juice) Rusty Sputum (Prune-juice)Sputum Pneumococcal Pneumonia Pneumococcal Pneumonia Pneumococcal Pneumonia Pneumococcal Pneumonia Purulent Sputum (yellow, green, dirty- gray) Purulent Sputum (yellow, green, dirty- gray) Purulent Sputum Purulent Sputum Color alone does not distinguish bacterial infection Color alone does not distinguish bacterial infection

10. Sputum Turbidity Frothy Sputum (air bubbles, Hemoglobin) Frothy Sputum (air bubbles, Hemoglobin)Sputum HemoglobinSputum Hemoglobin Pulmonary edema Pulmonary edema Foamy, clear material Foamy, clear material Saliva Saliva Saliva Nasal secretions Nasal secretions

11. Sputum Viscosity Bloody Gelatinous Sputum (Currant-Jelly) Bloody Gelatinous Sputum (Currant-Jelly)Sputum Klebsiella Pneumonia Klebsiella Pneumonia KlebsiellaPneumonia KlebsiellaPneumonia Pneumococcal Pneumonia Pneumococcal Pneumonia Pneumococcal Pneumonia Pneumococcal Pneumonia Stringy Mucoid Sputum (may also appear frothy) Stringy Mucoid Sputum (may also appear frothy)Sputum Follows Asthma exacerbation Follows Asthma exacerbationAsthma Cloudy, mucoid Sputum Cloudy, mucoid SputumSputum Chronic Bronchitis Chronic Bronchitis Chronic Bronchitis Chronic Bronchitis Three layered appearance (stagnant, Purulent Sputum) Three layered appearance (stagnant, Purulent Sputum)Purulent SputumPurulent Sputum Bronchiectasis Bronchiectasis Bronchiectasis Lung Abscess Lung Abscess Lung Abscess Lung Abscess

12. Sputum with Feculent Odor Anaerobic infection Anaerobic infection Bronchiectasis Bronchiectasis Bronchiectasis

13. Example of case A. An overall view shows a circumscribed, non- encapsulated mass of mature adipose tissue traversed by small vessels. It is covered by normal respiratory epithelium, which is separated from the fat by a loose fibrous stroma that contains a large number of lymphocytes and plasma cells. The hemoptysis that can occur with these lesions is caused by the pneumonia secondary to the tumor and not by the tumor itself. A. An overall view shows a circumscribed, non- encapsulated mass of mature adipose tissue traversed by small vessels. It is covered by normal respiratory epithelium, which is separated from the fat by a loose fibrous stroma that contains a large number of lymphocytes and plasma cells. The hemoptysis that can occur with these lesions is caused by the pneumonia secondary to the tumor and not by the tumor itself.

14. B. At higher magnification, the subepithelial fibrous tissue is chronically inflamed. The irregular dark blue area is a lymphoid aggregate. The underlying fat and blood vessels blend in with the fibrous tissue rather than being sharply demarcated. B. At higher magnification, the subepithelial fibrous tissue is chronically inflamed. The irregular dark blue area is a lymphoid aggregate. The underlying fat and blood vessels blend in with the fibrous tissue rather than being sharply demarcated.

15. C. The subepithelial tissue has chronic inflammatory cells (lymphocytes (without visible cytoplasm) and plasma cells (with visible cytoplasm)). C. The subepithelial tissue has chronic inflammatory cells (lymphocytes (without visible cytoplasm) and plasma cells (with visible cytoplasm)).

16. What is the diagnosis? Endobronchial lipoma D. At the edge of the lesion, loose connective tissue and fat are mixed together. D. At the edge of the lesion, loose connective tissue and fat are mixed together.

17. Sputum Microscopy

18. Assessing Sputum Sample Quality Ideal Sputum Sample for Culture Ideal Sputum Sample for CultureSputum Under 10 squamous epithelial cell per low power field Under 10 squamous epithelial cell per low power field Many Neutrophils present (>5 per high power field) Many Neutrophils present (>5 per high power field)Neutrophil Bronchial epithelial cells present Bronchial epithelial cells present Alveolar Macrophages may be present Alveolar Macrophages may be presentMacrophage Inadequate Sputum Sample Inadequate Sputum SampleSputum Over 25 squamous epithelial cells/LPM Over 25 squamous epithelial cells/LPM

19. Sputum Sample Preparation Pull strand or plug of Sputum onto slide Pull strand or plug of Sputum onto slideSputum Consider buffered crystal violet to stain cells Consider buffered crystal violet to stain cells Apply cover slip Apply cover slip View under oil immersion View under oil immersion

20. Cytology Stains No Stain No Stain Blastomycosis Blastomycosis Blastomycosis Cryptococcosis Cryptococcosis Gram Stain Gram Stain Gram Stain Gram Stain Gram Positive Bacteria Gram Positive Bacteria Gram Positive Bacteria Gram Positive Bacteria Candida Candida Tuberculosis (weakly Gram Positive) Tuberculosis (weakly Gram Positive) TuberculosisGram Positive TuberculosisGram Positive Nocardia (weakly Gram Positive) Nocardia (weakly Gram Positive) NocardiaGram Positive NocardiaGram Positive Direct Fluorescent Antibody Staining Direct Fluorescent Antibody StainingAntibody Legionella Legionella Legionella Wright stain or Giemsa Stain Wright stain or Giemsa Stain Intracellular organisms Intracellular organisms

26. Special Staining Circumstances

27. Acid-fast Mycobacteria (Tuberculosis) Ziehl-Neelsen Stain (Red against blue background) Ziehl-Neelsen Stain (Red against blue background) Kinyoun stain Kinyoun stain Less reliable than Ziehl-Neelsen stain Less reliable than Ziehl-Neelsen stain Results in quickly stained sample Results in quickly stained sample Fluorochrome dyes (auramine, rhodamine) Fluorochrome dyes (auramine, rhodamine) Higher false positive rate than Ziehl-Neelsen stain Higher false positive rate than Ziehl-Neelsen stain Assist greatly in identifying organisms Assist greatly in identifying organisms

28. Fungal Organisms PAS staining or Methenamine silver staining PAS staining or Methenamine silver staining Histoplasmosis Histoplasmosis Histoplasmosis Coccidioidomycosis Coccidioidomycosis Coccidioidomycosis Aspergillus Aspergillus Aspergillus Mucor Mucor KOH Preparation KOH Preparation KOH Preparation KOH Preparation

29. Microscopic findings Caseous masses Caseous masses Dittrich's plugs Dittrich's plugs Curschmann's spirals (Asthma) Curschmann's spirals (Asthma)Asthma Charcot-Leyden Crystals (Asthma) Charcot-Leyden Crystals (Asthma)Asthma Bronchial casts Bronchial casts Concretions Concretions Broncholith Broncholith Calcified particles as seen in Broncholithiasis Calcified particles as seen in BroncholithiasisBroncholithiasis Lung Cancer cells Lung Cancer cells Lung Cancer Lung Cancer Central bronchus tumors Central bronchus tumors May require 4 samples to detect May require 4 samples to detect Eosinophils (>5%): identified with Wright's Stain Eosinophils (>5%): identified with Wright's Stain Eosinophil Allergy Allergy Asthma Asthma Asthma

30. Саrре diem