CLINICAL LABORATORY DIAGNOSTICS OF PATHOLOGICAL PROCESSESS IN LUNGS

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

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

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

Sputum Gross Exam

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

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

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

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

Sputum with Feculent Odor Anaerobic infection Anaerobic infection Bronchiectasis Bronchiectasis Bronchiectasis

Sputum Microscopy

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

Sputum Sample Preparation Pull strand or plug of Sputum on to slide Pull strand or plug of Sputum on to 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

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

Special Staining Circumstances

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

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

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

Pneumonia General General Lab Indications Lab Indications Moderate or severe community acquired pneumonia Moderate or severe community acquired pneumonia Patient with comorbid conditions Patient with comorbid conditions Efficacy of Labs Efficacy of Labs No value in non-severe community acquired pneumonia No value in non-severe community acquired pneumonia

SputumSputum Examination Sputum Tests Tests Sputum Gram Stain Sputum Gram Stain Sputum Gram Stain Sputum Gram Stain Sputum Culture Sputum Culture Sputum Culture Sputum Culture Efficacy of Sputum exam Efficacy of Sputum examSputum Sputum has low diagnostic yield in CAP Sputum has low diagnostic yield in CAP Sputum Not recommended in community acquired pneumonia Not recommended in community acquired pneumonia

Blood Culture Blood Culture Indications Indications Not indicated unless severe disease Not indicated unless severe disease Recommended by ATS in hospitalized CAP Recommended by ATS in hospitalized CAP Efficacy Efficacy Low sensitivity: Positive in only 5- 10% of cases Low sensitivity: Positive in only 5- 10% of cases Does not predict severity or outcome Does not predict severity or outcome

Specific Testing with reasonable efficacy Chlamydia pneumonia Chlamydia pneumonia Chlamydia Rapid PCR (>30% Test Sensitivity) Rapid PCR (>30% Test Sensitivity)Test SensitivityTest Sensitivity Influenza Influenza Influenza Rapid Influenza Test (Influenza DFA) Rapid Influenza Test (Influenza DFA) Rapid Influenza TestInfluenza Rapid Influenza TestInfluenza Legionella pneumophila Legionella pneumophila Legionella pneumophila Legionella pneumophila Rapid PCR of Sputum (80% Test Sensitivity) Rapid PCR of Sputum (80% Test Sensitivity)SputumTest SensitivitySputumTest Sensitivity Urinary antigen (>50% Test Sensitivity) Urinary antigen (>50% Test Sensitivity)Test SensitivityTest Sensitivity Mycoplasma pneumoniae Mycoplasma pneumoniae Mycoplasma pneumoniae Mycoplasma pneumoniae Rapid PCR of Sputum (>30% Test Sensitivity) Rapid PCR of Sputum (>30% Test Sensitivity)SputumTest SensitivitySputumTest Sensitivity

Chronic Bronchitis Labs Labs Arterial Blood Gas (ABG) Arterial Blood Gas (ABG) Arterial Blood Gas Arterial Blood Gas Markedly reduced arterial pO2 Markedly reduced arterial pO2 Elevated arterial pCO2 (40-50 mmHg) Elevated arterial pCO2 (40-50 mmHg) Pulmonary Function Tests Pulmonary Function Tests Pulmonary Function Test Pulmonary Function Test Residual Volume increased Residual Volume increased Residual Volume Residual Volume FEV1 decreased FEV1 decreased FEV1/FVC decreased FEV1/FVC decreased FEF (mid-flows) decreased FEF (mid-flows) decreased Diffusion capacity (DLCO) near normal Diffusion capacity (DLCO) near normalDLCO

Bronchiectasis Labs: Sputum Labs: Sputum Sputum forms layers on standing Sputum forms layers on standing Sputum Top: Mucus Top: Mucus Middle: Clear fluid Middle: Clear fluid Bottom: Pus Bottom: Pus Sputum Culture not diagnostic (mixture of organisms) Sputum Culture not diagnostic (mixture of organisms) Sputum Culture Sputum Culture Fungal Culture Fungal Culture Fungal Culture Fungal Culture

Lung Abscess Labs Labs Sputum examination Sputum examination Sputum Microscopy Microscopy Gram Stain Gram Stain Gram Stain Gram Stain Mycobacterial stains Mycobacterial stains Mycobacteria Fungal stains Fungal stains Sputum layers on standing Sputum layers on standing Sputum Sputum Cultures are usually not helpful Sputum Cultures are usually not helpful Sputum Culture Sputum Culture Complete Blood Count Complete Blood Count Complete Blood Count Complete Blood Count Leukocytosis Leukocytosis Leukocytosis

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