CLINICAL CLINICAL LABORATORY LABORATORY

DIAGNOSTICS OF DIAGNOSTICS OF PATHOLOGICAL PATHOLOGICAL PROCESSESS IN PROCESSESS IN

LUNGSLUNGSMarushchak MariaMarushchak Maria

Obtaining a sputum Obtaining a sputum samplesample

Mouth should be free of foreign objects Mouth should be free of foreign objects Remove food, gum, or 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 Nebulized hypertonic saline or distilled water distilled water

Chest percussion Chest percussion Postural drainage Postural drainage

CoughCough into sterile specimen cup into sterile specimen cup

Special Circumstances Special Circumstances TuberculosisTuberculosis suspected suspected

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 ThoracentesisThoracentesis Direct lung puncture Direct lung puncture

Viral Culture Specimens Viral Culture Specimens Patient gargles and expectorates with Patient gargles and expectorates with

nutrient broth nutrient broth Nasopharyngeal swab transported in viral Nasopharyngeal swab transported in viral

medium medium

Preparation of Sputum for Preparation of Sputum for Lab Lab

Fixation of sputum for cytology (prevents air drying) Fixation of sputum for cytology (prevents air drying) Patient expectorates into jar of 70% 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 StainSputum Gram Stain assesses sample for adequacy assesses sample for adequacy 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 Discard supernatant (non-viscous SalivaSaliva) )

TuberculosisTuberculosis culture culture May be stored at room temperature for up to 48 May be stored at room temperature for up to 48

hour hour

Diagnosis #1: Necrotizing Diagnosis #1: Necrotizing granulomas, cervical lymph granulomas, cervical lymph node, acid-fast organisms node, acid-fast organisms present. Non-necrotizing present. Non-necrotizing

granulomas, pleura, no acid-fast granulomas, pleura, no acid-fast organisms seen.organisms seen. Cervical lymph node:Cervical lymph node:

A low-power view of A low-power view of the cervical lymph the cervical lymph node shows necrotic node shows necrotic nodules surrounded by nodules surrounded by a layer of pale a layer of pale histiocytes. Giant cells histiocytes. Giant cells and non-necrotizing and non-necrotizing granulomas were granulomas were absent. An acid-fast absent. An acid-fast stain revealed beaded stain revealed beaded bacilli consistent with bacilli consistent with mycobacteria. mycobacteria.

Pleural biopsy: Pleural biopsy: Non-necrotizing Non-necrotizing granulomas with granulomas with

central, epithelioid central, epithelioid cells and cells and

surrounding surrounding lymphoid cells are lymphoid cells are

present. Giant present. Giant cells are absent. cells are absent.

No acid-fast No acid-fast organisms were organisms were

found.found.

Sputum Gross ExamSputum Gross Exam

Estimate daily volume of Estimate daily volume of SputumSputum

Small amounts Small amounts Lung AbscessLung Abscess PneumoniaPneumonia TuberculosisTuberculosis

Copious amounts (>200 cc/day) Copious amounts (>200 cc/day) BronchiectasisBronchiectasis Bronchopleural FistulaBronchopleural Fistula

Sputum Color Sputum Color

Bloody SputumBloody Sputum ( (HemoptysisHemoptysis) ) Rusty Rusty SputumSputum (Prune-juice) (Prune-juice)

Pneumococcal PneumoniaPneumococcal Pneumonia Purulent SputumPurulent Sputum (yellow, green, (yellow, green,

dirty-gray) dirty-gray) Color alone does not Color alone does not

distinguish bacterial distinguish bacterial infection infection

Sputum TurbiditySputum Turbidity

Frothy Frothy SputumSputum (air bubbles, (air bubbles, HemoglobinHemoglobin) ) Pulmonary edema Pulmonary edema

Foamy, clear material Foamy, clear material SalivaSaliva Nasal secretions Nasal secretions

Sputum Viscosity Sputum Viscosity Bloody Gelatinous Bloody Gelatinous SputumSputum (Currant-Jelly) (Currant-Jelly)

KlebsiellaKlebsiella PneumoniaPneumonia Pneumococcal PneumoniaPneumococcal Pneumonia

Stringy Mucoid Stringy Mucoid SputumSputum (may also appear frothy) (may also appear frothy) Follows Follows AsthmaAsthma exacerbation exacerbation

Cloudy, mucoid Cloudy, mucoid SputumSputum Chronic BronchitisChronic Bronchitis

Three layered appearance (stagnant, Three layered appearance (stagnant, Purulent SputumPurulent Sputum) )

BronchiectasisBronchiectasis Lung AbscessLung Abscess

Sputum with Feculent Sputum with Feculent OdorOdor

Anaerobic infection Anaerobic infection BronchiectasisBronchiectasis

Example of Example of casecase

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

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

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

What is the diagnosis? What is the diagnosis? Endobronchial lipoma Endobronchial lipoma

D. At the edge D. At the edge of the lesion, of the lesion, loose loose connective connective tissue and fat tissue and fat are mixed are mixed together. together.

Sputum Sputum MicroscopyMicroscopy

Assessing Sputum Sample Assessing Sputum Sample QualityQuality

Ideal Ideal SputumSputum Sample for Culture Sample for Culture Under 10 squamous epithelial cell Under 10 squamous epithelial cell

per low power field per low power field Many Many NeutrophilNeutrophils present (>5 per s present (>5 per

high power field) high power field) Bronchial epithelial cells present Bronchial epithelial cells present Alveolar Alveolar MacrophageMacrophages may be present s may be present

Inadequate Inadequate SputumSputum Sample Sample Over 25 squamous epithelial Over 25 squamous epithelial

cells/LPM cells/LPM

Sputum Sample Sputum Sample Preparation Preparation

Pull strand or plug of Pull strand or plug of SputumSputum onto slide onto slide

Consider buffered crystal Consider buffered crystal violet to stain cells violet to stain cells

Apply cover slip Apply cover slip View under oil immersion View under oil immersion

Cytology Stains Cytology Stains No Stain No Stain

BlastomycosisBlastomycosis Cryptococcosis Cryptococcosis

Gram StainGram Stain Gram Positive BacteriaGram Positive Bacteria Candida Candida TuberculosisTuberculosis (weakly (weakly Gram PositiveGram Positive) ) NocardiaNocardia (weakly (weakly Gram PositiveGram Positive) )

Direct Fluorescent Direct Fluorescent AntibodyAntibody Staining Staining Legionella Legionella

Wright stain or Giemsa Stain Wright stain or Giemsa Stain Intracellular organisms Intracellular organisms

Special Staining Special Staining Circumstances Circumstances

Acid-fast Mycobacteria Acid-fast Mycobacteria (Tuberculosis)(Tuberculosis)

Ziehl-Neelsen Stain (Red against blue Ziehl-Neelsen Stain (Red against blue background) 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, Fluorochrome dyes (auramine, rhodamine) rhodamine) Higher false positive rate than Ziehl-Higher false positive rate than Ziehl-

Neelsen stain Neelsen stain Assist greatly in identifying organisms Assist greatly in identifying organisms

Fungal OrganismsFungal Organisms

PAS staining or Methenamine PAS staining or Methenamine silver staining silver staining Histoplasmosis Histoplasmosis Coccidioidomycosis Coccidioidomycosis Aspergillus Aspergillus Mucor Mucor

KOH Preparation KOH Preparation

Microscopic findings Microscopic findings Caseous masses Caseous masses Dittrich's plugs Dittrich's plugs Curschmann's spirals (Asthma) Curschmann's spirals (Asthma) Charcot-Leyden Crystals (Asthma) Charcot-Leyden Crystals (Asthma) Bronchial casts Bronchial casts Concretions Concretions Broncholith Broncholith

Calcified particles as seen in Broncholithiasis Calcified particles as seen in Broncholithiasis Lung Cancer cells Lung Cancer cells

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 Allergy Allergy Asthma Asthma

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