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pres.title = 'Smear Technique and Staining – GI, Respiratory & Urinary Tracts';
pres.author = 'III BSc MLD – RGUHS';

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// ════════════════════════════════════════════════════════════════════════════
// SLIDE 1 – TITLE SLIDE
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// ════════════════════════════════════════════════════════════════════════════
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  { num:'01', label:'Introduction to Cytology & Smear Technique', color:C.midBg },
  { num:'02', label:'Fixatives and Staining Methods', color:C.teal },
  { num:'03', label:'Gastrointestinal Tract – Smear & Staining', color:C.green },
  { num:'04', label:'Respiratory Tract – Smear & Staining', color:C.purple },
  { num:'05', label:'Urinary Tract – Smear & Staining', color:C.orange },
  { num:'06', label:'Comparison & Quality Control', color:C.red },
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// ════════════════════════════════════════════════════════════════════════════
// SLIDE 3 – SECTION TITLE: Introduction to Cytology
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sectionTitle(s, 'Introduction to Cytology', 'Definition | Scope | Types of Specimens');

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 4 – Introduction to Cytology
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
contentSlide(s, 'Introduction to Cytology', [
  '## What is Cytology?',
  'Branch of pathology studying individual cells or cell groups obtained from tissues/fluids to make a diagnosis',
  'First described by George Papanicolaou (1917–1928); widely adopted after 1943 for cervical cancer screening',
  '## Scope in MLD / Cytopathology',
  'Detection of malignancies, infections, inflammatory conditions and pre-neoplastic changes',
  '## Types of Cytology Specimens',
  'Exfoliative – cells shed naturally (sputum, urine, cervical smear)',
  'Aspiration – FNA or body fluid aspiration (pleural, peritoneal)',
  'Imprint / Scrape – direct touch or scrape from cut surface of tissue',
  'Brushing – endoscopic brushes from GI/respiratory mucosa',
]);

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 5 – Smear Preparation – General Principles
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
contentSlide(s, 'General Principles of Smear Preparation', [
  '## Slide Preparation',
  'Use clean, grease-free, labelled glass slides (frosted end for marking)',
  'Cells must be thinly and evenly spread – too thick obscures nuclear details, too thin causes cellular loss',
  '## Smearing Techniques',
  'Slide-over-slide (push technique) – for viscous material like sputum, FNA',
  'Line smear technique – for fluids (drop at one end, spreader at 30-45° angle)',
  'Centrifuge/cytospin – concentrates cells from dilute specimens (urine, BAL fluid)',
  'Membrane filter – older method; traps cells on polycarbonate filter paper',
  'Liquid-based cytology (LBC) – monolayer automated technique (ThinPrep, SurePath)',
  '## Important Rules',
  'Fix smears IMMEDIATELY after preparation to avoid drying artefact',
  'Never heat-fix smears intended for Pap stain – use wet (alcohol) fixation',
]);

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 6 – SECTION TITLE: Fixatives & Staining
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
sectionTitle(s, 'Fixatives & Staining Methods', 'Wet Fixation | Air Drying | Papanicolaou | MGG | H&E');

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 7 – Fixatives
// ════════════════════════════════════════════════════════════════════════════
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  'Cytological Fixatives',
  'Wet (Alcohol) Fixation',
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    '95% Ethyl Alcohol – most common',
    'Carnoy\'s fixative (ethanol + acetic acid + chloroform) – for mucoid specimens',
    'Polyethylene glycol (Saccomanno\'s fixative) – for sputum collection',
    'CytoLyt, PreservCyt – liquid-based cytology fixatives',
    'Immediate fixation required – drop slide into jar within 2–3 seconds',
    'Preserves nuclear chromatin detail for Pap staining',
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  'Air-Dried (No Fixation)',
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    'Slides allowed to air-dry at room temperature for 10–15 min',
    'Used prior to Romanowsky stains (MGG, Giemsa, Diff-Quik)',
    'Better for demonstrating cytoplasmic granules, mucin, extracellular material',
    'Suitable for FNA smears, bone marrow, sputum (alternative)',
    'Avoid in hot/humid conditions – may cause rapid drying artefact',
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// ════════════════════════════════════════════════════════════════════════════
// SLIDE 8 – Papanicolaou Stain
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
contentSlide(s, 'Papanicolaou (Pap) Stain – Steps', [
  '## Principle',
  'Polychromatic stain highlighting nuclear detail; cells stained with Haematoxylin (nuclear) + OG-6 (keratin) + EA-36/50 (cytoplasmic)',
  '## Step-by-Step Procedure',
  'Step 1 – Fixation: Wet-fix smear in 95% ethanol for 15–30 min',
  'Step 2 – Rehydration: Pass through descending alcohol grades (100% → 95% → 70% → water)',
  'Step 3 – Nuclear staining: Harris Haematoxylin (5 min) → rinse in water',
  'Step 4 – Differentiation: 0.05% HCl in 70% ethanol (acid alcohol) until nuclei clear',
  'Step 5 – Bluing: Running tap water 5–10 min OR dilute ammonia water',
  'Step 6 – Cytoplasmic stain: OG-6 (2 min) → EA-36 or EA-50 (3–5 min)',
  'Step 7 – Dehydration: Ascending alcohols → 100% ethanol (2 changes)',
  'Step 8 – Clearing: Xylene (2 changes, 2 min each)',
  'Step 9 – Mounting: Dibutyl phthalate polystyrene xylene (DPX)',
  '## Results',
  'Nuclei – blue-black | Cytoplasm – pink/orange/green | Mucus – pale green',
]);

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 9 – MGG / Other Stains
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
twoColSlide(s,
  'Other Cytological Staining Methods',
  'May–Grünwald–Giemsa (MGG)',
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    'Used on air-dried smears',
    'Step 1: Cover slide with May-Grünwald solution (2 min)',
    'Step 2: Add equal volume buffer pH 6.8 (2 min)',
    'Step 3: Drain and apply Giemsa solution (diluted 1:10) for 15–20 min',
    'Step 4: Rinse in buffer, air-dry, mount',
    'Results: Nuclei – purple; cytoplasm – pink to lilac; RBCs – pink; mucus – pale blue',
    'Best for: FNA, sputum, body fluids',
  ],
  'H&E & Special Stains',
  [
    'Haematoxylin & Eosin (H&E): Used on cell block sections',
    'Nuclei – blue; cytoplasm – pink',
    'Ziehl-Neelsen (ZN) Stain: AFB organisms (Mycobacterium) – red rods',
    'PAS (Periodic Acid-Schiff): Fungi, glycogen, mucin – magenta',
    'Alcian Blue: Acid mucins – bright blue',
    'Mucicarmine: Mucin – red (detects Cryptococcus)',
    'Silver stains: Fungi (Pneumocystis), basement membranes',
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  C.purple, C.green
);

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 10 – SECTION TITLE: GI Tract
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
sectionTitle(s, 'Gastrointestinal Tract', 'Sample Collection | Smear Preparation | Staining | Normal & Abnormal Findings');

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 11 – GI Tract – Sample Collection
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
contentSlide(s, 'GI Tract – Specimen Collection Methods', [
  '## Types of Specimens',
  'Endoscopic brushing – most common; brush passed through endoscope to scrape mucosa',
  'Endoscopic FNA – fine needle aspiration under EUS guidance (submucosal/mural lesions)',
  'Imprint cytology – direct contact of biopsy tissue with slide',
  'Endoscopic washings – saline lavage collected during upper/lower GI endoscopy',
  'Stool specimens – occasionally used (Entamoeba histolytica, ova)',
  '## Clinical Indications',
  'Suspected oesophageal / gastric / colonic carcinoma',
  'Helicobacter pylori detection (brush + rapid urease test)',
  'Fungal infections (Candida), viral infections (CMV, HSV)',
  'Pre-neoplastic lesions – Barrett\'s oesophagus, dysplasia',
  '## Brushing Technique',
  'Brush vigorously over suspicious area 3–5 times → immediately smear onto 2 slides → fix one in 95% ethanol (Pap) + air-dry second (MGG)',
]);

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 12 – GI Tract – Smear Prep & Staining
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
contentSlide(s, 'GI Tract – Smear Preparation & Staining', [
  '## Smear Preparation',
  'Roll or stroke the brush gently along the slide in a continuous motion – do NOT press hard (crush artefact)',
  'Prepare at least 2 smears: one wet-fixed + one air-dried for complementary staining',
  'For washings: centrifuge at 1500 rpm × 5 min; discard supernatant; smear sediment',
  'For FNA material: express drop onto slide → spread with second slide at 30–45°',
  '## Recommended Stains for GI Cytology',
  'Papanicolaou stain – nuclear detail, dysplasia assessment, malignant cells',
  'MGG / Giemsa – Helicobacter pylori (curved bacilli, blue), inflammatory cells',
  'H&E (cell block) – glandular architecture, adenocarcinoma',
  'PAS ± diastase – mucin-secreting cells, Candida (red cell walls)',
  'Silver stain (Warthin-Starry or Gimenez) – H. pylori confirmation',
  'Alcian Blue + PAS – goblet cell metaplasia in Barrett\'s oesophagus',
  '## Normal GI Cytology Findings',
  'Regular columnar cells in orderly honeycomb sheets | goblet cells | no nuclear atypia',
]);

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 13 – SECTION TITLE: Respiratory Tract
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
sectionTitle(s, 'Respiratory Tract', 'Sputum | BAL | Bronchial Brushing | FNA | Staining');

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 14 – Respiratory – Specimen Types & Collection
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
contentSlide(s, 'Respiratory Tract – Specimen Types & Collection', [
  '## Types of Specimens',
  'Sputum – expectorated or induced (3 early-morning samples preferred for maximum yield)',
  'Bronchial washing – saline instilled and aspirated via bronchoscope',
  'Bronchoalveolar lavage (BAL) – 100–250 mL saline → washes alveolar compartment',
  'Bronchial brushing – cytology brush passed via bronchoscope over lesion',
  'Transbronchial / Percutaneous FNA – CT/EUS-guided; 19–22 gauge needle',
  'Pleural fluid – thoracocentesis (sent for cytology if malignancy suspected)',
  '## Sputum Collection Protocol',
  'Patient coughs deeply after physiotherapy / postural drainage',
  'Early morning (post-sleep) samples preferred – highest cell concentration',
  'Collect in wide-mouthed sterile container; process within 2–4 hours',
  'Mucolytic agent (N-acetylcysteine / Saccomanno\'s fixative) added if delayed processing',
  '## Saccomanno\'s Technique',
  'Equal volume 50% ethanol + 2% Carbowax added to sputum → homogenised → centrifuged → smear sediment → Pap stain',
]);

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 15 – Respiratory – Smear Prep & Staining
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
contentSlide(s, 'Respiratory Tract – Smear Preparation & Staining', [
  '## Smear Preparation',
  'Sputum: Select bloodstained/mucoid areas → place on slide → cover with second slide → pull apart in one smooth motion (slide-over-slide)',
  'BAL & washings: Centrifuge 1500 rpm × 10 min → prepare cytospin slides or direct smear from pellet',
  'FNA: Express small drop → spread at 30–45° → fix immediately (Pap) OR air-dry (MGG)',
  'Bloody specimens: Treat with 1% acetic acid to lyse RBCs before centrifugation',
  '## Staining Methods',
  'Papanicolaou – gold standard for respiratory cytology; nuclei blue-black; cytoplasm polychromatic',
  'MGG (Romanowsky) – air-dried smears; best for inflammatory cells, FNA direct smears',
  'H&E – cell block sections from pellet',
  'ZN stain – AFB in TB/MAI specimens (red beaded rods on blue background)',
  'PAS / Silver (GMS) – Pneumocystis jirovecii (cysts), fungal elements',
  '## Normal Respiratory Cytology',
  'Ciliated columnar cells | goblet cells | alveolar macrophages (smokers: anthracotic pigment) | squamous metaplastic cells',
]);

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 16 – SECTION TITLE: Urinary Tract
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
sectionTitle(s, 'Urinary Tract', 'Urine Cytology | Bladder Washing | FNA | Staining');

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 17 – Urinary – Specimen Types & Collection
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
contentSlide(s, 'Urinary Tract – Specimen Types & Collection', [
  '## Types of Specimens',
  'Voided urine (VU) – spontaneously passed; 50–100 mL midstream sample',
  'Catheterised urine – via indwelling or in-out catheter; higher cellular yield',
  'Bladder washing (barbotage) – 50 mL saline instilled and aspirated via cystoscope; best cellularity',
  'Ureteral brushing – brush advanced via ureteroscope; for upper tract lesions',
  'Renal pelvis FNA – CT-guided; for solid renal pelvis masses',
  '## Collection Protocol',
  'Second morning voided specimen preferred (first voiding has degenerated cells)',
  'Collect fresh; process within 1–2 hours OR fix immediately in equal volume 50% ethanol',
  'Avoid very dilute first-void samples – low cellular yield',
  '## Processing',
  'Centrifuge at 1500–2000 rpm × 10 min → discard most supernatant → resuspend pellet',
  'Prepare cytospin preparations (best cellular morphology) OR direct smears',
  'Liquid-based cytology (ThinPrep) now standard in many laboratories',
]);

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 18 – Urinary – Smear Prep & Staining
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
contentSlide(s, 'Urinary Tract – Smear Preparation & Staining', [
  '## Smear Preparation',
  'Cytospin: Load 200–400 µL of resuspended sediment into cytospin funnel → spin at 1000 rpm × 5 min → air-dry or wet-fix',
  'Direct smear: Place 1–2 drops of sediment on labelled slide → spread with pipette tip → fix or air-dry',
  'For bloody urine: Add equal volume 1% acetic acid → mix → centrifuge → smear pellet',
  '## Staining Methods',
  'Papanicolaou – standard for urothelial cytology; nuclear detail for grading atypia',
  'H&E – cell block, tissue fragments, histological assessment',
  'MGG / Diff-Quik – air-dried slides; cellular overview, inflammatory cells',
  '## Paris System for Reporting Urine Cytology (2022)',
  'Non-diagnostic | Negative for HGUC | Atypical urothelial cells (AUC) | Suspicious for HGUC | HGUC',
  '## Normal Urinary Cytology',
  'Urothelial (transitional) cells – umbrella cells, intermediate cells, basal cells',
  'Squamous cells (from distal urethra), renal tubular cells (if catheterised)',
  'WBCs (<5/HPF normal), RBCs absent in health',
]);

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 19 – Comparison Table
// ════════════════════════════════════════════════════════════════════════════
s = pres.addSlide();
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    { text: 'Feature', options: { bold:true, color:C.white, fill:C.darkBg, align:'center' } },
    { text: 'Gastrointestinal', options: { bold:true, color:C.white, fill:C.green, align:'center' } },
    { text: 'Respiratory', options: { bold:true, color:C.white, fill:C.purple, align:'center' } },
    { text: 'Urinary', options: { bold:true, color:C.white, fill:C.orange, align:'center' } },
  ],
  ['Specimen', 'Brushing, FNA, washing', 'Sputum, BAL, brushing, FNA', 'Voided urine, catheter, BBF'],
  ['Smear technique', 'Roll brush; cytospin for washings', 'Slide-over-slide (sputum); cytospin (BAL)', 'Cytospin; direct smear'],
  ['Primary fixation', 'Wet (95% EtOH) for Pap', 'Wet + air-dried (dual preparation)', 'Wet (95% EtOH) or LBC'],
  ['Main stain', 'Pap; Giemsa (H. pylori)', 'Pap (gold standard); MGG', 'Papanicolaou'],
  ['Special stains', 'PAS, Alcian Blue, WS silver', 'ZN, GMS, PAS', 'H&E (cell block); Diff-Quik'],
  ['Key normal cells', 'Columnar, goblet cells', 'Ciliated columnar, macrophages', 'Urothelial (umbrella) cells'],
  ['Key malignancy', 'Adenocarcinoma', 'Squamous / adenocarcinoma, SCLC', 'Urothelial (transitional cell) carcinoma'],
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// ════════════════════════════════════════════════════════════════════════════
// SLIDE 20 – Quality Control in Cytology
// ════════════════════════════════════════════════════════════════════════════
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contentSlide(s, 'Quality Control in Cytological Smear Preparation', [
  '## Common Causes of Unsatisfactory Smears',
  'Thick smears – cells overlap, nuclear detail obscured',
  'Air-drying artefact – cytoplasm over-eosinophilic; nuclear details lost (for Pap stain)',
  'Excessive blood – RBCs mask diagnostic cells; treat with acetic acid lysis',
  'Crush artefact – too much pressure during spreading (especially brush smears)',
  'Delayed fixation – autolytic cell degeneration',
  'Under-staining / Over-staining – incorrect timing or exhausted stain solutions',
  '## QC Measures',
  'Use positive and negative control slides with each staining run',
  'Regular maintenance of staining solutions (filter, replenish, replace)',
  'Proper labelling of slides (patient ID, date, site, fixation method)',
  'Reporting with standardised terminology (Bethesda / Paris System / Saccomanno classification)',
  '## Storage',
  'DPX-mounted slides can be stored indefinitely at room temperature away from direct light',
]);

// ════════════════════════════════════════════════════════════════════════════
// SLIDE 21 – Summary / Conclusion
// ════════════════════════════════════════════════════════════════════════════
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  'Smear preparation quality is the foundation of accurate cytological diagnosis',
  'Choose fixation method FIRST – wet (alcohol) for Pap; air-dry for Romanowsky stains',
  'GI: Endoscopic brushing → dual smears → Pap for malignancy; Giemsa for H. pylori',
  'Respiratory: Pap is gold standard; MGG complements; ZN/GMS for organisms',
  'Urinary: Cytospin gives best results; report using Paris System 2022',
  'Liquid-based cytology is replacing conventional smears in modern labs',
  'QC – control slides, proper labelling, standardised reporting are mandatory',
];
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// ════════════════════════════════════════════════════════════════════════════
// SLIDE 22 – Thank You
// ════════════════════════════════════════════════════════════════════════════
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s.addText('THANK YOU', { x:0.5, y:1.9, w:9, h:1.5, fontSize:46, bold:true, color:C.white, fontFace:'Calibri', align:'center', charSpacing:4 });
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s.addText('III BSc Medical Laboratory Technology\nRajiv Gandhi University of Health Sciences\nSubject: Pathology – Cytopathology', {
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// ════════════════════════════════════════════════════════════════════════════
// WRITE FILE
// ════════════════════════════════════════════════════════════════════════════
pres.writeFile({ fileName: '/home/daytona/workspace/smear-ppt/Smear_Preparation_and_Staining_RGUHS.pptx' })
  .then(() => console.log('SUCCESS: PPT created!'))
  .catch(e => { console.error('ERROR:', e); process.exit(1); });

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