Papanicolaou Pap staining procedure steps fixation hematoxylin OG EA stain cytology

https://pmc.ncbi.nlm.nih.gov/articles/PMC9345133

Leishman stain blood smear procedure steps methanol fixation malaria cells appearance

Giemsa staining procedure steps dilution buffer blood film malaria parasite appearance WBC colors

ls /home/daytona/workspace/ 2>/dev/null || echo "workspace not found"

mkdir -p /home/daytona/workspace/staining-guide && python3 -c "from reportlab.li…

~/staining-guide/generate_pdf.py

from reportlab.lib.pagesizes import A4
from reportlab.lib import colors
from reportlab.lib.styles import getSampleStyleSheet, ParagraphStyle
from reportlab.lib.units import cm, mm
from reportlab.lib.enums import TA_LEFT, TA_CENTER, TA_JUSTIFY
from reportlab.platypus import (
    SimpleDocTemplate, Paragraph, Spacer, Table, TableStyle,
    PageBreak, HRFlowable, KeepTogether
)
from reportlab.pdfbase import pdfmetrics
from reportlab.pdfbase.ttfonts import TTFont
import os

OUTPUT_PATH = "/home/daytona/workspace/staining-guide/Histology_Staining_Complete_Guide.pdf"

# ── Color palette ──────────────────────────────────────────────
C_BLUE       = colors.HexColor("#1a3a5c")   # dark navy – headings
C_LIGHT_BLUE = colors.HexColor("#dce9f5")   # light blue – header bg
C_ACCENT     = colors.HexColor("#2e86de")   # medium blue – sub-heads
C_PURPLE     = colors.HexColor("#6c3483")   # purple – H&E section
C_GREEN      = colors.HexColor("#1e8449")   # green – Giemsa section
C_RED        = colors.HexColor("#c0392b")   # red – AFB section
C_ORANGE     = colors.HexColor("#d35400")   # orange – Leishman section
C_PAP        = colors.HexColor("#0e6655")   # teal – Pap section
C_GRAY_LIGHT = colors.HexColor("#f4f6f7")
C_GRAY_MID   = colors.HexColor("#aab7b8")
C_WHITE      = colors.white
C_BLACK      = colors.black

W, H = A4
MARGIN = 2*cm

doc = SimpleDocTemplate(
    OUTPUT_PATH,
    pagesize=A4,
    rightMargin=MARGIN, leftMargin=MARGIN,
    topMargin=2.5*cm, bottomMargin=2.5*cm,
    title="Histology Staining – Complete Guide",
    author="Orris Medical"
)

styles = getSampleStyleSheet()

# ── Custom paragraph styles ────────────────────────────────────
def make_style(name, parent="Normal", **kw):
    s = ParagraphStyle(name, parent=styles[parent], **kw)
    styles.add(s)
    return s

title_style = make_style("MyTitle",
    fontSize=26, leading=32, textColor=C_BLUE,
    alignment=TA_CENTER, spaceAfter=6, fontName="Helvetica-Bold")

subtitle_style = make_style("MySubtitle",
    fontSize=13, leading=18, textColor=C_ACCENT,
    alignment=TA_CENTER, spaceAfter=4, fontName="Helvetica")

toc_head = make_style("TocHead",
    fontSize=14, leading=18, textColor=C_BLUE,
    fontName="Helvetica-Bold", spaceAfter=8)

toc_entry = make_style("TocEntry",
    fontSize=11, leading=16, textColor=C_BLACK,
    fontName="Helvetica", leftIndent=12)

sec_head = make_style("SecHead",
    fontSize=16, leading=20, textColor=C_WHITE,
    fontName="Helvetica-Bold", spaceAfter=2, spaceBefore=4,
    backColor=C_BLUE, leftIndent=-5, borderPadding=(4,8,4,8))

sub_head = make_style("SubHead",
    fontSize=13, leading=17, textColor=C_BLUE,
    fontName="Helvetica-Bold", spaceBefore=10, spaceAfter=4)

step_head = make_style("StepHead",
    fontSize=11, leading=15, textColor=C_WHITE,
    fontName="Helvetica-Bold", spaceBefore=4, spaceAfter=2)

body = make_style("Body",
    fontSize=10, leading=15, textColor=C_BLACK,
    fontName="Helvetica", spaceAfter=4, alignment=TA_JUSTIFY)

bullet_style = make_style("Bullet",
    fontSize=10, leading=15, textColor=C_BLACK,
    fontName="Helvetica", spaceAfter=2,
    leftIndent=18, bulletIndent=6)

note_style = make_style("Note",
    fontSize=9.5, leading=14, textColor=colors.HexColor("#555555"),
    fontName="Helvetica-Oblique", spaceAfter=4,
    leftIndent=12, rightIndent=12,
    backColor=colors.HexColor("#fdf6e3"),
    borderPadding=(4,6,4,6))

caption_style = make_style("Caption",
    fontSize=9, leading=12, textColor=colors.HexColor("#555555"),
    fontName="Helvetica-Oblique", alignment=TA_CENTER, spaceAfter=6)

# ── Helper functions ───────────────────────────────────────────
def sp(h=0.3):
    return Spacer(1, h*cm)

def hr(color=C_GRAY_MID, thickness=0.5):
    return HRFlowable(width="100%", thickness=thickness, color=color,
                      spaceAfter=4, spaceBefore=4)

def colored_box_para(text, bg_color, text_color=C_WHITE, font_size=11, bold=True):
    fn = "Helvetica-Bold" if bold else "Helvetica"
    st = ParagraphStyle("_cb", fontSize=font_size, leading=font_size+5,
                        textColor=text_color, fontName=fn,
                        backColor=bg_color, borderPadding=(5,10,5,10),
                        spaceBefore=8, spaceAfter=4)
    return Paragraph(text, st)

def step_box(num, title, color=C_ACCENT):
    st = ParagraphStyle("_step", fontSize=11, leading=15,
                        textColor=C_WHITE, fontName="Helvetica-Bold",
                        backColor=color, borderPadding=(4,8,4,8),
                        spaceBefore=8, spaceAfter=2)
    return Paragraph(f"● STEP {num}: {title}", st)

def bullet(text):
    return Paragraph(f"• {text}", bullet_style)

def note(text):
    return Paragraph(f"⚠ {text}", note_style)

def body_para(text):
    return Paragraph(text, body)

def make_table(data, col_widths, header_bg=C_BLUE, header_fg=C_WHITE,
               alt_bg=C_GRAY_LIGHT, font_size=9):
    ts = TableStyle([
        ("BACKGROUND", (0,0), (-1,0), header_bg),
        ("TEXTCOLOR",  (0,0), (-1,0), header_fg),
        ("FONTNAME",   (0,0), (-1,0), "Helvetica-Bold"),
        ("FONTSIZE",   (0,0), (-1,-1), font_size),
        ("LEADING",    (0,0), (-1,-1), font_size+4),
        ("ROWBACKGROUNDS", (0,1), (-1,-1), [C_WHITE, alt_bg]),
        ("GRID",       (0,0), (-1,-1), 0.4, C_GRAY_MID),
        ("VALIGN",     (0,0), (-1,-1), "MIDDLE"),
        ("TOPPADDING", (0,0), (-1,-1), 5),
        ("BOTTOMPADDING", (0,0), (-1,-1), 5),
        ("LEFTPADDING",  (0,0), (-1,-1), 7),
        ("RIGHTPADDING", (0,0), (-1,-1), 7),
        ("WORDWRAP",   (0,0), (-1,-1), True),
        ("ALIGN",      (0,0), (-1,-1), "LEFT"),
    ])
    t = Table(data, colWidths=col_widths, repeatRows=1)
    t.setStyle(ts)
    return t

# ── Header/Footer ──────────────────────────────────────────────
def header_footer(canvas, doc):
    canvas.saveState()
    W_p, H_p = A4
    # Header bar
    canvas.setFillColor(C_BLUE)
    canvas.rect(0, H_p - 1.5*cm, W_p, 1.5*cm, fill=1, stroke=0)
    canvas.setFillColor(C_WHITE)
    canvas.setFont("Helvetica-Bold", 11)
    canvas.drawString(MARGIN, H_p - 1.0*cm, "Histology Staining – Complete Guide")
    canvas.setFont("Helvetica", 9)
    canvas.drawRightString(W_p - MARGIN, H_p - 1.0*cm, "Orris Medical Reference")
    # Footer
    canvas.setFillColor(C_BLUE)
    canvas.rect(0, 0, W_p, 1.2*cm, fill=1, stroke=0)
    canvas.setFillColor(C_WHITE)
    canvas.setFont("Helvetica", 9)
    canvas.drawString(MARGIN, 0.4*cm, "For educational purposes only")
    canvas.drawRightString(W_p - MARGIN, 0.4*cm, f"Page {doc.page}")
    canvas.restoreState()

# ══════════════════════════════════════════════════════════════════
# BUILD CONTENT
# ══════════════════════════════════════════════════════════════════
story = []

# ─── COVER PAGE ───────────────────────────────────────────────
story.append(sp(3))
# Big colored banner
banner_data = [["HISTOLOGY STAINING"]]
banner_t = Table(banner_data, colWidths=[W - 2*MARGIN])
banner_t.setStyle(TableStyle([
    ("BACKGROUND", (0,0), (-1,-1), C_BLUE),
    ("TEXTCOLOR",  (0,0), (-1,-1), C_WHITE),
    ("FONTNAME",   (0,0), (-1,-1), "Helvetica-Bold"),
    ("FONTSIZE",   (0,0), (-1,-1), 32),
    ("ALIGN",      (0,0), (-1,-1), "CENTER"),
    ("VALIGN",     (0,0), (-1,-1), "MIDDLE"),
    ("TOPPADDING", (0,0), (-1,-1), 22),
    ("BOTTOMPADDING", (0,0), (-1,-1), 22),
]))
story.append(banner_t)
story.append(sp(0.5))

sub_banner_data = [["Complete Procedure Guide"]]
sub_t = Table(sub_banner_data, colWidths=[W - 2*MARGIN])
sub_t.setStyle(TableStyle([
    ("BACKGROUND", (0,0), (-1,-1), C_ACCENT),
    ("TEXTCOLOR",  (0,0), (-1,-1), C_WHITE),
    ("FONTNAME",   (0,0), (-1,-1), "Helvetica"),
    ("FONTSIZE",   (0,0), (-1,-1), 16),
    ("ALIGN",      (0,0), (-1,-1), "CENTER"),
    ("VALIGN",     (0,0), (-1,-1), "MIDDLE"),
    ("TOPPADDING", (0,0), (-1,-1), 10),
    ("BOTTOMPADDING", (0,0), (-1,-1), 10),
]))
story.append(sub_t)
story.append(sp(1.5))

# 5 stain boxes on cover
stains = [
    ("PAP STAIN",     "Cervical Cytology • FNA • Sputum",             C_PAP),
    ("H&E STAIN",     "Histopathology Tissue Sections",               C_PURPLE),
    ("GIEMSA STAIN",  "Blood Parasites • Malaria • Bone Marrow",      C_GREEN),
    ("AFB STAIN (ZN)","Mycobacteria • Tuberculosis • Leprosy",        C_RED),
    ("LEISHMAN STAIN","WBC Differential • Malaria • Blood Smear",     C_ORANGE),
]
cover_data = []
for name, desc, clr in stains:
    cover_data.append([
        Paragraph(f"<b>{name}</b>", ParagraphStyle("_ct", fontSize=13, textColor=C_WHITE,
            fontName="Helvetica-Bold", alignment=TA_CENTER, leading=17)),
        Paragraph(desc, ParagraphStyle("_cd", fontSize=10, textColor=C_WHITE,
            fontName="Helvetica", alignment=TA_LEFT, leading=14)),
    ])

ct = Table(cover_data, colWidths=[5.5*cm, W - 2*MARGIN - 5.5*cm])
row_styles = []
for i, (_, _, clr) in enumerate(stains):
    row_styles += [
        ("BACKGROUND", (0,i), (-1,i), clr),
        ("ROWHEIGHT",  (0,i), (-1,i), 1.1*cm),
    ]
ct.setStyle(TableStyle([
    ("VALIGN",      (0,0), (-1,-1), "MIDDLE"),
    ("TOPPADDING",  (0,0), (-1,-1), 8),
    ("BOTTOMPADDING",(0,0),(-1,-1), 8),
    ("LEFTPADDING", (0,0), (-1,-1), 12),
    ("RIGHTPADDING",(0,0), (-1,-1), 12),
    ("LINEABOVE",   (0,0), (-1,0), 0, C_WHITE),
    ("LINEBELOW",   (0,-1),(-1,-1), 0, C_WHITE),
    ("INNERGRID",   (0,0), (-1,-1), 0.5, C_WHITE),
] + row_styles))
story.append(ct)
story.append(sp(1.5))

story.append(Paragraph("Complete Step-by-Step Procedures, Principles & Cell Appearances",
    ParagraphStyle("_cov", fontSize=11, textColor=colors.HexColor("#555555"),
    fontName="Helvetica-Oblique", alignment=TA_CENTER)))
story.append(Paragraph("Orris Medical Reference   |   July 2026",
    ParagraphStyle("_cov2", fontSize=10, textColor=C_GRAY_MID,
    fontName="Helvetica", alignment=TA_CENTER, spaceBefore=4)))

story.append(PageBreak())

# ─── TABLE OF CONTENTS ────────────────────────────────────────
story.append(colored_box_para("TABLE OF CONTENTS", C_BLUE, font_size=16))
story.append(sp(0.5))
toc_items = [
    ("1.", "PAP STAINING (Papanicolaou Stain)", "3"),
    ("2.", "H&E STAINING (Hematoxylin & Eosin)", "7"),
    ("3.", "GIEMSA STAINING", "11"),
    ("4.", "AFB STAINING (Ziehl-Neelsen)", "14"),
    ("5.", "LEISHMAN STAINING", "17"),
    ("6.", "Comparison Table – All 5 Stains", "20"),
]
toc_data = [[Paragraph(n, toc_entry), Paragraph(t, toc_entry),
             Paragraph(p, ParagraphStyle("_pg", fontSize=11, fontName="Helvetica-Bold",
             alignment=TA_CENTER, textColor=C_ACCENT))]
            for n, t, p in toc_items]
toc_t = Table(toc_data, colWidths=[1*cm, 13*cm, 2*cm])
toc_t.setStyle(TableStyle([
    ("FONTSIZE", (0,0), (-1,-1), 11),
    ("LEADING",  (0,0), (-1,-1), 18),
    ("LINEBELOW",(0,0), (-1,-1), 0.3, C_GRAY_MID),
    ("TOPPADDING",(0,0),(-1,-1), 6),
    ("BOTTOMPADDING",(0,0),(-1,-1), 6),
    ("ROWBACKGROUNDS",(0,0),(-1,-1),[C_WHITE, C_GRAY_LIGHT]),
]))
story.append(toc_t)
story.append(PageBreak())

# ══════════════════════════════════════════════════════════════════
# SECTION TEMPLATE HELPER
# ══════════════════════════════════════════════════════════════════
def section_header(num, title, subtitle, color):
    data = [[
        Paragraph(f"{num}", ParagraphStyle("_n", fontSize=28, fontName="Helvetica-Bold",
            textColor=C_WHITE, alignment=TA_CENTER, leading=34)),
        Paragraph(f"<b>{title}</b><br/><font size='11'>{subtitle}</font>",
            ParagraphStyle("_t", fontSize=18, fontName="Helvetica-Bold",
            textColor=C_WHITE, leading=24)),
    ]]
    t = Table(data, colWidths=[1.8*cm, W - 2*MARGIN - 1.8*cm])
    t.setStyle(TableStyle([
        ("BACKGROUND", (0,0), (-1,-1), color),
        ("VALIGN",     (0,0), (-1,-1), "MIDDLE"),
        ("TOPPADDING", (0,0), (-1,-1), 12),
        ("BOTTOMPADDING",(0,0),(-1,-1), 12),
        ("LEFTPADDING",(0,0),(-1,-1), 10),
    ]))
    return t

def step_row(num, title, step_color, content_paras):
    """Returns a KeepTogether block for a single step."""
    elems = []
    elems.append(sp(0.2))
    data = [[
        Paragraph(str(num), ParagraphStyle("_sn", fontSize=14, fontName="Helvetica-Bold",
            textColor=C_WHITE, alignment=TA_CENTER, leading=18)),
        Paragraph(f"<b>{title}</b>", ParagraphStyle("_st", fontSize=11,
            fontName="Helvetica-Bold", textColor=C_WHITE, leading=15)),
    ]]
    st = Table(data, colWidths=[1.0*cm, W - 2*MARGIN - 1.0*cm])
    st.setStyle(TableStyle([
        ("BACKGROUND", (0,0), (-1,-1), step_color),
        ("VALIGN",     (0,0), (-1,-1), "MIDDLE"),
        ("TOPPADDING", (0,0), (-1,-1), 5),
        ("BOTTOMPADDING",(0,0),(-1,-1), 5),
        ("LEFTPADDING",(0,0),(-1,-1), 8),
    ]))
    elems.append(st)
    for p in content_paras:
        elems.append(p)
    return KeepTogether(elems)

def app_color(sec_color):
    """Returns a lighter version for step backgrounds."""
    return sec_color

# ══════════════════════════════════════════════════════════════════
# 1. PAP STAINING
# ══════════════════════════════════════════════════════════════════
story.append(section_header("1", "PAP STAINING", "Papanicolaou Stain – Cytology", C_PAP))
story.append(sp(0.4))

story.append(colored_box_para("Overview & Principle", C_PAP, font_size=12))
story.append(body_para(
    "The Papanicolaou (Pap) stain is a <b>polychromatic cytological staining technique</b> used to examine "
    "cells in cervical smears, sputum, urine, FNA, and other body fluids. It uses three dyes: "
    "<b>Hematoxylin</b> (nuclear stain), <b>Orange G / OG-6</b> (keratin stain), and "
    "<b>Eosin Azure / EA-50/65</b> (cytoplasmic counterstain). The combination produces excellent "
    "nuclear detail and differential cytoplasmic staining."))
story.append(sp(0.2))

story.append(colored_box_para("Dyes Used", C_PAP, font_size=11))
dye_data = [
    ["Dye", "Type", "Target", "Color Produced"],
    ["Hematoxylin (Harris/Gill's)", "Basic (cationic)", "DNA, RNA, chromatin (acidic)", "Blue-Purple"],
    ["Orange G (OG-6)", "Acid (anionic)", "Keratin, basic proteins", "Orange"],
    ["Eosin Y (in EA)", "Acid (anionic)", "Cytoplasmic proteins, nucleoli", "Pink-Red"],
    ["Light Green (in EA)", "Acid (anionic)", "Immature cell cytoplasm", "Blue-Green"],
]
story.append(make_table(dye_data, [5*cm, 3*cm, 4.5*cm, 3*cm], header_bg=C_PAP))
story.append(sp(0.3))

story.append(colored_box_para("Step-by-Step Procedure", C_PAP, font_size=12))

pap_steps = [
    ("FIXATION", [
        body_para("Immerse wet smear <b>immediately</b> in <b>95% Ethyl Alcohol for 15–20 minutes</b>."),
        bullet("Preserves cell morphology by denaturing proteins"),
        bullet("Prevents autolysis and drying artifacts"),
        bullet("Wet fixation is critical — air drying causes cell shrinkage and distortion"),
        note("Most critical step. If slide dries before fixation, results are unreliable."),
    ]),
    ("HYDRATION (Graded Alcohol)", [
        body_para("Pass slide through descending alcohols: <b>80% → 70% → 50% → Distilled water</b> (10 dips each)."),
        bullet("Removes alcohol to allow water-based hematoxylin to penetrate"),
        bullet("Gradual hydration prevents cell distortion"),
    ]),
    ("HEMATOXYLIN (Nuclear Stain)", [
        body_para("Stain in <b>Gill's or Harris Hematoxylin for 10–15 minutes</b>."),
        bullet("Basic dye binds acidic nuclear components (DNA/RNA) via electrostatic attraction"),
        bullet("Aluminium mordant forms lake complex with hematoxylin — permanent staining"),
        bullet("Regressive method: overstain then differentiate | Progressive: exact timed staining"),
    ]),
    ("WASHING", [
        body_para("Running tap water for <b>2–3 minutes</b>. Removes excess hematoxylin."),
    ]),
    ("ACID ALCOHOL DIFFERENTIATION", [
        body_para("<b>0.5% acid alcohol — only 1 dip!</b> (Regressive method only)"),
        bullet("Removes non-specific cytoplasmic hematoxylin staining"),
        bullet("Nuclear stain is retained due to mordant binding"),
        note("Over-differentiation removes nuclear stain. Exactly 1 dip — no more."),
    ]),
    ("BLUING", [
        body_para("Running tap water or Scott's tap water substitute for <b>3–10 minutes</b>."),
        bullet("Acid treatment turns hematoxylin brown-red → alkaline environment converts to blue-purple"),
        bullet("Chemical shift: Red soluble salt → Blue insoluble aluminum-hematein lake"),
    ]),
    ("DEHYDRATION (Pre-counterstain)", [
        body_para("Pass through <b>50% → 70% → 95% alcohol</b> (10 dips each)."),
        bullet("Removes water — counterstains (OG, EA) are alcohol-based"),
        bullet("Water presence prevents proper counterstain binding"),
    ]),
    ("ORANGE G (OG-6) STAINING", [
        body_para("Dip in OG-6 solution for <b>1–2 minutes</b>. Then 95% alcohol 2–3 dips."),
        bullet("Acid dye binds basic cytoplasmic proteins, especially keratin"),
        bullet("Superficial squamous cells and RBCs stain bright orange"),
        bullet("Phosphotungstic acid in OG-6 prevents non-specific staining"),
    ]),
    ("EA STAINING (Eosin Azure)", [
        body_para("Stain in <b>EA-50 (gynecological) or EA-65 (non-GYN) for 3–5 minutes</b>."),
        bullet("EA is polychromatic: Eosin Y stains nucleoli, cilia pink-red"),
        bullet("Light Green stains immature/metabolically active cell cytoplasm blue-green"),
        bullet("Differential cytoplasmic staining allows maturation assessment"),
    ]),
    ("FINAL DEHYDRATION → CLEARING → MOUNTING", [
        body_para("95% alcohol × 2 → Absolute alcohol → Xylene × 3 → <b>DPX coverslip</b>."),
        bullet("Complete water removal required for resinous mounting medium"),
        bullet("Xylene clears tissue (same refractive index as glass/mountant)"),
        bullet("DPX provides permanent preservation for archival storage"),
    ]),
]

for i, (title, content) in enumerate(pap_steps):
    story.append(step_row(i+1, title, C_PAP, content))

story.append(sp(0.4))
story.append(colored_box_para("Cell Appearance in Pap Stain", C_PAP, font_size=12))
pap_result = [
    ["Structure", "Color", "Reason"],
    ["Nucleus / Chromatin", "Blue-Purple", "Hematoxylin + DNA/chromatin"],
    ["Nucleolus", "Dark blue-purple", "RNA-rich"],
    ["Superficial squamous cells", "Orange / Pink-orange", "Orange G binds keratin"],
    ["Intermediate cells", "Light green / Blue-green", "Light Green in EA"],
    ["Parabasal cells", "Dark green / Blue", "Immature, RNA-rich cytoplasm"],
    ["Cilia", "Pink-red", "Eosin Y"],
    ["Nucleoli", "Pink-red", "Eosin Y"],
    ["RBCs", "Orange-red", "OG strongly binds hemoglobin"],
    ["Neutrophils", "Small blue nuclei, pink cytoplasm", "Hematoxylin + Eosin"],
    ["Malignant cells", "Large dark nuclei, high N:C ratio", "Increased chromatin density"],
]
story.append(make_table(pap_result, [5.5*cm, 4*cm, 6*cm], header_bg=C_PAP))
story.append(sp(0.3))
story.append(note("Normal: Large cytoplasm, small pyknotic nucleus. Malignant: Large nucleus, scant cytoplasm, irregular chromatin. Bethesda system used for reporting."))
story.append(PageBreak())

# ══════════════════════════════════════════════════════════════════
# 2. H&E STAINING
# ══════════════════════════════════════════════════════════════════
story.append(section_header("2", "H&E STAINING", "Hematoxylin & Eosin – Histopathology Gold Standard", C_PURPLE))
story.append(sp(0.4))

story.append(colored_box_para("Overview & Principle", C_PURPLE, font_size=12))
story.append(body_para(
    "Hematoxylin & Eosin (H&E) is the <b>most widely used stain in histopathology</b>. It is applied to "
    "formalin-fixed, paraffin-embedded tissue sections. Hematoxylin (basic dye) stains nuclei blue-purple; "
    "Eosin (acidic dye) stains cytoplasm and extracellular matrix pink. This combination gives superb "
    "cellular detail for pathological diagnosis."))

story.append(colored_box_para("Dyes Used", C_PURPLE, font_size=11))
he_dye = [
    ["Dye", "Type", "Target", "Color Produced"],
    ["Hematoxylin + Al mordant", "Basic (cationic)", "DNA, RNA, chromatin", "Blue-Purple (Basophilic)"],
    ["Eosin Y (alcohol-based)", "Acid (anionic)", "Cytoplasmic proteins, collagen, muscle", "Pink-Red (Eosinophilic)"],
]
story.append(make_table(he_dye, [5*cm, 3*cm, 4.5*cm, 3*cm], header_bg=C_PURPLE))
story.append(sp(0.3))

story.append(colored_box_para("Step-by-Step Procedure", C_PURPLE, font_size=12))

he_steps = [
    ("FIXATION", [
        body_para("Immerse tissue <b>immediately</b> in <b>10% Neutral Buffered Formalin (NBF) for 24–48 hours</b>."),
        bullet("Terminates cell metabolism and enzyme activity"),
        bullet("Prevents autolysis (self-digestion by intracellular enzymes)"),
        bullet("Formaldehyde cross-links amino groups (mainly lysine) of proteins"),
        bullet("Kills pathogens — biosafety for lab handling"),
        note("Formalin does NOT fix lipids — fat cells appear as empty vacuoles after processing."),
    ]),
    ("TISSUE PROCESSING (Automated Processor)", [
        body_para("Sequential steps in tissue processor: Washing → Dehydration → Clearing → Infiltration."),
        bullet("Dehydration: Graded alcohol (70% → 80% → 95% → 100%) removes water"),
        bullet("Clearing: Xylene/Xylol replaces alcohol (miscible with both alcohol & paraffin)"),
        bullet("Paraffin infiltration: Molten paraffin (60°C) fills tissue spaces"),
        bullet("Paraffin provides solid support for thin sectioning"),
    ]),
    ("EMBEDDING & SECTIONING", [
        body_para("Tissue embedded in paraffin block. <b>Microtome</b> cuts <b>5–7 µm sections</b> mounted on glass slides."),
        bullet("5–7 µm thickness = single cell layer — essential for light microscopy"),
        bullet("Sections floated on warm water bath to flatten, then mounted on charged slides"),
    ]),
    ("DEPARAFFINIZATION", [
        body_para("<b>Xylene × 2 changes, 5 minutes each</b>."),
        bullet("Dissolves paraffin wax completely"),
        bullet("Stains cannot penetrate wax — must be removed first"),
    ]),
    ("REHYDRATION", [
        body_para("Descending alcohol series: <b>100% → 95% → 80% → 70% → Distilled water</b>."),
        bullet("Hematoxylin is water-based — tissue must be rehydrated"),
        bullet("Graded steps prevent osmotic shock to tissue"),
    ]),
    ("HEMATOXYLIN STAINING", [
        body_para("Immerse in <b>Mayer's or Harris Hematoxylin for 3–10 minutes</b>."),
        bullet("Hematoxylin oxidized to hematein (active form) binds aluminium mordant"),
        bullet("Al-hematein complex (positively charged) binds negatively charged DNA/RNA"),
        bullet("Nuclear chromatin, nucleoli, and mitotic figures stained blue-purple"),
    ]),
    ("WASHING & BLUEING", [
        body_para("Running tap water or Scott's solution for <b>5–10 minutes</b>."),
        bullet("Acid residues removed; alkaline water (pH 7-8) causes 'blueing' reaction"),
        bullet("Hematoxylin shifts from brown-red to characteristic blue-purple (bathochromic shift)"),
    ]),
    ("EOSIN STAINING", [
        body_para("Stain in <b>1% Eosin Y in 95% alcohol for 1–3 minutes</b>."),
        bullet("Eosin (anionic dye) binds positively charged cytoplasmic proteins"),
        bullet("Collagen, muscle fibers, RBCs all stain pink-red"),
        bullet("Alcohol-based eosin gives crisper results than aqueous eosin"),
    ]),
    ("DEHYDRATION → CLEARING → MOUNTING", [
        body_para("95% alcohol → 100% alcohol → <b>Xylene × 3 → DPX/Canada Balsam coverslip</b>."),
        bullet("Complete dehydration required — water causes cloudiness in permanent mount"),
        bullet("Xylene is transparent and miscible with mounting resin"),
        bullet("DPX mount is permanent — slides usable for decades"),
    ]),
]
for i, (title, content) in enumerate(he_steps):
    story.append(step_row(i+1, title, C_PURPLE, content))

story.append(sp(0.4))
story.append(colored_box_para("Cell Appearance in H&E", C_PURPLE, font_size=12))
he_result = [
    ["Structure", "Color", "Term"],
    ["Nucleus / Chromatin", "Blue-Purple", "Basophilic"],
    ["Nucleolus", "Dark blue/purple", "Basophilic (RNA)"],
    ["Cytoplasm (most cells)", "Pink", "Eosinophilic"],
    ["RBCs", "Bright pink/orange-red", "Eosinophilic (Hemoglobin)"],
    ["Collagen fibers", "Pale pink", "Eosinophilic"],
    ["Skeletal/cardiac muscle", "Deep pink-red", "Eosinophilic (Myosin)"],
    ["Cartilage matrix", "Blue-purple", "Basophilic (Proteoglycans)"],
    ["Fat cells (adipocytes)", "Empty white vacuoles", "Lipid dissolved in processing"],
    ["Calcification (calcium)", "Dark blue-purple", "Basophilic"],
    ["Mitotic figures", "Dark purple", "Condensed chromosomes"],
    ["Plasma cells", "Clockface purple nucleus, pink cytoplasm", "Eccentric nucleus, RER-rich"],
    ["Mast cell granules", "Purple/metachromatic", "Heparin/histamine granules"],
]
story.append(make_table(he_result, [5.5*cm, 4.5*cm, 5.5*cm], header_bg=C_PURPLE))
story.append(sp(0.3))
story.append(note("Basophilic = Blue-purple (DNA, RNA, GAGs, calcium). Eosinophilic = Pink-red (proteins, collagen, RBCs). These terms describe affinity for the respective dyes."))
story.append(PageBreak())

# ══════════════════════════════════════════════════════════════════
# 3. GIEMSA STAINING
# ══════════════════════════════════════════════════════════════════
story.append(section_header("3", "GIEMSA STAINING", "Romanowsky Stain – Blood Parasites & Haematology", C_GREEN))
story.append(sp(0.4))

story.append(colored_box_para("Overview & Principle", C_GREEN, font_size=12))
story.append(body_para(
    "Giemsa stain is a <b>Romanowsky-type stain</b> used for blood smears, bone marrow, and parasite detection. "
    "It is the <b>WHO-recommended stain for malaria diagnosis</b>. Composed of Azure B (polychromed methylene blue) "
    "and Eosin Y dissolved in methanol and glycerol. The Romanowsky effect (azure-eosin complex) produces the "
    "characteristic purple color of nuclei distinct from pink cytoplasm. pH 7.2 is critical for optimal results."))

story.append(colored_box_para("Composition", C_GREEN, font_size=11))
gi_comp = [
    ["Component", "Amount", "Role"],
    ["Giemsa powder (Azure B + Eosin Y)", "3.8 g", "Primary staining dyes"],
    ["Methanol (anhydrous)", "250 mL", "Solvent + fixative action"],
    ["Glycerol", "250 mL", "Stabilizer, extends shelf life"],
    ["Phosphate buffer pH 7.2", "As working diluent", "Critical for Romanowsky effect"],
]
story.append(make_table(gi_comp, [6*cm, 3*cm, 6.5*cm], header_bg=C_GREEN))
story.append(sp(0.3))

story.append(colored_box_para("Step-by-Step Procedure", C_GREEN, font_size=12))
gi_steps = [
    ("SMEAR PREPARATION", [
        body_para("Make <b>thin film</b> (individual cell morphology) or <b>thick film</b> (parasite sensitivity) on clean glass slide. Air dry completely."),
        bullet("Thin film: 1 drop of blood, spread at 45° angle — single cell layer"),
        bullet("Thick film: 2-3 drops, spread in circular motion — 3x more sensitive for parasites"),
    ]),
    ("FIXATION (THIN FILM ONLY!)", [
        body_para("<b>Absolute methanol — 2-3 dips (30 seconds)</b> in Coplin jar."),
        bullet("Methanol denatures proteins and fixes cellular morphology"),
        bullet("Adheres cells firmly to glass slide"),
        note("THICK FILM IS NOT FIXED. RBCs must lyse during staining to expose parasites. Methanol prevents lysis."),
    ]),
    ("PREPARE GIEMSA WORKING SOLUTION", [
        body_para("Dilute stock Giemsa in <b>phosphate buffer pH 7.2</b>:"),
        bullet("Thin film: 1:20 dilution → 20-30 minutes staining"),
        bullet("Thick film: 1:50 dilution → 45-60 minutes (or 10% solution for 8-10 min rapid)"),
        note("pH 7.2 is critical. Alkaline pH (>7.4) = over-blue, obscures detail. Acidic pH (<7.0) = Schuffner's dots lost."),
    ]),
    ("STAINING", [
        body_para("Immerse slides in working Giemsa solution for specified time (see above)."),
        bullet("Azure dyes (negatively charged) bind positively charged nuclear components → purple"),
        bullet("Eosin (negatively charged) binds positively charged cytoplasmic proteins → pink"),
        bullet("Parasite nucleus stains red/magenta (Romanowsky effect = azure-eosin complex)"),
    ]),
    ("WASHING", [
        body_para("Dip gently in <b>buffered water pH 7.2 (3-5 dips)</b>. Float off iridescent scum."),
        note("Excessive washing decolorizes the film. 3-5 gentle dips only."),
    ]),
    ("AIR DRY & EXAMINE", [
        body_para("Stand vertically to dry. Examine under oil immersion (100x)."),
        bullet("Thin film: Examine first at 40x for overview, then 100x oil for detail"),
        bullet("Thick film: Used for screening — quantify parasites per 1000 RBCs"),
    ]),
]
for i, (title, content) in enumerate(gi_steps):
    story.append(step_row(i+1, title, C_GREEN, content))

story.append(sp(0.4))
story.append(colored_box_para("Cell Appearance in Giemsa", C_GREEN, font_size=12))
gi_result = [
    ["Cell / Structure", "Color", "Notes"],
    ["Leukocyte nucleus", "Purple/Dark blue", "All WBCs — Azure dyes + DNA"],
    ["Neutrophil granules", "Neutral pink-purple", "Mixed charge — both dyes bind"],
    ["Eosinophil granules", "Bright red-orange", "Eosin strongly binds specific proteins"],
    ["Basophil granules", "Deep purple/violet-black", "Azure strongly binds heparin"],
    ["Monocyte cytoplasm", "Grey-blue, vacuolated", "Azure dyes, phagocytic vacuoles"],
    ["Lymphocyte cytoplasm", "Scant, sky blue", "Azure, minimal cytoplasm"],
    ["RBCs (normal)", "Pale pink/salmon", "Hemoglobin, eosinophilic"],
    ["Platelets", "Purple granules", "Azurophilic granules"],
    ["Malaria parasite nucleus", "Red/Magenta dot", "Romanowsky effect, eosin-azure"],
    ["Malaria cytoplasm", "Blue ring", "Azure dyes bind RNA"],
    ["Schüffner's dots (P. vivax)", "Pink stippling in RBC", "Characteristic of P. vivax/ovale"],
    ["Malarial pigment (hemozoin)", "Brown-black granules", "Hematin byproduct in parasites"],
    ["Histoplasma in macrophages", "Blue intracellular yeasts", "Fungal organisms"],
]
story.append(make_table(gi_result, [5*cm, 4*cm, 6.5*cm], header_bg=C_GREEN))
story.append(PageBreak())

# ══════════════════════════════════════════════════════════════════
# 4. AFB STAINING (ZIEHL-NEELSEN)
# ══════════════════════════════════════════════════════════════════
story.append(section_header("4", "AFB STAINING", "Ziehl-Neelsen Stain – Mycobacteria / Tuberculosis", C_RED))
story.append(sp(0.4))

story.append(colored_box_para("Overview & Principle", C_RED, font_size=12))
story.append(body_para(
    "The Ziehl-Neelsen (ZN) stain detects <b>Acid-Fast Bacilli (AFB)</b> such as <i>Mycobacterium tuberculosis</i>, "
    "M. leprae, and other mycobacteria. The key principle: mycobacterial cell walls contain "
    "<b>mycolic acids</b> (long-chain C60-C90 fatty acids), forming a waxy lipid layer. "
    "Carbol fuchsin + heat penetrates this layer. Once inside, the dye CANNOT be removed "
    "by acid-alcohol — hence 'acid-fast'. Non-AFB organisms are decolorized and "
    "counterstained blue."))

story.append(colored_box_para("Reagents", C_RED, font_size=11))
afb_reagents = [
    ["Reagent", "Composition", "Role"],
    ["Carbol Fuchsin", "Basic fuchsin + 5% phenol in ethanol/water", "Primary dye — penetrates mycolic acid wall"],
    ["Acid Alcohol", "3% HCl in 95% ethanol", "Decolorizer — removes dye from non-AFB"],
    ["Methylene Blue (1%)", "Aqueous solution", "Counterstain — stains non-AFB blue"],
]
story.append(make_table(afb_reagents, [4*cm, 6*cm, 5.5*cm], header_bg=C_RED))
story.append(sp(0.3))

story.append(colored_box_para("Step-by-Step Procedure (Ziehl-Neelsen)", C_RED, font_size=12))
afb_steps = [
    ("SMEAR PREPARATION & FIXATION", [
        body_para("Spread sputum/specimen on slide, air dry, then <b>heat fix</b> (pass over flame 3 times)."),
        bullet("Heat fixation adheres cells to slide"),
        bullet("Inactivates pathogenic organisms — biosafety for TB specimens"),
        bullet("Specimen can also be fixed at 80°C for 15 minutes (safer method)"),
    ]),
    ("CARBOL FUCHSIN (Primary Stain)", [
        body_para("Flood slide with <b>Carbol Fuchsin</b>. Apply <b>gentle heat until steaming</b>. Leave <b>5 minutes</b>. Cool."),
        bullet("Phenol (5%) disrupts the lipid-rich mycolic acid cell wall"),
        bullet("Heat further increases dye penetration into waxy wall"),
        bullet("Basic fuchsin (positively charged) binds nucleic acids inside organism"),
        note("Kinyoun modification: No heating — higher phenol (8%) compensates. Safer but slightly less sensitive."),
    ]),
    ("WASHING", [
        body_para("Running water wash. Removes unbound surface carbol fuchsin."),
    ]),
    ("ACID ALCOHOL DECOLORIZATION", [
        body_para("<b>3% HCl in 95% alcohol for 1–3 minutes</b> until no more red color runs off."),
        bullet("Removes carbol fuchsin from NON-acid-fast organisms (their walls lack mycolic acids)"),
        bullet("AFB RETAIN the dye — mycolic acids form insoluble dye-lipid complex"),
        bullet("This is the diagnostic key: retained red = acid-fast = mycobacterium"),
        note("Modified AFB (Nocardia, weakly acid-fast): Use 0.5% H₂SO₄ instead of acid-alcohol."),
    ]),
    ("WASHING", [
        body_para("Running water wash. Prepares slide for counterstain."),
    ]),
    ("METHYLENE BLUE COUNTERSTAIN", [
        body_para("<b>1% Methylene Blue for 1–2 minutes</b>."),
        bullet("Stains decolorized background cells, neutrophils, and non-AFB bacteria blue"),
        bullet("Creates color contrast: red AFB against blue background"),
        bullet("Without counterstain, AFB would be invisible against colorless background"),
    ]),
    ("WASH, DRY & EXAMINE", [
        body_para("Wash, air dry, examine under <b>oil immersion (1000x)</b>. Scan 100 fields minimum."),
        bullet("Report as: No AFB / 1+ / 2+ / 3+ / 4+ per WHO grading"),
        bullet("At least 300 fields should be examined before reporting negative"),
    ]),
]
for i, (title, content) in enumerate(afb_steps):
    story.append(step_row(i+1, title, C_RED, content))

story.append(sp(0.4))
story.append(colored_box_para("Cell Appearance in AFB (ZN) Stain", C_RED, font_size=12))
afb_result = [
    ["Structure", "Color", "Notes"],
    ["Acid-Fast Bacilli (M. tuberculosis)", "Bright red/magenta rods", "Retained carbol fuchsin"],
    ["Non-AFB bacteria", "Blue", "Methylene blue counterstain"],
    ["Neutrophils / Host cells", "Blue background", "Non-acid-fast"],
    ["Background", "Pale blue", "Methylene blue"],
    ["M. kansasii", "Red, cross-barred 'shepherd's crook'", "Distinctive banded morphology"],
    ["Nocardia", "Weakly pink/red (partial)", "Weakly acid-fast — needs modified AFB"],
    ["M. leprae", "Red, globi in macrophages", "Modified Fite-Faraco stain preferred"],
]
story.append(make_table(afb_result, [5*cm, 4*cm, 6.5*cm], header_bg=C_RED))
story.append(sp(0.3))
afb_grade = [
    ["Grading (Carbol Fuchsin, 1000x)", "Report"],
    ["0 AFB in 300 fields", "No AFB seen"],
    ["1-2 per 300 fields", "Doubtful — repeat"],
    ["1-9 per 100 fields", "1+"],
    ["1-9 per 10 fields", "2+"],
    ["1-9 per field", "3+"],
    [">9 per field", "4+"],
]
story.append(colored_box_para("AFB Grading (WHO/IUATLD)", C_RED, font_size=11))
story.append(make_table(afb_grade, [8*cm, 7.5*cm], header_bg=C_RED))
story.append(PageBreak())

# ══════════════════════════════════════════════════════════════════
# 5. LEISHMAN STAINING
# ══════════════════════════════════════════════════════════════════
story.append(section_header("5", "LEISHMAN STAINING", "Romanowsky Stain – WBC Differential & Malaria", C_ORANGE))
story.append(sp(0.4))

story.append(colored_box_para("Overview & Principle", C_ORANGE, font_size=12))
story.append(body_para(
    "Leishman stain is a <b>Romanowsky-type stain</b> named after Scottish pathologist William Boog Leishman. "
    "It is widely used for <b>WBC differential counts, malaria diagnosis, and trypanosomiasis</b>. "
    "Composed of polychromed methylene blue (azure mixture) + Eosin Y dissolved in methanol. "
    "Key advantage: <b>methanol in the stain acts as both fixative and solvent</b>, combining the "
    "fixation and primary staining into one step. Similar to Giemsa but faster."))

story.append(colored_box_para("Composition", C_ORANGE, font_size=11))
lei_comp = [
    ["Component", "Role"],
    ["Polychromed Methylene Blue (Azure mixture)", "Nuclear stain — blue-purple; Romanowsky effect"],
    ["Eosin Y", "Cytoplasmic stain — pink-red; granule staining"],
    ["Methanol (anhydrous)", "Solvent + FIXATIVE — combined in one step"],
    ["Phosphate Buffer pH 6.8-7.0", "Dilutant — activates ionic dissociation for staining"],
]
story.append(make_table(lei_comp, [8*cm, 7.5*cm], header_bg=C_ORANGE))
story.append(sp(0.3))

story.append(colored_box_para("Step-by-Step Procedure", C_ORANGE, font_size=12))
lei_steps = [
    ("BLOOD SMEAR PREPARATION", [
        body_para("Prepare <b>thin blood smear</b> from EDTA blood or finger prick. Spread evenly, <b>air dry completely</b>."),
        bullet("Thin film: single cell layer for individual morphology assessment"),
        bullet("Must be completely dry before staining — trace moisture causes artifacts"),
        note("Do NOT shake stain bottle before use — precipitates cause artifacts mimicking platelets."),
    ]),
    ("FIXATION + PRIMARY STAINING (Combined Step!)", [
        body_para("Flood slide with <b>undiluted Leishman stain for 1–2 minutes</b>. Do NOT rinse."),
        bullet("Methanol in stain = FIXATIVE: preserves morphology, adheres cells to slide"),
        bullet("Methanol also serves as solvent carrying the dye"),
        bullet("This combined fixation-staining step distinguishes Leishman from Giemsa"),
        note("This is Leishman's major advantage — no separate fixation step needed."),
    ]),
    ("BUFFER DILUTION (Critical Step)", [
        body_para("WITHOUT washing, add <b>double volume of phosphate buffer pH 6.8</b>. Mix gently by rocking. Leave <b>3 minutes</b>. A greenish metallic sheen should appear on surface."),
        bullet("Buffer provides water — essential for ionic dissociation of dyes"),
        bullet("Romanowsky effect activated: azure-eosin complex forms → purple color for nuclei"),
        bullet("pH 6.8: slightly acidic → better eosinophilic structures (eosinophil granules, malaria)"),
        bullet("Metallic sheen on surface = positive quality control indicator"),
    ]),
    ("WASHING", [
        body_para("Rinse slide with <b>deionized water for 10–15 seconds</b>. Drain."),
        bullet("Removes excess stain and buffer"),
        note("Excessive washing removes stain. 10-15 seconds only. If over-washed, restain."),
    ]),
    ("AIR DRY & EXAMINE", [
        body_para("Stand slides upright to drain and <b>air dry away from dust</b>. Examine under oil immersion (100x)."),
        bullet("Do not blot — causes artifacts"),
        bullet("Total staining time: 5–8 minutes (much faster than Giemsa)"),
    ]),
]
for i, (title, content) in enumerate(lei_steps):
    story.append(step_row(i+1, title, C_ORANGE, content))

story.append(sp(0.4))
story.append(colored_box_para("Cell Appearance in Leishman Stain", C_ORANGE, font_size=12))
lei_result = [
    ["Cell / Structure", "Color", "Notes"],
    ["RBCs", "Pale pink/salmon", "Hemoglobin eosinophilic"],
    ["Neutrophil nucleus", "Purple (multilobed)", "Azure + DNA"],
    ["Neutrophil granules", "Neutral pink-lilac", "Mixed charge, both dyes"],
    ["Eosinophil nucleus", "Purple (bilobed)", "Azure"],
    ["Eosinophil granules", "Bright orange-red", "Eosin strongly binds MBP"],
    ["Basophil granules", "Deep purple/violet-black", "Azure + heparin"],
    ["Monocyte nucleus", "Purple, kidney/horseshoe-shaped", "Azure"],
    ["Monocyte cytoplasm", "Grey-blue, vacuolated", "Azure, phagocytic vacuoles"],
    ["Lymphocyte nucleus", "Deep purple, dense", "Azure, condensed chromatin"],
    ["Lymphocyte cytoplasm", "Scant, sky blue", "Azure dyes"],
    ["Platelets", "Purple granules, pale blue area", "Azurophilic granules"],
    ["Malaria ring stage", "Blue cytoplasm + red nucleus dot", "Azure + eosin (Romanowsky)"],
    ["Malarial pigment (hemozoin)", "Brown-black coarse granules", "Hematin byproduct"],
    ["Trypanosoma", "Purple nucleus/kinetoplast, blue cytoplasm", "Blood parasites"],
    ["Leishman-Donovan bodies", "Blue oval amastigotes in macrophages", "Kala-azar diagnosis"],
]
story.append(make_table(lei_result, [5*cm, 4*cm, 6.5*cm], header_bg=C_ORANGE))
story.append(PageBreak())

# ══════════════════════════════════════════════════════════════════
# 6. MASTER COMPARISON TABLE
# ══════════════════════════════════════════════════════════════════
story.append(colored_box_para("MASTER COMPARISON – ALL 5 STAINS", C_BLUE, font_size=14))
story.append(sp(0.3))

comp_data = [
    ["Feature", "PAP", "H&E", "Giemsa", "AFB (ZN)", "Leishman"],
    ["Primary Use", "Cytology\n(cervical,\nFNA, sputum)", "Histopathology\ntissue biopsy", "Blood parasites\nmalaria\nbone marrow", "Mycobacteria\nTB, Leprosy\nNocardia", "WBC differential\nmalaria\nblood smear"],
    ["Specimen", "Cytology smear\n(wet)", "Paraffin tissue\nsections", "Blood/BM smear", "Sputum, tissue,\nBAL", "Blood smear"],
    ["Fixative", "95% Ethyl\nAlcohol (wet)", "10% NBF\n(formalin)", "Methanol\n(separate)", "Heat fixation", "Methanol\n(in stain!)"],
    ["Primary Stain", "Hematoxylin\n+ OG + EA", "Hematoxylin\n+ Eosin", "Azure B\n+ Eosin Y", "Carbol\nFuchsin (red)", "Polychromed\nMethylene Blue\n+ Eosin"],
    ["Counterstain", "None\n(EA acts as\ncounterstain)", "Eosin\ncounterstains\nhematoxylin", "None needed", "Methylene\nBlue (blue)", "Buffer activates\nRomanowsky\neffect"],
    ["Nucleus Color", "Blue-Purple", "Blue-Purple", "Purple", "Blue\n(methylene blue)", "Purple"],
    ["Cytoplasm", "Pink/Green\n(differential)", "Pink", "Pink-grey-blue", "Blue\n(background)", "Pink-grey"],
    ["Special Feature", "Diff cytoplasmic\nstaining by\nmaturation", "Universal tissue\nstain, gold\nstandard", "Schüffner's dots\nin P.vivax", "AFB = bright\nRED rods on\nblue background", "Combined\nfix+stain\nstep"],
    ["Total Time", "20–30 min", "~2 hours\n(with processing)", "30–60 min", "20–30 min", "5–8 min"],
    ["Reporting", "Bethesda\nsystem", "Histopathology\nreport", "Parasite count\nper 1000 RBCs", "WHO AFB\ngrading 1+–4+", "WBC\ndifferential %"],
]

col_w = [(W - 2*MARGIN) / 6] * 6
col_w[0] = 3*cm
remaining = W - 2*MARGIN - 3*cm
for i in range(1, 6):
    col_w[i] = remaining / 5

comp_t = Table(comp_data, colWidths=col_w, repeatRows=1)
row_colors = [C_WHITE] * len(comp_data)
section_colors_map = [C_BLUE, C_PAP, C_PURPLE, C_GREEN, C_RED, C_ORANGE]

row_ts = [
    ("BACKGROUND", (0,0), (-1,0), C_BLUE),
    ("TEXTCOLOR", (0,0), (-1,0), C_WHITE),
    ("FONTNAME", (0,0), (-1,0), "Helvetica-Bold"),
    ("FONTSIZE", (0,0), (-1,-1), 8),
    ("LEADING", (0,0), (-1,-1), 11),
    ("GRID", (0,0), (-1,-1), 0.4, C_GRAY_MID),
    ("VALIGN", (0,0), (-1,-1), "MIDDLE"),
    ("TOPPADDING", (0,0), (-1,-1), 4),
    ("BOTTOMPADDING", (0,0), (-1,-1), 4),
    ("LEFTPADDING", (0,0), (-1,-1), 5),
    ("ROWBACKGROUNDS", (0,1), (-1,-1), [C_WHITE, C_GRAY_LIGHT]),
    ("FONTNAME", (0,1), (0,-1), "Helvetica-Bold"),
    ("TEXTCOLOR", (0,1), (0,-1), C_BLUE),
]
comp_t.setStyle(TableStyle(row_ts))
story.append(comp_t)
story.append(sp(0.5))

# Leishman vs Giemsa mini table
story.append(colored_box_para("Leishman vs Giemsa – Quick Comparison", C_GREEN, font_size=11))
lg_data = [
    ["Feature", "Leishman", "Giemsa"],
    ["Fixation", "Combined in stain (methanol)", "Separate methanol step"],
    ["Total time", "5–8 minutes", "30–60 minutes"],
    ["Buffer pH", "6.8–7.0", "7.2"],
    ["Stability (diluted)", "Unstable — use within 3 hours", "Stable — glycerol present"],
    ["Malaria diagnosis", "Good for thin smears", "WHO recommended (thin + thick)"],
    ["Common in", "India, UK haematology labs", "Parasitology, reference labs"],
    ["Artifact risk", "Higher (precipitation)", "Lower"],
]
story.append(make_table(lg_data, [4.5*cm, 7*cm, 4*cm], header_bg=C_GREEN))
story.append(sp(0.4))

story.append(colored_box_para("Romanowsky Effect – Explanation", C_BLUE, font_size=11))
story.append(body_para(
    "The <b>Romanowsky effect</b> is seen in Giemsa, Leishman, Wright, and other polychromatic blood stains. "
    "It produces a <b>purple color distinct from either blue or red alone</b>. The mechanism: "
    "polychromed methylene blue (oxidized to Azure B, Azure A etc.) combines with eosin to form "
    "an <b>azure-eosin complex</b>. This complex selectively stains certain structures (malaria parasite nucleus, "
    "Schüffner's dots) a distinct purple-red (magenta) that neither dye alone can produce. "
    "This is the basis for differentiating malaria parasite chromatin (red-magenta dot) from the blue cytoplasm."))
story.append(sp(0.3))

# References
story.append(hr(C_BLUE, 1))
story.append(colored_box_para("Sources & References", C_BLUE, font_size=11))
refs = [
    "Histology: A Text and Atlas with Correlated Cell and Molecular Biology (Wolters Kluwer)",
    "Junqueira's Basic Histology – Text and Atlas, 17th Edition",
    "Henry's Clinical Diagnosis and Management by Laboratory Methods (Elsevier)",
    "Tietz Textbook of Laboratory Medicine, 7th Edition",
    "Robbins & Kumar Basic Pathology (Elsevier)",
    "Jawetz, Melnick & Adelberg's Medical Microbiology, 28th Edition",
    "Nuances of the Papanicolaou Stain – PMC/NIH (PMID: PMC9345133)",
    "WHO Guidelines – Giemsa Staining of Malaria Blood Films (WHO WPRO SOP-07A)",
    "Newcomer Supply – Papanicolaou Stain Procedure (Technical Reference)",
]
for r in refs:
    story.append(bullet(r))

story.append(sp(0.3))
story.append(Paragraph(
    "This document is for educational/reference purposes only. Always follow your institution's SOPs for diagnostic staining.",
    ParagraphStyle("_disc", fontSize=8.5, textColor=colors.HexColor("#888888"),
    fontName="Helvetica-Oblique", alignment=TA_CENTER)))

# ── BUILD PDF ──────────────────────────────────────────────────
doc.build(story, onFirstPage=header_footer, onLaterPages=header_footer)
print(f"PDF saved to: {OUTPUT_PATH}")

cd /home/daytona/workspace/staining-guide && python3 generate_pdf.py 2>&1

~/staining-guide/generate_pdf.py

Successfully replaced 1 instance(s) of the string in '/home/daytona/workspace/staining-guide/generate_pdf.py'

cd /home/daytona/workspace/staining-guide && python3 generate_pdf.py 2>&1

ls -lh /home/daytona/workspace/staining-guide/Histology_Staining_Complete_Guide.…