Microscopy and Types of Microscopes

magnification of tiny object; more distinct virtual image; study in more detail

2 lens systems; objective + eyepiece; visible light through specimen; brightfield; virtual image

brightfield; dark-field; phase-contrast; fluorescence; electron; compound

light passes through thin specimen; standard in hematology

makes sample look larger than real life

how close 2 objects can be before they look like 1; clear + distinct + sharp image

enhances parts within a cell

shorter wavelength = greater resolution

light-gathering ability of objective; larger NA = greater resolution; engraved on objective; related to angle of light collected

NA = n x sin(u); n = refractive index of medium; u = half the angle of aperture

d = 0.61 x wavelength / NA; d = distance between 2 points seen as separate

how much a substance slows light; light bends between media;

eyepiece x objective; 10x eyepiece x 100x objective = 1000x

usually 10x; magnifies intermediate image from objective; limits area of visibility; binocular at Michener

10x / 0.25 NA; largest field of view

40x / 0.65 NA

50x / 0.90 NA; 100x / 1.25 NA

lower magnification = larger field; higher magnification = smaller field

distance from objective front lens to slide when in focus; decreases as magnification increases; can crush slide

low magnification = large depth of field; high magnification = very limited depth of field

colour distortion; unwanted colour outline; ex: red focuses farther than blue

center in focus; periphery blurred; spherical lens surface

corrects chromatic + spheric aberration; 2 colours to 1 focal point; center in focus not periphery

corrects chromatic + spheric; also corrects field curvature; flat uniform focus

stays in focus when objective changed; minimal fine focus needed
focal points all in the same plane

object in the center of the field of view remains centered when another objective is rotated into use

same RI effect as immersion oil; protects smear; objectives list max coverslip thickness in mm

under stage; gathers light; cone of light through specimen; uniform intensity; can be raised/lowered + centered

in condenser; controls angle + amount of light; balances contrast + resolution; close no more than 1/3

more contrast; less resolution; diffraction artifacts; halos

even brightness across specimen; uses diaphragms + condenser; improves light path; do every time you use microscope

slide on stage; lamp on; focus 10x; rack condenser up; open iris; close field diaphragm; lower condenser to sharpen circle; center circle; open field until circle just disappears; close iris no more than 1/3

one hand on base; one hand around arm; never drag; never carry by other parts

lens paper + lens cleaner or 70% isopropyl (sparingly); dry right away; remove oil + debris; no fingers on lenses

Kimwipes; paper towel; tissue / Kleenex; scratch or leave lint

turn off; stage lowest; lowest power objective in place (10x); wrap cord; cover

eyepieces clean/loose; objectives clean/loose; condenser + Koehler; slide upside down; coverslip wrong side; 2 coverslips

musculoskeletal disorders; poor posture; neck + back + forearm + wrist + hand + leg strain

carpal tunnel (wrist); tenosynovitis (thumb); cubital tunnel (elbow); neck + shoulder + back strain

repetitive motion; awkward position; forceful exertion; contact stress

at or just below eyes; angle no more than 30 degrees above horizontal; avoid forward head

bend as little as possible; no more than 10-15 degrees below horizontal; chin tucked

upright; flat against backrest; lumbar support; feet supported

upper arms perpendicular to floor; elbows close to body; forearms parallel to floor; wrists neutral

feet flat on floor or footrest; even pressure on back of thighs; adjust footrest ring

adequate legroom; height-adjustable chair; lumbar + thigh support; check glare + reflections

every 15 min look away or close eyes; every 30-60 min stand + stretch; rotate tasks

watch posture; no slouching; rotate tasks; take breaks

study of blood cells + blood-forming tissue; diagnose + monitor treatment

liquid part of blood; transports + nourishes cells

RBCs; WBCs; PLTs

WBCs

platelets

RBCs

whole blood; automated cell counter

plasma + platelets

order CBC; collect; run analyzer; flags or abnormal = make + stain + examine PBF; coverslip; microscopic evaluation

abnormal or flagged CBC; confirm counts; check RBC + WBC + PLT morphology; estimates; WBC differential

venipuncture whole blood; capillary (finger or heel stick); minimum 2 patient identifiers

stops clotting; clotted sample = cells trapped; cannot assess

lavender top; anticoagulant of choice; chelates calcium; preserves cells + morphology

mix immediately; 8-10 end-to-end inversions (venous); 10 inversions (microcollection)

analyze within 6 h at room temp; microcollection within 4 h; smears within 4 h; store at 4 C

too much EDTA; low counts + low Hct; RBC + WBC shrinkage; crenated RBCs; WBC membrane damage; stain changes

within +/- 10% of stated draw volume

insufficient EDTA; poor mixing; PLT clumping; clots; reject

crenated or spherocytic RBCs; MCV + Hct up; necrobiotic WBCs; vacuolated neuts + monos; neut swelling + granule loss; eos + baso degranulate; large PLTs

pie-crust membrane; excess EDTA or old sample

dying; nucleus shrinking + fragmenting

pale clear spaces in cytoplasm; aged EDTA sample

slow draw; poor mixing; PLT clumps; microclots; fibrin; falsely low PLT count

reject; request recollection; obvious in tube

clots; fibrin; PLT clumps

2 beveled slides; 1 drop 2-3 mm; spreader 30-45 degrees; pull back into drop; push smooth + quick

smaller angle = longer thinner smear; larger angle = shorter thicker smear

raise spreader angle; smaller drop

lower spreader angle; larger drop

long thin smear; raise angle

short thick smear; lower angle

mechanical device; consistent quality; less variability

part of analyzer; one aspiration; Hct sets drop size; stains + dries slide

head (thick); body / monolayer; feathered edge / tail (thin)

2/3 to 3/4 slide length; not bullet-shaped; lateral edges visible; smooth no streaks or holes; rainbow feathered edge

unacceptable; hard to examine

banding on smear

long smear; large WBCs pushed to sides + end

jagged smear edge

streaks or holes; high lipids can also cause holes

drop began to dry before smearing; poor smear

air dry fast; no heat; no blowing or waving; completely dry before staining

slow drying or humidity; moth-eaten RBCs; crenated RBCs; punched-out central pallor; refractile rings

head of smear washes off; artifacts

Wright; Wright-Giemsa; Jenner; Leishman; polychromatic; alcohol-based

many colours; red to blue range

eosin (acid); methylene blue (basic); methanol

has methylene blue azure; best for early RBCs with RNA

shows morphology; RBC shape + Hgb; separates WBC types; shows PLTs

fixes cells to slide

staining starts when added; pH 6.4-6.8; pH dependent reaction

oxidized methylene blue + eosin; stains neutral components; neutrophil granules

negative (anionic); binds positive tissue; tissue called acidophilic (eosinophilic)

positive (cationic); binds negative tissue; tissue called basophilic

basic dye; stains acidic parts; nucleic acids (DNA + RNA); nuclear + cytoplasmic proteins; basophil granules; blue-grey to dark blue

acid dye; stains basic parts; hemoglobin; eosinophil granules; light pink to red-orange

neutral pH; lavender to lilac; thiazine-eosinate complex

flood with filtered stain 4 min; add equal buffer mix to green metallic sheen 8 min; rinse; wipe back; air dry

sign stain + buffer mixed properly during manual staining

more eosin binding; RBCs too red; WBCs too pale

more methylene blue binding; nuclei very dark; RBCs + eos grey

charge changes with pH; changes dye binding

pink to purple

RBCs pink to salmon; nuclei dark blue-purple; neut granules lavender; eos granules red-orange; baso granules dark purple-black; clean background

pink to salmon

dark blue to purple

blue

grey-blue

pink

pink-violet (lavender to lilac)

red to orange

dark purple-black

purple or red

light blue to colourless cytoplasm; red-violet granules

filter stain; poor rinsing; stain dried on slide

RBCs grey (brown or green); eos granules grey; WBC nuclei too dark; prolonged staining; thick film; heparin

RBCs too red; WBC nuclei pale

stainuffer too acidic; under-buffering; over-rinsing; thin film; old stain

poor stain technique; old smear or reagent; excess protein (rouleaux with myeloma)

consistent staining; handles many slides; less hands-on time

benchtop; holds 25 slides; continuous feed; 1 min between slides

all-in-one Wright or Wright-Giemsa; about 1 min; low volume labs; fast TAT; quality concern

facility dependent; normal patient PBF; keep with patient slides; log compliance; manual + automated

glass coverslip; mounting media (Permount); protects smear; lowers biohazard; same RI as slide

naked eye; check label + patient ID; smear quality; stain quality; tail direction

patient ID matches requisition + tube; legible

pink to purple overall

high lipids or too little blood

RBC agglutination

WBCs pushed to tail + edges

confirm smear + stain quality; scan tail for clumps + fibrin; excess WBCs at edges; parasites; RBC arrangement; find monolayer

reject sample; recollect; seen in feathered edge; can look like large WBC to analyzer

purple amorphous material; traps PLTs + WBCs + RBCs

smear not acceptable; make new smear; mix sample well

heavy; settle in tail/edges; Loa loa; Trypanosoma; Babesia; report to physician

RBC stacking; real in monolayer; not head of smear

RBC clumping; real in monolayer; not feathered edge

thinnest area; RBCs flat + large + distorted; honeycomb clumped; WBCs spread + smudged; not for assessment

between middle + last third; RBCs just touching or 2-3 overlap; biconcave + central pallor; assess here

pale center of RBC; about 1/3 of diameter; fades gradually

thick; RBCs stacked; WBCs + PLTs shrunken + dark; drying artifact; lymphs mistaken for basos; false hypochromia

where drop of blood was placed

start at feathered edge; move in toward body; confirm 10x then 40x; if stacked go back

monolayer only; 40x objective

debris; stain deposit; scratch; smudge cells; bacteria; nuclear degeneration; improper drying

mistaken for cells or organisms; can falsely raise WBC estimate; mistaken for hypochromia or inclusions

different focal plane; sit on top of RBCs; not in every field; random

artifact; on top of RBCs; can look like inclusion or PLT

fragile cells broken in smearing; no distinct membranes; do not count

confirm CBC; find sample problems; aid diagnosis; abnormal morphology; immature cells

any measured or calculated result

total WBCs; x10^9/L

separates 5 WBC types; relative + absolute

% or fraction of 1.0 of each WBC type

number of each WBC type; x10^9/L; relative x total WBC count

0.50 neuts x WBC 20.0 = 10.0 x10^9/L

x10^9/L; also MPV (size fL) + PDW (variation %)

same every time; WBC 5.0 x10^9/L not 5 000 000 000/L; unit does not change if high or low

about 5 L in average human

results of most healthy people; replaces normal range; by sex + age; use correct units

international system; Canada uses; US did not convert; Hgb 120-150 g/L

results needing attention; repeat; more tests; call result; not all CBC parameters have critical values

RI = healthy majority; action limit = significantly abnormal

3.6-10.6 x10^9/L

150-450 x10^9/L

WBC above 10.6 x10^9/L

WBC below 3.6 x10^9/L

PLT above 450 x10^9/L

PLT below 150 x10^9/L

quality assurance; confirm automated count; find errors or wrong patient smear; monolayer only

scan 10x; 40x objective; count WBCs in 10 fields; average per HPF

under 2 WBC/HPF; under 2.6 x10^9/L

2-3 WBC/HPF; 2.6-3.6 x10^9/L

3-7 WBC/HPF; 3.6-10.6 x10^9/L

7-10 WBC/HPF; 10.6-12.0 x10^9/L

over 10 WBC/HPF; over 12.0 x10^9/L

decreased; slightly decreased; normal; slightly increased; increased

scan 10x for clumps; 40x; count PLTs in 10 fields; average per HPF x 2.5 x10^9/L

under 40/HPF; under 100 x10^9/L

40-60/HPF; 100-150 x10^9/L

60-180/HPF; 150-450 x10^9/L

180-220/HPF; 450-550 x10^9/L

over 220/HPF; over 550 x10^9/L

wrong area; poor distribution; NRBCs counted as WBCs; smudge cells; pale PLTs; fibrin; PLT clumps

proportion of each WBC type; normal count can hide abnormal diff

measurement criteria; 5 mature types; counts 1000s of cells; flags odd cells; guesses abnormal type

visual morphology; 5 mature + most immature forms; detects abnormal morphology; counts 100 WBCs

100 sequential WBCs; monolayer; high power; do not skip hard cells; no artifacts

start near edge of monolayer; follow path; move exactly 1 field at each end

too deep in thick area; poor smear; inconsistent battlement; not 100 cells; wrong cell ID

smudge cells; damaged cells; dying (pyknotic) cells; badly distorted cells

counted with neutrophils

count all sizes together

neuts; bands; lymphs; monos; eos; basos; early neuts; NRBCs

lab worksheet = relative %; patient CBC report = absolute

neuts highest; then lymphs; then monos; eos + basos least

increased neutrophils

decreased neutrophils

increase in bands + less mature neuts; report with comment

increased lymphocytes

decreased lymphocytes

increased monocytes

increased eosinophils

increased basophils

continuous regulated blood cell production; renewal + proliferation + differentiation + maturation

oxygen exchange in tissues

immune function; fight infection

hemostasis; clotting

early cells in PB are abnormal; baby vs adult blood differs; find which cell lines are affected

multipotent; undifferentiated; self-renewing; not recognizable by morphology

divides to make more stem cells

gives rise to all blood lineages

top of hierarchy; to short-term stem cell

gives granulocytes + monocytes + RBCs + PLTs

gives lymphocytes (B + T + NK) + dendritic cells

all blood cells from one stem cell; most accepted

each cell line has own stem cell

neutrophils + monocytes

eosinophils + basophils

RBCs + platelets

multipotent HSC to myeloid or lymphoid progenitor; cytokines

progenitors to lineage-specific precursors; first recognizable cells; cytokines

stem + progenitor not recognizable; precursors first recognizable; all in bone marrow

about 6 billion cells per kg per day

soluble proteins; secreted by cells; regulators; growth factors + CSFs + interleukins

stimulates granulocyte + monocyte colonies

main cytokine for neutrophils

main cytokine for monocytes

RBC production; erythroid progenitors; prevents CFU-E apoptosis

platelet production; megakaryocytes

eosinophil production

basophil production

lymphoid cells

culture-derived progenitors; defined by colony growth in vitro

myeloid stem cell; granulocyte + erythrocyte + megakaryocyte + monocyte

lymphoid stem cell

erythrocyte

megakaryocyte

monocyte

granulocyte + monocyte

neutrophil

eosinophil

basophil

T lymphocyte

B lymphocyte

yolk sac; starts about day 19; primitive RBCs only; in developing blood vessels; hemoglobin made

starts 5-7 weeks; liver; also spleen + kidney + thymus + lymph nodes; RBCs + granulocytes + monocytes + lymphoid + megakaryocytes

starts 4th-5th month; bone marrow; all lineages; primary site by end of 6th month + at birth

bone marrow; from birth for life

bone marrow; thymus

spleen; lymph nodes; MALT

mucosa-associated lymphoid tissue

hematopoietically active; arranged in cords; mostly red at birth

inactive; adipocytes (fat); scattered within red marrow

red marrow replaced by yellow; starts age 5-7; continues with aging

skull; sternum; vertebrae; scapulae; ribs; pelvis; proximal ends of long bones

about 50:50 active cells to fat

increased chronic demand; e.g. chronic hemolytic anemia

blood cell production outside marrow; when marrow overwhelmed or damaged; liver + spleen main sites; also thymus + lymph nodes

marrow failure; hemolysis (thalassemia); neoplasms; infection; low oxygen; bleeding

dense outer layer

spongy; network of trabeculae; spaces hold marrow

extravascular tissue; hold all developing lineages + support cells

stem + progenitor + precursor cells; mature cells; adipocytes; macrophages; adventitial cells; connective tissue; endothelial cells

erythroid islands; megakaryocytes by sinuses; granulocytes deeper; lymphoid at endosteum

RBC precursors around macrophage; near sinuses

next to sinus walls; releases PLTs into sinus

fat cells; no stain on H&E; white spaces

support + anchor developing cells; guard sinus openings

anchor hematopoietic cells; need silver stain

lined by endothelium; exit route for blood cells

nutrient + periosteal arteries

central longitudinal vein; via venous sinusoids

cord; adventitial layer; basement membrane; endothelium; sinus lumen

smear on slide; Wright-Giemsa; shows spicules; hematology dept

bony pieces; dark blue-purple on smear

histology; shows trabeculae + cords + fat

cytokines stimulate; immature cells released in extreme cases

immature neuts in infection (left shift); immature RBCs in severe anemia

WBC production; myeloid (granulocytes + monocytes); lymphoid (lymphocytes)

neutrophil; lymphocyte; monocyte; eosinophil; basophil

3.6-10.6 x10^9/L

bands with neuts; all lymphs together

movement from marrow to blood to tissue

nonspecific; generic response; no memory; phagocytosis by neuts + monos + macrophages

specific; memory; antibodies from lymphocytes + plasma cells

neutrophil; eosinophil; basophil; secondary granules

monocyte; lymphocyte

myeloblast; promyelocyte; myelocyte; metamyelocyte; band; segmented

N:C ratio falls; chromatin coarser; nucleoli disappear; nucleus indents then segments; cytoplasm less blue; granules 1 then 2

N:C 8:1-4:1; 2-5 nucleoli; fine chromatin; blue cytoplasm; 0-20 primary granules; divides

N:C 3:1; 1-3 nucleoli; fine chromatin; blue cytoplasm; abundant primary granules; can be bigger than blast; divides