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4. Histopathologic- Techniques-MTLB-and-LM-

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 HISTOLOGY, PATHOLOGY AND HISTOPATHOL TECHNIQUE
by: Renz Jethro M. Ortega RMT, MLS (ASCPi)™

RECALL QUESTIONS
1. Removal of burrs ISTROPPING
2. First line in Panunumpa ng Propesyunal |AKO SI “PANGALAN” NG “TIRAHAN”
3. This is the most rapid embedding technique VACUUM EMBEDDING
4. This chemical is explosive when dry IPICRIC ACID
5. Polyclonal antibodies RABBIT
6. Clearing IDEALCOHOLIZATION
7. General purpose fixative 10% NBF
8. Included in COC IDRUG TESTNG
9. Suffix —itis INFLAMMATION
10. Antigen determinant IEPITOPE
11. Black bag IDRY NON-INFECTIOUS
12. Term of PRC Commissioner 7 YEARS
13. Routine H&E IREGRESSIVE STAINING
14. Independent variable IHAXI(Horizontal Abscissa X-axis Indep. Variable) & VDOY(Vertical Dep. var.
Ordinate Y-axis)
15. Mandatory requirement for potable water ILEVEL OF MINERALS IS WTIHIN TOLERABLE LEVELS
16. Prosector IPATHOLOGIST
17. Proper transport of microscope 1 HAND ON THE ARM, | HAND ON THE BASE
18. First 6 steps in H&E IDEPARAFFINIZATION
19. Methyl Green — Pyronin IDNA & RNA
20. Hormonal evaluation IPAP SMEAR (SPX.: UPPER LATERAL THIRD OF VAGINAL WALL)
21. Aminoethylcarbazole (AEC) IPOSITIVE RESULT: RED
22. Surgical connection between two structures |ANASTAMOSIS
23. Instrument for Pap smear except SYRINGE
24. Sterilization of vacutainer IGAMMA IRRADIATION
25. Lungs with pneumonia placed in water SINK
26. Patient has high bilirubin, yellow sclera and jaundiced |SIGN: SEEN BY THE DOCTOR IN THE PX. (SYMPTOM: WHAT PX. FEELS)
2/. Cursing of co-workers IRESTRICT BY PHRASES, CRITICISMS WITHIN CONSTRUCTIVE LIMIT
28. Chloroform ICLEARING AGENT
29. Double embedding IEMBED SAMPLE TWICE (1ST: CELLOIDIN, 2ND: PARAFFIN)
30. Knife used in rotary microtome STEEL KNIFE
31. Bevel angle 127°-32°
32. Malachite green ASCARIS EGG
33. Stropping IREMOVAL OF BURRS
34. Benzene ICAUSE APLASTIC ANEMIA
35. Revocation of license IUNANIMOUS DECISION (3/3)
36. Necrosis IPATHOLOGIC CELL DEATH
37. Members of the BMT 1 PATHOLOGIST, 2 MEDTECHS
38. _ Fixative for Immunohistochemistry +
39. Delayed transport and leakage of specimen GROUNDS FOR REJECTION
40. FDCI (FIXATION, DEHY, CLEARING, IMPREG) IPROCESSES PERFORMED BY AUTOTECHNICON
41. Fixative for tissue photography IMERCURIC CHLORIDE (HgC12)
42. Pink death certificate INFANTS (BLUE: ADULT)
43. Cardiac muscle INTERCALATED DISC

GERM LAYERS:
HISTOLOGY — f
Ectoderm
(forms the
Four Classification of Tissues and its Origin exoskeleton)
Epithelial Tissue Ectoderm, Mesoderm, Endoderm Mesoder
(develops
Muscular Tissue Mesoderm
© Buzzle.com

Connective Tissue Mesoderm

Nervous Tissue Ectoderm

1. Epithelial Tissue

Covering Epithelia
— tissues lining
the surfaces
Cellular Arrangement Cellular Shape
Simple Single layer | Squamous Flat
Stratified Many layers | Cuboidal Cube
Pseudostratified | Common BM] Columnar Taller than they are witeHT by RJMO 2020 | 1
Transitional | Changes shape when stretched

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 - Alveoli: site of gas exchange
- Glomerulus: site of exchange between wastes in the kidney
- Endothelium: lining of Blood vessel
- Mesothelium: lining of cavities
Locations of Covering Epithelia
Simple squamous Alveoli, glomerulus, endothelium, mesothelium
- site of exchange
Simple cuboidal Wall of thyroid follicles ~ Colloid: inside of Thyroid follicles | | Types of Signaling |
> Regaud’s/Muller’s — fixative of choice for
Simple columnar With cilia: oviducts - W/ Cilia: allow passage of
Autocrine Autoregulation
colloid/thyroid follicles 7]
egg cell towards uterus
Without cilia: gallbladder W/out cilia: for
secretion of mucus Paracrine Neighboring cells
Stratified squamous Keratinized: skin

Nonkeratinized: vagina Juxtacrine Secretions in cell

Stratified cuboidal Sebaceous gland ducts membrane
Stratified columnar Male urethra
Apocrine Apical portion
Transitional Urinary tract linings- (Urinary Bladder, Urethra, Ureters)

Pseudostratified columnar With cilia: trachea/Windpipe

Merocrine Vesicle formation
Glandular Epithelia
Exocrine With glands and ducts
Holocrine Drying of cells
Endocrine Secretions are directed to blood vessels
- Pancreas: ONLY organ BOTH exocrine and endocrine! ‘cz Dead cals ted
- e — = with keratin
> Exocrine: Ex. Amylase (earliest marker for pancreatitis)
& Lipase Stratum comeum
2. Connective Tissue > Endocrine: Islet of Langerhans - Upper most layer, 7
(BIGADS = Beta-Insulin,Glucagon-Alpha, - Composed of DEAD skin cells!! =
Stratum lucidum
_ Delta-Somatostatin)
General Connective Tissue
Loose = Wharton’s Jelly in Umbilical cord Stratum granulosum ———
Lamellar granules
(ISBB: Prepare by Washing 6 — 8 times)
Dense _[Tendons
Special Connective Tissue SS

Bone Stratum spinosum ——— Keratinocyte

Cartilage
Blood
Lymph
Hematopoietic Tissue
Stratum basale

ee Merkel cell

Stains for Collagen - produced by Fibroblast!! Melanocyte

Dermis Sejensory neuron

Mallory’s Aniline Blue
Masson’s Trichrome
Van Gieson Stain
Alcian Blue
Krajian’s Aniline Blue

A. Elastic cartilage

3. Muscular Tissue - in Outer ear

Skeletal :
striated, voluntary
| Muscle
B. Fibrocartilage

- in Vertebral disk
| Smooth nonstriated,
Muscle involuntary
WC. Hyaline cartilage

Cardiac
striated, involuntary in Trachea
Muscle
4. Nervous Tissue

Central Nervous System [Brain and Spinal cord

Peripheral Nervous System [Nerves outside CNS

Cell Cycle
State Phase Description
Resting GO Arrest state of division
C1 Increases in cell size
Preparation for DNA synthesis (10 hrs)
Synthesis | DNA replication (7.5 hrs)
G2 Continues growth
Preparation for cell division (3.5 hrs)
oy Mitosis Somatic cells H, P and HT by RJMO 2020 | 2
Cell Division Meiosis Sex cells

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 PATHOLOGY
Pathology — study of disease (pathos — suffering; logos — study)

Historical Insights
Hippocrates Father of Medicine
Rudolf Virchow Father of Cellular Pathology
Karl Landsteiner Father of Blood Transfusion
George Papanicolau Father of Exfoliative Cytology
Giovanni Battista Morgagni | Father of Modern Anatomical Pathology
Julius Conheim Introduced frozen sectioning
Cornelius Celsus Described the 4 cardinal signs of inflammation

| Branches of Pathology | Aspects of Pathology

Gross Pathology naked eye Etiology causative agent

Cellular Pathology microscopic study

development of the
Pathogenesis
General Pathology broad study of the field disease

Molecular Pathology ultramicroscopic structure .

Molecular and Morphologic .
xe start of injury
Clinical Pathology correlation of lab results Changes

Histopathology tissue changes .

Functional Derangement and .
t Ss = : signs and symptoms
Pathologic Anatomy in relation to anatomy —_ Clinical Manifestations

Two Ways of Obtaining Specimens for Pathologic Study

incisional
part only
1. Biopsy - living biopsy
Excisional . .
7 entire tissue
biopsy

Needle biopsy core biopsy

Aspiration
biopsy
fluid filled spx ; Ex. FNAB (Fine Needle Aspiration Biopsy)

Smear/
Exfoliative outer cell layer
Cytology

2. Autopsy - - dead Bite biopsy specialized

icli
forceps

Causes of Cellular Injury cylindrical

Punch biopsy Blade

Oxygen deprivation - Lead to HYPOXIA -> Tissue Damage Biologic/ Infectious agents
Reduced blood flow - Lead to NECROSIS Immunologic reactions- Ag/Ab complex deposition that lead to Injury
Physical agents - Too much HEAT/COLD -> Skin BURNS Genetic derangement — TrisOnoMy 21- most common geneticmutaion
Chemical agents and drugs Nutritional imbalance - Marasmus,
*Chloramphenicol Kwashworkor
* Cause Aplastic A.
* Gray baby syndrome

Abnormalities in Cell Growth

1. Retrogressive Changes

Developmental Defects
Aplasia incomplete or defective e development
- Usually occurs in PAIRED organs!
Agenesis complete non-appearance of organ
Atresia failure of an organ to fo rm an opening
Hypoplasia | fails to reach mature adult size

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Atrophy | Decrease in size of a normally mature organ
Physiologi
atrophy | Normal consequence of growth
fhaone _|Secondary to insulting agent

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 Types of Pathologic Atrophy | 2 SPECIAL TYPES OF ATROPHY:
Vascular atrophy interruption of blood — senile Atrophy — atrophy of the Sebaceous glands -> drying
of the Skin
supply BROWN Atrophy — Lipofuscin
Pressure atrophy persistent pressure
Starvation/ Hunger atrophy lack of nutrition
Atrophy of disuse inactivity
Exhaustion atrophy overuse
Endocrine atrophy lack of stimulus
2. Progressive Changes

Hypertrophy INCREASE CELL SIZE Hyperplasia INCREASE CELL NUMBER

Physiologic hypertrophy [Normal consequence of growth Physiologic hyperplasia Normal consequence of growth of
(Ex. Muscle, Uterus) ells multiplying (Ex. Breasts)
Compensatory hypertrophy _ {In paired organs, when 1 organ is | Pathologic hyperplasia Cells multiplying because of
removed (Ex. Kidneys) injury (Ex. Scrofula — cervical TB
- Ex. Barrett’s Esophagus Smoking — Pseudostrat(in Trachea) -> Squam.
3. Degenerative Changes > Always have Heart burn/Acid reflux
Metaplasia | transformation from one adult cell type to another —- Sava. Metaplasia .
> ri Fi 7 7 - Columnar epith. CONVERTS to Squam. bec. of Acidity
Dysplasia _| change in cellular shape, size, orientation _change in architecture
Anaplasia_ | from adult cell type to primitive form, irreversible
Neoplasia | tumor formation

Air Embolism Decompression Sickness

Definition [AIR BUBBLES IN THE BLOOD VESSELS NITROGEN OBSTRUCT THE BLOOD VESSELS
Introduction of air in IV lines Reduction in atmospheric
Bypass surgery, hemodialysis, pressure
Causes oral sex Scuba diving
Lung injury (decompression Driving a car up a mountain, flying
sickness in an unpressurized airplane

Neoplasia — tumor formation

Parts of Tumor
1. Parenchyma — neoplastic or proliferating cells
2. Stroma — blood vessels and supporting tissues

Types According to Clinical Behavior or the Capacity to Produce Death

1. Benign — tumors that form in only one spot without spreading to surrounding tissue, good prognosis, high
survival rate
2. Malignant - cancerous tumors can spread to nearby tissue, FATAL
A. Epithelial tissue — if tumor is derived from Epith. T. = CARCINOMA
B. Connective (or Mesenchymal) tissue - if tumor is derived from Conn. T. = SARCOMA
Types According to Histologic Characteristics
1. Medullary tumors — more cells
2. Scirrhous tumors — more supporting tissues

Grading of Tumor Staging of Tumor

Microscopic Macroscopic
Determine the extent/ degree by which neoplastic cell compare with Determine the spread of cancer in an
normal cells individual
Guide for treatment; used as a prognostic guide T — size of primary tumor
e T1,T2, T3 — increasing size of
BROADER’S CLASSIFICATION (GRADING) primary lesion
- cells look normal - aka Anaplastic cells N — regional lymph node involvement
nt ifferentiated Ce ndifferentiated
e NO, N2, N3 — indicates
100 - 75% — 25%
progressively advancing nodal
53-50% — 50%
disease
—25% — 75%
-0% 5 - 100%
M — metastasis — other organ involvement

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Characteristics of Tumor Cells
AJCC stage TNM stage TNM stage criteria for colorectal cancer

1. Changes in Intercellular Pattern Stage 0 Tis NO MO Tis: Tumor confined to mucosa; cancer-in-situ
° Increase in size, shape and pattern Stage I Tl NO MO T1: Tumor invades submucosa
e Anisocytosis and anisokaryosis : - .
e — Indistinct cell membrane Stage I T2 NO MO T2: Tumor invades muscularis propria

e Excessive grouping and crowding of Stage II-A T3 NO MO T3: Tumor invades subserosa or beyond
cells to form cluster (without other organs involved)

Stage II-B T4 NO MO T4: Tumor invades adjacent organs or

2. Cytoplasmic Changes
perforates the visceral peritoneum
e = Acidophilia
e Excessive cytoplasmic inclusion Stage III-A T1-2 N1 MO N1: Metastasis to 1 to 3 regional lymph nodes.

bodies Tl or T2.
e Abnormal vacuolation Stage III-B T3-4 NI MO NI: Metastasis to 1 to 3 regional lymph nodes.
T3 or T4.
3. Nuclear Changes : _
e Large, irregular and deeply Stage II-C any T, N2 MO N2: Metastasis to 4 or more regional lymph
pigmented nucleus nodes. Any T.
Multinucleated Stage IV any T, any N, M1 M1: Distant metastases present. Any T, any N.
Increased in the number and size of
nucleoli
Increased in the distribution and irregular size of chromatin material
Markly thickened nuclear membrane
e Necrotic or degenerative changes

Difference between Benign and Malignant Tumor

Point of
Benign Tumor Malignant Tumor
Differentiation
Age group Younger Older
Growth Slowly by Rapidly: infiltration
characteristic expansion and expansion
Adherence to
Not fixed More fixed
tissue
Surgery removed Difficult to remove
Recurrence Less chance High tendency

Death Metastasis Rarely observed Frequently observed

Cachexia Absent Present

1. Cellular Death — death of the cell Apoptosis a cell - dies but doesn’t grows back
eC

RaaerouiCcls physiologic cell _ dias and grows back

| death

Necrosis pathologic cell _ dies because of an INSULTING agent

death

Basic Morphologic Changes/ Hallmark Changes Seen in Necrosis

1. Nuclear Changes
2. Cytoplasmic Changes — granular, more
Pyknosis Condensation of the nucleus acidophilic, dense and opaque, cell

Karyorrhexis Fragmentation of the nucleus boundary Is lost, granular coagulation and

fragmentation
Karyolysis Dissolution of the nucleus
Types of Necrosis
1. According to location/ extent
A. Focal Necrosis - localized
B. Massive Necrosis - generalized

2. According to etiologic mechanism

Coagulation necrosis Preservation of architecture - MOST COMMON TYPE OF NECROSIS

Liquefactive necrosis Digestion of tissue
Combination of coagulative and liquefactive
Caseous necrosis
necrosis
Interruption of blood supply to lower extremities or
Gangrene necrosis
the bowel 20|5
Wet gangrene venous occlusion - Ex. Gas Gangrene (Causative agent: Clostridium perfringens)
Dry Gangrene arterial occlusion
Fat necrosis Release of pancreatic enzymes
Deposition of fibrin-like protein materials in the
Fibrinoid necrosis
arterial wall - Ex. Atherosclerosis

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 2. Somatic Death - death of the entire body

A. Primary Changes B. Secondary Changes — occurs AFTER death!!

- occurs DURING
° death!
. Pallor mortis Pale skin color |
|. Circulatory failure — heart stops — 3 re ,
: : . . Cooling of the = 7°F/hour, used to determine
Il. Respiratory failure — stops breathing Algor mortis :
f body time of death of px.
Ill. Nervous failure — loss of sense of aes
: . . Stiffening of
hearing Rigor mortis = for 3-4 days
muscle |
Livor mortis Purplish skin= aka “Post Mortem Hemolysis”
Putrefaction Bacterial action

ering Currant jelly-like

Dessication Drying
Autolysis Cell lysis
Skeletonization Bone formation

Difference between Livor Mortis and Ecchymosis

Livor Mortis Ecchymosis
disappears when Appears when pressure is
pressure is applied and applied and vanishes
reappears when removed when removed

oozing of blood upon No oozing of blood

incision

Postmortem Clot vs. Antemortem Clot

Postmortem Clot Antemortem Clot
Settling of RBCs from plasma Not readily detachable from the blood vessels
Chicken fat No chicken fat
Currant jelly No currant jelly
Assumes the shape of the vessel Seldom assumes the shape of blood vessels
Rubbery consistency Granular & friable

A healthy, relaxed, sedentary 70 kg man who was killed instantly in an accident will have organ weights in these
ranges:
e ~=Right lung: 300-400 g
e Left lung: 250-350 g
e Heart: 250-300 g
e Liver: 1100-1600 g — Largest INTERNAL Organ! (Skin: Largest Organ of the BODY)
e Adrenals: Ag
e =Thyroid: 10-50 g
e Spleen: 60-300 g
e = Brain: 1150-1450 g

process of taking pieces of tissues from a dead person to examine and investigate the cause of
Autopsy
death or extent of injury

Purpose of Autopsy
Determine actual cause of death Requirement for Autopsy
Determine final diagnosis e Consent from nearest kin
Determine extent of injury leading to death e Death certificate
of person e Medico-legal clearance
Preserve tissue for further testing

Types of Autopsy
1. According to Purpose
A. Routine hospital autopsy 2. According to Completeness of the Procedure
B. Medico-legal autopsy A. Complete autopsy
B. Partial autopsy
C. Selective

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 A. Y shaped incision
3. According to Manner of Incision B. Straight cut incision

Prosector One who dissects the cadaver

Diener Morgue/ autopsy assistant.
Coroner An official whose duty is to investigate sudden, suspicious or violent death to determine cause

Principal Techniques of Autopsy

Rudolf Virchow [Removal of organs ONE BY ONE
Carl Rokitansky [In SITU
Anton Ghon En Bloc
M. Letulle En Masses

| Inflammation | Vascular or a tissue response to injury in order to localize the insult

5 Cardinal Signs of Inflammation

Rubor REDNESS, “Ruby” bec. of Vasodilation -> Increase
blood flow (RBCs) to Injured area causing Redness Specialized Population of
Tumor SWELLING bec. of the escaping of fluids (plasma) Macrophages
Calor HEAT, “Calayo” Blood IMONOCYTE
Brain IMICROGLIAL cells
Dolor IPAIN as fluids escape and hits pain receptors
Bone IOsteoCLAST
Functio laesa LOSS OF FUNCTION bec of the swelling of the
larea, making it hard to move the area
Bone marrow = |PROmonocyte
Skin ILANGERHAN cells
Classification of Inflammation Connective IHISTIOCYTE
Tissue
Lungs [ALVEOLAR/DUST
1. According to Severity or Duration cell
A. Acute or Exudative Inflammation — vascular and exudative; PMN, Liver IKUPFFER cell
- Short term; PMN — dominant cell in Acute Inflam. Spleen LITTORAL cell
Kidney IMESANGIAL cell
Outcomes of Acute Inflammation Placenta IHOFBAUER cell
Lymph Node — DENDRITIC cell
e Complete resolution — there is recovery | best Ag presenting
e Healing by fibrosis — there is scarring cell

e Progression to chronic inflammation — if there is no proper recovery/healing

B. Subacute Inflammation — intermediate

C. Chronic Inflammation — proliferative response; Mononuclear cells — dominant cell in Chronic inflam.
- long term

2. According to Nature of Exudate

Serous Inflammation Increase of Plasma/tissue fluid

Fibrinous Inflammation Increase of Proteins
Catarrhal Inflammation IHypersecretion of the Mucosa
Hemorrhagic Inflammation [Presence of Blood
Suppurative Inflammation _ [Presence of Pus

3. According to Site Affected

Abscess Localized pus accumulation
Diffuse spreading injury in solid
Phlegmon or cellulitis e # eed
issue
- Most common cause of Peptic Ulcer
Ulcer Excavation in the surface
worldwide: Helicobacter pylori
Pseudomembranous False membrane - Ex. Clostridium difficile,
Corynebacterium diphtheriae
Granulomatous Granuloma formation - a ball of Macrophage

Diagnostic Cytology
| Diagnostic Cytology | Microscopic examination of cells from different body sites for diagnostic purpose

Two Divisions of Diagnostic Cytology

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1. Exfoliative Cytology - microscopic examination of desquamated cells from epithelial cells
2. Fine Needle Aspiration - a procedure in which a thin needle is used to draw cells or fluid from a lump or
mass under the skin

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 Specimen Mode of Collection

Pleural Fluid Thoracentesis

Pericardial Fluid Pericardiocentesis

Applications of Exfoliative Cytology

Peritoneal Fluid Paracentesis
: tas : /Ascite/Ascitic fluid
e Detect malignant condition (Cervical CA)
e Detect asymptomatic cancer Synovial Fluid Arthrocentesis

e Detect possible infection (Bacterial vaginosis) oe

e Evaluate vaginal hormonal activity Paneer ste ae artes
e Determine genetic sex
(Ex. Barr body— 2nd X chromosome in the female visible in the Nucleus, will tell px. is Female)

Specimens

1. Gynecological specimen

2. Non-gynecological specimens
e Respiratory Tract Specimens

A. Sputum — most commonly submitted NON-gyne. respiratory specimen in the Lab!!!

- Collection varies in the AGE of px.
> Infants/Babies-Throat swab;Adults-Sputum spx.;Debilitated px.-Endotracheal aspirate
- Deep cough (inhale 3x then cough); Collect 2 specimens (2-3 slides/spx.); Size:2x3cm
B. Bronchial brushings — spx. is PULL APART;spx. must be EQUALLY distrib. bet. 2 slides
C. BAL (Broncho Alveolar Lavage) — indication for dx. of P. jirovecii/carinii
- most common cause of Pneumonia
e Pleural, peritoneal and pericardial fluids in AIDS patients!!!
- add 300 units of Heparin/100 mL to prevent Jelly like clots!
e Smears of breast secretions
- secre. other than Milk! Ex. Blood, Pus
e Urinary tract specimen

Types of Urine Specimen

A. Voided urine — applicable for MALE px. (Midstream clean catch)

B. Catheterized specimen — for FEMALE px.
C. Washings from bladder or renal pelvis — for MALE and FEMALE px.

e Body cavity effusion

- 2 General types of Effusion: Exudates & Transudates
e GIT specimen — must be processed w/in 30 mins.!!! to avoid Cell digestion by the Gastric acidity

Points to Remember
e Smears are usually made from fresh material
e Smears must be prepared and immersed immediately in fixative
e Fluid specimen smear are made using sediments: Ex. Urine — Cytocentrifuge first, coll. sedi.(spx.) & smear

If smear cannot be made immediately:

e 50% ethanol Smear Preparation
e Saccomano’s fixative 1. Streaking — ZIGZAG direction
- BEST FIXATIVE FOR CYTOLOGY!!! 2. Spreading — spread in ALL DIRECTIONS
- Compo.: 50% Ethanol+2% Carbowax 3. Pull apart — Slide(w/ spx.)+Another slide on top
Common fixatives for Cytologic Smears 4. Touch preparation/ impression smears
e Equal parts of 95% ethanol and ether - Next best fixative to Saccomano’s - Moist sample + Touch slide in sample
e 95% ethanol — for General use - Disadvantage: Ether is FLAMMABLE!!!

e Spraycyte or cytospray - alternative is no available fixative; 1 foot/12 inches distance before spraying
e Carnoy’s fluid - FASTEST FIXATIVE!
e 50% alcohol — for Effusion
e 70% alcohol — for Sputum

Important points when mailing specimen — if Doctor (Pathologist) is not around/in another lab.
e Air dry after 2 hours of fixation then place in a wooden, cardboard or plastic container
e Glycerin technique — fix smear for 30 minutes, cover with glycerin then use another slide as coverslip
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Adhesives — permeable to fixative and stain (must not retain color of stain) Why can’t Mayer’s Egg
e Pooled serum/ plasma — BIOhazardous! Albumin be used in cytology?
e = Celloidin ether alcohol — FLAMMABLE!
e Leuconostoc culture — preferred in the lab, safer, NON-patho. bacteria - No, because it retains the
counterstain of Pap stain
- it stains GREEN in Pap stain
Specimens that require addition of adhesive
Concentrated sputum
Urinary sediments
BAL
Gastric lavage

Gynecological Specimen
PIL La ate)
Upper lateral 3"? of 7
a hormonal evaluation
vaginal wall
stratified squamous layer
pEndecervix MEO
most common site of cervical
T-zone
cancer
Vagina scrape px undergone hysterectomy
4 quadrant vaginal scrape localization of vaginal adenosis
Vulvar scrape herpetic lesions
Papanicolau’s/ Pap’s Smear — stain of choice for Tools for Gynecolologic Specimen Collection
cytology Glass pipette and
Vaginal smears
bulb
1. Harris Hematoxilyn — Primary/Nuclear stain! Ayre’s spatula Swab smear
2. OG 6 — Counterstain for Mature cell3 -*0G (Orange Green) Endocervical/ endometrial
. Laryngeal cannula
3. EA (Eosin Azure) 50 — Counterstain for aspiration
IMMATURE cell!
Components of Eosin Azure
e Light green SF Precautions during collection and smear
preparation
Bismarck brown
Eosin Y ° Patient should not be douched or undergone
vaginal exam for 1-2 days
Phosphotungstic acid
e No lubricant or powder on examiners oves
Lithium carbonate
° Smeat" IIS BBS yiRS SETS Mx.
motion

| Modified Pap’s Stain | a Pap Stain MINUS Bismarck brown |

Steps in PAP’s Smear Staining

1. Fix with 95 % alcohol - FIXATIVE 6. 70-95% Alcohol -DECOLORIZER

2. Stain with Harris Hematoxilyn- PRIMARY STAIN 7. Stain with EA 50 - COUNTERSTAIN (IMMATURE CELL)
3. Acid Alcohol - DECOLORIZER 8. Dehydrate
4. Blueing Agent 9. Xylene
5. Stain with OG-6 - COUNTERSTAIN (MATURE CELL) 10. Mount and label

Vaginal Hormonal Quantitative method of assessing hormonal activity; inexpensive and can be perform
Cytology even in pregnant women

1. Observe 100 cervicovaginal smear Shift to the left Parabasal cells

2. Classify cells as superficial, intermediate and parabasal
1. Manner of reporting: Shift to the Intermediate
A. Acidophilic Index — acidity of cytoplasm midzone cells
B. Maturation Index — most important
C. Pyknotic index — signs(?) of the nucleus Shift to the right Superficial cells

Cytohormonal Maturation Index (CHMI)

= %Parabasal Cells: %Intermediate Celis: %Superficial Cells

Day 14 Ovulation MI= 0:40:60 Mostly estrogen

Day 28 Premenstrual | MI= 0:70:30 Mostly progesterone
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 Menopausal Ml= 0:100:0 Progesterone
Atrophic Ml= 100:0:0 | None (NO hormone present)
Child Ml= 100:0:0 | None (NO hormone present)
Puberty Ml= 0:90:10 Mostly progesterone
Pregnancy Ml= 0:100:0 Progesterone
Postpartum Ml= 90:10:0 | Minimal hormonal stimulation
3 Types of Estrogen
- E1: Menopausal
Cells found in Cervicovaginal Smear ~ E2: Reproductive years
- E3: Pregnancy
1. Mature superficial cells — most mature, 45-50 um, polygonal cells with dark pyknotic nuclei; true
acidophilia
True acidophilia — cells appearing pink following estrogen effect
Pseudoacidophilia — drying of smear before fixation, prolapse and drying of epithelium, and
infection

2. Intermediate Cells — medium sized, polyhedral/elongated cells with basophilic and vacuolated
cytoplasm
A. Navicular cells — boat shape
B. Pregnancy cells — oval boat shape
C. Parabasal cells — fried egg appearance

Other Cells found in Cervicovaginal Smear

Endometrial cells less basophilic than parabasal
groups of 3 or more :
shed in response to ovarian hormone Ferning
found during and 1-10 days after menstruation Phenomenon where
Endocervical granular cells | honeycomb appearance, occurs in large groups or sheets _| | Ce'vical mucus
Doderlain Bacillus G (+) bacilli; Lactobacillus acidophilus eater on drying.
(Blue in Pap Stain) neracoed in 3T of pregnancy, DM, estrogen effect and (salt crystals)

: : mie Early pregnancy

Candida albicans increased in DM
Trichomonas vaginalis most common parasite isolated in urine
Gardnerella vaginalis clue cells
Koilocytes(HPV infected cell} perinuclear halo

Reporting Cancer Cytology Smears

Class | Absence of atypical cytologic picture
Class Il Atypical cytologic picture but no evidence of malignancy
Class Ill Cytologic picture suggestive but not conclusive of malignancy
Class IV Cytologic picture strongly suggestive of malignancy
Class V Cytologic picture conclusive of malignancy

Fine Needle Aspiration — safe, simple and rapid cytologic technique for cancer detection for superficial masses
1. Palpable mass — breast, thyroid, soft tissues and lymph nodes using gauge 22-23 needle
2. Non-palpable mass — aspirated under fluoroscopy, CT scan, UTZ
A. Non-cystic masses — remain in needle and expressed on to slide and fixed
B. Cystic lesions — fluid must be fresh in the syringe

Guidelines for Record and Specimen Retention

Records Reports
Instrument maintenance 2 years Clinical pathology reports 2 years
Quality Control 2 years Surgical pathology reports 10 years
Requisitions 2 years Cytogenetics reports 20 years
Blood bank QC 5 years Autopsy forensic reports Indefinitely
BB employee signatures 10 years
BB donor/recipient records Indefinitely

Specimen Forensic Cases

Serum and body fluids 48 hours Body fluids 1 year
Other body fluids 48 hours Tissue for toxicology 1 year
Blood smears 7 days Wet tissues 3 years
Microbiology smears 7 days Paraffin blocks Indefinitely
H, P and HT by RJMO 2020 | 10

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Blood bank donor and recipient 7 days post- Slides Indefinitely
specimens transfusion
Cytogenetic slides 3 years Reports Indefinitely
Pathology blocks 10 years Gross photographs and negatives Indefinitely
Pathology and bone marrow slides 10 years Dried blood films Indefinitely
Cytogenetics diagnostic images 20 years Frozen tissue for DNA indefinitely

Releasing of Results Copies of Report

e Surgical pathology and cytology — within 24 hours 3 copies!!!
e Frozen section — 5-15 minutes - 1 for Doctor
e Autopsy report — within 1 week 1 for Patient
- 1 for File/safe keeping
HISTOPATHOLOGIC TECHNIQUE
Methods of Fresh Tissue Examination - don’t use any preservative nor fixative, sample must be “fresh’!!

1. Teasing or Dissociation — tissue placed in a watch glass w/ Isotonic soln. & disintegrate/dissociate the fibers

2. Squash Preparation or Crushing — tissue less than 1mm is pressed between 2 slides; 1 slide serves as the
coverslip; sometimes a Vital dye is added to give color to the sample

3. Smear Preparation
ait lieM lm ec se lta
e Streaking - ZIGZAG direction 1. Rapid pathologic diagnosis
e Spreading - spread in ALL DIRECTIONS
2. Enzyme immunohistochemistry
e Pull-apart — Slide (w/ spx.) + Another slide on top
e Touch preparation ~ Moist sample
+ Touch slide insample 3+ Lipid and carbohydrate studies
-most common application of Touch Prep. 4, Immunofluorescent and
Lymph node biopsy
4. Frozen Section immunocytochemistry
5. Neuropathology (Silver staining)
Two Methods of Frozen Section
|. Cold Knife — uses “cold” knife for cutting tissue spx.; temp. should be -40 to -60C w/ the use of CO2 gas (freezing agent)
Il. Cryostat — a cabinet w/ a Microtome (Rotary) inside; optimum/best temperature for cryostat: -18 to -20C
Stains for Frozen Section
Freezing Agents 1. Progressive H & E-NO decolorizer used! VS Reg, H & E
|. Liquid nitrogen — MosT RAPID/COMMON FREEZING AGENT!!! | 2. Polychrome methylene blue
ll. lsopentane 3. Alcoholic pinacyanol
Ill. Carbon dioxide gas — freezing agent used in Cold knife method] 4, Thionine
IV. Aerosol spray

Mounting Media
|. Water
Il. 20-30% Bovine Albumin
Ill. Von Apathy’s G um Syrup
IV. Optimal Cutting Temperature (OCT) - BEST MOUNTING MEDIUM

Processing of Tissues FDDCIETSSML

F — Fixation T — Trimming
D — Decalcification S$ — Sectioning/ Microtomy
D — Dehydration S - Staining
C — Clearing/ Dealcoholization M — Mounting
| — Impregnation/ Infiltration L — Labeling
E — Embedding/ Casting/ Blocking *(S — Storage ...in some books)

FIXATION
AND FIXATIVES
e First and most critical step
The quality of the section in the slide is as good as the quality of the fixed tissue specimen
Ideal fixative: cheap, stable, safe to handle, can kill cell quickly to prevent distortion, inhibits
bacterial decomposition and autolysis, permit rapid and even penetration of tissue, must be
isotonic, must permit subsequent application of many staining procedures best fixative should
have a NEUTRAL (7) pH
e Primary purpose: preserve the morphological and chemical integrity of the cell
Secondary purpose: harden and protect the tissue H, P and HT by RJMO 2020 | 11
Most important reaction: stabilization of proteins by cross linking
e It prevents degeneration, decomposition, putrefaction and distortion of tissue after removal from the
body

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 Main Factors Involved in Fixation
Hydrogen ion concentration - should be between pH 6-8 Practical Consideration in Fixation
Oakwns

Temperature - Routine fixation (Room temp.); Electron Mx. (0-4C) 1. Speed - the faster the process, the more spx.
Thickness of section - the thinner section, the faster the fix. process you can process = more efficient
Osmolality 2. Penetration -1mm/hour (Ex. 10mm Lymph node is fixed
Concentration - too concentrated fixative causes damage/injury to tissue for 5 hrs. (get “Radius” of a round[diameter]
Duration of fixation -fix sample for 24 hrs. spx.) since spx. is penetrated in all directions)
3. Volume — 10-20x; if Ratio — 10-20:1
4. Duration — 24 hrs

Two Mechanisms of Fixation

1.
Additive fixation — chemical become “part” of tissue
2.
Non-additive fixation — removal of bound water attached to Hydrogen bonds
- chemical DOES NOT become part of tissue
Types of Fixative
1. According to Composition
A. Simple fixative — composed of only 1 chemical component
*tip: name of the fixative is usually a CHEMICAL; Ex. Formaldehyde, Mercuric Chloride, Chromate
B. Compound fixative — composed of 2 or more components
*tip: usually named after a person; Ex. Regaud’s, Muller’s, Helly’s, Bouin’s

2. According to Action
A. Microanatomical — general microscopic study
- Ex. 10% Formol saline, 10% NBF, Heidenhain’s susa, Formol sublimate, Zenker, Zenker-Formol, Brasil, Bouin
B. Cytological — preserves specific parts and particular microscopic elements
|. Nuclear — contains glacial acetic acid (enhance Nuclear staining), pH 4.6 or less, 20-22°C
- Ex. Bouin’s, Newcomer, Carnoy’s, Heidenhain’s susa, Flemming’s
Il. Cytoplasmic — wiTHOUT glacial acetic acid (destroy Mitochondria and Golgi bodies), pH 4.6 above
- Ex. Regaud’s, Orth’s, Helly’s, Flemming’s W/OUT acetic acid, Formalin w/ post chromatization
Ill. Histochemical — preserve the chemical constituents
- Ex. Formol Saline, Acetone, Absolute alcohol, “Newcomer (fix. for BOTH Nuclear
and histochemicall!!)

I. Formaldehyde Fixative

1. Formaldehyde
e¢ Most common fixative
e Working solution: 10%
e Stock solution: 37-40%
e Produced by the oxidation of methyl alcohol
e 24 hours usual fixation time

Formalin Pigments
Paraformaldehyde Acid Formaldehyde Hematin
| White crystalline precipitate Black to brown deposits
Due to prolonged storage Seen in tissue under the microscope
Removed by addition of 10% methanol or via filtration | N/A

10% Formol Saline — for CNS (Brain & Spinal cord) spx.
WN

10% Neutral Buffered Formalin — general purpose fixative

Formol Corrosive (Formol-Sublimate[HgCl2]) — formalin w/ Mercuric chloride
NOaR

Alcoholic formalin (Gendre’s) — for Sputum/mucus containing spx.

Formol-Calcium — for Lipids
Glutaraldehyde — has two reactive aldehyde groups separated by 3 carbon atoms; for Electron Microscopy (EM)
¢ 2.5% for small tissue for 2-4 hours
e 4% for larger tissue for 6-24 hours
8. Karnovsky Paraformaldehyde Glutaraldehyde — electron histochemistry (EM) and immunocytochemistry
9. Acrolein — for Surgical biopsy; for EM

Methods of Removing Acid Formaldehyde Hematin Left by Formalin

1. Kardesewitsch Method — specimen is placed in mixture of 70% ethanol and 30% ammonia water then
wash with water
2. Lillies Method — specimen is placed in mixture of acetone, hydrogen peroxide and ammonia water then
was in 70% alcohol and water
3. Picric Acid Method — specimen is placed in saturated picric acid then wash with run Wate 2020 | 12
Alcoholic KOH (Potassium Hydroxide) , y |

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ll. Metallic Fixative
1. Mercuric chloride — can bind to ALL substance; TOXIC
e Most common metallic fixative
Recommended for renal tissue, fibrin, connective tissues and muscles
Brilliant metachromatic staining — color of the dye is DIFFERENT from the color result of the tissue
Tissue photography
Causes lysis of RBC
Causes tissue to shrink — most dreaded disadvantage of HgCl2
Corrodes all metal except monel (nickel alloy)
Produces black precipitates of mercury — remove by 0.5% iodine solution in 70% ethanol then
decolorize iodine using absolute alcohol through the process of DEZENKERIZATION

Zenker’s fluid — for Trichrome staining

OW PY

Zenker-Formol (Helly’s) — for pituitary glands and bone marrow

. Heidenhain’s Susa — for tumor biopsies especially the skin; contains 2 chemicals
(Sublimate[Su] — a Mercuric chloride, Saure[Sa] — an “acid”)
. B-5 fixative — used for bone marrow
Qommo

Ohlmacher’s fluid
Schaudinn’s fluid
. Carnoy-Lebrum solution

2. Chromate Fixative

A. Chromic acid — preserves carbohydrates

B. Potassium dichromate — for mitochondria; if acid is added, this fixes nucleoproteins
C. Regaud’s fluid (Muller’s) — for chromatin, mitochondria, golgi bodies and colloid containing
tissue
D. Orth’s fluid — for early degenerative process and tissue necrosis; for spx. from a Rickettsia
infected px.!!

3. Lead Fixative (4% Aqueous soln. Lead Acetate) — for acid mucopolysaccharide

Picric Acid Fixatives

e Explosive when dry
e Excellent for glycogen demonstration
e May impart yellow color to tissue — remove by 70% ethanol followed by 5% sodium thiosulfate then
running water

1. Bouin’s Solution — for embryo and pituitary biopsies

2. Brasil’s Alcoholic Picroformol — for glycogen

. Glacial Acetic Acid

e Fixes nucleoproteins
e Destroys mitochondria and golgi bodies; Acetic acid causes tissue to SWELL, thats why it is used in
together w/ another fixative (Zenker/HgCl2) to counteract the Shrinkage effect!!!
e Normally used in conjunction with another fixative: Zenker/Mercuric chloride (causes tissue to SHRINK)
Solidifies at 17°C

Alcohol - widely used in Cytology; major disadvantage: leads to POLARIZATION (movement of

glycogen granules towards the periphery)
Methanol - dry and wet smears, blood smears and bone marrow tissue
Oakwns

Isopropyl alcohol — touch preparation

Ethanol — most commonly used alcohol fixative!
Carnoy’s Fluid — most rapid fixative; used for chromosomes, lymph glands and urgent biopsies
Newcomer's Fluid — for BOTH Nuclear & Histochemical fixation
Alcoholic formalin (aka Gendre’s) — for Mucus containing sample

Vi. Osmium tetroxide (Osmic acid) — can FIX, DIFFERENTIATE & STAIN tissue; major disadvantage: it is
NOT compatible w/ H & E staining!!!
e = Inhibits hematoxilyn
e Causes conjunctivitis or blindness
e Kept in dark colored, chemically clean bottle to prevent evaporation and reduction by sunlight

1. Flemming’s Solution — nuclear stain

2. Flemming’s Solution without Acetic Acid — cytoplasmic stain
H, P and HT by RJIMO 2020 | 13
VII. Trichloroacetic acid
e Weak decalcifying agent
e Precipitates proteins
e Swelling effect (similar to Glacial Acetic acid!!!)

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Vill. Acetone
e For water diffusible enzyme studies (Ex. ACP, ALT) and for brain studies e.g., rabies (specifically in the
form of Negri bodies)

IX. Heat fixation — use of a Physical agent (Heat) that can fix/stabilize the sample
e Usually employed for frozen tissue sections and bacteriologic smears (Ex. Gram staining)

Fixatives Used for Electron Microscopy

1. Glutaraldehyde Stains for Electron Microscopy

2. Platinic chloride 1. Uranyl acetate _
3. Platinic chloride formalin 2. Phosphotungstic acid
4. Gold chloride 3. Lead
5. Osmium tetroxide
6. 10% Neutral buffered formalin

Secondary Fixation {FIXING AN ALREADY FIXED SAMPLE

Post- Chromatization |A form of secondary fixation which uses 2.5-3% Potassium dichromate (mordant)
Washing out IRemoval of excess fixative

Pigment Color How to remove

Kardasewitsch method
Black to brown Lillie’s method
Acid formaldehyde hematin
granules Saturated picric acid
Alcoholic KOH
Mercuric chloride pigment Black granules Alcoholic iodine
Chromate pigment Fine, yellow brown Acid-alcohol
Osmic acid Black precipitate crystals Cold water

Factors that Affect Fixation Time Note: Mucus, Fats & Blood
retards the Fixation process!!!
Size and thickness of tissue — thicker (longer fixation time); thinner (faster fixation time)
Presence of mucus — removed by washing with NSS
Presence of fats — for demonstration, this is cut in thin sections and fixed in longer duration
Presence of blood — removed by the use of NSS
Temperature — high temp. will accelerate the fixation process; too.much will cook/destroy the tissue!
Agitation — will also accelerate the fixation process; in Auto Technicon (has constant agitation)

Methods used if chemical fixation is to be avoided

1. Freeze-drying — quenching (rapid freezing: -160°C) and desiccation (rapid drying) by a physical
process from still frozen tissue block without the use of any chemical fixative

2. Freeze-substitution — fixed in Rossman’s fluid or Osmium tetroxide in 1% acetone for 1-6 days at
temperature -60 to -70°C and dehydrated in 70% absolute alcohol

3. Fresh frozen tissue sectioning

DECALCIFICATION — removal of calcium ions from a bone or calcified tissue through a histological
process that makes them flexible and easier to cut; performed right after fixation,
before dehydration; Volume: 20x; Decalcification time: 24 — 48 hrs. (replace decal.
agent if tissue spx. is still NOT decalcified after 48 hrs.)

1. Acid Decalcifying Agents

A. Nitric Acid — most common and fastest; Color of Nitric acid: yellow
l. 10% Aqueous Nitric Acid
Il. Formol-Nitric Acid
Ill. Perenyi’s Fluid — tissue softener and decalcifying agent
IV. Phloroglucin-Nitric Acid — most rapid (specific) Nitric acid decalcifying agent!!!

B. Hydrochloric Acid
|. Von Ebner’s Fluid — teeth and small pieces of bones, surface decalcification
P and HT by RJMO 2020 | 14
5% Formic Acid — both a FIXATIVE & DECALCIFYING AGENT? 2" y |
Trichloroacetic Acid — both a FIXATIVE & DECALCIFYING AGENT!
"moo

Chromic Acid — both a FIXATIVE & DECALCIFYING AGENT!

Sulfurous Acid

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 2. Chelating Agents — combine with calcium ions to form weakly dissociated complex to facilitate removal
A. EDTA - 2 forms of EDTA; K2 (Versene) — powder/spray; K3 (Sequestrene) - liquid

3. lon Exchange Resin — removes calcium ions from formic acid-containing decalcifying solution

4. Electrophoresis — calcium (cation) migrates towards the negative pole (cathode)

Factors Influencing Rate of Decalcification
Concentration and volume (20x) of Tissue Softeners
decalcifying agent 1. 4% aqueous phenol
Temperature — should be Room temp. 2. Molliflex
Mechanical agitation — accelerate the 3. 2% HCl
decalcification process 4. 1% HCl in 70% alcohol
Size of the tissue — dictate the duration of
the decalcification process
= Thin/small tissue — faster
decal.
« Thick/big tissue — slower
decal.

Test to Measure Completeness of Decalcification

1. Physical or Mechanical — poke the tissue with a “stick” to check if its soft already; disadvantage: inaccurate
2. X-ray — most accurate; most ideal; disadvantage: expensive
3. Chemical Method (Calcium Oxalate Test) - most commonly used; disadvantage: inaccurate, reagent itself
has calcium
DEHYDRATION
Removal of intercellular and extracellular water from the tissue
Increasing concentration of alcohol
* Routine - starts with 70% ethanol
« Embryonic tissue — starts with 30% ethanol
* 10:1 ratio of dehydrating agent and tissue; Volume: 10x
Alcohol
Ethanol — best dehydrating agent
VOWP>

Methanol
Butanol — for plants and animals microtechnique
Denatured alcohol — composed of Ethanol & Methanol
Acetone — urgent biopsies (Ex. Lymph node biopsy)
Ooakwn

Cellosolve (Ethylene glycol monoethyl ether)

Triethyl phosphate
Diethylene dioxide (Dioxane) — dehydrating and clearing agent
Tetrahydrofuran — dehydrating and clearing agent
Additives to Dehydrating Agents
Anhydrous copper sulfate Indicator of water content
Presence of water = blue
Absence of water =
colorless
4% phenol + 95% ethanol Softener

CLEARING (DEALCOHOLIZATION)
Removal of alcohol from tissue and replaced with a substance that will dissolve the wax where the
tissue is to be impregnated
Ideal clearing agent:
© Should be miscible with ALCOHOL
Should be miscible with PARAFFIN WAX
Should not produce excessive shrinkage, hardening or damage to the tissue
000000

Should not dissolve out aniline dyes

Should not evaporate quickly
Should make tissue TRANSPARENT
High refraction index — which should be similar to RFI of glass (1.518)
Refractive index — the ratio of the velocity of light in a vacuum to its velocity in a specified medium

Xylene/Xylol — most rapid clearing agent; turns “milky” if there is incomplete dehydration
ORWN>

Toluene — substitute for xylene

Benzene — aka Aromatic hydrocarbon; a chemical known to cause Aplastic anemia
Chloroform — for nervous tissue, lymph nodes and embryo H, P and HT by RJMO 2020 | 15
Cedarwood Oil - for cytologic specimen

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 6. Aniline Oil — for embryo & fine, very delicate specimen
7. Clove Oil — easily adulterated
8. Methyl benzoate and Salicylate — for double embedding technique
IMPREGNATION
AND EMBEDDING
Impregnation (Infiltration) — removal of clearing agent and replaced by a medium that will completely fill all the
tissue cavities; Volume of impregnation = 25:1 (25x)

Embedding (Casting or Blocking) — impregnated tissue is placed into a precisely arranged position in a mold
containing a medium

Methods of Impregnation and Embedding 4 Types of Impregnating and Embedding Media

1. Manual Processing — 4 changes 1. Paraffin
of wax at 15 mins. interval 2. Celloidin
2. Automatic Processing — use of 3. Gelatin
Autotechnicon 4. Plastic
3. Vacuum Embedding — use of
Negative atmospheric pressure
(faster penetration of substance)
Paraffin Wax Impregnation — simplest, most common, best embedding medium; introduced by Butschlii; Melting
point = 56°C; Temp. of Paraffin oven (used to dissolve excess wax) = 2-5°C ABOVE the melting point of wax;
Temp. of Floatation bath (used to flatten out tissue ribbons after microtomy) = 6-10°C BELOW the melting point
of wax; Replace/change water in flotation bath if water is already visibly dirty, whenever necessary (everyday)

Factors Affecting Paraffin Wax impregnation

e Nature and size of tissue (the smaller the tissue, the faster the impregnation and vice versa)
e Type of clearing agent used (Xylene: best clearing agent for paraffin wax)
Substitute for Paraffin Wax
Paraplast Melting point at 56-57°C, more elastic and resilient
Embeddol Melting point at 57-58°C
Bioloid For the eyes
Tissue Mat Contains rubber
Ester Wax Melting point at 46-48°C, can be used for impregnation without clearing
Water soluble wax | No dehydration and clearing; Ex. Carbowax — a type of Polyethylene
glycol (PEG)

Celloidin Impregnation — specimens with large hollow cavity; a nitrocellulose; never use celloidin if spx. is a
carbohydrate (lead to false result, colors/stains on the celloidin instead of the carbohydrate specimen)

1. Wet Celloidin method — for bones, teeth & brain

2. Dry Celloidin Method — for eyes

Gelatin Impregnation — used when dehydration is to be avoided; for histochemical and enzyme studies
3 TYPES OF PLASTIC EMBEDDING
Plastic Embedding — light microscopic studies -1.Acrylic
- 2. Polyester
- 3. Epoxy Thickness of tissue sections
> Epoxy is further divided into 3 PARAFFIN section 4 to 6u
- Cyclohexene dioxide (Spurr) CELLOIDIN section 10-15u

~ Bisphenol A (Araldite) Ultrathin sections | 0.5u

TRIMMING AND CUTTINGISECTIONING
- removal of excess wax
Disposable blades | 2-40
Disposable Embedding Molds
Types of Blocking-out Molds Paper boats
1. Leuckhart’s Embedding Mold — L-shaped strip of brass or metal Peel-away
2. Compound Embedding Mold — a series of interlocking plates Plastic ice trays
3. Plastic Embedding Rings and Base Mold
4. Disposable Embedding Mold

Orientation — tissue is arranged in precise position in the mold during embedding, on the microtome before
cutting, and on the slide before staining

MICROTOMY - tissue is trimmed and cut into uniformly thin slices or sections
Three essential parts
1. Block holder
2. Knife carrier and knife
3. Pawl, ratchet feed wheel and adjust H, P and HT by RJMO 2020 | 16

Types of Microtome
1. Rocking Microtome — aka Cambridge microtome; simplest type of microtome; invented by Paldwell Trefall

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 2. Rotary Microtome — most commonly used microtome; invented by Minot
3. Sliding Microtome — invented by Adams; most dangerous type of microtome due to the movable exposed
knife; 2 Types: 1. Base sledge (Block: moving, Knife: stationary), 2. Standard sliding (Block: stationary,
Knife: MOVING) hence, the MOST DANGEROUS type of sliding microtome is Standard sliding!!!
4. Freezing Microtome — invented by Queckett
5. Ultrathin Microtome — cuts 0.5 um of tissue
6. Vibrotome — for UNFROZEN, UNFIXED spx. for enzyme demonstration

Microtome Knives
1. Plane-Concave Knife
* One side is flat, the other is concave
* Less concave — celloidin embedded tissue using sliding microtome
* More concave -— paraffin embedded tissue using rotary and rocking microtome
* Size: 25mm

2. Biconcave Knife
* Both sides concave
* For paraffin embedded sections using rotary microtome
* Size: 120 mm

3. Plane-Wedge Knife
* Both sides straight
* For frozen sections, extremely hard and tough specimen in paraffin blocks using base-
sledge microtome
* Size: 100 mm

Bevel Angle — angle formed between the cutting edge, normally 27°to 32°

Wedge Angle — angle formed by the sides of the wedge knives, normally 14° to 15°

Clearance Angle — angle formed between the cutting facet presenting to the block and the surface of the block,
normally 5° to 15°

Honing
* Removal of gross nicks
* Heel to toe (Edge first)
* Purpose: to remove irregularities from the knife

Hones
1. Belgium Yellow — gives the best result
2. Arkansas — more polishing effect
3. Fine Carborundum — for badly nicked knives
4. Plate-glass hone
5. Machine hone

Common Lubricant Used for Honing

1. Mineral oil
2. Clove oil
3. Xylene
. see paraffin Remedy for Broken Slides
- soapy water 1. Use xylene to remove coverslip
. 2. Incubate to remove mountant
Stropping 3. Place in 6 parts butyl acetate and 1 part durofix
* Removal of burr 4. Incubate
* Toe to heel (Edge last) 5. Cut the film around the section
* Purpose: polish and sharpen the cutting edge 6. Place in cold water to float the film and the section
7. Film with section
8. Mount
STAINING - the addition of color to the tissue 9. Incubate
10. Place in butyl acetate
1. Hi : a : 11. Xylene
. Histological Staining — staining the tissue DIRECTLY 12. M
: . . Mount
* Tissue constituents are demonstrated
in sections by direct interaction with dye
* Also called as micro-anatomical staining

2. Histochemical Staining — staining the tissue via chemical reaction H, P and HT by RJIMO 2020 | 17
* Various constituents of the tissues are studied through chemical reactions

3. Immunohistochemical Staining — staining the tissue via chemical reaction AND immunologic reaction
* Combination of immunologic and histochemical techniques

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Methods of Staining
1. Direct Staining
* Giving color to the sections using aqueous or alcoholic dye solution
* Staining the tissue directly! NO mordant is used!!

2. Indirect Staining
* The action of the dye is intensified by adding another agent (Mordant)
* Mordant — the link or bridge between the tissue and the dye
* Accentuator

3. Progressive Staining
* Tissue elements are stained in definite sequence; stain is applied until the desired intensity of
color is attained
* Ex. Frozen section

4. Regressive Staining
* The tissue is first overstained and decolorized until the desired intensity of color is obtained
* Ex. Routine H & E staining

5. Metachromatic Staining
* Entails the use of dyes which differentiate particular substances by staining them with the
color that is different from that of the stain itself

6. Orthochromatic Staining — staining color of the tissue is the same as the stain/dye itself

7. Counterstaining
* Application of a different color or stain to provide contrast and background
* Ex. Safranin (counterstain for Gram stain), Methylene blue (counterstain for Ziehl
Neelsen/Kinyoun in AFB staining), Malachite green (counterstain for Cold method Kinyoun)

Nuclear Counterstains Cytoplasmic Counterstains

Neutral red, safranin O, carmine, hematoxilyn Eosin Y, eosin B, Phloxine B
Picric acid, orange G, rose Bengal
Methylene blue, toluidine blue, celestine blue
Light green SF, Lissamine green

8. Vital Staining
* Selective staining of living cell constituents
A. Intravital Stain
* Staining of living cells is done by injecting the dye into any parts of
the animal body
¢ Ex. Lithium, carmine, india ink (inject inside proglottids of Taenia to
visualize the branches and Identify/differentiate the Taenia spp.;
Few branches (T. solium), Many branches (T. saginata)
B. Supravital Stain
* Used to stain living cells immediately after removal from the living
body; coating the surface of the cell
* Ex. Neutral red, Janus green, Trypan blue, Nile blue, Thionine,
Toluidine blue, Reticulocyte staining
Stains and Staining Solutions
1. Natural Dyes

A. Hematoxylin
* Derived from Mexican tree (Hematoxylin campechianum)
* Hematein — active coloring agent formed by the oxidation (ripening) of hematoxylin
= Natural ripening — exposing substance to air or sunlight
« Artificial ripening — uses substance that will accelerate the process
¥ Hydrogen peroxide, mercuric oxide, potassium permanganate,
sodium perborate, sodium iodate
* Ripened hematoxylin + alum, iron, chromium or copper salts (MORDANTS)
*NOTE: usually, the mordant is the chemical named/cited before the name of the
stain/dye!!! Ex. Aluminum Hematoxylin, so Aluminum is the MORDANT, while
Hematoxylin is the STAIN/DYE)

B. Cochineal Dye (Carmine) H, P and HT by RJMO 2020 | 18

* Dye extracted from the female cochineal bug
* Treated with alum to produce dye
* Carmine + picric acid = picrocarmine
* Carmine + aluminum chloride = best carmine

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 C. Orcein
* Vegetable dye extracted from lichens which are normally colorless — treated with
ammonia and exposed to air to produce blue or violet color
D. Saffron
* From a very expensive plant
2. Synthetic Dyes
* Coal Tar Dyes

Chromophores JCOLORING PROPERTY|

PROPERTY
Auxochromes Dyeing property: One that gives color
Benzene and Chromophore
Chromogen
Imparts temporary control
Chromogen and Auxochrome
Dye
Imparts color to tissue almost permanently

A. Acid Dyes
* Active coloring substance is found in the acid component and the inactive base
Will bind to the part of the cell that is BASIC (Cytoplasm is made up of proteins that are
BASIC)
Ex. Eosin
B. Basic Dyes
Active coloring substance is found in the basic component
Will bind to the ACIDIC components of the cell (Nucleus (full of DNA) is an ACIDIC
component)
Ex. Hematoxylin
C. Neutral Dyes
Capable of staining cytoplasm and nucleus
Will bind to BOTH nucleus and cytoplasm

Nuclear Stains | Cytoplasmic Stains | Metachromatic Stains

Methyl violet
Crystal violet
Neutral red Eosin Y
: Eosin B Cresyl blue
Safranin O :
: Phloxine B Safranin
Carmine .
: Picric acid Basic fuchsin
Hematoxilyn
Bismarck brown
Methylene blue Orange G
Rose bengal Methylene blue
Toluidine blue
Celestine blue Light green SF Thionine
Lissamine green Toluidine blue
Azure A, B, C

ROUTINE STAINS
Hematoxylin
* Most common (primary stain) for routine histology

1. Aluminum (mordant) Hematoxylin

* Recommended for progressive staining

A. Ehrlich’s Hematoxilyn
Ripening agent: Sodium iodate
Stabilizer: Glycerin
Harris’ Hematoxilyn
Ripening agent: Mercuric Chloride
Stabilizer: 4% Glacial Acetic acid
Cole’s Hematoxilyn
Ripening agent: Alcoholic iodine
D. Mayer's Hematoxilyn
Ripening agent: Sodium iodate
Regressive and progressive staining
Cytoplasmic glycogen

2. Iron (mordant) Hematoxylin

A. Weigert’s Hematoxilyn
e Mordant: Iron Ammonium chloride (NH4Cl)
e Standard iron hematoxylin H, P and HT by RJMO 2020 | 19
B. Heidenhain’s
e Mordant: Iron Ammonium sulfate (NH4S04)

3. Copper Hematoxilyn — for spermatogenesis

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 4. Tungsten Hematoxilyn — for muscle striations

Eosi n
Routinely used as counterstain

1. Yellowish (Eosin Y) — most commonly used

2. Bluish (Eosin B, Erythrosin B)
3. Ethyl eosin (Eosin S, eosin-alcohol soluble)

Hem atoxylin and Eosin Technique

e Most common method for tissue examination
Fixative — except Osmic Acid because it inhibits hematoxylin
Harris Hematoxylin — primary stain
Acid alcohol — differentiator
Ammonia water — blueing agent
Eosin — counterstain

Steps in Hematoxylin and Eosin Technique

Two changes of xylene *1—3: first 6 steps (2 Xylene, 3x Alcohol, 1 water)

WON >

(3) Descending grades of alcohol M ps (2x

are involved in DEPARAFFINIZATION!!!
SONAR

Water
Stain with hematoxilyn
Rinse slides in tap water
Acid alcohol
Ammonia water
Wash with running water
Stain with eosin
0. Ascending grade of alcohol
1. Xylene
2. Mount then label
3. Adhesive and Mounting Media

Staining Result of H and E Technique

Nuclei Blue to blue black
Karyosome Dark blue
Cytoplasm, proteins in edema fluid Pale pink
RBCs, eosinophilic granules, keratin Bright orange red
Basophil cytoplasm, plasma cells and osteoblast | Purplish pink
Cartilage Pink or light to dark blue
Calcium and calcified bone Purplish bone
Decalcified bone matrix, collagen and osteoid Pink
Muscle fibers Deep pink

Common Stains
Slide Holders for Staining
1 Benzidine
2 — hemoglobin Coplin jar 5-9 slides
2 Acridine Orange - DNA (green) and RNA (red) Slotted staining disher | 5— 19 slides
3 Crystal violet - amyloid and platelets Metal or glass racks 10 — 30 slides
4. Gentian violet — crystal violet + methyl violet + dextrin
5. Congo red — axis cylinders in embryos
6 lodine — oldest stain Restaining of Old Sections
7 Malachite green — Ascaris eggs, RBC and bacterial spores 1. Use xylene/xylol to remove coverslip
8 Janus green — intravital stain for mitochondria 2. Place in 0.5% K2MnO4
9. Night blue — substitute for carbol fuchsin in AFB 3. 5% Oxalic acid to decolorize
1 0. Victoria blue— neuroglia in frozen section 4. Restain

Basic Fuchsin Derivatives

Carbol fuchsin | Basic fuchsin + phenol crystal
Coleman’s fuelgen | Basic fuchsin + sodium metabisulfite
Schiff’s reagent | Basic fuchsin + sodium metabisulfite anhydrous
Mallory’s fuchsin | Basic fuchsin + 95% ethyl alcohol
Aldehyde fuchsin | Basic fuchsin in 70% alcohol + paraldehyde
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Stains of Carbohydrates
1.
Result: PAS (+) — red or magenta
Nuclei — blue
. PAS with Diastase — method of choice for glycogen demonstration
Result: Nuclei — blue-black
Glycogen — red
Control — only the nuclei are stained
Best Carmine — for glycogen demonstration
Result: Nuclei — blue or grayish-blue (Erlich’s hematoxylin as counterstain)
Glycogen — bright red granules
Mucin, fibrin — weak red
Langhan’s lodine — oldest stain (obsolete)
Result: Glycogen — mahogany brown
Tissue constituents - yellow
Fresh frozen Azure A — Metachromatic staining for glycosaminoglycans
Result: Glycosaminoglycans — red-purple
Tissue background - blue
Metachromatic Toluidine Blue Staining
Result: Glycosaminoglycans — red-purple
Tissue background — blue
Alcian Blue technique
Result: Acid mucin — blue
Nuclei — red
Combined Alcian Blue — PAS technique for acid and neutral mucin
Result: Acid mucin — blue
Neutral mucin — magenta
Nuclei — pale blue
Mucicarmine stain
Result: Mucins — red
Nuclei — blue
Background — unstained
10. Hale’s Dialyzed Iron Technique
Result: Acid mucin — dark blue
Nuclei — red
11. Fluorescent Acridine Orange
Result: Acid mucopolysaccharides — black
Fungi — greenish red fluorescent
Background — reddish orange fluorescent

Stains of Fats
1. Sudan Black B — most sensitive; *B for BLACK
Result: Lipids — blue black
Nuclei — red
2. Sudan
IV (Scharlach R); “*R for RED
Result: Lipids (mainly triglycerides) — red
Nuclei — blue or black
3. Qil Red QO in Dextrin
Result: Fats — brilliant red
Nuclei — blue
4. Osmic Acid
Result: Fats — black
Nuclei — yellow orange

Stains of Protein, Enzymes and Nucleic Acid

1. Alkaline Fast-Green
Result: Histones and Protamines — green
2. Gomori calcium
Result: Alkaline phosphatase activity — brownish black
Nuclei — green
3. Eeulgen — for nuclear DNA
Result: DNA — red-purple
Cytoplasm — green

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 4. Methyl Green-Pyronin — for RNA and DNA
Result: DNA — green or blue-green
RNA -— rose red
Plasma cell cytoplasm — purple
5. i
Result: DNA — yellow green
RNA: brick to orange-red

Stains of Connective Tissue

1. Collagen
A. Gomori’s Silver Impregnation Stain — for reticulin
Result: Reticulin fibers — black
B. Van Gieson’s Stain — for collagen
Result: Collagen (fibrous connective tissue) — pink, deep red
Nuclei — brownish black
Muscle, cytoplasm, RBC and fibrin — yellow
C. Masson’s Trichrome Stain- for collagen
Result: Collagen and mucus — blue
Muscle, RBC and Keratin — red
Nuclei — blue-black
D. Mallory’s Aniline Blue
E. Azocarmine
F
. Krajian’s Aniline Blue

2. Amyloid
A. Gram’s iodine
B. Congo Red
C. Methy violet-Crystal violet

3. Elastic Fibers
A. Weigert’s Elastic Tissue Stain
Result: Elastic fiber — dark blue, blue black
B. Verhoeff's
Result: Elastic fibers — black
Nuclei — gray to black
Collagen — red
Cytoplasm — yellow
C. Taenzer-Unna Orcein
D. Gomori’s Aldehyde-Fuschin
E. Krajian’s

4. Fibrin:
A. Mallory’s PTAH

Stains of Muscles and Bones

1. Muscle
A. Modified Gomori’s Trichrome Stain
Result: Muscle fiber — red
Collagen — green
Nuclei — blue to black
Mallory’s PTAH
UO

Heidenhain’s lron_ Hematoxylin

Lissamine Fast Red — tartrazine method for muscles and bones

2. Bone
A. Schmorl’s Picro-Thionin Method

Stains of Bone Marrow and Blood Elements

Rapid Toluidine-Eosin Stain
oOkwWN>

Romanowsky
Wright's stain
Giemsa stain
Peroxidase Reaction — for myeloid cells

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Stains of CNS
1. Bielschowsky’s Technigue — for neurons, axons and neurofibrils
Bodian’s Stain — nerve fibers and nerve endings
wN

Siever-Munger Technique- Neural tissues

Cresyl Fast Violet — NissI bodies
ok

Myelin Sheath
A. Weigert-Pal Technique
B. Kluver and Barrera Luxol Fast Blue Stain
C. Weil’s Method
6. Astrocytes
A. Cajal’s Gold Sublimate
B. Modified PTAH
C. Modified Holzer’s Method

Stains of Tissue Pigments and Deposits

Types of Pigment
A. Endogenous -— found inside the body
|. Hemosiderin — iron storage in tissue
ll. Hematoidin — bilirubin-like crystalline pigment free from iron
lll. Hematin — related to formalin
Iv. Hemozoin — malarial pigment from the destruction of RBC
Vv. Hemofuscin — yellow-brown pigment from Hemoglobin decomposition

B. Exogenous — most common exogenous pigment: CARBON pigment

C. Artifacts

1. Hemosiderin
A. Perl's Prussian Blue — stains FERRIC iron
B. Gomori’s Prussian Blue
C. Turnbull’s Blue Reaction — stains FERROUS iron

2. Hemoglobin
A. Benzidine-Nitroprusside Stain

3. Bile Pigments and Hematoidin

A. Modified Fouchet’s Technique
B. Gmelin’s Technique
C. Stein’s lodine

4. Lipofuschin
A. Gomori’s Aldehyde Fucshin Technique
B. Mallory’s Fuschin Stain

5. Melanin
A. Masson Fontana Technique — also for Argentaffin granules

6. Calcium
A. Von Kossa’s Silver Nitrate Method
7. Copper
A. Lindquist Modified Rhodanine Technique

Stains of Microorganisms
1. Bacteria
A. Gram’s Method
Brown and Brenn— for Nocardia and Actinomyces
Ziehl Neelsen— for Mycobacterium
Wade-Fite Technique — M. leprae, Nocardia
Hee

Auramine-Rhodamine— Mycobacteria
Toluidine Blue and Cresyl Violet Acetate — H. pylori
Dieterle Method — L. pneumophilia
Levaditi’s— Spirochete
Modified Steiner and Steiner — Spirochete
Warthin-Starry— Spirochete
Orcein method - HBsAg
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 2. Fungi
A. Grocott Methamine Silver

3. Virus
A. Lendrum’s Phloxine-Tartrazine Method — viral inclusion
B. Orcein Method — HBsAg

4. Protozoa
A. Giemsa

ADHESIVES
AND MOUNTING MEDIA
Adhesives - essential for methods that require Mounting Medium — added to slide before the
exposure of sections to acids and alkalis application of coverslip for protection

2. Mayer’s Egg Albumin 2. Aqueous (water-based) Mounting Media

e Glycerin A. Water
e Thymol crystal — to B. Glycerine
prevent/inhibit growth of C. Farrant’s Medium
molds D. Apathy’s Medium
3. Dried Albumin E. Brun’s Fluid
4. 1% Gelatin
5. Gelatin-Formaldehyde mixture 3. Resinous (derived from Resin of a Tree)
6. Starch Paste Mounting Media
7. Plasma A. Canada Balsam
8. Poly-L-Lysine B. DPX
9. APES Cc. XAM
D. Clarite

Ringing Sealing of margins of the coverslip to prevent evaporation of fluid

Kronig cement | 2 parts of paraffin and 4-9 parts of colophonium resin
Durofix Cellulose adhesive

IMMUNOHISTOCHEMISTRY
Polyclonal antibody — rabbit, goat, pig, sheep, horse, guinea pig
Monoclonal antibody — mice

Preparing Tissue for Immunohistochemistry

1. Proteolytic enzyme digestion — for complement, heavy chain immunoglobulin and specific antigens; uses
trypsin and protease
2. Microwave antigen retrieval — boiling formalin fixed deparaffinized section in 0.01 M citrate buffer (pH
6.0), EDTA (pH 8.0) and tris-EDTA (pH 9.9 or 10.0)
3. Microwave and trypsin antigen retrieval
4. Pressure cooker antigen retrieval — less time consuming and more consistent recovery of many antigens

Antigens
1. Epithelial Tumor Marker
A. Keratin — highly sensitive marker for epithelial cells/ carcinoma
|. Cytokeratin (CK) 7 — lung, breast, uterus and ovaries
Il. CK 20 —colon and stomach
Ill. CK 7 and 20 — transitional cell carcinoma of the bladder and mucinous ovarian
tumor
Epithelial Membrane Antigen (EPA) — adenocarcinoma of the breast, lungs and kidneys
VOD

Carcinoembryonic Antigen (CEA) — GIT, pancreas, lungs, breast, ovary, uterus and cervix
Thyroid Transcription Factor 1 (TTF-1) — thyroid, lung and neuroendocrine tumor to
differentiate adenocarcinoma and mesothelioma
E. Prostate Specific Antigen (PSA) — prostate, pancreas and salivary gland

2. Intermediate Filament Markers

A. Actin — smooth, skeletal and cardiac muscle H, P and HT by RJMO 2020 | 24
B. Vimentin — melanomas and schwannomas

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 Desmin — leiomyoma and rhabdomyosarcoma

™moOO
Glial Fibrillary Acidic Protein (GFAP) — astrocytoma
Neurofilament — neural or neuroendocrine differentiation
$100 Protein — CNS glial cells, melanocytes, histiocytes, chondrocytes etc.

3. Neuroendocrine Markers
A. Neuron Specific Enolase (NSE) - neural or neuroendocrine differentiation
B. Chromogranin — marker of neuroendocrine differentiation
C. Synatophysin

4. Germ Cell Tumor Markers

A. Human Chorionic Gonadotrophin (HCG) — choriocarcinoma
B. Alpha-fetoprotein (AFP) — endodermal sinus tumors showing yolk sac differentiation;also used
as a SCREENING test for Neural tube defects (CONFIRMATORY for
Neural tube defects: Acetyl Cholinesterase (AchE))
C. Placenta — like Alkaline Phosphatase (PLAP) — germinomas

5. Mesenchymal Tumor Marker

A. Myogenic Tumors — muscle markers
B. Fribrohistiocytic tumor
C. Vascular tumor— Factor VII, CD 31, UEA
D. Melanomas
E. Lymphomas — LCA (CD 45)

6. Cell Proliferation Marker

A. Ki67
B. Proliferating Cell Nuclear Antigen (PCNA)
FAULTS REASON REMEDY
Prolonged fixation
Prolonged dehydration
Prolonged clearing Tissue may be softened by
soaking in a small dish or bowl
Brittle or hard tissue Prolonged paraffin infiltration
containing water with
Overheated paraffin oven
detergent, phenol or Molliflex
Drying out of tissue before
actual fixation
Repeat dehydration with
Clearing agent turns milky as Water not completely removed
absolute alcohol, then clear
soon as tissue is placed in it due to incomplete dehydration
again
Block is trimmied down nearest to
the tissue. Remaining wax is
Clearing agent not completely
melted on embedding oven and
On trimming, tissue smells of removed due to insufficient
clearing agent paraffin impregnation is repeated,
impregnation changing the paraffin at least once
before blocking

Tissue is opaque, section cutting Repeat clearing; if object has

is difficult due to presence of Insufficient clearing already been embedded, prolong
clearing up to 12 hours, then re-
alcohol
embed
Insufficient dehydration, therefore
Tissue shrinks away from wax
incomplete clearing and Repeat the whole procedure
when trimmed
impregnation
Tissue is soft when block is
trimmed Incomplete fixation Repeat the whole procedure
Air holes found on tissue
during trimming Incomplete impregnation Repeat impregnation
Contaminated wax
On trimming, wax appears Re-embed in freshly filtered
crystalline Block not cooled rapidly wax
enough
Paraffin block, after cooling, is | Insufficient paraffin Repeat paraffin impregnation,
moist and crumbles impregnation then re-embed
Surfaces and the edges of the
Re-trim the block
block are not parallel
Horizontal surface of the block is
Readjust and reorient the block
Section fails to ribbon not parallel to the knife

Paraffin wax is too hard Coat Reviontal

with wax of lower
pan 6z8Ipo%
melting point

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 Knife is tilted too much Reduce the tilt
Readjust the thickness of the
Sections are too thick
section
Knife is dull Hone and strop
Sections roll up on cutting so that Knife is blunt Sharpen the knife
they adhere and get broken Tilt of knife is too great Reduce the tilt
against the knife edge Knife edge is dirty Clean the knife
Adjust the knife so that knife edge
Blunt of dull spot on the knife,
will present a uniformly sharp
producing an irregular knife edge
edge to the block or sharpen
Ribbon is curved, crooked or Edges of the block are not parallel
Re-trim the block
uneven instead of straight but round or wedge shape
Knife is not parallel to the block Readjust the knife and block
Repeat impregnation using pure
Paraffin is impure
wax
Knife is blunt or dull Re-sharpen the knife
Cool the block on ice water until
Paraffin block is warm and soft
firm
Sections are compressed,
Knife edge is coated with paraffin Clean the knife edge
wrinkled or jammed
Sections are too thin Readjust the thickness of section
Microtome set screw is loose Tighten the screw
Tilt of knife is too vertical Reduce the tilt
Sections are squashed (width of
Bevel of knife is lost due to Re-sharpen, using a knife back or
each section is less than that of
incorrect sharpening automatic knife sharpener
block)
Bubble or dirt formed in the Re-embed in freshly filtered
embedding medium wax if necessary
Once embedded in paraffin
A hole is formed in the section
Hard spot on the tissue due to wax, decalcification is
calcium impractical; use a base-sledge
microtome with a wedge knife
Tilt of knife is too great or bevel is
not cleared, hence object is
Reduce the tilt
compressed against the knife
edge
Sections of unequal thickness are
Clamp set screw on knife or block
produced Tighten the screw
holder is loose
Blocks are too large Cut blocks into smaller fragments
Soften the blocks in detergent or
Blocks are too hard
phenol
Breathe out or blow gently on the
Static electricity due to low block and knife to breakup static
atmospheric humidity electricity or boil water in the room
Sections adhere to the knife or
to increase humidity
other parts of the microtome
Knife edge is dirty Clean the knife edge
Knife edge is dull Sharpen the knife
Knife tilt is too great Reduce the tilt
Nicks or damage on the knife
Sharpen the knife
Ribbon is split or lengthwise edge
vertical scratches are seen on Dirty embedding Re-embed in filtered wax
sections Knife edge is dirty Clean the knife edge with xylene
Tilt of knife is too great Reduce the tilt
Knife tilt is too great Reduce the tilt
Sections are lifted from the knife Knife is dull Sharpen the knife
on upstroke Paraffin is too soft or room
Cool paraffin wax in ice water
temperature is warm
Tilt of knife is too small, paraffin
Resistance is felt on the lower block is therefore compressed
Increase the tilt
part of the section during cutting against the base of the knife
towards the end stroke
Horizontal or parallel lines or Knife edge vibrates due to Treat with phenol during
furrows across the section are hardness of tissue processing or collodionize
seen forming thin and thick zone Tilt of knife is too great Reduce the tilt

H, P and HT by RJMO 2020 | 26

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 Knife is blunt Sharpen the knife
Knife is not properly clamped Adjust the knife
Tilt of knife is too great Reduce the tilt
Section cut is sometimes thin, Tighten adjusting and locking
Knife or block holder is loose
sometimes thick screw
Knife tilt is too small that block is
compressed by bevel and section Increase the tilt
nis not cut
Readjust the angulation of the
Tilt of knife is too slanted
Knifes make a hard metallic knife
scraping or ringing sound on Take fresh block treated with
Tissue is too hard
backstroke, when section is cut phenol during processing
Knife blade is too thin Change the knife
Frozen tissue crumbles and
comes off the block holder Freezing is not adequate Refreeze the block
when cut
Frozen tissue chips into
Tissue is frozen too hard Warm the tissue with fingers
fragments when cut

Trust in the Lord with all your heart and lean not on your own understanding; in all your ways
submit to him, and he will make your paths straight.”

Proverbs 3:5-6

For questions and concerns, reach me at [email protected] ©

H, P and HT by RJMO 2020 | 27

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 Medical Technology Laws and Bioethics
Renz Jethro M. Ortega, RMT, MLS (ASCPi)©M
Information Description
MEDICAL TECHNOLOGY Auxiliary branch of laboratory medicine which deals with the examination of tissues,
secretion and excretion of the human body and body fluids by various electronic,
chemical, microscopic and other medical laboratory procedures or techniques either
manual or automated which will aid the physician in the diagnosis study and
treatment of disease and in the promotion of health in general

PATHOLOGIST or Heads the clinical laboratory

ANY DOCTOR W/ TRAINING IN
LABORATORY MEDICINE
FROM DOH
MEDICAL TECHNOLOGIST Medical detectives
VIVIAN HERRICK Identified intestinal parasites such as Taenia and Ascaris
IAANTON VAN LEEUWENHOEK Invented the microscope

IDR. WILLIAM WELCH Gave the 15 laboratory course in American School

IDR. WILLIAM OSLER Opened the first clinical laboratory at John Hopkins Hospital
JAMES CAMPBELL TODD & Author of Clinical Diagnosis by Laboratory Methods
ARTHUR HAWLEY SANFORD
26TH MEDICAL LABORATORY First laboratory in the Philippines
OF THE 6TH US ARMY
QUIRICADA, STA. CRUZ, Location of the first laboratory in the Philippines
MANILA
MANILA PUBLIC HEALTH Current name of the 26th Medical Laboratory of the 6th US Army
LABORATORY
IDR. PIO DE RODA Reorganized the laboratory of the 6th us Army
Offered a training program for aspiring laboratory workers
IDR. MARIANO ICASIANO Manila City Health Officer who assisted Dr. Pio de Roda
IDR. PRUDENCIO STA. ANA Helped Dr. Pio de Roda in the training program
Madea formal syllabus about the training program
IPHILIPPINE UNION COLLEGE Known today as Adventist University of the Philippines, first offered BSMT course
because of DR. WILLA HILGERT HENDRICK
IDR. JESSE UMALI First graduate of BSMT
IDR. ANTONIO GABRIEL & Faculty of UST who offered medical technology as elective course for Pharmacy students
DR. GUSTAVO REYES
IPASMETH National organization of schools of BSMT
UST Location of first meeting of PASMETH
(Philippine Association of Schools of MEdical Technology/Public Health)
IDR. GUSTAVO REYES First president of PASMETH
IPAMET National organization of all registered medical technologists in the Philippines
MR. CRISANTO ALMARIO Father of PAMET (Philippine Association of MEdical Technologists)
FEU (FAR EASTERN UNIV.) Location of the first national convention of PAMET
MR. CHARLEMAGNE First PAMET president
(TAMONDONG
ROMMEL SACEDA Current President of PAMET (Preceding president: RONALDO PUNO)
MS. ROSE MYLENE Founding/Current President of PSMLS (Philippine Society of Medical Laboratory
SOTOCINAL Scientists); Established in 2016
IDR. PROSPERO DE VERA Current CHED (Commission on Higher Education) Chairman
DR. TEOFILO PILANDO Current PRC (Professional Regulation Commission) Chairman;
Before PRC was founded, the Organization that handled Professionals was CIVIL
SERVICE COMMISSION (CSC)
IPAREDES STREET Location of PRC Manila
IDR. MARILYN BARZA (1) Chairperson of the Board of Medical Technology
PATHOLOGIST)
IHON. MARILYN ATIENZA & (2) Members of the BMT
IHON. MARIAN TANTINGCO (Board of Medical Technology)
IRA 5527 The Philippine Medical Technology Law
PHILIPPINE MEDICAL
(TECHNOLOGY ACT OF 1969)
IRA 6138 AMENDMENTS Sec. 16, 21, 22
PD 498 AMENDMENTS Sec. 2, 3, 4, 7, 8, 11, 13, 16, 17, 21, 29
IPD 1534 AMENDMENTS Sec. 3, 8, 13

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 Republic Act No. 5527
21 June 1969
Philippine Medical Technology Act of 1969

Section 1: Title (Philippine Medical Technology Act Section 8: Qualification of Examiners

of 1969)
1. Filipino citizen
Section 2: DEFINITION OF TERMS 2. Good moral character
Section 3: Council of Medical Technology Education, 3. Qualified pathologist or registered medical
Its Composition technologist
4. Practice of laboratory medicine or medical
= Chairman: SECRETARY OF EDUCATION or technology for 10 YEARS
DIRECTOR OF PRIVATE EDUCATION
5. Nota member of the faculty of any medical
=" Vice-Chairman: DIRECTOR OF THE
technology school - *to prevent leakages!!!
BUREAU OF RESEARCH LABORATORIES
OF DOH Section 9: EXECUTIVE OFFICER OF THE BOARD
Section 4: Compensation and Traveling Expenses of Section 10: Compensation of Members of the Board
Council Members of Examiners for Medical Technology
Section 5: FUNCTIONS OF THE COUNCIL of Section 11: FUNCTIONS AND DUTIES OF THE
Medical Technology Education BOARD

= Recommend curriculum = Administer provisions of the act

= The number of students = Administer oaths
" Approve MT schools = Issue, suspend and revoke Certificate of
= Report from medical technology schools Registration
= Inspect to determine high standard of = Look into conditions affecting the practice of
education medical technology
=" Admission to internship = Investigate violations of the act
=" Refresher course for those who failed for the
" Rules and regulations
third time Section 12: REMOVAL OF MEMBERS
=" Rules and regulations = Neglect of duty
= Incompetency
Section 6: Minimum Required Course
" Malpractice or unprofessional, unethical,
=" English
immoral or dishonorable conduct
=" Spanish
Section 13: Accreditation of Schools of Medical
" Social Science Technology and of Training Laboratories
= General Zoology
= Botany Section 14: Inhibition Against the Practice of Medical
=" Mathematics Technology
=" College Physics " Duly registered physician
=" General Chemistry =" MT from other country practicing for a
= Qualitative Chemistry specific function
= Quantitative Chemistry =" Medical technologist in the service of US
= Biochemistry Armed Forces in the Philippines
=" Gross Anatomy’
Section 15: Examination
" Histology
= Physiology Section 16: QUALIFICATION FOR EXAMINATION
= Clinical Parasitology = Good health and good moral character
= General Pathology = Completed the course
=" Microbiology
Section 17: Scope of Coverage and Examination
= Statistics
" Clinical Laboratory Methods = Clinical Chemistry: 20%
=" Microbiology and Parasitology: 20%
Section 7: BOARD OF EXAMINERS _for
= Hematology: 2Q4,L8 and LM by RIMO 2020 | 2
Medical Technology
= Blood Banking and Serology: 20%
= Chairman: PATHOLOGIST
=" Members (2): REGISTERED MED. TECHS.

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 = Clinical Microscopy: 10% Section 28: Roster of Medical Technology -
= Histopathologic technique: 10% list of all Med. Techs. compiled by the
Section 18: Report of Rating Secretary of the Board of Medical Technology
Section 19: Ratings in the Examination
Section 29: Penal Provisions
In order to pass the examination, a candidate must
obtain a general average of at least seventy-five = Practice without being registered or
percent in the written test, with no rating below fifty per exempted
cent in any of the major subjects: Provided, That the = Practice without supervision
candidate has not failed in at least sixty percent of the =" Madea fraudulent report
subjects computed according to their relative weights. = Failure to display certificate of registration
Section 20: OATH TAKING =" Presenting or attempting to use as his own
the Certificate of Registration of others
Section 21: Issuance of Certificate of Registration
= Give any false or fraudulent device to obtain
Section 22: FEES certificate of registration
= IJmpersonate any registrant of a fake or the
Section 23: REFUSAL TO ISSUE CERTIFICATE
same name
Section 24: Administrative Investigation-Revocation or = Attempt to use a revoked or suspended
Suspension of Certificates - at least 2 members of the certificate of registration
board w/ 1 legal council =" Convey the impression that he is a medical
Section 25: APPEAL technologist
= Any person who shall violate this Act
Section 26: Reinstatement, Reissue or Replacement of
= Violate the rules and regulations of Board
Certificates - 2/3 votes = license will be suspended;
3/3 votes = license will be revoked Section 30: Separability Clause

Section 27: FOREIGN RECIPROCITY Section 31: Repealing Clause

Section 32: EFFECTIVITY

RELATED LAWS
Law Date Approved Description
IRA 6138 August 31, 1970
IPD 498 June 28, 1974 Amendments to RA 5527
IPD 1534 June 11, 1978
IPD 223 June 22, 1973 Creation of PRC
IRA 8981 Dec. 5, 2000 PRC Modernization Act of 2000
IRA 4688 June 18, 1966 Clinical Laboratory Law
*2 Types of (Clinical) Centrifuge: Category of Laboratory Space in sq. m.
> 1. FIXED Angle — angle is fixed at 45°, most common ‘i
> 2. SWINGING Bucket Primary 10
- Moving/in Motion: tube is horizontal Secondary 20
- Stationary: tube is vertical
- Advantage: Sediment is flat, has cooling system Tertiary 60
- Disadvantage: once unbalanced = breakage of tube
> Stop watch: to calibrate timer Primary Secondary Tertiary
> Tachometer/Strobe light: to calibrate rotor of the centrifuge (quarterly)
> Cleaning/Disinfection of centrifuge (weekly/when needed (visibly dirty)) Clinical centrifuge Primary + Secondary +
HEPA Filter : High Efficiency Particulate Air filter. Removes particles, Hemacytometer Refrigerator Incubator
including microorganisms, from the air.
Microhematocrit
u i Photometer Balance scale
Biological safety cabinet. All biological safety cabinets use centrifuge
HEPA filters to treat air --- inflow/ exhaust. Two types: Microscope with O1IO Water bath Rotator

Class | : provide worker and environmental protection, but no product t

protection. Exhaust air filtered. Hemoglobinometer Timer Serofuge
WEC differential
Class II : filter both exhaust and intake air to protect the worker and Autoclave
the environment from contamination as well as to protect product in counter
the cabinet. Suitable for microorganisms assigned to bio-safety levels *
1,2 and 3... Drying oven
Class III : gas-tight provides the highest attainable level of protection BSC
to personnel and the environment.

A 7719 May 5, 1994 National Blood Services Act

IRA 1517 June 16, 1956 ‘Blood Banking Law
IRA 8504 Feb. 13, 1998 Philippine HIV/ AIDS Law
IRA 6425 April 4, 1972 Dangerous Drug Act of 1972
IRA 9165 June 7, 2002 Dangerous Drug Acts of 2002
IPD 856 Dec. 23, 1975 Code Sanitation of the PMP PAYS by RIMO 2020 | 3
AO No. 31 S. of 1979 1979 Accreditation of Water Analysis Laboratories
IRA 9288 . Newborn Screening Act of 2004
April 7, 2004

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NEWBORN SCREENING &

Bakit kailangang magpa-NBS test ang inyong sanggol?

Conditions Screened
Kandisyong sinusurt Epekto pag HINDinaNes EPekto pag na-NBS
nagamot kaagad at CONGENITAL HYPOTHYROIDISM (CRE TINISM)
alubhang Mental Retardation CONGENITAL ADRENAL HYPERPLASIA
GALACTOSEMIA
PHENYLKETONURIA
G6PD DEFICIENCY
MSUD (DOH Mem. #2012-0154)

May 25 2007 Anti-Rabies Act of 2007

CMO No.14 s.2006 2006 Policies, standards and guidelines for MT education
CMO No. 6 s.2008 2008 Accreditation of clinical laboratory as training lab for MT/MLS interns
Revised Rules and Regulations Governing the Licensure and Regulation
of Clinical Laboratories in the Philippines

Primary Secondary Tertiary

Routine Hematology Primary + Secondary +
Qualitative Platelet Routine Clinical Special Chemistry
Determination Laboratory
AO No 2007-0027 Routine Urinalysis Quantitative Platelet Special Hematology
Determination (+ Coagulation
assays)
Routine Fecalysis Crossmatching (hospital Immunology
based)
Blood typing (hospital Gram staining (hospital Microbiology (CS)
based) based)
KOH (hospital based)

National Reference Laboratories

BIOCHEMISTRY LUNG CENTER OF THE PHILIPPINES
MICROBIOLOGY AND INFECTIOUS DISEASES RITM (RESEARCH INSTITUTE OF TROPICAL
MEDICINE)
HEMATOLOGY AND IMMUNOHEMATOLOGY NKI (NATIONAL KIDNEY AND TRANSPLANT
INSTITUTE)
TOXICOLOGY, DRUG TESTING & EAST AVENUE MEDICAL CENTER
WATER ANALYSIS
HIV/ STDs SAN LAZARO HOSPITAL - SACCL (STI, AIDS
COOPERATIVE CENTRAL LABORATORY)
HISTOPATHOLOGY PHILIPPINE HEART CENTER
NEWBORN SCREENING NATIONAL INSTITUTE OF HEALTH - UP-PGH

PANUNUMPA NG PROPESYONAL

Ako, si » ng

a (Pook na Sinilangan, Bayan/Lungsod, Probinsya) _ i

ay taimtim na nanunumpa na itataguyod ko at ipagtatanggol ang Saligang Batas ng Pilipinas, na
ako ay tunay na mananalig at tatalima rito; na susundin ko ang mga batas, mga utos na legal, at
mga atas na ipinahayag ng mga sadyang itinakdang may kapangyarihan ng Republika ng Pilipinas;
at kusa kong babalikatin ang pananagutang ito, na walang ano mang pasubali o hangaring umiwas.

Taimtim pa rin akong manunumpa na sa lahat ng panahon at pook na kinaroroonan ay

mahigpit akong manghahawakan sa mga etikal at tuntuning propesyonal ng
mga sa Pilipinas, at marapat kong gagampanan ng
(Propesyon)
buong husay sa abot ng aking makakaya ang mga tungkulin at pananagutang iniatang sa isang
itinakdang
(Propesyon)

Kasihan Nawa ako ng Diyos.

(Lagda) LM by RJMO 2020 | 4

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 Board of Medical Technology
Code of Ethics

As I enter into the practice of Medical Technology, I shall accept the responsibilities
inherent to being a professional; | shall uphold the law and shall not engage in illegal work nor
cooperate with anyone so engaged; I shall avoid associating or being identified with any
enterprise of questionable character;

I shall work and act ina strict spirit of fairness to employer, clients, contractors,
employees and ina spirit of personal helpfulness and fraternity toward other members of the
profession;

I shall use only honorable means of competition for professional employment or services
and shall refrain form unfairly injuring, directly or indirectly, the professional reputation, projects or
business of a fellow medical technologist; I shall accept employment from more than one
employer only when there in no conflict of interest;

I shall perform professional work in a manner that merits full confidence and trust carried
out with absolute reliability, accuracy, fairness and honesty; I shall review the professional work of
other medical technologists, when requested, fairly and in confidence whether they are
subordinates or employees, authors of proposals for grants or contracts, authors of technical
papers or other publications or involved in litigation;

I shall advance the profession by exchanging general information and experience with
fellow medical technologists and other professionals and by contributing to the work of
professional organizations;

I shall restrict my praises, criticisms, views and opinions within constructive limits and
shall not use the knowledge I know for selfish ends; I shall treat any information I acquired about
individuals in the course of my work as strictly confidential, and may be divulged only to
authorized persons or entities or with consent of the individual when necessary;

I shall report any infractions of these principles of professional conduct to the authorities
responsible of enforcement of applicable laws or regulations, or to the Ethics Committee of the
Philippine Association of Medical Technologists as may be appropriate.

To these principles, I hereby subscribe and pledge to conduct myself at all times ina
manner befitting the dignity of my profession.

Medical Technologist's Prayer

God, who by calling us to the vocation ofa medical technologists, has placed upon us the obligation of being a
constant help in the scientific care of the sick, grant us by thy divine light a deep insight into the serious
responsibilities of our task.

By thy divine wisdom awaken in us a growing zeal and determination to increase our knowledge of how to search
for the underlying causes of sickness and disease; how to recognize the evidence of physical changes; how to make
important chemical analyses, and other valuable test so helpful in caring for the sick.

By thy divine love permit us in this way to share with those who directly care for the sick, that thus we may be of
constantly working through the eternal physician, Christ our Lord, Amen.

MTLB and LM by RJMO 2020 | 5

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 Laboratory Management and Bioethics

te a Subject
LEADERSHIP a ake up of
role
Listening Dacian Reward
Enthusiasm eae Shallow Mission/Vision
Awareness Approach Hypocrite
Decisive Vision Isolate
Equal re Positive Communication
Appeals to
Cutture
Action
Risk
Rules

Direction

Values
Concern
Focus
Human
Resource

Effective Management

MANAGEMENT is the act of organizing, planning, staffing, leading or directing and controlling
laboratory staff or entities for the purpose of accomplishing a goal.

1. TIME MANAGEMENT
2. HUMAN RESOURCE MANAGEMENT
3. FINANCIAL MANAGEMENT

Level of Management

1. TOP management
e Board of directors, president, vice president and chief financial officer
e Responsible for controlling and overseeing the entire organization

2. MIDDLE management
e General managers, branch managers and department managers
e Accountable to the top management for their department’s function

3. LOWER management
e Chief of staff and clinical instructors
e Focuses on controlling and directing
e Have the responsibility of assigning tasks to employees, guiding and supervising them.

Types of Clinical Laboratories

1. HOSPITAL Laboratory
¢ Operates within a hospital
e Managed by a pathologist or by physician (with training in lab medicine and management,
authorized by BRL)

2. FREE STANDING Laboratory

e Private laboratory that operates independently and receives samples from general practitioners,
insurance companies, etc.
e Managed by physician certified by PBP
Pre-Analytical Variables Analytical Variables Post-Analytical Variables
=Selection of assay/test = Assay/test validation = Accuracy of the result
=Patient ID & Prep. - most = Instrument selection = Data of the result
common pre-analytical = Quality control = Timeliness (TAT -
error: Mislabeling = Laboratory staff confidence Turnaround time)
= Specimen collection - most important due to = Cost analysis
= Specimen storage, prep. & human error factor - Post-analytical occurs AFTER
transport - Analytical occurs DURING testing! testing!
=" Monitoring of specimen
collection
- Pre-Analytical occurs BEFORE
testing!

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** LABORATORY MANAGEMENT QC TERMINOLOGIES
> SENSITIVITY C OUT
Quantity
= Able to detect the lowest possible concentration of the analyte Abilityto rule OUT disease
> SPECIFICITY alan | OF ie test i wagative)
Sensitivity of thi

= Quality
= Able to identify the SPECIFIC analyte being detected
> ACCURACY Ability to rule IN disease
= “bull's eye” Specificity of the test
(if the test is positive)
= True or target value
> PRECISION |
= Ability of the test to give the same result iN

Disease Status

> | b i
Subjects with
disease
| Subjects without)
i disease

peciive True Positive se Positive

(TP) (FP)
Test ; ; 7
Negative alse Negative | True Negative
(FN) (TN)
Sensitivity (Se) Specificity (Sp)
TP TN
TP +FN FP +TN

Moral Principles in Medical Technology Ethics

1. AUTONOMY _____ 5. RESPECT FOR DIGNITY

e Right to refuse or choose their e All the necessary means of care, high
treatment regard for the person or the patient
2. .BENEFICENCE 6. ‘TRUTHFULNESS AND HONESTY
e Acting in the best interest of the ¢ Dedication ofa person to his job and
patient is reflective of being honest and
3. .NONMALEFICENCE concerned
e Evil or harm should not be inflicted 7. STEWARDSHIP
either or oneself or on others e Expression of one’s responsibility to
4. JUSTICE nurture and cultivate what has been
e Distribution and decision regarding entrusted to him.
the scarce health resources in terms
of fairness and equality

Problems and Concerns in Medical Technology Practice

1. .NEGLIGENCE
e Failure to act and use reasonable care
2. .MALPRACTICE
e Act of negligence or omission ofa healthcare service
e More specific term that pertains to both the standard of care and professional status of the health
care provider

Trust in the Lord with all your heart and lean not on your own understanding; in all your ways
submit to him, and he will make your paths straigh@7LB and LM by RJMO 2020 | 7

Proverbs 3:5-6

For questions and concerns, reach me at [email protected] @

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