Akash medical College dept of dermatology

1. SIDE LAB INVESTIGATIONS IN
DERMATOLOGY
By:Dr. Shashank Royal T
Junior resident
Dept of Dermatology
AIMS&RC

2. Side lab : Defined as medical diagnostic testing at or near the point of care i.e at the time and place
of patient care

3. List of tests done in side lab
• Dark ground microscopy
• KOH mount
• Scraping for scabies
• Slit skin smear
• Tzank test
• Gram’s staining
• Ziehl neelsen staining
• Giemsa staining
• Wood’s lamp
• Patch test
• Dermoscopy/Dermatoscopy

4. Microscope

5. Mounting
• 3 types
1) Dry mount: simplest, specimen is merely placed on slide and cover
slip maybe placed on top. Ex: Hair
2) Wet mount: specimen is suspended in some type of liquid between
slide and coverslip. Ex: KOH mount
3) Prepared mount: specimen needs to be sliced ,dehydrated and
stained . Fixative also used to protect specimen from decay.
Ex: Hematoxylin &Eosin staining

6. Dark ground microscopy

7. • To observe unstained samples , appear brightly against dark
background
• Ideal for objects with refractive values similar to background
• Specially sized disc or patch blocks some light from centre of light
source , leaving only outer ring of illumination (cone of light)
• Condenser doesnot allow light to pass directly through specimen but
allow light to hit at oblique angle
• Light enters sample most of light is directly transmitted some is
scattered
• Only the light that hits object in specimen is deflected into objective
rest will miss making background dark

8. Bright field microscopy
1. Normal wide field illumination
2. Bright back ground
3. Low contrast
4. Usually for stained specimen
Dark ground microscopy
1. Opaque disc is placed in condenser
2. Dark background
3. High contrst (structural details)
4. unstained

9. Syphilis sample collection
• Lesion firmly grasped between thumb and index finger of left hand
• Small drop of serous exudate is taken on coverslip
• If blood stained mop till lesion exudes clear serous fluid
• Cover slip applied upside down on glass slide
• Edges are sealed with Vaseline to avoid drying up of exudate
• Apply oil on condenser and raise till it touches under surface of glass
slide another on cover slip and observe under oil immersion lens
• Dimensions : 5-6microns x 1microns
• Mobility : corkscrew ,to & fro ,bending

12. KOH mount
• Microscopic examination of stratum corneum to visualize fungal
elements
• KOH separation and destructuion of stratum corneum cells
(keratin)
• Hyphae and spores are unaffected by KOH
• 10% KOH solution
• Indications : dermatophytosis, candidiasis, Pityriasis versicolor

13. Sample collection
Skin: lesion is cleaned with alcohol
skin scraped with scalpel edge
collected in black paper/directly on glass slide
Hair : Plucked with foreceps
Nail: Nail clippings , scrape undersurface of nail plate , may include
subungual debris

14. Procedure
Specimen on glass slide
Cover with 2-4 drops of 10% KOH [20% for nails]
Place coverslip and gently heat
Examine after 20 min(overnight for nails)
10x for branching septate hyphae/ pseudohyphae & spore
40x for confirmation of diagnosis

15. False positive:
• Clothing fiber
• Hair Hyphae
• Cellwall keratinocytes
• Air bubble
• Oil droplets spores
Duration:
skin – 20min
Hyperkeratotic specimens- 30min-2hrs
Nails – 24hrs-48hrs

16. Diagnostic use
• Dermatophytic infection of skin, hair, nails – retractile, long, branching & septate
hyphae seen with or without spores

17. • Candidiasis – budding yeast cells & pseudohyphae

18. • Pityriasis versicolor – thickwalled spherical yeast cells , spaghetti &
meatballs or banana and grapes appearance

19. • Tinea nigra, deep fungal infections, Sarcoptes scabie & Demodex
folliculorum
• Bacterial vaginosis- fishy odour when KOH is added to vaginal
discharge

21. Scraping for scabies
• To demonstrate mites, ova, fecal pellets (Scybala)
• Scraping from papules or burrows
• Sites : anterior surface of wrist, Ulnar borders of hand, finger webs
Procedure- Drop of mineral oil on sterile scalpel blade apply on lesion
Scrape vigorously till tiny flecks of blood are visible in oil
Transfer material onto glass slide

23. Slit skin smear
• Test in which sample of material is collected from a tiny cut in skin &
then stained for Acid fast bacilli
• To confirm diagnosis of skin smear +ve leprosy is suspect
• To help diagnose leprosy relpase in patient who has previously been
treated
• To help with classification of new patients

24. Sites: 4 routine sites
1. Right earlobe
2. Chin
3. Forehead
4. Left buttock(M) /left upper thigh(F)
• Active/ suspicious lesions must be included if disease spectrum is
close to Paucibacillary
• In borderline lepromatous , 4 additional active lesions

25. Procedure
Clean the lesion with spirit
Pinch the fold of skin between thumb and index finger till it blanches
Incision 5mm long, 2mm deep (BP blade no.15)
Turn blade 90degree & scrape out fragments of tissue & fluid from
bottom & side of cut
Transfer material onto the slide – smear about 8-10mm diameter
Dry & heat fix

28. • Ziehl neelsen staining is done
• Examine under light microscopy in oil immersion
Live Mycobacterium leprae – solid stained bacilli
Dead Mycobacterium leprae – granular, broken & fragmented

29. Ziehl neelsen staining
• Divides bacteria into 2 groups
1. acidfast
2. non acidfast
• Resist decolourisation by both acid and
alcohol due to mycolic acid in their cellwall
• Reagents –
1. 1° stain Carbol fuchsin
2. Decolouriser:- 20% For M.Tuberculosis , 5% H2SO4 OR 1% HCl for
M.Leprae , 1%H2SO4 for Nocardia
3. Counterstain:- Methylene blue(0.2%)

30. Procedure
Airdry heatfix
Flood Carbol fuchsin+ heat till steam rises for 10-15min
------wash with water
Tilt slide add acid alcohol drop by drop until red colour stops streaming
from smear
------wash with water
Methylene blue for 1min
------wash with water
Examine under oil immersion

31. ZN staining results
• Pink bacilli - Acidfast bacilli
- MTB - Long slender bacilli
- M. Leprae- short thick bacilli
• Blue bacteria - Non acidfast,other bacteriaEpithelial cells,pus cells
• Other tissues -pale blue
• Caseous material - Very pale grayish blue

35. Tzanck smear
• Cytodiagnosis of infective, immunobullous conditions & cutaneous
tumours
• Procedure
Open intact roof of vesicle
Excess fluid blotted
Scrape base with scalpel (avoid bleeding)
Transfer material onto glass slide
&
Airdry

36. • Fixation :- formalin, Glutaraldehyde or formol denker solution
• Stains used :- Giemsa, H&E, Wright, Methylene blue, Papanicolaou & Toluidine
blue
• : Bulla fluid & blood may lead to inappropriate results
• For tumors ulcerated- Crust to be removed
non ulcerated- incise with sharp, pointed scalpel(blade no. 11)
Sample obtained with blunt/small curette
Tissue obtained is pressed between 2 slides

37. Cytological findings in
Tzanck

38. Disease Cytological findings
Pemphigus Acantholytic cells (rounded cells with a relatively large nucleus and a condensed
cytoplasm) with hazy nucleoli
Bullous pemphigoid Nonspecific findings. Scarcity of epithelial cells and an abundance of leukocytes,
particularly eosinophils with leukocyte adherence is seen
Stevens-Johnson syndrome No acantholytic cells, but plenty of leukocytes
Toxic epidermal necrolysis Necrotic basal cells and leukocytes
Staphylococcal scalded skin syndrome Minimal or no inflammation, dyskeratotic acantholytic cells
Herpes simplex, varicella, herpes zoster Ballooning multinucleate giant cells
Molluscum contagiosum Henderson-Patterson bodies
Leishmaniasis Leishman-Donovan bodies
Hailey–Hailey disease Acantholytic cells with normal nucleoli
Darier’s disease Corps ronds, and grains
BCC Basaloid cells
Paget’s disease of breast Paget cells
Mastocytoma Mast cells
Histiocytosis Atypical Langerhan cells

39. Staining

40. Staining type
1)Simple staining- single stain , provides colour contrast but gives same
colour to all bacteria and cells Eg:- Methylene blue, carbol fuchsin
2)Differential staining:- 2 contrast staining Decolourising agent is used
,different colours to differentiate bacteria Eg:- Gram staining, Acid
fast staining
3) Special staining:- used to visualise special structure Eg:- Capsule
staining, Spore staining, Auramine Rhodamine staining

41. Gram staining
Empherical method
Differentiate based on chemical and physical properties of cellwall
• Gram positive:
• Thick layer of peptidoglycan
• Negligible amount of lipids
• Numerous teichoic acid cross
linkages Resist decolourisation
• Gram negative:
• Very negligible peptidoglycan
• High amounts of lipids
• Gets decolourised

42. Reagents
• 1° stain - Gentian violet (1g/100ml)- hexamethyl para rosaniline
chloride
• Mordant:- Gram's Iodine (1g iodine+ 2g KI in 300ml)
• Decolouriser:- Acetone/ Alcohol
• Counterstain:- Safranine

43. Procedure
Smear
Cover with Gentian violet for 60s
rinse of with water
Gram's Iodine for 60s
rinse of with water
Alcohol/Acetone wash for 10-20s
rinse of with water
Safranine for 60s
wash with water
Airdry, blotdry and observe under microscope

50. In NTEP Rhodamine Auramine staining - UV microscope , Flurocent
stain Yellow-orange rods

52. Giemsa staining
• Is a buffered thiazine eosinate solution
• To differentiate nuclear and cytoplasmic morphology of platelets,
RBC, WBC, Bacteria & Parasites
Indications:- Blood film staining, bone marrow, malaria,protozoan
blood parasites, Enatmoeba histolytica, Toxoplasma gondii
• Preparation
Stock solution of giemsa stain prepared by mixing 4g of giemsa powder
in 250ml of glycerin and 250 ml of methyl alcohol

53. Procedure
Fix smear immediately in Dilute stock giemsa with 9
pure methyl or ethyl alcohol times volume of diluted
for 3-5 min Allow it to dry buffered water
Stain the smear with diluted giemsa stain for 30-45min
Wash in flowing water, dry and examine under oil immersion lens
Thick film : Air dry overnight, no fixing, use 1:50 dilution , Wash by placing in
buffered water for 3-5min

55. Wood’s lamp
• Mercury vapor UV lamp with a filter opaque to all wavelengths except
those between 320-400nm
• Mainly emits UV rays of 360nm-365nm
• Examine in dark room with wood’s lamp held about 10cm from skin
• Lesions with melanin in epidermis appear darker than surrounding
skin, while those confined to dermis donot

56. Color with Wood's Light Examination, Disease
Bright white to off-white Vitiligo, post inflammatory depigmentation, halo nevus, tuberous
sclerosis (ash leaf macules)
Darker than surrounding Melasma (melanin in epidermis), lentigo, freckles, caféau lait spots
No change from surrounding skin Melasma(melanin in dermis). Mongolian spot
Blue-green Tinea capitis due to Microsporum species
Yellow-white or copper-orange Tinea (pityrosporum) versicolor
Blue-white around hair follicles Pityrosporum folliculitis
Coral red (porphyrins produced by
Corynebacterium minutissimum)
Erythrasma
Green (pyocyanin) Pseudomonas
Red-pink Porphyrins in urine, blood,and teeth

59. Pityriasis versicolor

61. Used to identify causes of allergic contact
dermatitis-
Procedure
• Various patch test allergens (contained within small chambers i.e Finn
chamber method) are held against the the patient's back in cups using
a paper tape
• Remains on the skin for 48 hours during which the person cannot get
the tape wet.
• The results are read at 48, 72, and 96 hours after application
• The presence of erythema, papules, and/or vesicles indicates a
positive test

63. Angry back syndrome is seen in irritant reaction, a strong positive
patch test reaction could create an angry back which becomes hyper-
reactive to other patch test challenges

64. Dermoscopy
• Non invasive diagnostic technique that allows for the observation of
morphologic features that are not visible to naked eye
• A link between macroscopic clinical dermatology and microscopic
dermatopathology
• Enhance clinical assessment by providing new diagnostic criteria for
the differentiation of melanoma from other benign and malignant
neoplasms, both melanocytic and non-melanocytic
• The technique of dermoscopy classically involves applying a liquid or
gel to the skin surface and then inspecting the lesion using a hand-
held, illuminated microscope
• Upto 10x magnification can be obtained, sufficient for routine
assessment of skin tumors

65. • The fluid placed on lesion eliminates surface reflection and renders
cornified layer translucent allowing better visualisation of pigmented
structures within epiderms, dermoepidermal junction and superficial
dermis
• Recently hand held devices with polarized light are available , with
these use of liquid medium is no longer required
• Helps in early detection of melanoma
• It is also used in recognition of non pigmented skin conditions like:-
scabies, pediculosis, phthiriasis, tinea nigra, molluscum contagiosum,
psoriasis(red dot pattern), lichen planus(whitish striae pattern)

67. Pigmented basal cell carcinoma
Angiokeratoma with red-blue lacunas

68. Amelanotic melanoma
Spitz nevus

69. Nodular basal cell carcinoma
Bowen disease with clusters of
dotted/glomerular vessels

70. Scabies :- jet with contrail sign
Lichen planus

71. Plaque psoriasis with dotted vessels
Alopecia areata

72. Melanoma

73. References
• IADVl textbook of dermatology 4th edition
• Dermatology 4th edition Jean.L.Bolognia
• Fitzpatrick’s Dermatology 9th edition
• https://microbenotes.com/darkfield-microscopy/
• Mahajan V K , Slit skin smear in leprosy: lest we forget it! Indian
journal of leprosy 2013 , 85 : 177-183

74. THANK YOU