MORPHOLOGIC CHANGES DUE TO AREA OF SMEAR

Thin area- Spherocytes which are really "spheroidocytes" or flattened red cells. True spherocytes will be found in other (Good) areas of smear.

Thick area - Rouleaux, which is normal in such areas. Confirm by examining thin areas. If true rouleaux, two-three RBC's will stick together in a "stack of coins" fashion..

Common causes of a poor blood smear1. Drop of blood too large or too small.

2. Spreader slide pushed across the slide in a jerky manner.

3. Failure to keep the entire edge of the spreader slide against the slide while making the smear.

4. Failure to keep the spreader slide at a 30° angle with the slide.

5. Failure to push the spreader slide completely across the slide.

6. Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide

7. Holes in film: Slide contaminated with fat or grease

8. Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated with water.

Biologic causes of a poor smear

1. Cold agglutinin - RBCs will clump together. Warm the blood at 37° C for 5 minutes, and then remake the smear.

2. Lipemia - holes will appear in the smear. There is nothing you can do to correct this.

3. Rouleaux - RBC’s will form into stacks resembling coins. There is nothing you can do to correct this

Principle of Romanowsky Stain

Giemsa stain : polychromatic stain

Methanol : fixes cells to slide also use as solvent

methylene blue stains RNA,DNA blue-grey color

Eosin stains hemoglobin orange-red color

parasites cytoplasm: blue

nuclear material: purple-red

pigment : depending on type& species

pH value of phosphate buffer is very important

Staining Procedure

Thin smear are air dried after fixation with absolute alcohol.

Dilute Giemsa 10%

Flood the smear with stain.

Leave the stain on the slide for 10 min.

Wash off by running water directly to the centre of the slide to prevent a residue of precipitated stain.

Stand slide on end, and let dry in air.

too acidic suitable too basicStaining result

Causes and correctionToo Acid Stain:

1. insufficient staining time

2. prolonged buffering or washing

3. old stain

Correction:

1) lengthen staining time

2) check stain and buffer pH

3) shorten buffering or wash time

Too Alkaline Stain:

1. thick blood smear

2. prolonged staining

3. insufficient washing

4. alkaline pH of stain components

 Correction :

1) check pH

2) shorten stain time

3) prolong buffering time

Life cycle of Plasmodium

Components of the Malaria Parasite

Malaria in Thin Blood Film

Morphology of erythrocytic stages of Plasmoduim falciparum

Trophozoitesize: small to mediumnumber: often numerousshape: ring and comma forms commonchromatin: often 2 dotscytoplasm: regular, fine to fleshy

Morphology of erythrocytic stages of Plasmodium falciparum

Schizont

size: small

number: few

shape: compact

uncommon, usually seen in severe malaria

mature forms: 12-30 or more merozoits

pigment: single dark mass

Morphology of erythrocytic stages of Plasmodium falciparum

Gametocyte

shape:

-mature, banana-shaped or round

-immature, pointed-end (uncommon)

chromatin: single, well defined

pigment: scattered, coarse, rice-grain-like

Malaria in Thin Blood Film

Plasmodium malariae

Morphology of erythrocytic stages of P. malariaeTrophozoitesize:smallnumber:usually fewshape:ring to rounded, compact formschromatin:single, largecytoplasm:regular, densepigment:scattered, abundant, with yellow tinge in older forms

Morphology of erythrocytic stages of Plasmodium malariae

Schizontsize:small, compactnumber:usually fewmature forms:6-12 merozoits, usually 8, in loose cluster, some apparently without cytoplasmpigment:concentrated

Morphology of erythrocytic stages of Plasmodium malariae

Gametocyte shape:-immature:difficult to distinguish from mature trophozoit-mature:round, compactchromatin:single, well definedpigment:scattered, coarse, may be peripherally distributedEroded formswith only chromatin and pigment present

Malaria in Thin Blood Film

Morphology of erythrocytic stages of Plasmodium vivax

Trophozoite

size: small to large

number: few to moderate

shape: broken ring to irregular forms common

chromatin: single, occasionally 2

cytoplasm: irregular or fragmented (amoeboid)

pigment: scattered, fine

Morphology of erythrocytic stages of Plasmodium vivax

Schizont

size: large

number: few to moderate

shape: compact

mature forms: 12-24 merozoits (usually 16)

pigment: loose mass

Morphology of erythrocytic stages of Plasmodium vivax

Gametocyteshape: -immature: difficult to distinguish from mature trophozoit-mature: round, largechromatin: single, well definedpigment: scattered, fineEroded forms with scanty or no cytoplasm and only chromatin and pigment present

Malaria in Thin Blood Film

Morphology of erythrocytic stages of Plasmodium ovale

Trophozoite

size:may be smaller than P. vivax

number:usually few

shape:ring to rounded, compact forms

chromatin:single, prominent

cytoplasm:fairly regular, fleshy

pigment:scattered, coarse

Morphology of erythrocytic stages of Plasmodium ovale

Schizont

size:rather like P. malariae

number:few

mature forms:6-16 merozoits, usually 8, in loose cluster

pigment:concentrated mass

Morphology of erythrocytic stages of Plasmodium ovale

Gametocyteshape:-immature: difficult to distinguish from mature trophozoit-mature:round may be smaller than P. vivaxchromatin: single, well definedpigment: scattered, fineEroded forms with only chromatin and pigment present

Morphologic forms of haemoflagellate

There are 4 morphologic forms seen in hemoflagellates:

1) Amastigote2) Promastigote3) Epimastigote4) Trypomastigote - They can exist in two or more of the 4

morphologic forms depending on the species.

Amastigotes of Leishmania species

Structurally the amastigotes of Leishmania species that causing visceral, cutaneous and mucocutaneous are similar.

With variations in size between species.

Small, round to oval measuring 2–4 um.

Can be seen in groups inside blood monocytes ,less commonly in neutrophils, in macrophages in aspirates or skin smears, or lying free between cells.

The nucleus and kinetoplast stain dark reddish-mauve.

The cytoplasm stains palely and is difficult to see when the amastigotes are ingroups.

Promastigote of Leishmania species

Size: 9-15µmShape: long and slender.Nucleus: one, central.Kinetoplast: Anterior end of the organism, no undulating membrane.Flagellum: Single, anterior free flagellum.

Is the infective stage

Also result from culture (NNN)media.

Cutaneous leishmaniasis

Infection is often referred to as wet or dry oriental

sore. The early papule is often inflamed and resembles

a boil of 5–10 mm in diameter which rapidly develops

into a large uneven ulcer which is self-healing

in as little as 3–6 months. Multiple lesions may occur

Mucocutaneous leishmanisis

is the most severe and destructive form of

cutaneous leishmaniasis in South America.

Lesions are similar in development to those of oriental sore and the resulting ulcers may become very large and

long-lasting.

Post kalazar dermal leishmaniasis

occur about 2 years after treatment and recovery from visceral leishmaniasis.

Affects about 20% of patients in India.

Hypopigmented and raised erythematous patches can be found on the face, trunk of the body, and limbs.

These may develop into nodules and resemble those of lepromatous leprosy, fungal infections or other skin disorders.

Amastigotes are present in the papules and nodules.

Trypanosoma (cruzi,bruzei) trypomastigote

Extracellular Usually C-shaped.

measuring 12–30m in length

A narrow membrane and free flagellum.

large, round to oval, dark-red staining kinetoplast at the posterior end.

Nucleus is centrally placed and stains red mauve.

Disease: trypanosomiasis

Sleeping sickness in africa

Chagas disease in america

Sample: Blood or Cerebrospinal fluid

Life Cycle African Trypanosomiasis

Life cycle of Trypanosoma brucei gambiense & T. b. rhodesiense

Cerebrospinal fluid Lymph node aspiration

CATT test trypanosoma

Blood CoccidiaTachyzoites of Toxoplasma gondii

Parasites are frequently seen in neutrophils and mononuclear cells.

They are crescent shaped and small,

measuring about 37m. One end is

rounded and the other end more pointed.

Nucleus is situated towards the roundedend and stains dark red.

Cytoplasm stains blue.

Diagnosis of Free-living Amoebae

They are amoebae that normally inhabit:- Water (lakes, swimming pools, air-conditioning units)- Moist soil.- Decaying vegetations.

Potentially Pathogenic Free-living amoebae

Amoeboid form

Trophozoite form

Naegleria fowleri Acanthamoeba species

In water

Flagellate form

Cyst form Cyst form

Trophozoite

In water or air

Diagnosis of Free living amoeba: Naegleria fowleri

Geographical Distribution: some parts of the world.

Mode of infection:

- Swimming and sniffing (inhalation) in contaminated water.

- Sappinia sp cause nonlethal amoebic encephalitis

إستنشاق

Disease

Meningoencephalitis

Sample: CSF

Free living amoeba: Acanthamoeba species Balamuthia mandrillaris

Granulomatous Amoebic Encephalitis

Acanthamoeba Keratitis

Mode of infection

Nasal to Lower respiratory tract to Blood to Brain

Ulcerated skin and mucosa to Blood to Brain

Through corneal traumaExposure to contaminated waterWearing contaminated contact lenses

Morphology of free living amoebae

Naeglaria Acanthoamoeba Balamuthia

TROPHOZOITE

CYST

Urinogenital protozoa Trichomonas vaginalis

vaginal, urethral and prostatic tissue

only in humans; no animal

Structuer contain Fg=flagella

Bb=basal body

Nu=nucleus

Ax=axostyle

um=undulating membrane

Cy=cytostomal groove

Cs=costa

No cyst stage.

Multiplies by binary fission

Sample: urine sample,vaginal or urethral swab

Class insecta Anopheles

Morphology and medical importance

Female are blood feeder

Spotted wings

Maxillary palps as long as proboscis

Egg: floated, layed single

Proboscis and body in same straight line

Malaria transmition

In some areas it can also transmit filariasis

Class insectaCulex

Morphology and medical importance

Female are blood feeder

Uniform wings

Blunt tip abdomen

Maxillary palps shorter than proboscis

Egg: not floated, layed in group

Proboscis and body at an angle

vectors of filariasis and some viral

diseases

Class insectaAedes

Morphology and medical importance

Female are blood feeder

Uniform wings

Maxillary palps shorter than proboscis

Pointed tip abdomen

Egg: not floated, layed singly

Proboscis and body at an angle

vectors of dengue

yellow fever and other viral diseases

Class insectaPhlebotomus

morphology and medical importance

Also known as Sand fly

Vector of Leishmaniasis

Minute in size1.5–3 mm

yellowish in colour with black eyes

Hairy body, wings and legs.

The oval lanceolate wings are carried

erect on the humped thorax

Class insectaSimulium(Black fly)

morphology and medical importance

Transimitte onchocerciasis

Known as (river blindness)

small 1.5-5mm

humped thorax

short antennae

without hair

Class insectaCulicoides

morphology and medical importance

AnnoyanceFilarial disease1.5-5 mmPair antennaesmall headcoered by black

spott

Class insectaGlossina

Other name Tsetse fly

adults are relatively large flies

Measuring 6-15mm

have forward projecting proboscis

hatchet cell wing venation.

African trypanosoma

Order hemipteraFamily:Reduviidae

Triatomine bug

Measure 1-4 cm

Elongate snout-like

head with two eye

4 segment antennae

3 pairs of legs

Vector of Chagas disease