blood film staining protocol

8/10/2019 Blood Film Staining Protocol

1/7

Staining of Blood Film

BLOOD

Blood is sometimes considered to be a fluid connective tissue because of the mesenchymal origin of its cells anda low ratio of cells to liquid intercellular substance, the blood plasma. In human adults about 5 liter of bloodcontribute 7-8 % to the body weight of the individual. The contribution of red blood cells (erythrocytes) to the totalvolume of the blood (haematocrit) is about 43%.

Erythrocytes are the dominant (99%) but not the only type of cells in the blood. We also find leukocytesand, inaddition, blood platelets. Erythrocytes, leukocytes and blood platelets are also being referred to as the formedelements of the blood. Erythrocytes and blood platelets perform their functions exclusively in the blood stream. Incontrast, leukocytes reside only temporarily in the blood. Leukocytes can leave the blood stream through thewalls of capillaries and venules and enter either connective or lymphoid tissues.

BLOOD FILM PREPARATION

Microscope Slides

Slides should be made from the highest purity, corrosion-resistant glass; other material such as plastic is mostlynot acceptable. Glass slides typically measure 75 25 mm, are approximately 1 mm in thickness, must be flatand free from distortions and ripples, and

must be clear and colorless (water-white).

It is preferable to use pre-cleaned slides, but at minimum, it must be ensured that slides are free from scratches;are clean; are free of dust, lint, and fat (from fingerprints); and are dry. This may mean that in humidenvironments, slides must be hydration resistant and, after their sealed container is opened, should be kept in adesiccator or container with water-free (methyl) alcohol or a mixture of 3 parts alcohol and 1 part acetone untilused. Slides should always be stored in sealed containers only to be opened immediately before use.

Cleaning of Slides

It is essential to use clean, dry, dust-free slides: grease and residual detergent are liable to spoil a blood film. Ifclean grease-free slides are not available, they are left overnight in a detergent solution, then washed thoroughlyin running tap-water, rinsed in distilled water and wiped dry with a clean linen cloth. Before use, the surface waswiped with 95% ethanol or methanol and dried with a clean cloth; then kept covered to avoid having dust settle onthe surface.

Used slidesare discarded in detergent solution, heated to about 60oC for 20 minutes, then washed in running tap-water, rinsed in distilled water and treated as described above.

At the lower end of the slide, the student's identification was written with glass marking pencil on the edge of thefilm.

Blood

Blood should be used within one hour of blood collection. For the experiment, blood was obtained by fingerpricking method.

The tip of the left finger was cleansed with antiseptic and punctured with a new #23 hypodermic needle.

The finger was massaged from the bottom to the top to encourage blood flow. The blood was pressed towardsfingertip. Repeated pressing until a drop with a 3 to 4 mm (1/8 inch) diameter was seen.

Transferred one blood drop to the each of the three glass slides, approached from beneath the finger, avoidingsmearing the blood over the slide.

Adequate mixing of the specimen is necessary prior to film preparation, using a wooden stick or glass capillary. It

is important that the film of blood finishes at least 1 cm before the end of the slide.

Fixing the film


2/7

To preserve the morphology of the cells, films must be fixed as soon as possible after they have dried. It isimportant to prevent contact with water before fixation is complete.

STAINING

Romanowsky stains are universally employed for staining blood films and are generally very satisfactory. Themain components of a Romanowsky stain are:

1

Leishman's staining method was followed.

Air dry slides

Flood with neat Leishman's stain for three minutes; as the stain's formulation includes methanol, this willfix the cells.

Dilute the stain on the slide with an equal amount of buffered water, pH 6.8, adding the water slowly witha plastic Pasteur pipette and mixing by sucking the stain up and down with the pipette.

Leave the slide for approximately 12 minutes; the appearance of a polychromatic 'scum' on the surfaceof the slide is merely a result of oxidation of the dye components and can be ignored.

Wash off excess stain with slowly-running tap water and flood slide for one minute with buffered water,pH 6.8.

Dry the slide and mount with a coverslip if required, using glycerol.

The density of cells varies across the smear. Cells will be "heaped and piled" close to the point were the drop ofblood was placed on the slide. White blood cells appear shrunken, and some types are difficult to distinguish fromeach other. There are fewer cells close to the tip of the smear. In this region, white blood cells are sometimesdamaged and erythrocytes may be deformed. The best area to look at is between these two regions. Where it islocated exactly and how wide it is will depend on the smear, but the middle of the smear is a good starting point.

Microscopic Examination of Blood Films

Staining characteristics of a correctly stained normal film

Nuclei Purple

Cytoplasm

Erythrocytes Deep pink

Reticulocytes Grey-blue

Neutrophils Orange-pink

Lymphocytes Blue; some small lymphocytes deep blue

Monocytes Grey-blue

Basophils Blue

Granules

Neutrophils Fine purple

Eosinophils Red-orange

Basophils Purple-black

Monocytes Fine reddish (azurophil)

Platelets Purple


3/7

Sources of error

Film preparation

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

Holes in film: Slide contaminated with fat or grease

Irregular leucocyte and platelet distribution, especially in tail: poor film-making technique

Film too short and too thick: spreader held at incorrect angle

Film extending to end of slide: blood drop too large

Short thin film: blood drop too small

Film extends to edge of slide: spreader too wide or not positioned correctly

Cellular degeneration: delay in fixing, inadequate fixing time or methanol contaminated with water

Staining faults

Too blue: Smear too thick

Inadequate time in buffer

Buffer pH too high

Staining time too long

Diluted stain overused, requires replenishment

Stock MGG solution incorrectly made or made from impure dye

Stock solution left exposed to bright daylight

Too pink: Excessive time in buffer

Buffer pH too low

Stock MGG solution incorrectly made or made from impure dye

Coverslip mounted before film is completely dry

Too faint: Staining time too short

Excessive washing after staining


4/7

Stain deposit: Stain solution left in uncovered jar or tray

Stain solution not filtered

Dirty slides

Blue background: Inadequate fixation

Prolonged storage before fixation

Blood anticoagulated with heparin

Erythrocytes

Erythrocytes do not contain a nucleus. They do contain haemoglobin, which fills almost the entire cytoplasm.Erythrocytes are unable to move actively, but they are remarkably elastic and can withstand deformation. Theyare typically biconcave disks although their shape is influenced by osmotic forces. The average diameter of thedisk is ~7 m. Since erythrocytes can be found in the vast majority of histological sections - in small numberseven in perfused tissues - they will often allow us to estimate the size of other structures or cells. Mature

erythrocytes do not contain organelles, and their cytoplasm looks fairly homogenous - even in the EMFunctions

Erythrocytes function in the transport of oxygen. Haemoglobin, the oxygen binding protein in erythrocytes,contributes about 30% of the weight of an erythrocyte. The lifespan of an erythrocyte in the bloodstream is 100-120 days.About 51011erythrocytes are formed/destroyed each day.

Leukocytes

Leukocytes can be further subdivided into granular leukocytes, i.e. neutrophils, basophils and eosiniphils, andnon-granular leukocytes, i.e. monocytes and lymphocytes.

In healthy individuals the relative numbers of circulating leukocyte types are quite stable. A differential leukocytecountwould typically produce the following cell frequencies (numbers in parentheses are the range of normalfrequencies reported in different texts):

~ 60% neutrophils (50% - 70%)

~ 3% eosinophils (>0% - 5%)

~ 0.5% basophils (>0% - 2%)

~ 5% monocytes (1% - 9%)

~ 30% lymphocytes (20% - 40%)

Changes in their relative numbers indicate that something abnormal is happening in the organism. A larger thanusual number of neutrophils (neutrophilia) would indicate e.g. an acute or chronic infection. The number ofbasophils and eosinophils may increase (eosinophiliaor basophilia) as a consequence of e.g. allergic disorders.

Granular Leukocytes

Granular leukocytes are all approximately the same size - about 12-15 m in diameter. Their nuclei form lobes,and nucleoli cannot be seen. The number of nuclear lobes varies according to cell type. All granulocytes aremotile.

The term granulocytes refers to the presence of granules in the cytoplasm of these cells. The granulescorrespond to secretory vesicles and lysosomes. Specific granulesare the granules which are only found in oneparticular type of granulocytes.

Neutrophil granulocytes (or neutrophils)

have a very characteristic nucleus. It is divided into 3-5 lobes which are connected by thin strands of chromatin.The number of lobes increases with cell age. Up to 7 lobes can be found in very old neutrophils (hypersegmented

cells).

Neutrophils (like all other granulocytes, monocytes and lymphocytes) contain all the organelles that make up atypical cell. In addition to the usual complement of organelles, they also contain two types of granules. Primary


5/7

granules (or A granules) contain lysosomal enzymes and are likely to be primary lysosomes, although they arelarger (0.4 m) than the "ordinary" primary lysosome. Secondary granules(or B granules), the specific granulesof the neutrophils, contain enzymes with strong bactericidal actions.The specific granules of neutrophils stainonly weakly if they are at all visible - they are "neutral", hence the term neutrophil.

Functions

Neutrophils play a central role in inflammatory processes. Large numbers invade sites of infection in response to

factors (e.g. cytokines) released by cells which reside at an infection site. Neutrophils are the first wave of cellsinvading infection sires. Receptors in their plama membrane allow them to recognise foreign bodies, e.g. bacteria,and tissue debris, which they begin to phagocytose and destroy. The phagocytotic activity of neurophils is furtherstimulated if invading microorganisms are "tagged" with antibodies (or opsonised). Neutrophils cannot replenishtheir store of granules. The cells die once their supply of granules has been exhausted. Dead neutrophils andtissue debris are the major components of pus. Their lifespan is only about one week.

Lost neutrophils are quickly replenished from a reservepopulation in thebone marrow.Because theyare younger,

their nuclei havefewer lobes thanthe "average"neutrophil. Ahigh proportionof neutrophils,with few nuclearlobes indicates arecent surge intheir releasefrom the bonemarrow.

Eosinophil

granulocytes

(or eosinophils)

Nucleus usually has two lobes. Cytoplasm appears filled with the specific granules which stain red or pink.Eosinophils contain some large rounded vesicles in their cytoplasm. These granules correspond to the

eosinophilic grains. The specific granules contain an electron-dense, proteinaceous crystal composed of major basic protein(MBP).

Functions

The presence of antibody-antigen complexes stimulates theimmune system. Eosinophils phagocytose these complexes andthis may prevent the immune system from "overreacting". Theirgranules also contain the enzymes histaminaseandarylsufatase.These enzymes break down histamine and leukotrienes, whichagain may dampen the effects of their release by basophils ormast cells. MBP, which can also function as a cytotoxin, and itsrelease by eosinophils may be involved in the response of thebody against parasitic infections, which are accompanied by anincrease in the number of eosinophils.

Basophil granulocytes (or basophils)

Nucleus has a 2 or 3 lobes. The lobes are usually not as welldefined as in neutrophils and the nucleus may appear S-shaped.The specific granules of basophils are stained deeply bluish or


6/7

reddish-violet. Their colour corresponds closely to the colour of the nucleus which sometimes is difficult to seeamongst or behind the granules. The granules are not as numerous as in eosinophils. The specific granules ofbasophils contain heparin, histamine lysosomal enzymes and leukotrienes (the slow-reactingsubstance ofanaphylaxis or SRS-A).

Functions

Heparin and histamine are vasoactive substances. They dilate the blood vessels, make vessel walls more

permeable and prevent blood coagulation. As a consequence, they facilitate the access of other lymphocytes andof plasma-borne substances of importance for the immune response (e.g. antibodies) to e.g. a site of infection.The release of the contents of the granules of basophils is receptor-mediated. Antibodies produced by plasmacells (activated B-lymphocytes;) bind to Fc-receptors on the plasma membrane of basophils. If these antibodiescome into contact with their antigens, they induce the release of the contents of the basophil granules.

Non-granular leukocytes

Monocytes

Slightly larger than granulocytes (12-18 m in diameter). Cytoplasm stains usually stronger than that ofgranulocytes but does not contain any granular structures. The "textbook" monocyte has a C-shaped nucleus.Monocytes contain ultramicroscopic granules which correspond to lysosomes. Whatever the shape of the

nucleus, it is usually not lobed, and it is bound, at least on its concave side, by a wide rim of non-granularcytoplasm.

Functions

Once monocytes enter the connective tissue they differentiate into macrophages. They phagocytosemicroorganisms, tissue debris and the dead neutrophils. Monocytes also give rise to osteoclasts, which are ableto dissolve bone. They are of importance in bone remodelling.

Lymphocytes

Very variable in size. The smallest may be smaller than erythrocytes (~5 m in diameter) while the largest mayreach the size of large granulocytes (up to 15 m in diameter). Amount of cytoplasm depends on the size of thelymphocyte. In small ones, which are the majority in the blood, the nucleus appear to fill the entire cell. Largelymphocytes have a wider rim of cytoplasm which surrounds the nucleus. Both the nucleus and the cytoplasmstain blue (and darker than most other cell types in the blood). The typical lymphocyte only contains the usualcomplement of cellular organelles. The appearance of lymphocytes may change drastically when they areactivated.


7/7

Functions

Most lymphocytes in the blood stream belong to either the group of B-lymphocytes(~5%) or the group of T-lymphocytes(~90%). Unless they become activated, the two groups can not be distinguished using routine lightor electron microscopy.Upon exposure to antigens by antigen-presenting cells (e.g. macrophages) and T-helper cells (one special groupof T-lymphocytes) B-lymphocytes differentiate into antibody producing plasma cells.

Blood Platelets (or thrombocytes)

Platelets are about 3 m long. Platelets do not containa nucleus.Unlike erythrocytes, which also lack anucleus, the blood platelets of mammals have neverbeen nucleated cells. Instead, blood platelets arefragments of the cytoplasm of very large thrombocyteprecursor cells, megakaryocytes.

Different types of vesicles contain either serotonin(electron-dense delta granules; few) or compoundsimportant for blood coagulation(alpha granules - theyalso contain platelet-derived growth factor (PDGF)

which may play a role in the repair of damaged tissue).The hyalomere contains cytoskeletal fibres, which include actin and myosin.

Functions

Platelets assist in haemostasis, the arrest of bleeding. Serotonin is a potent vasoconstrictor. The release ofserotonin from thrombocytes, which adhere to the walls of a damaged vessels, is sufficient to close even smallarteries. Platelets, which come into contact with collagenous fibers in the walls of the vessel (which are notusually exposed to the blood stream), swell, become "sticky" and activate other platelets to undergo the sametransformation. This cascade of events results in the formation of a platelet plug (or platelet thrombus). Finally,activating substances are released from the damaged vessel walls and from the platelets. These substancesmediate the conversion of the plasma protein prothrombininto thrombin. Thrombin catalyzes the conversion offibrinogen into fibrin, which polymerizes into fibrils and forms a fibrous net in the arising blood clot. Platelets

captured in the fibrin net contract leading to clot retraction, which further assists in haemostasis.
http://openutility%28%27addons/blo01.htm')http://openutility%28%27addons/blo01.htm')

blood film staining protocol

Documents