Hematology - Peripheral blood smear

Because staining reactions are pH dependent, the buffer that is added to the stain should be __________ or ____________.

0.05 M sodium phosphate (pH 6.4) or aged distilled water (distilled water placed in a glass bottle for at least 24 hours; pH 6.4 to 6.8)

Fixation period should be in minimum of ___________

1 minute

More recently, stain manufacturers have used ___________ to minimize water or drying artifact.

10% volume-to-volume methanol

provides the highest magnification on most standard binocular microscopes

100x oil immersion objective

Rouleaux formation or RBC agglutination is easy to recognize at this power

10x Objective Examination

High-quality blood films can be made from the blood in the EDTA tube, provided that they are made within ______ of drawing the specimen

2 to 3 hours

Size of coverslip

22 mm

WBC estimate also can be performed with the 50x objective, but the multiplication factor is __________.

3000

If the leukocyte differential count reveals reveals greater than __________, the differential should be repeated on another slide

40% lymphocytes

Magnification used to perform a WBC estimate, the evaluator selects an area in which the RBCs are separated from one another with minimal overlapping (where only two or three RBCs can overlap).

40x High-Dry or 50x Oil Immersion Objective Examination

The presence of more than _______ the number of cells per field at the edges or feather compared with the monolayer area of the film indicates that the film is unacceptable (i.e., a "snowplow" effect), and the film should be remade

4x

Blood films from EDTA tubes that remain at room temperature for more than _________ often have unacceptable blood cell artifacts (echinocytic red blood cells [RBCs], spherocytes, necrobiotic leukocytes, and vacuolated neutrophils

5 hours

if the number of monocytes, eosinophils, or basophils exceed the normal reference ranges, what should you do?

A second 100 cells are to be differentiated on the same slide

Neutrophils and monocytes have greater tendency to appear in the feathered end of the preparation which may lead in:

Artifactually increased lymphocyte counts when performing leukocyte differential count

Disadvantages of centrifugal type

Automated films require longer preparation time Blood films cannot be made in the laboratory, and capillary blood cannot be used Small number of leukocytes appears on the monolater smear No specific location exists to search for immature, abnormal, or clustered cells Occasional cellular morphology is distorted by centrifugal force

superior than other azures

Azure B

Why are most blood smears are prepared from EDTA tubes?

Cellular morphology in fresh samples are well preserved and normal cells continue to demonstrate satisfactory morphology at least 5 hours

Used extensively for bone marrow aspiration

Coverslip method

Disadvantages of coverslip method

Difficulty in learning the technique Must be manually cleaned of dirt, dust, grease, and fingerprints prior to use Difficulty in labeling, transporting, and staining the small easily broken cover slip Use of mounting media must be kept matched for estimating platelet numbers Lack of specific areas to examine unusual cells

Several dishes (large enough to contain a portable slide holder and an adequate amount of solution to cover the slides) are required

Dip (incubation) method

In addition, spuriously low platelet counts and falsely increased WBC counts (pseudoleukocytosis) can result from

EDTA induced platelet clumping

Advantages of wedge type

Easier to master Commercially precleaned slides are adequate Slides are not easily broken Labeling, transporting, and staining are easier Least expensive type of preparation The tendency of larger cells to settle at the slide edges and feathered end makes it easier to find abnormal cells

In this type of smear, plasma causes nucleated cells to shrink and stain intensely.

Extremely thick smear

In this type of smear preparation, smudge cells are increased, and red blood cells become artificially spheroid with a distorted shape

Extremely thin smear

In this type of smear preparation, there is a tendency for more nucleated cells to be carried out to the edges of smears that have been made with two slow, a stroke, and this affects the accuracy of the differential count

Extremely thin smear

is acidic and stains basic (and eosinophilic) components, such as hemoglobin and eosinophilic granules.

Free eosin

___________ is basic and stains acidic (and basophilic) cellular components, such as ribonucleic acid (RNA).

Free methylene blue

Indicates an accumulation of nucleated cells, which may be attributable to a large number of leukocytes, to slow spreading or to delay in spreading

Gritty appearance of feathered or tail areas

_____ on the slide may represent material that will interfere with proper fixation or disturb the acid-base relations needed for good quality testing

Haze

An excellent spreader slide is a

Hemocytometer coverglass

With this anticoagulant, there is a tendency for leukocytes to accumulate at the tail end

Heparin

________ in the air as the slide dries may add to the punched-out, motheaten, or echinocytic appearance of the RBCs.

Humidity

Advantage of two glass slide method

Larger examination area Slides are not as breakable as coverslips Ease of labeling the blood smears

a mixture of Methylene blue, and Eosin dye, prepared in Alcohol medium and diluted with buffer or distilled water during staining procedure.

Leishman stain

one of the best stains for routine blood stain to stain the Peripheral blood smear for the examinations of blood film under the microscope and is satisfactory for malaria and other blood parasites

Leishman stain

is often preferred to the powdered form because it mixes more easily with blood.

Liquid tripotassium EDTA

Examining the film before placing it on the microscope stage sometimes can give the evaluator an indication of abnormalities or test results that need rechecking

Macroscopic examination

are so named because they have cytoplasmic granules that have a neutral pH and pick up some staining characteristics from both stains.

Neutrophils

constitutes combination of a Romanowsky stain with another stain; such that it improves staining of cytoplasmic granules and other bodies

Panoptic staining

What would you expect if films are made directly from a drop of finger-stick or heel-stick blood or if blood is collected in heparinized microhematocrit tubes?

Platelet clumping

Happens when blood is anticoagulated with EDTA where the platelets surround or adhere to neutrophils, which potentially causes pseudothrombocytopenia when counting is done by automated methods

Platelet satellitosis

that react best at room temperature are one of the mechanisms known to cause pseudoleukocytosis.

Platelet-specific autoantibodies

Disadvantages of wedge type

Poor distribution of nucleated cells There is greater trauma to the cells during the blood film

What is the prime function of methylene blue?

Provide oxidative products, the azure

occurs when platelet agglutinates are similar in size to WBCs and automated analyzers cannot distinguish the two. The platelet clumps are counted as WBCs instead of platelets.

Pseudoleukocytosis

These are considered polychrome stains because they contain both eosin and methylene blue.

Pure Wright stain or a Wright-Giemsa stain (Romanowsky stain)

is used for staining peripheral blood films and bone marrow smears

Pure Wright stain or a Wright-Giemsa stain (Romanowsky stain)

A grainy appearance to the film may indicate____________ as found in the cold hemaglutinin diseases

RBC agglutination

uses rods overlying a sink or dish that hold glass slides or coverslips in horizontal position during staining

Rack method

Demethylation and deamination of methylene blue in alcoholic solution produces various oxidized products and ________ will inhibit this change

Refrigeration

RBC form more __________ in thick areas of the smear and cannot be evaluated

Rouleaux

it is the tendency for platelets to adhere to neutrophils in EDTA which is falsely interpreted as thrombocytopenia by particle counters and can be recognized only by visual smear evaluation

Satellitism

Helpful in identifying artifact

Sharp transition between hemoglobinized rim and clear center

Reasons for blue stains:

Slides stained after 1 week Increased level of proteins, as in plasma cell myeloma, and rouleaux may be seen

Can cause drying or moisture red-cell artifact, hairy appearance of cytoplasm of normal lymphocytes, and shrinkage of normal leukocytes

Slow air drying of fresh prepared blood smear

makes it difficult to mix stain and buffer adequately, as well as to wash off the stain solution

Small surface area of coverslip

represent leukocytes ruptured during preparation of the smear

Smudge cells

Significant advantage of coverslip preparation

Superior leukocyte distribution

Advantages of centrifugal type

Superior, even cell distribution Consistency of preparations Large examination area (monolayer) Fewer broken (smudge) lymphoid cells in chronic lymphocytic leukemia

Extremely thick smears are caused by

Too large drop of blood Too fast spread Too high angle of spreader slide

Extremely thin smears are caused by:

Too small drop of blood Too slow spread Too low angle of spreader slide

Jerky pulling movement of coverslip causes

Uneven distribution of cellular elements

Causes of gritty appearance of feathered or tail areas

Using only a part drop of the blood Rough edge or dirty spreader

____________ normally occurs almost immediately with EDTA but causes no evaluation problems.

Vacuolization of monocytes

Most widely used for smear preparation

Wedge type

a polychromatic stain consisting of a mixture of Eosin and Methylene blue. As the Wright stain is methanol based, it doesn't require a fixation step prior to staining. However, fixation helps to reduce water artefact that can occur on humid days or with aged stain

Wright's Stain

Fixative agent

acetone-free methanol

the specimen of choice for evaluation of blood cell morphology.

anticoagulant-free blood

Why does blood staining results from fresh blood are the best?

blood itself acts as a buffer in the staining process

Eosinophils should have __________ refractile granules.

bright orange

Actual staining of cells or cellular components does not occur until the _______ is added.

buffer

forced rapid drying may

color intensities

includes enumeration of cellular elements, quantitation of hemoglobin, and statistical analyses that provide a snapshot of cell appearances.

complete blood count (CBC)

The peripheral film evaluation is the capstone of a panel of tests called the ___________

complete blood count (CBC) or hemogram

A delay in the separation of the coverslip may cause

coverslip to be stuck to each other Platelet clumping Uneven distribution of leukocytes

Regardless of film preparation method, before staining, all blood films and bone marrow smears should be dried as quickly as possible to avoid ________

drying artifact

Water contamination of the fixative may lead to

drying artifact

Blowing breath on a slide is counterproductive because the moisture in breath causes RBCs to become ___________

echinocytic (crenated) or to develop water artifact

Markedly increased WBC counts and platelet counts can be detected from the blue specks out at the _______.

feather edge

Under the 10x objective, it is possible to check for the presence of ________ strands; if they are present, the sample should be rejected

fibrin

Replacement of 50% methanol by __________ will retard the oxidative process, however, it produces viscous stain solution that does not mix easily with the buffer and may lead to uneven staining

glycerol

Drying artifact may render RBC as falsely ____________

hypochromic

holes all over the film could mean that the patient has _________ and some of the automated CBC parameters should be rechecked for interferences from lipemia.

increased lipid levels,

Essentially all specimens received for routine testing in the hematology section of the laboratory have been collected in ________

lavender (purple)-topped tubes

The most efficient check is _______________ of the peripheral areas for a disproportionate number of neutrophils

low power inspection

At the ___________, overall film quality, color, and distribution of cells can be assessed.

low-power magnification

When the hematocrit is higher than normal (i.e., .60%), as is found in patients with polycythemia or in newborns, the angle should be _________ so the film is not too short and thick

lowered

The purpose of staining blood films is simply to

make the cells more visible and to allow their morphology to be evaluated

Giemsa stains also contain __________.

methylene blue azure

Wedge-type smears prepared from day-old EDTA blood will show distribution defects with many _______________

nucleated cells being carried to the tail area

Microscopically, the RBCs should appear ________, and WBC nuclei should be _________.

orange to salmon pink, purple to blue

Macroscopically, a well-stained blood film should be ________

pink to purple

The cytoplasm of neutrophils should be _________.

pink to tan with violet or lilac granules

For extremely low hematocrits, the angle may need to be _______.

raised

Metallic elements serves as __________ to extend shelf life, but their presence may change stain quality

stabilizer

The oxidized methylene blue and eosin form a _________ complex, which stains neutral components

thiazine-eosinate

Pulling the coverslips apart prematurely or too quickly causes

thick or uneven spread

Moving the pusher slide forward __________ accentuates poor leukocyte distribution by pushing larger cells, such as monocytes and granulocytes, to the very end and sides of the film

too slowly

It is difficult to avoid drying artifact on films from extremely anemic patients because of the _________.

very high ratio of plasma to RBCs