Chapter 15 – Examination of the Peripheral Blood Film and Correlation with the Complete Blood Count

A well stained peripheral blood can provide information about:

a) WBC and platelet estimatesb) Relative proportions of the

different types of WBCc) Evaluation for the abnormalities

of the morphology of all three cell lines

The peripheral blood film evaluation is the capstone of a panel of test called the complete blood count (CBC) or hemogram

The CBC includes:a) Enumeration of cellular elementsb) Quantification of hemoglobinc) Statistical analysis that provides

a snapshot of cell appearances

Peripheral Blood FilmsSpecimen Collection

EDTA Specimen for peripheral blood film is

collected in lavender (purple) top tubes which contains Etyhylenediaminetetraacetic acid (EDTA)

EDTA anticoagulates the blood by chelating the calcium that is essential for coagulation

Specimen from EDTA tubes should be done within 2 to 3 hours of drawing the specimen

Blood film from EDTA tubes that remains in room temperature for more than 5 hours have unacceptable blood artifacts

Main advantages of making blood film for EDTA tubes:

a) Multiple slides can be made if necessary

b) Do not have to be prepared immediately after draw

c) Generally prevents platelets from clamping to the slide

Platelet satellitosis is a phenomenon where in the platelets surround or adhere to neutrophils, which potentially causes pseudo-thrombocytopenia when counted is done by automation

Low platelet counts and falsely increased WBC counts can result from EDTA induced platelet clumping

Sodium citrate EDTA induced problems can be

eliminated by recollecting specimens in light blue top tube which contains sodium citrate and ensuring the proper ratio of nine parts blood to one part anticoagulant is observed

Platelet counts and WBC counts from sodium citrate specimens must be corrected for the dilution of blood with the anticoagulant

Platelet counts and WBC counts from sodium citrate specimens, are multiplied to 1.1 to obtain accurate results

Finger and heel puncture Platelet clumping must be expected if

films are made directly from drop of finger-stick or hell stick or if blood is collected in heparinized microhematocrit tubes

Only a few films can be made directly from blood from a skin puncture before site stops bleeding

Peripheral Film Preparation

Types of films

Manual Wedge Technique Easiest to master Most convenient and commonly used

method for making peripheral blood film

Requires at least two 3-inch by 1-inch clean glass slides

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High quality beveled edge microscopic slides with chamfered corners for good lateral borders are recommended

The size of the blood is important: a) Too large a drop creates a long or

thick filmb) Too small a drop creates a short

or thin blood film It is important that the whole drop be

picked up and spread Moving the pusher slide forward too

slowly accentuates poor leukocyte distribution by pushing larger cells to the very end of the slide of the film

Maintaining an even, gentle pressure on the side is essential

It is crucial to keep the same angle all the way to the end of the film

When the hematocrit is higher than normal the angle should be lowered so the film is not too short and thick

For extremely low hematocrit, the angle may be raised

Procedure A drop of blood about 2-3mm is placed

at one end of the slide The drop may be delivered using a

Diff-Safe dispenser The Diff-Safe dispenser is inserted into

the rubber stopper of the EDTA tube which eliminates the need to remove the rubber stopper

Push type wedge preparation where in spreader slide is pulled into the drop of blood then the film is made by pushing the blood along the slide

Pull type wedge preparation where in spreader slide is pushed into the drop of blood then the film is made by pulling the blood along the slide

Features of a well made wedge peripheral blood film

1. The film is two thirds to three fourths the length of the slide

2. The film is finger shaped, very slightly rounded at the feathery edge, not

bullet shape, this provides the widest area for examination

3. The lateral edge is visible4. The film is smooth without

irregularities (holes or streaks)5. Thin feathery edge has rainbow

appearance when held against the light6. Whole blood is picked up and spread

Common causes of 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 cause of poor blood smear1. 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

Coverslip Technique Older technique compared to wedge

technique Rarely used for peripheral blood films

but is sometimes used for making bone marrow aspirate smears

Advantage is that this method produces excellent leukocyte distribution, which leads to more

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accurate determination of differential counts

Procedure The technique requires one drop of

blood or bone marrow aspirate placed on a clean coverslip (22X22mm) and another coverslip placed on top, with the blood allowed to spread across the two slides

Slight pressure is applied to the cover slip between the index finger and the thumb to help spread the bone marrow spicule before the two smear are pulled apart (crush preparation)

Too much pressure on the cover slip can cause cell rupture

Inadequate pressure prevents the spicule from spreading to a satisfactory monolayer

Automated Slide Making and Staining Sysmex SP-1000i

An automated slide making and staining machine

Films can be produced approximately every 30 seconds

Drying Films Before staining all blood films and

bone marrow smears should be dried as quickly as possible to avoid drying artifacts

Blowing breath on a slide is counter productive, because the moisture in breath causes RBCs to become echinocytic (crenated) or to develop water artifact (also called drying artifacts

Staining of Peripheral Blood Films Staining method used for staining

peripheral blood: Pure wright stain or Wright

Giemsa stain (Romanowsky stain) Considered polychrome stains because

of eosin and methylene blue content May also contain methylene blue azure

Purpose of staining is to makes cells more visible and allows their morphology to be evaluated

Methanol in the stain fixes the cell to the slide

Actual staining occurs when the buffer is added which is 0.05M sodium phosphate (pH 6.4) or aged distilled water

Oxidized methylene blue and eosin form a thiazine-eosinate complex, which stains neutral components

Staining reaction are pH dependent Free methylene blue is basic and stains

acidic cellular components Free eosin is acidic and stains basic

cellular components Water or drying artifact has been a

long-lived nuisance to hematology laboratories

Water or drying artifacts can appear as:a) Mouth eaten look to the RBCb) Heavily demarcated central pallorc) Refractile blotch on the RBCsd) Echinocytes (crenation) seen in

the areas of the slides that dries most slowly

Factor that contribute to water or drying artifact problem: Humidity in the air as the slide

dries may add to the punched out, moth eaten or echinocytic appearance of the RBC

Manufacturers have used 10% volume to volume methanol to minimize water or drying artifact

Wright Staining Method

Manual technique Performed over a sink or a pan with a

staining rack It is important to flood the slide

completely Stain should remain at least 1 to 3

minutes to fix the cells to the glass Approximate equal amount of buffer is

added to the slide

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A metallic sheen (green scum) should appear on the slide if mixing is correct

Bone marrow smears take longer to stain than peripheral blood film

When staining is complete, slide is rinsed with a steady but gentle stream of neutral pH neutral then the slide is air dried in a vertical position

Use of manual Wright staining technique is desirable for staining peripheral blood films containing very high WBC counts

Under staining is common when a leukemia slide is placed on an automated slide stainer

Disadvantage of manual technique are the increased risk of spilling stain and the longer time required to complete the procedure

Automated slide stainers It takes 5 to 10 minutes to stain a batch

of slides The slides may be automatically

dipped in stain and then in buffer and a series of rinses or propelled along a platen surface by two conveyors

In a Hema-Tek device, stain, buffer and rinses are pumped through holes in the platen surface flooding the slide at the appropriate time

Slides can be added anytime to Hema-Tek stainer, and stains package are stable for only6 months

Disadvantage of the dip-type batch stainersa) Stat slides can not be added to the

batch once staining process had begun

b) Working or aqueous solution of stain are stable for only 3 to 6 hours

Quick stains Fast and easy which takes about 1

minutes Modified Wright or Wright –Giemsa

stain

Required quantity can be filtered into a coplin jar or a staining dish, depending on the quantity of slides to be stained

Quantity is often the concern of quick stain, however color quantity is acceptable

Features of a well-stained peripheral blood film Should be pink to purple RBC should appear orange to salmon

pink WBC nuclei should be purple to blue Cytoplasm of neutrophils should be

pink to tan with violet or lilac granules Eosinophils should have bright orange

refractile granules Best staining result are obtained on

fresh slides because the blood itself acts as a buffer in the staining process

Slides stained after 1 week or longer turn out blue

Specimen that have increased levels of protein produce bluer staining blood film

Peripheral Film Examination Evaluates the platelet and WBC count

and differential, along with WBC, RBC, and platelet morphology

Macroscopic Examination Film that is bluer overall than normal

may indicate that the patient has increased blood proteins, as in multiple myeloma and that rouleaux may be seen in the film

A grainy appearance to the film may indicate RBC agglutination

Holes all over the film may indicate increased lipid levels

Increased WBC counts and platelet counts may be detected from the blue specks out at the feathery edge

Microscopic Examination Proper adjustment of the microscope

is done for proper blood film examination

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10X Objective (LPO) Examination 10X or low power objective with a

total magnification of 100X Not much time needs to be spent at

this magnification Things observed under LPO:

a) Overall film qualityb) Colorc) Distribution of cells

“Snow-plow effect” - poor leukocyte distribution; large cells such as monocytes and neutrophils are pushed to the end of the slide; "snowplow effect"

Check to see if there are good counting areas available free of ragged edges and cell clumps.

Check the WBC distribution over the smear.

Check that the slide is properly stained.

Check for the presence of large platelets, platelet clumps, and fibrin strands.

40X Objectives (HPO) Examination 40X high dry objective lens with a total

magnification of 400X Choose a portion of the peripheral

smear where there is only slight overlapping of the RBCs.

Count 10 fields take the total number of white cells and divide by 10.

To do a WBC estimate by taking the average number of white cells and multiplying by 2000.

100X Oil Immersion Objectives (OIO) Examination

100X oil immersion objectives provides the highest magnification on most standard binocular microscope which has a total magnification of 1000X

WBC differential count is usually done under OIO

RBC, WBC and platelet morphology evaluation and platelet estimate are done under OIO

RBC inclusions such as Howell-Jolly bodies, and WBC inclusions such as Döhle bodies can be seen easily if present

50X Oil Immersion Objectives (OIO) Examination

Larger view allows cells to be evaluated faster

Optimal Assessment area Observation is done between the thick

area (heel) and the thin area (feathery edge)

Microscopically the RBCs are uniformly and singly distributed with few overlapping and have normal biconcave appearance

Normal RBC count per 100X OIO is 200 to 250 RBC

Performance of the White Blood Cell differential

These counts are done in the same area as WBC and platelet estimates with the red cells barely touching

This takes place under × 100 (oil) using the back and forth serpentine or battlement tract pattern method of counting

Count 100 WBCs including all cell lines from immature to mature.

To increase accuracy it is advisable to count 200 cells when WBC is higher than 40 X109L

Results are reported in percentage In addition to counting the cells,

evaluators assesses their appearance Because differential alone provides

only partial information, the absolute cell counts are calculated for each cell type

Red blood Cell Morphology Includes the assessment of cell size,

variability in size (anisocytosis), cell color, cell shape (poikilocytosis) and cellular inclusions

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Degree of abnormal morphology is reported as:

a) Slight, moderate or markedb) Scale form (1+ to 3+)

Platelet Estimate Performed under 100X OIO 10 OIO field is counted in the same

area as WBC and platelet estimates with the red cells barely touching

Formula:

Average¿of platelet per field X total RBC count ¿200 RBCs per field

200 id the average number if RBCs per OIO field in the optimal assessment area

Summarizing Complete Blood Count Result CBC (complete blood count) includes

the assessment of WBCs, RBCs and platelets

CBC provides information about hematopoietic system

CBC plays a role in screening of those organs for disease

One of the most frequently ordered laboratory test

The process of interpreting the CBC test results has two phasesa) PHASE 1: The numbers and

description generated by the testing are summarized using appropriate terminology

b) PHASE 2: Is to recognize consistent with various disease and to be able to narrow the diagnosis for the given patient or perhaps even to pinpoint it so that appropriate follow up testing can be recommended

Organization of Complete Blood Count Results The CBC panel is essentially divided

into WBC, RBC, and platelet parameters

Generally all the parameters interpreted together provide the best

information, so a complete summary of the results should be generated

Assessing Hematology Results Relative to Reference Ranges

Strategies that can help determine the significance of the result:a) If the result are very far from the

reference range, it is more likely that they are truly outside range and represent pathologic process

b) If two or more diagnostically related parameters are slightly or moderately out of range in the same direction this suggests that the results are clinically significant and associated with some diagnostic process

Some healthy individuals always have results slightly outside the range, the best comparison for their result is their results from prior time when they where healthy

Summarizing White Blood Cell Parameter

The WBC-related parameters of a routine CBC include the following:

1. Total WBC count (WBCs X 109/L)2. WBC differential count values

expressed as percentages (relative count)

3. WBC differential count values expressed as the actual number of each type of cell (absolute count)

4. WBC morphology

STEP 1: Ensure that the WBC count is accurate. The presence of NRBC may require correction of WBC count

STEP 2: Examine the WBC count for variation in the total number of WBCs; compare the patients’ count with the laboratory’s established reference range

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STEP 3 and 4: Examine the differential information (relative and absolute) on variations in the distribution of WBCs

STEP 5: Make note of immature cells in any cell line reported in the differential that should not appear in normal peripheral blood

STEP 6: Make note of any morphologic abnormalities and correlate film findings with numerical values

Summarizing Red Blood Cell Parameter

The RBC-related parameters of a routine CBC include the following:

1. RBC count (RBCs X 1012/L)2. Hb (g/dL)3. Hct (% or L/L)4. MCV (fL)5. MCH (pg)6. MCHC (% or g/dL)7. RBC distribution width (RDW, %)8. Morphology

STEP 1: Examine the Hb concentration to assess anemia

STEP 2: Examine the MCV to assess cell size

STEP 3: Examine the MCHC to assess cell Hb concentration

STEP 4: Examine the RDW to assess anisocytosis

STEP 5: Examine the morphologic description and correlate with the numerical value. Look for evidence of a reticulocyte response

STEP 6: Review remaining information (RBC count and MCH)

Summarizing Platelet Parameter

The platelet-related parameters of a routine CBC include the following:

1. Platelet count (platelets X 109/L)2. Mean platelet volume (MPV, fL)

3. Morphology

STEP 1: Examine total platelet count

STEP 2: Examine the MPV to assess platelet size

STEP 3: Examine platelet morphology and correlate with the numerical values

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