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Examination of Peripheral Blood Smear

Examination of Peripheral Blood Smear

A well Made and well Stained Smear can provide:

Estimates of cell count

Proportions of the different types of WBC

Morphology

Peripheral Blood Smear

Objective

1. Specimen Collection

2. Peripheral Smear Preparation

3. Staining of Peripheral Blood Smear

4. Peripheral Smear Examination

Specimen Collection

Venipuncture

should be collected on an EDTA Tube

EDTA liquid form preferred over the powdered form

Chelates calcium

Disodium or Tripotassium ethylenediamine tetra-acetic acid

Specimen Collection

Advantages

Many smears can be done in just a single draw

Immediate preparation of the smear is not necessary

Prevents platelet clumping on the glass slide

Specimen Collection

Disadvantages:

PLATELET SATELLITOSIS

causes pseudothrombocytopenia and pseudoleukocytosis

Cause: Platelet specific auto antibodies that reacts best at room temperature

Specimen Collection

Platelet satellitosis

Specimen Collection

Solution

recollect specimen using Sodium Citrate in a 9:1 dilution

Correction for dilution

2.7 ml blood

0.3 ml anticoagulant

9/10 dilution is reciprocal 10/9 = 1.1

all computations for WBC and Platelet should be multiplied to 1.1

Peripheral Smear Preparation

Wedge technique

Coverslip technique

Automated Slide Making

and Staining


Wedge technique

Easiest to master

Most convenient and most commonly used technique

Material needed

Glass slide 3 in X 1in

Beveled/chamfered edges



Procedures:

Drop 2-3 mm blood at one end of the slide

Diff safe can be used

a. Easy dropping

b. Uniform drop


Precaution: Too large drop = too thick smear

Too small drop = too thin smear


The pusher slide be held securely with the dominant hand in a 30-45 deg angle.

- quick, swift and smooth gliding motion to the other side of the slide creating a wedge smear



Wedge Technique

Push Type wedge preparation

Pull Type wedge prepartion


Precautions:

Ensure that the whole drop of blood is picked up and spread

Too slow a slide push will accentuate poor leukocyte distribution, larger cells are pushed at the end of the slide

Maintain an even gentle pressure on the slide

Keep the same angle all the way to the end of the smear.


Precautions:

Angle correction:

1. In case of Polycythemia: high Hct angle should be lowered

- ensure that the smear made is not to thick

2. Too low Hct: Angle should be raised

Feature of a Well Made Wedge Smear

Smear is 2/3 or the entire slide

Smear is finger shaped, very slightly rounded at the feathery edge: widest area of examination

Lateral edges of the smear visible

Smear is smooth without irregularities, holes or streaks

When held up in light: feathery edge should show rainbow appearance

Entire whole drop of blood is picked up and spread


Cover Slip Technique

rarely used

used for Bone marrow aspirate smears

Advantage: excellent leukocyte distribution

Disadvantage: labeling, transport, staining and storage is a problem


22 x 27mm clean coverslip

More routinely used for bone marrow aspirate

Technique:

1. A drop of marrow aspirate is placed on top of 1 coverslip

2.Another coverslip is placed over the other allowing the aspirate to spread.

3. One is pulled over the other to create 1 thin smears


4. Mounted on a 3x1 inch glass slide

Precautions:

Very lgiht pressure should be applied between the index finger and the thumb

Crush preparation technique

Too much pressure causes rupture of the cells making morphologic examination impossible

Too little pressure prevents the bone spicules from spreading satisfactorily on the slide


Automatic Slide Making and Staining

SYSMEX 1000i


Drying of Smears

Fan

Heating pans

No breath blowing of smears may produce crenated RBCs or develop water artifact (drying artifact)

Staining of Peripheral Blood Smear

Wright Staing Method

Automated Slide Stainers

Quick Stains


Pure Wright stain or Wright Giemsa stain

Blood smears and bone marrow aspirate

Polychrome stains: Eosin and Methylene blue stains

Purpose: see and evaluate cell morphology


Eosin + Methylene Blue = thiazine eosinate complex

The complex will not stain any color unless a buffer is added: 0.05M sodium phosphate (pH 6.4) and aged distilled water (pH 6.4-6.8)

Methanol is added to fix the cells on the slide


Free Methylene Blue:

- basic

- stains acidic cellular components such as RNA

Free Eosin

- acidic

- stains basic cellular components such as Hgb and eosinophilic granules


Problem encountered during staining

Water artifact: moth eaten RBC, heavily demarcated central pallor on the RBC surface, crenation, refractory shiny blotches on the RBC


What contributes to the problem:

humidity in the air as you air dry the slides.

Water absorbed from the humid air into the alcohol based stain

Solution:

Drying the slide as quickly as possible.

Fix with pure anhydrous methanol before staining.

Use of 20% v/v methanol


AUTOMATED SLIDE STAINERS

It takes about 5-10 minutes to stain a batch of smears

Slides are just automatically dipped in the stain in the buffer and a series of rinses

Disadvantages:

Staining process has begun, no STAT slides can be added in the batch

Aqueous solutions of stains are stable only after 3-6 hours


HEMA-TEK STAINER


QUICK STAINS

Fast, convenient and takes about 1 minute to be accomplished

Modified Wrights-Giemsa Stain, buffer is aged distilled water

Cost effective

Disadvantage:

Quality of stains especially on color acceptance

For small laboratories and for physicians clinic only

Features of a well-stained PBS

Macroscopically: color should be pink to purple

Microscopically:

RCS: orange to salmon pink

WBC: nuclei is purple to blue

cytoplasm is pink to tan

granules is lilac to violet

Eosinophil: granules orange

Basophil: granules dark blue to black


Troubleshooting:

RBC gray, WBC too dark Eosinophil granules are gray

Cause: stain or buffer is to alkaline

inadequate rinsing

Prolonged staining

heparinized sample


Troubleshooting:

RBC too pale, WBC barely visible

Causes:Stain or buffer is too acidic

Underbuffering

Over rinsing

Peripheral Smear Examination

Macroscopic

Overall bluer color: increased blood proteins (multiple myeloma, rouleaux formation)

Grainy appearance: RBC agglutination (cold hemagglutinin diseases)

Holes: increased lipid

Blue specks at the feathery edge: Increased WBC and Platelet counts


Microscopic:

10x Objective

Assess overall quality of the smear i.e feathery edge, quality of the color, distributin of the cells and the lateral edges can be checked for WBC distribution

Snow-plow effect: more than 4x/cells per field on the feathery edge: Reject

Fibrin strands: Reject

Rouleaux formation, large blast cell assessment


Microscopic:

40x Objective

Correct area where to star counting is determined

WBC estimate: internal quality control


Microscopic:

100x Objective; OIO

Highest magnification

WBC differential counting


Optimal Assessment Area:

RBCs are uniformly and singly distributed

Few RBC are touching or overlapping

Normal biconcave appearance

200 to 250 RBC per 100x OIO


Too thin

Too thick

Performance of a White Blood Cell Differential Count

Systematic

Choose the best area for assesment

Back and forth serpentine or battlement track patters in preferred


100 WBCs are counted using a push down counters (Clay Admas Laboratory counters,Biovation diff counters

Accuracyof Diff Count:

Count 200 WBC if WBC>40 x 109/L

Extremely low WBC counts, do the Diff count under 50X OIO


Extremely low WBC counts, do the Diff count under 50X OIO

Extremely low WBCs: WBC are concentrated, buffy coat smears are made

RBC Morphology

Anisocytosis

Poikilocytosis

Cellular Inclusions

Platelet Estimate

Choose an area where RBC barely touch

No. of platelet in 10 OIO fields is counted multiplied by 20,000

Anemia or Erythrocytosis

Average No. of Plts/field x total RBC count

200 RBCs/field

(200 is the average number of RBC/field)

Summarizing WBC parameters

Total WBC counts per (WBC x 109/L)

WBC differential counts are percentages

WBC differential count values expressed as actual number of each type of cell

WBC morphology


STEP 1

WBC increased : leukocytosis

WBC decreases: leukopenia


STEP 2

Relative differential count

Cell TypeIncreasesDecreasesNeutrophilNeutrophiliaNeutropeniaEosinophilEosinophiliaN/A

BasophilBasophiliaN/ALymphocyteLymphocytosisLymphopeniaMonocyteMonocytosisMonocytopenia


STEP 3

Absolute Cell Counts

Ex. WBC 13.6

PMNs 67

Lym 26

Eos 3

Baso 3

Mono 1

Absolute Neutrophil Count: 9.1 (NV: 2.4-8.2)

Absolute Lymphocyte Cout: 3.5 ((NV: 1.4-4.0)

Absolute Monocyte Count: 0.4 (NV: 0.1-1.2)


STEP 4

Examination for immature cells

Young cells should not be seen in the peripheral blood smear

Immature cells: possess a nucleus

do not lyse during testing

can be counted as WBC and falsely elevate WBC results

LEFT SHIFT

Summarizing RBC Parameters

RBC Count )RBC x 1012/L)

Hb (g/dl)

Hct (5 or L/L)

Mean Cell Volume (MCV. Fl)

Mean Cell Hb (MCH, pg)

Mean Cell Hb Concentration (MCHC. %, g/dl)

RBC distribution

Morphology


Step1

Examne Hb an Hct for anemia or polycythemia

If the RBC morphology is normal: Use rule of three to estimate the Hct

Step 2

MCV: to check and correlate to the morpholic apperance of the cells


Step 3

Examine MCHC

Describes how well the cells are filled with Hb

Hypochromic, normochromic

2 conditions when MCHC should be evaluated:

1. spherocytosis: slight elevation

2. lipemia/icterus: markedly increase


Step 4

Examine MCHC

Describes how well the cells are filled with Hb

Hypochromic, normochromic

2 conditions when MCHC should be evaluated:

1. spherocytosis: slight elevation

2. lipemia/icterus: markedly increase


Step 5

Morphology

Size

Shape

Inclusions

Young rbcs

Color

Arrangement

Summarizing Platelet Parameters

Platelet count (x 109/L)

Mean Platelet Volume MPV, fl

Morphology

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