hapusan darah

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

Advantages

1. Many smears can be done in just a single draw

2. Immediate preparation of the smear is not necessary

3. 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

Ergo, all computations for WBC and Platelet should be multiplied to 1.1

Specimen Collection

Peripheral Smear Preparation

Wedge technique

Coverslip technique

Automated Slide Making

and Staining


Wedge technique

1. Easiest to master

2. Most convenient and most commonly used technique

Material needed

1. Glass slide 3 in X 1in

2. Beveled/chamfered edges

Procedures:

1. 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


2. 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

1. Push Type wedge preparation

2. 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 ¾ the entire slideSmear is finger shaped, very slightly rounded at the feathery edge: widest area of examinationLateral edges of the smear visibleSmear is smooth without irregularities, holes or streaksWhen held up in light: feathery edge should show rainbow appearanceEntire 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 coverslipMore routinely used for bone marrow aspirateTechnique: 1. A drop of marrow aspirate is placed on top of 1 coverslip2. 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 slidePrecautions:

Very lgiht pressure should be applied between the index finger and the thumbCrush preparation techniqueToo much pressure causes rupture of the cells making morphologic examination impossibleToo 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)


Wright Staing Method

Automated Slide Stainers

Quick Stains

Staining of Peripheral Blood Smear

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 RNAFree 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:1. humidity in the air as you air dry the slides.2. Water absorbed from the humid air into the

alcohol based stainSolution:

1. Drying the slide as quickly as possible.2. Fix with pure anhydrous methanol before

staining.3. Use of 20% v/v methanol


AUTOMATED SLIDE STAINERS1. It takes about 5-10 minutes to stain a batch of

smears2. Slides are just automatically dipped in the

stain in the buffer and a series of rinsesDisadvantages:

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

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



HEMA-TEK STAINER

QUICK STAINSFast, convenient and takes about 1 minute to be accomplishedModified Wrights-Giemsa Stain, buffer is aged distilled waterCost effectiveDisadvantage:

Quality of stains especially on color acceptanceFor small laboratories and for physician’s 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:

1. RBC gray, WBC too dark Eosinophil granules are gray

Cause: stain or buffer is to alkaline

inadequate rinsing

Prolonged staining

heparinized sample


Troubleshooting:

2. RBC too pale, WBC barely visible

Causes:Stain or buffer is too acidic

Underbuffering

Over rinsing

Peripheral Smear Examination

Macroscopic

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

2. Grainy appearance: RBC agglutination (cold hemagglutinin diseases)

3. Holes: increased lipid

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

Peripheral Smear ExaminationMicroscopic:10x Objective

1. 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

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

3. Fibrin strands: Reject4. Rouleaux formation, large blast cell

assessment


Microscopic:

40x Objective1. Correct area where to star counting is

determined

2. WBC estimate: internal quality control

Peripheral Smear ExaminationMicroscopic:

100x Objective; OIO1. Highest magnification

2. WBC differential counting

Peripheral Smear ExaminationOptimal Assessment Area:

1. RBCs are uniformly and singly distributed

2. Few RBC are touching or overlapping

3. Normal biconcave appearance

4. 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

1. Total WBC counts per (WBC x 109/L)

2. WBC differential counts are percentages

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

4. WBC morphology


STEP 1

WBC increased : leukocytosis

WBC decreases: leukopenia


STEP 2

Relative differential count

Cell Type Increases Decreases

Neutrophil Neutrophilia Neutropenia

Eosinophil Eosinophilia N/A

Basophil Basophilia N/A

Lymphocyte Lymphocytosis

Lymphopenia

Monocyte Monocytosis Monocytopenia

Summarizing WBC parametersSTEP 3Absolute Cell CountsEx. WBC 13.6

PMNs 67Lym 26Eos 3Baso 3Mono 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)

Summarizing WBC parametersSTEP 4Examination for immature cellsYoung cells should not be seen in the peripheral blood smearImmature 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 distributionMorphology

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 3Examine MCHCDescribes how well the cells are filled with HbHypochromic, normochromic2 conditions when MCHC should be evaluated:1. spherocytosis: slight elevation2. 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

1. Size

2. Shape

3. Inclusions

4. Young rbcs

5. Color

6. Arrangement


Summarizing Platelet Parameters

Platelet count (x 109/L)

Mean Platelet Volume MPV, fl

Morphology

hapusan darah

Documents

peripheral

peripheral

peripheral

peripheral

peripheral

peripheral

diff count

wbc morphology