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 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

Peripheral Smear Preparation

Wedge technique Coverslip technique Automated Slide Making and Staining

Peripheral Smear Preparation

1. 2.
Wedge technique Easiest to master Most convenient and most commonly used technique Material needed Glass slide 3 in X 1in Beveled/chamfered edges

1. 2.

Peripheral Smear Preparation

Peripheral Smear Preparation

Procedures: 1. Drop 2-3 mm blood at one end of the slide Diff safe can be used a. Easy dropping b. Uniform drop

Peripheral Smear Preparation

Precaution: Too large drop = too thick smear Too small drop = too thin smear

Peripheral Smear Preparation

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

Peripheral Smear Preparation

Peripheral Smear Preparation

Wedge Technique 1. Push Type wedge preparation 2. Pull Type wedge prepartion

Peripheral Smear Preparation

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.

Peripheral Smear Preparation

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

Peripheral Smear Preparation

Cover Slip Technique rarely used used for Bone marrow aspirate smears Advantage: excellent leukocyte distribution Disadvantage: labeling, transport, staining and storage is a problem

Peripheral Smear Preparation

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

Peripheral Smear Preparation

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

Peripheral Smear Preparation

Automatic Slide Making and Staining SYSMEX 1000i

Peripheral Smear Preparation

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

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

Staining of Peripheral Blood Smear

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

Staining of Peripheral Blood Smear

Free Methylene Blue: - basic - stains acidic cellular components such as RNA Free Eosin - acidic - stains basic cellular components such as Hgb and eosinophilic granules

Staining of Peripheral Blood Smear

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

Staining of Peripheral Blood Smear

1. 2.
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

1. 2. 3.

Staining of Peripheral Blood Smear

1.
2. 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

1. 2.

Staining of Peripheral Blood Smear

HEMA-TEK STAINER

Staining of Peripheral Blood Smear

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

Features of a well-stained PBS

Troubleshooting: 1. RBC gray, WBC too dark Eosinophil granules are gray Cause: stain or buffer is to alkaline inadequate rinsing Prolonged staining heparinized sample

Features of a well-stained PBS

Troubleshooting: 2. RBC too pale, WBC barely visible Causes: Stain or buffer is too acidic Underbuffering Over rinsing

Peripheral Smear Examination

1.
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

2.
3. 4.

Peripheral Smear Examination

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 2. Snow-plow effect: more than 4x/cells per field on the feathery edge: Reject 3. Fibrin strands: Reject 4. Rouleaux formation, large blast cell assessment
1.

Peripheral Smear Examination

Microscopic: 40x Objective
Correct area where to star counting is determined 2. WBC estimate: internal quality control
1.

Peripheral Smear Examination

Microscopic: 100x Objective; OIO
Highest magnification 2. WBC differential counting
1.

Peripheral Smear Examination

1. 2. 3. 4. 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

Peripheral Smear Examination

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

Performance of a White Blood Cell Differential Count

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

Performance of a White Blood Cell Differential Count

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

Summarizing WBC parameters

STEP 1 WBC increased : leukocytosis WBC decreases: leukopenia

Summarizing WBC parameters

STEP 2 Relative differenti al count Cell Type Increases Decreases

Neutrophil
Eosinophil Basophil

Neutrophilia Neutropenia
Eosinophilia N/A Basophilia N/A

Lymphocyte Lymphocyto Lymphopeni sis a Monocyte Monocytosis Monocytope nia

Summarizing WBC parameters

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)

Summarizing WBC parameters

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

Summarizing RBC Parameters

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

Summarizing RBC Parameters

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

Summarizing RBC Parameters

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

Summarizing RBC Parameters

Step 5 Morphology Size Shape Inclusions Young rbcs Color Arrangement

1. 2. 3. 4. 5. 6.

Summarizing Platelet Parameters

Platelet count (x 109/L) Mean Platelet Volume MPV, fl Morphology