BLOOD SMEAR EXAMINATION Making Blood smear

PREPARATION OF BLOOD SMEAR

There are three types of blood There are three types of blood smears: smears:

1.1. The cover glass smear.The cover glass smear.

2.2. The wedge smear .The wedge smear .

3.3. The spun smear.The spun smear.

The are two additional types of blood The are two additional types of blood smear used for specific purposessmear used for specific purposes

1.1. Buffy coat smear for WBCs < 1.0×10Buffy coat smear for WBCs < 1.0×1099/L /L

2.2. Thick blood smears for blood Thick blood smears for blood parasites .parasites .

WEDGE BLOOD SMEAR

Specimen Specimen : EDTA blood within 2 to 3 : EDTA blood within 2 to 3 hours & collected to the mark on hours & collected to the mark on tube.tube.

Not's : Not's : May change RBCs morphology May change RBCs morphology such as Spiculated (crenated) cells if :such as Spiculated (crenated) cells if :

1.1. Excessive amount of anticoagulant to Excessive amount of anticoagulant to specimenspecimen

2.2. Old blood - long standing. Old blood - long standing.

3.3. Warm environment (room Warm environment (room temperature) may hasten changes.temperature) may hasten changes.

PROCEDUREPROCEDURE

placing a drop of blood placing a drop of blood from mixed from mixed sample sample on a clean glass slide.on a clean glass slide.

Spreader slide using another clean Spreader slide using another clean glass slide at 30-40 degree angle.glass slide at 30-40 degree angle.

Control thickness of the smear by Control thickness of the smear by changing the angle of spreader slidechanging the angle of spreader slide

Allow the blood film to air-dry Allow the blood film to air-dry completely before staining. (Do not completely before staining. (Do not blow to dry. The moisture from your blow to dry. The moisture from your breath will cause RBC artifact.)breath will cause RBC artifact.)

large angle

low HCT

small angle

high HCT

CHARACTERISTICS OF A GOOD SMEAR

1.1. Thick at one end, thinning out to a Thick at one end, thinning out to a smooth rounded feather edge.smooth rounded feather edge.

2.2. Should occupy 2/3 of the total slide Should occupy 2/3 of the total slide area.area.

3.3. Should not touch any edge of the slide.Should not touch any edge of the slide.

4.4. Should be margin free, except for point Should be margin free, except for point of application.of application.

tail body head

MORPHOLOGIC CHANGES DUE TO AREA OF SMEAR

Thin areaThin area- Spherocytes which - Spherocytes which are really "spheroidocytes" or are really "spheroidocytes" or flattened red cells. True flattened red cells. True spherocytes will be found in spherocytes will be found in other (Good) areas of smear.other (Good) areas of smear.

Thick area Thick area - Rouleaux, which is - Rouleaux, which is normal in such areas. Confirm by normal in such areas. Confirm by examining thin areas. If true examining thin areas. If true rouleaux, two-three RBC's will rouleaux, two-three RBC's will stick together in a "stack of stick together in a "stack of coins" fashion.. coins" fashion..

COMMON CAUSES OF A POOR BLOOD SMEAR

1.1. Drop of blood too large or too small.Drop of blood too large or too small.

2.2. Spreader slide pushed across the slide in a jerky manner.Spreader slide pushed across the slide in a jerky manner.

3.3. Failure to keep the entire edge of the spreader slide Failure to keep the entire edge of the spreader slide against the slide while making the smear.against the slide while making the smear.

4.4. Failure to keep the spreader slide at a 30° angle with the Failure to keep the spreader slide at a 30° angle with the slide.slide.

5.5. Failure to push the spreader slide completely across the Failure to push the spreader slide completely across the slide.slide.

6.6. Irregular spread with ridges and long tail: Edge of Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slidespreader dirty or chipped; dusty slide

7.7. Holes in film: Slide contaminated with fat or greaseHoles in film: Slide contaminated with fat or grease

8.8. Cellular degenerative changes: delay in fixing, Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated with inadequate fixing time or methanol contaminated with water.water.

BIOLOGIC CAUSES OF A POOR SMEAR

1.1. Cold agglutinin Cold agglutinin - RBCs will clump - RBCs will clump together. Warm the blood at 37° together. Warm the blood at 37° C for 5 minutes, and then C for 5 minutes, and then remake the smear.remake the smear.

2.2. Lipemia Lipemia - holes will appear in the - holes will appear in the smear. There is nothing you can smear. There is nothing you can do to correct this.do to correct this.

3.3. Rouleaux Rouleaux - RBC’s will form into - RBC’s will form into stacks resembling coins. There stacks resembling coins. There is nothing you can do to correct is nothing you can do to correct thisthis

SLIDE FIXATION & SLIDE FIXATION & STAININGSTAININGLEISHMAN'S STAINLEISHMAN'S STAIN

PRINCIPLE LIKE ROMANOWSKY PRINCIPLE LIKE ROMANOWSKY PRINCIPLE PRINCIPLE

Leishman's stain : a polychromatic stainLeishman's stain : a polychromatic stain

Methanol : fixes cells to slideMethanol : fixes cells to slide

methylene blue stains RNA,DNAmethylene blue stains RNA,DNA

=== blue-grey color === blue-grey color

Eosin stains hemoglobin, eosin Eosin stains hemoglobin, eosin granulesgranules

====== orange-red color orange-red color

pH value of phosphate buffer is very pH value of phosphate buffer is very importantimportant

STAINING PROCEDURE STAINING PROCEDURE

Thin smear are air dried.Thin smear are air dried.

Flood the smear with stain. Flood the smear with stain.

Stain for 1-5 min. Experience will indicate Stain for 1-5 min. Experience will indicate the optimum time. the optimum time.

Add an equal amount of buffer solution Add an equal amount of buffer solution and mix the stain by blowing an eddy in and mix the stain by blowing an eddy in the fluid.the fluid.

Leave the mixture on the slide for 10-15 Leave the mixture on the slide for 10-15 min. min.

Wash off by running water directly to the Wash off by running water directly to the centre of the slide to prevent a residue of centre of the slide to prevent a residue of precipitated stain.precipitated stain.

Stand slide on end, and let dry in air.Stand slide on end, and let dry in air.

TOO ACIDIC SUITABLE TOO TOO ACIDIC SUITABLE TOO BASICBASIC

CAUSES & CORRECTION

Too Acid Stain:Too Acid Stain:

1.1. insufficient staining timeinsufficient staining time

2.2. prolonged buffering or washingprolonged buffering or washing

3.3. old stainold stain

Correction:Correction:

1)1) lengthen staining timelengthen staining time

2)2) check stain and buffer pHcheck stain and buffer pH

3)3) shorten buffering or wash timeshorten buffering or wash time

Too Alkaline Stain:Too Alkaline Stain:

1.1. thick blood smearthick blood smear

2.2. prolonged stainingprolonged staining

3.3. insufficient washinginsufficient washing

4.4. alkaline pH of stain componentsalkaline pH of stain components   Correction :Correction :

1)1) check pHcheck pH

2)2) shorten stain timeshorten stain time

3)3) prolong buffering timeprolong buffering time

PERFORMING A MANUAL PERFORMING A MANUAL DIFFERENTIAL AND DIFFERENTIAL AND

ASSESSING RBC ASSESSING RBC MORPHOLOGY MORPHOLOGY

PRINCIPLE PRINCIPLE

White Blood Cells.White Blood Cells.

1.1. Check for even distribution and Check for even distribution and estimate the number present estimate the number present (also, look for any gross (also, look for any gross abnormalities present on the abnormalities present on the smear).smear).

2.2. Perform the differential count. Perform the differential count.

3.3. Examine for morphologic Examine for morphologic abnormalities. abnormalities.

PRINCIPLE PRINCIPLE

Red Blood CellsRed Blood Cells, Examine for, Examine for: :

1.1. Size and shape. Size and shape.

2.2. Relative hemoglobin content. Relative hemoglobin content.

3.3. Polychromatophilia. Polychromatophilia.

4.4. Inclusions. Inclusions.

5.5.   Rouleaux formation or Rouleaux formation or agglutinationagglutination

PRINCIPLE

Platelets. Platelets.

1.1. Estimate number present. Estimate number present.

2.2.   Examine for morphologic Examine for morphologic abnormalities. abnormalities.

PROCEDURES

Observations Under ×10Observations Under ×10

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

2.2. Check the WBC distribution over Check the WBC distribution over the smear.the smear.

3.3. Check that the slide is properly Check that the slide is properly stained.stained.

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

Observing Observing direction:direction:

Observe one field and record the number of WBC Observe one field and record the number of WBC according to the different type then turn to another according to the different type then turn to another field in the snake-liked directionfield in the snake-liked direction**avoid repeat or miss some cellsavoid repeat or miss some cells

OBSERVATIONS UNDER× 40X : WBC OBSERVATIONS UNDER× 40X : WBC ESTIMATESESTIMATES

Using the × 40 high dry with no oil.Using the × 40 high dry with no oil.

Choose a portion of the peripheral Choose a portion of the peripheral smear where there is only slight smear where there is only slight overlapping of the RBCs. overlapping of the RBCs.

Count 10 fields, take the total Count 10 fields, take the total number of white cells and divide by number of white cells and divide by 10.10.

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

OBSERVATIONS UNDER × 100: OBSERVATIONS UNDER × 100: PLATELET ESTIMATESPLATELET ESTIMATES

1.1. Use the oil immersion lens estimate Use the oil immersion lens estimate the number of platelets per field.the number of platelets per field.

2.2. Look at 5-6 fields and take an average.Look at 5-6 fields and take an average.

3.3. Multiply the average by 20,000.Multiply the average by 20,000.

4.4. Note any macroplatelets.Note any macroplatelets.

Platelets per oil immersion field (OIF)Platelets per oil immersion field (OIF)

1)1) <8 <8 platelets/OIF = platelets/OIF = decreaseddecreased

2)2) 8 to 20 8 to 20 platelets/OIF = platelets/OIF = adequateadequate

3)3) >20 >20 platelets/OIF = platelets/OIF = increasedincreased

EVALUATE WBC MORPHOLOGY

Note if any abnormal white cell morphology is present

o Hypersegmented poly's (5 or more lobes)

o Vacuolation of neutrophils

o Toxic granulation of neutrophils

o Dohle bodies

o Atypical Lymphocytes

o Smudge cells

MANUAL DIFFERENTIAL COUNTSMANUAL DIFFERENTIAL COUNTS

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

This takes place under × 100 (oil) This takes place under × 100 (oil) using the zigzag method.using the zigzag method.

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

Reporting resultsReporting results

Absolute number of cells/µl = % of cell Absolute number of cells/µl = % of cell type in differential x white cell type in differential x white cell countcount

OBSERVING AND RECORDING NUCLEATED RED BLOOD CELLS (NRBCS)

If 10 or more nucleated RBC's (NRBC) If 10 or more nucleated RBC's (NRBC) are seen, correct theare seen, correct the

White Count using this formula:White Count using this formula:

Corrected WBC Count = Corrected WBC Count =

WBC x 100/( NRBC + 100)WBC x 100/( NRBC + 100)

Example Example : : If WBC = 5000 and 10 If WBC = 5000 and 10 NRBCs have been countedNRBCs have been counted

Then Then 5,000× 100/110 = 4545.505,000× 100/110 = 4545.50

The corrected white count is The corrected white count is 4545.50.4545.50.

TIPS ON DIFF'STIPS ON DIFF'S

Do not count cells that are Do not count cells that are disintegratingdisintegrating

• smudge cellssmudge cells

• eosinophil with no cytoplasmic eosinophil with no cytoplasmic membrane and with scattered granulesmembrane and with scattered granules

• Pyknotic cell (nucleus extremely Pyknotic cell (nucleus extremely condensed and degenerated, lobes condensed and degenerated, lobes condensed into small, round clumps condensed into small, round clumps with no filaments interconnecting).with no filaments interconnecting).

• Basket cellsBasket cells

ABNORMAL DIFFERENTIALS

1. 200 Cell diff:

a. WBC > 15.0 (>20.0 for babies under 1 month and labor unit)

b. Three or more basophils seen.

2. If more than five immature WBC's are seen (or any blasts) let someone else diff slide and average results.

3. Correct WBC for NRBC's if you seen ten or more NRBCs/100 WBC.

4. Always indicate number of cells counted on diff. 

5. If any cell type is extremely elevated (such as bands, monos, or eos > 20) indicate that you are aware of the abnormality by circling or checking on the card next to the results.

RECORDING RBC MORPHOLOGY1.1. Scan area using ×100 (oil immersion).Scan area using ×100 (oil immersion).

2.2. Observe 10 fields.Observe 10 fields.

3.3. Red cells are observed for Red cells are observed for sizesize, , shapeshape, , hemoglobin hemoglobin contentcontent, and the presence or absence of , and the presence or absence of inclusions. inclusions.

4.4. Abnormal morphology: Red cell morphology is assessed Abnormal morphology: Red cell morphology is assessed according to See the following sample grading system. Note according to See the following sample grading system. Note that red cell morphology must be scanned in a good counting that red cell morphology must be scanned in a good counting area.area.

Two questions should be askedTwo questions should be asked

1.1. Is the morphology seen in every field? Is the morphology seen in every field?

2.2. Is the morphology pathologic and not artificially induced? Is the morphology pathologic and not artificially induced?

Table 1 & 2 represents a system derived to determine a Table 1 & 2 represents a system derived to determine a

quantitative scalequantitative scale..

RED BLOOD CELL MORPHOLOGY

A normal red blood cell should be approximately the same size as a normal lymphocyte nucleus or 2 normal sized red blood cells should fit side by side across a normal sized poly (not a hypersegmented poly).

NO. of Field/ Oil imm.

Grade Degree of abnormality

1-6 per oil imm. field 1+

7-10 per OIF2+

11-20 per OIF3+

> 20 per OIF4+

REPORTING RESULTS

Where possible use macrocytic and Where possible use macrocytic and microcytic, rather than simply anisocytosis microcytic, rather than simply anisocytosis alone, when describing red cell morphology.alone, when describing red cell morphology.

  Use specific cell morphology when possible, Use specific cell morphology when possible, rather than simply reporting poikilocytosis.rather than simply reporting poikilocytosis.

  When red cells are normocytic, When red cells are normocytic, normochromic, report out as NORMAL. When normochromic, report out as NORMAL. When abnormal morphology has been noted, DO abnormal morphology has been noted, DO NOT indicate normal on the report form.NOT indicate normal on the report form.

EXAMPLE: 7-10 microcytic RBC's/OIF is EXAMPLE: 7-10 microcytic RBC's/OIF is reported out as: 2+ microcytosis or reported out as: 2+ microcytosis or Moderate microcytosis.Moderate microcytosis.

DETERMINE A QUANTITATIVE DETERMINE A QUANTITATIVE SCALESCALE

11

GRADING INCLUSIONS

22

MORPHOLOGY OF MORPHOLOGY OF WBCWBC IN PERIPHERAL IN PERIPHERAL BLOODBLOOD Normal Normal

NORMAL PERIPHERAL BLOOD NORMAL PERIPHERAL BLOOD SMEARSMEAR

STAB NEUTROPHIL

Diameter:12-16

Cytoplasm : pink

Granules: primary

secondary

Nucleus: dark purple blue

dense chromatin

BAND NEUTROPHIL

SEGMENTED NEUTROPHIL

Diameter: 12-16

Cytoplasm : pink

Granules: primary

secondary

Nucleus: dark purple blue

dense chromatin

2-5 lobes

SEGMENTED NEUTROPHIL

EOSINOPHIL

Diameter: 14-16Diameter: 14-16

Cytoplasm : full of granulesCytoplasm : full of granules

Granules: large refractile, orange-Granules: large refractile, orange-red red

Nucleus: blueNucleus: blue

dense chromatindense chromatin

2 lobes like a pair of 2 lobes like a pair of glassglass

EOSINOPHIL

BASOPHIL

Diameter: 14-16Diameter: 14-16

Cytoplasm : pinkCytoplasm : pink

Granules: dark blue –black Granules: dark blue –black obscure nucleus obscure nucleus

Nucleus: blueNucleus: blue

BASOPHIL

LYMPHOCYTE

DiameterDiameter: small 7-9: small 7-9

large 12-16large 12-16 CytoplasmCytoplasm: medium blue: medium blue Granules: Granules: small agranularsmall agranular

large a few large a few primary primary

granulesgranules Nucleus: Nucleus: dark blue \rounddark blue \round

dense dense chromatinchromatin

LYMPHOCYTE

MONOCYTE

DiameterDiameter: 14-20: 14-20

CytoplasmCytoplasm : grey blue : grey blue

Granules: Granules: dust-like dust-like lilac color granuleslilac color granules

Nucleus: Nucleus: blue blue

large irregularly large irregularly shaped and foldedshaped and folded

MONOCYTE

ABNORMAL CHANGES OF WBC MORPHOLOGY

LEFT-SHIFT AND RIGHT-SHIFT OF NEUTROPHIL:

Left-shiftLeft-shift: non-segmented : non-segmented neutrophil neutrophil > 5%> 5%

Right-shiftRight-shift: : hypersegmented neutrophil hypersegmented neutrophil >3%>3%

TOXIC GRANULATION

AUER BODIES(AUER ROD)

HYPERSEGMENTATION

Anisocytosis of neutrophil

vacuolization

Degeneration of nucleus

Dohle body