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Peripheral blood Smear Preparation

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This document provides information about preparing and examining peripheral blood smears. It discusses how to make a good blood smear by ensuring the smear is evenly spread and covers most of the slide. The document also describes staining blood smears using the Leishman's stain method and examining smears under a microscope. Key things to observe during examination include the different types of white blood cells, red blood cell morphology, and any abnormal findings. Performing a manual differential count involves identifying 100 white blood cells and categorizing them by type.

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BLOOD SMEAR EXAMINATION Making Blood smear Raihan Mannan JR-1, Deptt. of Physiology JNMCH, AMU, Aligarh
A WELL MADE AND WELL STAINED SMEAR CAN PROVIDE:  Estimates of cell count  Proportions of the different types of WBC  Morphology  Objective: 1. Specimen Collection 2. Peripheral Smear Preparation 3. Staining of Peripheral Blood Smear 4. Peripheral Smear Examination
PREPARATION OF BLOOD SMEAR  There are three types of blood smears: 1. The wedge smear. 2. The cover glass smear. 3. The spun smear.  The are two additional types of blood smear used for specific purposes 1. Buffy coat smear for WBCs < 1.0×109 /L 2. Thick blood smears for blood parasites .
WEDGE BLOOD SMEAR  Specimen : venipuncture, EDTA blood within 2 to 3 hours & collected to the mark on tube or by pricking finger.  May change in RBCs morphology such as Spiculated (crenated) cells if : 1. Excessive amount of anticoagulant to specimen 2. Old blood - long standing. 3. Warm environment (room temperature) may hasten changes.
PROCEDUREPROCEDUREEE  Prick the finger or placing a drop of blood from mixed sample on a clean glass slide.  Place the Spreader slide on the surface of slide just infront of the drop of blood at an angle of 45 degree.  Draw the spreader backward so as to touch the drop and hold there tilll the blood runs along the full width of spreader  Spreader is then moved slowly and smoothly to the other end of slide maintaining the angle of 45°  Allow the blood film to air-dry completely before staining. (Do not blow to dry. The moisture from your breath will cause RBC artifact.)
Peripheral blood Smear Preparation
Peripheral blood Smear Preparation
Peripheral blood Smear Preparation
CHARACTERISTICS OF A GOOD SMEAR  Smear should cover 2/3 of the entire slide  Smear is tongue shaped with no tails at the end.  Neither too thin nor too thick (uniform)  Smear is smooth without irregularities, holes or striations or air-gap  When held up in light: feathery edge should show rainbow appearance
tail body head
MORPHOLOGIC CHANGES DUE TO AREA OF SMEAR  Thin area- Spherocytes which are really "spheroidocytes" or flattened red cells. True spherocytes will be found in other (Good) areas of smear.  Thick area - Rouleaux, which is normal in such areas. Confirm by examining thin areas. If true rouleaux, two-three RBC's will stick together in a "stack of coins" fashion..
COMMON CAUSES OF A POOR BLOOD SMEAR 1. 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.
PERIPHERAL BLOOD SMEAR  Examples of unacceptable smears
PERIPHERAL BLOOD SMEAR Examples of unacceptable smears
SLIDE FIXATION &SLIDE FIXATION & STAININGSTAINING LEISHMAN'S STAINLEISHMAN'S STAIN
ROMANOWSKY PRINCIPLEROMANOWSKY PRINCIPLE Leishman's stain : a polychromatic stain  Acetone free methyl alcohol : as a fixative  fixes cells to slide (precipitation of protein)  Preseves the cells  Methylene blue: basic dye and stains cytoplasm, nuclei of WBCs and granules of basophils === blue color  Eosin: acidic dye and stains RBCs and granules of eosinophils === orange-red color  pH value of phosphate buffer is very important
FIXING AND STAINING PROCEDUREFIXING AND STAINING PROCEDURE  Fixing the blood smear:  Place the smear across two parallel glass rods.  Pour 8-10 drops of leishman’s stain. Leave it for about 2 min (fixation time)  Staining the smear:  After 2 min add an equal amount of buffer solution and mix the stain by blowing air intermittently with the help of dropper.  Leave the mixture on the slide for 10-15 min.  Wash the slide by running water, hold the slide slanted and water is allowed to flow on thumb until film gets a pinkish tinge.  Wipe clean the back of slide  Stand slide upright and let dry in air.
QUALITY OF STAINED SMEARQUALITY OF STAINED SMEAR  Smear is single-cell thick, no overlapping of cells and uniformly distributed  Atleast 1 WBC per high power field (100x)  RBCs are stained light pink  Overstained smear:RBCs look bluish & WBCs look purple  Understained smear: RBCs look very pale & WBCs almost colourless
CAUSES & CORRECTION  Too Acid Stain: 1. insufficient staining time 2. prolonged buffering or washing 3. old stain  Correction: 1) lengthen staining time 2) check stain and buffer pH 3) shorten buffering or wash time
Too Alkaline Stain: 1. thick blood smear 2. prolonged staining 3. insufficient washing 4. alkaline pH of stain components  Correction : 1)check pH 2)shorten stain time 3)prolong buffering time
TOO ACIDIC SUITABLE TOO BASICTOO ACIDIC SUITABLE TOO BASIC
PRECAUTIONS:  Slides should be clean & grease free.  Spreader should have a smooth clean edge.  Do not heat dry the smear.  Stored blood should not be used for making smear.  Assess the quality of smear both grossly & microscopically.
PERFORMING APERFORMING A MANUALMANUAL DIFFERENTIAL ANDDIFFERENTIAL AND ASSESSING RBCASSESSING RBC MORPHOLOGYMORPHOLOGY
ObservingObserving direction:direction: Observe one field and record the number of WBC according to the different type then turn to another field in the snake-liked direction *avoid repeat or miss some cells
MANUAL DIFFERENTIAL COUNTSMANUAL DIFFERENTIAL COUNTS  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 zigzag method.  Count 100 WBCs including all cell lines from immature to mature.  Reporting results Absolute number of cells/µl = % of cell type in differential x white cell count
N M N N N L N L N N: NEUTROPHIL L: LYMPHOCYTE M: MONOCYTE E: EOSINOPHIL B: BASOPHIL
MORPHOLOGYMORPHOLOGY OF WBCOF WBC IN PERIPHERALIN PERIPHERAL BLOODBLOOD NormalNormal
NORMAL PERIPHERAL BLOODNORMAL PERIPHERAL BLOOD SMEARSMEAR
Peripheral blood Smear Preparation
NEUTROPHIL
NEUTROPHIL
Peripheral blood Smear Preparation
EOSINOPHIL
EOSINOPHIL
Peripheral blood Smear Preparation
BASOPHIL
BASOPHIL
LYMPHOCYTE
LYMPHOCYTE Ly
Peripheral blood Smear Preparation
MONOCYTE
MONOCYTE
Peripheral blood Smear Preparation
Peripheral blood Smear Preparation
ABNORMAL CHANGES OF WBC MORPHOLOGY
ARNETH COUNT:  Left to shift or regenerative shiftLeft to shift or regenerative shift::  If N1+N2+N3 exceeds 80%If N1+N2+N3 exceeds 80%  Indicates hyperactive bone marrowIndicates hyperactive bone marrow  Causes:Causes: 1. Acute pyogenic infections. 2. Tuberculosis: Though there is lymphocytosis, a shift to the left may be due to removal of older neutrophils from the blood. 3. Hemorrhage. 4. Low-dosage irradiation is said to stimulate bone marrow while heavy doses cause a shift to the right.
 Right to shift or degenerative shift:Right to shift or degenerative shift:  If N4+N5+N6 exceeds 20%If N4+N5+N6 exceeds 20%  Indicates hypoactive bone marrowIndicates hypoactive bone marrow  Causes:Causes: 1. Bone marrow depression (hypoplasia and aplasia) due to any factor. 2. Drugs, toxins, chemical poisons. 3. Megaloblastic anemia. 4. Septicemia. 5. Uremia.
LEFT SHIFT N1 N2 N3 N4 N5&6
TOXIC GRANULATION
AUER BODIES(AUER ROD)
HYPERSEGMENTATION
Anisocytosis of neutrophil
vacuolization
Degeneration of nucleus
Dohle body

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Peripheral blood Smear Preparation

  • 1. BLOOD SMEAR EXAMINATION Making Blood smear Raihan Mannan JR-1, Deptt. of Physiology JNMCH, AMU, Aligarh
  • 2. A WELL MADE AND WELL STAINED SMEAR CAN PROVIDE:  Estimates of cell count  Proportions of the different types of WBC  Morphology  Objective: 1. Specimen Collection 2. Peripheral Smear Preparation 3. Staining of Peripheral Blood Smear 4. Peripheral Smear Examination
  • 3. PREPARATION OF BLOOD SMEAR  There are three types of blood smears: 1. The wedge smear. 2. The cover glass smear. 3. The spun smear.  The are two additional types of blood smear used for specific purposes 1. Buffy coat smear for WBCs < 1.0×109 /L 2. Thick blood smears for blood parasites .
  • 4. WEDGE BLOOD SMEAR  Specimen : venipuncture, EDTA blood within 2 to 3 hours & collected to the mark on tube or by pricking finger.  May change in RBCs morphology such as Spiculated (crenated) cells if : 1. Excessive amount of anticoagulant to specimen 2. Old blood - long standing. 3. Warm environment (room temperature) may hasten changes.
  • 5. PROCEDUREPROCEDUREEE  Prick the finger or placing a drop of blood from mixed sample on a clean glass slide.  Place the Spreader slide on the surface of slide just infront of the drop of blood at an angle of 45 degree.  Draw the spreader backward so as to touch the drop and hold there tilll the blood runs along the full width of spreader  Spreader is then moved slowly and smoothly to the other end of slide maintaining the angle of 45°  Allow the blood film to air-dry completely before staining. (Do not blow to dry. The moisture from your breath will cause RBC artifact.)
  • 9. CHARACTERISTICS OF A GOOD SMEAR  Smear should cover 2/3 of the entire slide  Smear is tongue shaped with no tails at the end.  Neither too thin nor too thick (uniform)  Smear is smooth without irregularities, holes or striations or air-gap  When held up in light: feathery edge should show rainbow appearance
  • 11. MORPHOLOGIC CHANGES DUE TO AREA OF SMEAR  Thin area- Spherocytes which are really "spheroidocytes" or flattened red cells. True spherocytes will be found in other (Good) areas of smear.  Thick area - Rouleaux, which is normal in such areas. Confirm by examining thin areas. If true rouleaux, two-three RBC's will stick together in a "stack of coins" fashion..
  • 12. COMMON CAUSES OF A POOR BLOOD SMEAR 1. 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.
  • 13. PERIPHERAL BLOOD SMEAR  Examples of unacceptable smears
  • 14. PERIPHERAL BLOOD SMEAR Examples of unacceptable smears
  • 15. SLIDE FIXATION &SLIDE FIXATION & STAININGSTAINING LEISHMAN'S STAINLEISHMAN'S STAIN
  • 16. ROMANOWSKY PRINCIPLEROMANOWSKY PRINCIPLE Leishman's stain : a polychromatic stain  Acetone free methyl alcohol : as a fixative  fixes cells to slide (precipitation of protein)  Preseves the cells  Methylene blue: basic dye and stains cytoplasm, nuclei of WBCs and granules of basophils === blue color  Eosin: acidic dye and stains RBCs and granules of eosinophils === orange-red color  pH value of phosphate buffer is very important
  • 17. FIXING AND STAINING PROCEDUREFIXING AND STAINING PROCEDURE  Fixing the blood smear:  Place the smear across two parallel glass rods.  Pour 8-10 drops of leishman’s stain. Leave it for about 2 min (fixation time)  Staining the smear:  After 2 min add an equal amount of buffer solution and mix the stain by blowing air intermittently with the help of dropper.  Leave the mixture on the slide for 10-15 min.  Wash the slide by running water, hold the slide slanted and water is allowed to flow on thumb until film gets a pinkish tinge.  Wipe clean the back of slide  Stand slide upright and let dry in air.
  • 18. QUALITY OF STAINED SMEARQUALITY OF STAINED SMEAR  Smear is single-cell thick, no overlapping of cells and uniformly distributed  Atleast 1 WBC per high power field (100x)  RBCs are stained light pink  Overstained smear:RBCs look bluish & WBCs look purple  Understained smear: RBCs look very pale & WBCs almost colourless
  • 19. CAUSES & CORRECTION  Too Acid Stain: 1. insufficient staining time 2. prolonged buffering or washing 3. old stain  Correction: 1) lengthen staining time 2) check stain and buffer pH 3) shorten buffering or wash time
  • 20. Too Alkaline Stain: 1. thick blood smear 2. prolonged staining 3. insufficient washing 4. alkaline pH of stain components  Correction : 1)check pH 2)shorten stain time 3)prolong buffering time
  • 21. TOO ACIDIC SUITABLE TOO BASICTOO ACIDIC SUITABLE TOO BASIC
  • 22. PRECAUTIONS:  Slides should be clean & grease free.  Spreader should have a smooth clean edge.  Do not heat dry the smear.  Stored blood should not be used for making smear.  Assess the quality of smear both grossly & microscopically.
  • 23. PERFORMING APERFORMING A MANUALMANUAL DIFFERENTIAL ANDDIFFERENTIAL AND ASSESSING RBCASSESSING RBC MORPHOLOGYMORPHOLOGY
  • 24. ObservingObserving direction:direction: Observe one field and record the number of WBC according to the different type then turn to another field in the snake-liked direction *avoid repeat or miss some cells
  • 25. MANUAL DIFFERENTIAL COUNTSMANUAL DIFFERENTIAL COUNTS  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 zigzag method.  Count 100 WBCs including all cell lines from immature to mature.  Reporting results Absolute number of cells/µl = % of cell type in differential x white cell count
  • 26. N M N N N L N L N N: NEUTROPHIL L: LYMPHOCYTE M: MONOCYTE E: EOSINOPHIL B: BASOPHIL
  • 27. MORPHOLOGYMORPHOLOGY OF WBCOF WBC IN PERIPHERALIN PERIPHERAL BLOODBLOOD NormalNormal
  • 28. NORMAL PERIPHERAL BLOODNORMAL PERIPHERAL BLOOD SMEARSMEAR
  • 46. ARNETH COUNT:  Left to shift or regenerative shiftLeft to shift or regenerative shift::  If N1+N2+N3 exceeds 80%If N1+N2+N3 exceeds 80%  Indicates hyperactive bone marrowIndicates hyperactive bone marrow  Causes:Causes: 1. Acute pyogenic infections. 2. Tuberculosis: Though there is lymphocytosis, a shift to the left may be due to removal of older neutrophils from the blood. 3. Hemorrhage. 4. Low-dosage irradiation is said to stimulate bone marrow while heavy doses cause a shift to the right.
  • 47.  Right to shift or degenerative shift:Right to shift or degenerative shift:  If N4+N5+N6 exceeds 20%If N4+N5+N6 exceeds 20%  Indicates hypoactive bone marrowIndicates hypoactive bone marrow  Causes:Causes: 1. Bone marrow depression (hypoplasia and aplasia) due to any factor. 2. Drugs, toxins, chemical poisons. 3. Megaloblastic anemia. 4. Septicemia. 5. Uremia.
  • 48. LEFT SHIFT N1 N2 N3 N4 N5&6