Peripheral blood smeared examination test

All about blood film and diagnosis the result

1. Peripheral blood smear
examination
RakheeKar
Additional Professor
JIPMER, Puducherry

4. Blood film examination may still help to..
•Diagnose a disease
•Monitor response
•Detect pre-analytical or
analytical error in output
of automated analyzer
•Correlate instrument flag
•Additional information

5. Layout
Examination of a peripheral smear-general
RBC morphology
WBC morphology
Platelet morphology
Hemoparasites
Artifacts and trouble shooting

6. Window to the bone marrow
Examination of a peripheral smear-general

7. A: Good look at the slide-
Quality of smear and staining

8. B: At scanner/low power-Choose optimal
assessment area and technique
1.RBCs are uniformly and
singly distributed
2.Few RBCs are touching or
overlapping
3.Normal biconcave RBC
appearance
4. 200 to 250 RBC per 100x

9. Optimal Assessment Area
Thin area
"Spheroidocytes" or flattened
red cells. True spherocyteswill
be found in other (good) areas
of smear.
Thick area
Rouleauxis normal in
such areas. Confirmed
in thin areas.

10. A word of caution:
•Beware of:
Tailing
Low WBC counts
•You are likely to find suspects in the tail end
and edges of smear

11. Importance of low power (10x)
Fibrin strands
Platelet distribution/
clumps

12. C: At high power (40x), observe
•RBC -Morphology
•WBC-Estimate of TLC, DC
•Choose a portion of the PS where there is only slight
overlapping of the RBCs.
•Count 10 fields, take the average number of WBC/ hpf.
•Multiply the average number of white cells by 2000.

13. D: In oil immersion (100x)
•RBC, WBC, Platelet morphology
•RBC inclusions
•Hemoparasites
•Estimate of platelet count
1.Estimate the number of platelets per oil immersion field.
2.Take an average of at least 5-6 fields.
3.Multiply the average by 15,000.

14. Platelet count
Alaska Med.2004 Oct-Dec;46(4):92-5.
Platelet count assessment from peripheral blood smear (PBS).
Webb DI
1
,Parker L,Webb K.
Abstract
OBJECTIVE:
To visually count platelets in a peripheral blood smear and compare with an automated machine platelet count.
METHODS:
Thirty-five peripheral blood smears were made from blood specimens counted on an automated blood cell machine: twenty-three
thrombocytopenic specimens, 1 with high platelet count and 11 with normal counts. Ten and 25 high-power fields were microscopically
averaged and then multiplied by 15,000 and 20,000 to arrive at a platelet count in 1,000 per microliter. Comparisons between visual and
machine counts were drawn.
RESULTS:
There was fair concordance in 27 specimens. In three specimens underestimation was found, overestimation in five. A 15,000 multiplier gave
slightly better results than 20,000. Average in 10 high-power fields was as good as 25. Abnormal counts could be assessed as well as normal.
CONCLUSION:
Average in 10 high-power field on a blood film microscopically and
multiplying by 15,000 gives a platelet count reasonably close to automated
machine counts in thousands per microliter

15. RBC Morphology

16. Assessment of RBCs
1.Size and shape and their variation
(Anisocytosis,Poikilocytosis)
2.Relative hemoglobin content.
3.Polychromatophilia.
4.Rouleauxformation or agglutination
5.Inclusions.

17. RBC size
Microcytic
MCV
Normocytic Macrocytic
Hypochromic Normochromic

18. Morphologic classification of anemia
Microcytic
hypochromic
Normocytic
normochromic
Macrocytic
normochromic
•Iron Deficiency
(IDA)
•Chronic
Infections
•Thalassemias
•Hemoglobino-
pathies
•Sideroblastic
Anemia
•Diseases of kidney,
liver, endocrine
organs
•Early IDA
•Marrow infiltration
•Marrow hypoplasia
•Haemolyticanemias
•Megaloblastic
anemia
•Liver
disease/alcoholism
•Hypothyroidism
•MDS
•Aplasticanaemia
•Myelopthisic
anaemias

19. Hemoglobin
Heme Globin
Porphyrin Iron
Deficiency Defective utilization
Thal/ hemoglobinopathies
IDA ACD
Sideroblastic
anemia
MicrocytichypochromicAnemias

20. D/D of microcytic hypochromic
anemia
Iron deficiency anemia Thalassemia trait
Moderate anisopoikilocytosis,
pencil cells, elongated cells
thrombocytosis+/-
Mild Anisocytosis, target cells

21. Homozygous thalassemia
(ThalMajor, intermedia)

22. D/D of microcytic hypochromic
anemia
Sideroblasticanemia-
Dimorphic anemia
Lead poisoning
basophilic stippling

23. Dimorphic anemia
•Two different population of RBC
–Macrocyteswith normocytes
–Microcyteswith normocytes
–Macrocyteswith microcytes
•MCV –low, normal or high

24. Dimorphic anemia
Causes
•Response to iron or vitamin therapy
•Blood transfusion in a pt. with
microcytic/macrocyticanemia
•Myelodysplasticsyndromes
•Sideroblasticanemias

25. MacrocyticAnemias
Megaloblastic
•MCV > 110 fl
•Folate or B
12deficiency
Non-megaloblastic
•Liver disease/alcoholism
•Hypothyroidism
•MDS
•Aplastic anaemia
•Myelopthisicanaemias
•Hemolytic anemiaswith
florid reticresponse
•Neonates

26. MegaloblasticAnemia
Pancytopenia,
macrovalocytes,
HSP, cabotrings,
nRBCswith
megaloblastic
maturation

27. Other macrocyticanemias
Anemia associated with Liver diseaseReticresponse (Polychromatophils)
AplasticAnemia

28. Normocytic normochromic anemia
•Diverse group
•Both hypo-and hyper-
proliferative anemias
•Reticcount important
to delineate the type

29. Clues to a hemolytic process
•Polychromasia
•n-RBC
•Specific morphologic
abnormalities

31. Clues to inherited hemolytic anemias
and other stories that RBCs tell..
Membranopathies
Enzymopathies
Hemoglobinopathies

32. Spherocyticanemia
Coombs test
History

33. Spherocyteson PS
Causes
•Hereditary Sherocytosis
•Auto Immune Hemolytic
Anemia
•Hemolytic Ds of Newborn
•Transfused RBC
•MAHA
•Burns
Clues
•Uniform-sized spherocytes
•Variable spheros, nRBCs,
Agglutination +/-
•Many nRBCs
•Spheroidocytes
•Microspherocytes, schistocytes
•Spheros, smaller RBC fragments

34. Clues to other membranopathies..
Elliptocytes Stomatocytes

35. Clues to G6PD Deficiency
Blister cells Bite cells
Dapsone-induced hemolysis

36. Pyrimidine5’ nucleotidasedeficiency

37. All enzymopathiesdo not have
characteristic morphology
•PK deficiency

38. Sickle cell anemia
Sickle beta thalassemia
Thalassemias and hemoglobinopathies

39. Thalassemiasand hemoglobinopathies
Homozygous HbC disease

40. Target cells
Microcytic Normocytic Macrocytic
•Severe Iron
Deficiency (IDA)
•Thalassemia trait
•HbE disease
•HbC disease
•Sickle cell anemia
•Sickle thalassemia
•Liver disease
•Post splenectomy
•Preterm infants

41. Clues to acquired hemolytic anemias
and other stories that RBCs tell..

42. Agglutination
Cold
agglutinin
disease
Addlclues:
Tight clusters
Polychromatophils
Spherocytes
n-RBCs

43. Rouleauxformation
Addlclues:
Proteinaceous
background
Plasmacytoidcells

44. Schistocytes

45. Microangiopathichemolyticanemia
Thrombotic thrombocytopenic purpura(TTP)
Hemolyticuremic syndrome (HUS)
HUS/TTP-related disorders
Disseminated carcinoma
Chemotherapy/drugs
Transplant-associated microangiopathy
Pregnancy and postpartum period
HELLP syndrome
TTP/HUS
Malignant hypertension
Disseminated intravascular coagulation
Schistocytes
•Well hemoglobinised
broken cells
•Predominant finding in
absence of other red cell
abnormality
•Look for low platelets,
and other signs of
hemolysis

46. KERATOCYTE
TRIANGLE
SCHISTOCYTE
HELMET CELL
MICROSPHEROCYTES

47. Some more poikilocytes..

48. Spur cell (Acanthocyte)
Longer and lesser
number of spikes

49. Burr cell (Echinocyte)
Shorter and more
number of spikes
Anemia of renal
disease

50. Tear drop cell (Dacryocyte)
MA

51. RBC inclusions on Romanowsky
stains
Basophilic stippling
Howell Jolly bodies
Pappenheimerbodies

52. Howell Jolly bodies

54. Int. Jnl. Lab. Hem. 2015, 37, 1–7

55. WBC Morphology

56. n-RBCs give spurious WBC count
•Look at the counts (TLC).
•Does it need any correction?
Hemolytic anemia, LEB picture etc
Eg: TLC-15000
nRBC: 50/100 WBC
Corrected TLC??

57. Distribution (DLC)
•Look at the distribution (DLC).
•Any peniasor philias? (look at absolute count)
•Any morphologic abnormality?
•Any abnormal cells ?

58. Nucleus
Pelger-Huetanormaly
Hypersegmentation
Hereditary
Myelodysplasticsyndrome
Megaloblastic anemia
Morphologic abnormalities of
neutrophils

59. Cytoplasm
Hypogranulation
Toxic granule
Vacuolization
Myelodysplastic syndrome
Bacterial infection
Bacterial infection
Neutrophil-morphology

60. Pseudo Pelger-Huetand hypogranulated
neutrophils
MDS

61. Toxic Granulation
•Increased basophilic
granules
in neutrophils.
•Seen in severe infections,
burns, malignancies, and
pregnancy.
•Distinguish from
basophils.

62. DohleBodies
•Sky blue inclusions in
cytoplasm of
neutrophils.
•Seen in infections,
burns, myleproliferative
disorders, and
pregnancy.
•Composed of RER and
glycogen granules.

63. ChediakHigashi syndrome

64. Leukocyte vacuolation
Lipid Storage DiseaseChanarin–Dorfmansyndrome

65. Any left shift?

66. Neutrophils& Precursors

67. Grade 4
Grade 0
Leukemoidreaction CML
LAP Score

68. Normal Lymphocyte
morphology

69. Transformed lymphocyte
(atypical lymphocyte)
Dengue
IM

70. Abnormal
Lymphoid cell
morphology
Mantle cell
lymphoma
Blastoid
variant

71. Eosinophils
Nuclear hypersegmentationof neutrophils,
eosinophils, and basophilsdue to hydroxyurea

72. Time for a blast!
Myeloblast Lymphoblast
Lineage is often made out on PS morphology.

73. What else?
AML M2 AML M5
Subtype is often made out on PS morphology.

74. What is the emergency?
APL-variant
APL-variant with DIC

75. But we may not be lucky always…
Blasts/ abnormal cells may not stare at you; you
might have to look for them
Acute promyelocyticleukemia-classical
Sub-leukemic leukemia
HypoplasticAML
MDS (RAEB 1/2)
Associated myelofibrosis/ myelonecrosis(LEB)

76. Platelet morphology

77. What does the PS tell?
•Estimate of count.
(normal, thrombocytopenia, thrombocytosis)
•Does it tally with counter value.
•Any spurious low or high count.
•Any morphologic abnormalities

78. Spurious low counts
(counts low, appear adequate on smear and
normal in morphology)
1. EDTA induced
pseudothrombocytopenia
1.Exposure of cryptic Ag on
IIb/IIIaby EDTA
2. Platelet satellitism
•EDTA
•Uncertain mechanism

79. Spurious low counts
3. Non-EDTA
•Procedural
4. Partial clotting of sample

80. Low counts/ Spurious low counts
(platelets large in size)
•Large platelets
–ITP
–Bernard Soulier
Syndrome
–May Hegglinanomaly
–Immature reactive
platelets
• ITP on steroids
• Post chemotherapy
recovery

81. D: Giant platelets in primary myelofibrosis, cellular phase.
E: Bizarre plateletsinessential thrombocythemia.
F: Giant platelets and a megakaryoblastin acute megakaryoblastic
leukemia.
G: Stripped megakaryocytic nucleus.
H: Bernard-Souliersyndrome.
I: May-Hegglinanomaly. Large Döhle-like inclusions (arrow) and giant platelets
Large platelets

82. Bacterial overgrowth in stored sample
Cryoglobulins
MAHA
Burns patient
Spurious high counts

83. Spurious high counts
Rarer than pseudothrombocytopenia
• Microcyticred cells –HbH
• Fragmented red cells –TTP, DIC
• Leukemic fragments, apoptotic cells -TLS
• Cryoglobulinemia
• Hyperlipidemia
• Bacteria, fungi, parasitisedred cells
• Inadvertantheating of sample

84. Hemoparasites

85. Any infectious organism
spending a part of its life cycle
in blood with a demonstrable
life form in any of the blood
cells or their marrow
precursors
HEMOPARASITE

86. Peripheral smear in Malaria
THICKSMEARS
•2-3 drops of blood
•Increased sensitivity
•Decreased specificity
•All parasites
extracellular
THINSMEARS
•1 drop of blood
•Decreased sensitivity
•Increased specificity
•Intracellular forms also
well made out.

87. Plasmodium–ring forms
Pl. falciparum
Pl. vivax
Pl. knowlesi
Pl. ovale
Pl. malariae

88. Plasmodium –trophozoiteform
Pl. falciparum
Pl. knowlesi
Pl. vivax
Pl. ovale
Pl. malariae

89. Plasmodium-schizont
Pl. falciparum
Pl. knowlesi
Pl. ovale
Pl. vivax
Pl. malariae

90. Plasmodium-gametocytes
Pl. falciparum
Pl. malariae
Pl. ovale
Pl. vivax
Pl. knowlesi

91. Other diagnostic clues
•Monocytosis
•Pigments in monocytes, neutrophilseither in
peripheral smear or marrow
•Sometimes features of hemolysis

92. Malarial pigment

93. Wuchereriabancrofti-Microfilaria
•PERIPHERAL BLOOD :
Direct wet mount with no staining
Giemsaor Leishmanstained thick blood smear
Post DEC provocation test
•Examine the extreme tail end of a smear under scanner view
to screen

94. Artefactsand trouble shooting

95. Features of a well-stained PBS
•Macroscopically: color should be pink to purple
•Microscopically:
RBCs: Orange to salmon pink
WBC: Nuclei is purple to blue
Cytoplasm is pink to tan
Granules are lilac to violet
Eosinophil: Granules orange
Basophil: Granules dark blue to black

96. Pale staining
Too Acidic Stain:
RBC pale, WBC barely visible
1.Insufficientstainingtime
2.Prolongedbufferingorwashing
3.Oldstain
Correction:
1)Lengthenstainingtime
2)CheckstainandbufferpH
3)Shortenbufferingorwashtime

97. Dark staining
Too Alkaline Stain:
RBC gray, WBC too dark, Eogranules gray
1.Thick blood smear
2.Prolonged staining
3.Insufficient washing
4.Alkaline pH of stain components
5.Heparinizedsample
Correction :
1.Check pH
2.Shorten stain time
3.Prolong buffering time

98. Water Artifact
•Moth eaten RBC,
•Heavily demarcated central pallor
•Crenation
•Refractory shiny blotches on the RBC
What contributes to the problem:
1.Humidity in the air during air drying
2.Water absorbed from the humid air into the alcohol based
stain
Solution:
1.Drying the slide as quickly as possible.
2.Fix with pure anhydrous methanol before staining.
3.Use of 20% v/v methanol

99. Summary

100. A drop of blood tells a story
•A simple tool; yet exceedingly important.
•Can tell some things that no automated
hematology analyzer can.
–Type/ cause of anemia
–Likely nature of leukemia / lymphoma
–Cause of thrombocytopenia
–Bugs in the scene
•Guide to do or not to do a BM examination

101. Thank you