1. Introduction to the Peripheral Blood Smear
Rafi Ahmed
Hematology Oncology

2. To smear or not to smear? Powerful diagnostic tool
A window into the functional status of the bone marrow
A noninvasive view into the factory of all blood elements.
Most important when assessing cytopenias
Anemia
Leukopenia
Thrombocytopenia
Not required in all situations
(i.e slam dunk situations iron deficiency with low MCV)
Extremely helpful in evaluating:
Hemolytic anemia – Review of red cell morphology may identify the cause of erythrocyte destruction
Thrombocytopenia – Distinguishing between increased platelet consumption and reduced platelet production can often be made through review of platelet size
White cell disorders – presence of blasts, auer rods etc.

4. How a PBS is made A drop of blood is placed on one end of a slide
A spreader slide is used to disperse the blood over the slide's length.
Ideally would want a monolayer, where the cells are spaced far enough apart to be counted and differentiated.

5. Peripheral Blood Smear
The slide is left to air dry
Then fixed by immersing it briefly in methanol. The fixative is essential for good staining and presentation of cellular detail.
After fixation, the slide is stained: Wright-Giemsa

6. Finding an Optimal Viewing Area
Scan entire slide under lower power (10x-40x)
Enables selection of an optimal area
Look for an area where the red blood cells are just touching and demonstrate central pallor
Then move to higher power (100x with oil)

7. What is consider and ideal viewing area?
Individually separated from one another and fairly uniformly dispersed
Blood cells are clumped or unevenly dispersed.
Jumbled together and touching one another.

8. Viewing Area on Higher Power (100x)

9. Times when viewing area is abnormal
Rouleaux formation due to elevated levels of plasma proteins
Peripheral blood smear from a patient with multiple myeloma shows red blood cell rouleaux giving the appearance of stacked coins.
Cold agglutinin hemolytic anemia shows marked red blood cell agglutination into irregular clumps
Or Sometimes you just have a bad smear….

10. Overview of hematopoeisis

11. RBC Maturation

12. Red Blood Cells Look at typical cell size
Microcytosis
Small RBC with MCV <80fL
Macrocytosis
Abnormally large RBC with MCV >100fL.
Anisocytosis: Is there variation in cell size?
Are all cells nearly the same?  
Are many cells bigger, or smaller, than the typical cell?  Are there cells in both bigger and smaller categories?
Poikilocytosis: Is there variation in cell shape?
Do many cells appear in any shape that is dramatically different from round, such as pointy?
Is there variation in cell texture/color. 
Do all cells stain similarly pink, or do some contain basophilic patches or granules?
Hypochromia
Enlarged area of pallor due to cell’s decreased hemoglobin content
Polychromasia
Bluish/purple hue in young RBCs due to residual RNA

13. Abnormalities in RBC Shape

14. Abnormalities in RBC Shape

15. Slightly smaller than a mature lymphocyte
Normal Red Blood Cell
Slightly smaller than a mature lymphocyte

16. Microcytosis
"Puny" for Pyridoxine deficiency "T" for Thalassemia "I" for Iron deficiency anemia "C" for Chronic disease anemia (sometimes): AKA Anemia of Chronic Disease "S" for Sideroblastic anemia (e.g. Lead poisoning): sometimes
Slightly smaller than a normal mature RBC which correlates to the MCV of <80 fL
Pallor is usually present
Can be hypochromic (central pallor greater than 1/3 of cell diameter)

17. Macrocytosis
Vitamin B12 or folate deficiency Chemotherapy (e.g. Methotrexate) Liver disease Thyroid disease MDS
Slightly larger than a normal mature RBC which correlates to the MCV of >100 fL
Round to oval in shape

18. Spur Cell /Acanthocyte
Increased in: End stage Liver disease (Cirrhosis) Abetalipoproteinemia McLeod or Lu blood group phenotype Post-splenectomy Artifact Etc.
Irregular narrow-base sharp projections of red cell membrane (looking like cowboy boot spurs)
Rare Spur cells may be seen in a blood smear which may be due to the very old RBCs that are reaching their end of life. (life span of an RBC is ~ 4 months)

19. Burr Cell / Echinocyte
Increased in: Artifact (e.g. older blood specimen)  Uremia (e.g. chronic renal disease) Liver disease Hyperlipidemia Etc.
Regular broader-base short blunt projections of red cell membrane
Most often they are due to an artifact of smear preparation. Also one must inquire about the age of the blood sample.

20. Schistocytes
Increased in: Microangiopathic Hemolytic Anemia (e.g. as seen in DIC, TTP, or HUS) Severe burns Mechanical valve-induced Uremia Malignant hypertension
Different RBC fragment variants (helmet cell, irregular fragments, etc.)
Usually associated with an intravascular hemolytic process (as opposed to spherocytes which are usually associated with an extravascular hemolytic process)

21. Bite Cell
Increased in: G6PD deficiency Unstable hemoglobin disorders Oxidative drug-effect
Usually slightly smaller than a normal RBC
The RBC has a "bitten" appearance (like a bitten apple)

22. Elliptocyte / Ovalocyte
Increased in: Severe iron deficiency anemia Hereditary elliptocytosis
Same size or slightly larger than a normal RBC
Usually longer and narrower than normal RBC
Usually has a central pallor

23. Increased in: Hereditary spherocytosis Autoimmune hemolytic anemia
Spherocyte
Increased in: Hereditary spherocytosis Autoimmune hemolytic anemia
Very Round
No central pallor

24. Target Cell
Increased in: Thalassemia Hemoglobinopathies (C, SC, or E disease) Post-splenectomy Liver disease Artifact of slow drying of blood smear
Bull's eye appearance (resembles the Target sign)
Increased surface membrane to volume ratio gives rise to a central darker hemoglobinized region within the area of central pallor

25. Sickle Cell Banana-shaped RBC with two pointed ends
An underlying mutation in the hemoglobin Beta chain (The hydrophilic residue Glutamate is replaced with the hydrophobic residue Valine) which leads to aggregation of the hemoglobin molecules which ultimately twists the RBC into a sickled shape

26. Tear Drop
Increased number of tear drop RBCs is a clue to a possible underlying marrow process
Increased in: Myelofibrosis Bone marrow infiltration by hematologic or non-hematologic malignancy Artifact of slide preparation (in which all the tails usually point in the same direction)
Increased number of tear drop RBCs is a clue to a possible underlying marrow process

27. Red Cell Agglutination
Paroxysmal cold hemoglobinuria Cold agglutinins Elevated serum IgM M-spike as in Waldenstom 's macroglobulinemia Plasma cell neoplasm associated with monoclonal IgM
A cluster of overlapping RBCs (Non-linear overlapping RBCs)
Must be evaluated in the appropriate section of the peripheral blood smear (where RBCs are usually not touching each other).
Mostly due to reduced zeta potential (Note: the zeta potential between RBCs keeps them apart.
Positively charged molecules such as immunoglobulins, that reduces this zeta potential will facilitate RBCs to aggregate or stack on top of each other).

28. Roleaux
Plasma cell Neoplasms (e.g. plasma cell myeloma) Chronic liver disease with hypergammaglobulinemia Chronic infections Chronic inflammation Evaluating the wrong area of a slide (e.g. thick area of a blood smear with pseudo-rouleaux)
Mostly due to reduced zeta potential (Note: the zeta potential between RBCs keeps them apart.
Positively charged molecules such as immunoglobulins, that reduces this zeta potential will facilitate RBCs to aggregate or stack on top of each other).

29. RBC Maturation

30. Polychromasia
Increased in: In response to some anemias (e.g. hemolytic anemia or blood loss) Reactive Erythroid hyperplasia Post erythropoeitin therapy Polycythemia Vera
Slightly larger than normal mature RBCs
Cytoplasm is greyish-blue in color

31. Red Blood Cell Inclusions
Not all of them!

32. Howell Jolly Asplenism
Smooth round dark blue-purple cytoplasmic inclusion
May be centrally located or sometimes in the periphery
Composed of fragments of DNA

33. Basophillic Stippling
Lead poisoning Thalassemia Sickle cell anemia MDS
Very small (dot-like) Blue or blue-gray granules distributed throughout the cytoplasm of RBCs
Composed of ribosomes

35. Segmented Neutrophil
Increased in:  Infections (especially bacterial infections) Myeloproliferative disorders (e.g. CML and CNL) Leukemoid reaction Drug-induced
2-3x larger than a mature RBC
Nucleus is mature and divided into 3 to 5 lobes connected by thin chromatin filaments

36. Band Neutrophil
Increased in:  Infections (especially bacterial infections) Myeloproliferative disorders (e.g. CML) Leukemoid reaction Drug-induced
2-3x larger than a mature RBC
Kidney bean-shaped Nucleus with nucleus indentation (more than 1/2 the diameter indented) and mature chromatin (clumped)

37. Usually associated with: Infection Inflammatory conditions
Toxic Granulation
Usually associated with: Infection Inflammatory conditions
Darker (red/purple) granules are easily noted (similar to the primary granules noted in promyelocytes)

38. Hypersegmented Neutrophil
Usually associated with: Vitamin B12 deficiency Folate deficiency
Nucleus is mature and has >5 lobes connected by thin chromatin filaments

39. Myeloblast
If in blood or increased numbers in bone marrow: Acute myeloid leukemia (AML) Myelodysplastic Syndromes (e.g. RAEB-I or II) Myeloproliferative Neoplasms (e.g. CML with increased blasts) Leukemoid reactions Post GCSF therapy
High nuclear to cytoplasmic ratio
Round Nucleus with immature chromatin (not clumped)
Prominent nucleoli
Cytoplasm is scant, gray to pale blue and lacks granules

40. How can you tell its acute leuk if you see a blast?
Hiatus Leukemicus: jump in cell development from an early stage to a late stage with nothing in between
Leukemoid Reaction: Full spectrum of granulocytes noted

41. Normal Lymphocyte
Slightly (1.5x) larger than a mature RBC

42. Reactive Lymphocyte
Increased numbers in blood may be associated with: Epstein-Barr virus infection (Infectious mononucleosis) CMV infection Other viral Infections Drug reaction Chronic inflammatory disorders (e.g.systemic lupus erythematosis, rheumatoid arthritis)
2-4x larger than a mature RBC
Increased amount of light blue cytoplasm
Cytoplasm is usually indented by surrounding RBCs

43. Large Granular Lymphocyte
If increased in blood or bone marrow: Infection Neoplastic (LGL leukemia)
2-4x larger than a mature RBC
Cytoplasm is abundant, light blue
Pink/red granules in the cytoplasm (hence the term "granular")

44. Chronic Lymphocytic Leukemia
Increased number of Small to intermediate sized lymphocytes in the peripheral blood (usually >5000 absolute count)
Background smudge cells (smeared partially broken up cells) are common but not specific to CLL

45. Acute Lymphoblastic Leukemia
3-4x larger than a mature RBC
High nuclear to cytoplasmic ratio
Cytoplasm is scant, light blue and lacks granules

47. Monocyte 3-4x larger than a mature RBC
Kidney shaped Indented Nucleus with mature chromatin (clumped)
Cytoplasm is abundant, gray to pale blue and with rare to no granules
vacuoles are commonly noted in cytoplasm

48. Eosinophil
Increased in:  Allergy-related (e.g. Asthma) Drug reactions Invasive parasitic infections Myeloproliferative neoplasms Hodgkin's and non-Hodgkin's lymphomas Autoimmune disorders (some)
2-3x larger than a mature RBC
Nucleus is mature and usually with 2 lobes connected by thin chromatin filament
More cytoplasm with only secondary granules
Secondary (specific) granules are Eosinophilic (Red-Orange)

49. Basophil 2-3x larger than a mature RBC
More cytoplasm with only secondary granules
Secondary (specific) granules are Basophilic (Blue-Violet)
The granules obscure the underlying nucleus
Increased in CML

50. Platelets Increased in Iron deficiency Anemia Splenectomy
Acute or chronic inflammation
Myeloproliferative Neoplasm (e.g. Essential Thrombocythemia, etc.)
Drug reaction

51. Platelet Count is Low on Auto Diff… What’s going on here?
Platelet clumping in an EDTA-anticoagulated blood sample.
This patient had an EDTA-dependent platelet agglutinin which caused platelet clumping, resulting in low platelet count (ie, "pseudothrombocytopenia").
Platelet count was normal, in a blood sample with sodium citrate.

52. Ig A Lambda Multiple Myeloma
Case 1
60 year old male presents with fatigue and weakness. Is found to have a normal WBC and Plt count. He has normocytic anemia with Hb 9. Other abnormalties on labs include a corrected calcium of 13.

53. Macrocytosis: B12 deficiency
Case 2
74 year old male comes to your office after seeing another doctor. He states that he was told he was anemic and needed a medication to fix his anemia. While waiting for his CBC to come back, you look at his smear.

54. Case 3
MAHA/ TTP
A 35 year old female with a history of SLE presents with confusion. Her CBC comes back first. Her WBC is 8K, Hb is 7.5, MCV is 86, and her platelets are 20K.

55. AIHA: Spherocytes Case 5
A 27 year old female presents to the ED with scleral icterus and fatigue. Her WBC and Plt count is normal. Her hemoglobin is found to be 7.3

56. Case 6
Megaloblastic Anemia
What type of anemia does this person have?

57. Case 7
Acute Leukemia
18 year old male presents with gingival bleeding. Found to have a WBC count of 2, Hb of 7, Plts of 10K.

58. Encapsulated Organisms
SS with Asplenia
Case 8
Encapsulated Organisms
What infections is this person at increased risk for?

59. Case 9
AML-M3 (APML)
55 year old male presents with significant fatigue, nose bleeding and easy bruising. His CBC shows: WBC of 0.5k, Hb 7, Plt 15k. Found to have an INR of 3

60. Case 10
CLL
34 year old asymptomatic male was told to follow up because his recent labs showed he had a high white count. He’s found to have a WBC of 45k. What’s going on?

61. Case 11
Myelofibrosis
What do you think is going on in this patient with anemia, weight loss, night sweats and significantly enlarged spleen?

62. Case 12
CML: BCR/ABL
30 year old female presents with elevated WBC of 50K. What simple blood test would confirm the diagnosis?

63. Case 13
Cold Agglutin HA
45 year old with history of follicular lymphoma presents with fatigue. Found to have a hemoglobin of 5.7. What’s going on?

64. So next time you order a peripheral blood smear…..

65. Lets make sure you look at it.
Peripheral Blood Smear