Peripheral smear

PERIPHERAL SMEAR IN A NUT SHELL. TRIED MAKING IT SIMPLE.

1. Peripheral blood smear examination DR. MITHILA MODERATOR : DR MANJUNATH

2. Introduction Peripheral blood smear is a very important tool in the hematology lab It provides rapid, reliable access to information about a variety of hematologic disorders Examination of the peripheral blood smear is an inexpensive but powerful diagnostic tool in both children and adults The smear offers a window into the functional status of the bone marrow Review of the smear is an important adjunct to other clinical data; in some cases, the peripheral smear alone is sufficient to establish a diagnosis

3. INDICATION FOR PERIPHERAL SMEAR Features suggestive of anemia, unexplained jaundice, or both . Features suggestive of thrombocytopenia (e.g., petechiae or abnormal bruising ) or neutropenia (e.g., unexpected or severe infection ). Features suggestive of a lymphoma or leukaemia lymphadenopathy, splenomegaly , bone pain, and systemic symptoms such as fever, sweating, and weight loss . Features suggestive of a myeloproliferative disease — splenomegaly, plethora , itching, or weight loss .

4. General ill health, often with malaise and fever, suggesting infectious mononucleosis or other viral infection or inflammatory or malignant disease . Suspicion of a bacterial or parasitic disease that can be diagnosed from a blood smear

5. Peripheral Smear Preparation Staining of Peripheral Blood Smear Peripheral Smear Examination objectives

6. SMEAR PREPARATION Place a drop of blood, about 2-3 mm in diameter approximately 1 cm from one end of slide. Place the slide on a flat surface, and hold the other end between your left thumb and forefinger. With your right hand, place the smooth clean edge of a second (spreader) slide on the specimen slide, just in front of the blood drop. Hold the spreader slide at a 30°- 45 angle, and draw it back against the drop of blood Allow the blood to spread almost to the edges of the slide. Push the spread forward with one light, smooth moderate speed. A thin film of blood in the shape of tongue. Label one edge with patient name, lab id and date. 9. The slides should be rapidly air dried by waving the slides or using an electrical fan .

7. A well made peripheral smear is thick at one end and progressively thinner at the opposite end. The "zone of morphology" (area of optimal thickness for light microscopic examination) should be at least 2 cm in length. The smear should occupy the central area of the slide and be margin-free at the edges

8. PBS examination requires a systematic approach in order to gather all possible information. In addition, all specimens must be evaluated in the same manner, to assure that consistent information is obtained.

9. Slide fixation and staining

10. Romanowsky staining It includes: May- Grunwald – Geimsa stain, Jenner’s stain, Wright’s stain, Leishman’s stain and, Field’s stain .

11. Color responses of blood cells to Romanowsky staining Cellular component Color Nucle i Chromatin Purple Nucleoli Light blue Erythroblast Dark blue Erythrocyte Dark pink Reticulocyte Grey–blue

12. Cytoplasm color Lymphocyte Blue Metamyelocyte Pink Monocyte Grey–blue Myelocyte Pink Neutrophil Pink/orange Promyelocyte Blue Basophil Blue

13. 1. Macroscopic view : quality of the smear 2.The microscopic analysis begins on lower power (10x), Determine good distribution of the cells Scans the edges for abnormal cells Find a optimal area in the smear for detailed examination. PBS examination - preliminary

14. Hi-power (40x) : To obtain a WBC estimate. All of the detailed analysis of the cellular elements using high power or oil immersion. Evaluate the morphology of the WBC and record any abnormalities such as toxic granulation or Dohle bodies. The WBC estimate can be performed using a factor which is based on the fact that WBC seen in 40x is approx equivalent to 2000 cells /micro litre . F or example if the average number of WBC counted per high power field is 5, the estimate WBC is 5 x 2000 = 10000

15. Oil immersion Perform a 100 WBC differential count , counting is done in zig zag motion. All WBC have to included until a total of 100 have been counted Evaluate RBC for anisocytosis , poikilocytosis , hypochromasia , polychromasia , and inclusions. Perform platelet estimate and platelet morphology Count the number of platelets in 10 OIF. Divide by 10

16. Ten microscopic fields are examined in a vertical direction from bottom to top or top to bottom slide is horizontally moved to the next field Ten microscopic fields are counted vertically. procedure is repeated until 100 WBCS have been counted (zig zag motion) Scanning technique for WBC differential count and morphologic evaluation

17. 1. RBC Size Shape Color Arrangement Inclusions Abnormal cells 2. WBC Total counts Differential counts Abnormal WBC 3. Platelets Counts Abnormality 4. Parasite s Evaluation of PBS

18. RED CELL MORPHOLOGY

19. Morphology of Normal Red Blood Cells Biconcave disc Diameter : 7 ~ 8 μm Average volume : 90 fl. Central pallor occupy 1/3 rd of total size Approx same as nucleus of mature lymphocyte

20. RED CELL ABNORMALITY Normal MCV is -80-100 fl Microcytes –MCV<80 fl Macrocytes – MCV> 100 fl Anisocytosis - variation in the size of the RBC Poikilocytosis – Variation in the shape of RBC

21. Variation In Size Anisocytosis - Variation in size of the red blood cells The severity of the variation corresponds to increased RDW. Anisocytosis results from the abnormal cell development ( deficiency of iron , B12, Folic acid) Normal MCV is -80-100 fl Microcytes ( MCV <80 fl ) Macrocytes (MCV >100fl)

22. Microcytes A M icrocyte is a small cell having a diameter less <7 & MCV < 80fl. Anemia associated with microcytes is said to microcytic Expanded central zone of pallor anemia Decreased or defective globin synthesis also presents as Microcytic hypochromic anemia.

23. MORPHOLOGY - MICROCYTE IR IRON DEFICIENCY THALASSEMIA SIDEROBLASTIC LEAD POISONING CHRONIC DIESEASE PO POSSIBLE PATHOLOGY

24. Macrocytes When MCV of RBC is Increased(>100fl) The common cause of macrocytes is due to the impaired DNA synthesis, RNA synthesis is unaffected resulting in the asynchrony between the cytoplasmic and nuclear maturation . Neutrophillic hypersegmentation is typically seen.

25. MORPHOLOGY - MACROCYTE MEGALOBLASTIC PROCESS HIGH RETICULOCYTE COUNT LIVER DIESEASE HYPOTHYROIDISM POST SPLENECTOMY OVAL MACROCYRTES HEMOLYTIC ANEMIA / ACUTE BLOOD LOSS CHRONIC ALCOHOLISM FOLATE AND B12 DEFICIENCY

26. normocytes The average size of the erythrocyte is indicated by the measurement of the MCV A Normal MCV would corresponds to the MCV reference range ( 80 -100 ) fl Subsequent review of the peripheral smear reveals no abnormality in the size variation. This scenario is referred to as NORMOCYTIC and red cells are referred as normocytes .

27. HEMOGLOBIN CONTENT – COLOR VARIATION NORMOCHROMIA HYPOCHROMIA HYPERCHROMIA POLYCHROMASIA

28. NORMOCHROMIA The term Normochromic indicates the red cell is essentially high in color A normochromic erythrocyte has a well hemoglobinized cytoplasm with a small but distinct zone of central pallor. The central pallor does not exceed 3µm . The term normochromic is used to describe the anemia with a normal MCHC, and MCH and when used in conjunction with MCV the anemia is described as NORMOCYTIC / NORMOCHROMIC anemia .

29. HYPOCHROMIA Any RBC having a central area of pallor of greater than 3µm is said to be hypochromic There is a direct relationship between the amount of hemoglobin deposited in the RBC and the appearance of red cell when stained. The term Hypochromia indicates low color and indicates that the cells have less hemoglobin. MCHC < 32% the anemic process is described as hypochromic .

30. Hypochromia grading 1 + AREA OF CENTRAL PALLOR IS ONE HALF OF CELL DIAMETER 2 + AREA OF CENTRAL PALLOR IS TWO THIRDS OF CELL DIAMETER 3 + AREA OF CENTRAL PALLOR IS OF THREE QUARTERS 4 + THIN RIM OF HEMOGLOBIN

31. polychromasia When RBC are delivered to the peripheral circulation prematurely appearing diffusely basophilic and are gray blue in color and usually larger than normal red cell. The basophilic color is due to the RNA residue involved in hemoglobin synthesis. Polychromatic cells are actually reticulocytes. Any clinical condition in which marrow is stimulated particularly RBC regeneration will produce a polychromatic blood picture . The degree of polychromasia is a excellent indicator of therapeutic effectiveness when patient is given iron or vitamin therapy as treatment of anemia

32. POIKILOCYTOSIS Variation In shape is called Poikilocytosis . It is of following types- Spherocytes Elliotocytes Target cells Schistocytes Acanthocytes Keratocytes Echinocytes Bite cells Howel jolly bodies

33. Spherocytosis Spherocytes are small dense spheroidal RBC with absence of central pallor . Because of their density they are easily seen in the peripheral smear. This abnormality is due to the abnormality of the red cell membrane . The detailed mechanism for sphering is the congenital condition known as hereditary spherocytosis. This is an inherited , autosomal dominant condition and is due to the deficiency of the membrane proteins , spectrin and ankyrin . Acquired causes of spherocytes are ABO incompatibility Autoimmune hemolytic anemia (warm antibody type) Infections (e.g., EBV, CMV, E. coli, Sepsis/ sepsis) Severe burns DIC and HUS

34. Elliptocytes or ovalocytes Ovalocytes / elliptocytes are due to the result of morphological abnormality due to the result of mechanical weakness or fragility of the membrane skeleton that may be acquired or hereditary.

35. Stomatocytes Red cells with central biconcave area appears slit like in dried film. Wet film it appears as cup-shaped. The abnormal morphology is due to the Membrane defect. Seen in Artifact Hereditary stomatocytosis liver disease , Alcoholic cirrhosis Hemolytic anemia

36. Tear drop cells / dacrocytes Tear drop cells appear in the peripheral circulation as tear drop or pear shaped red cells. Exact mechanism not known. It is seen in : Myelofibrosis Bone marrow infiltrated with hematological or non-hematological malignancies Iron deficiency anemia megaloblastic anemia

37. Target cells Cells in which central round stained area and peripheral rim of cytoplasm. Seen in Thalassaemia Chronic liver disease Hereditary hypo- betalipoproteinemia Iron deficiency anemia Hemoglobinopathies ( Hb C, Hb H, Sickel cell anemia Post splenectomy

38. Acanthocytes or spur cells , are spherical cells with blunt-tipped or club-shaped spicules of different lengths projecting from their surface at irregular intervals . Acanthocytes Acanthocytes are seen in Hereditary Abetalipoproteinemia Hereditary acanthocytosis End stage liver disease Micro angiopathic hemolytic anemia Malnutrition Post splenectomy it is the hallmark in the diagnosis of the neuro acanthocytosis syndrome such as Chorea-acanthosis and Mcleod syndrome

39. Schistocytes These are fragmented erythrocytes . Smaller than normal red cells and of varying shape resulting from some trauma to the cell membrane. Triggering events within the circulation leading to fragmentation of RBC. Fluid alteration results in development of fibrin strands, damaged endothelium. The flow of the blood in the circulation sweep the RBC through the fibrin strands splitting the red cells Acquired disorder of RBC formation Megaloblastic Dyserythropoietic Mechanical stress MAHA DIC Heart valve surgery HUS / renal graft rejection Direct thermal injury / Severe burns/

40. Sickle cell Cells are sickle (boat shape) or crescent shape Present in film of patient with homozygosity for Hb S. Usually absent in neonates and rare in patients with high Hb F percentage

41. RED CELL INCLUSION Basophilic stippling (Punctate basophilia ) Howell – jolly Bodies Heinz body Cabot Rings Protozoan inclusions Rouleaux formation

42. ROULEAUX Rouleaux is a condition in which red cells appear as stacks of coins on the peripheral smear . The stacks of RBC are evenly distributed through out the smear , rouleaux formation is the result of elevated globulins or fibrinogens in the plasma where the RBC has been “bathed “ in the abnormal plasma giving sticky consistency. It is seen in multiple myeloma and W aldenstroms macroglobulinemia , intra venous administration of plasma volume expanders like dextran.

43. Howell Jolly bodies Howell-Jolly bodies are small round bodies composed of DNA, about 1 µm in diameter, usually single and in the periphery of a red cell. They are readily visible on the Wright-Giemsa-stained smear . The spleen is responsible for the removal of nuclear material in the red cells, so in absence of a functional spleen, nuclear material is removed ineffectively. Howell-Jolly bodies are seen in : Post splenectomy Functional asplenia Anatomical absence of spleen

44. Presence of irregular basophilic granules with in Rbc which are variable in size . Stain deep blue with W right’s stain Fine stippling seen with I ncreased polychromatophilia Increased production of red cells. Coarse stippling Lead and heavy metal poisoning Disturbed erythropoiesis Megaloblastic anemia Thalassaemia infection liver disease Unstable Hb Pyrimidine-5’-nucleotidase defiency Basophilic Stippling

45. Heinz bodies S een on supravital stains N ot seen on Romanowsky stain. P urple, blue , large, single or multiple inclusions attached to the inner surface of the red blood cell. R epresent precipitated normal or unstable hemoglobins . seen – Post splenectomy Oxidative stress Glucose-6-phosphate dehydrogenase deficiency, Glutathione synthetase deficiency Drugs Toxins Unstable hemoglobins

46. Cabot Rings These are Ring shaped figure of eight or loop shaped Red or Reddish purple with Wright’s stain and have no internal structure Observed rarely in P ernicious anemia , L ead poisoning ,

47. White blood cells

48. Types Granulocyte ( polymorphonuclear ) Agranulocyte (mononuclear) Contain membrane bound granules, which stains differently with stains Apparently absent granules, but contain non specific azurophilic granules E.g. Neutrophils Basophil Eosionophil E.g. Lymphocyte Monocyte Macrophage

49. POLYMORPHONUCLEAR NEUTROPHILS 40 to 80 percent of total WBC count( 2.0–7.0 × 10 9 /l ) D iameter - 13 µm segmented nucleus and pink/orange cytoplasm with fine granulation(0.2-0.3µm) stain tan to pink with Wright’s L obes -2-5 Neutrophils usually have trilobed nucleus. small percent has four lobes and occasionally five lobes.

50. Band forms neutrophils has either a strand of nuclear material thicker than a filament connecting the lobes, or a U-shaped nucleus of uniform thickness. Up to 8% of circulating neutrophils are unsegmented or partly segmented (‘band’ forms )

51. Band cells constitute <5-10% of white blood cells An increase in number of band cell and other immature neutrophils is called a “ shift to left” can be seen in Severe infections, sepsis Non infectious inflammatory disease Pregnancy

52. Granules Toxic granulation- increase in staining density and number of granules S een with Bacterial infections and other inflammation A dministration of G-CSF Anaplastic anemia

53. Dohle Bodies S mall , round or oval, pale blue-grey structure F ound at periphery of neutrophil. C ontains Ribosomes and Endoplasmic reticulum S een in – Bacterial infection inflammation administration of G-CSF during pregnancy Pernicious anemia Myeloproliferative disorders Myelodysplastic disorders Cancer chemothrapy

54. EOSINOPHILS Normally 1-6 ( 0.02–0.5 × 10 9 /l) Size- 12–17 µm Nucleus- Bilobed (spectacle shaped) Cytoplasm- Pale blue Granules - Coarse spherical gold/orange  

55. Eosinopenia - seen with prolonged steroid administration. Eosinophilia- allergic conditions hay fever, asthma severe eosinophilia- parasitic infection reactive eosinophilia E osinophilic leukaemia I diopathic hypereosinophilic syndrome T-cell lymphoma, B-cell lymphoma and acute lymphoblastic leukaemia .

56. BASOPHILS Rarest <1 Nucleus segments fold up on each other resulting compact irregular dense nucleus(closed lotus flower like) Granules-large, variable size dark blue or purple often obscure the nucleus G ranules are rich in histamine, serotonin and heparin I ncrease in myeloproliferative disorder-CML  

57. MONOCYTES 2-10% of total wbc count Size- largest circulating leucocyte, 15–18µm in diameter Cytoplasm- grey blue Nucleus- large , curved , horse shoe shape No segmentation occur Chromatin- fine evenly distributed Increase in chronic infections and inflammatory conditions such as Tuberculosis and Crohn’s disease, Chronic myeloid leukaemias Acute leukaemias with a monocytic component Infectious mononu cleosis

58. LYMPHOCYTES 20-40% of total WBC count It is of two types 1. Small lymphocyte(6-10µm) 2. Large lymphocyte(12-15µm) Nucleus- single, sharply defined, stain dark blue on Wright’s stain Cytoplasm- Pale blue Large lymphocytes less densely stain nuclei & abundant cytoplasm F ew round purple(azure) granules are present Lymphocytes predominate in the blood films of infants and young children.

59. Reactive lymphocytes H ave slightly larger nuclei with more open chromatin A bundant cytoplasm that may be irregular. S een in infectious mononucleosis viral infections

60. Platelets morphology

61. Platelets Thrombopoiesis take place in bone marrow 1 megakaryocyte produce 4000 platelets Normal platelet are about 1.3 micron, blue grey, contain fine, purple to pink granules Red cell to platelet ratio : 10-40:1 Life span 9-12 days Range : 1.5-4.5 lakhs/ microL

62. Platelets Neubars chamber : count platelets in 64 small squares Counts * 250 = total platelets Normal counts 4.5 to 5.5 lakh Common Causes of Thrombocytopenia • Decreased production − Aplastic anemia − Acute leukemia − Viral infections *Parvovirus *CMV − Amegakaryocytic thrombocytopenia (AMT) • Increased destruction − Immune thrombocytopenia * Idiopathic thrombocytopenic purpura (ITP ) *Neonatal alloimmune thrombocytopenia (NAITP) − Disseminated intravascular coagulation (DIC ) − Hypersplenism Thrombocytosis Reactive thrombocytosis Post infection Inflammation Juvenile rheumatoid arthritis Collagen vasvular disease Essential thrombocythemia

63. EXAMINATION OF BLOOD FILMS FOR PARASITES

64. Malaria Giemsa stain are used, identifies species and life cycle stages Parasitemia is quantifiable Threshold of detection thin film: 100 parasites/ L, thick film: 2-20 parasite/L Thick film Thin film Lysed RBCs Larger volume 0.25microliter / 100 fields blood element more concentrated Good screening for positive or negative parasitemia and parasite density difficult to diagnose species Fixed RBCs Single layer Smaller volume 0.005 microliter blood required Good species differentiation Requires more time to ready Peripheral smear

65. Appearance of P falciparum in the blood films Ring or trophozoit e Many cells infected – same with more than one parasite Red cell size unaltered Parasite is often attatch to the margin of the host cell: called as accole form (arrow) Schizont Very rarely seem except in cerebral malaria A single brown pigment dot along with 18-32 merozoites Gamatocyte Sickle shape “ cresent ” Matuer gametocyte is about 1.5 times larger than RBC harbouring it Microgamatocyte : Broader, shorter, blunt ends. Cytoplasm light blue Macrogamatocytes : Longer, narrower, pointed ends. Cytoplasm deep blue

66. Appearance of P vivex in film Ring or trophozoit e Many cells infected – same with more than one parasite Unoccupied portion by parasite shows a dotted or stripped appearance “ Schuffner’s dot” Schizont Represent the full grown trophozoite Contain 12-24 merozoits Arranged in the form of rosette with yellow brown pigment at the center Gamatocyte Certain schizont get modified and result in sexual forms. Merozoite arising from single schizont are either all males or females Microgamatocyte : Spherical. Cytoplasm light blue Macrogamatocytes : spherical. Cytoplasm deep blue

67. Disadvantages of the Peripheral Blood Smear Provides information that cannot be obtained from automated cell counting. However, some limitations are: Experience is required to make technically adequate smears. There is a non-uniform distribution of white blood cells over the smear, with larger leukocytes concentrated near the edges and lymphocytes scattered throughout. There is a non-uniform distribution of RBCs over the smear, with small crowded red blood cells at the thick edge and large flat red blood cells without central pallor at the feathered edge

68. Thank you