1_Dr Jasmita Das Peripheral blood smear examination with Romanowsky stains.pptx

Staining

1. Peripheral blood smear examination with Romanowsky stains Dr. Jasmita, Assistant Professor (Hematopathology) Department of Hematology , All India Institute of Medical Sciences, New Delhi [email protected] , [email protected] 10 th Basic Hematopathology Course, 2023

2. Outline of today’s talk Utility of peripheral smear examination Romanovsky stains & staining principle Things to observe on various powers Cases to show abnormalities on Peripheral smear

3. Evaluation of a patient Thorough clinical history Clinical examination Lab investigations

4. Peripheral smear

5. Peripheral smear Components Head Body Tail Stain: Romanovsky stains Junction of body and tail: best for morphology Battlement method of counting

6. Common Romanowsky stains Wright stain Leishman stain Giemsa stain Jenner’s stain Panoptic stains: combination of two stains Improves the staining of cytoplasmic granules and other bodies like nucleoli of blast cells MGG WG JG Optimal for staining quality

7. Romanowsky stains Generic description of the azure B/polychromed methylene blue – eosin stains family Polychromy : Red+blue+purple+magenta colours Basic dye: Azure B>Azure A Acidic dye: Eosin Y=Eosin B Polychrome methylene blue- mix of dyes including Azure B; it is sufficient to ensure that the stains contain at least 80% of the appropriate dye Polychroming: oxidative treatments such as exposure to atmospheric oxygen under alkaline conditions or acidic dichromate solution Demethylation and eventually deamination of methylene blue

8. Mechanism of Romanowsky stains

9. Gross appearance  Too pink Correct stain Too blue

10. Microscopic appearance Less staining time Increased staining time

11. Water effect and overstaining

12. Low power examination General assessment of TLC 10X: General assessment of DLC; especially useful in samples with leucopenia Large platelet clumps; platelet size Fibrin threads Screening for filaria 20X objective: useful tool for DLCs in patients on chemotherapy Red cell morphology

13. Low power examination 40X DLC Red cell morphology Screening for parasites Auer rods in blasts Assessment of dysgranulopoiesis 100X Estimated platelet count in thrombocytopenic samples Basophilic stippling in red cells & other red cell inclusions Confirmation of Auer rods

14. Utility of P/S examination in Hematology

15. Most P/S evaluations are for checking low Platelet counts Pseudothrombocytopenia

16. Other reasons of pseudothrombocytopenia

17. Case 1: 35-year-old male Carrying an outside report of thrombocytopenia Referred to us for preoperative approval for cholecystectomy Etiology of giant platelets Recovery from thrombocytopenia MYH11 related disorders Bernard Soulier syndrome

18. Our patient Working in Delhi Resident of Kolkata Giant platelets on the smear Inherited giant plt disorder described in West Bengal and Bangladesh called Harris syndrome IPF: 28%; manual Plt 100X10^9/L Highly active platelets No bleeding and none expected Naina VKH, Nair SC, Daniel D, George B,  Chandy M. Asymptomatic constitutional macrothrombocytopenia among West Bengal blood donors.  Am J Med  2002;  112 : 742–3.

19. Other abnormalities that may be seen MYH9-RD WAS GPS BSS

20. Identify the abnormality on this smear Thrombocytosis Basophilia

21. Case 2 21-year-old male Symptomatic anemia for 2 years. Required 6 units of PRBC transfusions in the last 2 years There is a history leg cramps He also reports few episodes of abdominal pain.

22. Peripheral smear Megaloblastic anemia

23. Bone marrow: nearly always avoidable in classical cases of megaloblastic anemia If done: Perl’s stain should always be performed to rule out concomitant IDA

24. Other investigations Baseline reticulocyte count: 4.57%; another report prior to transfusion- 13.6% Very unusual in a case of megaloblastic anemia Workup for hemolysis initiated: No specific features of any hemolytic anemia on peripheral smear LDH: 670U/L (upto 180U/L) Bil (T/I): 4.0/ 3.0 mg/dL AST and ALT WNL Haptoglobin: Undetectable Urine dipstick: positive for blood Urine microscopy: only 2RBCs/ hpf Urine for Hemosiderin positive G6PD normal DAT negative HbHPLC done earlier normal Viral markers: Negative for HIV/ HCV/ HBV Hemoglobinuria

25. Hemolytic anemia with secondary folate deficiency and depleted iron stores Suspect: Paroxysmal nocturnal hemoglobinuria

26. Unexplained hemolysis: Flow cytometry for PNH performed Megaloblastic anemia may occur in case of hemolytic anemias due to an increased requirement. May mask the cause of hemolysis and the patient requires workup after correction of folate deficiency Rule out PNH in case of unexplained hemolysis

27. Case 3A BM from a 50-year-old lady Complaints of mild hyperbilirubinemia off and on, Spleen 1cm bcm Suspected auto-immune disease/ MDS Hb: 90g/L, TLC: 8.5X10 9 /L, Platelets: 350X10 9 /L Bone marrow received Normoblastic erythroid hyperplasia, mild dyserythropoiesis Myeloid series and megakaryocytes normal in morphology

28. Going back to the history History of weakness off and on after birth of 2 nd child Similar symptoms in patient’s son and daughter AD pattern of inheritance with spherocytes: Hereditary spherocytosis Missed P/S examination led to an unnecessary bone marrow

29. HS: Peripheral smear spherocytosis + AD history sufficient to make the diagnosis

30. MFI: 5513; mean of 5 ctrls MFI: 3889 %↓ in EMA: 29% %residual cells: <1% EMA dye binding test Flow cytometric OFT

31. Case 3B: 50-year-old male with jaundice since 1week with increasing fatigue and mild splenomegaly

32. Autoimmune hemolytic anemia

33. Polyspecific DAT

34. Case 4 55-year-old female R/O Delhi presents in December with Complaints of Fatigue for 2 weeks Jaundice for 2 weeks Painful dark fingertips No history of fever No past H/O blood transfusions An attempt to transfuse her was made but Transfusion could not be given due to inability of blood bank to find an exact match Multiple samples hemolysed O/E- Pallor+/ Icterus+/ no palpable LAP/ no Petechiae/ Spleen 2cm bcm CT chest and abdomen- Multiple intra-abdominal LAP largest measuring 2X3cm

35. Peripheral smear

36. Jenner Giemsa X1000

37. Smears prepared after warming Jenner Giemsa X100- Post-warming Jenner Giemsa X100- Pre-warming

38. Case 4: 56-year-old male presenting with anemia in winter months Peripheral smear Bone marrow Cold type AIHA secondary to WM

39. After warming at 37degC for 1 hour Multiple samples may be reported as hemolysed for these patients

40. Cold type AIHA Children Mycoplasma pneumonie / influenza infection Self resolving Middle-aged and older Adults Usually associated with a CLPD specially WM CAD recognized as a distinct entity May be the first presenting sign Should trigger Bone marrow examination and flow cytometry to exclude a clonal B-cell population

41. Bone marrow done in this case revealed WM CD20

42. Monospecific DAT

43. Case 5 32-year female PLHA on HAART Referred for unexplained anemia and thrombocytopenia from Bihar

44. Peripheral smear

45. What are schistocytes/ schizocytes RBC fragments produced by extrinsic mechanical damage within the circulation. Derived from the Greek word schisto, broken or cleft, or the correspondent verb schizo Whenever present: should trigger workup for TMA (TTP/ HUS) Important for monitoring of transplant associated TMA Critical alert- Inform the treating team at the earliest. Also occur with a multitude of causes: Structural abnormalities of the heart and great vessels (often a malfunctioning prosthetic valve (Marsh & Lewis, 1969) HELLP syndrome Malignant hypertension Metastatic cancer.

46. Pseudothrombotic microangiopathy non-TMA-related genetic or acquired RBC disorders RBC membrane defects Thalassemia Megaloblastic anemia Primary myelofibrosis Thermal injuries Key differentiating finding: Marked anisopoikilocytosis ; additional RBC size and morphological changes, not specific for the diagnosis of TMA Additional: DIC autoimmune atrophic gastritis splenic angiosarcoma COVID-19 with high levels of VWF, LDH, Low ADAMTS and decreased platelet counts

47. Total/ Indirect Bilirubin : 1.67/ 1.06 mg/ dL LDH: 855U/L (<420) Serum B12: >2000 pg/ mL (197-771) Serum folate : >20 ng / mL (3.1-17.5)

48. Morphology of schistocytes

49. Changes in the 2021 update Schistocyte range for healthy term neonate: 1% Pre-term neonates: normal range <5% Counting at 40X included formally Slight attenuation of staining intensity in central part accepted Lack of schistocytes does not exclude TMA diagnosis Confirmation of high negative predictive value of FRC TMA- MAHA, MAHAT Transplant associated TMA: requires further evaluation Pre-analytic variables: Smear should be prepared within 3 hours of collection if at RT and for 8 hours if stored at 4˚C. Standardised staining protocol

50. Thrombotic microangiopathy TTP HUS- Typical and atypical DIC HELLP syndrome Antiphosphopholipid syndrome Heparin induced thrombocytopenia with thrombosis Drug associated/ cancer associated/ transplant associated/ autoimmune disease associated TMA Malignant hypertension HIV Drug induced Cyclosporine Mitomycin C Gemcitabine Tacrolimus Clopidogrel estrogen/progesterone Interferon Quinine Ticlopidine Our patient: HIV associated TMA

51. Other hemolytic anemias where P/S is diagnostic G6PD deficiency Sickle cell anemia in sickle crisis; autosplenectomy

52. Peripheral smear: thalassemia major Wright Giemsa X400 Wright Giemsa X1000

53. Case 6: 60-year male with massive splenomegaly

54. Smear review

55. Differentials of hairy cell leukemia

56. Case 7 45-year-old male, k/n c/o Rheumatoid arthritis Generalised weakness for 3-4 months Parameter CBC 03/04/2017 Value Hemoglobin 70g/L TLC 11.82X10^9/L Platelets 755X10^9/L MCV 68.6 fl RET –He 20.8pg Corrected reticulocyte count 0.86% DLC - N - 23%, L-72%, M-3%, E-2% Absolute lymphocyte count 6146/µL

57. Peripheral blood flow cytometry

58. LGL leukemia with IDA and thalassemia trait Thrombocytosis secondary to IDA Large granular lymphocytic leukemia Serum ferritin: 8 ng/mL Thalassemia trait confirmed on HPLC STAT3 p.Y640F mutation positive https://doi.org/10.3324/haematol.2017.186338

59. Other abnormalities Cleaved cells: Follicular Mantle Some cases of CLL

60. CLL

61. DLBCL

62. ALCL T-PLL

63. Case 8 54-year-old female Admitted with Swine Flu, bilateral pneumonia Hb: 7.1 g/dl; RBC: 2.17 mill/µl; PCV: 21.2%; MCV: 97.8 fl ; MCH: 32.7 pg ; MCHC: 33.4 g/dl; RDW:20.9%; platelets:100,000/µl and TLC:5,800/µl

64. Plasma cell leukemia

65. Myeloma panel on PB 22% circulating PCs CD56+ CD45-/lo CD19 partial loss C λ restricted Plasma cell leukemia

66. Case 9A 56-year-old lady with symptomatic anemia No cause identified Progressively developed thrombocytopenia (Lowest platelet count-50X10 9 /L) but no manifestations of bleeding

67. Leucoerythroblastic blood picture Myelophthisis Myeloproliferative neoplasms-PMF Acute Hemolysis Infections Granulomatous inflammation of BM Severe trauma/ blood loss Small child with HSM: Storage disorders/ Osteopetrosis/ Vitamin D deficiency

68. Case 9A: Bone marrow Metastatic carcinoma

69. Case 9B: 60-year female, anemia req PRBC

70. Hemogram

71. Overt PMF Primary myelofibrosis P/S clues: Basophilia with leucoerythroblastosis and prominent tear drops

72. Case 10: 45-year-old female with abdominal pain and splenomegaly

73. Chronic myeloid leukemia in chronic phase

74. Case 11: 30-year female with bleeding per vaginum

75. Hypogranular APL Suspicion of APL on P/S: raise a critical alert Therapy is to be instituted with ATRA without waiting for PML::RARA and BM

76. 21/F Pallor, fatigue x 2 weeks Skin bleeds, epistaxis, menorrhagia x 2 weeks

77. Jenner Giemsa X400

78. MPO cytochemistry X400 MPO cytochemistry X1000

79. Jenner Giemsa X400 Look for schistocytes when you see APL & monocytic leukemias

80. Blasts in megakaryoblastic leukemia

81. Case 12: 65-yr-male with Anemia not responding to therapy MDS: Dysgranulopoiesis

82. Features of dysplasia Dysmyelopoiesis Dyserythropoiesis Dysmegakaryopoiesis Pseudo Pelger-Huet abnormality and hyposegmented neutrophils Hypogranularity Small or unusually large size Nuclear hypersegmentation Dohle bodies Auer rods Pseudo Chediak Higashi granules Nuclear budding Irregular nuclear membrane Internuclear bridging Karyorrhexis Multinuclearity Megalobalstosis Ring sideroblasts Cytoplasmic vacuolization Per-iodic acid Schiff positivity Micromegakaryocytes Nuclear hypolobation and monolobation Multinuclear megakaryocytes with presence of widely separated equally sized nuclei

83. MDS with defining genetic abnormalities Hb<12/13g/dL; ANC<1800/µL, Platelets <150,000/µL (BMA, BMBx )+CTG+NGS Biallelic TP53 mutation + blasts <20% on PB & BM; proerythroblasts <30% Biallelic TP53 mutation: NGS detected: VAF ≥50% (presumptive) ≥2 TP53 mutations 1 TP53 mutation with e/o TP53 copy number loss or cnLOH PB blasts <2%, BM blasts <5% CTG: del5q/ del5q with 1 anomaly except monosomy 7 or 7q deletion MDS with biallelic TP53 inactivation (usually a/w complex karyotype) MDS with low blasts and 5q deletion (MDS-5q) SF3B1 mutation (VAF≥5%) & RS ≥5% & CTG: Absence of 5q deletion, monosomy 7/ or complex karyotype MDS with low blasts and SF3B1 mutation (MDS- SF3B1 ) ≥15% RS & CTG: Absence of 5q deletion, monosomy 7/ or complex karyotype MDS with low blasts and ring sideroblasts

84. MDS morphologically defined Hb<12/13g/dL; ANC<1800/µL, Platelets <150,000/µL Blast %age on BMA, BMBx PB blasts <2% and BM blasts <5% Check age adjusted cellularity Age adjusted Cellularity ≤25% MDS Hypoplastic (MDS-h) Age adjusted Cellularity >25% MDS with low blasts (MDS-LB) MDS-LB-SLD/ MDS-LB-MLD PB blasts ≥2% & <20%, BM blasts ≥5% & <20% MDS with increased blasts (MDS-IB) PB blasts 2-4% &/or BM blasts 5-9 % MDS-IB1 PB blasts 5-19% &/or BM blasts 10-19% &/or Auer rods MDS-IB2 BM fibrosis MDS with fibrosis (MDS-f) MDS-U no longer exists as CCUS is now recognised

85. Case 13 85-year-old retired doctor Father of a Radiologist Shoulder joint pains for which he received steroids CBC showed leucocytosis and monocytosis 3 months down the line, monocytosis persisted and he developed anemia and Intermittent fever

86. Peripheral smear

87. CMML

88. Case 14: 9-month-old girl with fever, pallor, hepato-splenomegaly and cervical lymphadenopathy. JMML

89. Case 14: A 7-year-old female presented with high fever (102.4°F ) with chills & cough – for 5 days No hepatosplenomegaly CBC: Hb : 8.2 g/dl, TLC: 3600/Cu.mm, Platelet count : 1,79,000/cu.mm Chest x-ray –Lower lobe consolidation-Right lung Past history : Four similar episodes in the last two years Bone marrow: Reported as HLH elsewhere

90. Chediak Higashi syndrome Case courtesy: Prof. Sunita Sharma, LHMC

91. Case 15: A 23-year-old male, referred with no history

92. CBC

93. Other cases

94. Take home message Peripheral smear is a basic yet indispensable first-line investigation It is as important to a hematopathologist as a careful physical examination to a physician. A good 100-200 cell differential should be performed when WBC differential is flagged Polychromasia provides a useful hint to an underlying hemolytic anemia/ anemia on to therapy Diligent P/S examination can reveal the diagnosis in many hematological diseases as well as guide further work-up and avoid un-necessary invasive investigations Important to raise critical alerts ex suspicion of APL/ blasts/ schistocytes/ P. falciparum

95. Thank You