laboratory investigations in pancytopenia
TRANSCRIPT
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LABORATORY INVESTIGATIONS IN PANCYTOPENIA
MODERATOR- DR. HEMALATHA A.PRESENTER- DR. ANKITA BAGHEL
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OBJECTIVES-
1. DEFINITION OF PANCYTOPENIA2. CAUSES3. INVESTIGATIONS4. BLOOD EXAMINATION5. BONE MARROW EXAMINATION6. OTHER TESTS7. CASES8. FEW INDIVIDUAL DISEASES9. SUMMARY
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WHAT IS PANCYTOPENIA?
1.Hemoglobin concentration < 10 g/dL, or Absolute reticulocyte count < 40,000/μL (40 ×
109/L).
2. Total leucocyte count <4000/μL(4x109/L)Absolute Neutrophil count < 1500/μL (1.5 ×
109/L), 3.Platelet count < 150,000/μL (150 × 109/L)
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CAUSES OF PANCYTOP
ENIA
INHERITED ACQUIRED
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INHERITED CAUSES-
1. FANCONI ANEMIA2. DYSKERATOSIS CONGENITAL3. SHWACHMAN-DIAMOND
SYNDROME4. AMEGAKARYOCYTIC
THROMBOCYTOPENIA5. DIAMOND BLACKFAN
SYNDROME
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CAUSES OF PANCYTOPEN
IA
HYPOCELLULAR BONE
MARROW
CELLULAR BONE
MARROW WITH PRIMARY MARROW
DISORDERS
CELLULAR BONE
MARROW WITH SYSTEMIC
DISORDERS
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HYPOCELLULAR BONE MARROW CAUSES
1.APLASTIC ANEMIA2.HYPOPLASTIC MYELODYSPLASTIC
SYNDROME3.LYMPHOMA IN HYPOPLASTIC BONE MARROW4.CYTOTOXIC AGENTS AND RADIOTHERAPY
VERY RARE- ACUTE LEUKEMIA IN HYPOPLASTIC BONE MARROW
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HYPOCELLULAR
NORMOCELLULAR
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CELLULAR BONE MARROW WITH PRIMARY MARROW DISORDERS
1. ACUTE LEUKEMIA/ LYMPHOMA2. HAIRY CELL LEUKEMIA3. MYELOFIBROSIS4. MYELODYSPLASTIC LEUKEMIAS5. PAROXYSMAL NOCTURNAL
HAEMOGLOBINURIA6. MULTIPLE MYELOMA7. HAEMOPHAGOCYTIC
LYMPHOHISTIOCYTOSIS
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CELLULAR BONE MARROW WITH SYSTEMIC DISORDERS
1. METASTATIC SOLID TUMOURS2. HYPERSPLENISM3. DEFICIENCY OF VITAMIN B12 AND FOLIC ACID
4. INFECTIONS: -SEPSIS - TB - KALA AZAR - BRUCELLOSIS5. OVERWHELMING INFECTIONS6. ALCOHOL7. AUTOIMMUNE DISORDERS- SLE, SJOGREN’S8. SARCOIDOSIS9. STORAGE DISEASE- GAUCHER, NIEMANN-PICK
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NORMOCELLULAR
HYPERCELLULAR
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MOST COMMON CAUSES IN DEVELOPING COUNTRY
1. Megaloblastic anemia2. Infections3. Hypersplenism4. Aplastic anemia5. Drug induced pancytopenia6. Acute myeloid leukemia
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INVESTIGATIONS??1. HISTORY
2. PHYSICAL EXAMINATION
3. LABORATORY INVESTIGATIONS
4. SPECIFIC INVESTIGATIONS
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HISTORY
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SYMPTOMS OCCURENCEDuration of symptoms MDS (long)
Severe Aplastic Anemia (short history)
Family history Congenital or Hereditary disease
Age of the patients MDS in Adult
Previous treatments/ Exposures Radiotherapy or ChemotherapyMDS, AML
Drugs Toxic effect
Chronic alcohol abuse Hepatopathy
Pain crisis, black urine crisis Paroxysmal nocturnal haemoglobinuria
Bleeding, infections Estimation of the degree of pancytopenia.Eventually additional hemostatic problems.
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CLINICAL FINDINGS
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FINDINGS OCCURENCELymphadenopathy Lymphoma, Hodgkin lymphoma,
also viral infection.
Splenomegaly Not found in MDS or Aplastic anemia.Typical in Myeloproliferative disorders, sometimes in acute leukemia.
Hepatomegaly Myeloproliferative disorders, Lymphoma
Mediastinal bulk Lymphoma,Hodgkin’s disease
Excessive bleeding Decreased Platelets
Bony tenderness Multiple Myeloma, Metastasis
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LABORATORY INVESTIGATIONS??
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FOR ALL CASES :
1. PERIPHERAL BLOOD SMEAR
2. BONE MARROW ASPIRATION AND TREPHINE BIOPSY
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PERIPHERAL BLOOD SMEAR-
1. ANISOCYTOSIS AND POIKILOCYTOSIS.
2. WBC AND RBC PRECURSORS.3. PLATELETS.4. ABNORMAL INCREASED OR
DECREASED GRANULATION IN NEUTROPHILS.
5. HYPO/HYPERSEGMENTATION IN NEUTROPHILS.
6. ESR
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1. ANISOCYTOSIS AND POIKILOCYTOSIS-
MODERATE DEGREE IS COMMON
• Very marked Poikilocytosis- MYELOFIBROSIS
• Less degree- APLASTIC ANEMIA, MARROW INFILTRATION BY LYMPHOMA/ MULTIPLE MYELOMA.
• Conspicuous- METASTASIS BONE CARCINOMA
• Invariably absent- ACUTE LEUKEMIA
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ROULEAUX FORMATION:MULTIPLE MYELOMA
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RBC INDICES
• RDW- Usually increased• MCV- maybe increased or normal• RETICULOCYTE COUNT- Define severity and differentiate production vs. destruction.
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2. WBC AND RBC PRECURSORS
• BLAST CELLS- MYELOFIBROSIS SUBLEUKAEMIC LEUKEMIA• METASTATIC CARCINOMA IN BONE
LESS CHARACTERISTIC: - PLASMACYTIC CELLS- MULTIPLE MYELOMA - IMMATURE LYMPHOCYTES- MARROW INVOLVEMENT BY LYMPHOMA
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WBC AND RBC PRECURSORS ARE NOT TYPICAL OF APLASTIC ANEMIA.
SO THEIR PRESENCE IN PANCYTOPENIA SUGGEST DIAGNOSIS OTHER THAN
APLASTIC ANEMIA.
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RBC INCLUSIONS- HOWEL JOLLY BODIES
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3. PLATELETS
• NORMAL PLATELETS- APLASTIC ANEMIA• GIANT PLATELETS- LEUKEMIA - MDS
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4. ABNORMAL GRANULATION IN NEUTROPHILS-
• TOXIC GRANULES- INFECTIONS.
• HYPOGRANULAR NEUTROPHILS- MYELODYSPLASTIC SYNDROME
- ACUTE NON-LYMPHOBLASTIC LEUKEMIA
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5. HYPOSEGMENTATION AND HYPERSEGMENTATION IN
NEUTROPHILS-
• PELGER HUET LIKE CELLS- MDS - SOME LEUKEMIAS
• HYPERSEGMENTATION- MEGALOBLASTIC LEUKEMIA..
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HYPERSEGMENTATION
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6. ESR
• INCREASED
• >150 mm/hr - MULTIPLE MYELOMA - MACROGLOBULINEMIA - INFECTIONS
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BONE MARROW EXAMINATION
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FEATURES OCCURRENCECELLULARITY HYPERCELLULAR- Myeloproliferative
disorders, HyperslenismDRY TAP- Myelofibrosis, Carcinoma, Non-Hodgkin’s lymphomaHYPOPLASTIC- Myelodysplastic syndromes.
ERYTHROPOIESIS NONE- ErythroaplasiaDYSPLASTIC- MDS, some AMLINCREASED- Hemolysis
MYELOPOIESIS DYSPLASTIC- Myelodysplastic syndromeDominating,mrophologically normal in Myeloproliferating disorders.
BLASTS Counting for Myelodysplastic disorders
MEGAKARYOPOIESIS DYSPLASTIC- Myelodysplastic disorder
OTHER CELLS Reedsternberg cell and Hodgkin cellBacteria, Fungus, Parasite, VirusesLD bodies
ALIP(ABNORMAL LOCATION OF IMMATURE PRECURSORS)
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HYPOCELLULAR
HYPERCELLULAR
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SPECIFIC INVESTIGATIONS
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TEST RATIONALEBONE X-RAYS Multiple myeloma, metastasis.
BLOOD CULTURE Infectious agent- Tuberculosis or virus.
VITAMIN B12 AND FOLATE ASSAYS Megaloblastic anemia
ASPARTATE AMINOTRANSFERASE, ALANINE AMINOTRANSFERASE, GAMMA GLUTAMYL TRANSFERASE, BILIRUBIN
Evaluate hepatitis
BLOOD UREA NITROGEN, CREATININE Assess for Chronic Renal Failure
SEROLOGY For HIV, EBV, Hepatitis
HAM’S TEST Paroxysmal Nocturnal Haemoglubinuria
CHROMOSOMAL BREAKAGE STUDIES Fanconi anemia
DNA ANTIBODY, LUPUS ERYTHEMATOSUS CELL TEST
Systemic Lupus Erythematosus
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PANCYTOPENIA AT INITIAL EVALUATION
REFER PATIENT FOR URGENT EVALUATION
REPEAT CBC AND BLOOD SMEAR
BONE MARROW ASPIRATE ANDTREPHINE BIOPSY
BONE MARROW CYTOGENETICS BONE MARROW IMMUNOPHENOTYPING
LFT,B12 AND FOLATE,COAGULATION PROFILE,VIRAL ETIOLOGY,AUTOIMMUNE
PROFILE
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CASE 1
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HISTORY
•A 30yrs old male,presented to OPD with malaise, tiredness and weakness.•He is a known alcoholic.
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PHYSICAL FINDINGS
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INVESTIGATIONS• PERIPHERAL BLOOD SMEAR-
Anisopoikilocytosis - Macro-ovalocytes - Hypersegmented
Neutrophil
• RBC INDICES- MCV- 110 - RETICULOCYTE COUNT- low
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DIAGNOSIS??
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MEGALOBLASTIC ANEMIAS
DEFINITION-
• Impaired DNA synthesis due to
deficiency of vitamin B12
and folic acid.
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Biochemical Assays-
• Serum B12 & Folate levels – Automated chemiluminescence
• Serum LDH levels
• Serum Methylmalonic acid & Homocysteine levels – HPLC
• Intrinsic factor antibody test
• Serum gastrin or gastric juice Ph
Upper GI endoscopy and biopsy – Villous atrophy
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•Increase in Homocysteine and
Methy malonic acid – Vit B 12
Deficiency
•Only increase in Homocysteine :
Folate deficiency
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ATROPHIC GLOSSITIS KNUCKLE HYPERPIGMENTATION
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“BONE MARROW EXAMINATION IS NOT REQUIRED FOR THE DIAGNOSIS OF MEGALOBLASTIC ANEMIA”
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CASE 2
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HISTORY
• A 10yrs old girl presents with pallor and weakness.• Congenital anomalies seen• Family history of cancer.
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PHYSICAL FINDINGS
EPICANTHAL FOLDS
ABSENT THUMB
SHORT STATURE
MICROCEPHALY
HYPERPIGMENTATION
HYPOGONADISM
ABSENT RADIUS
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DIAGNOSIS??
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FANCONI ANEMIA
• Inherited syndrome• Autosomal recessive• Includes- Pancytopenia - Congenital anomalies - Cancer susceptibility
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FURTHER WORK-UP
• Demonstration of increased chromosomal breakage in the presence of DNA cross-linking agents such as MITOMYCIN C or DIEPOXYBUTANE
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“No other constitutional pancytopenia is
associated with an abnormal chromosomal
breakage study”
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CASE 3
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HISTORY
•A 55yrs old male on chemotherapy presents with pallor and dyspnea.•Also complains of petechiae and frequent minor infections.
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INVESTIGATIONS
• PERIPHERAL BLOOD SMEAR- Pancytopenia• BONE MARROW ASPIRATION- Dry tap
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DIAGNOSIS??
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FURTHER WORK-UP
•BONE MARROW BIOPSY- Hypocellular marrow•No Splenomegaly
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HYPOCELLULAR BONE MARROW BIOPSY
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CRITERIA FOR SEVERE APLASTIC ANEMIA
At least 2 of the following peripheral blood findings:• Reticulocytes <1%, corrected for hematocrit• Absolute neutrophil count <500/μL (0.5 × 109/L)• Platelets <20,000/μL (20 × 109/L)• AND• Bone marrow biopsy with <25% normal cellularity• OR• Bone marrow biopsy with <50% normal cellularity
in which less• than 30% of the cells are hematopoietic
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CAUSES OF APLASTIC ANEMIA
ACQUIRED(80%)
• Idiopathic• Drug induced• Viral (hepatitis, EBV)• Ionising radiation• Toxins (pesticides,
benzene, arsenic)
• Pregnancy • Leukaemia
INHERITED(20%)
• Fanconi Anaemia• Dyskeratosis
congenita• Shwachman-Diamond
syndrome• Diamond-Blackfan
anemia
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DRUGS CAUSING APLASTIC ANEMIA
• Anti cancer drugs :Alkylating agents Antimetabolities Antimitotics
• Antibiotics : Streptomycin Tetracycline Methicillin Chloramphenicol
• Anti inflammatory drugs : Indomethacin Ibuprofen Aspirin
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• Anti thyroid : Methimazole Methylthiouracil Propylthiouracil
• Anti hypertensive : Methyldopa
• Anticonvulsants : Hydantoins Carbamazepine
• Antihistaminics : Cemitidine Chlorpheniramine
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“Most common cause Of Aplastic Anemia is IDIOPATHIC”
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CASE 4
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HISTORY
• A 20yr old male presents with sudden onset malaise and fatigue with recurrent abdominal pain.• He also complains of dark color urine on waking up.
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INVESTIGATIONS
• PERIPHERAL BLOOD EXAMINATION- Hemolytic picture seen• BONE MARROW EXAMINATION- Hypoplastic
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DIAGNOSIS??
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FURTHER WORK UP• HAM’S TEST
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FLOW CYTOMETRY
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PAROXYSMAL NOCTURNAL HAEMOGLUBINURIA
• PNH arises as a result of nonmalignant clonal expansion of one or more hematopoietic stem cells that have acquired somatic mutation of the X-chromosome gene PIGA (phosphatidylinositol glycan class A)
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“ Best Diagnostc test of Paroxysmal Nocturnal Haemoglobinuria is by FLOW CYTOMETRY ”
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CASE 5
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HISTORY
• A 8yr old child comes with sudden onset fever and fatigue.• Also gives history of recurrent pneumonia.
• On examination- generalised lymphadenopathy present.
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INVESTIGATIONS
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DIAGNOSIS??
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FURTHER WORK-UP• CYTOGENETIC STUDY- t(12;21) present.
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ACUTE LYMPHOBLASTIC LEUKEMIA
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ACUTE LYMPHO--BLASTIC LEUKEMIA
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CASE 6
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HISTORY• A 40 yrs old male complains of fever and malaise.
• On examination had gum hypertrophy and splenomegaly.
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INVESTIGATIONS• PERIPHERAL BLOOD EXAMINATION-
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DIAGNOSIS??
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ACUTE MYELOID LEUKEMIA- M5
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FURTHER WORK-UP• BONE MARROW EXAMINATION-
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ACUTE MYELO-
-BLASTIC LEUKEMIA
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ACUTE LEUKEMIAS
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SUBLEUKAEMIC LEUKEMIA
•MORE COMMON CAUSE OF PANCYTOPENIA THEN APLASTIC ANEMIA• PROBLEM IN DIAGNOSIS- VERY FEW OR NO BLAST CELLS IN BLOOD FILM.•DIAGNOSIS BY EXAMINATION OF BONE MARROW
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CASE 7
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HISTORY
• A 70 yrs old male patient complains of fever and weakness.• He has history of recurrent infections.• History of unprovoked bleeding from skin and gums.
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INVESTIGATIONS• PERIPHERAL BLOOD EXAMINATION-
Pancytopenia - Nucleated RBC’s - Neutrophil with
two lobes
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DIAGNOSIS??
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FURTHER WORK-UP
• BONE MARROW EXAMINATION- Ring sideroblasts seen
- Megakaryocytes with multiple nuclei.
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MYELODYSPLASTIC SYNDROMES
• The myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by cytopenias with cellular marrow and a risk for leukemic transformation.
• Features of dysplasia of hematopoietic cell lines with impairment of proliferation and differentiation of these cells.
• Hallmark – Ineffective hematopoiesis
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WHO Classification
• Refractory Anemia (RA)• Refractory Anemia with Ring Sideroblasts (RARS)• Refractory cytopenia with multilineage dysplasia
(RCMD)• MDS associated with isolated del(5q) • Childhood myelodysplastic syndrome• Refractory anemia with excess blasts-1 (RAEB-1)• Refractory anemia with excess blasts-2 (RAEB-2)• Myelodysplastic syndrome, unclassified (MDS-U)
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CASE 8
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HISTORY
• A 40 yr old male presents with fever and cough.• He also complains of fatigue and weakness.
• On examination shows enlarged cervical lymph nodes.
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INVESTIGATIONS
• CHEST X-RAY- Shows pleural effusion.• PERIPHERAL BLOOD SMEAR- Pancytopenia.
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DIAGNOSIS??
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FURTHER WORKUP
• BONE MARROW EXAMINATION- Granuloma• ZN STAIN- Shows acid fast bacilli.• HIV POSITIVE
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DISSEMINATED TUBERCULOSIS
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Granuloma in a trephine biopsy section of bone marrow from apatient with AIDS and disseminated atypical mycobacterial infection. H&E
Bone marrow granuloma from a patient with AIDS and disseminated Mycobacterium avium intracellulare infection. The macrophages contain many acid-fast bacillli. Ziehl–Neelsen stain
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CASE 9
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HISTORY
•Mr. A 55yrs old male patient, a railway worker, consulted his doctor for tiredness, malaise and anorexia.• He was found to be mildly jaundiced with an enlarged irregular hepatomegaly and considerable ascites.
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INVESTIGATIONS• PERIPHERAL BLOOD SMEAR-
Anisopoikilocytosis - Macrocytosis - Target cells - Stomatocytes
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DIAGNOSIS??
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FURTHER WORKUP
• SONOGRAPHY- Enlarged spleen• BONE MARROW EXAMINATION- Normocellular marrow with hematopoeisis• HEPATITIS VIRUS STUDIES- Negative for A, B and C.
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HYPERSPLENISM• Splenic hyperactivity with increased blood
cell destruction.
Diagnostic criteria 1. Splenomegaly 2. Pancytopenia 3. Normal or hypercellular bone marrow 4. Reversibility by splenectomy
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MYELOFIBROSIS• Fibrosis of the bone marrow
• Etiology : Due to dysregulated production PDGF and TGF.
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INVESTIGATIONS•PERIPHERAL BLOOD SMEAR- Tear drop cells.•BONE MARROW ASPIRATION- Dry tap•BONE MARROW BIOPSY- Hypocellular and fibrotic obliteration of marrow space.• JAK2 mutations present.
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HAEMOPHAGOCYTIC SYNDROME
• Also called Hemophagocytic lymphohistiocytosis
Clinical features• Fever • Hepatosplenomegaly • Jaundice • Lymphadenopathy • Rash
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LABORATORY FINDINGS
• Histiocytosis
• Hemophagocytosis
• Pancytopenia
• Eleveted serum ferritin
• Elevated liver enzymes
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DYSKERATOSIS CONGENITA
• RARE inherited disorder.• X-linked Recessive, autosomal dominant, autosomal recessive.• Pancytopenia + dematological manifestation.• Nail dystrophy and leukoplakia.
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Mutations in DKC1 at band Xq28
Dermatological manifestations
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INFECTIONS• HIV • Infectious mononucleosis• Hepatitis B• Hepatitis C• Measles• Hepatitis A• Parainfluenza• Influenza
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KALAZAR
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Parvovirus B19-induced pure red cell aplasia. BM aspirate smear - giant erythroblast with intranuclear viral inclusion, resembling a large nucleolus, and the cytoplasm may be dark blue and contain vacuoles. BM biopsy with early erythroid precursors showing glassy intranuclear inclusions ( �lantern cells).
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SARCOIDOSIS- Non caseating granulomas
BONE MARROW BIOPSY
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METASTATIC SOLID TUMORS
TUMOURS METASTASIZING TO BONE MARROW
Adults• Breast • Lung • Prostate• Kidney• Thyroid
Children• Neuroblastoma• Ewing’s sarcoma• Wilms’ tumor• Retinoblastoma• Rhabdomyosarcoma• Germ cell tumor
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DIAGNOSIS
1. Peripheral blood findings• Anemia
2. Bone marrow aspiration & biopsy• Cells not constituent to marrow.• Myelofibrosis & Necrosis• Special stains & IHC
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3. BIOCHEMICAL INVESTIGATIONS-
• Hypercalcemia
• Elevated Alkaline phosphatase
• Lactate dehydrogenase
• Parathyroid hormone related protein
• Specific tumor markers
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Bone marrow biopsy
Metastasis from an adenocarcinoma. The carcinoma cells are arranged in a well-defined tubular pattern
Showing myelofibrosis and osteosclerosis secondary to the metastatic tumor cells from unidentified primary tumor
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Metastasis melanoma- bone marrow biopsy
Metastasis PROSTATE- bone marrow biopsy
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MAIN CAUSES OF MEDICAL ERRORS IN HEMATOLOGICAL DIAGNOSTICS
• Incomplete information on the case• Bad quality of the material- blood fils, aspirates, biopsies, colorations.• No integrated diagnosis- speak together.• Confirmatory bias- to seek data that confirm a favorite hypothesis and ti interpret even the low-relevance data as being supportive of a hypothesis.
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SUMMARY
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REFERENCES
• Clinical Laboratory Hematology – Mckenzie.• De Gruchy’s Clinical Haematology in Medical Practice• Dacie and Lewis Practical hematology• Wintrobe’s Atlas of Clinical Haematology• Atlas and Textbook of Hematology
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THANK YOU
“ LOOK CAREFULLY ,
YOUR PATIENTS AND
THEIR BLOOD FILMS ”