LABORATORY INVESTIGATIONS IN PANCYTOPENIA

MODERATOR- DR. HEMALATHA A.PRESENTER- DR. ANKITA BAGHEL

OBJECTIVES-

1. DEFINITION OF PANCYTOPENIA2. CAUSES3. INVESTIGATIONS4. BLOOD EXAMINATION5. BONE MARROW EXAMINATION6. OTHER TESTS7. CASES8. FEW INDIVIDUAL DISEASES9. SUMMARY

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)

CAUSES OF PANCYTOP

ENIA

INHERITED ACQUIRED

INHERITED CAUSES-

1. FANCONI ANEMIA2. DYSKERATOSIS CONGENITAL3. SHWACHMAN-DIAMOND

SYNDROME4. AMEGAKARYOCYTIC

THROMBOCYTOPENIA5. DIAMOND BLACKFAN

SYNDROME

CAUSES OF PANCYTOPEN

IA

HYPOCELLULAR BONE

MARROW

CELLULAR BONE

MARROW WITH PRIMARY MARROW

DISORDERS

CELLULAR BONE

MARROW WITH SYSTEMIC

DISORDERS

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

HYPOCELLULAR

NORMOCELLULAR

CELLULAR BONE MARROW WITH PRIMARY MARROW DISORDERS

1. ACUTE LEUKEMIA/ LYMPHOMA2. HAIRY CELL LEUKEMIA3. MYELOFIBROSIS4. MYELODYSPLASTIC LEUKEMIAS5. PAROXYSMAL NOCTURNAL

HAEMOGLOBINURIA6. MULTIPLE MYELOMA7. HAEMOPHAGOCYTIC

LYMPHOHISTIOCYTOSIS

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

NORMOCELLULAR

HYPERCELLULAR

MOST COMMON CAUSES IN DEVELOPING COUNTRY

1. Megaloblastic anemia2. Infections3. Hypersplenism4. Aplastic anemia5. Drug induced pancytopenia6. Acute myeloid leukemia

INVESTIGATIONS??1. HISTORY

2. PHYSICAL EXAMINATION

3. LABORATORY INVESTIGATIONS

4. SPECIFIC INVESTIGATIONS

HISTORY

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.

CLINICAL FINDINGS

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

LABORATORY INVESTIGATIONS??

FOR ALL CASES :

1. PERIPHERAL BLOOD SMEAR

2. BONE MARROW ASPIRATION AND TREPHINE BIOPSY

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

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

ROULEAUX FORMATION:MULTIPLE MYELOMA

RBC INDICES

• RDW- Usually increased• MCV- maybe increased or normal• RETICULOCYTE COUNT- Define severity and differentiate production vs. destruction.

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

WBC AND RBC PRECURSORS ARE NOT TYPICAL OF APLASTIC ANEMIA.

SO THEIR PRESENCE IN PANCYTOPENIA SUGGEST DIAGNOSIS OTHER THAN

APLASTIC ANEMIA.

RBC INCLUSIONS- HOWEL JOLLY BODIES

3. PLATELETS

• NORMAL PLATELETS- APLASTIC ANEMIA• GIANT PLATELETS- LEUKEMIA - MDS

4. ABNORMAL GRANULATION IN NEUTROPHILS-

• TOXIC GRANULES- INFECTIONS.

• HYPOGRANULAR NEUTROPHILS- MYELODYSPLASTIC SYNDROME

- ACUTE NON-LYMPHOBLASTIC LEUKEMIA

5. HYPOSEGMENTATION AND HYPERSEGMENTATION IN

NEUTROPHILS-

• PELGER HUET LIKE CELLS- MDS - SOME LEUKEMIAS

• HYPERSEGMENTATION- MEGALOBLASTIC LEUKEMIA..

HYPERSEGMENTATION

6. ESR

• INCREASED

• >150 mm/hr - MULTIPLE MYELOMA - MACROGLOBULINEMIA - INFECTIONS

BONE MARROW EXAMINATION

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)

HYPOCELLULAR

HYPERCELLULAR

SPECIFIC INVESTIGATIONS

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

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

CASE 1

HISTORY

•A 30yrs old male,presented to OPD with malaise, tiredness and weakness.•He is a known alcoholic.

PHYSICAL FINDINGS

INVESTIGATIONS• PERIPHERAL BLOOD SMEAR-

Anisopoikilocytosis - Macro-ovalocytes - Hypersegmented

Neutrophil

• RBC INDICES- MCV- 110 - RETICULOCYTE COUNT- low

DIAGNOSIS??

MEGALOBLASTIC ANEMIAS

DEFINITION-

• Impaired DNA synthesis due to

deficiency of vitamin B12

and folic acid.

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

•Increase in Homocysteine and

Methy malonic acid – Vit B 12

Deficiency

•Only increase in Homocysteine :

Folate deficiency

ATROPHIC GLOSSITIS KNUCKLE HYPERPIGMENTATION

“BONE MARROW EXAMINATION IS NOT REQUIRED FOR THE DIAGNOSIS OF MEGALOBLASTIC ANEMIA”

CASE 2

HISTORY

• A 10yrs old girl presents with pallor and weakness.• Congenital anomalies seen• Family history of cancer.

PHYSICAL FINDINGS

EPICANTHAL FOLDS

ABSENT THUMB

SHORT STATURE

MICROCEPHALY

HYPERPIGMENTATION

HYPOGONADISM

ABSENT RADIUS

DIAGNOSIS??

FANCONI ANEMIA

• Inherited syndrome• Autosomal recessive• Includes- Pancytopenia - Congenital anomalies - Cancer susceptibility

FURTHER WORK-UP

• Demonstration of increased chromosomal breakage in the presence of DNA cross-linking agents such as MITOMYCIN C or DIEPOXYBUTANE

“No other constitutional pancytopenia is

associated with an abnormal chromosomal

breakage study”

CASE 3

HISTORY

•A 55yrs old male on chemotherapy presents with pallor and dyspnea.•Also complains of petechiae and frequent minor infections.

INVESTIGATIONS

• PERIPHERAL BLOOD SMEAR- Pancytopenia• BONE MARROW ASPIRATION- Dry tap

DIAGNOSIS??

FURTHER WORK-UP

•BONE MARROW BIOPSY- Hypocellular marrow•No Splenomegaly

HYPOCELLULAR BONE MARROW BIOPSY

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

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

DRUGS CAUSING APLASTIC ANEMIA

• Anti cancer drugs :Alkylating agents Antimetabolities Antimitotics

• Antibiotics : Streptomycin Tetracycline Methicillin Chloramphenicol

• Anti inflammatory drugs : Indomethacin Ibuprofen Aspirin

• Anti thyroid : Methimazole Methylthiouracil Propylthiouracil

• Anti hypertensive : Methyldopa

• Anticonvulsants : Hydantoins Carbamazepine

• Antihistaminics : Cemitidine Chlorpheniramine

“Most common cause Of Aplastic Anemia is IDIOPATHIC”

CASE 4

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.

INVESTIGATIONS

• PERIPHERAL BLOOD EXAMINATION- Hemolytic picture seen• BONE MARROW EXAMINATION- Hypoplastic

DIAGNOSIS??

FURTHER WORK UP• HAM’S TEST

FLOW CYTOMETRY

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)

“ Best Diagnostc test of Paroxysmal Nocturnal Haemoglobinuria is by FLOW CYTOMETRY ”

CASE 5

HISTORY

• A 8yr old child comes with sudden onset fever and fatigue.• Also gives history of recurrent pneumonia.

• On examination- generalised lymphadenopathy present.

INVESTIGATIONS

DIAGNOSIS??

FURTHER WORK-UP• CYTOGENETIC STUDY- t(12;21) present.

ACUTE LYMPHOBLASTIC LEUKEMIA

ACUTE LYMPHO--BLASTIC LEUKEMIA

CASE 6

HISTORY• A 40 yrs old male complains of fever and malaise.

• On examination had gum hypertrophy and splenomegaly.

INVESTIGATIONS• PERIPHERAL BLOOD EXAMINATION-

DIAGNOSIS??

ACUTE MYELOID LEUKEMIA- M5

FURTHER WORK-UP• BONE MARROW EXAMINATION-

ACUTE MYELO-

-BLASTIC LEUKEMIA

ACUTE LEUKEMIAS

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

CASE 7

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.

INVESTIGATIONS• PERIPHERAL BLOOD EXAMINATION-

Pancytopenia - Nucleated RBC’s - Neutrophil with

two lobes

DIAGNOSIS??

FURTHER WORK-UP

• BONE MARROW EXAMINATION- Ring sideroblasts seen

- Megakaryocytes with multiple nuclei.

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

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)

CASE 8

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.

INVESTIGATIONS

• CHEST X-RAY- Shows pleural effusion.• PERIPHERAL BLOOD SMEAR- Pancytopenia.

DIAGNOSIS??

FURTHER WORKUP

• BONE MARROW EXAMINATION- Granuloma• ZN STAIN- Shows acid fast bacilli.• HIV POSITIVE

DISSEMINATED TUBERCULOSIS

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

CASE 9

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.

INVESTIGATIONS• PERIPHERAL BLOOD SMEAR-

Anisopoikilocytosis - Macrocytosis - Target cells - Stomatocytes

DIAGNOSIS??

FURTHER WORKUP

• SONOGRAPHY- Enlarged spleen• BONE MARROW EXAMINATION- Normocellular marrow with hematopoeisis• HEPATITIS VIRUS STUDIES- Negative for A, B and C.

HYPERSPLENISM• Splenic hyperactivity with increased blood

cell destruction.

Diagnostic criteria 1. Splenomegaly 2. Pancytopenia 3. Normal or hypercellular bone marrow 4. Reversibility by splenectomy

MYELOFIBROSIS• Fibrosis of the bone marrow

• Etiology : Due to dysregulated production PDGF and TGF.

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.

HAEMOPHAGOCYTIC SYNDROME

• Also called Hemophagocytic lymphohistiocytosis

Clinical features• Fever • Hepatosplenomegaly • Jaundice • Lymphadenopathy • Rash

LABORATORY FINDINGS

• Histiocytosis

• Hemophagocytosis

• Pancytopenia

• Eleveted serum ferritin

• Elevated liver enzymes

DYSKERATOSIS CONGENITA

• RARE inherited disorder.• X-linked Recessive, autosomal dominant, autosomal recessive.• Pancytopenia + dematological manifestation.• Nail dystrophy and leukoplakia.

Mutations in DKC1 at band Xq28

Dermatological manifestations

INFECTIONS• HIV • Infectious mononucleosis• Hepatitis B• Hepatitis C• Measles• Hepatitis A• Parainfluenza• Influenza

KALAZAR

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).

SARCOIDOSIS- Non caseating granulomas

BONE MARROW BIOPSY

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

DIAGNOSIS

1. Peripheral blood findings• Anemia

2. Bone marrow aspiration & biopsy• Cells not constituent to marrow.• Myelofibrosis & Necrosis• Special stains & IHC

3. BIOCHEMICAL INVESTIGATIONS-

• Hypercalcemia

• Elevated Alkaline phosphatase

• Lactate dehydrogenase

• Parathyroid hormone related protein

• Specific tumor markers

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

Metastasis melanoma- bone marrow biopsy

Metastasis PROSTATE- bone marrow biopsy

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.

SUMMARY

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

THANK YOU

“ LOOK CAREFULLY ,

YOUR PATIENTS AND

THEIR BLOOD FILMS ”