Normal Haemopoiesis Bone Marrow Failure Iheanyi Okpala FRCPath

Normal Haemopoiesis & Bone Marrow Failure Iheanyi Okpala FRCPath. FWACP

Normal Components of Blood: consists of cells suspended in the liquid called plasma Plasma: water with carbohydrates, lipids, proteins, salts and various substances dissolved or suspended in it. Blood Cells: erythrocytes, contain Hb & transport oxygen. leucocytes, fight infections. platelets, enhance blood clotting /prevent bleeding

Normal Blood and Bone Marrow Cells

Sites of Blood Production or Haemopoiesis at different stages of development Adults: in bones near to, or in the midline: skull, sternum vertebrae, pelvis, ribs and proximal femur.

Production of blood cells begins in the haemopoietic stem cell Can reproduce itself, or differentiate to a more mature cell. Can differentiate to all types of blood cells; pluripotent (PSC) Relatively rare. 1 PSC / 104 -106 nucleated marrow cells PSCs cannot be identified by distinctive structural features. Look like small-medium sized lymphocytes. Evidence for PSC existence is functional – reconstitution of haemopoietic system after marrow transplantation.

Normal Haemopoiesis

Marrow Haemopoietic Microenvironment Stromal Cells: fibroblasts, macrophages, endothelial & fat cell

Glycoproteins That Stimulate Production and Function of Haemopoietic Cells Growth Factors Target Cells Stem Cell Factor Pluripotent Haemopoietic Stem Cell Flt-Ligand Pluripotent Haemopoietic Stem cell Interleukin-3 Multipotent Progenitors Interleukin-6 Multipotent Progenitors Megakaryocytes, B-lymphocytes Thrombopoietin Multipotent Progenitors, BFU-E Megakaryocytes effects of SCF & Flt-L on PSC

Growth Factors Erythropoietin GM-CSF Target Cells Erythroid Progenitors to Reticulocytes Multipotent Progenitors, CFU-GM, CFU-G Monocytes /Granulocytes & their precursors G-CSF CFU-G, Granulocytes & Precursors effects of SCF, Flt-L, GM-CSF on PSC M-CSF CFU-M, Monocytes & Precursors Interleukin-5 CFU-Eo, Eosinophils & Precursors

Haemopoietic Cell Kinetics Leucopoiesis: 6– 10 dys from CFU-GM to mature granulocytes Neutrophils: ~ 6 hrs in blood, diapedesis to tissues die after 2 -4 dys; or sooner if they engulf and kill microbes. Erythropoiesis: 6 -7 dys from proerythroblast to reticulocyte 2 -3 dys from retic to fully mature rbc Life-span of rbc 100 -120 dys. Platelets: bud off megakaryocyte cytoplasm Life-span 7 -10 dys; initial 3 dys in the spleen Clinical applications in anaemia, neutropenia or low plt count

Reference Values of Blood Cell Counts In Various Adult Populations Blood Cell Blacks Caucasians All Leukocytes 2. 6 – 10. 2 x 109/L 4 – 11 x 109/L Neutrophils 1. 3 – 3. 3 x 109/La 2. 5 – 7. 5 x 109/L Platelets 100 – 300 x 109/Lb 150 – 400 x 109/L Erythrocytes 3. 9 – 6. 5 x 1012/L Hb Level 11. 5 – 17. 5 g/dl a: Anyaegbu et al, 1998 b: Essien et al, 1973. 11. 5 – 17. 5 g/dl

Bone Marrow Failure Inability of the bone marrow to produce a normal number of normal red blood cells, granulocytes or platelets. Analogous to the concept of any other organ failure A classical example is aplastic anaemia. Marrow Failure also occurs in AML, ALL, CML, CLL & MDS. Usually, anaemia due to deficiency of haematinics like iron or folate, are not included in bone marrow failure syndromes.

Aplastic Anaemia Is pancytopenia with a hypocellular bone marrow in the absence of an abnormal infiltrate and with no increase in reticulin (fibrous tissue) Types 1. Congenital e. g Fanconi’s Anaemia, Dyskeratosis Congenita 2. Acquired Aplastic Anaemia

Acquired Aplastic Anaemia Incidence: 1 -2 /million population /yr 2 -3 x higher in Southeast Asia & Japan Bimodal Age Distribution: 10 -25 yrs and > 60 yrs No sex preponderance Associated Factors: Drugs, e. g chloramphenicol Autoimmunity e. g SLE Chemicals e. g benzene PNH. ? Viral Hepatitis

Clinical Features Signs and symptoms of anaemia e. g tiredness pallor Signs of thrombocytopenia e. g mucosal or skin petechial bleeding visual disturbance from retinal bleed Features of neutropenia e. g infections No splenomegaly or hepatomegaly; suggest other diagnosis Jaundice 2 -3 months previously

Consider Congenital AA if the patient… 1. Is a child or young adult 2. Has short stature, skeletal anomalies, or skin pigmentation (café-au-lait spots). Above are features of Fanconi Anaemia; commonly presents at 3 -14 yrs; may be up to 32 y (M) or 48 y (F). Dyskeratosis Congenita (DC) is characterised by: Leucoplakia Nail dystrophy Skin pigmentation DC presents from 6 mths – 26 yrs; median 7 yrs.

Investigations Are done with the following objectives: 1. Confirm diagnosis of AA 2. Detect or Exclude other causes of pancytopenia with a hypocellular bone marrow 3. Detect or Exclude congenital aplastic anaemia 4. Screen for an underlying cause of acquired AA 5. Detect co-existing PNH or abnormal cytogenetics

1. FBC + Retics, Blood Film and % Hb. F (MDS in children) 2. Marrow Aspiration & Biopsy 3. Liver Function Tests & Hepatitis Viral Screen 4. Autoantibody Screen, for SLE, etc 5. Folate & Vitamin B 12 Levels 6. CD 59 Expression on rbc/wbcs (or other test for PNH) 7. Mitomycin-C or Diepoxybutane-induced chromosomal breakage ( in Fanconi Anaemia) 8. Abdominal Ultrasound re: hepato/splenomegaly

Differential Diagnosis of Pancytopenia with Hypocellular Marrow 1. Myelodysplasia 2. Acute Myeloid Leukaemia 3. Hypocellular Acute Lymphoblastic Leukaemia 4. Tuberculosis; marrow may be fibrotic 5. Lymphoma or Hodgkin’s Disease 6. Anorexia Nervosa or Prolonged Starvation

Degrees of Severity In Aplastic Anaemia Non-Severe Retics Does Not meet Neuts criteria for Plts severe or very severe Marrow Severe <20% <0. 5 x 109/L < 20 x 109/L Very Severe < 20% <0. 2 x 109/L <20 x 109/L <25% cellularity, or 25 -50% cellularity with <30% haemopoietic cells

Normal Marrow ~ 50% Cellularity Aplastic Anaemia Marrow <25% Cellularity

Buccal Mucosal Bleeding in Aplastic Anaemia

Dr Guido Fanconi. Swiss Pediatrician, described Fanconi Anaemia In 1927. Skeletal anomalies of the hips, spine, or ribs Kidney malformations Skin discoloration Digital Anomalies Small head or eyes Low birth weight Small male genitalia G. I. Malformations Ventricular septal defects Mental retardation or learning disabilities

Abnormalities of digits and skeleton in a child with Fanconi Anaemia

A B C D Children with Fanconi Anaemia showing Short stature (A&B) and small eyes (C&D)

Chromosome Abnormalities in Fanconi Anaemia Radial chromosome formations (red arrows) Chromosome breakages (blue arrows)

A Dyskeratosis Congenita (A) Dysplastic toe nails (B) Skin pigmentaion and (C) Dysmorphism B C

Anorexia Nervosa & Starvation: Hypoplastic and gelatinous bone marrow with pancytopenia.

Treatment Options In Aplastic Anaemia 1. Transfusion of rbcs and/or platelets 2. Prevention & Treatment of infections 3. Immunosuppressive Agents: Anti-thymocyte Globulin (ATG) Cyclosporin Oxymethalone not preferred=> liver dysfunction liver cell cancer 4. Haemopoietic stem cell transplantation

Paroxysmal Nocturnal Haemoglobinuria (PNH) A chronic haemolytic anaemia due to acquired deficiency of plasma membrane proteins that protect erythrocytes from complement-mediated lysis. Why the misleading description paroxysmal & nocturnal? Now clear that haemolysis occurs continually day & night though concentrated urine is passed early in the morning

Pathogenesis of PNH Structure of red cell plasma membrane Molecular Structure of GPI-linked complement regulatory proteins CD 59 & CD 55 are reduced bc of somatic mutation of PIG-A gene which codes for an enzyme involved in GPI synthesis. CD 55: decay accelerating facor. CD 59: membrane inhibitor of reactive lysis.

The Plethora of GPI-linked Membrane Proteins ! C 8 bp rbc complement factor C 8 binding protein Acetylcholinesterase rbc enzyme Alkaline phosphatase neutrophil enzyme CD 14 monocyte receptor for the LPS-binding protein CD 16 Fcg RIII neutrophils. Low affinity receptor for Ig. G CD 24 neutrophils CD 48 monocytes CDw 90 CDw 108 CDw 109 CD 52 Campath-1 Antigen. Immunoregulatory protein CD 55 DAF rbc/wbc maturation independent, good for dx. CD 58 LFA 3 expressed on many cells in the body CD 59 MIRL rbcs maturation independent, good for dx. CD 66 b & CD 67 granulocyte. CD 73 lymphocyte ecto-5`-nucleotidase. Combined effects of deficiencies likely => clinical problems

Diagnostic Tests For PNH

Acidified Serum Lysis (Ham) Test for PNH rbcs are lysed by complement in acidified normal serum at room temp (RT) & 37°C (red supernatant). Normal rbcs are not lysed; tubes with clear supernatant. Principle of Ham Test Complement-mediated cell lysis is enhanced in acidified environments

Clinical Presentation of PNH Intravascular haemolytic anaemia; neg anti-globulin test. Features of bone marrow failure or marrow hypoplasia. Pancytopenia, bilineage or single lineage cytopenia. Aplastic Anaemia (GPI defect detected in 50% of AA pts) Iron deficiency anaemia bc of recurrent haemoglobinuria. Inexplicable thrombosis in unusual sites, e. g spleen, brain, git, liver (Budd-Chiari Syndrome) Abdominal or low back pain No sex preponderance; though genetic defect is X-linked.

Normal Male Karyotype with One X Chromosome

The Reason PNH Has No Male Preponderance PIG-A gene is in the x-chromosome. The PNH mutation is acquired after birth, and occurs in somatic cells (PSCs). Mutation not congenital. Does not occur in germ cells (sperm or ova). NOT like haemophilia or G 6 PD deficiency After birth, one of the 2 x-chromosomes in somatic cells of females is permanently inactivated (lyonisation). So, each has only one active x-chromosome; like in males. PIG-A mutation causes PNH in females only if it affects the active x-chromosome. Therefore, risk of PNH in females = males

Treatment Options in PNH Folic acid 5 mg dly. Fe. SO 4 200 mg t. d. s if iron deficient Red cell transfusion if Hb < 7 g/dl Treatment of thrombotic events with anticoagulants Preventive treatment with warfarin if previous thrombosis Anti-C 5 monoclonal antibody (eculizumab) reduced blood transfusion requirements and improved quality of life in a recent clinical trial. Dose 900 mg IV over 30 mins 2 wkly. Licensed by USA Food & Drug Admin in March 2007. If marrow hypolasia or AA dominant feature, ATG and haemopoietic stem cell transplantation are beneficial.