Paediatric Morphology

Transcription

1 Robyn Wells Core Haematology Pathology Queensland Central Laboratory 1

2 Normal paediatric films: Red blood cells are slightly hypochromic and microcytic in normal infants and children, compared to adult cells. Even when iron stores are normal, cells have this appearance Lower ranges for MCV and MCH for these age groups. Mild iron deficiency is common The lymphocyte count is often higher than the neutrophils (reversed differential) Majority of these are small dark normal lymphocytes. The larger, paler, granulated lymphocytes (large granular lymphocytes) are more numerous but remain at constant levels throughout childhood. Reactive lymphocytes may occur in small numbers in apparently healthy children especially in the first three years Response to subclinical viral infections and other immunological stimuli. The platelet morphology is similar to adult films. Red cell abnormalities: Low production nutritional, viral, hypoplasia Abnormal production - haemoglobinopathies, sideroblastic anaemia, CDAs, Haemolysis - AIHA, membrane disorders (HS, HE, MO), oxidative, enzyme deficiencies 2

3 Nutritional Fe deficiency Commonest anaemia of childhood Usually dietary but can be due to blood loss If not eating iron containing foods, will start at 5 6 months when iron from mum runs out This coincides with a 2-3 year period of rapid growth with peak iron requirements Megaloblastic anaemia is uncommon Can occur if nursing mother is strict vegan Also if intrinsic factor deficiency or Ab s for B12 deficiency Malabsorption may also be a cause for either B12 or folate deficiency 3

4 Parvovirus or other viruses can cause a red cell aplasia (and often neutropenia as well) EBV, viral hepatitis, HIV, varicella, and CMV also implicated Hypoplasia due to immune diseases such as SLE, syndromes such as Fanconi s and Diamond Blackfan Abnormal production includes the thalassemias and Hb defects, sideroblastic anaemia and CDA α chain disorders are detectable at birth whereas β chain variants will not manifest until 4-6 months. Other Hb opathies will be apparent at different ages moderate to severe anaemia will occur in HbSS from 4 months onwards, HbC will only cause a slight anaemia 4

5 Hereditary sideroblastic anaemia is rare with anaemia occurring from a few months to 7-8 years. It is either X-linked or autosomal recessive Acquired SA can be due to toxins such as lead, drugs or secondary to immune disorders CDA very rare, structural abnormalities of the rbc membrane Any increased RBC turnover is haemolysis may be slight (HE) or acute (AIHA) Inherited membrane disorders such as HE, HS and MO may have chronic haemolysis but also acute episodes HE spectrin defect. Precursor cells are normal in shape Usually mild except homozygous state HS - instability of the skeletal lipid bilayer results in loss of membrane as the red cell circulates through the microvasculature. Red cell picture of a mixture of normal looking cells and spherocytes it is not a really homogenous looking population unless the patient is asplenic. Can be hard to differentiate from AIHA especially in children in a haemolytic crisis 5

6 Melanesian Ovalocytosis Melanesia, Indonesia, SE Asia Oval stomatocytes with 1 or 2 transverse slits Cells are more rigid gives some protection against malaria 6

7 AIHA Relatively rare in children 1 in 80,000 Primary or secondary Primary warm reactive, PCH, cold agglutinin disease Secondary generalized auto-immune disease, immune deficiency, malignancy, medication, infection The most common primary AIHA is the warm reactive form Autoantibodies, usually IgG bind to RBC at 37 C May fix complement Extravascular haemolysis mainly in spleen Leads to splenomegaly, jaundice and anaemia Mechanism warm reactive IgG autoantibodies coat autologous red cells and may fix complement. Sensitized cells pass through spleen and RES Interact with Fc receptors and complement on macrophages Ingested by macrophages or part membrane removed spherocytes Paroxysmal cold haemoglobinuria (PCH) occurs after a viral-like illness IgG autoantibodies bind preferentially at colder temperatures Fix complement efficiently Cause intravascular haemolysis with haemoglobinuria, haemoglobinaemia and anaemia 7

8 Cold agglutinin disease Rare can be after Mycoplasma infection IgM autoantibodies bind I/i antigens at colder temperatures Fix complement Complement-mediated intravascular or immune-mediated extravascular clearance Mechanism cold reactive (PCH and CAD) Antibody binds preferentially at 4 C and fixes complement efficiently If complement is activated to completion, intravascular haemolysis occurs If complement is deposited on the red cell but not fully activated, macrophages in the RES bind the rbc using complement receptors Clearance in the liver, rather than the spleen 8

9 Oxidative haemolysis can be due to chemicals, drugs, heavy metals Denatures the Hb which precipitates and then removed from the red cell by the RES Uncommon in children more in babies or adults Unstable Hb gives similar morphology but no oxidative cause as such Enzyme deficiencies - PK, G6PD G6PD X-linked Can have haemolytic crises triggered by oxidants or infections Favism ingestion of fava beans acute haemolysis PK highly variable with chronic haemolysis Prickle cells in film but many more after splenectomy 9

10 Childhood ALL can be very difficult to pick as the blasts can be small even microblasts Children s lymphocytes often have little cytoplasm, an inactive nucleoli or look a little clefted or convoluted 10

11 The WHO classification of precursor B- lymphoblastic leukaemia is equivalent to B-cell ALL L1 and L2 in the FAB classification and precursor T-lymphoblastic leukaemia encompasses the T-cell All L1 and L2 (FAB) Pre B-ALL occurs most commonly in children <6 years The B-cell type is the one that is harder to distinguish as it is more homogenous in appearance, small to moderately sized blasts, high N/C ratio with only a little scanty basophilic cytoplasm and inconspicuous nucleoli The T lineage blasts are larger and more heterogenous with more obvious nucleoli, varied chromatin patterns from fine to coarsely condensed and variable amounts of cytoplasm T-cell blasts can have very folded and cleaved nuclei The count is often high and the patients can present with a tumour mass The full blood count results and clinical notes may or may not be helpful. Usually at least one cell line is suppressed and anaemia and/or thrombocytopenia is a presenting feature. White cell counts can be anywhere from 1.0 to 1500 x 10 9 /L There is nearly always neutropenia 11

12 Differential diagnosis for children who present with severe cytopenias include such things as viral infections, red cell aplasia, aplastic anaemia, solid tumours, lymphomas or the rarer conditions such as Diamond Blackfan anaemia, Fanconi s anaemia or congenital dyserythropoietic anaemia 12

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17 Reactive lymphocytes can vary from slightly larger round cells with basophilic cytoplasm to very large with abundant cytoplasm with basophilic rim. The chromatin pattern also varies from being diffuse to partly condensed There is always a range or spectrum of cells not a homogenous or clonal population 17

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19 Acute myeloid leukaemias Morphology often misleading Flow, cytogenetics, molecular Sometimes clues in the company they keep Age, WCC, history can help 19

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22 Definition of promyelocyte?? The malignant promyelocyte will have very variable granularity of the cytoplasm even the microgranular variant will have occasional heavily granulated cells and Auer rods or Faggott cells present. The majority of the promyelocytes will have some very fine granules often need to check under oil Nuclear shape that is distinctive (can be confused with monocytic) The microgranular variant will often have a higher WCC, but all have the tendency to DIC/bleeding presentation and complications. 22

23 The typical or hypergranular APML features promyelocytes with densely packed granular cytoplasm and cells with Auer rods often multiple or in bundles (Faggot cells) Both types have reniform or bilobed nuclei that can vary a lot in size The WCC for hypogranular variant can be quite high up to 100 x 10 9 /L, so a lot of these reniform and bilobed cells are seen The typical form is easier to pick, but does present a challenge as they are nearly always pancytopenic on presentation, with the WCC below 4 x 10 9 /L 23

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30 PAEDIATRIC MORPHOLOGY WORKSHOP Case studies 1-12 Wednesday 10 th August 2011 Case 1: 12 mth M, gastro (D&V) Case 1: Other results: DAT neg, B. pertussis NR, CMV past infection, parvovirus B19 NR, toxoplasmosis NR 30

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32 Blood film: Does it agree with the parameters? Yes - hypochromic, microcytic Other significant cells??elliptocytes?pencil cells?tear drops?polychromatic cells Possible causes & differential diagnosis: Fe deficiency Haemoglobinopathy 32

33 - Haemoglobinopathy RCC high/n Hb - N RDW - N MCHC - N - Iron deficiency RCC low Hb - low RDW - high MCHC - low - Fe studies: Iron 3 umol/l (9 22) Transferrin BC 109 umol/l (40-75) Transferrin 4.3 g/l ( ) Transferrin satn 3 % (115-45) Ferritin 6 ug/l (6-60) Case 2 2 yr F, CN haemolytic anaemia 33

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36 - Case 2 What cells are present in the film? Do they match the FBC results? What can be the cause of anaemia in a 2 yr old child? What does the presence of so many NRBC mean? - - Microcytes: Hb opathy or iron deficiency Stippling: Lead poisoning, Hb opathy, ineffective erythropoiesis Dimorphic Post transfusion, sideroblastic anaemia Spherocytes: Haemolysis, rbc membrane instability 36

37 - Increased polychromasia and NRBC: BM response, haemolysis, ineffective erythropoiesis Tear drops: Myelofibrosis, MDS, megaloblastic anaemia, HbH disease Hypochromia: Fe deficiency, Hb opathy - Fe studies: - Hb studies: Case 3: 5 y male Referred to paediatric haematology clinic No family history of anything haematological Mother: β thal minor Father: β thal minor 37

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39 - Case 3: What are the significant abnormalities in the film? What might you see in the biochem results? 39

40 - Other results: Bili 34 umol/l (<20) Bili (conj) 5 umol/l (<4) LD 369 U/L ( ) - DAT: Poly 3+, IgG 3+, C3d negative Eosin-5-maleimide: Normal staining pattern consistent with haemolysis? further tests? Case 4: 2 yr F pancytopenia, Hb 30 yesterday?leukaemia 40

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42 - Case 4: Causes of pancytopenia in a 2 yr old leukaemia viral aplastic anaemia nutritional drugs - - Any blasts? Any reactive lymphs? Any features of aa? Any signs of iron deficiency or megaloblastic anaemia? Any history of drugs? LD 5310 U/L ( ) 42

43 - - Extremely rare condition Imerslund- Gräsbeck syndrome Autosomal recessive Identical twin sister also has it Vitamin B12 deficiency due to selective malabsorption Megaloblastic anaemia occurs after 4 months (transplacental transfer runs out) - In about half the cases proteinuria also occurs with no signs of kidney disease Cause is defect in the receptor of the vitamin B12-intrinsic receptor complex of the ileal enterocyte. This receptor is also essential for renal protein reabsorption. Mutation is either cubilin (CUBN) gene on chr 10 or amnionless (AMN) on chr14. Responds to life-long B12 injections Case 5 2 yr M, chronic urticaria, vasculitis 43

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45 - Case 5 2 year old male presented with angioedema and hepatomegaly Hb 72 g/l and platelets 10 x 10 9 /L LD 1160 U/L ( ), total bilirubin 55 umol/l (<20) mild numbers of spherocytes, moderate polychromasia and slight red cell agglutination - - What could be causing this thrombocytopenia and haemolysis? Evan s syndrome is the combination of immune thrombocytopenic purpura (ITP) and AIHA. Autoimmune neutropenia also occurs in a minority of cases 45

46 - The clinical course is variable Often prolonged, with chronic haemolysis Relapses are common and significant mortality of 13% in a review of 99 patients Underlying causes have been proposed and include a variety of immune defects SLE, CVID and autoimmune lymphoproliferative syndrome (ALPS). - Recent evidence suggests that ALPS may be the underlying cause in a substantial proportion of cases. In ALPS there is a defect in the programmed cell death of lymphocytes which leads to massive lymphadenopathy and abnormal function of the T-cells. Other lymphoid malignancies (HD and lymphomas) and chronic infections (eg HIV and Hep C) need to be considered - Rx with T-cell immunosuppressive (mycophenolate) Now quite stable and well Blood film only occasional spherocyte and slight polychromasia Platelets normal Case 6 CN haemolysis and acute liver impairment 46

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49 - Case 6 Film consistent with liver failure? Consistent with haemolysis? Which are the significant features in the film? Polychromasia, elliptocytes, target cells, spherocytes, Pappenheimer bodies, basophilic stippling, acanthocytes, blister cells, bite cells - Which are the significant features in the film? Polychromasia, elliptocytes, target cells, spherocytes, Pappenheimer bodies, basophilic stippling, acanthocytes, blister cells, bite cells What causes spherocytes, blister and bite cells? Oxidative haemolysis - - Oxidative haemolysis G6PD def, unstable Hb, chemicals, drugs, membrane damage Liver failure?wilson s disease 49

50 - Case 7 10 mth old, Haematology clinic patient First presented with pseudomonas sepsis when 8 weeks old 50

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52 - Case 7 Severe neutropenia in an infant?cause - Leukaemia Viral Infection Toxins BM failure Familial/cyclical neutropenia Immune - Leukaemia blasts? Viral reactive lymphs? Infection any response? Toxins other cytopenias? BM failure other cytopenias? Familial/cyclical neutropenia -?? Immune underlying autoimmune disease? Age group? (alloimmune neonatal period) 52

53 - In this case there was infection, but no BM response G-CSF therapy response - Therefore there are stem cell precursors that are responsive to G-CSF BM no maturation beyond promyelocyte/myelocyte stage. No malignancy Kostmann s syndrome (infantile genetic agranulocytosis) Often compensatory monocytosis Infection is usual cause of death - Rx massive doses of G-CSF 30mg/kg (normal dose 5mg/kg) BMT No sibling Parents are talking about trying for nonaffected baby for cord cell donation Case 8 3y M fever, rash FBC results: Hb 118 g/l Ne x 109/L RCC 4.35 x 1012/L Ly x 109/L MCV 86 fl Mo x 109/L Hct 0.37 Eo x 109/L WBC 7.9 x 109/L Ba x 109/L Plats 104 x 109/L Meta x 109/L RDW 16.2 % Mye x 109/L MCH 27.1 pg Pro x 109/L MCHC 317 g/l Blasts x 109/L 53

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55 - Case 8 What are the cells in the film? Homogeneous or pleomorphic population? Variable amounts of cytoplasm? Variable sized nucleus? Nasty or nice? 55

56 - FBC results: Hgb 118 g/l Ne 1.7 x 109/L RCC 4.35 x 1012/L Ly 6.0 x 109/L MCV 86 fl Mo 0.2 x 109/L Hct 0.37 Eo 0.1 x 109/L WBC 7.9 x 109/L Ba x 109/L Plats 104 x 109/L Meta x 109/L RDW 16.2 % Mye x 109/L MCH 27.1 pg Pro x 109/L MCHC 317 g/l Blasts x 109/L - Diagnosis? - - Diagnosis? Viral infection (EBV negative) 56

57 Case 9 14 mth F CN anaemia, Hx viral illness 57

58 - Case 9 Usual questions!! Do the cells look normal? Any signs of infection/malignancy/haemolysis/nutritional deficiency/viral infection? 58

59 - What can cause the anaemia and neutropenia??bm failure platelets normal?viral no reactive lymphs?drugs hope not BM aspirate Viral studies on BM: 59

60 - Case 10 2 y M, easy bruising Post transfusion and G-CSF Now completely well 6 months later TEC and immune neutropenia 60

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62 - Case 10 - Any signs of infection/reactive process/haem disorder? Any characteristic features of a particular disorder? What could be the differential diagnosis? What are the abnormal FBC parameters? Cause in a 2 yr old for these results? 62

63 - Acute or chronic? What WHO classification? Is this usual in a 2 yr old? Case day M bronchiolitis/pneumonia 63

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65 - Case 11 WCC 65.4, Hb 63, plts 173, neuts 30, lymphs 21.8 Film features? High WCC reactive or disorder? RBC changes what can cause these? - Renal function not too bad. Worst results: Urea 14.0 ( ) Creat 148 (36-99) Haemolysis occurring LD 3610 ( ) 3 days later plats were Diagnosis?? 65

66 - - NPA B. pertussis DNA Detected HUS/TTP can occur secondary to malignancy, infections etc Cells fragmented as they push through thrombus formed due to exotoxin-induced endothelial damage Usually diarrhoea associated E. coli or S. dysenteriae Occasionally URTI <4 yrs age Case 12 4y F, pale, unwell 66

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68 - FBC what is low? Neutrophils 9% or

69 - - What is most likely? Morphology? Appearance of cells?

70 - Precursor B-ALL Markers: positive for CD10, CD19, CD20 (v), CD34, CD38, HLA-DR, CD79a No cytogenetics abnormality detected, but very poor sample 70