23/10/2017 1 PNH Case Presentations STEPHEN J RICHARDS PhD FRCPath Consultant Clinical Scientist HMDS & PNH National Service (Leeds) Leeds Teaching Hospitals NHS Trust UK [email protected]Background • Paroxysmal Nocturnal Haemoglobinuria and aplastic anaemia are rare diseases. • Aplastic Anaemia is an exclusion diagnosis • PNH- has a diagnostic flow cytometry test • Clinical PNH • Can provide supportive information for a diagnosis of AA • Lots of requests for PNH testing. • Understanding the pathobiology of PNH and Aplastic Anaemia is essential for interpretation of flow cytometry data. Background • PNH and Aplastic Anaemia are two closely related rare conditions where the underlying defect is immune mediated bone marrow failure. • Bone marrow failure positively selects for haematopoietic stem cells that have a somatic mutation of the X-linked phosphatidylinositol glycan complementation class A (PIG-A) gene. • This results in a partial or absolute deficiency of all proteins normally linked to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. • The GPI deficient progeny of these stem cells repopulate haematopoiesis leading to many of the clinical features of PNH – haemolytic anaemia, thrombosis. Relationships between PNH/Aplastic Anaemia Initiating event(s)? Primary & secondary changes within the immune system Complex biology: Immune disorder/haemolytic anaemia/stem cell disorder/thrombotic Time scales: disease progression/spontaneous remission/secondary MDS/AML Failing haematopoiesis 100% PNH clone AA 0% PNH clone Immune mediated bone marrow failure Stem cell destruction/loss ± mutation & selection Managing cytopenia haemolysis+/- thrombosis & clinical sequela Managing cytopenias & clinical sequela Prodromal Clinical Disease Diagnosis Relationships between PNH/Aplastic Anaemia Stable disease /disease progression/spontaneous remission/secondary MDS/AML Bone marrow transplant Long term anticoagulation Long term Complement blockade treatment Immunosuppression/steroids. Transfusions/blood product support Complex clinical issues: renal damage/iron overload/pulmonary hypertension/QOL Failing haematopoiesis 100% PNH clone AA 0% PNH clone Managing cytopenia haemolysis+/- thrombosis & clinical sequela Managing cytopenias & clinical sequela Flow Cytometry Requests for PNH Screening 1) Haemolysis 2) Cytopenias of varying severities 3) Unexplained thrombosis 4) Lab generated requests 5) Monitoring requests 6) No idea what’s wrong with the patient: - test for PNH
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23/10/2017
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PNH Case Presentations
STEPHEN J RICHARDS PhD FRCPath
Consultant Clinical ScientistHMDS & PNH National Service (Leeds)
Background• Paroxysmal Nocturnal Haemoglobinuria and aplastic
anaemia are rare diseases.• Aplastic Anaemia is an exclusion diagnosis• PNH- has a diagnostic flow cytometry test
• Clinical PNH• Can provide supportive information for a
diagnosis of AA• Lots of requests for PNH testing.• Understanding the pathobiology of PNH and Aplastic
Anaemia is essential for interpretation of flowcytometry data.
Background• PNH and Aplastic Anaemia are two closely related rare
conditions where the underlying defect is immunemediated bone marrow failure.
• Bone marrow failure positively selects forhaematopoietic stem cells that have a somatic mutationof the X-linked phosphatidylinositol glycancomplementation class A (PIG-A) gene.
• This results in a partial or absolute deficiency of allproteins normally linked to the cell membrane by aglycosylphosphatidylinositol (GPI) anchor.
• The GPI deficient progeny of these stem cellsrepopulate haematopoiesis leading to many of theclinical features of PNH – haemolytic anaemia,thrombosis.
Relationships between PNH/Aplastic Anaemia
Initiating event(s)?Primary & secondary changes within the immune system
Case 1: Learning Points & Conclusions• Anaemia for 8 years
• Had attended a variety of hospital departments
• Be aware of all results
• Outcome
– Candidate for complement blockade therapy
– Clinical assessment for long term untreated haemolysis, evidence of thrombosis
– Regular monitoring
– Review of clinical history showed signs and symptoms of classical haemolytic PNH
Case 2
• Clinical History
• Results of PNH Screen
• Conclusions
• Learning Points
• Patient Age 77yrs with macrocytic anaemia
• WBC 4.8
• Neutrophils 3.6
• Lymphocytes 1.0
• Monocytes 0.2
• Haemoglobin 89g/L
• MCV 98.1
• Platelets 18
• Blood film:No evidence of dysplasia
Case 2: Leucocytes
0.23% NEUTROPHIL CLONE 0.83% MONOCYTE CLONE
Case 2: Leucocytes Case 2: Red cells
0.06% (145 events)
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Case 2: Learning Points & Conclusions
• Small PNH clones detected.
• Referred for PNH test
• Diagnosis of longstanding MDS with refractory cytopenia
• Review and investigation of bone marrow aspirates and trephine biopsy
– No evidence of dysplasia by morphology
– Normal Karyotype
– No detectable myeloid gene mutations by NGS.
– Progressive anaemia
Case 2: Additional testing
CD34+ = 0.36% of totalcells of which 46% are Bprogenitors and 54%myeloid progenitors with anormal phenotype.Increased mast cells andplasma cells
• Additional flow cytometry failed to detect any evidence of an LPD or abnormal progenitor cell population.– Low CD34+ progenitor compartment
– Increased Mast cells (CD117++)
• No evidence of MDS– More appropriate to treat as aplastic anaemia
– Monitor PNH clone 3 monthly initially, important if receiving immunosuppressive therapy
• The findings support a diagnosis of aplastic anaemia with small PNH clones
Case 2: Learning Points & Conclusions Case 3
• Bone marrow sent with clinical details of pancytopenia ?cause.
• Trephine biopsy– Reduced cellularity
– Lymphocytes (T cells)
– Plasma cells
– Mast cells
– Reduced myeloid and erythroid activity
• Consistent with Aplastic/hypoplasticanaemia.
• Recommended PNH testing
• Counts improved
• PNH screen performed 3 years later– Granulocyte clone 23.6%
– Monocyte clone 18.9%
– Red cell clone 1.2%
Case 3
• WBC 6.4
• Neutrophils 4.6
• Lymphocytes 1.0
• Monocytes 0.8
• Haemoglobin 144g/L
• MCV 99
• Platelets 96
• Blood film Unremarkable
Case 3: Leucocytes
Granulocyte PNH clone 65.13% Monocyte PNH 64.61%
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Case 3: Leucocytes Case 3: Red cells
Type III (CD59 negative) 0.01%, Type II (CD59 weak) 9.83%: Total Red cell PNH clone 9.84%
Case 3: Red cells
Type III (CD59 negative) 0.23%; Type II (CD59 weak) 71.23%Total Red cell PNH clone 71.46%
Case 3: Conclusions & Learning Points
• Based on a haematopathology bone marrow report finding of a hypoplastic/aplastic anaemia.
– PNH testing should be carried out a soon as convenient
– Repeat testing for should also be undertaken (even if no clones are found initially).• If counts improve rapidly on immunosuppressive or other
treatments.
• If no PNH clones are found then repeat test annually, but also be aware that a small proportion can develop MDS/AML.
Case 3: Conclusions & Learning Points
• GPI antigen staining can be complex to interpret.
– Three lineages of cells should be assessed (neutrophils, monocytes and red cells (together with immature fraction if possible)
– Mixtures of type III, type II and normal cells can be seen in many PNH patients• The proportions of cells and the strength of GPI antigen
expression can vary widely
– Due to cells studied
– Target antigen
– Multiple different PIG-A mutations in individual patients
– Single mutation that allows partial GPI synthesis in some cells but not others
Red Cell Analysis
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Red Cell Analysis Red Cell Analysis
1. Patients can have multiple PIG-A mutations2. Red cells haemolyse: Type III most sensitive3. Nucleated red cells more resistant to lysis4. Transfused red cells5. CD59 expression can decrease with age of red cell.6. Essential to know size of leucocyte PNH clones7. Technical variables: cytometer, antibody, fluorchrome
Should be the most straightforward (single antigen expression)
Red Cell Analysis: Transfused cases
Tota
l red
cel
ls
CD
71
+ re
d c
ells
Summary
What can we do to improve testing?
1. Understanding the pathology/biology of this rare disease.2. Understand what we are testing for and the limitations of
the procedure.3. New ICCS guidelines provide, in great detail, what is
required of a PNH assay and what is required to ensure compliance with accreditation standards.