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PNH Case Presentations

STEPHEN J RICHARDS PhD FRCPath

Consultant Clinical ScientistHMDS & PNH National Service (Leeds)

Leeds Teaching Hospitals NHS TrustUK

stephen.richards2@nhs.net

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

Complex biology: Immune disorder/haemolytic anaemia/stem cell disorder/thromboticTime scales: disease progression/spontaneous remission/secondary MDS/AML

Failing haematopoiesis

100%PNH clone

AA0% PNH clone

Immune mediated bone marrow failure

Stem cell destruction/loss ± mutation & selection

Managing cytopeniahaemolysis+/-

thrombosis & clinical sequela

Managing cytopenias& clinical sequela

Pro

dro

mal

Clin

ical Dise

ase

Diagn

osis

Relationships between PNH/Aplastic Anaemia

Stable disease /disease progression/spontaneous remission/secondary MDS/AML Bone marrow transplantLong term anticoagulationLong term Complement blockade treatmentImmunosuppression/steroids.Transfusions/blood product supportComplex clinical issues: renal damage/iron overload/pulmonary hypertension/QOL

Failing haematopoiesis

100%PNH clone

AA0% PNH clone

Managing cytopeniahaemolysis+/-

thrombosis & clinical sequela

Managing cytopenias& clinical sequela

Flow CytometryRequests 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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Case Studies

• Clinical History

• Results of PNH Screen

• Other Investigations

• Conclusions

• Learning Points

• Flow Cytometry

• Leucocytes• FLR/CD24/CD16/CD33/CD15/CD14

• FLAER/CD157/CD45/CD64/CD15

• FLAER/CD157/CD123/CD64/CD15/HLADR/CD45/CD16.

• CD55/CD14

• Red Cells

• CD71/CD59/CD235a• Clone: MEM43 PE

Case 1

• Results of PNH Screen

• Conclusions

• Clinical History

• Learning Points

• WBC 4.5

• Neutrophils 2.1

• Lymphocytes 1.9

• Monocytes 0.5

• Haemoglobin 105g/L

• MCV 100.3

• Platelets 174

• Blood film Unremarkable

92.2% NEUTROPHIL CLONE

89.4% MONOCYTE CLONE

Case 1: Peripheral blood PNH screen

Type III (CD59 negative) 23.63%Type II (CD59 weak) 5.81%

Total Red cell PNH clone 29.44%

Case 1: Peripheral blood PNH screen

Type III (CD59 negative) 67.27%Type II (CD59 weak) 3.06%

CD71+ Red cell PNH clone 70.33%

Case 1: Peripheral blood PNH screenCase 1

• Sample not sent for PNH screen.

• Clinical Information provided: ‘anaemia.’

• Female, age 18

• Investigations undertaken.

– Lymphocyte subsets were normal.

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Case 1: T cell screen

CD16-ve granulocytes

1. Eosinophils2. CD16 polymorphism3. Immature/dysplastic

granulocytes4. PNH cells

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.