Slides from a Microsoft PowerPoint® presentation I delivered covering the basic clinical presentation, diagnosis, pathogenesis/pathophysiology, treatment, and prognosis of paroxysmal nocturnal hemoglobinuria (PNH). This presentation was given on October 3, 2018 at the Medical College of Georgia, Augusta Campus to an audience of clinical pathologists and second-year MD candidates.

1. Paroxysmal Nocturnal
Hemoglobinuria (PNH)
Jackson David Reynolds, BS, AS, AA
Medical College of Georgia at Augusta University
October 3, 2018

2. Case Presentation: HPI & Physical Exam
• A 36 y/o M presents to his primary care physician c/o
fatigue, SOB, abdominal pain, & passing red-tinged
urine in the AM for the past month
• Pain does not accompany micturition & he denies
any other physical complaints
• He reports no major illnesses nor family history of
similar issues
• He is taking no medications
• Upon PE, he appears pallid & has mild non-tender
splenomegaly by palpation
• Vitals: P = 90; RR = 17; BP: 119/76 mmHg
Besa, 2018; Danier, 2018; Maloy, 2017; Schrezenmeier, et al., 2014.
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3. Differential Diagnosis
• Hemolytic Anemia very likely (red urine & fatigue)
• Paroxysmal Nocturnal Hemoglobinuria suspected & should be tested for
in pts w/:
• Hemoglobinuria
• Evidence of hemolysis
• Abdominal/cerebral vein thrombosis
• Thrombocytopenia
• Macrocytosis
• Possible bleed in kidney(s), ureter(s), or urinary bladder
• UTI
• Malignancy
Mayo Clinic, 2017.
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4. Case Presentation: Pertinent Labs
• CBC:
• Normocytic anemia
• Neutropenia
• Flow cytometry of peripheral blood:
• Decreased expression of protectin (CD59) & DAF (CD55)
• Serum LDH: Elevated (suggestive of hemolysis)
• Serum haptoglobin: Low (suggestive of hemolysis)
• Bilirubin: Raised (suggestive of hemolysis)
• Urinalysis: Blood ++
Besa, 2018; Correia, et al., 2016; Ostler, 2006; Schrezenmeier, et al., 2014.
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5. Diagnosis: PNH
• Anti-CD59 mAb flow cytometry of peripheral blood:
• Gold standard
• Confirmatory
• Acidified serum lysis (Ham test):
• Hemolysis prevented in heated serum (56 ºC), b/c complement
activation cannot be achieved
• Formerly classic Dx test for PNH
• (Performed infrequently today)
• Sugar water / sucrose lysis test:
• (Performed infrequently today)
• Complement lysis sensitivity test (of Rosse & Dacie)
Besa, 2018; Danier, 2018.
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Selected tubes from a
Ham test

6. PNH Pathogenesis & Pathophysiology I
• Decay Accelerating Factor (CD55/DAF), Homologous Restriction Factor (HRF), &
Protectin (CD59) are expressed on self-cells & function to prevent self-targeting of the
complement system
• DAF & CD59 possess lipid tails, impaired synthesis of which prevents their anchoring to
the surfaces of RBCs
Besa, 2018; Brodsky, 2014; Danier, 2018; Emw, 2009; Parham, 2015; Takeda et al., 1993.
• Complement system can now target the “naked” self-
RBCs  intravascular hemolysis:
• Worsened by the slight drop in pH that accompanies deep
sleep
• PIGA gene somatic mutation responsible for impaired
lipid tail synthesis:
• Somatic mutation = acquired disease
• (Phosphatidylinositol N-acetylglucosaminyltransferase
subunit A)
CD55/DAF
(Decay Accelerating Factor)
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7. PNH Pathogenesis & Pathophysiology II
Parham, 2015.
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In PNH, failure of CD59 (protectin) allows C9 to facilitate the formation of the MCH in self-RBCs

8. PNH Histopathology
• Peripheral blood smear:
• Normocytic anemia
• Neutropenia
• Bone marrow:
• Usually hypercellular
• NML cellularity (%) = 100 - age
• Normoblastic erythroid hyperplasia
• (C/w intravascular hemolysis)
Ostler, 2006.
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Bone marrow histology in PNH

9. PNH: Additional Clinical Features & Prognosis
• Intravascular hemolysis
• As RBC are lysed, risk for clot formation rises
& multiorgan damage 2º to thrombotic
events can result:
• Sometimes unusual sites (e.g., hepatic v.)
• Median survival: 10-15 years
• 25% surviving @ ≥ 25 years
• 33% of pts experience spontaneous remission
• 33% of pts experience severe thrombotic
complications
• Potential sequelae: aplastic anemia, acute
leukemia
Agolini et al., 1997; Ostler, 2006.
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Splenic infarct 2º to
intravascular thrombosis

10. PNH Treatment
• Blood transfusions (sx-based):
• *Additional complement may pose risks!
• Androgenic steroids  increase hematopoiesis
• Eculizumab:
• MOA: Binds C5 & prevents MAC formation
• Only FDA-approved Rx Tx
• Improves quality of life
• Reduces the frequency of need for blood transfusions
• Does not reduce the risk of death
• Does not reduce the incidence of blood clots
• Extremely expensive: ~$440,000/year
• Only cure is HPSC bone marrow allograft:
• Mixed success rates
• Still carries high risk of major complications &/or death
• Usually reserved for pts w/poor response to eculizumab
Alexion, 2017; Danier, 2018; Hoffman, 2018; Martí-Carvajal et al., 2017.
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11. Summary
Diagnosis
• Paroxysmal Nocturnal Hemoglobinuria
(PNH)
Treatment
• Eculizumab
• Sx-based transfusions
• HPSC allograft
Prognosis
• 10-15 years median survival
• Quality of life dependent on response to Tx
Conclusion
• Carefully considered, global Tx paramount
• Monitor closely for potential PNH sequelae
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12. Question 1
A 39 y/o F pt presents to the primary
care clinic c/o dark urine in the AM for
the past two weeks which progressively
lightens in color throughout the day.
Upon physical exam, general pallor and
mild scleral icterus are noted. The
physician suspects PNH. Which
laboratory test(s) should be performed
to best confirm PNH?
A. Anti-C5 mAb flow cytometry
B. Serum bilirubin & LDH
C. Sucrose lysis test of AM urine sample
D. Anti-CD59 (protectin) mAb flow
cytometry
E. Sucrose lysis test of peripheral blood
F. CD55 (DAF) & CD59 (protectin)
Western blot
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13. Question 1
A 39 y/o F pt presents to the primary
care clinic c/o dark urine in the AM for
the past two weeks which progressively
lightens in color throughout the day.
Upon physical exam, general pallor and
mild scleral icterus are noted. The
physician suspects PNH. Which
laboratory test(s) should be performed
to best confirm PNH?
A. Anti-C5 mAb flow cytometry
B. Serum bilirubin & LDH
C. Sucrose lysis test of AM urine sample
D. Anti-CD59 (protectin) mAb flow
cytometry
E. Sucrose lysis test of peripheral blood
F. CD55 (DAF) & CD59 (protectin)
Western blot
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Besa, 2018; Danier, 2018.

14. Question 2
David Blamed is 41 year-old M patient
with recently-diagnosed PNH who is set
to begin eculizumab Tx next week.
Which of the following
physical/laboratory findings (directly
related to his PNH) would one expect
to be present in Mr. Blamed at this
time?
A. Macroovalocytes on peripheral blood
smear
B. Hypercellular bone marrow
C. Markedly elevated MCV & mild
neutropenia
D. Thrombocytosis & markedly depressed
serum LDH
E. Absence of CD4+ T cell precursor
production in the bone marrow
F. Maculopapular rash of the abdomen
spanning multiple dermatomes
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15. Question 2
David Blamed is 41 year-old M patient
with recently-diagnosed PNH who is set
to begin eculizumab Tx next week.
Which of the following
physical/laboratory findings (directly
related to his PNH) would one expect
to be present in Mr. Blamed at this
time?
A. Macroovalocytes on peripheral blood
smear
B. Hypercellular bone marrow
C. Markedly elevated MCV & mild
neutropenia
D. Thrombocytosis & markedly depressed
serum LDH
E. Absence of CD4+ T cell precursor
production in the bone marrow
F. Maculopapular rash of the abdomen
spanning multiple dermatomes
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Ostler, 2006.

16. Question 3
Maria J. is a 39 y/o F pt in her first
month of eculizumab Tx for PNH.
Which of the following processes
does eculizumab target to interfere
with PNH pathophysiology?
A. MAC formation on RBC membranes
B. Erythropoiesis
C. CD59 (protectin) binding to the RBC
membrane
D. CD55 (DAF) binding to the RBC
membrane
E. Synthesis of C5
F. C5a anaphylatoxin-mediated
chemotaxis
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17. Question 3
Maria J. is a 39 y/o F pt in her first
month of eculizumab Tx for PNH.
Which of the following processes
does eculizumab target to interfere
with PNH pathophysiology?
A. MAC formation on RBC membranes
B. Erythropoiesis
C. CD59 (protectin) binding to the RBC
membrane
D. CD55 (DAF) binding to the RBC
membrane
E. Synthesis of C5
F. C5a anaphylatoxin-mediated
chemotaxis
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Alexion, 2017; Danier, 2018; Hoffman, 2018; Martí-Carvajal et al., 2017.

18. References I
Agolini, Stefano F., et al. “Splenic Infarction Caused by a Large Thoracic Aortic Thrombus.” Journal of Vascular Surgery,
vol. 26, no. 6, 1997, pp. 1069–1072., doi:10.1016/s0741-5214(97)70022-3.
Alexion. Soliris. 2017, mms.businesswire.com/media/20171023006539/en/619703/5/Alexion_Soliris_Bottle_English.jpg.
Besa, Emmanuel C. “Paroxysmal Nocturnal Hemoglobinuria Clinical Presentation: History, Physical.” Sickle Cell Anemia
Differential Diagnoses, 16 Sept. 2018, emedicine.medscape.com/article/207468-clinical.
Brodsky, R. A. “Paroxysmal Nocturnal Hemoglobinuria.” Blood, vol. 124, no. 18, 2014, pp. 2804–2811., doi:10.1182/blood-
2014-02-522128.
Correia, Rodolfo Patussi, et al. “Technical Advances in Flow Cytometry-Based Diagnosis and Monitoring of Paroxysmal
Nocturnal Hemoglobinuria.” Einstein (São Paulo), vol. 14, no. 3, 2016, pp. 366–373., doi:10.1590/s1679-45082016ao3641.
Danier, Paul M. “Hemolytic Anemias.” 21 Sept. 2018.
Emw. Protein CD55 PDB 1h03. 15 Dec. 2009, en.wikipedia.org/wiki/File:Protein_CD55_PDB_1h03.png.
Hill, Michael. “Paroxysmal Nocturnal Hemoglobinuria (PNH): Johns Hopkins Sidney Kimmel Comprehensive Cancer Center.” Is
There Really Any Benefit to Multivitamins?, 18 July 2017,
www.hopkinsmedicine.org/kimmel_cancer_center/types_cancer/paroxysmal_nocturnal_hemoglobinuria_PNH.html.
Krauss, Jonathan S. “The Laboratory Diagnosis of Paroxysmal Nocturnal Hemoglobinuria (PNH): Update 2010.” Laboratory
Medicine, vol. 43, no. 1, 2012, pp. 20–24., doi:10.1309/lmr59zn0mfzmgqrb.
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19. References II
Maloy, Olivia. “Causes Of Blood In Your Urine (Hematuria).” SteadyHealth.com, 29 Jan. 2017,
www.steadyhealth.com/articles/red-urine-does-it-mean-i-have-blood-in-my-urine-and-i-have-a-kidney-problem/causes-of-
blood-in-your-urine-hematuria.
Martí-Carvajal, Arturo J, et al. “Eculizumab for Treating Patients with Paroxysmal Nocturnal Hemoglobinuria.” Cochrane
Database of Systematic Reviews, 2013, doi:10.1002/14651858.cd010340.
Mayo Clinic. “Blood in Urine (Hematuria).” Mayo Clinic, Mayo Foundation for Medical Education and Research, 17 Aug. 2017,
www.mayoclinic.org/diseases-conditions/blood-in-urine/symptoms-causes/syc-20353432.
Ostler, Daniel. Paroxysmal Nocturnal Hemoglobinuria. 10 July 2006, hemepathreview.com/Heme-Review/Part12-11-
PNH.pdf.
Parham, Peter. The Immune System. 4th ed., Garland Science, 2015.
“Paroxysmal Nocturnal Hemoglobinuria.” Hematology: Basic Principles and Practice, by Ronald Hoffman, Elsevier, 2018.
Schrezenmeier, H., et al. “Baseline Characteristics and Disease Burden in Patients in the International Paroxysmal Nocturnal
Hemoglobinuria Registry.” Haematologica, vol. 99, no. 5, 2014, pp. 922–929., doi:10.3324/haematol.2013.093161.
Takeda, Junji, et al. “Deficiency of the GPI Anchor Caused by a Somatic Mutation of the PIG-A Gene in Paroxysmal Nocturnal
Hemoglobinuria.” Cell, vol. 73, no. 4, 1993, pp. 703–711., doi:10.1016/0092-8674(93)90250-t.
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