Clinical Pathological Conference

  Elizabeth Ross, M.D.

 Chief Resident Department of Medicine  October 12 th , 2007

Chief Complaint

 A 46 year old Dominican woman presents with 3 months of increasing abdominal distention and one month of diffuse epigastric pain

History of Present Illness

 2-3 years prior to admission: patient first noticed easy bruisability, she was diagnosed with “anemia” and iron supplementation was started.  3 months pta: she noticed abdominal distention and was started on a “water pill”.  1-2 months pta: Her abdominal distention progressed, she felt like she looked pregnant.  2-3 weeks pta: unrelenting diffuse epigastric pain and discomfort.

HPI, continued

 Her pain persisted so she sought medical attention and was admitted to an outside hospital  Imaging and lab studies revealed abnormal LFTs and portal and splenic vein thrombosis  She was started on a heparin drip and transferred to Bellevue  Repeat imaging confirmed IVC and hepatic vein thrombosis and also showed portal and splenic vein thrombosis

Additonal History

Past Medical History: As above Past Surg History: Tuboligation 15 years ago Medications: iron, multivitamin On transfer: heparin drip Allergies: none Family History: Denies history of: clotting disorders, bleeding disorders, malignancy Social History: Born in Dominican Republic, has lived in the US for 10 years, no recent travel. Ten pack-year tobacco history, quit 9 years ago. No etoh, no illicit drug use. Lives with husband. Worked as HHA until four months ago.

Review of Symptoms

 Monthly, regular menstruation since menarche, with heavy bleeding

Physical Exam

 General: well-developed woman with apparent ascites, moaning in pain, appears stated age, mildly jaundice  Vital signs: BP 127/82, HR 108 and regular, RR 18, Temp 97.6, SpO2 97% room air  HEENT: oropharynx dry, mild scleral icterus  Lymph: no cervical, axillary or inguinal lymphadenopathy  Neck: supple, no jugular venous distension  Pulmonary: clear to auscultation bilaterally

 Physical Exam, continued Heart: tachycardic, regular rhythm, normal heart sounds, no murmurs  Abdominal: Distended, diffusely tender, shifting dullness present, fluid wave present, no masses palpable  Extremities: trace lower extremity edema bilaterally, 2+ peripheral pulses  Skin: no rashes  Rectal: guaiac negative  Neuro: Alert and oriented to person, place and time  Asterixis present

Hematology

11.7

9.3 59 MCV 85 (80-100) 34.9 MPV 9.9 (7.4-10.4)  Differential - wnl INR 1.67, PT 21, PTT 66 HIT Antibody – Positive Thrombin Time 133.6 (21.5 –29.9) RVVT – No Inhibitor Detected

Chemistry

130 95 13 4.6 26 0.5 90 Ca 8.0

Mg 1.7

Phos 2.0

Chemistry/Serology

311 129 6.8 6.0

193 4.3 3.0

LDH – 783 (110-225) ANA – positive Hep Bs Ab – positive Hep Bs Ag – negative Hep Bc Ab – positive Hep C Ab – negative

 Urinalysis:  orange colored, clear; no glucose, moderate (2+) bilirubin, no ketones, Specific gravity 1.048, trace blood, trace protein, pH 6.5, Urobilinogen 4.0 eu/dL (0-2), no nitrite, trace leukocyte esterase, WBC 0-2, RBC 0-2

EKG, sinus tachycardia

Abd/Pelvic CT with contrast



Hepatic vein, portal vein, splenic vein and IVC thromboses



… A diagnostic procedure was performed

Student Discussants

 Marty Wolf – Paroxysmal Nocturnal Hematuria  Leo Drozhinin – Anti-phospholipid Antibody Syndrome  Marci Cherit – Autoimmune Hepatitis  Cristobal Gao – Gastric Cancer

Abdominal/pelvic CT with IV contrast

Dr. Kay Cho

Faculty Discussant

Dr. Mitchell Charap

A DIAGNOSTIC PROCEDURE WAS PERFORMED

Hematologist/Hematopathologist

Dr. David Araten

WBC

PNH 06-113.001

FLOW CYTOMETRY 68%

R1 Patient Normal

99%

100 101 102 CD45 PerCP 103 104 100 M1 M2 101 102 103 CD1 6- FITC M2 M1 104 100 101 102 CD55 PE 103 104 100 M2 M1 101 102 CD5 9 PE 103 104

RBC

PNH 06-11 3.00 1 R2 0

75%

200 400 600 800 100 0 Forward Sca tter 100 M2 M1 101 102 103 104 CD5 5 PE 100

88%

101 102 CD59 PE

CD 59

103 M2 M1 104 100 101 102 103 104 Glycop horin A FITC

IR CD59 FITCÉFSC-H, SSC-H subset 500 400

Flow Analysis of Red Cells Patient Normal Control

PCV-Y CD59 FITC rbcÉFSC-H, SSC-H subset 2000 1500 300 200 100 0 10 0 10 1 1000 500 10 2 FL1-H 10 3 10 4 0 10 0 10 1 10 2 FL1-H

CD59 Expression

10 3 10 4

Flow Analysis of Red Cells Patient

IR CD59 FITCÉFSC-H, SSC-H subset 500 25% 65% 400

Normal Control

10% PCV-Y CD59 FITC rbcÉFSC-H, SSC-H subset 2000 1500 300 1000 200 500 100 0 10 0 10 1 10 2 FL1-H 10 3 10 4 0 10 0 10 1 10 2 FL1-H

CD59 Expression

10 3 10 4

Flow Analysis of Red Cells Patient

IR CD59 FITCÉFSC-H, SSC-H subset 500 25% 65% 400 10%

Normal Control

PCV-Y CD59 FITC rbcÉFSC-H, SSC-H subset 2000 1500 300 200 100 0 10 0 1000 PNH III PNH II 10 1 500 10 2 FL1-H 10 3 10 4 0 10 0 10 1 10 2 FL1-H

CD59 Expression

10 3 10 4

Paroxysmal Nocturnal Hemoglobinuria

More Typical PNH Patient MA CD59 FITC rbcÉFSC-H, SSC-H subset 500 400 300 200 100 0 10 0 PNH III 10 1 10 2 FL1-H 10 3 10 4

CD55 CD59 CD14 CD48 CD87 TFPI monocytes stem cell red cell CD55 CD59 acetylcholinesterase neutrophil CD55 CD59 CD66 CD24 CD16 lymphocyte CD55 CD59 CD48 CD52 CD87 platelets CD55 CD59

C5 Eculizumab Classical pathway C5 convertase C3b C4b C2a C5b C3b Bb C3b Eculizumab Alternative Pathway C5 convertase C3b C3b Bb C3 Classical Pathway C3 convertase C4b C2a X X CD55 C4 C2 C1r/C1s Classical pathway C1q Antibody GPI GPI CD59 X C5b C6 C7 C8 C9 Membrane Attack Complex (MAC)

Immune Lysis Test

Rosse 1974

Two populations of cells

Immune Lysis Test

Rosse 1974 Three

populations of cells

Immune Lysis Test PNH II

Rosse 1974

PNH III

Three

populations of cells

Loss of all GPI-linked proteins Extracellular trans membrane protein O=C N GPI GPI-linked protein CD48 CD55 CD59 Intracellular

Large deletion del 735 bp Acquired Somatic Mutations in PIG-A (Xp22.1) in PNH patients del exons 3-4-5 Null mutations

1

1

2

H

* * * * *

716

3 * * * *

849

* * * * * * * * *

982

4 5 * * * * **

1189

** 6

1452

Ava

I polymorphism 50 bp

*

in frame deletion missense mutation inherited mutation nonsense mutation splice site mutation deletion insertion insertion/deletion insertion/duplication non coding region coding region TM, Transmembrane domain (nt 1246-1326, aa 415-442) GlcNAc transferase homologous region (nt 912-1185, aa 304-395) changes very close to each other, presumably resulting from a single mutational event H: Hot spot reported by Mortazavi et al 2003 From Luzzatto & Nafa 2000

Marked Geographical Disparity

 High rates of thrombosis reported in the United States, Europe and India  Much lower rates in Mexico, Japan, China, and Thailand

Ethnicity as a Risk Factor for Thrombosis and Death

Araten et al 2005

Wrap-Up

Dr. Elizabeth Ross

Final Diagnosis:

Paroxysmal Nocturnal Hemoglobinuria

-

with granulocyte and erythrocyte clone size greater than 50%

PNH

 An acquired hemolytic anemia  somatic mutation of the PIG-A gene in a multipotent hematopoietic stem cell  This results in the impaired synthesis of the GPI anchor so proteins (CD 55, 59) can’t link antigens to cell surface  Used to be diagnosed with Ham’s test, illustrating red cells’ susceptibility to lysis by complement, now flow cytometry is used

PNH

 See intravascular hemolysis, cytopenias and venous thrombosis  Venous thrombosis occurs in ~50% of patients which is the most common cause of death  When granulocyte clone size is larger than 50%, patients are at increased risk of thrombosis (Hall et al, Blood, 2003)  PNH accounts for 15-25% of patients with hepatic venous thrombosis

Hillmen et al, NEJM, 1995

PNH

 Management:  Note – 15% spontaneous remission (Hillmen, 1995)  Thrombolysis for hepatic vein thrombosis as soon as possible – case reports with TPA (McMullin et al, Jrnl of Int Med, 1994)  Warfarin should be used as primary prophylaxis in patients if platelets >100 (Hall et al, Blood, 2003)  Monoclonal Ab, Eculizumab – inhibit lytic effect of complement by binding to C5 (Hillmen et al, NEJM, 2006)  Cure?: bone marrow transplant, gene therapy

Pathogenesis of Patient’s Disease Acquire PIG-A mutation  Defect of GPI anchor on cell surface  GPI-linked proteins (CD 55, 59) can’t link antigens to cell surface  Multipotent hematopoietic stem cells are susceptible to complement Platelets RBCs Platelet activation Intravascular hemolysis Thrombosis Thrombocytopenia Anemia Venous Thrombosis (Budd Chiari) Heparin gtt Abdominal distention and ascites Diffuse epigastric pain Elevated aminotransferases and INR HIT Ab Progression of thrombosis

Follow-up

 The patient’s abdominal pain and distention continued to cause her distress as an inpatient  She was evaluated by the transplant service for liver transplant and GI for a portocaval shunt but given the presence of extrahepatic thrombosis, neither was pursued  Once the HIT Ab was found to be positive she was taken off of heparin and transferred to the MICU so that she could receive TPA based on hematology’s consultation

Follow-up

 She received 6 cycles of tissue plasminogen activator (Alteplase) – which was stopped secondary to a thigh hematoma and she was started on lepirudin (Refludan) and started on warfarin with a goal INR 2.8-3.4

 Abd/Pel CT and doppler ultrasound 2 weeks after admission (approximately 1 week after the TPA) revealed some flow through the hepatic vein

Follow-up

 Once her ascites and epigastric pain improved, and her INR was at goal, she was discharged  Approximately one year later she had an abd ultrasound with doppler which revealed: -patent main portal vein, collateral flow in the porta hepatis supplying the left portal vein, patent hepatic and splenic veins. No ascites.

 Abd/pel CT showed: portal venous hypertension  Her platelets remain near 90,000

Thank You

Martin Blaser, MD Mitchell Charap, MD David Araten, MD Kay Cho, MD Josh Olstein, MD