Clinical Pathological Conference

Elizabeth Ross, M.D.Elizabeth Ross, M.D. Chief Resident Chief Resident

Department of MedicineDepartment of Medicine October 12October 12thth, 2007, 2007

Chief Complaint

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

History of Present Illness 2-3 years prior to admission: patient first noticed 2-3 years prior to admission: patient first noticed

easy bruisability, she was diagnosed with easy bruisability, she was diagnosed with “anemia” and iron supplementation was started. “anemia” and iron supplementation was started.

3 months pta: she noticed abdominal distention 3 months pta: she noticed abdominal distention and was started on a “water pill”. and was started on a “water pill”.

1-2 months pta: Her abdominal distention 1-2 months pta: Her abdominal distention progressed, she felt like she looked pregnant. progressed, she felt like she looked pregnant.

2-3 weeks pta: unrelenting diffuse epigastric pain 2-3 weeks pta: unrelenting diffuse epigastric pain and discomfort. and discomfort.

HPI, continued

Her pain persisted so she sought medical attention Her pain persisted so she sought medical attention and was admitted to an outside hospitaland was admitted to an outside hospital

Imaging and lab studies revealed abnormal LFTs Imaging and lab studies revealed abnormal LFTs and portal and splenic vein thrombosis and portal and splenic vein thrombosis

She was started on a heparin drip and transferred She was started on a heparin drip and transferred to Bellevue to Bellevue

Repeat imaging confirmed IVC and hepatic vein Repeat imaging confirmed IVC and hepatic vein thrombosis and also showed portal and splenic thrombosis and also showed portal and splenic vein thrombosisvein thrombosis

Additonal History

Past Medical HistoryPast Medical History: : As aboveAs above

Past Surg HistoryPast Surg History: : Tuboligation 15 years agoTuboligation 15 years ago

MedicationsMedications: : iron, multivitaminiron, multivitamin

On transferOn transfer: : heparin dripheparin drip

AllergiesAllergies: : nonenone

Family HistoryFamily History: : Denies history of: clotting disorders, Denies history of: clotting disorders, bleeding disorders, malignancybleeding disorders, malignancy

Social HistorySocial History: : Born in Dominican Republic, has lived in Born in Dominican Republic, has lived in the US for 10 years, no recent travel. Ten pack-year tobacco the US for 10 years, no recent travel. Ten pack-year tobacco history, quit 9 years ago. No etoh, no illicit drug use. Lives history, quit 9 years ago. No etoh, no illicit drug use. Lives with husband. Worked as HHA until four months ago.with husband. Worked as HHA until four months ago.

Review of Symptoms

Monthly, regular menstruation since Monthly, regular menstruation since menarche, with heavy bleedingmenarche, with heavy bleeding

Physical Exam General: well-developed woman with apparent General: well-developed woman with apparent

ascites, moaning in pain, appears stated age, ascites, moaning in pain, appears stated age, mildly jaundicemildly jaundice

Vital signs: BP 127/82, HR 108 and regular, RR Vital signs: BP 127/82, HR 108 and regular, RR 18, Temp 97.6, SpO2 97% room air18, Temp 97.6, SpO2 97% room air

HEENT: HEENT: oropharynx dry, mild scleral icterusoropharynx dry, mild scleral icterus Lymph: no cervical, axillary or inguinal Lymph: no cervical, axillary or inguinal

lymphadenopathy lymphadenopathy Neck: supple, no jugular venous distensionNeck: supple, no jugular venous distension Pulmonary: clear to auscultation bilaterallyPulmonary: clear to auscultation bilaterally

Physical Exam, continued Heart: tachycardic, regular rhythm, normal heart Heart: tachycardic, regular rhythm, normal heart

sounds, no murmurssounds, no murmurs Abdominal: Distended, diffusely tender, shifting Abdominal: Distended, diffusely tender, shifting

dullness present, fluid wave present, no masses dullness present, fluid wave present, no masses palpablepalpable

Extremities: trace lower extremity edema Extremities: trace lower extremity edema bilaterally, 2+ peripheral pulsesbilaterally, 2+ peripheral pulses

Skin: no rashesSkin: no rashes Rectal: guaiac negativeRectal: guaiac negative Neuro: Alert and oriented to person, place and Neuro: Alert and oriented to person, place and

timetime Asterixis presentAsterixis present

Hematology

11.711.7 9.3 59 9.3 59 MCV 85 (80-100)MCV 85 (80-100)

34.9 34.9 MPV 9.9 (7.4-10.4)MPV 9.9 (7.4-10.4)

Differential - wnlDifferential - wnl

INR 1.67, PT 21, PTT 66INR 1.67, PT 21, PTT 66HIT Antibody – PositiveHIT Antibody – PositiveThrombin Time 133.6 (21.5 –29.9)Thrombin Time 133.6 (21.5 –29.9)RVVT – No Inhibitor DetectedRVVT – No Inhibitor Detected

Chemistry

130 95 13130 95 13

90 Ca 8.090 Ca 8.0

4.6 4.6 26 26 0.5 0.5 Mg 1.7 Mg 1.7

Phos 2.0Phos 2.0

Chemistry/Serology

311 129 6.8 6.0311 129 6.8 6.0 193 4.3 3.0193 4.3 3.0

LDH – 783 (110-225)LDH – 783 (110-225)ANA – positiveANA – positiveHep Bs Ab – positiveHep Bs Ab – positiveHep Bs Ag – negativeHep Bs Ag – negativeHep Bc Ab – positiveHep Bc Ab – positiveHep C Ab – negativeHep C Ab – negative

Urinalysis:Urinalysis: orange colored, clear; no glucose, orange colored, clear; no glucose,

moderate (2+) bilirubin, no ketones, moderate (2+) bilirubin, no ketones, Specific gravity 1.048, trace blood, trace Specific gravity 1.048, trace blood, trace protein, pH 6.5, Urobilinogen 4.0 eu/dL protein, pH 6.5, Urobilinogen 4.0 eu/dL (0-2), no nitrite, trace leukocyte esterase, (0-2), no nitrite, trace leukocyte esterase, WBC 0-2, RBC 0-2 WBC 0-2, RBC 0-2

EKG, sinus tachycardia

Abd/Pelvic CT with contrast

Hepatic vein, portal vein, splenic Hepatic vein, portal vein, splenic vein and IVC thrombosesvein and IVC thromboses

… … A diagnostic procedure was A diagnostic procedure was performedperformed

Student Discussants

Marty Wolf – Paroxysmal Nocturnal Marty Wolf – Paroxysmal Nocturnal HematuriaHematuria

Leo Drozhinin – Anti-phospholipid Leo Drozhinin – Anti-phospholipid Antibody SyndromeAntibody Syndrome

Marci Cherit – Autoimmune HepatitisMarci Cherit – Autoimmune Hepatitis Cristobal Gao – Gastric CancerCristobal Gao – Gastric Cancer

Abdominal/pelvic CT with Abdominal/pelvic CT with IV contrastIV contrast

Dr. Kay ChoDr. Kay Cho

Faculty Discussant

Dr. Dr. Mitchell CharapMitchell Charap

A DIAGNOSTIC PROCEDURE A DIAGNOSTIC PROCEDURE WAS PERFORMEDWAS PERFORMED

Hematologist/Hematopathologist

Dr. David AratenDr. David Araten

100 101 102 103 104

CD16- FITC

M1M2

100 101 102 103 104

CD55 PE

M1M2

100 101 102 103 104

CD59 PE

M1M2

PNH 06-113.001

100 101 102 103 104

CD45 PerCP

R1

PNH 06-113.001

0 200 400 600 800 1000Forward Scatter

R2

100 101 102 103 104

Glycophorin A FITC

M1M2

WBC

RBC

CD 59

Normal

Patient

68% 99%

100 101 102 103 104

CD55 PE

M1M2

75%

100 101 102 103 104

CD59 PE

88%

FLOW CYTOMETRY

100 101 102 103 104

0

100

200

300

400

500

IR CD59 FITCÉFSC-H, SSC-H subset

FL1-H

# C

ells

100 101 102 103 104

0

500

1000

1500

2000

PCV-Y CD59 FITC rbcÉFSC-H, SSC-H subset

FL1-H

# C

ells

Normal ControlPatient

# E

vent

s

CD59 Expression

Flow Analysis of Red Cells

100 101 102 103 104

0

100

200

300

400

500

IR CD59 FITCÉFSC-H, SSC-H subset

FL1-H

# C

ells

100 101 102 103 104

0

500

1000

1500

2000

PCV-Y CD59 FITC rbcÉFSC-H, SSC-H subset

FL1-H

# C

ells

Normal ControlPatient

25% 65%10%

# E

vent

s

CD59 Expression

Flow Analysis of Red Cells

100 101 102 103 104

0

100

200

300

400

500

IR CD59 FITCÉFSC-H, SSC-H subset

FL1-H

# C

ells

100 101 102 103 104

0

500

1000

1500

2000

PCV-Y CD59 FITC rbcÉFSC-H, SSC-H subset

FL1-H

# C

ells

Normal ControlPatient

25% 65%10%

# E

vent

s

CD59 Expression

Flow Analysis of Red Cells

PNH III

PNH II

Paroxysmal Nocturnal Hemoglobinuria

100 101 102 103 104

0

100

200

300

400

500

MA CD59 FITC rbcÉFSC-H, SSC-H subset

FL1-H

# C

ells

More Typical PNH Patient

PNH III

red cell neutrophilplatelets

monocytes

stem cell lymphocyte

CD55CD59acetylcholinesterase

CD55CD59CD66CD24CD16

CD55CD59

CD55CD59CD48CD52CD87

CD55CD59CD14CD48CD87TFPI

CD59

C6

C7C8

C9 Membrane AttackComplex (MAC)

Antibody

C1q

C1r/C1s

C4 C2

C4b C2a CD55

XC3

C3bBb

Classicalpathway

Alternative Pathway

C3 convertase

C4b C2a

C3b

Classical Pathway C3 convertase

BbC3b C3b

AlternativePathwayC5 convertase

Classical pathwayC5 convertase

C5b

C5b

C5

C5

XX

GPI GPI

C3b

Eculizumab

C3

C3b

Eculizumab

Immune Lysis Test

Two populations of cellsRosse 1974

Immune Lysis Test

Three populations of cellsRosse 1974

Immune Lysis Test

Three populations of cellsRosse 1974

PNH III

PNH II

Loss of all GPI-linked proteins

Extracellular

Intracellular

GPI

O=CN

GPI-linkedprotein

CD48 CD55CD59

trans-membrane

protein

4

982 11898497161

1

1452

3

Ava Ipolymorphism

*

****

*

** *

*

***

**

50 bp

**

652

H

** *** **

**

** **

GlcNAc transferase homologous region (nt 912-1185, aa 304-395)

non coding region coding regionTM, Transmembrane domain (nt 1246-1326, aa 415-442)

deletion insertioninsertion/deletioninsertion/duplicationinherited mutation

missense mutationin frame deletion

nonsense mutationsplice site mutation

* changes very close to each other, presumablyresulting from a single mutational event

.

Null mutations

del exons 3-4-5del 735 bp

Large deletion

H: Hot spot reported by Mortazavi et al 2003

Acquired Somatic Mutations in PIG-A (Xp22.1) in PNH patients

From Luzzatto & Nafa 2000

Marked Geographical Disparity

High rates of thrombosis reported in the High rates of thrombosis reported in the United States, Europe and India United States, Europe and India

Much lower rates in Mexico, Japan, Much lower rates in Mexico, Japan, China, and ThailandChina, and Thailand

0

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ombo

sis-

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AA/PNH

Classic PNH

Other patientsLatin-

American

African-American

A

B

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COtherPatients

Latin-American

African-American

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Years

Thr

ombo

sis-

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hrom

bosi

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AA/PNH

Classic PNH

Other patientsLatin-

American

African-American

A

B

0

0.1

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5 10 15 20 25

Sur

viva

l

COtherPatients

Latin-American

African-American

Ethnicity as a Risk Factor for Thrombosis and Death

Araten et al 2005

Wrap-Up

Dr. Elizabeth RossDr. Elizabeth Ross

Final Diagnosis:

Paroxysmal Nocturnal HemoglobinuriaParoxysmal Nocturnal Hemoglobinuria

--with granulocyte and erythrocyte clone with granulocyte and erythrocyte clone size greater than 50% size greater than 50%

PNH An acquired hemolytic anemia An acquired hemolytic anemia somatic somatic

mutation of the PIG-A gene in a multipotent mutation of the PIG-A gene in a multipotent hematopoietic stem cellhematopoietic stem cell

This results in the impaired synthesis of the This results in the impaired synthesis of the GPI anchor so proteins (CD 55, 59) can’t GPI anchor so proteins (CD 55, 59) can’t link antigens to cell surfacelink antigens to cell surface

Used to be diagnosed with Ham’s test, Used to be diagnosed with Ham’s test, illustrating red cells’ susceptibility to lysis illustrating red cells’ susceptibility to lysis by complement, now flow cytometry is usedby complement, now flow cytometry is used

PNH See intravascular hemolysis, cytopenias and See intravascular hemolysis, cytopenias and

venous thrombosisvenous thrombosis Venous thrombosis occurs in ~50% of Venous thrombosis occurs in ~50% of

patients which is the most common cause of patients which is the most common cause of deathdeath

When granulocyte clone size is larger than When granulocyte clone size is larger than 50%, patients are at increased risk of 50%, patients are at increased risk of thrombosis thrombosis (Hall et al, Blood, 2003)(Hall et al, Blood, 2003)

PNH accounts for 15-25% of patients with PNH accounts for 15-25% of patients with hepatic venous thrombosishepatic venous thrombosis

Hillmen et al, NEJM, 1995

PNH Management: Management:

Note – 15% spontaneous remission Note – 15% spontaneous remission (Hillmen, 1995)(Hillmen, 1995)

Thrombolysis for hepatic vein thrombosis as soon Thrombolysis for hepatic vein thrombosis as soon as possible – case reports with TPA as possible – case reports with TPA (McMullin et al, Jrnl (McMullin et al, Jrnl of Int Med, 1994)of Int Med, 1994)

Warfarin should be used as primary prophylaxis in Warfarin should be used as primary prophylaxis in patients if platelets >100 patients if platelets >100 (Hall et al, Blood, 2003)(Hall et al, Blood, 2003)

Monoclonal Ab, Eculizumab – inhibit lytic effect Monoclonal Ab, Eculizumab – inhibit lytic effect of complement by binding to C5 of complement by binding to C5 (Hillmen et al, NEJM, (Hillmen et al, NEJM, 2006)2006)

Cure?: bone marrow transplant, gene therapyCure?: bone marrow transplant, gene therapy

Pathogenesis of Patient’s DiseaseAcquire PIG-A mutation Acquire PIG-A mutation Defect of GPI anchor on cell surface Defect of GPI anchor on cell surface GPI-linked proteins (CD 55, 59) can’t link antigens to cell surface GPI-linked proteins (CD 55, 59) can’t link antigens to cell surface Multipotent hematopoietic stem cells are susceptible to complementMultipotent hematopoietic stem cells are susceptible to complement

PlateletsPlatelets RBCs RBCs

Platelet activationPlatelet activation Intravascular hemolysisIntravascular hemolysis

AnemiaAnemia

ThrombosisThrombosis ThrombocytopeniaThrombocytopenia

Venous ThrombosisVenous Thrombosis Heparin gtt Heparin gtt HIT Ab HIT Ab

(Budd Chiari)(Budd Chiari)

Abdominal distention and ascitesAbdominal distention and ascites ProgressionProgression

Diffuse epigastric pain Diffuse epigastric pain of of thrombosisthrombosis

Elevated aminotransferases and INRElevated aminotransferases and INR

Follow-up The patient’s abdominal pain and distention The patient’s abdominal pain and distention

continued to cause her distress as an continued to cause her distress as an inpatientinpatient

She was evaluated by the transplant service She was evaluated by the transplant service for liver transplant and GI for a portocaval for liver transplant and GI for a portocaval shunt but given the presence of extrahepatic shunt but given the presence of extrahepatic thrombosis, neither was pursuedthrombosis, neither was pursued

Once the HIT Ab was found to be positive Once the HIT Ab was found to be positive she was taken off of heparin and transferred she was taken off of heparin and transferred to the MICU so that she could receive TPA to the MICU so that she could receive TPA based on hematology’s consultationbased on hematology’s consultation

Follow-up She received 6 cycles of tissue plasminogen She received 6 cycles of tissue plasminogen

activator (Alteplase) – which was stopped activator (Alteplase) – which was stopped secondary to a thigh hematoma and she was secondary to a thigh hematoma and she was started on lepirudin (Refludan) and started on started on lepirudin (Refludan) and started on warfarin with a goal INR 2.8-3.4warfarin with a goal INR 2.8-3.4

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

Follow-up Once her ascites and epigastric pain improved, and Once her ascites and epigastric pain improved, and

her INR was at goal, she was dischargedher INR was at goal, she was discharged Approximately one year later she had an abd Approximately one year later she had an abd

ultrasound with doppler which revealed:ultrasound with doppler which revealed:

-patent main portal vein, collateral flow -patent main portal vein, collateral flow in the porta hepatis supplying the left portal in the porta hepatis supplying the left portal vein, patent hepatic and splenic vein, patent hepatic and splenic veins. No veins. No ascites.ascites.

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

Thank You

Martin Blaser, MDMartin Blaser, MD

Mitchell Charap, MDMitchell Charap, MD

David Araten, MDDavid Araten, MD

Kay Cho, MDKay Cho, MD

Josh Olstein, MDJosh Olstein, MD