HEMOGLOBINURIA

BY SHASHIDHAR PATIL

Heamoglobinuria
Hemoglobinuria is a condition in which the oxygen transport protein hemoglobin is found in high concentrations in the urine. The condition is often associated with hemolytic anemia, in which red blood cells are destroyed, thereby increasing levels of free plasma hemoglobin. The excess hemoglobin is filtered by the kidneys, which release it into the urine, giving urine a red colour.

Classification of Hemolytic anemias

I. Red cell abnormality (Intracorpuscular factors) A. Hereditary 1. Membrane defect (spherocytosis, elliptocytosis) 2. Metabolic defect (Glucoze-6-Phosphate-Dehydrogenaze (G6PD) deficiency, Pyruvate kinase (PK) deficiency) 3. Hemoglobinopathies (unstable hemoglobins, thalassemias, sickle cell anemia )
B. Acquired 1. Membrane abnormality-paroxysmal nocturnal hemoglobinuria (PNH)

II. Extracorpuscular factors

A. Immune hemolytic anemias
1. Autoimmune hemolytic anemia - caused by warm-reactive antibodies - caused by cold-reactive antibodies 2. Transfusion of incompatible blood

B. Nonimmune hemolytic anemias

1. Chemicals 2. Bacterial infections, parasitic infections (malaria), venons 3. Hemolysis due to physical trauma - hemolytic - uremic syndrome (HUS) - thrombotic thrombocytopenic purpura (TTP) - prosthetic heart valves 4. Hypersplenism

Metabolic Defect
G6PD deficiency Hexose monophosphate shunt Most common RBC enzyme defect, >50 variants X-linked Low glutathione due to low NADPH Oxidative lysis, Heinz bodies, spherocytic Primaquine, fava beans Pyruvate kinase deficiency Glycolysis Low RBC ATP level Non-spherocytic B12 and folate deficiency Macrocytic HJ bodies Hemoglobinopathies Poikilocytosis Abnormal Hb

Extracorpuscular Factors
Antibodies Autoimmune Isoimmune Drugs, antibiotics Fresh water Abnormal plasma lipids Acanthocytosis Venom Snake Spider Bee

Extracorpuscular Factors
Trauma DIC Hemolytic uremic syndrome (HUS) TTP Angiopathy Heat Heart valves March hemoglobinuira

Microorganisms Malaria Babesia Clostridium Gram negative endotoxin

Mechanisms of hemolysis:
- intravascular - extravascular

Intravascular hemolysis :
- red cells destruction occurs in vascular space

- clinical states associated with Intravascular hemolysis: acute hemolytic transfusion reactions severe and extensive burns paroxysmal nocturnal hemoglobinuria severe microangiopathic hemolysis physical trauma bacterial infections and parasitic infections (sepsis)

Intravascular hemolysis :
- laboratory signs of intravascular hemolysis:

indirect hyperbilirubinemia erythroid hyperplasia hemoglobinemia methemoalbuminemia hemoglobinuria absence or reduced of free serum haptoglobin hemosiderynuria

Extravascular hemolysis :
- red cells destruction occurs in reticuloendothelial system - clinical states associated with extravascular hemolysis : autoimmune hemolysis delayed hemolytic transfusion reactions hemoglobinopathies hereditary spherocytosis hypersplenism hemolysis with liver disease - laboratory signs of extravascular hemolysis: indirect hyperbilirubinemia increased excretion of bilirubin by bile erythroid hyperplasia hemosiderosis

Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal Nocturnal Hemoglobinuria (PNH)

PNH is an acquired chronic hemolytic anemia which arises from a somatic mutation in a hematopoietic stem cell. Most hematopoitic cell lines may be affected by the intrinsic membrane defect. This defect renders the red cells highly susceptible to complement mediated lysis resulting in the characteristic hemolysis.

History
Investigator Year
Gull Strubing 1866 1882

Contribution
Described nocturnal and paroxysmal nature of intermittent haematinuria in a young tanner. Distinguished PNH from paroxysmal cold haemoglobinuria and march haemoglobinuria. Attributed the problem to the red cells. Red cells lysed in acidified serum. Suggested a role for complement. Coined the name paroxysmal nocturnal haemoglobinuria. Described perpetual hemosiderinemia in absence of hemolysis. Their names became eponymous for PNH in Europe. Identified the role of complement in lysis of PNH red cells. Developed the acidified serum test, also called the Ham test, which is still used to diagnose PNH. Demonstrated that only a portion of PNH red cells are abnormally sensitive to complement. Suggests defect in membrane protein anchoring system responsible Flow cytometry for the diagnosis of PNH

van den Burgh 1911 Enneking Marchiafava and Micheli Ham 1928 19281931 19371939

Davitz

1986

Hall & Rosse

1996

Epidemiology
Rare disease frequency unknown thought to be on the same order as aplastic anemia (2-6 per million)

Median age at diagnosis

~ 35 yrs PNH reported at extremes of age

Female:Male ratio = 1.2:1.0 No increased risk of PNH in patient relatives Median Survival after diagnosis ~ 10-15 yrs

Paroxysmal Nocturnal Hemoglobinuria

Caused from a defect in the production of GPI protein anchors on the surface of all blood cells produced by the PNH bone marrow stem cells This is an acquired mutation of PIG-A, a gene on the X chromosome important in making GPI protein anchors.
Only blood cells have the defect. Defect makes the red cells in particular susceptible to destruction by the complement

PNH
There are 2 main ways to attach proteins to the surface of cells-either through transmembrane attachments or GPI-anchors. Many proteins are attached to the surface by GPI anchors. PIG-A gene is vital to the production of GPI anchors. In PNH, you have a mutation in PIG-A so that it has reduced activity or no activity at all.

Pathogenesis - The Defect

Defect - Somatic mutation of PIG-A gene (phosphatidylinositol glycan complementation group A) located on the X chromosome in a clone of a hematopoietic stem cell
>100 mutations in PIG - A gene known in PNH The mutations (mostly deletions or insertions) generally result in stop codons - yielding truncated proteins which may be non or partially functional - explains heterogeneity seen in PNH

Pathogenesis - The Defect

GPI Anchor

PIG - A gene codes for 60 kDa protein glycosyltransferase which effects the first step in the synthesis of the glycolipid GPI anchor (glycosylphosphatidylinositol). Results in clones lacking GPI anchor - in turn, attached proteins
PIG - A protein

Pathogenesis - The Defect GPI Anchor deficiency

PNH blood cells deficient in GPI anchor lack membrane proteins linked via the anchor
Membrane proteins w/o anchor degraded in ER

Severity & size of deficiency - variable clinical/diagnostic implications GPI anchor highly conserved in all eukaryotic cells
Variant surface proteins of Trypanosomes - GPI linked Shed by cleavage of GPI anchor - immune system avoid Swapping GPI linked proteins - CD55 complement resistance - Schistosoma mansoni In Humans
signal transduction, co-receptors advantage to this type of anchor?

Surface Proteins Missing on PNH Blood Cells Antigen Enzymes Acetylcholinesterase (AchE) Ecto-5'-nucleotidase (CD73) Neutrophil alkaline phosphatase(NAP) ADP-rybosyl transferase

Proteins anchored by GPI Anchor and

Expression Pattern

Red blood cells Some B- and T-lymphocytes Neutrophils Some T-lymphs, Neutrophils
Lymphocytes All blood cells Neutrophils

Adhesion molecules Blast-I/CD48 Lymphocyte functionassociated antigen-3(LFA-3 or CD58) CD66b

Complement regulating surface proteins Decay accelerating factor (DAF or CD55) Homologous restriction factor, Membrance inhibitor of reactive lysis (MIRL or CD59)

All blood cells All blood cells

Surface Proteins Missing on PNH Blood Cells Antigen Receptors Fc- receptor III (Fc Rlll or CD16) Monocyte differentiation antigen (CD14) Urokinase-type Plasminogen Activator Receptor (u-PAR, CD87) Expression Pattern Neutrophils, NK-cells, macrophages, some T-lymphocytes Monocytes, macrophages Monocytes, granulocytes

Blood group antigens Comer antigens (DAF) Yt antigens (AchE) Holley Gregory antigen John Milton Hagen antigen (JMH) Dombrock reside
Neutrophil antigens NB1/NB2

Red blood cells Red blood cells Red blood cells Red blood cells, lymphocytes Red blood cells Neutrophils

Surface Proteins Missing on PNH Blood Cells

Antigen
Other surface proteins of unknown functions CAMPATH-1 antigen (CDw52) CD24 p5O-80 GP500 GPI75

Expression Pattern

Lymphocytes, monocytes B-lymphocytes, Neutrophils, eosinophils Neutrophils Platelets Platelets

Pathogenesis - Functional consequences of lack of GPI linked proteins

In vivo function of many of these membrane proteins not fully understood However, CD55 and CD59 functions are well known
CD55 (decay accelerating factor) inhibits the formation or destabilizes complement C3 convertase (C4bC2a) CD59 (membrane inhibitor of reactive lysis, protectin, homologous restriction factor) Protects the membrane from attack by the C5-C9 complex Inherited absences of both proteins in humans have been described
Most inherited deficiencies of CD55 - no distinct clinical hemolytic syndrome Inherited absence of CD59 - produces a clinical disease similar to PNH with hemolysis and recurrent thrombotic events

Mechanism for hemolysis in PNH via lack of CD59

(CD59)

(CD59)

Pathogenesis - Clonal evolution and cellular selection

Expansion of abnormal hematopoietic stem cell required for PNH disease expression
Theories for expansion
Blood cells lacking GPI-linked proteins have intrinsic ability to grow abnormally fast
In vitro growth studies demonstrate that there are no differences in growth between normal progenitors and PNH phenotype progenitors In vivo - mice deficient for PIG -A gene also demonstrates no growth advantage to repopulation of BM.

Additional environmental factors exert selective pressure in favor of expansion of GPI anchor deficient blood cells
PNH hematopoitic cells perferentially engraft SCID mice compared to phenotypically hematopoitic cells Close association with AA - PNH hematopoitic cells cells may be more resistant to the IS than normal hematopoitic cells. Evidence in AA is that the decrease in hematopoitic cells is due to increased apoptosis via cytotoxic T cells by direct cell contact or cytokines (escape via deficiency in GPI linked protein???)

Normal Hematopoietic Cells

CD55

CD59

Transmembrane protein Membrane lipid bilayer

GPI anchors

Lectin Pathway Carbohydrate structure Pathogen/Damaged cell

Classical Pathway Antibody bound to antigen Immune complexes

Alternative Pathway Microbial membranes Bacterial LPS

C3 convertase C4b2a, C3bBb C3 C3b C3a CD55 Weak anaphylatoxin

C5 convertases C4b2a3b, C3bBb3b CD55 C5 C6,C7,C8,C9 CD59 C5b-9 Microorganism Destruction Red Blood Cell Lysis Platelet Activation C5a Inflammation Cell Activation

MA

PNH
PNH red cells are deficient in all GPI anchored protein, but 2 are important in protecting red cells from destruction: CD55 (DAF) and CD59 (MIRL). Without these proteins, red cells dont have their normal protection against the complement system. In PNH, you have uncontrolled, complement mediated hemolysis (destruction of red cells). This happens all the time, and is accelerated when you have an event that activates the complement system (infection).

How do you get PNH?

This is an acquired disease. We think PIG-A mutations can happen spontaneously, but unless the environment is supportive of those mutations they never develop into PNH. In a bone marrow under attack or failing, PIG-A mutations have an advantage-they survive the attack better (for some reason). Therefore, the PNH cells have an advantage and can expand to become a significant portion of the bone marrow cells.

Two-Step Model of Developing PNH

Normal Bone Marrow

MARROW INJURY
Step I. Clonal Selection

Normal bone marrow with normal HSCs and rare PNH mutant HSCs

Bone marrow damage (likely immune mediated) selects for clones.

Step II. Clonal Dominance

After selection, expansion of PIG-A mutant HSCs varies depending on other characteristics of the affected cells. These other characteristics may be determined by genetic (mutational), epigenetic (nonmutational), or environmental factors.

How do we know PNH cells have an advantage?

PNH is found in diseases where the bone marrow is under attack or damaged:
Aplastic anemia (up to 60% of patients with aplastic anemia have a detectable PNH clone). Myelodysplastic syndrome (MDS)-up to 20% of patients with MDS have an identifiable PNH clone Other immune-mediated diseases: ITP Blood cancers: leukemias-both chronic and acute Why the PNH cells have an advantage is unknown. Why the PNH cells expand to produce more blood cells is unknown.

How do you get PNH?

Patients with PNH often have a history of aplastic anemia or other bone marrow injury PNH can come on later, after they have recovered from the initial bone marrow insult. You can have a little or a lot of PNH cells, and that can effect how the disease impacts the health of the patient.

Who Should be Tested for PNH?

Patients with unexplained hemolytic anemia Patients with bone marrow failure, including aplastic anemia and MDS Patients with hemoglobinuria Patients with unusual/repetitive thrombosis, and arterial thrombosis otherwise unexplained. Patients with episodic swallowing problems or abdominal pain of unclear etiology with associated hemolysis

PNH symptoms
FATIGUE Anemia Decreased stamina Shortness of breath Abdominal pain Back pain Difficulty swallowing Chest pain Erectile dysfunction Decreased libido or interest in intimacy Headaches Swelling related to blood clots Increased risk for infections Increased risk for bleeding Depression, frustration, feeling lack of control over life Weight changes, body changes

Presenting features in 80 patients with PNH

Signs and Symptoms Symptoms of anemia Hemoglobinuria Hemorrhagic signs and symptoms Aplastic anemia Gastrointestinal symptoms # of Px (%) 28 (35) 21 (26) 14 (18) 10 (13) 8 (10)

Hemolytic anemia and jaundice Iron-deficiency anemia

Thrombosis or embolism Infections Neurologic signs and symptoms

7 (9) 5 (6)
5 (6) 4 (5) 3 (4)

Dacie and Lewis, 1972

Clinical Features
Major symptoms (Hemolysis, Cytopenia, and tendency to thrombosis)
chronic hemolysis with acute exacerbations (hallmark)
most patient at some stage only 1/3 exhibit hemolysis at diagnosis Recurrent attacks of intravascular hemolysis are usually associated with;
hemoglobinuria abdominal pain dysphagia

Clinical Features
cytopenia (varying severity)
isolated subclinical thrombocytopenia classical severe aplastic anemia

tendency to thrombosis
venous thrombosis (40%) of patients, main cause of morbidity

Variable expression of above often causes considerable delay in the diagnosis Major cause of death
venous thrombosis complications from progressive pancytopenia

Clinical Features - Long term

25% of PNH patients survive >25 years - one half of these go on to spontaneous remission Remission patients
hematological values revert to normal no PHN rbcs or granulocytes detected PNH lymphocytes - still detected but no clinical consequence

Higher incidence of acute leukemia (6%)

preleukemic condition most likely bone marrow failure not PNH

Clinical Features - Relationship to aplastic anemia (AA)

AA described as pancytopenia with nonfunctioning bone marrow. Cytopenia in one or all cell lineages also common to PNH High percentage of patients with AA develop clinical PNH or have lab evidence of PNH abnormality at some point (52%) Supports the theory that bone marrow failure supports the abnormal PNH cells - more later

Natural History of PNH

Long term study of 80 patients with PNH seen at one institution between 1940 and 1970 Results median age at diagnosis: 42 (16-75) median survival: 10 years 28% survived more than 25 years 39% had one or more episodes of venous thrombosis 12 experienced spontaneous clinical recovery leukemia did not develop in any of the patients

Hillmen et al, NEJM, 1995

Laboratory Evaluation of PNH

Acidified Serum Test (Ham Test 1939)
Acidified serum activates alternative complement pathway resulting in lysis of patients rbcs May be positive in congenitial dyserythropoietic anemia Still in use today

Sucrose Hemolysis Test (1970)

10% sucrose provides low ionic strength which promotes complement binding resulting in lysis of patients rbcs May be positive in megaloblastic anemia, autoimmune hemolytic anemia, others Less specific than Ham test

Laboratory Evaluation of PNH

PNH Diagnosis by Flow Cytometry (1986) Considered method of choice for diagnosis of PNH (1996) Detects actual PNH clones lacking GPI anchored proteins More sensitive and specific than Ham and sucrose hemolysis test

PNH Diagnosis by Flow Cytometry

Of the long list of GPI anchored protein, monoclonal antibodies to the following antigens have been used in the diagnosis of PNH
The most useful Abs are to CD14, 16, 55, 59, and 66. Are all required? Probably not - more studies needed

Antigen Cell Lineage CD14

Function LPS receptor, MDF

monocytes

CD16
CD24 CD55 CD58 CD59 CD66b

neutrophils
neutrophils all lineages all lineages all lineages neutrophils

FcIII receptor
B-cell differentiation marker DAF possible adhesion MIRL, HRF, protectin CEA-related glycoprotein

PNH Diagnosis by Flow Cytometry Antigen expression is generally categorized into three antigen density groups
type I Normal Ag expression type II Intermediate Ag expression type III No Ag expression

Patient samples that demonstrate cell populations with diminished or absent GPI-linked proteins (Type II or III cells) with multiple antibodies are considered to be consistent with PNH. Should examine multiple lineages (ie granulocytes & monocytes)

PNH Diagnosis by Flow Cytometry

Examples of variable GPI linked CD59 expression on granulocytes on four PNH patients

PNH Diagnosis by Flow Cytometry

Example of variable expression of several GPI linked Ags on several lineages

From Purdue Cytometry CD-ROM vol3 97

PNH Diagnosis by Flow Cytometry Flow Cytometry is method of choice but only supportive for/against diagnosis More studies are needed to better define whether the type (I, II, or III), cell lineage, and size of the circulating clone can provide additional prognostic information.

Theoretically - should be very valuable

PNH Complications
Bone marrow failure-all the blood counts are low, bone marrow is not producing cells as it should be. Clots! Infections-either related to disease or complications of treatment (prednisone, eculizumab) Bleeding-either from low blood counts or blood thinners for treatment/prevention of clots Risk of blood transfusions-luckily the blood product pool is extremely safe at this time Cardiopulmonary issues related to nitric oxide scavenging by free hemoglobin-can cause high pressure in the lung system (pulmonary hypertension) that can damage the heart. Some can be reversed by eculizumab. Pregnancy

Implications of Living with PNH

Quality of life issues Financial impacts
Ability to work Costs of treatments/medical care

Unpredictability Fear of complications

Am I going to clot (again)?

Burden of treatments
Blood transfusions Eculizumab Bone marrow transplantation

Therapy
Bone Marrow Transplantation
Only curative treatment chronic condition (possiblity of spontaneous remission) BMT should be avoided

Immunosuppressive therapy
Antilymphocyte globulin &/or cyclosporine A
Does not alter proportion of PNH hemopoiesis

Steroids - experimental - controlled studies ??

Growth Factors
Some improvement no evidence that normal clones respond better than PNH clones

PNH: Management Guidelines

Luzzato, ASH, 2001

What are the treatments? Cures?

Treatment options depend on certain factors
What is the clone size? How does the marrow function? What are all the blood counts? Clot risk

Short-term vs. long-term treatment

Blood transfusions and pulse prednisone often used in short-term Long-term-vitamin replacement, low-dose prednisone, eculizumab, bone marrow transplant

Cures? Yes, with bone marrow transplant Control? Yes.

Eculizumab
First/only drug targeted to PNH Monoclonal antibody against complement protein 5 (C5). Binds this protein and halts the rest of the complement cascade. Protects PNH cells from destruction by halting the complement cascade. Very effective at reducing hemolysis, reducing transfusion needs, improving QOL. Early evidence suggests it may reduce clots.

Lectin Pathway Carbohydrate structure Pathogen/Damaged cell

Classical Pathway Antibody bound to antigen Immune complexes

Alternative Pathway Microbial membranes Bacterial LPS

C3 convertase C4b2a, C3bBb C3 C3b C3a CD55 Weak anaphylatoxin

Eculizumab
C5 convertases C4b2a3b, C3bBb3b CD55 C5 C6,C7,C8,C9 CD59 C5b-9 Microorganism Destruction Red Blood Cell Lysis Platelet Activation C5a Inflammation Cell Activation

Eculizumab-Pros and Cons

Pros
Very effective at reducing hemolysis Well tolerated Improvements in QOL, reduction in transfusions proven Reduction in burden of disease Infusion weekly X5, then every 2 weeks Probable reduction in clots

Cons
$$$$ Infusion weekly X5, then every 2 weeks Infection risk: meningococcal meningitis Burden of treatment Plan for lifetime therapy Does not improve other blood counts

Eculizumab in Pregnancy
Pregnancy in PNH is very risky for both the mother and fetus, due to risk of clotting, infection, and fetal loss. Is eculizumab safe in pregnancy? Could this help reduce the risks of pregnancy? Report of 7 pregnancies in patients that received eculizumab at some point during pregnancy.

Outcomes in Pregnancy with Eculizumab

Patient Clone size
Use in pregnancy Complications in Outcomes pregnancy

1
2 3 4 5 6 7

70%
93% 96% 88% 99% 98% 98%

Up to 5 weeks
Up to 5 weeks Up to 14 weeks Up to 4 weeks All of pregnancy From week 27 All of pregnancy

No
No
Post-partum fever

Elective termination
Healthy baby Healthy baby Healthy baby Healthy baby Healthy baby twins at 35 wks Healthy baby at 28 wks

No
Hemolysisbreakthough
Post-partum hemorrhage

Pre-eclampsia

Bone Marrow Transplant for PNH

This is very effective at curing PNH Risks include toxicity from the transplant and graft vs. host disease (GVHD) In patients with aplastic anemia or MDS/leukemia and PNH transplant is driven by other disease Transplantation continues to improve over time, in particular transplants from unrelated donors

From Italian group, 26 transplants for PNH 1988-2006

Matched sibling 10 year survival 65%

All transplants 10 year survival 56%

Treatment Options
Classical PNH
High clone size (>50%) Risk of clot highest Bone marrow functions well and tries to keep up (high reticulocyte count) May or may not need red cell transfusions

Treatments
Folic acid 3-5 mg per day Iron supplements Prophylactic coumadin? Transfusions as needed Eculizumab Bone marrow transplant Prednisone

Treatment Options
PNH/Aplastic Anemia
In addition to anemia, have low platelet count and/or low white count Bone marrow production is deficient Clone size may be large or small Clotting risk may be less (still increased compared to baseline)

Treatments
Immunosuppression (cyclosporine, ATG) Danazol (marrow stimulator) Folic acid and iron less important, but need to make sure adequate Erythropoietin supplementation Bone marrow transplant Prednisone Eculizumab (?)

Where is the field moving?

Many research questions still to be answered:
Why do PNH cells survive immune mediated insults better? Why clotting? Why does the PNH clone expand?

Better treatments? Improvement in supportive care and transplantation

Hemoglobinuria and ARF

It is one of the complications of hemoglobinuriaPigment Nephropathy Pathophysiology:

Hypovolemia and renal ischemia Direct hemoglobin nephrotoxicity Tubular obstruction Hemolysis(DIC-glomerular fibrin deposit) Risk factors: Older age, hypotension,dehydration and severe acidosis with low urinary ph

Myglobinuria v/s Hemoglobinuria

SL No 1 Factors Myoglobinuria Hemoglobinuria Urine colour Brown Reddish Brown

Serum colour

Clear

Pink

Orthotoludine test

Positive

Positive

Muscle pain

Present

Absent

CPK levels

Normal/Absent

Serum Haptaglobin

Normal

Decreased

March hemoglobinuria
March hemoglobinuria, also known as march hematuria, occurs when blood is seen in the urine after strenuous exercise, particularly affecting the feet (such as running on a hard roa d or Kendo) and hands (e.g. Conga or Candomb e drumming). The word "march" is in reference to the conditi on arising in soldiers who have been marching for long periods

 The repetitive nature of these types of activities cause mechanical trauma to the red blood cells causing hemolysis The situation usually lasts for a day or two. No specific medication is required. This hematuria is usually unnoticed by common man, and is figured out usually by one's who've had a history with hematuria. Usually March hematuria isn't life threatening.