Table of Contents
Epidemiology of PNH
Paroxysmal nocturnal hemoglobinuria as a rare disorder
Paroxysmal nocturnal hemoglobinuria typically presents in males and females in early adulthood and manifests throughout. It has an incidence of 10 cases per million with a 50 % mortality.
Etiology of PNH
PNH is an acquired defect in the myeloid stem cells with a mutation in the PIG-A gene located in the stem cells of bone marrows. With a PIG mutation affected red blood cells are known as „PNH RBCs“. The PNH RBCs lack the shield of proteins that protect normal red blood cells from the complement system. This is all due to deficiency in glycosylphosphatidylinositol (GPI). GPI are linked proteins on RBCs, neutrophils and platelets. If absent it renders cells susceptible to destruction.
Pathophysiology and Pathogenesis of PNH
The predisposing factor in anyone with PNH is the inability to synthesize GPI. GPI is located on the X chromosome, rendering only one mutation necessary to eliminate the expression of GPI linked proteins. One essential GPI membrane linked protein is decay accelerating factor (DAF). This protein interacts with complement proteins to neutralize the compliment attached to RBCs, neutrophils and platelets. In the absence of this protein, RBCs are susceptible to complement-mediated intravascular hemolysis. The destruction of RBC membranes by the complement releases hemoglobin into the bloodstream and circulation.
The body has a certain threshold in which it can degrade plasma hemoglobin, however once levels are reached, any extra leads to increased heme in the urine and plasma. This sequelae leads to further pathologies as increased hemoglobin levels deplete nitric oxide levels in circulation. Nitric oxide is needed for vasodilation, smooth muscle relaxation and vascular homeostasis. Decreased levels of nitric oxide leads to vasoconstriction, smooth muscle contractions and spasms.
When it comes to mortality and morbidity, PNH unfortunately has higher incidences, almost 50 %. This is due to pathophysiology of thrombosis secondary to PNH. Hemolysis increase the number of thrombotic events which when logged in multiple organs increases organ failure and insufficiency. Thrombotic events is also due to hypercoagulable states induced by free hemoglobin in the bloodstream.
Paroxysmal nocturnal hemoglobinuria is named due to episodic (paroxysmal) hemolysis occuring usually at night due to acidotic activation. It is believed mild respiratory acidosis develops with shallow breathing during sleep. This activates the complement mechanism leading to RBC, WBC and platelet lysis. This lysis leads to hemoglobinemia and hemoglobinuria in the morning.
Symptoms of PNH
Signs of paroxysmal nocturnal hemoglobinuria
Associated with a plethora of clinical findings, PNH is usually symptomatic, with a small percentage being asymptomatic. The first classic sign of PNH is hemolysis due to red blood cells lysis. This leads to symptoms of fatigue, jaundice and hematuria.
The side effects of hemolysis can be broken down by different manifestations in the body’s organs and pathways. Renal insufficiency arises due to intravascular hemolysis in PNH. Toxicity increases due to direct free heme in the kidney. Chronic hemolysis increases renal iron deposition leading to cortical scarring and infarcts.
Additionally, lysis leads to depletion of nitric oxide levels. Symptoms of decreased nitric oxide leads to smooth muscle dystonia, abdominal pain, cramping and finally erectile dysfunction. Abdominal pain is due to smooth muscle dystonia, leading to contraction of the visceral organs and vasculature leading to spasms and pain. Lack of nitric oxide also leads to decrease vascular dilation in genital tissue, specifically the corpora cavernosa leading to erectile dysfunction.
The most detrimental complication with PNH is thrombosis. It is the primary cause of death in these afflicted patients, occurring in over 40 % of individuals. Thrombi may lodge in the venous or arterial system such as hepatic, portal or cerebral veins causing cirrhosis, splenic congestion and cerebral strokes respectively. Symptoms may be insidious as clots aren’t found immediately. Skin necrosis, vein thrombosis and pulmonary embolisms to name a few. Some clots are found incidentally and unfortunately many go undetected leading to increased mortality.
Iron deficiency from blood loss leads to anemic like signs and symptoms i.e., fatigue, pallor (Image 2). dyspnea, headaches, tachycardia. Patients with aplastic anemia (a disorder where the body halts production of new red blood cells due to bone marrow damage), studies show, are more prone to develop PNH.
Diagnosis of PNH
Paroxysmal nocturnal hemoglobinuria detected by flow cytometry
Several laboratory measures are used to detect PNH. Typically, an evaluation consists of testing the patient for hemolytic anemia and to rule out other causes of hemolysis. This includes autoimmune pathologies or injuries. Most common performed analysis are:
- RBC smear
- Reticulocyte count
- Direct Coombs testing
- Urine hemoglobin or hemosiderin
Flow cytometry (Image 3), is the most specific and used to confirm PNH. Patients at risk for PNH, aplastic anemia or myelodysplastic disorder patients, are screened yearly to monitor development of subclinical PNH. The patient’s blood is incubated with tagged fluorescent antibodies that bind to GPI-linked proteins. The most commonly used antibodies are Fluorescent AERolysin (FLAER). This state-of-the-art laboratory test sends the patient’s blood for flow cytometry to detect CD59 (MIRL), a glycoprotein, and CD55 (DAF) in regulation of complement action. Absence or reduced expression of both CD59 and CD55 on PNH RBCs is
A second diagnostic test would be the sugar water or sucrose lysis test. The sugar water or sucrose lysis test uses the ionic strength of serum that is reduced by adding an iso-osmotic solution of sucrose, which then activates the classic complement pathway, and complement-sensitive cells are lysed.
Lab values typically show anemia, increased reticulocyte count, increased LDH, free serum hemoglobin with red serum, a negative Coombs test and iron deficiency. Paroxysmal nocturnal hemoglobinuria (PNH) leukocytes have a low leukocyte alkaline phosphatase (ALP) score.
Therapy of PNH
Therapy aimed at treated PNH is focused on targeting the underlying hemolytic defect and monitoring for progression. Patients with PNH have yearly screens for increased or decreased PNH clone sizes. As of now, the only therapies for classic PNH include hematopoietic cell transplantation (HCT) or complement inhibition with medications such as eculizumab. Those with multiple thrombi are treated with anticoagulation therapeutically. Iron supplementation is used for iron deficient patients in conjunction with folate. Those with bone marrow failures or malignancies are treated with immunosuppressive therapy and weekly WBC monitoring.
Opioids and analgesics are used for those suffering from smooth muscle dystonia and spasms. Women suffering from PNH interested in pregnancy pose a risk of increased maternal and fetal morbidity and mortality. Therefore strict iron and folate supplementation along with transfusions may be needed during pregnancy.
Overall, paroxysmal nocturnal hemoglobinuria (PNH) is a rare disorder yet life threatening disorder. From unexplained hemolytic anemia, thrombi and hematuria, all these lead to a painful increase in mortality. All patients should have a baseline testing and annually after. Management may be aggressive or mild depending on symptoms and severity. Overall, there is no cure for PNH at the moment but management and research is being done to help treat this hemolytic disease.
The answers are below the references.
1. A 26 year old male comes into the clinic for lab work due to suspected PNH. Past medical history shows recent onset of labored breathing, stomach pains and neuropathies. If diagnostic, his lab results would read all as follows except?
- Hemolytic anemia
2. A 56 year old female comes into the hospital for hemolytic anemia, red urine and difficulty breathing. She was diagnosed with paroxysmal nocturnal hemoglobinuria. Red blood cells in PNH patients are sensitive to which of the following?
- Complement proteins
- Warm antibodies
- Cold antibodies
- Nonoxidative stress
3. A 37 year old male is seen in your office for dyspnea and fatigue for 1 month. Past medical history is significant for hospitalization for hepatic vein thrombosis. On physical exam, patient was seen to have slight skin necrosis. Lab studies showed the following:
HGB: 5.9 gm%
MCV 80 fl
Platelets : 82 k/µl
Reticulocyte count 4.3 %
Haptoglobin : Undetectable
Lactic Dehydrogenase (LDH) 900 U/L
Direct Coombs Test : Negative
Which is the next best step in diagnosing the suspected condition?
- urine analysis
- flow cytometry
- blood smear
- PT/PTT/INR analysis