Erythropoietin (EPO)

The role of red blood cells is to carry oxygen. Just like anything in the body, this is tightly regulated by a mechanism that monitors whether or not there is adequate oxygen getting to tissues and other cells. Hypoxia is detected by the peritubular fibroblasts of the kidneys which causes erythropoietin (EPO) to be released. The EPO gene has a hypoxia-sensing region in its 3’ regulatory component which causes hypoxia inducible factor-1 (HIF-1) transcription factor to be assembled and it interacts with the 3’ enhancer of the gene causing increased EPO mRNA and production of more EPO.  EPO is a true hormone, being produced in the kidneys, and acting upon another distant location being the bone marrow. When EPO binds to its ligand (receptor) on red blood cell progenitors it initiates a cascade which is mediated through the JAK2 signal transducers which ultimately effects the gene expression. EPO has three main physiological effects on the body; it allows early release of reticulocytes from the bone marrow, prevents apoptosis, and reduces the time needed for cells to mature in the bone marrow before release into the periphery. 

There are two mechanism for which EPO stimulates early release of red cell precurors into the bone marrow. It induces changes in the adventitial cell layer of the marrow sinuses that increases the width of the spaces that the red cells squeeze out of. It also down regulates red blood cell surface receptors for adhesive molecules that are located on the bone marrow stroma. As a result the red cells are able to pass through without the receptor so that they won’t bind to the stroma and delay release.

Apoptosis is programmed cell death. EPO inhibits apoptosis by removing the induction signal. Under normal physiology the bone marrow produces more CFU-Es than needed that are stored in the bone marrow which have a “head start” in the maturation process. About a 10 day head start in maturation. The CFU-Es (Colony-forming unit-erythroid) are red blood cell progenitor cells that develop from BFU-Es (Burst-forming unit-erythroid). Both BFU-E and CFU-E are red blood cell progenitor cells that develop into the pronormoblast, which is the first morphologically identifiable red blood cell precursor. If healthy, those cells live out there life span and undergo apoptosis. If there is a deficiency of red blood cell mass, those cells undergo maturation to be released, while simultaneously the apoptosis induction signal is inhibited. The normal death signal consists of a death receptor being FAS, on the membrane of the earliest red blood cell precursors (CFU-Es/BFU-Es), and FASL ligand on the maturing red blood cells precursors. When EPO levels are low, because there is adequate oxygen delivery the older FASL bearing cells cross-link with earlier FAS precursors which stimulates apoptosis. EPO is able to subdue apoptosis by stimulating the more mature precursors to be released from the marrow, especially in times of hypoxia. At which point there will no FASL bearing cells to cross-link the early FAS bearing precursors. Its a two fold effect, the more mature cells are released to help increase red cell mass in times of need, and the early precursor are allowed to mature and be released without undergoing apoptosis. When EPO binds to its ligand on the red blood cell activates the JAK2-STAT pathway, which ends in and up-regulation of transcription for BCL-2, which is an anti-apoptotic protein. This anti-apoptotic protein rests on the cell membrane and prevents the release of cytochrome c, which initiates apoptosis. 

JAKSTAT

EPO has an effect on the bone marrow transit time of a red blood cell precursor in two different ways; increased rate of cellular processes, and decreased cell cycle times. What this means is that EPO stimulates synthesis of red cell RNA, such as the production of hemoglobin. It also stimulates the production of egress-promoting surface molecules within the bone marrow which allow the red blood cells to flow through the marrow easier. EPO stimulates cells to enter cell cycle arrest earlier than normal, and as a result, spend less time maturing and are able to be released. These cells may appear larger in size and have a bluish tinge to their cytoplasm because of this.

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Donath-Landsteiner Antibodies

The history of the DL antibody goes back to the 1900’s. It was one of the first recognized forms of immune mediated hemolysis and responsible for inducing Paroxysmal Cold Hemoglobinuria (PCH). PCH is a transient condition, meaning that it comes on when immunoglobulins (Antibodies) are formed in response to a viral, bacterial, or spirochete infection. Its history will suggest that there is an association between PCH and syphilis. In over 90% of the cases of PCH in early history, the patient was co-diagnosed with syphilis. Throughout the 1900’s the condition began to evolve and is now seen most commonly in children following some sort of infection. Although it should be noted that PCH is not limited to those of adolescent age. So what really is the Donath-Landsteiner antibody and how does it contribute to PCH?

Clinical Presentation

Paroxysmal Cold Hemoglobinuria (PCH) is an autoimmune hemolytic anemia (AIHA). Autoimmune meaning that they are antibodies that have cross-reacted to attack the individuals own cells. Hemoglobinuria means that there will be hemoglobin present in the blood, which suggests intravascular hemolysis. PCH is one of the more common intravascular hemolytic anemias. Typical patients present with fever, chills, abdominal and back pain, and pronounced hemoglobinuria. PCH typically presents in children following and upper respiratory infection or immunization. These patients often have a rapidly progressing anemia with hemoglobins that can fall as low as 2.5 g/dL. Peripheral blood smears show significant red blood cell agglutination and anisocytosis and poikilocytosis. Anisocytosis indicating variance in size of the red blood cells and poikilocytosis indicating variance in structure to the red blood cells. Schistocytes, spherocytes, and polychromasia are common findings. The spherocytes and polychromasia are indicative of the bone marrow trying to replenish the red cell population as best it can so it forces out immature erythrocytes into the peripheral blood. Its an effort to sustain the hemoglobin as best it can. One distinguishing peripheral blood smear finding in patients with PCH is erythrophagocytosis. Lets break this word down. Erythro- short for erythrocyte meaning red blood cells. Phagocytosis is mediated by neutrophils and monocytes as a way to kill foreign pathogens. In the case of erythrophagocytosis in PCH, neutrophils are characteristically seen engulfing red blood cells, which is diagnostic for AIHA.

The Donath-Landsteiner Antibody

The DL antibody, although being recognized as an cold autoantibody, is an IgG antibody that has developed P antigen specificity and it is a biphasic hemolysin. What that means is that when someone has the DL antibody and is exposed to cold temperatures, it will bind to the individuals red blood cells through the P antigen, but does not cause hemolysis until the coated red blood cells are heated to 37 degrees Celsius as they (RBC:antibody complex) travel from the peripheral fingertips and toes to the core of the human body.   At cold temperatures, the IgG molecule is able to recruit complement (C3), and at the higher temperatures, activates the membrane attack complex (C5-C9) and lyses the red blood cells. One very interesting piece of information regarding the difference between Cold Agglutinin Syndrome (CAS), another autoimmune hemolytic anemia caused by Anti-I, is that the hemolysis from PCH is stronger and more severe because of the DL antibodies ability to detach from lysed red blood cells and reattaching to other cells. 

Laboratory Diagnosis

There are a few different ways to pinpoint PCH in the blood bank. One is by use of a Direct Coombs test (DAT). This test provides information regarding the type of hemolysis, whether it be acquired or inherited. It also tests for antibodies that have are bound in vivo. The most common DAT result in PCH is red blood cells coated with C3d causing a positive reaction. This is sensitive in 94-99% of cases. The other way to diagnosis DLAIHA (Donath-Landsteiner Autoimmune Hemolytic Anemia) is by the indirect DL test. This process involves collection of a fresh serum specimen that is strictly maintained at 37 degrees Celsius from collection all the way through to testing. If the sample is allowed to cool or is refrigerated, there could potentially be autoadsorption of the DL anti-P antibodies onto the patients autologous red blood cells. This could cause a false negative result. Upon testing, the patients serum is mixed with P antigen positive, group O red blood cells, and fresh donor serum. The fresh donor serum is added because the complement level within the patients may be low due to consumption. The patient and donor serum mixture is incubated in a melting ice bath (O degrees Celsius) for 30 minutes, then warmed to 37 degrees Celsius for one hour. The specimen is then centrifuged and examined for hemolysis. If hemolysis is present then this constitutes a positive result for DL antibody.

Indirect-Donath-Landsteiner-test-Tube-1-OP-red-cells-suspension-patients-serum

Indirect DL test: As you can see in tubes 1 and 4, the presence of hemolysis indicates a positive test result for the DL antibody.

Treatment

There is unfortunately no cure for PCH, and very little reliable treatment options for those with the DL antibody. It is recommended to avoid cold climates as much as possible and when inside to have the temperature at 30 degrees Celsius to keep the hemoglobinuria low. This doesn’t treat the PCH, but it will minimize the recurrence and induced anemia. Steroids have been through extensive trials for treatment of PCH and there are mixed results. Theory is that steroids are better at clearing red blood cells coated with IgG, and less effective at clearing red blood cells that are coated with complement. More aggressive treatment such as splenectomy and Rituximab, which is an monoclonal antibody that targets the transmembrane protein CD20 present on B cells has been found effective for those patients with refractory PCH.

Blood System Portfolio: Rhesus System

The Rhesus blood system is arguably the second most important blood system behind the ABO system. There are 50 defined blood group antigens, among which the five antigens; D, C, E, c, e are the most significant. Individuals who are Rh positive possess the D antigen and those who are Rh negative lack the D antigen. Antibodies to Rh antigens play a major role in hemolytic transfusion reactions and cause significant risk for hemolytic disease of the fetus and newborn (HDFN).

Biochemistry

The gene locus for the Rh system antigens is located on chromosome 1. There are two genes that are closely related. RHD is a 417 amino acid sequence membrane protein that encodes for the D antigen. RHCE codes for a different membrane protein that carries the C/c and E/e antigens. A third gene, RHAG, located on chromosome 6 is associated with the expression of RHD and RHCE membrane proteins. RHAG NEEDS to be expressed for RHD and RHCE to be expressed. The Rh antigens are membrane bound non-glycosylated proteins (meaning that there is no carbohydrate attached) involved with membrane transport of cations. An individual who is C instead of c has a difference found in amino acid position 103, where C has a serine and c has a proline. An individual who has E antigen possesses a proline at amino acid position 226, and an individual who has the e antigen has an alanine at amino acid position 226.

History

The Rhesus blood system was discovered in 1937 by Karl Landsteiner and Alexander S. Wiener who named it the “Rhesus factor” because they believed it resembled an antigen found on rhesus monkey red cells. It was soon after that it was discovered that the human factor (Rh) is not at all similar to antigens found on the red cells of the rhesus monkey, although it stands today as a misnomer. Today in the United States 85% of the population are Rh positive and 15% are Rh negative. 70% of the population has the C antigen, 30% have the E antigen, 80% have the c antigen, and 98% have the e antigen. The Rh system currently has two sets of nomenclatures, one which was discovered by Ronald Fisher and R.R. Race, and the other by Alexander Wiener. Both systems are based on alternate theories which have both been since proved partially correct. The Fisher-Race system operates on the theory that separate genes control the product of each corresponding antigen. The Wiener system is based on the theory that there was a single gene on a single locus on each chromosome that gave rise to multiple antigens. Testing today shows that there are two genes that control the Rh system. The first one; RHD gene which produces a single antigen (D) and immune anti-D, and the RHCE gene which synthesizes the C, c, E, e antigens and corresponding antibodies.

Rh Testing

Some individuals can have a weak expression of the D antigen. They are Rh positive, but it is difficult to detect the presence of the antigen on the red cells. They require more sensitive methods of detection using anti-human globulin which is a poly specific CD3-IgG antibody reagent. It enhances the antigen-antibody complex formed so that agglutination is detected. Its important to detect weak D is cross-matching the donor and recipient blood samples especially when the recipient has anti-D in the serum. There are a few mechanisms for weak D expression. There can be a genetic weak D where a genetic variation of the D antigen is inherited. A partial D where the structure of the D antigen is made up of antigenic subparts where different D epitopes are missing or genetically altered.

Rh null is when there is absence of the RHAG gene. If individuals do not have a functioning RHAG gene there is no expression of genes RHD and RHCE and the corresponding antigens do not get expressed on the red cells. Red cell abnormalities have been observed with the phenotype including hemolytic anemia, decreased cell survival, stomatocytosis, spherocytosis, and altered activity of other blood group systems, most notably the MNS blood system.

Rh antibodies are IgG and are not detected at room temperature and need incubation at 37 degrees C. and the addition of a protein enhancement such as albumin or LISS to make detection more reliable. Anti-D is the most important antibody that can be formed. It takes just one exposure as the D antigen is extremely immunogenic. This typically happens through transfusion of antigen positive blood to an antigen negative recipient or through pregnancy and birth where there is maternal and fetal blood exchange where the mother gets sensitized. This is the basis of HDFN.

Important reminders regarding transfusion practice for the Rh system; Rh negative individuals should never receive Rh positive donor units. Rh positive individuals can receive Rh positive, but can in emergencies receive Rh negative. If there are Rh antibodies present, transfuse blood units that lack the Rh antigens to those antibodies. Sometimes its appropriate to phenotype and genotype a recipient or a donor. To do that the five different specific antisera is used to test for the five antigens that can be expressed. The purpose of Rh phenotypic and genotyping is to identify unexpected Rh antibodies, estimate the risk of HDFN in women, and in some cases can be used to exclude the male in paternity testing.